Histology Atlas of the Developing Mouse Respiratory System From Prenatal Day 9.0 Through Postnatal Day 30.

Respiratory diseases are one of the leading causes of death and disability around the world. Mice are commonly used as models of human respiratory disease. Phenotypic analysis of mice with spontaneous, congenital, inherited, or treatment-related respiratory tract abnormalities requires investigators to discriminate normal anatomic features of the respiratory system from those that have been altered by disease. Many publications describe individual aspects of normal respiratory tract development, primarily focusing on morphogenesis of the trachea and lung. However, a single reference providing detailed low- and high-magnification, high-resolution images of routine hematoxylin and eosin (H&E)-stained sections depicting all major structures of the entire developing murine respiratory system does not exist. The purpose of this atlas is to correct this deficiency by establishing one concise reference of high-resolution color photomicrographs from whole-slide scans of H&E-stained tissue sections. The atlas has detailed descriptions and well-annotated images of the developing mouse upper and lower respiratory tracts emphasizing embryonic days (E) 9.0 to 18.5 and major early postnatal events. The selected images illustrate the main structures and events at key developmental stages and thus should help investigators both confirm the chronological age of mouse embryos and distinguish normal morphology as well as structural (cellular and organ) abnormalities.

Investigating the differential therapeutic efficacy and mechanisms of human umbilical cord mesenchymal stem cells at various passages in osteoarthritis treatment.

This study aimed to assess the clinical efficacy of umbilical cord mesenchymal stem cells (hUC-MSCs) from different passages (P3, P8, and P13) in the treatment of knee osteoarthritis (OA) and explore the underlying mechanisms. The hUC-MSCs from each passage were characterized and evaluated for their stemness, migration, proliferation, and marker expression. Rats with OA were treated with hUC-MSCs from each passage, and the therapeutic effects were assessed based on knee swelling, discomfort, and pathological examination of the knee joint. Co-culture experiments were conducted to examine the ability of hUC-MSCs to stimulate type II collagen synthesis and inhibit MMP13 expression in chondrocytes. Telomere length and telomerase activity of hUC-MSCs from each passage were measured to investigate the reasons for the observed differences in clinical efficacy. The results revealed that P3 and P8 hUC-MSCs exhibited superior osteogenic and chondrogenic differentiation potential compared to P13, while P13 demonstrated stronger adipogenic differentiation. The wound healing rate was significantly higher in the P3 and P8 groups compared to P13. All hUC-MSC groups expressed high levels of CD90 and CD105, indicating their mesenchymal stem cell characteristics, while CD31 and CD45 were not expressed. CD105 expression was significantly reduced in the P13 group. In the treatment of rat osteoarthritis, there were no significant differences in knee swelling, discomfort, Mankin scores, and pathological findings between P3 and P8 hUC-MSC treatments. However, there was a significant difference between the 8th and 13th passages. Co-culture experiments showed that hUC-MSCs from P3 and P8 enhanced type II collagen synthesis and reduced MMP13 expression in chondrocytes. Although no significant difference was observed between the P3 and P8 groups, a significant difference was found between the P13 and P8 groups. Telomere length analysis revealed that P13 samples had significantly shorter telomeres compared to both P3 and P8. The telomerase activity was positive in P3 and P8 hUC-MSCs, indicating no significant difference between these passages, while it was negative in P13 hUC-MSCs. In conclusion, P3 and P8 hUC-MSCs exhibited superior therapeutic potential for knee osteoarthritis compared to P13, possibly due to their enhanced differentiation capacity and telomerase activity.

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance.

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

An Analysis of Positional Generic and Individualized Speed Thresholds Within the Most Demanding Phases of Match Play in the English Premier League.

OBJECTIVES: To analyze the positional distances covered above generic and individualized speed thresholds within the most demanding phases of match play. Categorized by position, 17 English Premier League players' match data were analyzed over 2 consecutive seasons (2019-20 and 2020-21). The most demanding phases of play were determined using a rolling average across 4 periods of 1, 3, 5, and 10 minutes. Distance covered in the time above the standard speed of 5.5 m/s was analyzed, with individualized metrics based on the maximal aerobic speed (MAS) test data. RESULTS: Central defenders displayed lower values for high-intensity periods when compared with fullbacks, midfielders, and wide midfielders for both generic and individualized metrics. MAS during 1-minute periods was significantly higher for forwards when compared with central defenders (82.9 [18.9] vs 67.5 [14.8] for maximum high-speed running [HSR] and 96.0 [15.9] vs 75.7 [13.8] HSR for maximum MAS activity). The maximum effect size differences between the central midfielders, wide midfielders, and fullbacks for HSR and MAS measures under the maximum HSR criterion was 0.28 and 0.18 for the 1-minute period, 0.36 and 0.19 for the 3-minute period, 0.46 and 0.31 for the 5-minute period, and 0.49 and 0.315 for the 10-minute period. CONCLUSIONS: Individualized speed metrics may provide a more precise and comparable measure than generic values. Data appear to be consistent across playing positions except for central defenders. This information may allow practitioners to directly compare individualized physical outputs of non-central defenders during the most demanding phases of play regardless of the players' positional group. This may provide coaches with important information regarding session design, training load, and fatigue monitoring.

Border fences reduce potential for transboundary migration of Marco Polo Sheep (Ovis ammon polii) in the Pamir Plateau.

Border fences have severely impeded the transboundary migration of a number of large mammals worldwide, with central Asia being one of the most impacted. The Marco Polo sheep (Ovis ammon polii), an iconic species of Pamir, is threatened in its transboundary movement by increasing border fencing among their five distributed countries, including Tajikistan, Kyrgyzstan, China, Afghanistan, and Pakistan. In this study, by building ensemble species distribution models, we found that eastern Tajikistan had the largest suitable Macro Polo sheep habitat (about 42 % of the total suitable habitat), followed by China (about 32 %). We used least-cost paths to identify 51 ecological corridors including 5 transboundary ecological corridors, which may be important to maintain connectivity in both domestic and transboundary regions. To assess the potential barrier effect of border fences, we assessed four scenarios (30, 40, 50 and 60°) corresponding to the upper limit of the slope for the construction of fences. In areas too steep for fencing, these could be used by wild sheep to cross barriers or borders and may represent migration or movement routes, defined as natural passages. In the most pessimistic Scenario 60, only 25 migratory passages along the border fences were identified, compared to 997 in the most optimistic scenario (Scenario 30), indicating a strong negative effect of intensive border fencing on the transboundary movement of Marco Polo sheep. The establishment of transnational conservation parks, and ensuring permeability is maintained in key areas, could have a positive impact on the connectivity and persistence of Marco Polo sheep populations, and provide important lessons for other large migratory mammals in transboundary regions.

Are genetic drift and stem cell adherence in laboratory culture issues for cultivated meat production?

Mesenchymal stem cell-based cultivated meat is a promising solution to the ecological and ethical problems posed by traditional meat production, since it exhibits a protein content and composition that is more comparable to original meat proteins than any other source of cultivated meat products, including plants, bacteria, and fungi. Nonetheless, the nature and laboratory behavior of mesenchymal stem cells pose two significant challenges for large-scale production: genetic drift and adherent growth in culture. Culture conditions used in the laboratory expose the cells to a selective pressure that causes genetic drift, which may give rise to oncogene activation and the loss of "stemness." This is why genetic and functional analysis of the cells during culture is required to determine the maximum number of passages within the laboratory where no significant mutations or loss of function are detected. Moreover, the adherent growth of mesenchymal stem cells can be an obstacle for their large-scale production since volume to surface ratio is limited for high volume containers. Multi-tray systems, roller bottles, and microcarriers have been proposed as potential solutions to scale-up the production of adherent cells required for cultivated meat. The most promising solutions for the safety problems and large-scale obstacles for cultivated meat production are the determination of a limit number of passages based on a genetic analysis and the use of microcarriers from edible materials to maximize the volume to surface proportion and decrease the downstream operations needed for cultivated meat production.

Inactivated and Immunogenic SARS-CoV-2 for Safe Use in Immunoassays and as an Immunization Control for Non-Clinical Trials.

Successful SARS-CoV-2 inactivation allows its safe use in Biosafety Level 2 facilities, and the use of the whole viral particle helps in the development of analytical methods and a more reliable immune response, contributing to the development and improvement of in vitro and in vivo assays. In order to obtain a functional product, we evaluated several inactivation protocols and observed that 0.03% beta-propiolactone for 24 h was the best condition tested, as it promoted SARS-CoV-2 inactivation above 99.99% and no cytopathic effect was visualized after five serial passages. Moreover, RT-qPCR and transmission electron microscopy revealed that RNA quantification and viral structure integrity were preserved. The antigenicity of inactivated SARS-CoV-2 was confirmed by ELISA using different Spike-neutralizing monoclonal antibodies. K18-hACE2 mice immunized with inactivated SARS-CoV-2, formulated in AddaS03TM, presented high neutralizing antibody titers, no significant weight loss, and longer survival than controls from a lethal challenge, despite RNA detection in the oropharyngeal swab, lung, and brain. This work emphasizes the importance of using different techniques to confirm viral inactivation and avoid potentially disastrous contamination. We believe that an efficiently inactivated product can be used in several applications, including the development and improvement of molecular diagnostic kits, as an antigen for antibody production as well as a control for non-clinical trials.

Epigenetic Iinsights into the Senescence of Porcine Fetal Fibroblasts induced by Passaging.

Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were in vitro passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased ß-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower (p < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as METTL3, YTHDF2, and YTHDC1 were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.

[Disorders of lacrimal drainage: the way from theory to practice]. / Narushenie slezootvedeniya: put' ot teorii k praktike.

The article summarizes the results of research conducted over the past decade concerning the current problems of dacryology, analyzes the improvements made to the methods of diagnostics used in disorders of lacrimal passages on the basis of modern imaging and functional studies, describes the techniques aimed at improving the clinical effectiveness of the intervention, as well as drug and non-drug methods of intraoperative prevention of excessive scarring in the area of the artificial ostium. The article also analyzes the experience of using balloon dacryoplasty in relapses of tear duct obstruction after dacryocystorhinostomy, and presents the modern minimally invasive surgical techniques, including nasolacrimal duct intubation, as well as balloon dacryoplasty and endoscopic plastic surgery of the ostium of the nasolacrimal duct. Additionally, the work lists the fundamental and applied tasks of dacryology and outlines promising directions of its development.

Adiabatic Amplification of Energy and Magnetic Moment of a Charged Particle after the Magnetic Field Inversion.

We study the evolution of the energy and magnetic moment of a quantum charged particle placed in a homogeneous magnetic field, when this field changes its sign adiabatically. We show that after a single magnetic field passage through zero value, the famous adiabatic invariant ratio of energy to frequency is reestablished again, but with a proportionality coefficient higher than in the initial state. The concrete value of this proportionality coefficient depends on the power index of the frequency dependence on time near zero point. In particular, the adiabatic ratio of the initial ground state (with zero radial and angular quantum numbers) triplicates if the frequency tends to zero linearly as a function of time. If the Larmor frequency attains zero more than once, the adiabatic proportionality coefficient strongly depends on the lengths of the time intervals between zero points, so that the mean energy behavior can be quasi-stochastic after many passages through zero value. The original Born-Fock adiabatic theorem does not work after the frequency passes through zero. However, its generalization is found: the initial Fock state becomes a wide superposition of many instantaneous Fock states, whose weights do not depend on time in the new adiabatic regime.

Development and Characterization of Efficient Cell Culture Systems for Genotype 1 Hepatitis E Virus and Its Infectious cDNA Clone.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition.

Goblet cell-associated antigen passage: A gatekeeper of the intestinal immune system.

Effective delivery of luminal antigens to the underlying immune system is the initial step in generating antigen-specific responses in the gut. However, a large body of information regarding the immune response activation process remains unknown. Recently, goblet cells (GCs) have been reported to form goblet cell-associated antigen passages (GAPs). Luminal antigens can be transported inside GAPs and reach subepithelial immune cells to induce antigen-specific immune responses, contributing largely to gut homeostasis and the prevention of some intestinal diseases like allergic enteritis and bacterial translocation. In this article, we summarized recent observations on the formation of intestinal GAPs and their roles in mucosal immunity. We hope that this review can offer a fresh perspective and valuable insights for clinicians and researchers interested in studying the intestinal immune system.

Autofluorescence imaging permits label-free cell type assignment and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs).

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the study of three or four cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explants in real time. Furthermore, we find that this methodology for direct cell type-specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of SAPs and goblet cell associated antigen passages (GAPs) enable luminal antigen sampling. Airway secretory cells with SAPs are frequently juxtaposed to antigen presenting cells, suggesting that airway SAPs, like their intestinal counterparts, not only sample antigen but convey their cargo for immune cell processing.

Imaging several cell types, at the same time, within a living tissue is no small endeavor. To do so, scientists usually have to perform genetic manipulations that make certain proteins in each cell type fluorescent and therefore easy to track. However, these approaches are cumbersome, limited, and often not applicable to intact human tissues. A possible alternative would be to make use of autofluorescence ­ the fact that certain molecules in living cells naturally fluoresce when exposed to a particular wavelength of light. For example, this is the case for NAD(P)H and FAD, two small molecules necessary for life's biochemical processes, and whose intracellular levels and locations vary depending on cell type. In response, Shah, Hou et al. developed a new imaging technique that takes advantage of the unique autofluorescence signatures of NAD(P)H and FAD to distinguish between the seven different types of cells that line the surface of the airways of mice. Using their autofluorescence approach, Shah, Hou et al. were also able to discover a new role for secretory cells, which normally produce fluids, mucus and various proteins necessary for the lungs to work properly. The imaging experiments show that these cells could also sample material from the surface of the airway in a manner similar to how cells in the intestine take up material from the gut and pass their cargo to immune cells that mediate infection control or tolerance. Further studies should uncover more details about this new function of secretory lung cells. Other exciting discoveries may also emerge from researchers adopting the method developed by Shah, Hou et al. to examine a range of organs (both healthy and diseased), and attempting to apply it to human tissues.

The Study on the Identification of Musical Passages for an Emotion Perception Scale for People With Developmental Disabilities.

BACKGROUND: Emotion recognition is essential to the social adjustment and social interaction of people with intellectual and developmental disabilities (IDDs). Given that music is a medium for expressing and conveying human emotion, we conducted this preliminary study to identify musical passages representing the basic human emotions of happiness, sadness, and anger, with the goal of developing a music-based emotion perception scale for IDDs. METHODS: To identify musical passages for emotion perception, 20 certified music therapists evaluated 100 selected musical passages and established 60 pieces that yielded the highest agreement for each emotion category. During the second phase of this study, 300 neurotypical participants rated 60 passages in terms of the perceived type and intensity of emotions expressed. RESULTS: The 60 passages showed high reliability and were statistically classified into three factors: happiness, sadness, and anger. The k-means cluster analysis yielded a cut-off score of 41 for the low emotion perception group (F = 1120.63, P < 0.001). The hierarchical logistic regression analysis revealed that only model 3 (musical passages) was significantly associated with low emotion perception (step χ² = 227.8, P < 0.001). CONCLUSION: The selected musical passages demonstrated high reliability and established three factors for identifying perceptions of happiness, sadness, and anger. Neither psychological status nor individual demographic characteristics affected the emotion perception results.

A social-ecological engagement with reef passages in New Caledonia: Connectors between coastal and oceanic spaces and species.

Healthy and protected coral reefs help island systems in the tropics thrive and survive. Reef passages link the open ocean to lagoon and coastal areas in these ecosystems and are home to an exceptionally diverse and abundant marine life, hosting emblematic species and fish spawning aggregations. Their multiple benefits for the islands and their peoples (e.g., for transport, fishing, socio-cultural aspects) remain yet understudied. Drawing from qualitative interviews with fishers, scuba divers, and surfers along the coast of Grande Terre in New Caledonia, this study highlights the multi-faceted importance of these keystone places. It shows that reef passages are locally deemed 'communication zones' between coastal and oceanic spaces and species, and have significant un(der)explored ecological and socio-cultural roles. Understanding and protecting these ecological and cultural keystone places will strengthen both the reef ecosystems and the people dependent on them.

Corrigendum: Possible Involvement of Hsp90 in the Regulation of Telomere Length and Telomerase Activity During the Leishmania Amazonensis Developmental Cycle and Population Proliferation.

[This corrects the article DOI: 10.3389/fcell.2021.713415.].

A road map for in vivo evolution experiments with blood-borne parasitic microbes.

Laboratory experiments in which blood-borne parasitic microbes evolve in their animal hosts offer an opportunity to study parasite evolution and adaptation in real time and under natural settings. The main challenge of these experiments is to establish a protocol that is both practical over multiple passages and accurately reflects natural transmission scenarios and mechanisms. We provide a guide to the steps that should be considered when designing such a protocol, and we demonstrate its use via a case study. We highlight the importance of choosing suitable ancestral genotypes, treatments, number of replicates per treatment, types of negative controls, dependent variables, covariates, and the timing of checkpoints for the experimental design. We also recommend specific preliminary experiments to determine effective methods for parasite quantification, transmission, and preservation. Although these methodological considerations are technical, they also often have conceptual implications. To this end, we encourage other researchers to design and conduct in vivo evolution experiments with blood-borne parasitic microbes, despite the challenges that the work entails.

Zinc and hypoxic preconditioning: a strategy to enhance the functionality and therapeutic potential of bone marrow-derived mesenchymal stem cells.

The therapeutic use of bone marrow mesenchymal stem cells (BM-MSCs) requires a large number of cells (1-100 × 106 cells/kg of body weight). Extensive in vitro growth is limited due to the aging of cultured BM-MSCs which leads to abnormal morphology and senescence. Hypoxia increases BM-MSC proliferation, but the question of whether hypoxia preconditioning is safe for clinical application of BM-MSCs remains to be answered. Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. It is a structural constituent of numerous proteins on a molecular level, including transcription factors and enzymes of cellular signaling machinery. All the tissues, fluids, and organs of the human body contain zinc. More than 95% of zinc is intracellular, of which 44% is involved in the transcription of DNA. We investigated the effects of ZnCl2 on proliferation, morphology, migration, population doubling time (PDT), and gene expression of BM-MSCs under hypoxic (1% O2) and normoxic (21% O2) environments. BM-MSCs were preconditioned with optimized concentrations of ZnCl2 under normoxic and hypoxic environments and further examined for morphology by the phase-contrast inverted microscope, cell proliferation by MTT assay, PDT, cell migration ability, and gene expression analysis. Zinc significantly enhanced the proliferation of BM-MSCs, and it decreases PDT under hypoxic and normoxic environments as compared to control cells. Migration of BM-MSCs toward the site of injury increased and expression of HIF1-α significantly decreased under hypoxic conditions as compared to non-treated hypoxic cells and control. At late passages (P9), the morphology of normoxic BM-MSCs was transformed into large, wide, and flat cells, and they became polygonal and lost their communication with other cells. Conversely, zinc-preconditioned BM-MSCs retained their spindle-shaped, fibroblast-like morphology at P9. The expression of proliferative genes was found significantly upregulated, while downregulation of genes OCT4 and CCNA2 was observed in zinc-treated BM-MSCs under both normoxic and hypoxic conditions. ZnCl2 treatment can be used for extensive expansion of BM-MSCs in aged populations to obtain a large number of cells required for systemic administration to produce therapeutic efficacy.

Comparative analysis of mouse embryonic palatal mesenchymal cells isolated by two primary culture methods.

Cell culture plays a vital role in mechanism research, as a supplement to experiments in vivo. At present, there are two main methods to obtain mouse embryonic palatal mesenchymal (MEPM) cells, while no systematic investigation about characteristics of cells using these two methods to get a clear application. In this study, using the traditional two-step primary culture method as the control group, we found that the MEPM cells of the simplified one-step method and the control group were both consistent with the surface markers of mesenchymal stem cells. The vimentin (mesenchymal marker) was positive, cytokeratin 14 (CK14, epithelium marker) was negative. In addition, along with the increasing passages, the cells cultured by two methods had similar changes with a decreasing proliferation activity, increasing apoptosis rate, declining migration, diminishing stemness, and obvious senescence. Moreover, the senescence of MEPM cells in the one-step group was lower than that in the control group. Our findings indicate that there are no significant differences between the two methods and one-step method can be a good method in investigating palatal development mechanisms by shortening the processing of the MEPM cell culture and may reduce the damage to the cells done by more enzymes and processing.