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.
Epithelial-stromal interactions play an essential role in regulation of mammary gland development, homeostasis, and tumorigenesis. Fibroblasts constitute a substantial proportion of mammary gland stromal cells in human breast and have been recognized for their paracrine signaling and extracellular matrix production and remodeling roles during normal breast development as well as in breast cancer. However, our current knowledge on human breast fibroblast functions is incomplete. Here we provide a detailed protocol for an organotypic human breast assay to facilitate research in the roles of human breast fibroblasts in mammary epithelial morphogenesis and early tumorigenesis.
In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.
- MeSH
- barvení a značení MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- organoidy * MeSH
- prsy MeSH
- zobrazování trojrozměrné * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- lidé MeSH
- mléčné žlázy lidské * MeSH
- mléčné žlázy zvířat * MeSH
- nádory mléčné žlázy u zvířat * MeSH
- nádory prsu * MeSH
- periodika jako téma normy trendy MeSH
- redakční směrnice * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- úvodní články MeSH
- úvodníky MeSH
- MeSH
- analýza jednotlivých buněk metody trendy MeSH
- lidé MeSH
- mikroskopie metody trendy MeSH
- mléčné žlázy lidské * cytologie patologie fyziologie MeSH
- mléčné žlázy zvířat * cytologie patologie fyziologie MeSH
- myši MeSH
- nádory mléčné žlázy u zvířat patologie MeSH
- nádory prsu patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- úvodní články MeSH
- úvodníky MeSH
Embryonic stem cells seem to have the intriguing capacity to divide indefinitely while retaining their pluripotency. This self-renewal is accomplished by specialized mechanisms of cell-cycle control. In the last few years, several studies have provided evidence for a direct link between cell-cycle regulation and cell-fate decisions in stem cells. In this review, we discuss the peculiarities of embryonic stem cell-cycle control mechanisms, implicate their involvement in cell-fate decisions, and distinguish centrosomes as important players in the self-renewal versus differentiation roulette.
- MeSH
- buněčná diferenciace MeSH
- buněčné dělení fyziologie MeSH
- buněčný cyklus fyziologie MeSH
- centrozom metabolismus MeSH
- embryonální kmenové buňky cytologie fyziologie MeSH
- lidé MeSH
- poškození DNA MeSH
- proliferace buněk MeSH
- signální transdukce fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyclin-dependent kinase two (Cdk2) is the major regulator of the G1/S transition and the target of an activated G1 checkpoint in somatic cells. In the presence of DNA damage, Cdk2 kinase activity is abrogated by a deficiency of Cdc25A phosphatase, which is marked by Chk1/Chk2 for proteasomal degradation. Embryonic stem cells (ESCs) lack a G1 checkpoint response. In this study, we analyzed the G1 checkpoint pathways in mouse ESCs (mESCs) in the presence of DNA double-strand breaks evoked by ionizing radiation (IR). We show that checkpoint pathways, which operate during G1 phase in somatic cells, are activated in mESCs after IR; however, Cdk2 activity is not abolished. We demonstrate that Cdc25A is degraded in mESCs, but this degradation is not regulated by Chk1 and Chk2 kinases because they are sequestered to the centrosome. Instead, Cdc25A degradation is governed by glycogen synthase kinase-3beta kinase. We hypothesize that Cdc25A degradation does not inhibit Cdk2 activity because a considerable proportion of Cdk2 molecules localize to the cytoplasm and centrosomes in mESCs, where they may be sheltered from regulation by nuclear Cdc25A. Finally, we show that a high Cdk2 activity, which is irresponsive to DNA damage, is the driving force of the rapid escape of mESCs from G1 phase after DNA damage.
- MeSH
- aktivace enzymů genetika MeSH
- buněčné linie MeSH
- buněčný cyklus genetika MeSH
- CDC geny fyziologie MeSH
- centrozom enzymologie MeSH
- cyklin-dependentní kinasa 2 genetika MeSH
- cytoplazma enzymologie MeSH
- DNA genetika účinky záření MeSH
- embryonální kmenové buňky cytologie enzymologie MeSH
- fosfatasy cdc25 genetika MeSH
- G1 fáze genetika MeSH
- ionizující záření MeSH
- kinasa 3 glykogensynthasy metabolismus MeSH
- myši MeSH
- oprava DNA MeSH
- poškození DNA genetika MeSH
- protein-serin-threoninkinasy genetika MeSH
- proteinkinasy genetika MeSH
- signální transdukce genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Embryonic stem cells (ESCs) proliferate rapidly and have a unique cell-cycle structure with a very short G1 phase. Previous reports suggested that the rapid G1 phase progression of ESCs might be underpinned by high and precocious Cdk2 activity and that Cdk2 activity might be crucial for both cell-cycle regulation and cell-fate decisions in human ESCs. However, the actual role of Cdk2 in cell-cycle progression of mouse ESCs (mESCs) has not been elucidated. In this study, we investigated the effects of down-regulation of Cdk2 activity by olomoucine II in 2 mESC lines. Olomoucine II treatment significantly increased the G1 phase cell numbers, decreased the S phase cell numbers, and inhibited DNA replication in mESCs. In nocodazole-synchronized mESCs, we show that specific down-regulation of Cdk2 activity prolongs G1 phase progression. In addition, down-regulation of Cdk2 activity in mESCs established a somatic cell-like cell cycle and induced expression of differentiation markers. Our results suggest that high Cdk2 activity is essential for rapid G1 phase progression and establishment of ESC-specific cell-cycle structure in mESCs and support the hypothesis of a link between cell-cycle regulation and pluripotency maintenance in ESCs. This study reveals olomoucine II to be an effective tool for manipulation of the cell cycle and pluripotency in ESCs and very likely also for the manipulation of other stem cell types, including cancer stem cells.
- MeSH
- buněčné linie MeSH
- buněčný cyklus účinky léků MeSH
- buňky HT-29 MeSH
- časové faktory MeSH
- cyklin-dependentní kinasa 2 antagonisté a inhibitory genetika metabolismus MeSH
- cyklin-dependentní kinasa 9 antagonisté a inhibitory genetika metabolismus MeSH
- embryonální kmenové buňky cytologie účinky léků metabolismus MeSH
- G1 fáze účinky léků MeSH
- inbrední kmeny myší MeSH
- inhibiční koncentrace 50 MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteinkinasa CDC2 antagonisté a inhibitory genetika metabolismus MeSH
- průtoková cytometrie MeSH
- puriny farmakologie MeSH
- replikace DNA účinky léků MeSH
- viabilita buněk účinky léků MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
