Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only ...indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell‐based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony‐forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75NTR to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real‐time quantitative polymerase chain reaction indicated that NGF and p75NTR are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti‐NGF antibody showed decreased colony‐forming efficiency, DNA replication and expression of putative LSC markers ABCG2 and C/EBPδ. Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers ΔNp63α and ABCG2. Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony‐forming efficiency, and a maintainer of the LSC phenotype. Stem Cells 2019;37:139–149
Limbal stem cells (LSCs) harvested from human cadaveric corneoscleral rims show increased expression of putative LSC markers (ABCG2 and ΔNp63α) and greater life spans in vitro when cultured in medium supplemented with recombinant human nerve growth factor (NGF). Those cultured with NGF‐blocking antibodies show reduced expression of putative LSC markers (ABCG2 and C/EBPδ), decreased colony‐forming efficiency and proliferation.
Uniform pressure actuators (UPAs) have unique advantages for electromagnetic forming, and they have excellent prospects in specific applications. The typical structure of conventional UPAs consists ...of an internal coil and an external conductive channel; this leads to certain limitations such as poor electrical contact and low coil strength. In the present study, in order to resolve these shortcomings, a new type of UPA is proposed, in which the induced circuit consists of a conductive channel and a workpiece that is placed inside a spiral coil. Benefitting from the innovation of the special structure, the proposed UPA can improve the electrical contact by producing a squeezing force between the conductive channel and workpiece during the forming process. Moreover, it provides an open reinforced environment for increasing the coil strength without affecting the forming efficiency. Numerical and experimental studies demonstrate that forming efficiency of the proposed UPA is higher than that of the conventional UPA. Based on these advantages and the new proposed uniform-velocity method, the proposed UPA is applied to fabricate a fuel cell titanium bipolar plate in a single discharge process.
Display omitted
•Inner-field uniform pressure actuator (UPA) solved arcing issues by squeezing force.•The coil strength of Inner-field UPA can be improved by open reinforcement space.•The Inner-field UPA has higher forming efficiency than the conventional UPA.•The fully coupled finite element models of two types of UPAs are first established.•The complex titanium bipolar plate was formed by the new uniform-velocity method.
Wire arc additive manufacturing (WAAM) has the potential to become a method to production large and complex metal components, but there is a lot of material waste and energy consumption in the ...forming of thin-walled metal parts with variable-size. To solve this problem, this paper proposes a method for forming thin-walled parts with variable width and height is proposed, where metal samples are prepared by Variable Polarity (VP) TIG & Fused-deposition Additive Manufacturing (FDAM). This method adopts multi node subsection control strategy. Under the real-time acquisition system of composite dual-vision sensors, through the on-line control of deposition layer size is realized by matching each piece with different process parameters of thin-walled parts. The thin-wall parts with controllable range of 5.5–10 mm and the heights controllable range of 6.5–13 mm. The results show that the multi-node piecewise strategy achieves a stable and convergent control. Compared to the conventional method, the material utilization increases by 51.4%. With a 128% shortening of producing time. The proposed control method can remarkably reduce the frequencies of arc initiation and extinction, improve the production efficiency, and save materials and energy.
Display omitted
•Electrochemical study of interaction of three toxicants with lipid monolayer.•Effect of three toxicants on cell viability and genotoxicity.•Benchmarking of model membrane results ...with live cell assays.•Electrochemical characterisation of membrane interaction mechanism of toxicants.•Identification of biomembrane damage using fluorescent probe assay.
This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.
Objective
Glucose concentrations used in current cell culture methods are a significant departure from physiological glucose levels. The study focuses on comparing the effects of glucose ...concentrations on primary human progenitors (connective tissue progenitors CTPs) used for cartilage repair.
Design
Cartilage- (Outerbridge grade 1, 2, 3; superficial and deep zone cartilage), infrapatellar fatpad-, synovium-, and periosteum-derived cells were obtained from 63 patients undergoing total knee arthroplasty and cultured simultaneously in fresh chondrogenic media containing 25 mM glucose (HGL) or 5 mM glucose (NGL) for pairwise comparison. Automated ASTM-based quantitative image analysis was used to determine colony-forming efficiency (CFE), effective proliferation rates (EPR), and sulfated-proteoglycan (GAG-ECM) staining of the CTPs across tissue sources.
Results
HGL resulted in increased cell cultures with CFE = 0 compared with NGL in all tissue sources (P = 0.049). The CFE in NGL was higher than HGL for superficial cartilage (P < 0.001), and contrary for synovium-derived CTPs (P = 0.046) when CFE > 0. EPR of the CTPs did not differ between the media in the 6-day assay time period (P = 0.082). The GAG-ECM area of the CTPs and their progeny was increased in presence of HGL (P = 0.027).
Conclusion
Glucose concentration is critical to progenitor’s physiology and should be taken into account in the setting of protocols for clinical or in vitro cell expansion strategies.
Abstract At present, laser deposition forming technology of titanium alloy is widely used based on the flexibility of laser additive manufacturing technology. However, the mechanism and law of ...influence on the forming accuracy and efficiency need further study. Based on the orthogonal experiment, the analysis of the results shows that with the increase of energy density, the forming accuracy and efficiency are improved. There is a negative correlation between precision and efficiency. The experimental data are fitted and analyzed by establishing a statistical regression analysis model, a regression prediction model is established, and the regression prediction model is comprehensively calculated and analyzed, so as to optimize the appropriate parameter interval. During the forming process, the energy density is maintained in the range of 300 J/mm2–400 J/mm2, the forming accuracy is at a high level of 80%, the forming efficiency can be maintained in a high range of 0.35 g/s-0.40 g/s, and the whole forming process is in a state of high precision and high efficiency, so that the matching strategy of precision and efficiency in the forming process is the best. In order to improve the laser deposition forming titanium alloy.
The aim of the present study was to characterize human side population (SP) epidermal keratinocytes isolated from primary cell cultures. For that purpose, keratinocytes were isolated from normal ...adult breast skin samples and the Hoechst 33342 exclusion assay described for hematopoietic cells was adapted to keratinocytes. Three types of keratinocytes were studied: the SP, the main population (MP), and the unsorted initial population. SP keratinocytes represented 0.16% of the total population. In short-term cultures, they exhibited an increased colony-forming efficiency and produced more actively growing colonies than did unsorted and MP keratinocytes. In long-term cultures, SP cells exhibited an extensive expansion potential, performing a mean of 44 population doublings for up to 12 successive passages after cell sorting. Moreover, even in long-term cultures, SP keratinocytes were able to form a pluristratified epidermis when seeded on a dermal substrate. Unsorted and MP keratinocytes promoted a reduced expansion: mean values of 14 population doublings for five passages and 12 population doublings for four successive passages, respectively. To further characterize SP cells, cDNA microarrays were used to identify their molecular signature. Transcriptome profiling showed that 41 genes were differentially expressed in SP (vs. MP) cells, with 37 upregulated genes and only four downregulated genes in SP cells. The majority of these genes were functionally related to the regulation of transcription and cell signaling. In conclusion, SP human keratinocytes isolated from primary cultures exhibited both short- and long-term high proliferative potential, formed a pluristratified epidermis, and were characterized by a specific gene expression profile.
Cigarette smoke (CS) is associated with chronic obstructive pulmonary disease (COPD) and cancer. However, the underlying pathological mechanisms are not well understood. We recently reported that ...mice exposed to long-term intermittent CS for 3 months developed more severe emphysema and higher incidence of adenocarcinoma than mice exposed to long-term continuous CS for 3 months and long-term continuous CS exposure activated alveolar stem cell proliferation. However, the influence of variations in the CS exposure pattern in alveolar stem cell in unknown. Here, we exposed mice to 3 weeks of continuous or intermittent CS to identify whether different CS exposure patterns would result in differential effects on stem cells and the mechanisms underlying these potential differences.
Female mice expressing GFP in alveolar type 2 (AT2) cells, which are stem cells of the alveolar compartment, were exposed to mainstream CS via nasal inhalation. AT2 cells were collected based on their GFP expression by flow cytometry and co-cultured with fibroblasts in stem cell 3D organoid/colony-forming assays. We compared gene expression profiles of continuous and intermittent CS-exposed AT2 cells using microarray analysis and performed a functional assessment of a differentially expressed gene to confirm its involvement in the process using activator and inhibitor studies.
AT2 cells sorted from intermittent CS-exposed mice formed significantly more colonies compared to those from continuous CS-exposed mice, and both CS-exposed groups formed significantly more colonies when compared to air-exposed cells. Comparative microarray analysis revealed the upregulation of genes related to fatty acid oxidation (FAO) pathways in AT2 cells from intermittent CS-exposed mice. Treatment of intermittent CS-exposed mice with etomoxir, an inhibitor of the FAO regulator Cpt1a, for 5 weeks resulted in a significant suppression of the efficiency of AT2 cell colony formation. In vitro treatment of naïve AT2 cells with a FAO activator and inhibitor further confirmed the relationship between FAO and AT2 stem cell function.
Alveolar stem cell function was more strongly activated by intermittent CS exposure than by continuous CS exposure. We provide evidence that AT2 stem cells respond to intermittent CS exposure by activating stem cell proliferation via the activation of FAO.
PDF
