Cells Tissues Organs最新文献

Cell morphology is an important regulator of cell function. Many abnormalities in cellular behavior can be discerned from changes in the shape of the cell and its organelles, typically the nucleus. Two major challenges for developing such phenotypic assays are reconstructing 3D surfaces of individual cells and nuclei from confocal images and developing characterizations of these surfaces for comparisons. We demonstrate two algorithms - 3D active contours and 3D condensed-attention UNet - to segment cells and nuclei from confocal images. The cell and nuclear surfaces are then converted into vectors using a reversible, spherical transform - i.e., shapes can be recovered from the vectors. Typical methods for characterizing shapes using size, shape, and image parameters such as area, volume, shape factor, solidity, and pixel intensities are not amenable to such reverse transformation. Our vector representation's principal component analysis shows that the significant modes of variability among cell and nucleus shapes are scaling and flattening. We benchmark these modes using a known mechanical model for nucleus morphology. Subsequent modes alter the eccentricity of the nucleus and translate and rotate it with respect to the cell. Our vector-space representation of cell and nucleus shape helps physically interpret the variability sources. It may further help to guide mechanical models and identify molecular mechanisms driving cell and nuclear shape changes.

Introduction: Digital dermatitis (DD) in cattle appears with high prevalence; nevertheless, the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD.

Methods: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, readout parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (days 7, 14, and 21), cultured skin samples were taken for (immuno)histological analysis.

Results: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially, basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin, and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture.

Conclusion: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as, e.g., DD.

Harsh local microenvironment, such as hypoxia and lack of instructive clues for transplanted stem cells, presents the serious obstacle for stem cell therapies' efficacy. Therefore, continued efforts have been taken to improve stem cells' viability and plasticity. Hepatocyte growth factor (HGF) has previously been reported to mitigate the complications of various human diseases in animal model studies and in some clinical trials. Besides, human stem cells from the root apical papilla (SCAP) are deemed a better resource of mesenchymal stem cells due to derived stem cells holding greater amplification ability in vitro compared with those from other dental resources. To move forward, evaluating effects and understanding underlying molecular mechanisms of HGF on SCAP for periodontal regeneration are needed. In this study, HGF was transgenically expressed in SCAP, and it was found that HGF enhanced osteo/dentinogenic differentiation capacity of SCAP compared with those of non-treated control in an ectopic mineralization model. Moreover, HGF reduced the apoptosis of SCAP under both normoxic and hypoxic conditions, whereas the combination of HGF and hypoxia exposure had inhibitory effects on cell proliferation during an 8-day in vitro culture period. Transcriptome analysis further revealed that suppressed cell cycle progression and activated BMP/TGFβ, Hedgehog, WNT, FGF, HOX, and other morphogen family members result upon HGF overexpression, which may render SCAP recapitulate part of neural crest stem cell characteristics. Moreover, strengthened stress response modulation such as unfolded protein response, macroautophagy, and anti-apoptotic molecules might explain the increased viability of SCAP. In all, our results imply that these potential mechanisms underlying HGF-promoting SCAP differentiation could be further elucidated and harnessed to improve periodontal tissue regeneration.

Momordica charantia (MC) is a traditional plant widely used since ancient times for wound healing. This study evaluated its potential effects on tendon healing. Adult male Wistar albino rats (n = 32, 8 rats in each group) were anesthetized, and their Achilles tendons were prepared for surgical procedures. Group 1 (Cont = control group) was not subjected to any surgery and was used as a control group for baseline values. Group 2 (PR = primary repair group) underwent primary repair (PR) with a monofilament suture after a full-thickness incision of the Achilles tendon. A full-thickness incision was also made to the Achilles tendon of group 3 (CT = collagen tube-administered group), followed by PR and collagen tube insertion. In group 4 (MC = M. charantia-administered group), 1 mL of MC extract was applied locally on the collagen tube in addition to the surgical procedure applied to group 3. The Achilles tendons were excised on the postoperative 40th day and examined stereologically, histologically, and bioinformatically. Data showed that the total volume of the collagen fibers was higher in MC and CT groups than in the PR group. The total volume of the tendon was decreased in MC and CT groups than in the Cont group. The ratios between the volumes of the collagen fibers and total tendon in the MC and CT groups were significantly different from PR, but not different from the Cont group. Additionally, MC improved tenoblastic activity, collagen production, and neovascularization. Bioinformatic interactions showed that the proteases of MC could trigger the signals playing a role on vasculogenesis, reducing inflammation, and contributing to tenoblast activation and collagen remodeling. MC extract ameliorates the healing of injured tendon and can provide satisfactory tendon repair. Further works are recommended to explore the healing capacity of MC.

Introduction: The decellularized extracellular matrix (dECM) from ovarian tissue could be the best scaffold for the development of a transplantable artificial ovary. Typically, dECM from ovarian tissue has been obtained using sodium dodecyl sulfate (SDS), at a concentration of 1% for 24 h. However, SDS can leave residues in the tissue, which may be toxic to the seeded cells. This study aimed to obtain dECM from bovine ovarian tissue using SDS and NaOH at a minimum concentration in the shortest incubation time.

Methods: The respective SDS and NaOH concentrations investigated were 1% and 0.2 m; 0.5% and 0.1 m; 0.1% and 0.02 m; and 0.05% and 0.01 m, with 24-, 12-, and 6-h incubation periods. After the incubation time, the tissue was washed in 50 mL of distilled water for 6 h.

Results: Histological analysis confirmed decellularization and showed the conservation of collagen fibers in all samples following treatment. Furthermore, the lowest SDS and NaOH concentrations that showed no DNA remaining during electrophoresis analysis were 0.1% and 0.02 m when incubated for 24 and 12 h. DNA quantification resulted in <0.2 ng DNA/mg ovarian tissue using these protocols. Additionally, the coculture of dECM (obtained by 0.1% SDS and 0.02 m NaOH for 12 h) with ovarian cells showed that there was no toxic effect for the cells for up to 72 h.

Conclusion: The protocol involving 0.1% SDS and 0.02 m NaOH for 12-h incubation decellularizes bovine ovarian tissue, generating a dECM that preserves the native ECM morphology and is nontoxic to ovarian cells.

In our series of studies, the changes in the skin characteristics of mice caused by aging were investigated in correlation with the stem cells for keratinocytes and melanocytes in the natural hair cycle until middle age. The aim of the present review was to investigate these characteristics of hair follicles (HFs) at older age and complete the analysis of these changes as a study throughout the mouse lifetime. In addition, stem cells for keratinocytes and melanocytes were evaluated for changes in skin characteristics caused by aging. Postnatal day 200 (P200) appears to be the age of complete maturation of skin and the onset of aging with regard to HFs. Keratin 15-positive keratinocyte stem cells complete their localization as a quantitatively sufficient amount of progenitor in the hair bulge region and orchestrate the regeneration of hairs in every anagen phase thereafter. Although their frequency is low, an unusual structure of HFs, curved HFs, appear for the first time at P200. Thereafter, abnormal hair curvature continues to increase throughout life. In contrast, HF characteristics derived from melanocytes begin to show a high frequency of hypopigmented hair bulbs at P200 and appear to lead to a significant increase in the number of white hairs. Curved HFs and white hairs were considered biomarkers of aging in mice. The number of tyrosinase-related protein 2-positive melanocyte stem cells in the hair bulge is extremely low and may be one cause underlying not only the induction of melanocyte-derived characteristics by aging but possibly also that of keratinocyte-derived characteristics. These results provide insight into the mechanisms of the actions of stem cells on hair regeneration through the aging process.

Lung development is impaired in mice generated through transfer of in vitro-derived blastocysts. The main objective of the current study was to determine if the composition of epithelial cells in the fetal and adult lung tissue is altered in mice generated through transfer of in vitro-derived blastocysts. The study comprised two experimental (EGs) and two control (CGs) groups. Fetuses (18.5 d.p.c.) and adult mice (8 weeks old) of the EGs (EGfetus, n = 18; EGadult, n = 15) were produced by the transfer of day 5 F2 blastocysts to pseudo-pregnant females. F2 fetuses and adult mice derived from naturally ovulating females served as the CGs (CGfetus, n = 18; CGadult, n = 15). The expression of Tuba-1a (a marker of ciliated cells), Foxj-1 (a marker of motile ciliated cells), Uch-L1 (a marker of neuroendocrine cells), Cldn-10 (a marker of club cells), Aqp-5 (a marker of type I alveolar cells), and Sp-C (a marker of type II alveolar cells) was determined using Western blot, immunohistochemistry/immunofluorescence, and quantitative RT-PCR analyses. Weight of fetuses as well as adult mice is decreased in mice comprising the EGs. Impaired lung development observed in EGfetus was associated with altered expression of Tuba-1a, Foxj-1, Cldn-10, Uch-L1, Sp-C, and Aqp-5. Morphology of the adult lung tissue was similar between the groups except for a significant increase in the thickness of the epithelia in EGadult. The expression of Cldn-10 and Sp-C was also altered in EGadult. It remains to be determined whether altered expression of these genes has any long-term impact on epithelial cell functions in the adult lung tissue.

Cell therapy is one of the promising approaches used against type 1 diabetes. Efficient generation of human embryonic stem cell (hESC)-derived pancreatic progenitors (PPs) is of great importance. Since signaling pathways underlying human pancreas development are not yet fully understood, various differentiation protocols are conducted, each considering variable duration, timing, and concentrations of growth factors and small molecules. Therefore, we compared two PP differentiation protocols in static suspension culture. We tested modified protocols developed by Pagliuca et al. (protocol 1) and Royan researchers (protocol 2) until early PP stage. The morphological changes of hESC aggregates during differentiation, and also gene and protein expression after differentiation, were evaluated. Different morphological structures were formed in each protocol. Quantitative gene expression analysis, flow cytometry, and immunostaining revealed a high level of PDX1 expression on day 13 of Royan's differentiation protocol compared to protocol 1. Our data showed that using protocol 2, cells were further differentiated until day 16, showing higher efficiency of early PPs. Moreover, protocol 2 is able to produce hESCs-PPs in a static suspension culture. Since protocol 2 is inexpensive in terms of media, growth factors, and chemicals, it can be used for massive production of PPs using static and dynamic suspension cultures.

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Introduction: Ascending aortic aneurysm is a serious health risk. In order to study ascending aortic aneurysms, elastase and calcium ion treatment for aneurysm formation are mainly used, but their aneurysm formation time is long and the aneurysm formation rate is low. Thus, this study aimed to construct a rat model of ascending aorta aneurysm with a short modeling time and high aneurysm formation rate, which may mimic the pathological processes of human ascending aorta aneurysm.

Methods: Cushion needles with different pipe diameters (1.0, 1.2, 1.4, and 1.6 mm) were used to establish a human-like rat model of ascending aortic aneurysm by narrowing the ascending aorta of rats and increasing the force of blood flow on the vessel wall. The vascular diameters were evaluated using color Doppler ultrasonography after 2 weeks. The characteristics of ascending aortic aneurysm in rats were detected by Masson's trichrome staining, Verhoeff's Van Gieson staining, and hematoxylin and eosin staining, while real-time polymerase chain reaction was utilized to assess the total RNA of cytokine interleukin-1β, interleukin 6, transforming growth factor-beta 1, and metalloproteinase 2.

Results: Two weeks after surgery, the ultrasound images and the statistical analysis demonstrated that the diameter of the ascending aorta in rats increased more than 1.5 times, similar to that in humans, indicating the success of animal modeling of ascending aortic aneurysm. Moreover, the optimal constriction diameter of the ascending aortic aneurysm model is 1.4 mm by the statistical analysis of the rate of ascending aortic aneurysm and mortality rate in rats with different constriction diameters.

Conclusions: The human-like ascending aortic aneurysm model developed in this study can be used for the studies of the pathological processes and mechanisms of ascending aortic aneurysm in a more clinically relevant fashion.