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  1. Article ; Online: Long-term differentiating primary human airway epithelial cell cultures: how far are we?

    Bukowy-Bieryłło, Zuzanna

    Cell communication and signaling : CCS

    2021  Volume 19, Issue 1, Page(s) 63

    Abstract: Background: Human airway epithelial (HAE) cellular models are widely used in applicative studies of the airway physiology and disease. In vitro expanded and differentiated primary HAE cells collected from patients seem to be an accurate model of human ... ...

    Abstract Background: Human airway epithelial (HAE) cellular models are widely used in applicative studies of the airway physiology and disease. In vitro expanded and differentiated primary HAE cells collected from patients seem to be an accurate model of human airway, offering a quicker and cheaper alternative to the induced pluripotent stem cell (iPSCs) models. However, the biggest drawback of primary HAE models is their limited proliferative lifespan in culture. Much work has been devoted to understand the factors, which govern the HAE cell proliferation and differentiation, both in vivo and in vitro. Here, I have summarized recent achievements in primary HAE culture, with the special emphasis on the models of conditionally reprogrammed cells (CRC), which allow longer in vitro proliferation and differentiation of HAE cells. The review compares the CRC HAE technique variants (feeder culture or HAE mono-culture), based on recently published studies exploiting this model. The advantages and limitations of each CRC HAE model variant are summarized, along with the description of other factors affecting the CRC HAE culture success (tissue type, sampling method, sample quality).
    Conclusions: CRC HAE cultures are a useful technique in respiratory research, which in many cases exceeds the iPSCs and organoid culture methods. Until the current limitations of the iPSCs and organoid culture methods will be alleviated, the primary CRC HAE cultures might be a useful model in respiratory research. Airway epithelium (AE) is a type of tissue, which lines the whole length of human airways, from the nose to the bronchi. Improper functioning of AE causes several human airway disorders, such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). Much work has been devoted to finding the best scientific model of human AE, in order to learn about its functioning in health and disease. Among the popular AE models are the primary in vitro cultured AE cells collected from human donors. Unfortunately, such human AE (HAE) cells do not easily divide (expand) in vitro; this poses a large logistic and ethical problem for the researchers. Here, I summarize recent achievements in the methods for in vitro culture of human AE cells, with special emphasis on the conditionally reprogrammed cell (CRC) models, which allow longer and more effective expansion of primary human AE cells in vitro. The review describes how the specific chemicals used in the CRC models work to allow the increased HAE divisions and compares the effects of the different so-far developed variants of the CRC HAE culture. The review also pinpoints the areas which need to be refined, in order to maximize the usefulness of the CRC AE cultures from human donors in research on human airway disorders. Video abstract.
    MeSH term(s) Cell Culture Techniques ; Cell Differentiation ; Epithelial Cells/cytology ; Humans ; Lung/cytology ; Models, Biological ; Time Factors
    Language English
    Publishing date 2021-05-27
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 2126315-2
    ISSN 1478-811X ; 1478-811X
    ISSN (online) 1478-811X
    ISSN 1478-811X
    DOI 10.1186/s12964-021-00740-z
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Long-term differentiating primary human airway epithelial cell cultures

    Zuzanna Bukowy-Bieryłło

    Cell Communication and Signaling, Vol 19, Iss 1, Pp 1-

    how far are we?

    2021  Volume 18

    Abstract: Abstract Background Human airway epithelial (HAE) cellular models are widely used in applicative studies of the airway physiology and disease. In vitro expanded and differentiated primary HAE cells collected from patients seem to be an accurate model of ... ...

    Abstract Abstract Background Human airway epithelial (HAE) cellular models are widely used in applicative studies of the airway physiology and disease. In vitro expanded and differentiated primary HAE cells collected from patients seem to be an accurate model of human airway, offering a quicker and cheaper alternative to the induced pluripotent stem cell (iPSCs) models. However, the biggest drawback of primary HAE models is their limited proliferative lifespan in culture. Much work has been devoted to understand the factors, which govern the HAE cell proliferation and differentiation, both in vivo and in vitro. Here, I have summarized recent achievements in primary HAE culture, with the special emphasis on the models of conditionally reprogrammed cells (CRC), which allow longer in vitro proliferation and differentiation of HAE cells. The review compares the CRC HAE technique variants (feeder culture or HAE mono-culture), based on recently published studies exploiting this model. The advantages and limitations of each CRC HAE model variant are summarized, along with the description of other factors affecting the CRC HAE culture success (tissue type, sampling method, sample quality). Conclusions CRC HAE cultures are a useful technique in respiratory research, which in many cases exceeds the iPSCs and organoid culture methods. Until the current limitations of the iPSCs and organoid culture methods will be alleviated, the primary CRC HAE cultures might be a useful model in respiratory research. Plain English summary Airway epithelium (AE) is a type of tissue, which lines the whole length of human airways, from the nose to the bronchi. Improper functioning of AE causes several human airway disorders, such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). Much work has been devoted to finding the best scientific model of human AE, in order to learn about its functioning in health and disease. Among the popular AE models are the primary in vitro cultured AE cells collected from human donors. Unfortunately, such human AE (HAE) cells do not easily divide (expand) in vitro; this poses a large logistic and ethical problem for the researchers. Here, I summarize recent achievements in the methods for in vitro culture of human AE cells, with special emphasis on the conditionally reprogrammed cell (CRC) models, which allow longer and more effective expansion of primary human AE cells in vitro. The review describes how the specific chemicals used in the CRC models work to allow the increased HAE divisions and compares the effects of the different so-far developed variants of the CRC HAE culture. The review also pinpoints the areas which need to be refined, in order to maximize the usefulness of the CRC AE cultures from human donors in research on human airway disorders. Video abstract
    Keywords Primary airway cell culture ; Air–liquid interface culture ; Conditional reprogramming ; ROCK inhibitor ; SMAD inhibitor ; TGF-β1 inhibitor ; Medicine ; R ; Cytology ; QH573-671
    Subject code 610
    Language English
    Publishing date 2021-05-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Perspectives for Primary Ciliary Dyskinesia.

    Bukowy-Bieryllo, Zuzanna / Witt, Michal / Zietkiewicz, Ewa

    International journal of molecular sciences

    2022  Volume 23, Issue 8

    Abstract: Primary ciliary dyskinesia (PCD) is a ciliopathy caused by genetically determined impairment of motile cilia-organelles present on the surface of many types of cells [ ... ]. ...

    Abstract Primary ciliary dyskinesia (PCD) is a ciliopathy caused by genetically determined impairment of motile cilia-organelles present on the surface of many types of cells [...].
    MeSH term(s) Cilia ; Ciliary Motility Disorders/genetics ; Humans ; Mutation
    Language English
    Publishing date 2022-04-08
    Publishing country Switzerland
    Document type Editorial
    ZDB-ID 2019364-6
    ISSN 1422-0067 ; 1422-0067 ; 1661-6596
    ISSN (online) 1422-0067
    ISSN 1422-0067 ; 1661-6596
    DOI 10.3390/ijms23084122
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Perspectives for Primary Ciliary Dyskinesia

    Zuzanna Bukowy-Bieryllo / Michal Witt / Ewa Zietkiewicz

    International Journal of Molecular Sciences, Vol 23, Iss 4122, p

    2022  Volume 4122

    Abstract: Primary ciliary dyskinesia (PCD) is a ciliopathy caused by genetically determined impairment of motile cilia–organelles present on the surface of many types of cells [.] ...

    Abstract Primary ciliary dyskinesia (PCD) is a ciliopathy caused by genetically determined impairment of motile cilia–organelles present on the surface of many types of cells [.]
    Keywords n/a ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Language English
    Publishing date 2022-04-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia.

    Dabrowski, Maciej / Bukowy-Bieryllo, Zuzanna / Jackson, Claire L / Zietkiewicz, Ewa

    International journal of molecular sciences

    2021  Volume 22, Issue 9

    Abstract: Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, ...

    Abstract Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.
    MeSH term(s) Aminoglycosides/pharmacology ; Cell Death/drug effects ; Cells, Cultured ; Cilia/drug effects ; Cilia/metabolism ; Ciliary Motility Disorders/genetics ; Codon, Nonsense/genetics ; Epithelial Cells/drug effects ; Epithelial Cells/metabolism ; HEK293 Cells ; Humans ; Mutation/genetics ; Nose/pathology ; Protein Biosynthesis/drug effects ; Protein Biosynthesis/genetics
    Chemical Substances Aminoglycosides ; Codon, Nonsense
    Language English
    Publishing date 2021-05-07
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2019364-6
    ISSN 1422-0067 ; 1422-0067 ; 1661-6596
    ISSN (online) 1422-0067
    ISSN 1422-0067 ; 1661-6596
    DOI 10.3390/ijms22094923
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia

    Maciej Dabrowski / Zuzanna Bukowy-Bieryllo / Claire L. Jackson / Ewa Zietkiewicz

    International Journal of Molecular Sciences, Vol 22, Iss 4923, p

    2021  Volume 4923

    Abstract: Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, ...

    Abstract Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.
    Keywords readthrough ; primary ciliary dyskinesia ; premature termination codon ; aminoglycosides ; STOP suppression ; rare disease ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 610
    Language English
    Publishing date 2021-05-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons.

    Dabrowski, Maciej / Bukowy-Bieryllo, Zuzanna / Zietkiewicz, Ewa

    Molecular medicine (Cambridge, Mass.)

    2018  Volume 24, Issue 1, Page(s) 25

    Abstract: Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translation and generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceutical compounds is a promising ... ...

    Abstract Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translation and generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceutical compounds is a promising way of restoring functional protein expression and reducing disease symptoms, without affecting the genome or transcriptome of the patient. While in some cases proven effective, the clinical use of readthrough-inducing compounds is still associated with many risks and difficulties. This review focuses on problems directly associated with compounds used to stimulate PTC readthrough, such as their interactions with the cell and organism, their toxicity and bioavailability (cell permeability; tissue deposition etc.). Various strategies designed to overcome these problems are presented.
    MeSH term(s) Animals ; Codon, Nonsense ; Drug Therapy ; Humans ; Protein Biosynthesis
    Chemical Substances Codon, Nonsense
    Language English
    Publishing date 2018-05-29
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 1283676-x
    ISSN 1528-3658 ; 1076-1551
    ISSN (online) 1528-3658
    ISSN 1076-1551
    DOI 10.1186/s10020-018-0024-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium - The framework for functional studies on airway differentiation in ciliopathies.

    Bukowy-Bieryłło, Zuzanna / Daca-Roszak, Patrycja / Jurczak, Joanna / Przystałowska-Macioła, Hanna / Jaksik, Roman / Witt, Michał / Ziętkiewicz, Ewa

    European journal of cell biology

    2021  Volume 101, Issue 1, Page(s) 151189

    Abstract: Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive ... ...

    Abstract Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used custom-designed Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells. Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.
    MeSH term(s) Cell Differentiation ; Cells, Cultured ; Cilia ; Ciliopathies ; Epithelial Cells ; Epithelium ; Humans
    Language English
    Publishing date 2021-12-02
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 391967-5
    ISSN 1618-1298 ; 0070-2463 ; 0171-9335
    ISSN (online) 1618-1298
    ISSN 0070-2463 ; 0171-9335
    DOI 10.1016/j.ejcb.2021.151189
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Translational readthrough potential of natural termination codons in eucaryotes--The impact of RNA sequence.

    Dabrowski, Maciej / Bukowy-Bieryllo, Zuzanna / Zietkiewicz, Ewa

    RNA biology

    2015  Volume 12, Issue 9, Page(s) 950–958

    Abstract: Termination of protein synthesis is not 100% efficient. A number of natural mechanisms that suppress translation termination exist. One of them is STOP codon readthrough, the process that enables the ribosome to pass through the termination codon in mRNA ...

    Abstract Termination of protein synthesis is not 100% efficient. A number of natural mechanisms that suppress translation termination exist. One of them is STOP codon readthrough, the process that enables the ribosome to pass through the termination codon in mRNA and continue translation to the next STOP codon in the same reading frame. The efficiency of translational readthrough depends on a variety of factors, including the identity of the termination codon, the surrounding mRNA sequence context, and the presence of stimulating compounds. Understanding the interplay between these factors provides the necessary background for the efficient application of the STOP codon suppression approach in the therapy of diseases caused by the presence of premature termination codons.
    MeSH term(s) Animals ; Codon, Nonsense ; Codon, Terminator ; Eukaryota/genetics ; Eukaryota/metabolism ; Humans ; Nucleotide Motifs ; Peptide Chain Termination, Translational ; Peptide Termination Factors/metabolism ; Poly A ; Poly(A)-Binding Proteins/metabolism ; Protein Biosynthesis ; RNA, Messenger/chemistry ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Transfer/genetics ; RNA, Transfer/metabolism
    Chemical Substances Codon, Nonsense ; Codon, Terminator ; Peptide Termination Factors ; Poly(A)-Binding Proteins ; RNA, Messenger ; Poly A (24937-83-5) ; RNA, Transfer (9014-25-9)
    Language English
    Publishing date 2015
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ISSN 1555-8584
    ISSN (online) 1555-8584
    DOI 10.1080/15476286.2015.1068497
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Aminoglycoside-stimulated readthrough of premature termination codons in selected genes involved in primary ciliary dyskinesia.

    Bukowy-Bieryllo, Zuzanna / Dabrowski, Maciej / Witt, Michał / Zietkiewicz, Ewa

    RNA biology

    2016  Volume 13, Issue 10, Page(s) 1041–1050

    Abstract: Translational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential ...

    Abstract Translational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential of different concentrations of several aminoglycosides (AAGs) for promoting PTC-readthrough in 5 genes involved in the pathogenesis of primary ciliary dyskinesia, an inherited disorder caused by the dysfunction of motile cilia and flagella. The efficiency of readthrough stimulation of PTCs cloned in dual reporter vectors was examined in 2 experimental settings: in vitro (transcription/translation system) and ex vivo (transiently transfected epithelial cell line). PTC-readthrough was observed in 5 of the 16 mutations analyzed. UGA codons were more susceptible to AAG-stimulated readthrough than UAG; no suppression of UAA was observed. The efficiency of PTC-readthrough in vitro (from less than 1% to ∼28% of the translation from the corresponding wild-type constructs) differed with the AAG type and concentration, and depended on the combination of AAG and PTC, indicating that each PTC has to be individually tested with a range of stimulating compounds. The maximal values of PTC suppression observed in the ex vivo experiments were, depending on AAG used, 3-5 times lower than the corresponding values in vitro, despite using AAG concentrations that were 2 orders of magnitude higher. This indicates that, while the in vitro system is sufficient to examine the readthrough-susceptibility of PTCs, it is not sufficient to test the compounds potential to stimulate PTC-readthrough in the living cells. Most of the tested compounds (except for G418) at their highest concentrations did not disturb ciliogenesis in the cultures of primary respiratory epithelial cells from healthy donors.
    MeSH term(s) Aminoglycosides/pharmacology ; Cells, Cultured ; Codon, Nonsense ; Codon, Terminator ; European Continental Ancestry Group/genetics ; Gene Regulatory Networks/drug effects ; HEK293 Cells ; Humans ; Kartagener Syndrome/genetics ; Mutation ; Poland
    Chemical Substances Aminoglycosides ; Codon, Nonsense ; Codon, Terminator
    Language English
    Publishing date 2016-10-02
    Publishing country United States
    Document type Journal Article
    ISSN 1555-8584
    ISSN (online) 1555-8584
    DOI 10.1080/15476286.2016.1219832
    Database MEDical Literature Analysis and Retrieval System OnLINE

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