The efficacy of N-acetylcysteine in decreasing airway inflammation and mucus accumulation in horses with 18 hours of head confinement.

BACKGROUND: During transportation many horses develop post-transportation infection, which can be life-threatening and end their sport career. Preventing mucus accumulation and inflammation during transportation is vital, emphasizing the need for effective strategies to enhance overall horse health welfare. OBJECTIVES: Assess the impact of N-acetylcysteine (NAC) on mucus accumulation and inflammation in horses subjected to 18 hours of head confinement. ANIMALS: Six healthy crossbred horses, 5.3 ± 2.1 years of age and weighing 387 ± 30 kg. METHODS: Prospective placebo-controlled cross-over design study. The horses' heads were restrained in their stalls for a period of 18 hours. They were studied under 4 conditions: Not confined (NC): before head confinement, placebo (P), and confined head (CH): 18 hours of head confinement without treatment, and N-Acetylcysteine (NAC): 18 hours of head confinement treated with NAC before confinement (15 mg/kg/day NAC PO for 3 days). Bronchoalveolar lavage (BAL) was performed in each condition. Mucus accumulation along the trachea was evaluated by endoscopy. RESULTS: Endoscopic scores were significantly different between CH and other conditions, whereas no significant differences were found among NC, P, and NAC. The BAL cell count (34 291 ± 2624 cells/µL), neutrophil and lymphocyte count (18 601 ± 3193 cells/µL and 3337.4 ± 593 cells/µL, respectively) in CH were significantly higher compared to NAC. Neutrophil percentage was significantly higher in CH (53.8 ± 8%) compared to horses that received NAC (20.08 ± 8%). Conversely, in comparison to NAC (66.33 ± 9%), the percentage of macrophages was significantly lower in CH (35.7 ± 10%). CONCLUSIONS: N-acetylcysteine was found to significantly decrease mucus accumulation and inflammatory cell counts in horses with head confinement.

Decellularized Lung Extracellular Matrix Scaffold Promotes Human Embryonic Stem Cell Differentiation towards Alveolar Progenitors.

OBJECTIVE: Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells. MATERIALS AND METHODS: This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments. RESULTS: We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker) genes. CONCLUSION: Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

Morphometric study of the testis in sheep embryos using unbiased design-based stereology.

Sheep have been used as translational models of human postnatal testicular development. However, the morphometric features of the normal developing testis in sheep embryos have not been previously investigated using stereology. The objective of the present work was to establish normal quantitative parameters for fetal testicular tissue components in sheep, using unbiased design-based stereological methods. Twenty-four sheep embryos were divided into four gestational age groups (9-11, 12-14, 15-17 and 18-20 weeks of gestation) on the basis of the embryos' crown-rump length. Isotropic, systematic uniform random sections of the left testes were obtained by employing the orientator method. Testicular total volume, the absolute and proportional volumes occupied by the seminiferous tubules and interstitial tissue, as well as the seminiferous tubule length, were estimated using the point-counting system and the unbiased counting frame principle. All the parameters, with the exception of the interstitial tissue's fractional volume, gradually increased along with gestational age, with the maximum increase especially seen in the late fetal stages. The proportional volume of the interstitial tissue, on the other hand, showed a decreasing trend along with increasing gestational age. The absolute volume of the testes, of the seminiferous tubules and of the interstitial tissue, and the length of the seminiferous tubules showed a significant (p< 0.05) positive linear correlation with gestational age. Several similarities were observed with human testicular embryogenesis. The stereological data emerging from the present study might prove useful as basic contribution to the fields of andrology and embryology and stimulate further research in these areas.

Hydrocortisone Promotes Differentiation of Mouse Embryonic Stem Cell-Derived Definitive Endoderm toward Lung Alveolar Epithelial Cells.

In this article which was published in Cell J, Vol 20, No 4, winter 2019, on pages 469-479, the authors regret to acknowledge that we failed to mention in our article that a patent based on this study had been filed by Royan Institute and Tehran University with S.S.C., M.R.M.D., H.B., and Y.T. as inventors. The authors would like to apologies for any inconvenience caused.

Prevention of LPS-induced acute respiratory distress syndrome in sheep by bone marrow-derived mesenchymal stem/stromal cells.

MATERIALS AND METHODS: In this study, 10 male Shall sheep were used in two groups and bone marrow samples were collected and BM-MSCs isolated. Then experimental model of ARDS was induced by intrapulmonary injection of LPS to dose of 400 µg/kg. Twenty-four hours after LPS injection, 5 × 107 cells of BM-MSCs were autologous transferred in the group of treatment and 1 ml PBS was infused in the group of control as intrapulmonary. Then, the symptoms of clinical, complete blood count, analysis of arterial blood gases and the concentrations of IL6,IL10,TNF-α,total protein, Ig M and albumin BAL were determined before and at times of 3,6,12,24,48,72, and 168 after transplantation/infusion. KEY FINDINGS: The results of the investigations 24 h post-LPS injection(time 0) indicated the occurrence of acute inflammation which confirmed ARDS model. These changes included increase in RR, HR and RT, decrease in PO2 and SatO2 and increase in PCO2, WBC, neutrophils, macrophages, total protein,IL6,IL10, TNF-α,Ig M and albumin. But the stem/stromal cells transplantation reduced the severity of clinical signs induced by LPS, caused significant increase in PO2, SatO2 and IL-10 and significant decrease in PCO2, the total protein, TNF-α,IL-6, Ig M, albumin, WBCs, neutrophils and macrophages at different times of sampling both in compared with before transplantation(time 0) and in compared with the group of control. While in the control group, inflammation continued until the end of the study. SIGNIFICANCE: These results showed that BM-MSCs are able to reduce inflammation and have an important role in reconstruction of the damaged lung.

Mesenchymal stem/stromal cells: the therapeutic effects in animal models of acute pulmonary diseases.

The pulmonary diseases are one of the most important causes of death in the world. The successful therapies in the field of lung diseases are very limited and the medical treatments available are ineffective in many of the lung diseases. Many studies have evaluated the new therapies in the acute pulmonary diseases, and the transplantation of mesenchymal stem/stromal cells (MSCs), which is a branch of cell therapy, has a special place among the new medical techniques. The MSCs are present throughout the body and are thought to play a role in tissue regeneration and inflammation control. In the event of injury, the local MSCs traverse the shortest possible distance from the tissue or blood vessels to reach the affected site. But, there are few undifferentiated cells in the tissues. The exogenous MSCs are used to immunity modify or regenerative treatments in preclinical models of acute pulmonary diseases. Several studies have shown the positive effects of MSCs replacement in the acute lung disorders. The effection mechanism of the MSCs include the differentiation ability and the secretion of paracrine agents such as the anti-inflammatory mediators. Many studies suggest that this treatment method is safe and is probably to be widely used in future clinical trials. This review will describe the therapeutic effects of the MSCs in the experimental models of the acute pulmonary diseases for use as a method of treatment in clinical trials in future.

Hydrocortisone Promotes Differentiation of Mouse Embryonic Stem Cell-Derived Definitive Endoderm toward Lung Alveolar Epithelial Cells.

OBJECTIVE: The ability to generate lung alveolar epithelial type II (ATII) cells from pluripotent stem cells (PSCs) enables the study of lung development, regenerative medicine, and modeling of lung diseases. The establishment of defined, scalable differentiation methods is a step toward this goal. This study intends to investigate the competency of small molecule induced mouse embryonic stem cell-derived definitive endoderm (mESC-DE) cells towards ATII cells. MATERIALS AND METHODS: In this experimental study, we designed a two-step differentiation protocol. mESC line Royan B20 (RB20) was induced to differentiate into DE (6 days) and then into ATII cells (9 days) by using an adherent culture method. To induce differentiation, we treated the mESCs for 6 days in serum-free differentiation (SFD) media and induced them with 200 nM small molecule inducer of definitive endoderm 2 (IDE2). For days 7-15 (9 days) of induction, we treated the resultant DE cells with new differentiation media comprised of 100 ng/ml fibroblast growth factor (FGF2) (group F), 0.5 µg/ml hydrocortisone (group H), and A549 conditioned medium (A549 CM) (group CM) in SFD media. Seven different combinations of factors were tested to assess the efficiencies of these factors to promote differentiation. The expressions of DE- and ATII-specific markers were investigated during each differentiation step. RESULTS: Although both F and H (alone and in combination) promoted differentiation through ATII-like cells, the highest percentage of surfactant protein C (SP-C) expressing cells (~37%) were produced in DE-like cells treated by F+H+CM. Ultrastructural analyses also confirmed the presence of lamellar bodies (LB) in the ATII-like cells. CONCLUSION: These results suggest that hydrocortisone can be a promoting factor in alveolar fate differentiation of IDE2-induced mESC-DE cells. These cells have potential for drug screening and cell-replacement therapies.

Intrapulmonary autologous transplant of bone marrow-derived mesenchymal stromal cells improves lipopolysaccharide-induced acute respiratory distress syndrome in rabbit.

BACKGROUND: Lung diseases such as acute respiratory distress syndrome (ARDS) have a high incidence worldwide. The current drug therapies for ARDS have supportive effects, making them inefficient. New methods such as stromal cell therapy are needed for this problem. METHODS: This research was performed with ten New Zealand rabbits in two groups. Bone marrow aspiration was performed on the treated group, and mesenchymal stem cells were isolated and cultured. The experimental model of ARDS was induced using LPS from Escherichia coli strain O55:B5. Then, 1010 bone marrow mesenchymal stem cells (BM-MSCs) were autologously transplanted intrapulmonary in the treatment group, and 1-2 ml of PBS in the control group. The clinical signs, computed tomographic (CT) scans, echocardiography, blood gas analysis, complete blood count, and cytokine levels were measured before and at 3, 6, 12, 24, 48, 72, and 168 h after BM-MSC transplant. Finally, the rabbits were killed, and histopathological examination was performed. RESULTS: The results showed that BM-MSCs decreased the severity of clinical symptoms, the number of white blood cells and heterophils in the blood, the total cell count, and number of heterophils and macrophages in bronchoalveolar lavage, and balanced the values of arterial blood gases (increase in partial pressure of oxygen and O2 saturation and decrease in the partial pressure of carbon dioxide). They also downregulated the tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations and increased the IL-10 concentrations at different times compared with time 0 and in the control group, significantly. In the CT scan, a significant decrease in the Hounsfield units and total lung volume was found by echocardiography, and in comparing the two groups, a significant difference in the parameters was noticed. The histopathology demonstrated that the BM-MSCs were able to reduce the infiltration of inflammatory cells and pulmonary hemorrhage and edema. CONCLUSIONS: This study indicated that BM-MSCs play a significant role in the repair of lung injury.

Generation of Lung and Airway Epithelial Cells from Embryonic Stem Cells In Vitro.

The epithelium covers the internal surface of the lung airways and is composed of numerous cell types that arise from the anterior foregut endoderm. The ability to generate pulmonary cells from embryonic stem cells will allow study of lung development, drug screening, regenerative medicine, and modeling of lung diseases. The aim of this review is to describe the methods used to produce lung cells in vitro, based on mechanisms of lung development in during embryology.