Uterine macrophages as treatment targets for therapy of premature rupture of membranes by modified ADSC-EVs through a circRNA/miRNA/NF-κB pathway.

BACKGROUND: Circular RNA (circRNA) is a type of stable non-coding RNA that modifies macrophage inflammation by sponging micro RNAs (miRNAs), binding to RNA-binding proteins, and undergoing translation into peptides. Activated M1 phenotype macrophages secrete matrix metalloproteinases to participate in softening of the cervix uteri to promote vaginal delivery. METHODS: In this study, the premature rupture of membranes (PROM) mouse model was used to analyze the role of macrophages in this process. Profiling of circRNAs was performed using a competing endogenous RNA microarray, and their functions were elucidated in vitro. Meanwhile, adipose tissue-derived stem cell-secreted extracellular vesicles (EVs) were applied as a vehicle to transport small interfering RNAs (siRNAs) targeting the circRNAs to demonstrate their biological function in vivo. RESULTS: The miRNA miR-1931 is dependent on the nuclear factor kappa-B (NF-κB) pathway but negatively regulates its activation by targeting the NF-κB signaling transducer TRAF6 to prevent polarization of M1 macrophages and inhibit matrix metalloproteinase (MMP) secretion. The host gene of circRNA B4GALNT1, also an NF-κB pathway-dependent gene, circularizes to form circRNA_0002047, which sponges miR-1931 to maintain NF-κB pathway activation and MMP secretion in vitro. In the PROM model, EVs loaded with siRNAs targeting circRNAs demonstrated that the circRNAs reduced miR-1931 expression to maintain NF-κB pathway activation and MMP secretion for accelerating PROM in vivo. CONCLUSIONS: Our data provide insights into understanding PROM pathogenesis and improving PROM treatment.

Role of microRNA-21 in the formation of insulin-producing cells from pancreatic progenitor cells.

MicroRNAs (miRNAs) regulate insulin secretion, pancreas development, and beta cell differentiation. In this study, to screen for miRNAs and their targets that function during insulin-producing cells (IPCs) formation, we examined the messenger RNA and microRNA expression profiles of pancreatic progenitor cells (PPCs) and IPCs using microarray and deep sequencing approaches, respectively. Combining our data with that from previous reports, we found that miR-21 and its targets play an important role in the formation of IPCs. However, the function of miR-21 in the formation of IPCs from PPCs is poorly understood. Therefore, we over-expressed or inhibited miR-21 and expressed small interfering RNAs of miR-21 targets in PPCs to investigate their functions in IPCs formation. We found that miR-21 acts as a bidirectional switch in the formation of IPCs by regulating the expression of target and downstream genes (SOX6, RPBJ and HES1). Small interfering RNAs were used to knock down these genes in PPCs to investigate their effects on IPCs formation. Single expression of si-RBPJ, si-SOX6 and si-HES1 in PPCs showed that si-RBPJ was an inhibitor, and that si-SOX6 and si-HES1 were promoters of IPCs formation, although si-HES1 induced formation of IPCs at higher rates than si-SOX6. These results suggest that endogenous miRNAs involved in the formation of IPCs from PPCs should be considered in the development of an effective cell transplant therapy for diabetes.

Cryopreservation and multipotential characteristics evaluation of a novel type of mesenchymal stem cells derived from Small Tailed Han Sheep fetal lung tissue.

Lung mesenchymal stem cells (L-MSCs) characterized by plasticity, reduced relative immune privilege and high anti-fibrosis characteristics play the crucial role in lung tissue regenerative processes. However, up to date, the multi-differentiation potentials and application values of L-MSCs are still uncertain. In the current study, the Small Tailed Han Sheep embryo L-MSCs line from 12 samples, stocking 124 cryogenically-preserved vials, was successfully established by using primary culture and cell cryopreservation techniques. Isolated L-MSCs were morphologically consistent with fibroblasts, could be passaged for at least 18 passages and more than 91.8% of cells were diploid (2n = 54) analyze by G-banding. The majority of cells were in the G0/G1 phase (70.5-91.2%), and the growth curves were all typically sigmoidal. Moreover, L-MSCs were found to express pluripotent genes Oct4, Nanog and MSCs-associated genes ß-integrin, CD29, CD44, CD71, CD73 and CD90, while the expressions of hematopoietic cell markers CD34 and CD45 were negative. In addtion, the L-MSCs could be differentiated into cells of three layers with induction medium in vitro, which confirmed their multilineage differentiation potential. The secretion of urea and ALB showed the differentiated hepatocytes still possessed the detoxification function. These results indicated that the isolated L-MSCs displayed typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their maintenance of stemness and their proliferation in vitro.

Combination of melatonin and Wnt-4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury.

Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promoting differentiation of neural cells remain unknown. Wnt signaling mediates major developmental processes during embryogenesis and regulates maintenance, self-renewal, and differentiation of adult mammalian stem cells. However, the role of the noncanonical Wnt pathway during neurogenesis remains poorly understood. In this study, the amniotic epithelial cells ( AECs) were isolated from bovine amnion and incubated with various melatonin concentrations (0.01, 0.1, 1, 10, or 100 µm) and 5 × 10(-5) m all-trans retinoic acid (RA) for screening optimum culture medium of neural differentiation, compared with each groups, 1 µm melatonin and 5 × 10(-5) m RA were selected to induce neural differentiation of AECs, and then siMT1, siMT2, oWnt-4, and siWnt-4 were expressed in AECs to research role of these genes in neural differentiation. Efficiency of neural differentiation was evaluated after expressed above genes using flow cytometry. Cell function of neural cells was demonstrated in vivo using spinal cord injury model after cell transplantation, and damage repair of spinal cord was assessed using cell tracking and Basso, Beattie, Bresnahan Locomotor Rating Scale scores. Results demonstrated that melatonin stimulated melatonin receptor 1, which subsequently increased bovine amniotic epithelial cell vitality and promoted differentiation into neural cells. This took place through cooperation with Wnt-4. Additionally, following cotreatment with melatonin and Wnt-4, neurogenesis gene expression was significantly altered. Furthermore, single inhibition of melatonin receptor 1 or Wnt-4 expression decreased expression of neurogenesis-related genes, and bovine amniotic epithelial cell-derived neural cells were successfully colonized into injured spinal cord, which suggested participation in tissue repair.

Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells.

Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 µm melatonin, and cell viability studies indicated that 10 µm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 µm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 µm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 µm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.

Multi-lineage potential research of bone marrow mesenchymal stem cells from Bama miniature pig.

Bone marrow mesenchymal stem cells (BMSCs) are easy to obtain and thought to be ideal candidate cells for reconstruction of tissues and organs. Pigs are an appropriate animal model because their physiological structure, organ size, nutritional metabolism, and pathological reactions are similar to those of humans. In this study, bone marrow was collection from Bama miniature pigs to isolate BMSCs (B-BMSCs) by whole bone marrow culture method. We then examined their biological characteristics such as growth kinetics, surface antigen, and multi-lineage potential. B-BMSCs could be cultured for 36 passages in vitro. Growth kinetics and colony forming assay analyses indicated that B-BMSCs had a strong capacity for self-renewal in vitro and their proliferation rate appeared to decrease with passaging. These findings were supported by the animal cytophysiology in vitro. Surface antigen detection showed that B-BMSCs expressed CD29, CD44, CD71, CD73, and CD90, but not the endothelial cell marker CD31 or hematopoietic cell-specific marker CD34. This result was consistent with the characteristics of B-BMSCs. Furthermore, under appropriate conditions for multidirectional differentiation, B-BMSCs were induced to differentiate into adipocytes, osteoblasts, neuron-like cells, islet cells, liver-like cells, and endothelial cells as indicated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. These results verified the differentiation potential of B-BMSCs. In this study, B-BMSCs were isolated from Bama miniature pigs, and the self-renewal ability and differential potential was evaluated in vitro. The present study has important bearing on the potential application of B-BMSCs as a stem cell source for regenerative therapies. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 671-685, 2015. © 2015 Wiley Periodicals, Inc.

Construction and analysis of Siberian tiger bacterial artificial chromosome library with approximately 6.5-fold genome equivalent coverage.

Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

Ginger-Derived Extracellular Vesicles: A Natural Solution for Alopecia.

BACKGROUND: Ginger (Zingiber officinale (L.) Rosc), as an edible plant-derived nanoparticle, offers several advantages, such as a high return rate, low budget, no ethical barriers, and good for health. Ginger-Derived Extracellular Vesicles (GDEVs) are nanoscale vesicles isolated from ginger. METHODS: In this study, GDEVs were used to treat the alopecia mouse model, and its main active components and potential mechanism of action were investigated. The LC-MS/MS analysis of GDEVs revealed the presence of 1299 chemical compounds, among which auxiliary components were identified. Interestingly, the crux of the analysis lies in the discovery of 13 specific ingredients that play a pivotal role in hair proliferation. The aim of this study was to investigate the protective effect of GDEVs on hair loss. These advantages make ginger-derived nanoparticles a promising solution to overcome technical limitations associated with mammalian nanoparticles. This study elucidates the mechanism of action of GDEVs in the treatment of alopecia. However, the active ingredients and mechanism of action of GDEVs in the treatment of hair loss are unknown. RESULTS: GDEVs were isolated from ginger using the differential centrifugal method. Network pharmacological analysis of the GDEVs revealed that the anti-hair loss effect of GDEVs on alopecia was closely linked to its ability to reduce inflammation and promote the proliferation of hair follicle stem cells. Subsequently, it was applied to the balding areas of hair-loss mice using a brush. The results demonstrated that the application of GDEVs led to a rapid recovery of the balding areas and promoted the growth of healthier hair. CONCLUSION: This experiment reported that GDEVs can effectively suppress the inflammatory activity in the alopecia model mice.

Energy Absorption and Failure Modes of Different Composite Open-Section Crush Elements under Axial Crushing Loading.

In order to study the energy absorption characteristics of the open-section thin-walled composite structures with different cross-sections, axial compression tests were carried out at loading speeds of 0.01 m/s, 0.1 m/s, and 1 m/s. Finite element models were built to predict the crushing response and energy absorption behaviors of these open-section structures. The effects of the cross-section's shape, cross-section aspect ratio, trigger mechanism, and loading speed on the energy absorption characteristics of the composite structures were analyzed. The results show that the average crushing loads of the hat-shaped and Ω-shaped open-section structures are 14.1% and 14.6% higher than those of C-shaped open-section structures, and the specific energy absorption (SEA) values are 14.3% and 14.8% higher than that of C-shaped open-section structures, respectively. For the C-shaped open-section structures, a 45° chamfer trigger is more effective in reducing the initial peak load, while a 15° steeple trigger is more appropriate for the hat-shaped open-section structures. The average crushing loads and SEA of C-shaped, hat-shaped, and Ω-shaped open-section structures are reduced when the loading speed is increased from 0.01 m/s to 1 m/s. The increase in loading speed leads to the splashing of debris and thus reduces the loading area and material utilization of open-section structures, leading to a decrease in energy absorption efficiency.

Upregulation of MMPs in placentas of patients with gestational diabetes mellitus: Involvement of the PI3K/Akt pathway.

In recent years, there has been a notable rise in the incidence of pregnancies complicated by gestational diabetes mellitus (GDM), characterized by glucose intolerance first identified during pregnancy. Analysis of placental tissue has revealed that placentas from women with GDM tend to be larger and heavier compared to control placentas, indicating potential changes in trophoblast proliferation, differentiation, and apoptosis. In this study, transcriptome sequencing was conducted on placentas obtained from both normal pregnancies and pregnancies with GDM to investigate the molecular mechanisms underlying this condition. The original sequencing data were subjected to sequencing analysis, resulting in the identification of 935 upregulated genes and 256 downregulated genes. The KEGG and GO analysis techniques on differential genes uncovered evidence suggesting that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway may contribute to the pathogenesis of GDM. Subsequent analysis indicated that the expression levels of matrix metalloproteinases (MMP) 11, MMP12, MMP14, and MMP15, which are regulated by the PI3K/Akt pathway, were upregulated in the placentas of patients with GDM when compared to those of individuals with normal placental function. Additionally, our investigation into alternative splicing patterns revealed an increase in exon skipping alternative splicing of CSF3R in the placenta of patients with GDM compared to that in the control group. The CSF3R-PI3K-MMP pathway is speculated to regulate the pathogenesis of GDM.

Transfer of inflammatory mitochondria via extracellular vesicles from M1 macrophages induces ferroptosis of pancreatic beta cells in acute pancreatitis.

Extracellular vesicles (EVs) exert a significant influence not only on the pathogenesis of diseases but also on their therapeutic interventions, contingent upon the variances observed in their originating cells. Mitochondria can be transported between cells via EVs to promote pathological changes. In this study, we found that EVs derived from M1 macrophages (M1-EVs), which encapsulate inflammatory mitochondria, can penetrate pancreatic beta cells. Inflammatory mitochondria fuse with the mitochondria of pancreatic beta cells, resulting in lipid peroxidation and mitochondrial disruption. Furthermore, fragments of mitochondrial DNA (mtDNA) are released into the cytosol, activating the STING pathway and ultimately inducing apoptosis. The potential of adipose-derived stem cell (ADSC)-released EVs in suppressing M1 macrophage reactions shows promise. Subsequently, ADSC-EVs were utilized and modified with an F4/80 antibody to specifically target macrophages, aiming to treat ferroptosis of pancreatic beta cells in vivo. In summary, our data further demonstrate that EVs secreted from M1 phenotype macrophages play major roles in beta cell ferroptosis, and the modified ADSC-EVs exhibit considerable potential for development as a vehicle for targeted delivery to macrophages.

Biological characterization of chicken mesenchymal stem/progenitor cells from umbilical cord Wharton's jelly.

Mesenchymal stem/progenitor cells derived from Wharton's jelly of the umbilical cord (UCMSCs/UCMPCs) are multipotent, and can be differentiated in vitro into many cell types. Much work has been done on UCMSCs/UCMPCs from humans, mice, rabbits, and other mammals, but the relatively little literature has been published about these cells in chickens. In our work, we isolated USMSCs/USMPCs from chicken embryos. We characterized the isolated cells using immunofluorescence and reverse transcription polymerase chain reaction techniques. Primary UCMSCs/UCMPCs were subcultured to passage 30 and growth curves for each passage determined. The growth curves at different passages were all typically sigmoidal. Isolated UCMSCs/UCMPCs were induced to differentiate into adipocytes, osteoblasts, myocardial cells, and neural cells, and we were able to detect characteristic CD44, CD29, CD73, and CD71 cell surface markers. Our results suggest that UCMSCs/UCMPCs isolated from chickens possess similar biological characteristics to those from other species. Their multi-lineage differentiation capabilities herald a probable application for cellular transplant therapy in tissue engineering.

Isolation and characterization of mesenchymal stem cells from chicken bone marrow.

The bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells, which can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit and other mammals, but rarely in poultry. So, in this study, Thirty- to sixty-day old chicken was chosen as experimental animal, to isolate and characterize BMSCs from them. To investigate the biological characteristics of chicken BMSCs, immunofluorescence and RT-PCR were used to detect the characteristic surface markers of BMSCs. Growth curves were drawn in accordance with cell numbers. To assess the differentiation capacity of the BMSCs, cells were induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The surface markers of BMSCs, CD29, CD44, CD31, CD34, CD71 and CD73, were detected by immunofluorescence and RT-PCR assays. The growth curves of different passages were all typically sigmoidal. Karyotype analysis showed that these in vitro cultured cells were genetically stable. In addition, BMSCs were successfully induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The results suggest that the BMSCs isolated from chicken possess similar biological characteristics with those separated from other species, and their multi-lineage differentiation potentiality herald a probable application for cellular transplant therapy in tissue engineering.

Corrigendum: miR-212/132-enriched extracellular vesicles promote differentiation of induced pluripotent stem cells into pancreatic beta cells.

[This corrects the article DOI: 10.3389/fcell.2021.673231.].

Extracellular Vesicle Content Changes Induced by Melatonin Promote Functional Recovery of Pancreatic Beta Cells in Acute Pancreatitis.

Aim: Acute pancreatitis is an inflammatory disorder of the pancreas, which causes abnormal activation of immune cells. The macrophages were accumulated in pancreas and infiltrated into islets during the AP process to induce abnormal glucose metabolism. However, the role of macrophages in abnormal glucose metabolism remains understood. Extracellular vesicles act in the regulation of intercellular function, but whether EVs secreted by macrophages contribute to ß cell failure and apoptosis in AP is unclear. Based on this, the aim of this study was to reveal the role of macrophages-EVs in AP and develop a treatment for symptoms of hyperglycemia in AP. Methods: The AP model was established and treated by various doses of melatonin to analyze the therapeutic effect. The accumulation and polarization of macrophages in the AP pancreas were observed, and the ß cells were incubated with pancreatic derived EVs to analyze the role in ß cell failure and apoptosis. Results: The results showed that macrophages were recruited and polarized to M1 phenotype macrophages in the pancreas of AP mice, which obtained inflammatory EVs that contained specific miRNAs to induce ß cell failure and apoptosis. Then, the EVs derived from M1 macrophages triggered ß cell failure and apoptosis. Melatonin prevented polarization of macrophages to the M1 phenotype in vivo, which reduced the secretion of inflammatory EVs, changed the abundance of miRNAs in EVs, and therefore decreased inflammatory EV-mediated ß cell failure and apoptosis. Conclusion: Our results demonstrate that similar to 20S proteasome inhibitor MG132, analyses indicated that melatonin prevented degradation of IκBα through the ubiquitylation pathway to restrict p50 subunits to the cytoplasm of macrophages, inhibited activation of the NF-κB pathway to downregulate the transcription of specific miRNAs, and reduced miRNA transport into EVs.

A Novel Variant in the Desmoplakin Gene in One Case of the Rare Carvajal Syndrome with Dilated Cardiomyopathy: A Case Report and Literature Review.

Carvajal syndrome is a rare hereditary cardiocutaneous syndrome caused by the variants of the desmoplakin (DSP) gene. In this study, we report a patient of Carvajal syndrome with a novel homozygous missense variant of DSP gene. We diagnosed a 7-year-old female patient with Carvajal syndrome characterized by dilated cardiomyopathy, palmoplantar keratoderma, woolly hair, and dental dysplasia, who disclosed a novel homozygous missense variant c.4597C > T (p.Q1533X) in exon 6 of the DSP gene found for the first time. Both her parents were heterozygous for the identified nonsense variant c.4597C > T (p.Q1533X) in DSP gene but neither showed evidence of Carvajal syndrome, indicating that this novel variant causes the disease in an autosomal recessive manner. Genotypes of Carvajal syndrome are even broader than so far anticipated. When patients with dilated cardiomyopathy, palmoplantar keratoderma, woolly hair, and dental dysplasia are found in clinical practice, Carvajal syndrome should be highly suspected, and family gene sequencing should be actively carried out.

Characterization of vascular endothelial progenitor cells from chicken bone marrow.

BACKGROUND: Endothelial progenitor cells (EPC) are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. RESULTS: We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P) 1, -5 and -9 were typically "S"-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. CONCLUSIONS: These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

Quantification and statistical analysis on the cranial vault morphology for Chinese children 3-10 years old.

BACKGROUND AND OBJECTIVE: Head injury is the leading cause of fatalities and disabilities in children. Characterizing the variation in cranial size/shape and thickness during growth is important for developing finite element models of child heads and evaluating head injury risk at different ages. However, the quantitative morphological features of the cranial vault (size/shape and non-uniform thickness distribution) have not been accounted for in children aged between 3 and 10 years old (YO). METHODS: Geometrically equivalent discrete points were identified on 42 head CT scans of 3-10 YO children by separation, curve dividing, and point fitting. Based on discrete points, the principal component analysis and regression (PCA&R) method was used to develop a statistical model of the cranial vault as a function of age and head circumference. RESULTS: The ontogeny of three-dimensional cranial morphology and non-uniform thickness from 3 to 10 years of age was quantified and cranial vault morphologies for 3-10 YO children were generated in 1 year intervals. CONCLUSIONS: The automatic method, the procedure of identifying discrete points from CT scans, and the developed quantitative cranial vault model are reliable and accurate.

Immune cell-derived extracellular vesicular microRNAs induce pancreatic beta cell apoptosis.

Background: Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by an autoimmune response against pancreatic islet ß cells. Increasing evidence indicates that specific microRNAs (miRNAs) from immune cells extracellular vesicles are involved in islet ß cells apoptosis. Methods: In this study, the microarray datasets GSE27997 and GSE137637 were downloaded from the Gene Expression Omnibus (GEO) database. miRNAs that promote islet ß cells apoptosis in T1DM were searched in PubMed. We used the FunRich tool to determine the miRNA expression in extracellular vesicles derived from immune cells associated with islet ß cell apoptosis, of which we selected candidate miRNAs based on fold change expression. Potential upstream transcription factors and downstream target genes of candidate miRNAs were predicted using TransmiR V2.0 and starBase database, respectively. Results: Candidate miRNAs expressed in extracellular vesicles derived from T cells, pro-inflammatory macrophages, B cells, and dendritic cells were analyzed to identify the miRNAs involved in ß cells apoptosis. Based on these candidate miRNAs, 25 downstream candidate genes, which positively regulate ß cell functions, were predicted and screened; 17 transcription factors that positively regulate the candidate miRNAs were also identified. Conclusions: Our study demonstrated that immune cell-derived extracellular vesicular miRNAs could promote islet ß cell dysfunction and apoptosis. Based on these findings, we have constructed a transcription factor-miRNA-gene regulatory network, which provides a theoretical basis for clinical management of T1DM. This study provides novel insights into the mechanism underlying immune cell-derived extracellular vesicle-mediated islet ß cell apoptosis.

Directed neural differentiation of duck embryonic germ cells.

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.