Desmin-positive Cells Research Articles

Bi-layered Adipose Mesenchymal Cell Sheets Improve Bladder Compliance in Spinal Cord-Injured Rats.

To improve bladder compliance in patients with low-compliance bladders, augmentation cystoplasty with the intestinal tract is performed. However, the use of the intestinal tract often leads to serious surgical complications. Tissue engineering technologies have the potential to improve bladder compliance without using the intestinal tract. In this study, we fabricated bi-layered adipose-derived mesenchymal cell (AMC) sheets and then determined whether the bi-layered AMC sheets could improve bladder compliance in rats with spinal cord injury (SCI). The abdominal adipose tissues of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were harvested, and the attached and proliferating cells on type I collagen were used as AMCs. The AMCs were then cultured on temperature-responsive culture dishes. After reaching over-confluence, the AMCs that maintained cell-cell contacts were detached from the dishes and applied to a gelatin hydrogel sheet. Then, another detached AMC monolayer was accumulated on the AMC monolayer-applied gelatin. Prior to 4 weeks of transplantation, the levels of T8-9 in the spinal cords of recipient SD rats were partially transected. After producing the bi-layered AMC sheets and the rats with SCI, the detrusor muscles of the anterior bladder walls of the rats with SCI were incised, and the bi-layered AMC sheet was patch-transplanted onto the exposed bladder epithelium (n = 8). As a control, the sham operation was performed (n = 7). Four weeks after the transplantation, bladder capacity and bladder compliance in AMC sheet-transplanted SCI rats were significantly higher than those in sham-operated control SCI rats. The smooth muscle layers in AMC sheet-transplanted bladders were significantly larger than those in control bladders. In addition, the collagen fibers in the AMC sheet-transplanted bladders were significantly smaller than those in the control bladders. Some GFP-positive transplanted AMCs differentiated into smooth muscle actin- or desmin-positive cells. Furthermore, GFP-positive cells secreted transforming growth factor-β1 or vascular endothelial growth factor. Therefore, this study showed that bi-layered AMC sheets could improve bladder compliance and bladder tissues in SCI rats.

Selective inhibition of hepatic stellate cell and fibroblast-derived LOXL1 attenuates BDL- and Mdr2-/--induced cholestatic liver fibrosis.

Lysyl oxidase-like 1 (LOXL1) proteins are amine oxidases that play a crucial role in extracellular matrix remodeling due to their collagen cross-linking and intracellular functions. The role of LOXL1 in cholestatic liver fibrosis remains unexplored. We measured LOXL1 expression in two murine models of cholestasis [Mdr2 knockout (Mdr2-/-) and bile duct ligation (BDL)]. We used adeno-associated virus (AAV) serotype 6-mediated hepatic delivery against LOXL1 (AAV2/6-shLoxl1) to investigate the therapeutic efficacy of targeting LOXL1 in cholestatic liver fibrosis. NIH-3T3 murine fibroblasts were used to investigate the function and regulatory mechanisms of LOXL1 in vitro. LOXL1 expression was significantly upregulated in Mdr2-/- and BDL mice compared with their corresponding controls, predominantly in collagen-rich fibrous septa and portal areas. AAV2/6-shLoxl1 significantly reduced LOXL1 levels in Mdr2-/- and BDL mice, mainly in desmin-positive hepatic stellate cells (HSCs) and fibroblasts. Concomitant with reduced LOXL1 expression, there was reduced ductular reaction, inflammation, and fibrosis in both Mdr2-/- and BDL mice. In addition, Loxl1 intervention decreased Ki-67-positive cells in the desmin-positive areas in both Mdr2-/- and BDL mice. Overexpression of LOXL1 significantly promoted fibroblast proliferation by activating the platelet-derived growth factor receptor and extracellular signal-regulated kinase signaling pathways in vitro. Our findings demonstrated that selective inhibition of LOXL1 derived from HSCs/fibroblasts attenuated cholestatic liver/biliary fibrosis, inflammation, ductal reaction, and HSC/fibroblast proliferation. Based on our findings, LOXL1 could be a potential therapeutic target for cholestatic fibrosis.NEW & NOTEWORTHY Selectively, inhibition of HSC/fibroblasts-derived LOXL1 by AAV2/6-shLoxl1 could reduce collagen deposition, HSC/fibroblasts proliferation, and cholestatic liver fibrosis progression. In addition, overexpression of LOXL1 significantly promoted HSC/fibroblast proliferation by activating the PDGFRß/PI3K and ERK signaling pathways in vitro.

Clinicopathologic and genetic characterization of angiofibroma of soft tissue: a study of 12 cases including two cases with AHRR::NCOA3 gene fusion.

Angiofibroma of soft tissue (AFST) is a benign tumour characterised by prominent arborizing blood vessels throughout the lesion. Approximately two-thirds of AFST cases were reported to have AHRR::NCOA2 fusion, and only two cases have been reported to have other gene fusions: GTF2I::NCOA2 or GAB1::ABL1. Although AFST is included in fibroblastic and myofibroblastic tumours in the World Health Organization's 2020 classification, histiocytic markers, especially CD163, have been reported to be positive in almost all examined cases, and it still remains the possibility of a fibrohistiocytic nature of the tumour. Therefore, we aimed to clarify the genetic and pathological spectrum of AFST and identify whether histiocytic marker-positive cells were true neoplastic cells. We evaluated 12 AFST cases, which included 10 cases with AHRR::NCOA2 and two with AHRR::NCOA3 fusions. Pathologically, nuclear palisading, which has not been reported in AFST, was detected in two cases. Furthermore, one tumour resected by additional wide resection revealed severe infiltrative growth. Immunohistochemical analysis indicated varying levels of desmin-positive cells in nine cases, whereas CD163- and CD68-positive cells were diffusely distributed in all 12 cases. We also performed double immunofluorescence staining and immunofluorescence in situ hybridisation in four resected cases with >10% desmin-positive tumour cells. The results suggested that the CD163-positive cells differed from desmin-positive cells with AHRR::NCOA2 fusion in all four cases. Our findings suggested that AHRR::NCOA3 could be the second most frequent fusion gene, and histiocytic marker-positive cells are not genuine neoplastic cells in AFST.

Establishment of a novel experimental system using single cell-derived pleomorphic rhabdomyosarcoma cell lines expressing K-RasG12V and deficient in p53.

Pleomorphic rhabdomyosarcoma (PRMS) predominantly arises in adult skeletal musculature and is usually associated with poor prognosis. Thus, effective treatments must be developed. PRMS is a rare tumor; therefore, it is critical to develop an experimental system to understand the cellular and molecular mechanisms of PRMS. We previously demonstrated that PRMS develops after p53 gene deletion and oncogenic K-Ras expression in the skeletal muscle tissue. In that study, oncogenic K-Ras-expressing cells were diverse and the period until disease onset was difficult to control. In this study, we developed an experimental system to address this problem. Single cell-derived murine cell lines, designated as RMS310 and RMSg2, were established by limiting the dilution of cells from a lung metastatic tumor colony that were positive for various cancer stem cells and activated skeletal muscle-resident stem/progenitor cell marker genes by RT-PCR. All cell lines stably recapitulated the histological characteristics of human PRMS as bizarre giant cells, desmin-positive cells, and lung metastases in C57BL/6 mice. All subclones of the RMSg2 cells by the limiting dilution in vitro could seed PRMS subcutaneously, and as few as 500 RMSg2 cells were sufficient to form tumors. These results suggest that the RMSg2 cells are multipotent cancer cells that partially combine the properties of skeletal muscle-resident stem/progenitor cells and high tumorigenicity. Thus, our model system's capacity to regenerate tumor tissue in vivo and maintain stable cells in vitro makes it useful for developing therapeutics to treat PRMS.

Spatiotemporal distribution analyses of Wnt5a ligand and its receptors Ror2, Frizzled2, and Frizzled5 during tongue muscle development in prenatal mice

BackgroundThe mammalian tongue is a highly specialized muscular organ. The Wnt5a ligand regulates muscle development by mediating the activation of several noncanonical Wnt signaling pathways in a receptor context-dependent fashion. However, there is poor information on the expression and behavior of Wnt5a proteins during muscle development of the embryonic tongue. MethodsThe spatiotemporal distribution profiles of the Wnt5a ligand and its receptors, receptor tyrosine kinase-like orphan receptor 2 (Ror2), Frizzled2 (Fzd2), and Frizzled5 (Fzd5), in the developing tongue muscles of prenatal mice from embryonic day 12.5–18.5 were analyzed using immunofluorescence (IF) double staining of a target protein and desmin, a marker protein of myogenic cells. Immunolabeling images were subjected to digital detection analysis using the WinROOF 2018 version 4.19.0 image processing software when needed. ResultsIF signals of the Wnt5a ligand protein and its receptors Ror2 and Fzd2 were detected in developing myoblasts and myotubes of the embryonic tongue, but they were undetectable in mature myofibers equipped with sarcomere structures. Fzd2 expression was specific for desmin-positive developing muscle cells, whereas those of Ror2 and the Wnt5a ligand were widespread and nonselective for desmin-positive cells and that of Fzd5 was predominant in desmin-negative cells of the epithelium and subepithelial mesenchyme. ConclusionDeveloping muscle cells but not mature myofibers of the mouse embryonic tongue express the Wnt5a ligand and its receptors Ror2 and Fzd2, which may mediate Wnt5a signaling in the development processes of tongue muscle fibers.

650. ROBOTIC ESOPHAGEAL LEIOMYOMA ENUCLEATION

Abstract Leiomyomas are rare benign esophageal neoplasms. Surgery is indicated in symptomatic patients or if malignancy is suspected. A 20-year-old female presented with dysphagia to solids. Contrast enhanced computed tomography showed a 33-mm by 30-mm by 51 mm well circumscribed lobulated mass arising from the right anterolateral wall of the upper thoracic esophagus, causing luminal narrowing and abutting the azygos vein. An esophageal ultrasound performed for confirmation showed the lesion arising from the muscularis propria. Patient was scheduled for a robotic leiomyoma excision. Patient was placed in left lateral position, lung isolation was obtained with a double lumen tube. Four standard ports were placed in the sixth intercostal space. Robotic instruments used were the monopolar scissors, Maryland bipolar forceps, fenestrated bipolar forceps for dissection and needle holder for suturing. The leiomyoma was identified, mediastinal pleura and muscle layer were incised longitudinally. The leiomyoma was dissected all around in the submuscular plane taking care not to injure the underlying mucosa. However, while delivering the deep lobulated portion, there was an inadvertent injury to the mucosa. The mucosal tear was repaired with interrupted sutures utilising 4-0 Polydiaxanone sutures and the muscle layer was closed with V-Lock sutures. A check endoscopy was performed on table along with air insufflation with saline in the pleural cavity to check for an air leak. A nasogastric tube was placed, specimen was retrieved in a bag through the assistant port. Surgery was completed in 155 minutes with 50 ml blood loss. Post-operative course was uneventful, and the patient was discharged on full diet on the fourth post-operative day. Histopathology showed a SMA and desmin-positive spindle cell neoplasm without conspicuous mitotic activity. Precise dissection in a confined space is possible due to wrist-like movements, three-dimensional view and magnification offered by the robot. Repair of the injured mucosa and myotomy closure to prevent future diverticulum formation is easily accomplished with robotic suturing. Intraoperative endoscopy is useful in evaluating the integrity of the mucosa and muscle repair. Hence a robotic approach may be superior to a thoracoscopic/open leiomyoma enucleation and can be utilised even in moderate size masses.

Immunohistochemical Study of Smooth Muscle Cells and Elastin in Goose Lungs

Abstract There are many differences (morphological, physiological and mechanical) between the lungs of birds and the lungs of mammals. Birds have a more efficient exchange of oxygen and carbon dioxide than mammals. In this article, we studied the presence of four antibodies (actin, α-smooth muscle actin, desmin and elastin) in the lungs of geese. Smooth muscle cells (SMCs) immunoreactive to actin, α-SMA and desmin were observed in the primary and secondary bronchi and arranged as a continuous layer. In the tertiary bronchus (parabronchus), immunoreactive cells on α-SMA and desmin were observed as aggregations of smooth muscle cells in the septum tips in atrial opening. A small number of α-SMA and desmin-positive cells were observed on the periphery of the parabronchi and between the air and blood capillaries. The elastic fibres were found in the large bronchi in connection with smooth muscle bands. In the parabronchi the elastic fibres form an elastic membrane lining the parabronchial lumen. In the blood vessels, the elastic fibres form the inner and outer elastic membrane. The individual elastic membranes connect neighbouring blood vessels.

Two distinct Notch signals, Delta-like 4/Notch1 and Jagged-1/Notch2, antagonistically regulate chemical hepatocarcinogenesis in mice

Notch signaling is one of the most common drivers of carcinogenesis in many types of cancers, including hepatocellular carcinoma (HCC); however, it occasionally suppresses tumor progression. Moreover, it is virtually unknown how different sets of Notch ligands and receptors regulate the HCC development. In this study, we demonstrate that the expression of the Notch ligands, Delta-like 4 (Dll4) and Jagged-1 (Jag1), is upregulated during diethylnitrosamine-induced hepatocarcinogenesis. Dll4 is detected in the preneoplastic hepatocytes and HCC cells, but not in the normal hepatocytes, while Jag1 is expressed in the desmin-positive mesenchymal cells. Hepatocyte-specific Dll4 knockout abolishes the Notch1 signaling and suppresses the tumor progression. In contrast, Jag1 deletion induces the ectopic expression of Dll4 in hepatocytes along with the loss of Notch2 signaling, leading to the tumor progression. These results indicate that the two distinct Notch signals, Dll4/Notch1 and Jag1/Notch2, are antagonistic to each other, exerting opposite effects on HCC progression.

Defined d-hexapeptides bind CUG repeats and rescue phenotypes of myotonic dystrophy myotubes in a Drosophila model of the disease

In Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic double-stranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects. In this paper, we characterize four d-amino acid hexapeptides identified together with abp1, a peptide previously shown to stabilize CUG RNA in its single-stranded conformation. With the generalized sequence cpy(a/t)(q/w)e, these related peptides improved three MBNL-regulated exon inclusions in DM1-derived cells. Subsequent experiments showed that these compounds generally increased the relative expression of MBNL1 and its nuclear-cytoplasmic distribution, reduced hyperactivated autophagy, and increased the percentage of differentiated (Desmin-positive) cells in vitro. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests. Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different association constant, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp1. Finally, molecular modeling suggests a detailed view of the interactions of peptide-CUG RNA complexes useful in the chemical optimization of compounds.

Highly feasible procedure for laparoscopic transplantation of cell sheets under pneumoperitoneum in porcine model.

Cell sheet technology is one of the most successful methodologies in regenerative medicine. Various applications of cell sheets have been introduced in first-in-human studies in several clinical fields. When transplanting a cell sheet into internal organs, a relatively large incision is required for delivery due to difficulty handling the sheet. We developed a laparoscopic delivery procedure for safe and easy transplantation of cell sheets in a porcine model. Pneumoperitoneum was established by inflation with CO2. First, to increase the strength during handling, fibrin was sprayed onto the surface of the cell sheet, and then a myoblast sheet was placed onto the newly developed carrier. The sheets were pinched with laparoscopic forceps to insert into the abdominal cavity through the laparoscopic port. Myoblast sheets were then applied to the surface of the liver, colon, small intestine, and stomach, and procedure times were measured. At three days post transplantation, a histopathological examination was performed to confirm engraftment of the sheet. The function and engraftment were also analyzed in a duodenal endoscopic submucosal dissection (ESD) model. The fibrin-processed myoblast sheet was able to be managed with conventional laparoscopic forceps without breaking. Despite the drastic change in air pressure by passing through the laparoscopic port, the sheets suffered no apparent damage. The transplantation procedure times did not markedly differ among transplant sites. A histopathological examination revealed thin-layered, desmin-positive cells at each transplant site. With transplantation following ESD, the engrafted myoblast sheets effectively prevented delayed perforation. Our procedure is simple, and the system involves a carrier made of medically fit silicon, commercially available fibrin glue and conventional laparoscopic forceps. Our procedure is a powerful tool for laparoscopical cell sheet transplantation.

Generation of myogenic progenitor cell-derived smooth muscle cells for sphincter regeneration

BackgroundDegeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells.MethodsMyogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice.ResultsMPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue.ConclusionsOur work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

Endoplasmic reticulum stress enhances tumor necrosis factor-α expression in rat Kupffer cells to trigger hepatic stellate apoptosis cell through TNFR/caspase-8 pathway

To investigate the role of endoplasmic reticulum (ER)-stress of Kupffer cells (KCs) and KCs-derived tumor necrosis factor-α (TNF-α) in medicating apoptosis of hepatic stellate cell (HSC). Sixty male SD rats were randomized into control group, model group, ER- stress group, depletion group and KCs block group (n=15). The 4 groups of rats were given intraperitoneal injections (twice a week for 8 weeks) of normal saline (2 mg/kg); 40% CCl4 solution (in peanut oil, 2 mg/kg); 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg); and 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg) followed by clodronate liposomes (50 mg/kg), respectively. After the treatments, samples of the liver tissue and serum were collected from the rats from the 4 groups to isolate KC cells, which were co-cultured with LX2 cells. In the depletion group, the rats were injected with anti-rat TNFR mAb (0.35 mg/kg) via the portal vein before isolating the KCs. Liver function examination, Eirius red staining, ELISA, immuno- histochemical staining, and RT-PCR were performed to assess the liver function, liver fibrosis, KC phenotypes, expression of the in fl ammatory factors, and the number of active HSC was detected. The isolated KCs were treated with tunicamycin before co-culture with LX2 cells, and ELISA, RT-PCR and Western blot were performed to examine KC phenotypes, in fl ammatory factors, LX2 cell apoptosis and TNFR/caspase8 pathway activity. Compared with the rats in the control group, the rats in the model group had significantly increased ALT and AST levels, Sirius red staining-positive area, and Desmin-positive cells (activated HSCs) (P < 0.05) with significantly lowered number of CD16-positive KCs (M1), and TNF-α protein and mRNA expression (P < 0.05). Compared with those in the model group, the rats in ER-stress group showed significantly decreased ALT and AST levels, Sirius red staining positivity and Desmin-positive cells (P < 0.05) and increased number of CD16-positive KCs and TNF-α expressions (P < 0.05). In the depletion group, compared with the ER-stress group, the rats had significantly increased ALT and AST levels of, Sirius red staining positivity and Desmin-positive cells (P < 0.05) and reduced CD16- positive KCs and TNF-αexpressions (P < 0.05). In the cell co-culture experiment, the model group showed significantly reduced TUNEL-positive LX2 cells, CD16-positive cells, and expressions of TNFR1, cleaved caspase- 8 and cleaved caspase- 3 in the KCs (P < 0.05) with increased Desmin-positive LX2 cells (P < 0.05). Compared with the model group, the ER- stress group exhibited significantly increased TUNEL-positive LX2 cells, CD16-positive cells and expressions of TNFR, cleaved caspase-8 and cleaved caspase-3 in the KCs (P < 0.05) and decreased Desmin-positive LX2 cells (P < 0.05). In the depletion group, blocking TNFR resulted in significantly decreased expressions of cleaved caspase-8 and cleaved caspase-3 compared with those in ER- stress group (P < 0.05) although there was no significant changed in TNFR expression. ER stress of KCs promotes the transformation of KCs towards M1 phenotype and increases the expression of TNF-α, which triggers the apoptosis of HSCs through the TNFR/caspase-8 pathway.

MyoD1 expression in fibroepithelial stromal polyps

Fibroepithelial stromal polyps (FESPs) are benign polypoid mesenchymal lesions thought to arise from desmin-positive specialized stromal cells of the female genital tract. Although most cases are easily diagnosed by morphology alone, the morphology of FESPs is variable and in some instances can contain hypercellular stroma with numerous atypical desmin-positive cells, simulating botryoid embryonal rhabdomyosarcoma (ERMS). Recently, we encountered a cellular FESP showing desmin expression as well as nuclear immunoreactivity for the skeletal muscle-associated transcription factor MyoD1. Although these lesions are widely known to express desmin, there are very few studies examining expression of the more specific markers of skeletal muscle differentiation, myogenin and MyoD1. The aim of our study was to examine desmin, MyoD1, and myogenin expression in a series of 25 FESPs. Of the 25 cases, desmin expression was present in 23 (92%), at least focal MyoD1 expression was present in 10 (40%), and all cases were negative for myogenin. Follow-up data were available for all 25 cases, and none recurred or behaved in a malignant fashion. Awareness of this potential immunohistochemical pitfall and careful morphologic evaluation should allow for the confident distinction of MyoD1-positive FESP from botyroid ERMS in almost all instances.

Pleuropulmonary blastoma type I might arise in congenital pulmonary airway malformation type 4 by acquiring a Dicer 1 mutation

Congenital pulmonary airway malformation (CPAM) occurs most commonly in infants. It is divided into 5 types. The most common types 1 and 2 are cystic, type 0 presents as bronchial buds without alveolar tissue, most likely corresponding to alveolar dysgenesis, while type 3 is composed of branching bronchioles and appears as a solid lesion. A defect in the epithelial-mesenchymal crosstalk might be the underlying mechanism for all. Type 4 is a peripheral cystic lesion with a thin cyst wall covered by pneumocytes. CPAM 4 has been mixed up with pleuropulmonary blastoma (PPB) type I and some authors question its existence. We investigated five cases of CPAM type 4 for the presence or absence of rhabdomyoblasts, and for markers associated with CPAM development. In addition, all cases were evaluated for mutations within the Dicer gene and for mutations of the RAS family of oncogenes. All five cases showed smooth muscle actin and desmin-positive cells; however, only one case showed a few cells positive for MyoD. The same case showed a mutation of Dicer 1. All cases were negative for mutations of the RAS family of genes. Fibroblast growth factor 10 was similarly expressed in all cases, and thus cannot be used to differentiate CPAM4 from PPB-I. Low expression of the proliferation marker Ki67 was seen in our CPAM 4 cases and the probable PPB-I case. YingYang-1 protein seems to play an active role in the development of PPB-I. CPAM 4 can be separated from PPB-I based on the presence of rhabdomyoblasts and mutations in Dicer 1 gene. These cells might not be numerous; therefore, all available tissue has to be evaluated. As CPAM 4 morphologically looks very similar to PPB-I, it might be speculated, that there exists a potential for progression from CPAM 4 to PPB-I, by acquiring somatic mutations in Dicer 1.

NLR Family Pyrin Domain-Containing 3 Inflammasome Activation in Hepatic Stellate Cells Induces Liver Fibrosis in Mice.

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC).Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.

Application of tissue-engineered pericardial patch in rat models of myocardial infarction.

Myocardial infarction (MI) is a major cause of mortality and morbidity in industrialized societies. Myocardial tissue engineering is an alternative and promising approach for substituting injured myocardium through development and seeding of appropriate scaffolds. In this study, we investigated the efficacy of using an acellular pericardium to deliver autologous mesenchymal stem cells (MSCs) to the infarcted site for regeneration of the myocardium. MI was induced in two groups of rats; G1 or MI group, and G2 or patch-implanted group. In G2 group, rats had undergone transplantation of a pericardial patch which was previously seeded with adipose tissue derived MSCs. To evaluate the efficacy of the pericardial patches, biopsies were taken one month after transplantation. In order to evaluate the extent of regeneration, inflammation and fibrosis, histopathological investigations including hematoxylin and eosin (H&E), Sirius Red and trichrome staining were performed. In addition, immunohistochemical investigations by Desmin as well as CD68, CD45 and CD34 antibodies were performed. Furthermore, Tunnel assay was performed to detect the extent of apoptosis. H&E assessments of biopsies from the patch-implanted group confirmed presence of pre-seeded pericardium containing MSCs along with neo-vessels. Immunohistochemical assessments demonstrated higher number of CD34 positive cells and Desmin-positive cells in the patch implanted group (p < 0.05); these findings are suggestive of cardiomyocyte regeneration in G2 rats. This study demonstrates the advantages of application of natural acellular scaffolds as cell delivery devices and it emphasizes neovascularization following this approach. However, further investigations are required to analyze long-term cardiac function in recipients. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2670-2678, 2018.

Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies.

We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

Hepatic stellate cells derived from the nestin-positive cells in septum transversum during rat liver development.

Hepatic stellate cells (HSCs) play a principal role in Vitamin A metabolism and are considered the major matrix-producing cell type in the diseased liver. Rat HSCs are identified by immunohistochemistry with myogenic or mesenchymal (desmin, vimentin, and alpha-smooth muscle actin) or neural (e.g., GFAP or neuronal cell adhesion molecule) markers. Embryonic origin of rat HSCs was determined using these markers. Nestin, an intermediate filament protein originally identified in neuronal stem or progenitor cells, is widely used as a stem cell marker, including hepatic stem cells in adult rat livers. Additionally, nestin is reportedly expressed in activated HSCs during liver injury and hepatic regeneration. However, little is known about nestin expression in rat fetal liver HSCs. The present study aimed to clarify nestin-positive HSC expression during rat liver development. At embryonic day (ED) 10.5, nestin expression in mesenchymal cells adjacent to the liver bud was detected by immunohistochemistry. At ED 11.5, nestin-positive cells were also detected in desmin-positive cells appearing and increasing in intensity by ED 16.5. However, nestin-positive cells in the parenchyma decreased by ED 20.5 or later. These findings reveal that the nestin-positive HSCs during rat liver development originate from nestin-positive mesenchymal cells in the septum transversum.

Circumferential Esophageal Replacement by a Tissue-engineered Substitute Using Mesenchymal Stem Cells

Tissue engineering appears promising as an alternative technique for esophageal replacement. Mesenchymal stem cells (MSCs) could be of interest for esophageal regeneration. Evaluation of the ability of an acellular matrix seeded with autologous MSCs to promote tissue remodeling toward an esophageal phenotype after circumferential replacement of the esophagus in a mini pig model. A 3 cm long circumferential replacement of the abdominal esophagus was performed with an MSC-seeded matrix (MSC group, n = 10) versus a matrix alone (control group, n = 10), which has previously been matured into the great omentum. The graft area was covered with an esophageal removable stent. A comparative histological analysis of the graft area after animals were euthanized sequentially is the primary outcome of the study. Histological findings after maturation, overall animal survival, and postoperative morbidity were also compared between groups. At postoperative day 45 (POD 45), a mature squamous epithelium covering the entire surface of the graft area was observed in all the MSC group specimens but in none of the control group before POD 95. Starting at POD 45, desmin positive cells were seen in the graft area in the MSC group but never in the control group. There were no differences between groups in the incidence of surgical complications and postoperative death. In this model, MSCs accelerate the mature re-epitheliazation and early initiation of muscle cell colonization. Further studies will focus on the use of cell tracking tools in order to analyze the becoming of these cells and the mechanisms involved in this tissue regeneration.

YAP/TAZ Regulate Hepatic Stellate Cell and Portal Fibroblast Proliferation After Hepatic Ischemia/Reperfusion Injury

IntroductionHepatic ischemia/reperfusion (I/R) injury is a major complication after liver resection, liver transplantation, and trauma, however, the precise mechanisms of liver recovery after I/R have not been elucidated. The Hippo signaling pathway and their downstream effectors, Yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ), have been identified as key regulators of regulating cell proliferation and organ size. In the current study, we sought to determine the expression of YAP and TAZ and their contribution to liver recovery after I/R.MethodsA murine model of partial hepatic ischemia/reperfusion (I/R) was used to study liver recovery after injury. Hepatic stellate cell and portal fibroblast in liver tissue were assessed by immunonhistochemical staining for desmin. Tissue levels of PDGF‐BB and TGF‐b1 were measured by enzyme‐linked immunosorbent assay (ELISA). The expression of YAP/TAZ and their related protein were measured by immunohistochemical staining and Western blot. Proliferation of desmin positive cell was assessed by dual immunofluorescence staining for desmin and proliferationg cell nuclear antigen (PCNA).ResultsStellate cells and portal fibroblasts, which stained positive for desmin, proliferated around portal veins during early recovery periods. Proliferation of these cells correlated with increased liver expression of PGDF‐BB and TGF‐b1. The expression of phosphorylated LATS1 and phosphorylated MOB1 which sequester YAP/TAZ in the cytoplasm were decreased in liver tissue and nuclear staining of both YAP and TAZ was evident in desmin‐positive cells during liver recovery after I/R. Pharmacological inhibition of YAP and TAZ using verteporfin attenuated desmin positive cell proliferation after I/R.ConclusionThe data suggest that hepatic stellate cell and/or portal fibroblasts expand during liver recovery after I/R and YAP/TAZ regulate their proliferation.