Establishment of cell strains from human urothelial carcinoma and their morphological characterization

We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain.     

3-Dimensional microscopy as a method for volume measurement in cells undergoing apoptosis

Different patterns of cell volume perturbations are commonly used for modes of cell death: necrosis (cell swelling) and apoptosis (cell shrinkage). In this study we employed recently developed three dimensional microscopy for the measurement of the volume of attached vascular smooth muscle cells transfected with E1A-adenoviral protein. These cells undergo rapid apoptosis in the absence of growth factors or in the presence of staurosporine. In 30–60 min of serum deprivation the volume of these cells is increased by ～40% that corresponds to the time point of maximal activation of caspase 3 and chromatin cleavage. In 10–15 min swollen cells exhibit morphological collapse indicated by formation of apoptotic bodies. In contrast to serum-deprived cells, staurosporine leads to attenuation of cell volume by 30%. In this case, apoptotic bodies are detected in ～2.5 h after maximal shrinkage. Thus, our results show that cell shrinkage can not be considered as universal hallmark of apoptosis. The role of stimulus-specific cell volume perturbation in the triggering of the cell death machinery should be examined further.     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

Establishment of cell strains from human urothelial carcinoma and their morphological characterization

Summary We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain. This work was supported in part by Grants 5319 and 5322 in aid for cancer research from the Ministry of Health and Welfare, Japan.     

Modeling cell proliferation for simulating three-dimensional tissue morphogenesis based on a reversible network reconnection framework

Tissue morphogenesis in multicellular organisms is accompanied by proliferative cell behaviors: cell division (increase in cell number after each cell cycle) and cell growth (increase in cell volume during each cell cycle). These proliferative cell behaviors can be regulated by multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze multicellular dynamics, a reversible network reconnection (RNR) model has been suggested, in which each cell shape is expressed by a single polyhedron. In this study, to apply the RNR model to simulate tissue morphogenesis involving proliferative cell behaviors, we model cell proliferation based on a RNR model framework. In this model, cell division was expressed by dividing a polyhedron at a planar surface for which cell division behaviors were characterized by three quantities: timing, intracellular position, and normal direction of the dividing plane. In addition, cell growth was expressed by volume growth as a function of individual cell times within their respective cell cycles. Numerical simulations using the proposed model showed that tissues grew during successive cell divisions with several cell cycle times. During these processes, the cell number in tissues increased while maintaining individual cell size and shape. Furthermore, tissue morphology dramatically changed based on different regulations of cell division directions. Thus, the proposed model successfully provided a basis for expressing proliferative cell behaviors during morphogenesis based on a RNR model framework.     

Cell Size and Cln-Cdc28 Complexes Mediate Entry into Meiosis by Modulating Cell Growth

In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G1-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size. In contrast,Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not beenestablished. Here, we report that entry in meiosis is also dependent upon cell growth. Moreover,sporulation and cell growth rates were proportional to cell size; large cells grew rapidly andsporulated sooner while smaller cells grew slowly and sporulated later. In addition, Cln2 proteinlevels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis insmaller cells by preventing cell growth. In support of this hypothesis, loss of Clns, or thepresence of a cdc28 mutation increased cell growth in smaller cells and accelerated meiosis inthese cells. Finally, over-expression of CLNs repressed meiosis in smaller cells, but not in largecells. Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis inpart by inhibiting cell growth.     

Fission Yeast Germinal Center (GC) Kinase Ppk11 Interacts with Pmo25 and Plays an Auxiliary Role in Concert with the Morphogenesis Orb6 Network (MOR) in Cell Morphogenesis

How cell morphology and the cell cycle are coordinately regulated is a fundamental subject in cell biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 and Nak1, play an essential role in septation/cytokinesis and cell separation/cell polarity control, respectively, as components of the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR). Here we show that a third GCK, Ppk11, is also required for efficient cell separation particularly, at a high temperature. Although Ppk11 is not essential for cell division, this kinase plays an auxiliary role in concert with MOR in cell morphogenesis. Ppk11 physically interacts with the MOR component Pmo25 and is localized to the septum, by which Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. The conserved C-terminal WDF motif of Ppk11 is essential for both septum accumulation of Pmo25 and efficient cell separation. In contrast its kinase activity is required only for cell separation. Thus, both interaction of Ppk11 with Pmo25 and Ppk11 kinase activity are critical for efficient cell separation.     

Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology

Summary We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter-and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single expreiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations, depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter-and intraspecific contaminations.     

Balance between cell division and differentiation during plant development

The processes which make possible that a cell gives rise to two daughter cells define the cell division cycle. In individual cells, this is strictly controlled both in time and space. In multicellular organisms extra layers of regulation impinge on the balance between cell proliferation and cell differentiation within particular ontogenic programs. In contrast to animals, organogenesis in plants is a post-embryonic process that requires developmentally programmed reversion of sets of cells from different differentiated states to a pluripotent state followed by regulated proliferation and progression through distinct differentiation patterns. This implies a fine coupling of cell division control, cell cycle arrest and reactivation, endoreplication and differentiation. The emerging view is that cell cycle regulators, in addition to controlling cell division, also function as targets for maintaining cell homeostasis during development. The mechanisms and cross talk among different cell cycle regulatory pathways are discussed here in the context of a developing plant.     

Production of interferon-beta upon induction with polyinosinic acid:polycytidylic acid during the cell cycle of human fibroblasts

The production of interferon-beta was examined at various stages of the cell cycle in synchronized and unsynchronized cell populations induced by poly(I):poly(C). Human fibroblasts were synchronized with mitotic detachment and, at different stages of the cell cycle, poly(I):poly(C) was added for induction of interferon-beta. One hour after induction, cell-free medium was collected and assayed for secreted interferon-beta. The cells were then fixed and stained with a DNA-specific fluorochrome, 4',6-diamidino-2-phenylindole (DAPI), for cell cycle analysis by microfluorometry. The data indicated that interferon-beta was produced in every stage examined of the cell cycle. In addition, the level of intracellular interferon-beta was quantitatively measured in single cells of an unsynchronized cell population using a specific antibody. In the same individual cell, DAPI fluorescence intensity was measured for determination of the cell cycle position. The results show that interferon-beta protein can be detected throughout the cell cycle.     

Multiple signalling pathways establish cell fate and cell number in Drosophila malpighian tubules

A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost (M. Hoch et al., 1994, Development 120, 3439-3450). In this paper we chart the mechanisms by which achaete expression is differentially maintained in the tip cell lineage to stabilise cell fate. First, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Second, Notch signalling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless after the critical period for tip cell allocation reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number.     

Insulin-dependent diabetes, an autoimmune disease

In type I (insulin-dependent) diabetes evidence for an autoimmune process is now fully established. This is also true for a similar disease observed in the NOD mouse and the BB rat. In addition to circulating antipancreatic antibodies, we demonstrated T-lymphocyte mediated cellular immunity in both these diabetic animals and in the human. Immunological abnormalities precede the development of diabetes and may be responsible for beta cell alteration. Evidence for this interpretation appears stronger for cell-mediated than for humoral immunity. However, full demonstration and understanding of the relationship between anti-beta cell immunity and beta cell alteration still raise many unresolved problems.     

一种细胞培养微芯片的制作及应用

细胞培养是细胞研究的基础, 微系统技术的发展给细胞培养提供了新的方法。在微系统平台上进行细胞研究,能够充分利用微流体和微结构的性质, 对细胞进行操控, 在细胞生物学、组织工程学、药物筛选等领域有广泛应用。介绍了一种利用SU-8负性光刻胶模具制作双层细胞培养微芯片的方法, 该芯片通过狭缝将细胞培养区和微通道区隔离, 既保证细胞培养区域的相对独立, 又可以利用微流体的特性调节细胞外基质的性质, 给基于微芯片进行细胞研究提供了一种新的平台。     

Cell wall integrity: Targeted post-synthetic modifications to reveal its role in plant growth and defense against pathogens

The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways.     

The specificity of Ca2+ signalling

A calcium signal is a sudden increase in concentration of calcium ions (Ca2+) in the cytosol. Such signals are crucial for the control of many important functions of the body. In the brain, for example, Ca2+ signals are responsible for memory, in muscle cells they switch on contraction, whereas in gland cells they are responsible for regulation of secretion. In many cases Ca2+ signals can control several different processes in the same cell. As an example, we shall deal with one particular cell type, namely the pancreatic acinar cell, which is responsible for the secretion of the enzymes essential for the digestion of food. In this cell, Ca2+ signals do not only control the normal enzyme secretion, but also regulate growth (cell division) and programmed cell death (apoptosis). Until recently, it was a mystery how the same type of signal could regulate such diverse functions in one and the same cell. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited.     

Cell wall alterations in the arabidopsis emb30 mutant

The Arabidopsis EMB30 gene is essential for controlling the polarity of cell growth and for normal cell adhesion during seedling development. In this article, we show that emb30 mutations also affect the growth of undifferentiated plant cells and adult tissues. EMB30 possesses a Sec7 domain and, based on similarities to other proteins, presumably functions in the secretory pathway. The plant cell wall depends on the secretory pathway to deliver its complex polysaccharides. We show that emb30 mutants have a cell wall defect that sometimes allows material to be deposited into the interstitial space between cells instead of being restricted to cell corners. In addition, pectin, a complex polysaccharide important for cell adhesion, appears to be abnormally localized in emb30 plants. In contrast, localization of epitopes associated with xyloglucan or arabinogalactan was similar in wild-type and emb30 tissues, and the localization of a marker molecule to vacuoles appeared normal. Therefore, emb30 mutations do not cause a general defect in the secretory pathway. Together, these results suggest that emb30 mutations result in an abnormal cell wall, which in turn may account for the defects in cell adhesion and polar cell growth control observed in the mutants.     

Differential molecular and anatomical basis for B cell migration into the peritoneal cavity and omental milky spots

The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that alpha(4)beta(7) integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, alpha(4)beta(1) integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.     

A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion.