Stromal Factor Research Articles

Gene Regulatory Network Analysis of Decidual Stromal Cells and Natural Killer Cells.

Human reproductive success relies on the proper differentiation of the uterine endometrium to facilitate implantation, formation of the placenta, and pregnancy. This process involves two critical types of decidual uterine cells: endometrial/decidual stromal cells (dS) and uterine/decidual natural killer (dNK) cells. To better understand the transcription factors governing the in vivo functions of these cells, we analyzed single-cell transcriptomics data from first-trimester terminations of pregnancy, and for the first time conducted gene regulatory network analysis of dS and dNK cell subpopulations. Our analysis revealed stromal cell populations that corresponded to previously described in vitro decidualized cells and senescent decidual cells. We discovered new decidualization driving transcription factors of stromal cells for early pregnancy, including DDIT3 and BRF2, which regulate oxidative stress protection. For dNK cells, we identified transcription factors involved in the immunotolerant (dNK1) subpopulation, including IRX3 and RELB, which repress the NFKB pathway. In contrast, for the less immunotolerant (dNK3) population we predicted TBX21 (T-bet) and IRF2-mediated upregulation of the interferon pathway. To determine the clinical relevance of our findings, we tested the overrepresentation of the predicted transcription factors target genes among cell type-specific regulated genes from pregnancy disorders, such as recurrent pregnancy loss and preeclampsia. We observed that the predicted decidualized stromal and dNK1-specific transcription factor target genes were enriched with the genes downregulated in pregnancy disorders, whereas the predicted dNK3-specific targets were enriched with genes upregulated in pregnancy disorders. Our findings emphasize the importance of stress tolerance pathways in stromal cell decidualization and immunotolerance promoting regulators in dNK differentiation.

Clinical Value of Stromal Cell Factor (Sdf-1) Determination in Chemotherapy-Induced Peripheral Polyneuropathy in Patients with Hematological Malignancies (Results of a Prospective Cohort Study)

Clinical Value of Stromal Cell Factor (Sdf-1) Determination in Chemotherapy-Induced Peripheral Polyneuropathy in Patients with Hematological Malignancies (Results of a Prospective Cohort Study)

Evaluation of Yukmijihwang-Tang as a Novel Preventive Agent in Ovalbumin-Induced Murine Asthma

Yukmijihwang-tang (YJT) is a blend of six herbal ingredients that has long been used in Asia for various diseases, including diabetes mellitus and mental decline. Here, we assessed the prophylactic efficacy of YJT in a murine model of asthma induced by ovalbumin (OVA). Male C57BL/6 mice underwent sensitization followed by an airway challenge with OVA for 1 h. YJT (100 or 300 mg/kg once daily) was administered orally for 6 days. Our analyses revealed that YJT significantly reduced inflammatory cell counts in bronchoalveolar lavage fluid (BALF), decreased the concentrations of interleukin (IL)-4, IL-5, and IL-13 in BALF, and lowered the relative mRNA levels of thymic stromal lymphopoietin (TSLP) and tumor necrosis factor α (TNFα) in lung tissues. Histological analyses showed that YJT substantially decreased inflammation and mucus hypersecretion in the bronchial airway. YJT also effectively reduced oxidative stress, as evidenced by improved levels of malondialdehyde (MDA), total glutathione, glutathione reductase (GR), and reduced glutathione (GSH). YJT also markedly suppressed the phosphorylation of nuclear factor-kappa B (NF-κB) in lung tissues. Together, these results show that YJT effectively inhibits respiratory inflammation, mucus hypersecretion, and oxidative stress induced by OVA, suggesting its potential for asthma treatment.

P146 Latent activation of the extrinsic coagulation pathway in patients with ulcerative colitis

Abstract Background Ulcerative colitis (UC) is associated with an increased risk of venous thromboembolism. We investigated the correlation of UC severity with latent activation of the extrinsic coagulation pathway and colonic stromal tissue factor (TF) expression. Methods Plasma exosomes, microparticles (EM), TF protein, activities of EM-bound TF (EM-TF) and FVIIa, as well as TF mRNA (F3) in primary colonic stromal cells in culture were measured. Disease extent by Montreal classification, medications, UC clinical activity by partial Mayo score (PMS), endoscopic severity by the MS endoscopic component (eMS; loss of response defines as ≥2), quality of life by the short IBD questionnaire- SIBDQ, complete blood count, CRP, ESR and albumin were retrieved. Statistical significance (p) was assessed with the following tests: Student’s t for 2 groups with normally distributed values, Mann-Whitney U if not, Wilcoxon test for paired data. Correlation of continuous against scale variables was assessed with Pearson’s or Spearman’s r and corresponding p values for linear or logarithmic continuous variables, respectively. Results Plasma was obtained from 38 UC patients (31 males, disease duration 151±25 months, 14 with extensive colitis, 5 current smokers) and 28 healthy blood donors (HBD; 20 males) who served as controls. TF mRNA (F3) was measured in primary colonic stromal cells in culture from 12 UC patients (11 males, disease duration 169±53 months, 8 with extensive colitis, 1 current smoker) and from 7 controls undergoing screening colonoscopy. UC patients had 4- and 3.7- times more exosomes and microparticles than controls, respectively (A). TF protein correlated with PMS (r 0.443), albumin (-0.362), ESR (0.353), PLT (0.575) and endoscopic UC severity (0.468; B). EM-TF activity was 1.6-times higher (C) and correlated to disease extent (E3: 1.9 x E2), albumin (-0.624), endoscopic severity (0.422; D) and SIBDQ (-0.64). Downstream, FVIIa activity was 2.2-times higher. Refractory-to-treatment patients had 5.8-, 1.5-, 2.1- times higher TF protein (E), MP-TF, FVIIa activity, respectively. Within responders, the need for steroids or biologics correlated with 2.2-times higher MP-TF activity. Colonic stromal cells from patients with UC maintained a 2.2-times higher F3 (F), which correlated to PMS (0.56), albumin (Spearman’s r -0.543), endoscopic severity (M3: 8.3 x M2). All the aforementioned were statistically significant with a < 0.05. Conclusion The extent of spontaneous activation of the extrinsic coagulation pathway is associated with clinical and endoscopic UC activity and response to treatment. TF in colonic stromal cells mirrors its systemic activity and indicates the possible contribution of such cells to increased TF plasma bioactivity.

Effects of platelet-rich plasma and stromal vascular factor combination on fibroblast count in anal trauma in Wistar rats

Effects of platelet-rich plasma and stromal vascular factor combination on fibroblast count in anal trauma in Wistar rats

SOCS3 inhibits the mesenchymal stromal cell secretory factor SDF-1-mediated improvement of islet function in non-obese diabetic mice

BackgroundIslet transplantation is used therapeutically in a minority of patients with type 1 diabetes (T1D). However, successful outcomes are hampered by early islet β-cell loss caused by immune rejection and autoimmunity. Recent studies have demonstrated that mesenchymal stromal cells can enhance islet function both in vitro and in vivo by secreting ligands that activate islet G-protein coupled receptors (GPCRs). Stromal cell-derived factor 1 (SDF-1) is an MSC-secreted GPCR ligand, whereas the suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of STAT3-activating cytokines. Here, we determined whether improvement in islet function mediated by exogenous SDF-1 is impaired by SOCS3 in experimental models of T1D.MethodsIsolated islets were cultured for 48 h with SDF-1. Cytokine-induced apoptosis was measured immediately. Islets from Socs3−/− mice were pre-cultured with exogenous SDF-1 and transplanted underneath the kidney capsule of C57BL/6 mice with streptozotocin-induced diabetes. Blood glucose levels were monitored for 28 days. AMD3100, an antagonist of the SDF-1 ligand CXCR4, was administered subcutaneously to islet transplanted mice to inhibit CXCR4 before and after transplantation.ResultsSDF-1 protected islet cells from cytokine-induced apoptosis in vitro. SOCS3-knockout (KO) islets pretreated with SDF-1 were effective in reducing blood glucose in non-obese diabetic mice in vivo. We found that SDF-1 elicits localized immunosuppression in transplanted SOCS3-KO islets. Immunomodulation was observed when SOCS-KO islets were preconditioned with SDF-1. Gene expression and flow cytometric analyses revealed significantly decreased immune cell infiltration, inflammatory cytokines, and concomitant increases in FOXP3+ regulatory T cells, alternatively activated M2 macrophages, and dendritic cell phenotypes. Administration of AMD3100 impaired the SDF-1-mediated improvement in SOCS3-KO islet function and local immune suppression.ConclusionSDF-1 improves the function of islet grafts in autoimmune diabetes through regulation by CXCR4; however, the presence of SOCS3 reverses the protective effect of SDF-1 on islet grafts. These data reveal a molecular pathway that can elicit localized immunosuppression and delay graft destruction in transplanted islets.

KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration

Upon transplantation, skeletal stem cells (also known as bone marrow stromal or mesenchymal stem cells) can regulate bone regeneration by producing secreted factors. Here, we identify KIAA1199 as a bone marrow stromal cell-secreted factor in vitro and in vivo. KIAA1199 plasma levels of patients positively correlate with osteoporotic fracture risk and expression levels of KIAA1199 in patient bone marrow stromal cells negatively correlates with their osteogenic differentiation potential. KIAA1199-deficient bone marrow stromal cells exhibit enhanced osteoblast differentiation in vitro and ectopic bone formation in vivo. Consistently, KIAA1199 knockout mice display increased bone mass and biomechanical strength, as well as an increased bone formation rate. They also exhibit accelerated healing of surgically generated bone defects and are protected from ovariectomy-induced bone loss. Mechanistically, KIAA1199 regulates osteogenesis by inhibiting the production of osteopontin by osteoblasts, via integrin-mediated AKT and ERK-MAPK intracellular signaling. Thus, KIAA1199 is a regulator of osteoblast differentiation and bone regeneration and could be targeted for the treatment or management of low bone mass conditions.

CLPB3 is required for the removal of chloroplast protein aggregates and for thermotolerance in Chlamydomonas.

In the cytosol of plant cells, heat-induced protein aggregates are resolved by ClpB/Hsp100 family member HSP101, which is essential for thermotolerance. For chloroplast family member CLPB3 this is less clear with controversial reports on its role in conferring thermotolerance. To shed light onto this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TIG1 and the small heat shock proteins HSP22E/F in vivo and for conferring thermotolerance under heat stress. Although CLPB3 accumulates to similarly high levels as stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the ribosomal subunit PRPL1 and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that reduction in chloroplast protein synthesis capacity and increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely to the thylakoid membrane occupied area. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.

MORPHOLOGICAL FEATURES OF THE ENDOMETRIUM TRANSFORMATION IN RECURRENT MISCARRIAGE

The problems of pathological diagnosis of recurrent miscarriage are relevant in modern reproductive medicine. One of the important molecular markers of endometrium is the progesterone-induced blocking factor, which is induced by progesterone under the influence of activated lymphocytes and has an immunomodulatory effect on the implantation characteristics of the endometrium. In the process of trophoblast invasion, a special role is also played stromal cell factor molecules synthesized by endometrial and trophoblast cells, as a mechanism that potentiates the susceptibility of the endometrium to the onset and development of pregnancy, trophoblast invasion and embryogenesis in general. The purpose of the study was the histological and immunohistochemical features of the transformation of the endometrium in recurrent miscarriage. It were studied 100 samples of endometrium in case of non-developing pregnancy at a development period of 5-8 weeks, 85 samples in habitual miscarriage and 15 samples in pregnancy interrupted surgically at the request of the woman. The presence of 2 non-developing pregnancies was verified in 57 cases (67,1%), the presence of 3 non-developing pregnancies in 28 cases (32,9%). A histological examination of the abortion material stained with hematoxylin and eosin was performed to verify the morphological transformation of the endometrium. Immunohistochemical study included detection of estrogen and progesterone receptors, stromal cell factor (SDF1), progesterone-induced blocking factor (PIBF). The results of histological and immunohistochemical studies showed that with a complete morphological transformation of the endometrium in the group of patients with habitual miscarriage, there is a violation of the receptor profile. The decrease in PIBF expression in the glands and the stromal component and the expression of SDF1 in the glands of the compact layer of the endometrium is a reflection of the immunological imbalance in the endometrium in recurrent miscarriage. The development of a unified morphological algorithm, taking into account the basic indicators of the processes of transformation of the endometrium with an assessment of its receptor profile and diagnostically significant immunological factors, makes it possible to verify the pathology of the endometrium at the molecular level and justify the need for using pathogenetically substantiated therapy at recurrent miscarriage.

A miniaturized screening platform to identify novel regulators of extracellular matrix alignment.

Extracellular matrix alignment contributes to metastasis in a number of cancers and is a known prognostic stromal factor; however, the mechanisms controlling matrix organization remain unclear. Cancer-associated fibroblasts (CAF) play a critical role in this process, particularly via matrix production and modulation of key signaling pathways controlling cell adhesion and contractility. Stroma normalization, as opposed to elimination, is a highly sought strategy, and screening for drugs that effectively alter extracellular matrix (ECM) alignment is a practical way to identify novel CAF-normalizing targets that modulate ECM organization. To meet this need, we developed a novel high-throughput screening platform in which fibroblast-derived matrices were produced in 384-well plates, imaged with automated confocal microscopy, and analyzed using a customized MATLAB script. This platform is a technical advance because it miniaturizes the assay, eliminates costly and time-consuming experimental steps, and streamlines data acquisition and analysis to enable high-throughput screening applications. As a proof of concept, this platform was used to screen a kinase inhibitor library to identify modulators of matrix alignment. A number of novel potential regulators were identified, including several receptor tyrosine kinases (c-MET, tropomyosin receptor kinase 1 (NTRK1), HER2/ERBB2) and the serine/threonine kinases protein kinase A, C, and G (PKA, PKC, and PKG). The expression of these regulators was analyzed in publicly available patient datasets to examine the association between stromal gene expression and patient outcomes.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Multipotent Progenitor Niche Factors

Signal transducer and activator of transcription 5 (STAT5a and STAT5b isoforms) are latent transcription factors that are intrinsically critical for normal hematopoiesis but are also expressed in bone marrow stromal cells. However, despite known STAT5a (prolactin) and STAT5b (growth hormone) functions in breast and liver tissue respectively, hematopoiesis-supporting stromal functions have not been explored. Here, floxed STAT5ab double knockout (KO) was generated with a variety of hematopoietic and stromal Cre-expressing transgenic mouse strains to test whether STAT5a or STAT5b play a role in extrinsic regulation of hematopoiesis. Pan-hematopoietic deletion with Vav1-Cre was the positive control for loss of multipotent hematopoietic function but surprisingly Vav1-Cre also deleted STAT5ab in CD45neg cells, dysregulated stromal niche factor mRNA expression, and altered MSC osteo/chondro-lineage differentiation priming. Single cell transcriptome analysis of bone marrow from wild-type or Vav1-Cre KO mice long-term repopulating hematopoietic stem cells (LT-HSC) showed upregulated oxidative phosphorylation (P<10-10) and protein translation (P<10-39) genes in LT-HSCs and throughout myeloid-primed clusters, while in contrast common lymphoid progenitor-like cells were completely eliminated. Similarly, like Vav1+ cells and based on scRNAseq analysis, Nes+ cells were also detected in both CD45neg and CD45+ cell clusters. Deletion of STAT5ab with Nes-Cre reduced the number of LT-HSCs (30% reduction compared to Nes-Cre control mice, P=0.02, N=4) and caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45+ and CD45neg bone marrow cell populations, additional stroma-specific Cre strains (LepR, Osx, Ocn, Prrx1, Nes-CreERT2) as well as endothelial-specific (Cdh5) strains were generated lacking STAT5ab. Viable mice were assessed for peripheral blood hematology, competitive repopulation, as well as hematopoietic stem/progenitor subsets (LT-HSC, MPP1, MPP2, MPP3, MPP4, KLS) using well-accepted flow cytometry antibody panels. Deletion of STAT5 in stromal cells with LepR-Cre significantly reduced MPP4 (35% reduction, P=0.025, N=4). Overall, extrinsic stromal (but not endothelial) cells were deficient in supporting multipotent and lineage-committed hematopoietic progenitors. However, peripheral blood hematology was rescued through homeostatic production. Gene regulation profiles resembled that of cytokine-starved MPPs. Functional support for niche-supporting activity was also assessed using STAT5ab KO MSCs in vitro. With Lepr-Cre mediated STAT5ab deletion, bone marrow niche factor mRNAs were downregulated (Cxcl12, Angpt1, Igfbp4, Igf1, Il34) and support of multipotent hematopoietic cells was significantly impaired. ELISA assay showed that SDF-1a was reduced 3-fold (P<0.001, N=5) and IGF-1 was reduced 2-fold (P<0.01, N=5) compared to wild type MSCs. Inversely, mRNAs for Kitl and Grem1 were increased in the same stromal cells, consistent with loss of multipotency and mixed HSC vs. MPP stromal support activities. Furthermore, novel scRNAseq analysis on simultaneous bone marrow hematopoietic and stromal cells compared wild-type and KO mice. Differentially expressed gene sets with Maximum Mean Discrepancy (MMD) co-expression revealed a key central role for coordinated STAT5ab/Cish balance as a biomarker of the cellular progenitor activity state (metabolic, protein translation, survival) associated with multipotency, comparing KO to wild-type cells. Importantly, Cish was strongly co-expressed in cells primed for lineage-specific differentiation (MSC - osteo/chondrogenic-primed; HSC - myeloid-primed) with STAT5ab co-expressed genes being associated with multipotent potential. Therefore, novel STAT5ab/Cish-mediated gene regulation programs operate within the bone marrow microenvironment to mediate cytokine production and responsiveness. This work highlights the importance of examining heterotypic cell types within bone marrow to elucidate functional cross-talk underlying hematopoietic homeostasis and defines conserved STAT5ab-mediated gene signatures as potential biomarkers to test in human hematopoietic and immune cell disorders.

Stromal FOXF2 suppresses prostate cancer progression and metastasis by enhancing antitumor immunity

Cancer-associated fibroblasts (CAFs) mediate an immunosuppressive effect, but the underlying mechanism remains incompletely defined. Here we show that increasing prostatic stromal Foxf2 suppresses the growth and progression of both syngeneic and autochthonous mouse prostate cancer models in an immunocompetent context. Mechanistically, Foxf2 moderately attenuates the CAF phenotype and transcriptionally downregulates Cxcl5, which diminish the immunosuppressive myeloid cells and enhance T cell cytotoxicity. Increasing prostatic stromal Foxf2 sensitizes prostate cancer to the immune checkpoint blockade therapies. Augmenting lung stromal Foxf2 also mediates an immunosuppressive milieu and inhibits lung colonization of prostate cancer. FOXF2 is expressed higher in the stroma of human transition zone (TZ) than peripheral zone (PZ) prostate. The stromal FOXF2 expression level in primary prostate cancers inversely correlates with the Gleason grade. Our study establishes Foxf2 as a stromal transcription factor modulating the tumor immune microenvironment and potentially explains why cancers are relatively rare and indolent in the TZ prostate.

EP16.04-001 The Role of Stromal PDGFRβ-activation in NSCLC

An increased expression of stromal platelet-derived growth factor receptor beta (PDGFRβ) has been associated with a poor prognosis in several solid tumor types. However, the prognostic relevance of PDGFRβ in NSCLC remains unclear. Since general PDGFRβ expression patterns do not reflect active PDGFRβ-signaling, it is likely that the activation status of the receptor would represent a more robust biomarker. The aim of this study is therefore to develop an in situ assay to detect activated PDGFRβ and to investigate its clinical relevance and prognostic potential for NSCLC patients.

Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/β-Catenin Signaling.

Kidney formation requires coordinated interactions between multiple cell types. Input from the interstitial progenitor cells is implicated in multiple aspects of kidney development. We previously reported that transcription factor 21 (Tcf21) is required for ureteric bud branching. Here, we show that Tcf21 in Foxd1+ interstitial progenitors regulates stromal formation and differentiation via interaction with β-catenin. We utilized the Foxd1Cre;Tcf21f/f murine kidney for morphologic analysis. We used the murine clonal mesenchymal cell lines MK3/M15 to study Tcf21 interaction with Wnt/β-catenin. Absence of Tcf21 from Foxd1+ stromal progenitors caused a decrease in stromal cell proliferation, leading to marked reduction of the medullary stromal space. Lack of Tcf21 in the Foxd1+ stromal cells also led to defective differentiation of interstitial cells to smooth-muscle cells, perivascular pericytes, and mesangial cells. Foxd1Cre;Tcf21f/f kidney showed an abnormal pattern of the renal vascular tree. The stroma of Foxd1Cre;Tcf21f/f kidney demonstrated marked reduction in β-catenin protein expression compared with wild type. Tcf21 was bound to β-catenin both upon β-catenin stabilization and at basal state as demonstrated by immunoprecipitation in vitro. In MK3/M15 metanephric mesenchymal cells, Tcf21 enhanced TCF/LEF promoter activity upon β-catenin stabilization, whereas DNA-binding deficient mutated Tcf21 did not enhance TCF/LEF promoter activity. Kidney explants of Foxd1Cre;Tcf21f/f showed low mRNA expression of stromal Wnt target genes. Treatment of the explants with CHIR, a Wnt ligand mimetic, restored Wnt target gene expression. Here, we also corroborated previous evidence that normal development of the kidney stroma is required for normal development of the Six2+ nephron progenitor cells, loop of Henle, and the collecting ducts. These findings suggest that stromal Tcf21 facilitates medullary stroma development by enhancing Wnt/β-catenin signaling and promotes stromal cell proliferation and differentiation. Stromal Tcf21 is also required for the development of the adjacent nephron epithelia.

Serum Levels of Stromal Cell-Derived Factor-1α and Vascular Endothelial Growth Factor Predict Clinical Outcomes in Head and Neck Squamous Cell Carcinoma Patients Receiving TPF Induction Chemotherapy.

Chemokines, such as stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF), are associated with clinical outcomes in several cancer types. This study aimed to investigate the role of SDF-1α and VEGF in the prognosis of patients with head and neck squamous cell carcinoma (HNSCC) who underwent TPF induction chemotherapy (docetaxel, cisplatin, and 5-fluorouracil). A total of 77 HNSCC patients were enrolled and circulating SDF-1α and VEGF values were examined at two time points for each patient, including pre-TPF treatment (treatment-naïve) and post-TPF treatment but before chemoradiotherapy. The median progression-free survival (PFS) and overall survival (OS) were 18.1 and 32.9 months, respectively. Decreased SDF-1α and VEGF levels after TPF treatment, post-TPF SDF-1α < 1500 pg/mL and VEGF value < 150 pg/mL were independent prognostic factors for better PFS and OS in univariate and multivariate analyses. A combination of SDF-1α and VEGF values may predict clinical outcomes significantly. Our study confirmed the role of SDF-1α and VEGF in the disease progression of HNSCC, and that decreased SDF-1α and VEGF after TPF treatment and lower post-TPF SDF-1α and VEGF values were associated with better prognosis in HNSCC patients who received induction chemotherapy with TPF followed by chemoradiotherapy.

Anti-fibrotic effect of adipose-derived stem cells on fibrotic scars.

BACKGROUNDSustained injury, through radiotherapy, burns or surgical trauma, can result in fibrosis, displaying an excessive deposition of extracellular matrix (ECM), persisting inflammatory reaction, and reduced vascularization. The increasing recognition of fibrosis as a cause for disease and mortality, and increasing use of radiotherapy causing fibrosis, stresses the importance of a decent anti-fibrotic treatment.AIMTo obtain an in-depth understanding of the complex mechanisms underlying fibrosis, and more specifically, the potential mechanisms-of-action of adipose-derived stomal cells (ADSCs) in realizing their anti-fibrotic effect.METHODSA systematic review of the literature using PubMed, Embase and Web of Science was performed by two independent reviewers.RESULTSThe injection of fat grafts into fibrotic tissue, releases ADSC into the environment. ADSCs’ capacity to directly differentiate into key cell types (e.g., ECs, fibroblasts), as well as to secrete multiple paracrine factors (e.g., hepatocyte growth factor, basis fibroblast growth factor, IL-10), allows them to alter different mechanisms underlying fibrosis in a combined approach. ADSCs favor ECM degradation by impacting the fibroblast-to-myofibroblast differentiation, favoring matrix metalloproteinases over tissue inhibitors of metalloproteinases, positively influencing collagen organization, and inhibiting the pro-fibrotic effects of transforming growth factor-β1. Furthermore, they impact elements of both the innate and adaptive immune response system, and stimulate angiogenesis on the site of injury (through secretion of pro-angiogenic cytokines like stromal cell-derived factor-1 and vascular endothelial growth factor).CONCLUSIONThis review shows that understanding the complex interactions of ECM accumulation, immune response and vascularization, is vital to fibrosis treatments’ effectiveness like fat grafting. It details how ADSCs intelligently steer this complex system in an anti-fibrotic or pro-angiogenic direction, without falling into extreme dilation or stimulation of a single aspect. Detailing this combined approach, has brought fat grafting one step closer to unlocking its full potential as a non-anecdotal treatment for fibrosis.

Aberrant stromal tissue factor localisation and mycolactone-driven vascular dysfunction, exacerbated by IL-1β, are linked to fibrin formation in Buruli ulcer lesions

Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues. Despite this, the role of the vasculature in BU pathogenesis remains almost completely unexplored. We hypothesise that fibrin-driven ischemia can be an ‘indirect’ route to mycolactone-dependent tissue necrosis by a mechanism involving vascular dysfunction. Here, we tracked >900 vessels within contiguous tissue sections from eight BU patient biopsies. Our aim was to evaluate their vascular and coagulation biomarker phenotype and explore potential links to fibrin deposition. We also integrated this with our understanding of mycolactone’s mechanism of action at Sec61 and its impact on proteins involved in maintaining normal vascular function. Our findings showed that endothelial cell dysfunction is common in skin tissue adjacent to necrotic regions. There was little evidence of primary haemostasis, perhaps due to mycolactone-dependent depletion of endothelial von Willebrand factor. Instead, fibrin staining appeared to be linked to the extrinsic pathway activator, tissue factor (TF). There was significantly greater than expected fibrin staining around vessels that had TF staining within the stroma, and this correlated with the distance it extended from the vessel basement membrane. TF-induced fibrin deposition in these locations would require plasma proteins outside of vessels, therefore we investigated whether mycolactone could increase vascular permeability in vitro. This was indeed the case, and leakage was further exacerbated by IL-1β. Mycolactone caused the loss of endothelial adherens and tight junctions by the depletion of VE-cadherin, TIE-1, TIE-2 and JAM-C; all Sec61-dependent proteins. Taken together, our findings suggest that both vascular and lymphatic vessels in BU lesions become “leaky” during infection, due to the unique action of mycolactone, allowing TF-containing structures and plasma proteins into skin tissue, ultimately leading to local coagulopathy and tissue ischemia.

VERIFICATION OF THE STROMAL CELL FACTOR SDF-1 IN THE GRAVIDAR ENDOMETRIUM IN MISSED MISCARRIAGE AFTER THE USE OF IN VITRO FERTILIZATION TECHNOLOGIES

The immunological factor in the genesis of reproductive losses, including the use of in vitro fertilization technologies, remains not completely clear due to the multiple pathogenetic mechanisms of immunological interactions in the mother-placenta-fetus system. The purpose of this study is to study the expression of the factor SDF-1 (stromal cell-derived factor) in the endometrium during missed miscarriage using in vitro fertilization technologies. Abortive material 5-8 weeks’ old was studied using in vitro fertilization technologies with different variants of gravidar transformation of the endometrium. A total of 92 samples were studied, of which 77 were abortive material from a missed miscarriage after the use of in vitro fertilization technologies and 15 were samples of abortive material from a pregnancy terminated surgically. A histological study was performed by staining with hematoxylin and eosin with a description of the variants of gravidar transformation of the stroma and endometrial glands and an immunohistochemical study of the SDF-1 factor in the endometrium of the examined groups. Based on the histological picture, depending on the morphological variant of the transformation of the endometrium, 5 groups of cases were formed: complete transformation of the stroma and glands of the endometrium and stroma with glands of the secretory type; incomplete transformation of the stroma with glands of the secretory type, proliferative type, with both types and the control group of observations. The expression area of ​​the marker in the endometrium (glands and stroma) was determined by morphometry. The results of the study showed a significant decrease in the expression of SDF-1 in the glands of the compact layer of the endometrium, regardless of the options for its transformation after the use of in vitro fertilization technologies, which is probably associated with a violation of the synthesis of the SDF-1 chemokine by glandulocytes at the stage of cyclic transformation of the endometrium. A significant increase in the expression of SDF-1 in the stroma of the endometrium is due to the initial violation of the its morphological and functional state in patients with infertility. Verification of stromal cell factor in the endometrium at the stage of pregravidar preparation will allow to correct this stage of the application of in vitro fertilization technologies for patients with endometrial dysfunction.

OR15. Cell Surface Markers on Adipose-Derived Mesenchymal Stem Cells: Isolation, Culture, and Identification for Translational Success

Abstract Aims The purpose of this study is determining that each part of adipose tissue will generate stem cells with similar adipose-derived mesenchymal stem cells (AMSCs) characteristic and specific cell-surface markers, thus there is no waster product of part adipose tissue in regenerative medicine. Methods An invitro experimental study, used approximately 20 grams of human subcutaneous adipose tissue were isolated and processed, until stromal vascular factor (SVF) and creamy supernatant separated. Both substances then divided into single filtration group and double filtration group consecutively. Each group and control group were sextuplicate before further analysis using immunocytochemistry. Single and double filter of AMSCs were applied to the subsequent experiments. AMSCs were observed under a microscope, the growth curves of the cells were assessed using a cell counting kit‑8 assay and the membrane expression of cell surface antigens, including cluster of differentiation (CD) 34, CD45, CD90, and CD105 were detected by immunocytochemistry. Results Flow cytometry demonstrated that the cells were highly positive for fourth cell surface markers. CD34 and CD45 expressions were detected in control group, and gradually decreased among another groups whereas lack of CD45 expression were consistent on other remaining groups. The expression of CD90 and CD105 were visibly increased significantly from control group to fourth group. Conclusion This study demonstrated that either SVF and/or supernatant generates fibroblastic-like cells and surface markers expression that matched the MSCs criteria. Therefore, all parts of adipose tissue are an appealing alternative of stem cell source regardless of culture method chosen.

Application of Human Adipose-Derived Stem cells for Bone Regeneration of the Skull in Humans.

Archeological archives report cranioplasty as 1 of the oldest surgical procedures; however, it was not until the last century that true advances have been made. Alternative approaches are necessary to achieve optimal closure of the defect with fewer adverse effects. We aim to evaluate the use of human adipose-derived stem cells (hADSCs) alone or seeded in scaffolds as the main treatment for cranial bone defects and to assess human patient outcomes. A systematic review was performed by querying PubMed, Ovid MEDLINE, EMBASE, and Cumulative Index to Nursing and Allied Health Literature databases with the MeSH terms: "adipose-derived stem cells," "cranial bone defect," "stromal vascular factor," "fat grafting," as well as synonyms in combinations determined by our search strategy. We included human models that used hADSCs as primary therapy. We excluded studies in languages other than English. One hundred ninety-four studies were identified after removal of duplicates. Four articles that used hADSCs as the main therapy to treat calvarial defects in humans were included. One article applied the cell therapy alone, and 3 used β-tricalcium phosphate granules as a scaffold to seed the hADSCs. Bone regeneration was reached in a short and intermediate period using autologous hADSCs in humans with no major adverse effects in all 4 articles included. A long-term follow-up study (6 years) exhibited late infections and reabsorption of the β-tricalcium phosphate scaffold seeded with hADSCs.