Collagenase Digestion Research Articles

An in vitro investigation into the impact of corneal rinsing on riboflavin/UVA corneal cross-linking

BackgroundCorneal cross-linking (CXL) using riboflavin and ultraviolet-A light (UVA) is a treatment used to prevent progression of keratoconus. This ex vivo study assesses the impact on CXL effectiveness, as measured by tissue enzymatic resistance and confocal microscopy, of including a pre-UVA corneal surface rinse with balanced salt solution (BSS) as part of the epithelium-off treatment protocol.MethodsSixty-eight porcine eyes, after epithelial debridement, were assigned to six groups in three experimental runs. Group 1 remained untreated. Groups 2–6 received a 16-min application of 0.1% riboflavin/Hydroxypropyl methylcellulose (HPMC) drops, after which Group 3 was exposed to 9 mW/cm2 UVA for 10 min, and Groups 4–6 underwent corneal surface rinsing with 0.25 mL, 1 mL or 10 mL BSS followed by 9 mW/cm2 UVA exposure for 10 min. Central corneal thickness (CCT) was recorded at each stage. Central 8.0 mm corneal buttons from all eyes were subjected to 0.3% collagenase digestion at 37 °C and the time required for complete digestion determined. A further 15 eyes underwent fluorescence confocal microscopy to assess the impact of rinsing on stromal riboflavin concentration.ResultsApplication of riboflavin/HPMC solution led to an increase in CCT of 73 ± 14 µm (P < 0.01) after 16 min. All CXL-treated corneas displayed a 2–4 fold greater resistance to collagenase digestion than non-irradiated corneas. There was no difference in resistance between corneas that received no BSS rinse and those that received a 0.25 mL or 1 mL pre-UVA rinse, but each showed a greater level of resistance than those that received a 10 mL pre-UVA rinse (P < 0.05). Confocal microscopy demonstrated reduced stromal riboflavin fluorescence after rinsing.ConclusionsAll protocols, with and without rinsing, were effective at enhancing the resistance to collagenase digestion, although resistance was significantly decreased, and stromal riboflavin fluorescence reduced with a 10 mL rinse. This suggests that a 10 mL surface rinse can reduce the efficacy of CXL through the dilution of the stromal riboflavin concentration.

Combining Riboflavin/UV-A Light and Rose Bengal/Green Light Corneal Cross-Linking Increases the Resistance of Corneal Enzymatic Digestion.

The purpose of this study was to determine if concurrent riboflavin/UV-A light (RF/UV-A) and rose Bengal/green light (RB/green) epi-off PACK-CXL enhances corneal resistance to enzymatic digestion compared to separate chromophore/light treatments. Ex vivo porcine corneas were allocated as follows. Group A corneas were soaked with riboflavin (RF) and were either not irradiated (A1, controls) or were irradiated with 10 (A2) or 15 J/cm² (A3) UV-A light at 365nm, respectively. Group B corneas were soaked with RB and either not irradiated (B1, controls) or were illuminated with 10 (B2) or 15 J/cm² (B3) green light at 525nm, respectively. Corneas in group C were soaked with both RF and RB and were either not irradiated (C1, controls) or were subjected to the same session consecutive 10 J/cm2 (C2) or 15 J/cm2 (C3) UV-A and green light exposure. Following treatment, all corneas were exposed to 0.3% collagenase A to assess digestion time until corneal button dissolution. A1 to A3 digestion times were 21.38, 30.5, and 32.25hours, respectively, with A2 and A3 showing increased resistance to A1. B1-3 had digestion times of 31.2, 33.81, and 34.38hours, with B3 resisting more than B1. C1 to C3 times were 33.47, 39.81, and 51.94hours; C3 exhibited superior resistance to C1 and C2 (both P < 0.05). Same-session combined RF/UV-A and RB/green PACK-cross-linking significantly increases corneal enzymatic digestion resistance over standalone treatments. Combining RF-based and RB-based PACK-CXL considerably increases corneal collagenase digestion resistance, potentially minimizing ulcer size in clinical contexts.

P119 Single-cell RNA sequencing in Inflammatory Bowel Disease in patients with Primary Sclerosing Cholangitis: a distinct form of colitis

Abstract Background Primary sclerosing cholangitis (PSC) is an inflammatory disorder of the bile ducts, in which Inflammatory bowel disease (IBD) concomitantly occurs (PSC-IBD). Substantial differences in clinical presentation are observed between PSC-IBD and ulcerative colitis (UC). In this study we aim to find distinct pathomechanisms for PSC-IBD using single-cell RNA sequencing. Methods Forty-seven colonic samples of PSC-IBD (n=24), UC (n=18) and non-IBD subjects (n=5) were collected. Whole biopsies were dissociated into single cells using collagenase digestion. Library preparation was done with 10x Genomics and sequencing was performed on an MGI2000 sequencer. Then, differential abundance, differential expression, and cell-cell interaction analyses were performed. Stainings for DUOX2 and HLA-DR were performed on paraffin embedded colon mucosal slides. Results In total, 71,798 high quality cells identified 54 distinct cell types, including a new type of epithelial cells: DUOX2+ enterocytes. DUOX2+ enterocytes are almost exclusively present in diseased inflamed colon and can act as antigen presenting cells through the expression of MHC2 on its surface. A general shift in cell type composition upon inflammation was observed, comparable between PSC and UC samples. However, an increased abundance was seen for stem cells in non-inflamed (NI) PSC, and not in UC-NI, compared to healthy controls. Additionally, we found distinct gene expression patterns between PSC and UC inflammation: while activation of inflammatory monocytes was observed in PSC inflammation, several types of fibroblasts were activated upon UC inflammation. Conclusion We describe DUOX2+ enterocytes, which may play a role in both PSC and UC colitis through antigen presentation. Furthermore, we show that intestinal inflammation in PSC-IBD, as compared to UC, is characterized by distinct mucosal cell composition and cell-specific gene expression patterns. PSC colitis, and not UC colitis, is driven by activation of inflammatory monocytes, with antigen presentation through HLA-DRB1, and activation of CD4+ T cells. Additionally, an increased abundance of stem cells in PSC-NI could be related with the increased colorectal cancer risk in PSC-IBD.

Effect of accelerated high-fluence riboflavin and rose bengal-mediated corneal cross-linking on resistance to enzymatic digestion

PurposeThis study evaluated the effect of high-fluence accelerated corneal cross-linking on the resistance to enzymatic digestion, assessing two chromophore/light combinations: riboflavin/UV-A light (RF/UV-A) and rose bengal/green light (RB/green).MethodsFreshly prepared ex-vivo porcine corneas (n = 189) were divided into 8 groups groups. Group A corneas were unirradiated controls without chromophore soaking (A0), or soaked with riboflavin (A1) or rose bengal (A2). Group B corneas underwent accelerated epi-off RF/UV-A CXL at fluences of 5.4 J/cm² (B1), 10 J/cm² (B2), or 15 J/cm² (B3). Group C corneas underwent accelerated epi-off RB/green CXL at fluences of either 10 J/cm² (C1) or 15 J/cm² (C2). Following CXL, all corneas were digested in 0.3% collagenase-A solution, and the time until complete dissolution was measured.ResultsNon-irradiated controls exposed to RF and RB enhanced corneal resistance to collagenase digestion, with RB having a stronger effect than RF. RF/UV-A-treated corneas showed significantly increased digestion resistance with increasing fluence levels. RB/green-treated corneas displayed enhanced digestion resistance with each increase in fluence up to 10 J/cm²; a 15 J/cm² fluence yielded similar digestion resistance times to a 10 J/cm² fluence, suggesting a plateau effect in accelerated RB/green CXL protocols.ConclusionsWhen compared to standard-fluence treatments, high-fluence accelerated epi-off CXL using both riboflavin and rose bengal significantly increases resistance to enzymatic digestion. The optimal settings for clinical protocols might be 15 J/cm² (30 mW/cm² for 8 min 20 s) for RF/UV-A and 10 J/cm² (15 mW/cm² for 11 min 7 s) for RB/Green Light.

DETERMINING THE FUNCTION OF MATRIX-BOUND AND SECRETED VESICLES IN MINERALIZATION

Matrix-bound vesicles (MBVs) are embedded within osteoid and function as the site of initial mineral formation. However, they remain insufficiently characterised in terms of biogenesis, composition and function while their relationship with secreted culture medium EVs (sEVs) such as exosomes remains debated. We aimed to define the biogenesis and pro-mineralisation capacity of MBVs and sEVs to understand their potential in regenerative orthopaedics.sEVs and MBVs isolated from conditioned medium (differential ultracentrifugation) and ECM (collagenase digestion and differential ultracentrifugation) of mineralising MC3T3 pre-osteoblast and human bone marrow MSC cultures were characterised by nanoparticle tracking analysis, western blotting, nano-flow cytometry, super resolution microscopy (ONI) and TEM. Immunoprecipitated populations positive for alkaline phosphatase (ALP), a putative marker of mineralisation capacity, were also characterised. Collagen binding efficiency was evaluated using MemGlow staining.Results reported were comparative across both cell lines. Western blots indicated MBV fractions were positive for markers of endosomal biogenesis (CD9, CD81, ALIX, TSG101) and pro-mineralising proteins (ALP, Pit1, Annexin II, Annexin V), with Annexin V and CD9 present in immunoprecipitated ALP-positive fractions. MBVs were significantly larger than sEVs (p&lt;0.05) and contained a higher amount of ALP (p&lt;0.05) with a significant increase from day 7 to day 14 of cellular mineralisation (p&lt;0.05). This mirrored the pattern of electron-dense vesicles seen via TEM. Super resolution single vesicle analysis revealed for the first-time co-expression of ALP with markers of endosomal biogenesis (CD9, CD63, CD81, ALIX) and Annexin II in both vesicle types, with higher co-expression percentage in MBVs than sEVs. MBVs also exhibited preferential collagen binding.Advanced imaging methods demonstrated that contrary to opinions in the field, MBVs appear to possess exosomal markers and may arise via endosomal biogenesis. However, it was evident that a higher proportion of MBVs possessed machinery to induce mineralisation and were enriched in mineral-dense material.

Isolation of Human Adipose-Derived Stromal/Stem Cells from Lipoaspirates.

Adipose tissue is an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Adipose-derived stromal/stem cells (ASCs) have been widely used in tissue engineering and cell therapy. In addition, the clinical application of ASCs in the treatment of inflammation and injury has been proven a success. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of Adipose-derived stromal/stem cells (ASCs). We present a large-scale procedure suitable for processing >100mL volumes of lipoaspirate tissue specimens by collagenase digestion, a related procedure suitable for processing adipose tissue aspirates without digestion, and a procedure suitable for intact human adipose tissue, such as buccal fat pads in the maxillofacial region.

Insulin release from isolated cat islets of Langerhans

Feline diabetes mellitus is a common endocrine disease with increasing prevalence. It shows similarities with human type 2 diabetes and is characterized by insulin resistance and deficient insulin secretion. Moreover, cats and humans belong to the very few species that form amyloid depositions in the pancreatic islets. However, little is known about cat islet function and no studies have addressed insulin secretion from isolated islets ex vivo. The aim of this study was to establish a protocol for isolation of islets of Langerhans from pancreata of cats euthanized due to disease, and to evaluate insulin secretion responses to various physiological and pharmacological stimuli. Collagenase digestion of pancreatic tissue from 13 non-diabetic cats and two cats with diabetic ketoacidosis yielded individual islets surrounded by a layer of exocrine tissue that was reduced after two days in culture. Histological examination showed islet amyloid in pancreatic biopsies from most non-diabetic and in one diabetic cat. Islets from non-diabetic cats cultured at 5.5 mM glucose responded with increased insulin secretion to 16.7 mM glucose, 30 mM K+ and 20 µM of the sulfonylurea glipizide (2-3 times basal secretion at 3 mM glucose). The glucagon-like peptide-1 receptor agonist exendin-4 (100 nM) had no effect under basal conditions but potentiated glucose-triggered insulin release. Only one of nine islet batches from diabetic cats released detectable amounts of insulin, which was enhanced by exendin-4. Culture of islets from non-diabetic cats at 25 mM glucose impaired secretion both in response to glucose and K+ depolarization. In conclusion, we describe a procedure for isolation of islets from cat pancreas biopsies and demonstrate that isolated cat islets secrete insulin in response to glucose and antidiabetic drugs. The study provides a basis for future ex vivo studies of islet function relevant to the understanding of the pathophysiology and treatment of feline diabetes.

MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization

As the only reliable treatment option for end-stage liver diseases, conventional liver transplantation confronts major supply limitations. Accordingly, the decellularization of discarded livers to produce bioscaffolds that support recellularization with progenitor/stem cells has emerged as a promising translational medicine approach. The success of this approach will substantially be determined by the extent of extracellular matrix (ECM) preservation during the decellularization process. Here, we assumed that the matrix metalloproteinase (MMP) inhibition could reduce the ECM damage during the whole liver decellularization of an animal model using a perfusion-based system. We demonstrated that the application of doxycycline as an MMP inhibitor led to significantly higher preservation of collagen, glycosaminoglycans, and hepatic growth factor (HGF) contents, as well as mechanical and structural features, including tensile strength, fiber integrity, and porosity. Notably, produced bioscaffolds were biocompatible and efficiently supported cell viability and proliferation in vitro. We also indicated that produced bioscaffolds efficiently supported HepG2 cell function upon seeding onto liver ECM discs using albumin and urea assay. Additionally, MMP inhibitor pretreated decellularized livers were more durable in contact with collagenase digestion compared to control bioscaffolds in vitro. Using zymography, we confirmed the underlying mechanism that results in these promising effects is through the inhibition of MMP2 and MMP9. Overall, we demonstrated a novel method based on MMP inhibition to ameliorate the ECM structure and composition preservation during liver decellularization as a critical step in fabricating transplantable bioengineered livers.

MUC-16, tumour and congestive heart failure marker, is expressed on epicardial mesothelial cells and is inversely associated with epicardial fat genesis

Abstract Background CA-125 or mucin-16 (MUC-16) is a protector protein on epithelial luminal surfaces against physical stress through hydration process. However, some evidence suggests a role in fibrogenesis. MUC-16 can be detected in plasma after proteolytic cleavage with a predictive value for heart failure progression. Our main objective was to study the expression levels of this glycoprotein on epicardial mesothelial cells, its association with epicardial fat genesis, and biochemical markers. Methods and results We have used epicardial and subcutaneous fat from patients(n = 29) undergoing open heart surgery under informed consent and after approval by Ethics Committee. MUC-16 expression levels were detected on formalin-fixed paraffin-embedded epicardial fat biopsies with CA-125 antibody or real-time PCR on stored biopsies (n = 18) at -80ºC. Epicardial cells scraped with a scalpel were cultured and their MUC-16 expression was confirmed with a specific antibody and secondary fluorescent antibody. Primary cultures from epicardial stromal cells of 8 patients were obtained with collagenase digestion. After 15 days, adipogenesis was induced for 15 days with an adipogenic cocktail. Adipogenesis (AdipoQ) and mesothelial (ITLN-1) markers were quantified by real-time PCR on cells with and without adipogenesis. CA-125 expression levels were analyzed by real time-PCR on epicardial fat from 18 patients, as well as inflammation, fibrosis, stretch and adiposity markers levels on plasma by multiplex assay. Our results showed that MUC-16 is localized on epicardial mesothelial cells but not on adipocytes. The MUC-16-stained cells can be cultured and isolated after scraping the epicardium. The isolated epicardial stromal cells with collagenase showed a positive relationship between mesothelial maker (ITLN-1) and CA125 (Figure below). The higher presence of MUC-16 levels was inversely associated with AdipoQ after adipogenesis induction. From 8 plasma studied proteins, thrombospondin-2 (TP-2), was the best associated protein with MUC-16 on epicardial fat (r = 0,472; p = 0,48). Conclusion Epicardial cells express MUC-16. These types of cells can be isolated and cultured for studying their cleavage regulation and modulation. Epicardial stromal cells with a higher percentage of MUC-16 reduce the epicardial fat genesis. Further studies are needed for finding the main circulating plasma levels associated with epicardial MUC-16.

High FABP4, associated with low-voltage areas, increases the epicardial fibrosis and its glucose metabolism

Abstract Background High plasma levels of FABP4 were associated with epicardial fat volume on persistent AF patients. FABP4 can be released by differentiated adipocytes and macrophages, which can be involved in fibrosis. Purpose Our main objective was to study the association between FABP4 and epicardial fibrosis, which can explain low-voltage areas (LVA) and atrial fibrillation (AF) recurrence. Material and methods Peripheral levels of FABP4 and Galectin-3 were analysed on AF patients undergoing pulmonary vein isolation (PVI) (n=299) and signed informed consent. During the intervention, LVA was evaluated by invasive electroanatomic voltage mapping (EAM). Epicardial (EAT) (n=6) and subcutaneous adipose tissue (SAT) (n=5) were obtained from undergoing open heart surgery patients. Stromal cells were isolated by collagenase digestion, cultured and treated with and without FABP4 100 ng/mL and/or galectin-3 30 ng/mL for 24 hours. The proliferation and migration rate were measured by wound healing assay; mitochondrial activity was studied by MTT assay; and glucose and lactate consumption were detected by colorimetric and fluorescence enzymatic assays, respectively. Gene expression levels were assessed by real-time PCR. Results Regression analysis determined that the main independent predictors of LVA were age, left atrial area and FABP4 levels. Preclinical "in vitro" studies have shown that FABP4 100 ng/mL induced fibrosis in epicardial stromal cells (p=0.0039) and subcutaneous stromal cells (p=0.0009), evidenced by wound healing assay and by the increment of associated fibroblast genes (COL1A2, VIM, TGF-β1). Moreover, the high FABP4 concentration modulates the protein levels and metabolic genes expression (CPT1, FASN, ATGL, related to transport, synthesis and degradation of fatty acids, respectively) on EAT stromal cells. Glucose metabolism without lactate production was also incremented after FABP4 treatment (p&amp;lt;0.0001). Even, on SAT cells, released lactate was reduced after FABP4 treatment (p=0.0218). Conclusions Plasma FABP4 was a predictor of LVA. Our results suggest that this association might be explained by the fibrotic and metabolic role of FABP4 in epicardial cells. Future studies will shed light on the benefits on atrial remodelling with FABP4 modulators.Figure 1Figure 2

Abstract 17456: Inositol 1,4,5-Trisphosphate Receptors (ITP3Rs) and Activation of Cardiac Fibroblasts in Ischemia/Reperfusion Injury

Introduction: Inositol 1,4,5-trisphosphate receptors (ITP3Rs) are Ca 2+ release channels on the endoplasmic reticulum (ER). The role of ITP3Rs in cardiomyocytes has been previously investigated for hypertrophy and arrhythmias, however, the exact function in the activation of cardiac fibroblasts (FBs) is yet to be determined. The main goal of this project is to investigate the mechanisms linking FB ITP3Rs to post-ischemia/reperfusion (I/R) cardiac fibrosis. Hypothesis: We hypothesize that ITP3Rs activate FB via a direct transcriptional regulation of αSMA and other genes involved in myoFB activation. Indeed, myoFB differentiation and persistence is regulated by a balance between pro-and anti-apoptotic signaling pathways. Methods: We generated myoFB-specific ITP3Rs KO mice (ITP3R KO ) and isolated FBs as well as cardiomyocytes via collagenase digestion and performed immunoprecipitation (IP) assays showing that ITP3Rs bind p53 upregulated modulator of apoptosis (PUMA). Cytosolic levels of Cytochrome C (Cyt C) were assessed in myoFB isolated from areas remote from injury and scar areas 7 d after I/R. After having immunoprecipitated for ITP3R1 we blotted the isolated FBs for PUMA and, vice versa. PUMA levels were measured in cytosol, ER, and mitochondria of FBs isolated from regions remote from injury and scar areas 7 d post I/R. Results: Our data show reduced levels of cytosolic Cyt C following I/R, which was prevented by IP3Rs ablation, providing an explanation for the persistence of myoFBs in remote areas. We found that the mitochondrial expression of PUMA was significantly decreased - while ER levels were markedly increased - in myoFBs isolated from areas remote from I/R injury, indicating a sequestration of PUMA by ITP3Rs; this phenotype was rescued by ITP3Rs ablation. Instead, in myoFBs isolated from the scar areas, mitochondrial PUMA levels were increased both in ITP3R fl/fl and ITP3R KO mice, and no sequestration was observed, consistent with the absence of ITP3R upregulation. Conclusions: Using a specific Cre/lox KO model, we provide the first assessment of functional contribution of ITP3R to post-I/R activation of cardiac FBs and understanding the molecular mechanisms underlying excessive reactive fibrosis in the heart.

Abstract 12147: Flow-Induced Cellular Reprogramming in Mouse Atherosclerosis Model

Introduction: Atherosclerosis, a leading cause of death worldwide, is a chronic inflammatory disease that occurs preferentially in the arterial regions exposed to disturbed flow (d-flow), while those exposed to stable flow (s-flow) are protected. Recently, we reported d- F low I nduced R eprogramming of E ndothelial cells (ECs) ( FIRE ), including endothelial inflammation, endothelial-to-mesenchymal transition (EndMT) and endothelial-to-immune-cell transition (EndIT). Hypothesis: D-flow plus hypercholesterolemia (HighChol) exacerbates reprogramming of arterial cells leading to atherosclerosis development compared to d-flow alone and HighChol alone. Methods: To test this hypothesis, we induced atherosclerosis in C57BL/6 mice by an AAV-PCSK9 injection and high-fat diet to induce HighChol and/or partial carotid ligation (PCL) surgery to expose left carotid artery (LCA) to d-flow compared to the right CA (RCA) exposed to s-flow. Single cells prepared from collagenase digestion of the LCAs and RCAs at 2 and 4 weeks post-PCL were analyzed by scRNA-seq and integrated with our prior scRNA-seq data obtained at 2 days and 2 weeks post-PCL under normal blood cholesterol levels. Results: Overall, our data showed that d-flow was the most predominant regulator of arterial cell characteristics while HighChol alone had relatively minor effects. In the RCA, homeostatic EC, smooth muscle cell (SMC), and macrophage (MΦ) clusters were dominant regardless of cholesterol levels. Additionally, for these cell types, we found clusters that increased in response to d-flow in the LCA, which further increased with HighChol and time. Surprisingly, diffusion map trajectory analysis revealed a clear convergence of transcriptomic profiles of EndIT ECs, SMC-derived foam cell SMCs, and foamy MΦs. Conclusions: These results suggest FIRE initiated by d-flow is exacerbated by HighChol. We also found that SMCs and MΦs transition toward proatherogenic phenotypes under d-flow, which is further exacerbated by HighChol. Furthermore, differential gene expression analysis showed that proatherogenic ECs, SMCs, and MΦs show common transcriptomic profiles, suggesting common set of genes and pathways that could be targeted as effective atherosclerotic therapeutics.

MSC Reprogramming and Aberrant Inflammatory Signaling in Myelofibrosis: Insights from Single-Cell RNA-Sequencing of Trephine Bone Marrow Cells

Myelofibrosis (MF) results from mutation-induced activation of JAK/STAT signaling in hematopoietic stem, progenitor and mature cells, and features a progressive, fibrosing stromal reaction, splenomegaly, constitutional symptoms and reduced life expectancy. Identification of disrupted cellular pathways within bone marrow (BM) is pivotal for therapeutic breakthrough, but fibrosis severely limits access to stromal elements for single-cell analysis. To overcome this, we utilized collagenase digestion of fresh BM trephine biopsy material surplus to diagnostic needs and profiled isolated cells using scRNAseq. 9 samples were obtained from 8 patients with MF (4, JAK inhibitor naïve; 4, on ruxolitinib); 4 samples were from 4 patients with MF 4, 10, 12 &amp; 12 months after allogeneic transplant; and 7 samples (termed “normal controls”) were from 6 patients who had received an allograft for myelodysplasia or acute leukemia, and 1 who had received CAR-T cells for B-ALL. Normal controls exhibited normal or near-normal blood counts and lacked BM fibrosis. Each sample was subjected to collagenase digestion and flow sorted to isolate non-erythroid hematopoietic (CD45+GlyA-) and microenvironmental (CD45+GlyA-CD71-) cells for single cell RNA sequencing and downstream comparative analysis. 105,231 transcriptomes met quality control criteria (median 4975/sample, range 1036-13277, no significant difference between sample categories) and 43 transcriptional clusters were identified. Compositional analysis identified the non-hematopoietic stromal cell fraction as harboring the biggest differences with, surprisingly, a proportional relative depletion of mesenchymal stromal cells (MSCs) and enrichment of endothelial cells (ECs) in MF vs. normal controls. While transcriptomes of ECs did not differ substantially, there were extensive transcriptional differences between MSCs recovered from MF vs. normal control samples. Subclustering revealed two subsets, MSC1 &amp; MSC2 asymmetrically distributed between MF and normal controls. Intriguingly, MSCs recovered from MF patients after allograft occupied an intermediate position in principal component space indicating that reversion of disease-related MSC transcriptional reprogramming post-allograft occurs over a timescale of months. The reprogrammed transcriptional state of MF MSCs is defined by reduced expression of hematopoietic support factor genes (eg, KITLG, CXCL12, ANGPTL2, NCAM2, CSF1) and increased expression of extracellular matrix (ECM) genes (eg, FN1, COL1A2, COL3A1, DCN, SPARC), features which suggest an explanation for the increased frequency of graft failure following allograft in MF versus other hematologic malignancies. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) indicated transcription factors with either increased (eg, NR2F2, RUNX2, FOXP2) or decreased (eg, CEBPA, FOXC1, and PPARG) transcriptional activity as candidate master transcriptional regulators of the observed MSC reprogramming in MF. Transcriptional change in MSCs correlated strongly with histopathological assessment of BM reticulin. Within the hematopoietic fraction, and agnostic of JAK inhibitor treatment status, comparison of MF vs. control cells unexpectedly revealed significant upregulation of interferon response genes, especially in CD14 + monocytes, but also in neutrophils and their progenitors, centred on expression of interferon-stimulated gene factor 3 (ISGF3) genes IRF9 and STAT1/2. Comparison of MF samples according to ruxolitinib treatment status indicated that ruxolitinib induced downregulation of an NFκB-centered inflammatory program, particularly in neutrophils. Finally, ligand-receptor analysis using NicheNet identified TGFB1, IL1B, and TNF as top predicted ligands for induction of the MF signature in MSCs. While myeloid cells were top producers of these factors, their expression did not vary between MF vs. controls, suggesting other mechanisms, such as prolonged retention of ligand in the ECM, are important. In summary, our comparative scRNA analysis of BM cells from MF patients and controls - the largest reported to date - reveals (i) transcriptional reprogramming of MSCs leading to reduced expression of hematopoietic support factor and increased expression of ECM genes; and (ii) increased expression of interferon and NFkB inflammatory pathways in myeloid cells.

Collagenase treatment reduces the anisotropy of ultrasonic backscatter in rat myocardium by reducing collagen crosslinks.

Dysregulation of collagen deposition, degradation, and crosslinking in the heart occur in response to increased physiological stress. Collagen content has been associated with ultrasonic backscatter (brightness), and we have shown that the anisotropy of backscatter can be used to measure myofiber alignment, that is, variation in the brightness of a left ventricular short-axis ultrasound. This study investigated collagen's role in anisotropy of ultrasonic backscatter; female Sprague-Dawley rat hearts were treated with a collagenase-containing solution, for either 10 or 30 min, or control solution for 30 min. Serial ultrasound images were acquired at 2.5-min intervals throughout collagenase treatment. Ultrasonic backscatter was assessed from anterior and posterior walls, where collagen fibrils are predominately aligned perpendicular to the angle of insonification, and the lateral and septal walls, where collagen is predominately aligned parallel to the angle of insonification. Collagenase digestion reduced backscatter anisotropy within the myocardium. Collagen remains present in the myocardium throughout collagenase treatment, but crosslinking is altered within 10 min. These data suggest that crosslinking of collagen modulates the anisotropy of ultrasonic backscatter. An Anisotropy Index, derived from differences in backscatter from parallel and perpendicularly aligned fibers, may provide a noninvasive index to monitor the progression and state of myocardial fibrosis.

A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis.

Although adenomyosis is a common and benign gynecological disease, the specific pathogenesis of this condition is yet to be fully elucidated. It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most of the previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions. In the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify, and evaluate the viability of isolated ectopic endometrial cells. Using our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37 and 93.49%, respectively. Our method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis.

Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVECs).

This protocol describes a simple and an economical method for isolation of endothelial cells from human umbilical vein. Umbilical cord is easily available postpartum following informed consent, and the method for its collection is noninvasive with few ethical concerns. Thus, umbilical vein is an ideal source for isolation of endothelial cells of human origin. Endothelial cells are isolated from umbilical vein by collagenase digestion. This is followed by their culture on extracellular matrix (ECM) protein coated tissue culture flasks in presence of endothelial cell growth medium. In the last few decades, human umbilical vein endothelial cells (HUVECs) have come to be regarded as a standard model system by vascular biologists to understand general principles of endothelial cell biology and dysfunction. Here, we describe isolation, culture, as well as validation of HUVECs.

2-(3-(chloromethyl)benzoyloxy)benzoic Acid Increases CD4+ Regulatory T-Cell Population and FoxP3 Expression in Lipopolysaccharide-induced Mice

BACKGROUND: Lipopolysaccharide (LPS) has been reported to increase CD4+ regulatory T-cell (CD4+ Treg) populations. Acetylsalicylic acid (ASA) has been reported to have immunomodulatory activity, but it may induce chronic gastric ulceration. Another salicylic acid-bearing compound, 2-(3-(chloromethyl)benzoyloxy)benzoic acid (3-CH2Cl), has been reported to have less gastric mucosal damage. However, the effect of 3-CH2Cl on CD4+ Tregs in LPS-induced mice is still unknown. Therefore, the present study was conducted to investigate the immunomodulatory effect of 3-CH2Cl on CD4+ T-cell and CD4+ Treg populations as well as FoxP3 expression in LPS-induced mice.METHODS: Synthesis of 3-CH2Cl was performed by mixing salicylic acid and chloromethylbenzoylchloride with the catalyzation of pyridine, acetone and heat. The 3-CH2Cl tablets were prepared using direct compression method. After intraperitoneal injection of 1 mg/kg BW LPS to mice, 60 mg/kg BW ASA or 60 mg/kg BW 3-CH2Cl was given orally for 3 days. The splenocyte was obtained through splenectomy and collagenase digestion. The population of CD4+ T-cells and CD4+ Tregs, as well as the splenic FoxP3 expression were determined using flow cytometry technique.RESULTS: CD4+ T-cell populations in mice treated with LPS and 3-CH2Cl or ASA were lower than those treated with LPS merely. Meanwhile, CD4+ Treg populations and FoxP3 expression levels in mice treated with LPS and 3-CH2Cl or ASA were higher than those treated with LPS merely.CONCLUSION: Since 3-CH2Cl could decrease CD4+ T-cell population and increase CD4+ Treg population mediated by the increase of FoxP3 expression in LPS-induced inflammation, it may act as a potential therapeutic drug to reduce inflammatory conditions.KEYWORDS: 2-(3-(chloromethyl)benzoyloxy)benzoic acid, acetylsalicylic acid, CD4, T-regulatory cells, FoxP3, LPS

In Vitro Human Fetal Pancreatic Islets to Redefine Pancreatic Research.

In vitro studies with human fetal islets of different gestational ages (GA) would be a great tool to generate information on the developmental process of the islets as this would help to recontextualize diabetes research and clinical practice. Pancreatic islets from human cadavers and other animal species are extensively researched to explore their suitability for islet transplantation procedure, one of the upcoming treatment strategies for insulin-dependent diabetes mellitus. Although human fetal islets are also considered for islet transplantation, ethical issues and limited knowledge constraints their use. The fetal islets could be explored to address the information lacunae on the maturity process of pancreatic islets and the endocrine-exocrine signaling mechanisms. This study aimed to assess the feasibility of isolating viable islets and study the cytoarchitecture of the fetal pancreas of GA22-29 weeks, not reported otherwise. Pancreas obtained from the aborted fetuses of GA 22-29 weeks were subjected to collagenase digestion and were further cultured to determine the viability in vitro. Parameters assessed were expression of markers for endocrine cell lineages and insulin release to glucose challenge. Islets were viable in vitro and islets were shown to maintain cues for post-digestion re-aggregation and expansion in culture. The immunofluorescent staining showed islets of varying sizes, homogenous cell clusters aggregating to form heterogenous cell clusters, otherwise not reported for this GA. On stimulation with different concentrations of glucose (2.8 and 28 mM), the fetal islets in the culture exhibited insulin release, and this response confirmed their viability in vitro. Our findings showed that viable islets could be isolated and cultured in vitro for further in-depth studies to explore their proliferative potential as well as for the identification of pancreatic progenitors, a good strategy to take forward.

1505-P: CD44 Knockdown in Human Adipocytes—Effect on Adipocyte Proliferation and Differentiation

Introduction: eGWAS implicates the immune-cell receptor CD44 as the top candidate gene in the molecular pathogenesis of T2D1. We have shown that in human adipose tissue, CD44 expression is associated with inflammatory gene expression, M1 macrophage phenotype, and systemic insulin resistance2. Here we sought to determine whether CD44 directly impairs adipocyte function via siRNA knockdown in human preadipocytes with quantification of proliferation, differentiation, and gene expression. Methods: Abdominal subcutaneous adipose tissue was harvested from five healthy nondiabetic obese individuals during bariatric surgery. Preadipocytes were isolated via collagenase digestion and seeded at 1×10^4 cells/well and treated with siRNA-CD44 or mock condition (control) using siRNA Maxi Transfection Reagents (Invitrogen). Cell proliferation was assayed by MTS (Promega) on Days 1, 2, 3 post transfection. Differentiation was induced at confluence (as described3), and cells were harvested at days 3, 6, 12 after confluence for gene expression and at day 12 for oil red O staining (total lipid uptake) and immunofluorescence microscopy (% differentiation = bodipy/dapi*100). Results: CD44 knockdown decreased preadipocyte proliferation and increased adipocyte differentiation and lipid uptake. Conclusion: CD44 may contribute to human insulin resistance by impairing adipocyte differentiation and fat storage. Disclosure H.Chen: None. N.R.Saini: None. E.M.Ayhan: None. T.Mclaughlin: Board Member; January, Inc., Research Support; Merck &amp; Co., Inc., Stock/Shareholder; Eiger Bio. Funding National Institutes of Health (1R21AI159024-01A1)

Primary culture and endocrine functional analysis of Leydig cells in ducks (Anas platyrhynchos).

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.