Epithelial cells in culture: injured or differentiated cells?

Isolation of epithelial cells for cell culture is based on destruction of epithelial integrity. The consequences are manifold: cell polarity and specific cell functions are lost; cells acquire non‐epithelial characteristics and start to proliferate. This situation may also occur in situ when parts of the epithelium are lost, either by apoptosis or necrosis by organ or tissue injury. During recovery from this injury, surviving epithelial cells proliferate and may restore epithelial integrity and finally re‐differentiate into functional epithelial cells. In vitro, this re‐differentiation is mostly not complete due to sub‐optimal culture conditions. Therefore cultured epithelial cells resemble wounded or injured epithelia rather than healthy and well differentiated epithelia. The value of an in vitro cell model is the extent to which it helps to understand the function of the cells in situ. A variety of parameters influence the state of differentiation of cultured cells in vitro. Although each of these parameters had been studied, the picture how they co‐ordinately influence the state of differentiation of epithelial cells in vitro is incomplete. Therefore we discuss the influence of the isolation method and cell culture on epithelial cells, and outline strategies to achieve highly differentiated epithelial cells for the use as an in vitro model.     

Prostaglandin E2 and T cells: friends or foes?

Our understanding of the key players involved in the differential regulation of T-cell responses during inflammation, infection and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. With respect to this, the inhibitory role of the lipid mediator prostaglandin E(2) (PGE(2)) in T-cell immunity has been documented since the 1970s. Studies that ensued investigating the underlying mechanisms substantiated the suppressive function of micromolar concentrations of PGE(2) in T-cell activation, proliferation, differentiation and migration. However, the past decade has seen a revolution in this perspective, since nanomolar concentrations of PGE(2) have been shown to potentiate Th1 and Th17 responses and aid in T-cell proliferation. The understanding of concentration-specific effects of PGE(2) in other cell types, the development of mice deficient in each subtype of the PGE(2) receptors (EP receptors) and the delineation of signalling pathways mediated by the EP receptors have enhanced our understanding of PGE(2) as an immune-stimulator. PGE(2) regulates a multitude of functions in T-cell activation and differentiation and these effects vary depending on the micro-environment of the cell, maturation and activation state of the cell, type of EP receptor involved, local concentration of PGE(2) and whether it is a homeostatic or inflammatory scenario. In this review, we compartmentalize the various aspects of this complex relationship of PGE(2) with T lymphocytes. Given the importance of this molecule in T-cell activation, we also address the possibility of using EP receptor antagonism as a potential therapeutic approach for some immune disorders.     

Annexins: putative linkers in dynamic membrane–cytoskeleton interactions in plant cells

Summary. The plasma membrane, the most external cellular structure, is at the forefront between the plant cell and its environment. Hence, it is naturally adapted to function in detection of external signals, their transduction throughout the cell, and finally, in cell reactions. Membrane lipids and the cytoskeleton, once regarded as simple and static structures, have recently been recognized as significant players in signal transduction. Proteins involved in signal detection and transduction are organised in specific domains at the plasma membrane. Their aggregation allows to bring together and orient the downstream and upstream members of signalling pathways. The cortical cytoskeleton provides a structural framework for rapid signal transduction from the cell periphery into the nucleus. It leads to intracellular reorganisation and wide-scale modulation of cellular metabolism which results in accumulation of newly synthesised proteins and/or secondary metabolites which, in turn, have to be distributed to the appropriate cell compartments. And again, in plant cells, the secretory vesicles that govern polar cellular transport are delivered to their target membranes by interaction with actin microfilaments. In search for factors that could govern subsequent steps of the cell response delineated above we focused on an evolutionary conserved protein family, the annexins, that bind in a calcium-dependent manner to membrane phospholipids. Annexins were proposed to regulate dynamic changes in membrane architecture and to organise the interface between secretory vesicles and the membrane. Certain proteins from this family were also identified as actin binding, making them ideal mediators in cell membrane and cytoskeleton interactions. Correspondence and reprints: Laboratory of Plant Pathogenesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland.     

Embryonic stem cells: are useful in clinic treatments?

It is not uncommon to find statements in the social media and even in some scientific journals declaring that embryonic stem cells can be used in human medicine for therapeutic purposes. In our opinion, this statement does not fit the medical reality. To go into this subject in depth, and if possible to clarify it, we reviewed the most recent literature on clinical trials conducted with embryonic stem cells, concluding that up to the present time, there is only one ongoing clinical trial being carried out with these types of cells to treat a small group of patients with spinal cord injury. The results of this trial have still not been published. In conclusion, at present, there is only evidence of one phase I clinical trial conducted with embryonic stem cells, in comparison to the numerous trials conducted with adult stem cells.     

Phenotype alteration in colon carcinoma cells: Effect of in vivo passage?

Summary A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell (“CaCo”) antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor reponsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-α, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression. Financial support was provided from the Richard Walter Gibbon Fund of the Faculty of Medicine, the University of Western Australia; and from the Sir Charles Gairdner Hospital Research Foundation.     

Endothelial progenitor cells: a new player in lupus?

Patients with systemic lupus erythematosus (SLE) have a greatly increased risk of cardiovascular disease. There is growing interest in the link between vascular damage and lupus-specific inflammatory factors. Impaired endothelial repair could account for the endothelial dysfunction in this patient group. This review describes the contribution that endothelial progenitor cells could play in the pathogenesis of premature vascular damage in this disease. The methods of isolation, detection, and characterization of endothelial progenitor cells, together with their potential role in repair of the endothelium and as a therapeutic target in SLE, are discussed.     

Choline phospholipid metabolism: A target in cancer cells?

The experience of treating cancer over the past several decades overwhelmingly demonstrates that the disease continues to evade the vast array of drugs and treatment modalities available in the twenty-first century. This is not surprising in view of the complexity of this disease, and the multiplicities of pathways available to the cancer cell to enable its survival. Although the progression of cancer arrives at a common end point of cachexia, organ failure, and death, common pathways are rare in cancer. Identifying and targeting common pathways that would act across these levels of multiplicity is essential for the successful treatment of this disease. Over the past decade, one common characteristic consistently revealed by magnetic resonance spectroscopic studies is the elevation of phosphocholine and total choline-containing compounds in cancer cells and solid tumors. This elevation has been observed in almost every single cancer type studied with NMR spectroscopy and can be used as an endogenous biomarker of cancer. In this article, we have summarized some of the observations on the choline phospholipid metabolism of cancer cells and tumors, and make a case for targeting the aberrant choline phospholipid metabolism of cancer cells.     

Interphase microtubules in cultured cells: long or short?

Presently, the question about the length of microtubules in the interphase cell became actual, since the parameters of dynamic instability of the plus end measured in vivo do not allow one to explain the rapid turnover of the long microtubule system. The problem may be solved if one of the following suppositions is assumed: either microtubules undergo rapid depolymerization from the minus end or they are on the average much shorter than it is usually considered. To check the last hypothesis, we have reconstructed microtubules using stereophotography of electron microscopic sections. Microtubules around the cell center in cultures of epithelial cells (kidney of pig embryo (PK) and bovine trachea (FBT)) and fibroblasts (MEF, primary mouse embryo fibroblasts, and L cells), as well as at the periphery of PK cells were studied. All in all, no less than 200 microtubules were found near the centrosome in each cell culture. From 2.5 to 8% microtubules were beyond the studied volume (4.0 x 5.5 x 1.5 microm). Most of microtubules in all studied cell lines were up to 1 microm and about 1/3 of them were 0.2-0.4 microm long. The mean length of microtubules surrounding the centrosome in different cell lines differed insignificantly and equalled 0.4-0.8 microm. In this case, the microtubules attached to the centrosome were on the average slightly shorter than the free ones. Thus, almost all microtubules around the centrosome are short, and the majority of those attached to it do not reach the cell periphery. A similar reconstruction of a part of the PK cell cytoplasm (10 x 35 microm) has shown that at the periphery, the mean length of microtubules is about 1.6 microm and most of them are 0.5 to 1.5 microm long. Thus, our data confirm the recent hypothesis of Vorobjev et al. (I. A. Vorobjev, T. M. Svitkina, and G. G. Borisy, J. Cell Sci. 110:2635-2645 (1997)) that most of microtubules in the cells are not connected with the centrosomes.     

Identification and characterization of small cells in the adult pancreas: potential progenitor cells?

In this report we describe the identification of a novel cell type in human and canine pancreas using tissue culture techniques. These cells, representing less than 1% of total islet cells, are of a small size (7-10 microm) and highly quiescent. They display a fairly immature morphology, which is characterized by a weakly developed protein synthesis machinery, a few mitochondria and a small number of neuroendocrine granules. These cells, which we have termed "small cells," are usually organized into small clusters, which can be identified within the islets of predominantly small size. They can also be collected as separate structures from preparations of freshly isolated islets. Immunohistochemically, small cells are positive for PDX-1, synaptophysin, insulin, glucagon, somatostatin, pancreatic polypeptide, alpha-fetaprotein and Bcl-2 and negative for cytokeratin 19 and nestin. Insulin secretion studies demonstrated that these cells secrete insulin in a glucose-responsive fashion, although do not respond to secretagogues such as IBMX and arginine as do mature beta cells. Although this study does not provide evidence of the proliferative and differentiation potential of small cells, their immature morphology, along with a small size and quiescence, let us hypothesize that these cells may serve as progenitors contributing to the islet growth.     

Target cells for HIV in the central nervous system: macrophages or glial cells?

Infection of foetal or embryonic brain cells and cell lines from human astrocytomas and gliomas with HIV1 derived from T-lymphoma cultures leads to the expression of HIV in about 1 to 2% of the cells in culture. Single-cell cloning of astrocytoma cells shortly after infection resulted in the establishment of persistently HIV1-infected cell lines. These cultures were characterized by low production of virus and moderate intra- and extracellular expression of structural proteins. However, high expression of the nef regulatory protein was found. The virus could be rescued by cocultivation with T cells and primary macrophages giving rise to typical syncytia formation.In contrast to infection with HIV-infected T-lymphoma lines, cocultivation with HIV1-infected primary macrophages or monocytic cell lines induced a reduction in the growth of astrocytes and failed to induce productive infection. These in vitro observations support the hypothesis that astrocytes and glial cells may be a reservoir for HIV in the central nervous system and that macrophages may not carry the virus to the brain, but rather may be infected in the brain after having penetrated the blood-brain barrier.     

Stem cells: is there a future in plastics?

The concept that ostensibly tissue-specific stem cells can give rise to cells of heterologous lineages has gained support from studies using purified hematopoietic stem cells and sensitive donor-cell tracking methods. The ability to exploit these findings in clinical settings will probably depend on new insights into the mechanisms by which such stem cells or their progeny migrate to sites of organ damage and differentiate to cell types competent to participate in tissue regeneration.     

CNS dendritic cells: critical participants in CNS inflammation?

Dendritic cells (DCs) are a heterogeneous population of migratory cells specialized for the uptake, processing, and presentation of antigen to T cells. They consist of a variety of mature subpopulations, classically divided into "lymphoid" and "myeloid" subsets. Although there likely exists significant plasticity and redundancy between DC subpopulations, unique differences have been noted in their abilities for T cell stimulation, tolerance induction, T helper cell polarization, cytokine secretion, and anatomic localization. Although DCs are conspicuously absent from the healthy CNS parenchyma, their presence in the vascular-rich regions of the healthy CNS has been well established and suggests they may have a role in immune surveillance. DCs do accumulate in the CNS parenchyma in a wide range of inflammatory responses including parasite, viral, or bacterial infection and CNS autoimmune disease. They also are present in CNS immune responses without overt T cell involvement, such as the inflammation accompanying CNS injury or neurodegeneration. Controversy remains on the role of CNS DCs during inflammation and whether they differentiate from CNS-resident microglia or infiltrate from a blood-borne population. This review will summarize DC subsets and function, overview the current research on DCs in the healthy and inflamed CNS, and address discrepancies between experimental studies.     

Photoreceptor cells in flies and mammals: Crumby homology?

Recently, two papers have revealed a new function for the fruit fly epithelial apical membrane protein Crumbs and its mammalian homolog CRB1 in photoreceptor cell morphogenesis. This supports the previous observation that disruption of CRB1 function can cause retinal degeneration in humans.     

Tumour-initiating cells vs. cancer 'stem' cells and CD133: what's in the name?

Recent evidence suggests that a subset of cells within a tumour have 'stem-like' characteristics. These tumour-initiating cells, distinct from non-malignant stem cells, show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumour cells, resistance to chemotherapy or radiation, and they are often characterised by elevated expression of the stem cell surface marker CD133. Understanding the molecular biology of the CD133(+) cancer cells is now essential for developing more effective cancer treatments. These may include drugs targeting organelles, such as mitochondria or lysosomes, using highly efficient and selective inducers of apoptosis. Alternatively, agents or treatment regimens that enhance sensitivity of these therapy-resistant "tumour stem cells" to the current or emerging anti-tumour drugs would be of interest as well.     

Murine gammadelta T cells in infections: beneficial or deleterious?

Although the importance of gammadelta T cells in pathogen-induced immune responses is becoming increasingly apparent, it is not clear that their involvement is always of benefit to the host. Here we review evidence for the protective and damaging roles of gammadelta T cells in infection and discuss how these disparate findings might be resolved by considering the nature and properties of the pathogen, the sites of infection and conditions under which gammadelta T cell responses are initiated, and the involvement of different subsets of gammadelta T cells.     

Epithelial stem cells in the esophagus: who needs them?

In their recent Science publication, Doupé et?al. (2012) demonstrate that a single population of proliferating progenitor cells is solely responsible for homeostatic self-renewal and repair of injured esophageal epithelium. These findings argue against an obligate requirement for long-lived (reserve) stem cells in adult epithelia.     

RNA interference: towards a functional genomics in mammalian cells?

The discovery of the induction of RNA degradation by double stranded RNA in C. elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells. Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation. Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing. The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach. However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells.     

Th17 cells: positive or negative role in tumor?

Th17 cells have been recently identified as a distinct Th cell lineage and found in an experimental animal model of cancer and in human cancers, but whether these cells promote tumor growth or regulate antitumor responses remains controversial. This review provides a summary of the current literature regarding interleukin (IL)-17/IL-23 and Th17 cells in cancer and discusses their potential roles in cancer development. Finally, we note several issues in this research area that must be resolved before the design of novel therapeutic approaches specifically targeting Th17 cells in cancer become feasible.