The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface in vitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.
Lately, the need for three-dimensional (3D) cell culture has been recognized in order to closely mimic the organization of native tissues. Thus, 3D scaffolds started to be employed to facilitate the 3D cell organization and enable the artificial tissue formation for the emerging tissue engineering applications. 3D scaffolds can be prepared by various techniques, each with certain advantages and disadvantages. Decellularization is an easy method based on removal of cells from native tissue sample, yielding extracellular matrix (ECM) scaffold with preserved architecture and bioactivity. This chapter provides a detailed protocol for decellularization of pig lung and also some basic assays for evaluation of its effectivity, such as determination of DNA content and histological verification of the selected ECM components. Such decellularized scaffold can subsequently be used for various tissue engineering applications, for example, for recellularization with cells of interest, for natural ECM hydrogel preparation, or as a bioink for 3D bioprinting.
- MeSH
- extracelulární matrix MeSH
- hydrogely MeSH
- plíce * MeSH
- prasata MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Inhalation of lead oxide nanoparticles (PbO NPs), which are emitted to the environment by high-temperature technological processes, heavily impairs target organs. These nanoparticles pass through the lung barrier and are distributed via the blood into secondary target organs, where they cause numerous pathological alterations. Here, we studied in detail, macrophages as specialized cells involved in the innate and adaptive immune response in selected target organs to unravel their potential involvement in reaction to subchronic PbO NP inhalation. In this context, we also tackled possible alterations in lipid uptake in the lungs and liver, which is usually associated with foam macrophage formation. RESULTS: The histopathological analysis of PbO NP exposed lung revealed serious chronic inflammation of lung tissues. The number of total and foam macrophages was significantly increased in lung, and they contained numerous cholesterol crystals. PbO NP inhalation induced changes in expression of phospholipases C (PLC) as enzymes linked to macrophage-mediated inflammation in lungs. In the liver, the subchronic inhalation of PbO NPs caused predominantly hyperemia, microsteatosis or remodeling of the liver parenchyma, and the number of liver macrophages also significantly was increased. The gene and protein expression of a cholesterol transporter CD36, which is associated with lipid metabolism, was altered in the liver. The amount of selected cholesteryl esters (CE 16:0, CE 18:1, CE 20:4, CE 22:6) in liver tissue was decreased after subchronic PbO NP inhalation, while total and free cholesterol in liver tissue was slightly increased. Gene and protein expression of phospholipase PLCβ1 and receptor CD36 in human hepatocytes were affected also in in vitro experiments after acute PbO NP exposure. No microscopic or serious functional kidney alterations were detected after subchronic PbO NP exposure and CD68 positive cells were present in the physiological mode in its interstitial tissues. CONCLUSION: Our study revealed the association of increased cholesterol and lipid storage in targeted tissues with the alteration of scavenger receptors and phospholipases C after subchronic inhalation of PbO NPs and yet uncovered processes, which can contribute to steatosis in liver after metal nanoparticles exposure.
- MeSH
- cholesterol MeSH
- fosfolipasy typu C * MeSH
- kovové nanočástice * chemie MeSH
- lidé MeSH
- makrofágy MeSH
- olovo MeSH
- oxidy MeSH
- zánět MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
TACSTD2 encodes a transmembrane glycoprotein Trop2 commonly overexpressed in carcinomas. While the Trop2 protein was discovered already in 1981 and first antibody-drug conjugate targeting Trop2 were recently approved for cancer therapy, the physiological role of Trop2 is still not fully understood. In this article, we show that TACSTD2/Trop2 expression is evolutionarily conserved in lungs of various vertebrates. By analysis of publicly available transcriptomic data we demonstrate that TACSTD2 level consistently increases in lungs infected with miscellaneous, but mainly viral pathogens. Single cell and subpopulation based transcriptomic data revealed that the major source of TACSTD2 transcript are lung epithelial cells and their progenitors and that TACSTD2 is induced directly in lung epithelial cells following infection. Increase in TACSTD2 expression may represent a mechanism to maintain/restore epithelial barrier function and contribute to regeneration process in infected/damaged lungs.
- MeSH
- antigeny nádorové * metabolismus MeSH
- epitelové buňky metabolismus MeSH
- molekuly buněčné adheze * metabolismus MeSH
- plíce metabolismus MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS: By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS: These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION: Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.
The human respiratory system is continuously exposed to varying levels of hazardous substances ranging from environmental toxins to purposely administered drugs. If the noxious effects exceed the inherent regenerative capacity of the respiratory system, injured tissue undergoes complex remodeling that can significantly affect lung function and lead to various diseases. Advanced near-to-native in vitro lung models are required to understand the mechanisms involved in pulmonary damage and repair and to reliably test the toxicity of compounds to lung tissue. This review is an overview of the development of in vitro respiratory system models used for study of lung diseases. It includes discussion of using these models for environmental toxin assessment and pulmonary toxicity screening.
- MeSH
- biologické modely * MeSH
- buněčné kultury MeSH
- dýchací soustava * anatomie a histologie MeSH
- laboratoř na čipu MeSH
- lidé MeSH
- mikrofluidika MeSH
- tkáňové podpůrné struktury MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.
- MeSH
- acetylcystein aplikace a dávkování MeSH
- aktinin genetika MeSH
- embryonální kmenové buňky cytologie MeSH
- homeoboxový protein Nkx-2.5 genetika MeSH
- kardiomyocyty cytologie MeSH
- kultivační média bez séra * MeSH
- kultivované buňky MeSH
- myosiny genetika MeSH
- myši MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Notch and gp130 signaling are involved in the regulation of multiple cellular processes across various tissues during animal ontogenesis. In the developing nervous system, both signaling pathways intervene at many stages to determine cell fate-from the first neural lineage commitment and generation of neuronal precursors, to the terminal specification of cells as neurons and glia. In most cases, the effects of Notch and gp130 signaling in these processes are similar. The aim of the current review was to summarize the knowledge regarding the roles of Notch and gp130 signaling in the maintenance of neural stem and progenitor cells during animal ontogenesis, from early embryo to adult. Recent data show a direct crosstalk between these signaling pathways that seems to be specific for a particular type of neural progenitors.
- MeSH
- cytokinový receptor gp130 metabolismus MeSH
- interakce mezi receptory a ligandy MeSH
- lidé MeSH
- nervové kmenové buňky metabolismus MeSH
- neurogeneze MeSH
- receptory Notch metabolismus MeSH
- signální transdukce * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Retinoic acid (RA) is able to induce the differentiation of embryonic stem cells into neuronal lineages. The mechanism of this effect is unknown but it has been evidenced to be dependent on the formation of floating spheroids called embryoid bodies. Results presented here show that the inhibition of phosphoinositide 3-kinase signaling pre-determines mouse embryonic stem cells to RA induced neurogenesis in monolayer culture with no need of embryoid bodies formation.
- MeSH
- 1-fosfatidylinositol-3-kinasa metabolismus MeSH
- buněčné kultury MeSH
- chromony farmakologie MeSH
- diferenciační antigeny genetika metabolismus MeSH
- embryonální kmenové buňky účinky léků metabolismus fyziologie MeSH
- genetická transkripce MeSH
- inhibitory fosfoinositid-3-kinasy MeSH
- kadheriny genetika metabolismus MeSH
- keratin-8 genetika metabolismus MeSH
- kultivované buňky MeSH
- luciferasy biosyntéza genetika MeSH
- molekuly buněčné adheze nervové genetika metabolismus MeSH
- morfoliny farmakologie MeSH
- myši MeSH
- neurogeneze účinky léků MeSH
- reportérové geny MeSH
- signální transdukce účinky léků MeSH
- tretinoin farmakologie MeSH
- tubulin genetika metabolismus MeSH
- tvar buňky účinky léků MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
