Mesenchymal Lineages Research Articles

Alpha terpineol directs bone marrow mesenchymal stem cells towardneuronal lineage through regulation of wnt signaling pathway.

Central nervous systemanomalies give rise to neuropathological consequences with immense damage to the neuronal tissues. Cell based therapeutics have the potential to manage several neuropathologies whereby the differentiated cells are explored for neuronal regeneration. The current study analyzes the effect of a bioactive compound, alpha terpineol (AT) on the differentiation of rat bone marrow derived mesenchymal stem cells (BM-MSCs) towardneuronal lineage, and explores regulation of differentiation process through the study of Wnt pathway mediators. BM-MSCs were cultured and characterized based on their surface markers and tri-lineage differentiation. Safe dose of AT as optimized by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide assay, was used for the treatment of MSCs. Treated cells were analyzed for the neuronal, astroglial and germ layer transition markers at the gene and protein levels, by quantitative polymerase chain reaction and immunocytochemistry, respectively. Temporal expression of Wnt pathway genes was assessed during the course of neuronal differentiation. AT treated group showed significant upregulation of neuron specific (NSE, MAP2, Tau, Nestin, and NefL) and astroglial (GFAP) genes with positive expression of late neuronal markers. Germ layer transition analysis showed the overexpression of ectodermal markers (NCAM, Nestin, and Pax6), whereas endodermal (AFP, MixL1, and Sox17), and mesodermal (Mesp1 and T Brachyury) markers were also found to be upregulated. Wnt signaling pathway was activated during the initial phase (30 min) of differentiation, which later was downregulated at 1, 3, and 5 h. AT efficiently induces neuronal differentiation of BM-MSCs by regulating Wnt signaling. Overexpression of both early and late neuronal markers indicate their neuro-progenitor state and thus can be utilized as a promising approach in cellular therapeutics to treat various neurodegenerative ailments. In addition, exploration of the molecular pathways may be helpful to understand the mechanism of cell-based neuronal regeneration.

A Cre knockin mouse reveals specific expression of Agouti gene in mesenchymal lineage cells in multiple organs and provides a unique tool for conditional gene targeting.

The mouse Agouti gene encodes a paracrine signaling factor which promotes melanocytes to produce yellow instead of black pigment. It has been reported that Agouti mRNA is confined to the dermal papilla after birth in various mammalian species. In this study, we created and characterized a knockin mouse strain in which Cre recombinase was expressed in-frame with endogenous Agouti coding sequence. The Agouti-Cre mice were bred with reporter mice (Rosa26-tdTomato or Rosa26-ZsGreen) to trace the lineage of Agouti-expressing cells during development. In skin, the reporter was detected in some dermal fibroblasts at the embryonic stage and in all dermal fibroblasts postnatally. It was also expressed in all mesenchymal lineage cells in other organs/tissues, including eyes, tongue, muscle, intestine, adipose, prostate and testis. Interestingly, the reporter expression was excluded from epithelial cells in the above organs/tissues. In brain, the reporter was observed in the outermost meningeal fibroblasts. Our work helps to illustrate the Agouti expression pattern during development and provides a valuable mouse strain for conditional gene targeting in mesenchymal lineage cells in multiple organs.

Osteoblast-Specific Overexpression of Nucleolar Protein NO66/RIOX1 in Mouse Embryos Leads to Osteoporosis in Adult Mice.

In previous study, we showed that nucleolar protein 66 (NO66) is a chromatin modifier and negatively regulates Osterix activity as well as mesenchymal progenitor differentiation. Genetic ablation of the NO66 (RIOX1) gene in cells of the Prx1-expressing mesenchymal lineage leads to acceleration of osteochondrogenic differentiation and a larger skeleton in adult mice, whereas mesenchyme-specific overexpression of NO66 inhibits osteochondrogenesis resulting in dwarfism and osteopenia. However, the impact of NO66 overexpression in cells of the osteoblast lineage in vivo remains largely undefined. Here, we generated osteoblast-specific transgenic mice overexpressing a FLAG-tagged NO66 transgene driven by the 2.3 kB alpha-1type I collagen (Col1a1) promoter. We found that overexpression of NO66 in cells of the osteoblast lineage did not cause overt defects in developmental bones but led to osteoporosis in the long bones of adult mice. This includes decreased bone volume (BV), bone volume density (bone volume/total volume, BV/TV), and bone mineral density (BMD) in cancellous compartment of long bones, along with the accumulation of fatty droplets in bone marrow. Ex vivo culture of the bone marrow mesenchymal stem/stromal cells (BMSCs) from adult Col1a1-NO66 transgenic mice showed an increase in adipogenesis and a decrease in osteogenesis. Taken together, these data demonstrate a crucial role for NO66 in adult bone formation and homeostasis. Our Col1a1-NO66 transgenic mice provide a novel animal model for the mechanistic and therapeutic study of NO66 in osteoporosis.

Changes in the mechanical properties of human mesenchymal stem cells during differentiation.

A thorough understanding of the changes in mechanical property behind intracellular biophysical and biochemical processes during differentiation of human mesenchymal stem cells (hMSCs) is helpful to direct and enhance the commitment of cells to a particular lineage. In this study, displacement creep of the mesenchymal cell lineages (osteogenic, chondrogenic and adipogenic hMSCs) were determined by using atomic force microscopy, which was then used to determine their mechanical properties. We found that at any stages of differentiation, the mesenchymal cell lineages are linear viscoelastic materials and well matched with a simple power-law creep compliance. In addition, the viscoelasticity of mesenchymal cell lineages showed different trends during differentiation. The adipogenic hMSCs showed continuous softening at all stages. The osteogenic and chondrogenic hMSCs only continuously soften and become more fluid-like in the early stage of differentiation, and get stiffened and less fluid-like in the later stage. These findings will help more accurately imitate cellular biomechanics in the microenvironment, and provided an important reference in the biophysics biomimetic design of stem cell differentiation.

Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification.

The expression of a mutant Lamin A, progerin, in Hutchinson-Gilford Progeria Syndrome leads to alterations in genome architecture, nuclear morphology, epigenetic states, and altered phenotypes in all cells of the mesenchymal lineage. Here, we report a comprehensive analysis of the transcriptional status of patient derived HGPS fibroblasts, including nine cell lines not previously reported, in comparison with age-matched controls, adults, and old adults. We find that Progeria fibroblasts carry abnormal transcriptional signatures, centering around several functional hubs: DNA maintenance and epigenetics, bone development and homeostasis, blood vessel maturation and development, fat deposition and lipid management, and processes related to muscle growth. Stratification of patients by age revealed misregulated expression of genes related to endochondral ossification and chondrogenic commitment in children aged 4-7 years old, where this differentiation program starts in earnest. Hi-C measurements on patient fibroblasts show weakening of genome compartmentalization strength but increases in TAD strength. While the majority of gene misregulation occurs in regions which do not change spatial chromosome organization, some expression changes in key mesenchymal lineage genes coincide with lamin associated domain misregulation and shifts in genome compartmentalization.

Nanohydroxyapatite-Chitosan Hydrogel Scaffold with Platelet Rich Fibrin and Buccal Fat Pad derived Stem Cell for Aggressive Periodontitis Treatment: A Narrative Review

Periodontal disease is oral health problem which attacks 20-50% of the global population. In Indonesia, 60% population suffers periodontal disease and become the second most common disease in the oral cavity. Aggressive periodontitis (AP) is one of periodontal disease, mainly attacks individual under 30 years-old, with rapid alveolar bone destruction. Bone graft implantation surgery is the current treatment for AP, but it is invasive and high risk infection. Nanohydroxyapatite-chitosan (nHPA-CS) hydrogel injectable scaffold is non-invasive treatment that contains bone mineral hydroxyapatite that derived from bovine bone and chitosan (CS) biopolymer with low immunogenicity. Platelet-Rich-Fibrin (PRF) contains huge amount growth factor which is biodegradable to human body. Buccal-Fat-Pad Stem-Cell (BFPSC) was chosen because it is easy to harvest and able to differentiate into mesenchymal lineage tissue. The aim of this narrative review is to describe the potential combination of nanohydroxyapatite-chitosan hydrogel scaffold, platelet-rich-fibrin, and buccal-fat-pad stem-cells for regenerating alveolar bone in aggressive-periodontitis patients. Data was obtained by literature study scientific research articles. In patients with AP, BFPSC can differentiate into osteoblast to improve osteogenesis, nHPA-CS scaffold creates host like tissue microenvironment to stimulate osteoinduction and osteoconduction activity of BFPSC, while PRF stimulates osteogenic differentiation and proliferation of BFPSC with insulin growth-factor, platelets derived growth-factor, transforming growth-factor, fibroblast growth-factor, vascular endothelial growth-factor. this combination increasing runt-related transcription-factor2, osterix, bone alkaline phosphatase, and controls pro-inflammatory cytokines such as interleukin-1beta (IL-1b) and tumor necrosis factor-alpha (TNF-a) so it can enhance bone regeneration and avoid bone destruction. The combination of nHPA-CS, PRF, BFPSC has the potential for alveolar bone regeneration in Aggressive-Periodontitis patients.

Discoidin domain receptor 2 regulates aberrant mesenchymal lineage cell fate and matrix organization.

Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)-mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.

Sarcomatoid carcinoma metastasis to bone marrow with unknown primary

Sarcomatoid carcinoma presenting as bone marrow metastasis with unknown primary is a rare entity. Usually patients present with widespread symptoms of extensive disease process. In the present case patient was evaluated for anemia and a subsequent bone marrow examination showed metastatic malignancy. All complimentary diagnostic tests were performed, Immunohistochemistry of bone marrow biopsy revealing presence of epithelia differentiation lineage markers with mesenchymal lineage markers also although routine biopsy showed only subtle mesenchymal component. The patient was referred to higher cancer centre and despite of elaborate laboratory work up including PET scan the primary origin of metastatic sarcomatoid carcinoma remained undetermined as patient unfortunately passed away within 10 days.

Soluble tumor endothelial marker 1 in heart failure with reduced ejection fraction: A pilot study.

Tumor endothelial marker 1 (TEM1/CD248) is a transmembrane protein that expresses in mesenchymal lineage derived cells during embryogenesis and becomes undetectable in normal adults after birth. Re-expression of TEM1 is found in organ fibrosis, wound healing and cardiac remodeling indicating its potential role in heart failure (HF). The purpose of this study is to explore the role of soluble TEM1 (sTEM1) in patients with HF with reduced ejection fraction. We examined endomyocardial biopsy specimens from three HF patients and blood samples from 48 patients admitted for acute decompensated HF (age 72 years, men 61.7%). The expression of TEM1 in cardiac tissue and concentrations of sTEM1 in plasma were evaluated. Cultured rat cardiomyocytes (H9c2) and human cardiac fibroblasts (HCF) were stimulated with hypoxia or transforming growth factor beta (TGF-β) to observe the release of sTEM1 into culture media. The conditioned media of hypoxia-stimulated H9c2 cells was harvested and added into cultured cardiac fibroblast to evaluate its biological effect. Immunofluorescence study of biopsy specimens from three HF patients showed TEM1 expression in cardiomyocytes and cardiac fibroblasts. The plasma level of sTEM1 was significantly higher in patients (0.90 ± 0.23 vs. 0.33 ± 0.10 ng/mL, p = 0.032) with LVEF ≤ 35% compared with those with LVEF 36-49%. The sTEM1 levels had correlations with HF biomarkers of cardiac fibrosis, including growth differentiation factor-15 (GDF-15) and galectin-3. There was a significant increase in sTEM1 levels in the cultured media of H9c2 and HCF after being stressed with hypoxia or TGF-β. The conditioned media derived from hypoxia-stimulated H9c2 cells significantly increased cell proliferation of cardiac fibroblasts. This effect was partially reversed by anti-TEM1 antibody. This pilot study demonstrated that cardiac TEM1 expression was upregulated in HF. The levels of sTEM1 were significantly higher in HF patients with LVEF ≤ 35% and correlated with other biomarkers of cardiac fibrosis. In vitro study proved that functional sTEM1 was released into cultured media after stressing cardiomyocytes and HCF.

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Repressive β-Catenin-Ikaros Complexes Are Essential to Prevent Clonal Evolution of Human Lymphopoiesis

Introduction β-catenin signaling represents a central driver of proliferation and survival in multiple epithelial, neuronal, and mesenchymal lineages, but is dispensable for hematopoietic development. Illegitimate activation of Wnt/β-catenin signaling induce development arrest at progenitor stages and underlies development of myeloid dysplastic syndrome (MDS). Moreover, β-catenin is required for emergence and self-renewal of leukemia stem cells (LSC), which are responsible for initiation and relapse of acute myeloid leukemia (AML). Experimental approach and results Our global interactome and gene editing experiments revealed that β-catenin engages lymphoid-specific Ikaros zinc finger (IKZF) factors to repress MYC and restrict lymphoid cell proliferation. Therefore, we reasoned that genetic deletion of β-catenin in human hematopoietic stem/progenitor cells (HSPC) could selectively benefit lymphoid development, which could be useful under lymphopenic conditions, immunosenescence and myeloid skewing in elderly. To this end, we introduced Crispr/Cas9 mediated deletion of β-catenin in CD34+ HSPCs from human cord blood and injected them into immune-deficient recipients for assessing multi-lineage reconstitution of human hematopoiesis. Compared to the controls, mice that received β-catenin-deleted progenitor cells showed increased human chimerism in the peripheral blood at 10 and 15 weeks after transplantation. Comparing absolute cell numbers, β-catenin-deficient progenitors gave rise to markedly increased human B-cell and T-cell production in the bone marrow and spleen, while myeloid populations were largely unchanged. Mice receiving β-catenin deficient cells contained significantly more human thymocytes compared to the control mice. These findings suggest that genetic deletion of β-catenin does not adversely impact human hematopoiesis and instead relieves lymphoid-intrinsic repression of B- and T-lymphopoiesis by β-catenin:IKZF complexes. Therefore, we hypothesized that deletion of β-catenin in pre-leukemic cells from MDS patients would favor generation of normal lymphocytes and prevent transformation into AML. To test this hypothesis, we deleted β-catenin in CD34+ HSPCs derived from bone marrow of MDS patients and injected them into immune-deficient recipients. 16 weeks after transplantation, we detected human B cells only in the blood of the recipients that received β-catenin deficient HSPCs but the mice that were injected with control cells only had myeloid cells in their blood. Interestingly, β-catenin deletion increased lymphoid cell generation by 5-and 2-fold in the bone marrow and spleen, respectively. Pseudo-bulk analysis of single-cell RNA sequencing of MDS-derived bone marrow cells revealed that upon β-catenin deletion, genes that define leukemic stem cell signature in AML (LSC17) such as CD34, SOCS2, EMP1 etc. were repressed. Strikingly, β-catenin deleted bone marrow progenitors from MDS patients could migrate to the thymus and give rise to de-novo thymopoeisis, while none of the mice that received control cells could generate human thymocytes. Although β-catenin deletion improved human lymphopoiesis from pre-leukemic HSPCs, we observed evolution into T-ALL and CD8+ TCR γδ T-cell lymphoma in two cases studied. In the first case human thymocytes generated from HSPCs with 5q- deletion, were arrested at CD4CD8 double positive stage (CD7+ CD3-), resembling a subgroup of T-cell acute lymphoblastic leukemia (T-ALL) with 5q- deletions. In the second case, deleting β-catenin in HSPCs carrying EZH2G660V mutation led to clonal expansion of CD8+ γδ-T cells in spleen of the mice. Conclusion In summary, deletion of β-catenin in human HSPCs improved lymphoid output in humanized mice. However, in the absence of β-catenin-IKZF mediated tumor-suppression, HSPCs carrying 5q- deletion and EZH2G660V mutation evolved into T-ALL and γδ-T cell lymphoma, respectively. These results suggest, although targeting Wnt/β-catenin pathway may be a promising strategy to treat MDS and AML and improve lymphopoiesis, interference with β-catenin-IKZF mediated restraint of lymphoid expansion may unleash pathological lymphocytes and lead to development of lymphoid malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unraveling and Visualizing the Interactions of Acute Myeloid Leukemia with the Bone Marrow Microenvironment in Vitro and In Vivo

The bone marrow (BM) is a dynamic stem cell microenvironment, harboring hematopoietic stem cells (HSCs) and various non-blood components such as endothelium, mural cells, multipotent stromal cells (MSCs) and their derivatives including osteoblasts, adipocytes, and fibroblasts. This stromal niche supports HSC-fate decisions and has been proposed as a major determinant of chemoresistance in hematological malignancies, such as acute myeloid leukemia (AML). Following chemotherapy, AML patients often suffer from relapse, in part due to chemoresistant leukemic stem cells (LSCs). There have been various indications in literature that leukemic cells remodel their niche, creating a permissive microenvironment for LSCs. However, which cell types respond and which underlying molecular mechanisms are involved skewing the compositional changes in the BM microenvironment remain unclear. Here, we aim to investigate how high LSC frequency (LSChigh) versus low LSC frequency (LSClow) burden AML initiates BM niche remodeling in the favor of AML expansion, with a particular focus on the MSC lineages. We further aim to determine which stromal cell types interact with LSCs and how their composition is altered upon AML development. Additionally, we aim to visualize which cell types interact with and are positioned in close proximity to LSCs. We combine single-cell transcriptomics of AML xenograft models and in vitro co-cultures of MSCs with primary AML. First, we set up clinically relevant murine xenograft models in which primary AML cells with either a low vs high LSC frequency (Garg et al., 2019) were injected in immune-deficient NSGW41 mice. As control, we injected cord blood-derived HSPCs. 6-7 weeks after engraftment, we harvested and digested the bones using our established protocol (adapted from Baccin et al., 2020) to recover the stromal cells with high efficiency. Besides, we also kept a femur per mouse for follow-up (multiplexed) immunofluorescence imaging. We enriched for human and mouse HSPCs, human AML cells, and mouse niche cells using FACS-based sorting, followed by scRNA-seq. Additionally, we isolated BM-derived mononuclear cells from healthy donors and AML patients and expanded the MSCs in vitro. We set up 4 independent co-cultures with MSCs and primary AML cells with LSChigh versus LSClow frequency. After 48 hours, we performed scRNA-seq to investigate transcriptomic changes and subsequent variant biological processes in the MSCs. scRNA-seq profiling of the BM niche of our AML xenograft models demonstrates that AML presence significantly impacts the niche composition, leading to the expansion of Cxcl12-abundant-reticular adipocytes (adipo-CAR) and the decline of osteoblasts. When comparing LSChigh versus LSClow, we detect significant differences in abundance of certain stromal cells, including endothelium, adipo-CARs, and fibroblast subtypes. This suggests that the LSC-burden plays a critical role in the extent of niche remodeling. To further uncover the direct effects of LSCs on MSCs, we have established an AML-MSC co-culture system and perform scRNA-seq on the MSCs. In line with our in vivo data, we observe that cultured BM MSCs, especially upon exposure to LSChigh AML, suppress osteoblast lineage genes while overexpressing fibrosis-related genes. We are validating our observations using multiplexed immunofluorescence to further investigate the direct cell-cell communication of LSCs with their microenvironment. Taken together, our data in murine AML models and human AML-MSC co-cultures unravel new insights into the BM stem cell niche composition and highlight a specific LSC-induced skewing of the mesenchymal lineage. Our data helps to better understand to which extent the BM microenvironment impacts AML disease progression and relapse due to residual LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 12013: Nuclear Localization of PDGFRa Tyrosine Kinase Represents a Novel Mechanism Controlling the Fibrotic Response of Mesenchymal Stromal Cells to Myocardial Ischemia

Background: We discovered a time dependent dual role role for mesenchymal stromal cells expressing the platelet-derived growth factor alpha (PDGFRα + MSCs), which play essential functions in tissue revascularization and in fibro-adipogenic repair. Hypothesis: Based on our preliminary data, we hypothesized that specific and distinct stromal cells exist in the vertebrate heart, which retain regenerative capacities relevant to the extension of disease. Methods: All animal studies were performed in compliance with the regulations of the NIH and the local institutional animal care. Myocardial ischemia/reperfusion (MI/R) was induced in mice (n=10, FVB female mice) and pigs (n=10 female Yorkshire pigs) with standard procedures. Control animals were not operated (n=3 mice and n=4 pigs). Bulk RNA sequencing (n=4) was performed using a HiSeq2500 (Illumina). Results: Here, we showed that cardiac PDGFRα + MSCs have the potential to revascularize cardiac tissue and likely control cardiac electromechanical properties only earlier after MI/R. During the subacute phase, we discovered nuclear shuttling of the receptor from the plasma membrane before lineage specification into fibrotic cells (nuclear PDGFRα in non-ischemic 3±1 versus ischemic 7.7±2.5 hearts; p<0.05). Interestingly, a similar response was observed in the heart of large animals (i.e., pigs), suggesting a cross-species mechanism controlling PDGFRα + MSCs commitment via intracellular shuffling of the tyrosine kinase receptor. A roadblock to the clinical translation of these discoveries is that there is no mechanism to manipulate PDGFRα + MSCs dual function. To cover this gap, we compared the transcriptomics of murine cardiac and skeletal muscle PDGFRα + MSCs using bulk RNA sequencing in physiological conditions. We discovered that key pathways, likely a signature of non-regenerating stromal cells, are differentially regulated in cardiac PDGFRα + MSCs, including Notch mediators of mesenchymal lineages and IGF-1 signaling. Conclusions: These data suggest that by modulating PDGFRα intracellular shuttling or altering the expression of specific genes, it may be possible to therapeutically direct PDGFRα + MSCs duality to reduce fibrosis, while harnessing their beneficial properties.

Osteocytes regulate senescence of bone and bone marrow.

The skeletal system contains a series of sophisticated cellular lineages arising from the mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) that determine the homeostasis of bone and bone marrow. Here, we reasoned that osteocyte may exert a function in regulation of these lineage cell specifications and tissue homeostasis. Using a mouse model of conditional deletion of osteocytes by the expression of diphtheria toxin subunit α in dentin matrix protein 1 (DMP1)-positive osteocytes, we demonstrated that partial ablation of DMP1-positive osteocytes caused severe sarcopenia, osteoporosis, and degenerative kyphosis, leading to shorter lifespan in these animals. Osteocytes reduction altered mesenchymal lineage commitment, resulting in impairment of osteogenesis and induction of osteoclastogensis. Single-cell RNA sequencing further revealed that hematopoietic lineage was mobilized toward myeloid lineage differentiation with expanded myeloid progenitors, neutrophils, and monocytes, while the lymphopoiesis was impaired with reduced B cells in the osteocyte ablation mice. The acquisition of a senescence-associated secretory phenotype (SASP) in both osteogenic and myeloid lineage cells was the underlying cause. Together, we showed that osteocytes play critical roles in regulation of lineage cell specifications in bone and bone marrow through mediation of senescence.

Immunological detection of Platelet-derived growth factor and Vascular endothelial growth factor in Iraqi women with Polycystic Ovarian Syndrome

Background: platelet-derived growth factor (PDGF) signaling controls a wide range of the mesenchymal cell origin lineages throughout the development and in the females with the polycystic ovarian syndrome. Vascular endothelial growth factor (VEGF) has an important impact on vascularization of the tissues generally, and in pathogenesis of a wide range of the illnesses. The PCOS has been found related to high VEGFexpression levels. Aims and Objectives: for the detection and studying of immunological effects of PDGF and VEGF in the Iraqi females with the PCOS. Materials and Methods: 250 Iraqi females, aged between 20 and 50 years have been divided to twogroups, which are: study group (n=125, PCOS) and control group (n=125, which mainly consisted from normal females), the samples of the blood have been obtained in 2ndmenstruate cycle day, VEGF and PDGF concentrations have been estimated for every one of the groups. Results: the PCOS patients have shown a considerable difference in comparison to the other group. The level has been 71.22±65.23Pg/ml and 243± 153.44Pg/ml, for the controls and PCOS patients respectively. In addition to that, the PCOS patients have shown a considerable difference in comparison to the other group. . The level has been 71.22±65.23Pg/ml and 243± 153.44Pg /ml, for the controls and PCOS patients respectively. Conclusions: Increases concentration of PDGF and VEGF in PCOS women related to ovulation lack and that lead to increases the risk of PCOS appearance.

In vitro effects of ascorbic acid on viability and metabolism of patients' osteosarcoma stem cells.

Stagnation in novelties of osteosarcoma (OS) treatment indicates the need for new therapeutic methods. OS cancer stem cells (OS-CSC) are taught to have the ability to self-renew and develop mechanisms of anticancer drug resistance, and this is why it is difficult to eradicate them. Their metabolism has been recognized as a potential target of therapeutic action. Ascorbic acid (AA) is considered to act pro-oxidative against OS-CSC in vitro by oxidative effect and by inhibition of glycolysis. This study examined an in vitro impact of AA on OS-CSC metabolism isolated from patients' biopsies, with the aim of better understanding of OS-CSC metabolism and the action of AA on OS-CSC. OS-CSC were isolated using a sphere culture system and identified as stem cells using Hoechst 33342 exclusion assay. Determination of the dominant type of metabolism of OS-CSC, parental OS cells, human mesenchymal stem cells (hMSC) and U2OS OS lineage before and after AA treatment was done by Seahorse XF (Agilent). Cytotoxicity of high-dose AA was confirmed by the MTT test and was proven for all the examined cell types as well as HEK293. Seahorse technology showed that OS-CSC can potentially use both glycolysis and oxidative phosphorylation (OXPHOS), and can turn to glycolysis and slow metabolic potential in unfavorable conditions such as incubation in AA.

Molecular Mechanisms Underlying Pathological and Therapeutic Roles of Pericytes in Atherosclerosis.

Pericytes are multipotent mesenchymal stromal cells playing an active role in angiogenesis, vessel stabilisation, maturation, remodelling, blood flow regulation and are able to trans-differentiate into other cells of the mesenchymal lineage. In this review, we summarised recent data demonstrating that pericytes play a key role in the pathogenesis and development of atherosclerosis (AS). Pericytes are involved in lipid accumulation, inflammation, growth, and vascularization of the atherosclerotic plaque. Decreased pericyte coverage, endothelial and pericyte dysfunction is associated with intraplaque angiogenesis and haemorrhage, calcification and cholesterol clefts deposition. At the same time, pericytes can be used as a novel therapeutic target to promote vessel maturity and stability, thus reducing plaque vulnerability. Finally, we discuss recent studies exploring effective AS treatments with pericyte-mediated anti-atherosclerotic, anti-inflammatory and anti-apoptotic effects.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Morphogenesis of the female reproductive tract along antero-posterior and dorso-ventral axes is dependent on Amhr2+ mesenchyme in mice†.

Morphogenesis of the female reproductive tract is regulated by the mesenchyme. However, the identity of the mesenchymal lineage that directs the morphogenesis of the female reproductive tract has not been determined. Using in vivo genetic cell ablation, we identified Amhr2+ mesenchyme as an essential mesenchymal population in patterning the female reproductive tract. After partial ablation of Amhr2+ mesenchymal cells, the oviduct failed to develop its characteristic coiling due to decreased epithelial proliferation and tubule elongation during development. The uterus displayed a reduction in size and showed decreased cellular proliferation in both epithelial and mesenchymal compartments. More importantly, in the uterus, partial ablation of Amhr2+ mesenchyme caused abnormal lumen shape and altered the direction of its long axis from the dorsal-ventral axis to the left-right axis (i.e., perpendicular to the dorsal-ventral axis). Despite these morphological defects, epithelia underwent normal differentiation into secretory and ciliated cells in the oviduct and glandular epithelial cells in the uterus. These results demonstrated that Amhr2+ mesenchyme can direct female reproductive tract morphogenesis by regulating epithelial proliferation and lumen shape without affecting the differentiation of epithelial cell types.

Abstract A011: Emergence of persister cells following bromodomain inhibition in Ewing sarcoma

Abstract Ewing sarcoma (ES) is driven by the tumor initiating fusion EWS::FLI1 that causes widespread transcriptional dysregulation via epigenetic reprogramming. ES cells are highly plastic and heterogenous and have features of both neural crest and mesenchymal lineages. Epigenetic plasticity is well established as a mediator of disease progression and drug resistance, leading to the creation of heterogeneous tumor cell subpopulations that can initiate metastasis and relapse. Successful therapeutic strategies for metastatic or relapsed ES patients are limited and unchanged in the last three decades. Identification of new therapeutic strategies, and defining the effect of new agents on ES biology, is critical to improving patient outcomes. Given the epigenetic dependencies of ES, epigenetic modifying agents are an important class of drugs to investigate. The class of bromodomain and extra-terminal domain protein inhibitors (BETi) have shown preclinical efficacy in ES models but drug combinations will be needed. We are testing the hypothesis that transcriptional rewiring downstream of BETi will create new dependencies in ES cells that could be therapeutically exploited with biologically-defined combinations. Our studies show that the BETi, BMS-986158, the first BETi to be tested in pediatric patients, has profound cytostatic effects in preclinical ES models in vitro and in vivo. Our data further reveal that long term exposure to BMS-986158 creates a persister population of cells that reacquires proliferative capacity. We performed RNA-seq studies on parent, acutely treated (72 hours), and persister populations from three ES cell lines to define the effects of short and long term BET inhibition. Exposure to BMS-986158 led to strong inhibition of the EWS::FLI1-activated gene signature that was evident both acutely and in persister cells. Thus, despite continued inhibition of EWS::FLI1 activity, persister cells restored their ability to survive and proliferate. Gene Set Enrichment Analysis of transcripts that were significantly altered in persister cells identified gene programs involved in development and lineage commitment, including polycomb targets, suggesting that persister cells may have been rewired to more stem-like states. In addition, in two of the three cell lines, persister cells demonstrated upregulation of MYC target genes and cell cycle programs that were initially repressed by BET inhibition. Ongoing studies are investigating the molecular mechanisms by which MYC programs are reactivated in persister cells. We anticipate that epigenetic reprogramming mediates the transcriptional rewiring of MYC programs in persister cells, thereby escaping the cytostatic effects of continued BET inihbition. By defining these mechanisms we aim to identify new vulnerabilities that can be exploited in the context of BETi combination trials in the future. Citation Format: Shireen S. Ganapathi, Nicolas M. Garcia, Veerin R. Sirihorachai, Elizabeth R. Lawlor. Emergence of persister cells following bromodomain inhibition in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A011.