Transcription profiling during the cell cycle shows that a subset of Polycomb-targeted genes is upregulated during DNA replication

Genome-wide gene expression analyses of the human somatic cell cycle have indicated that the set of cycling genes differ between primary and cancer cells. By identifying genes that have cell cycle dependent expression in HaCaT human keratinocytes and comparing these with previously identified cell cycle genes, we have identified three distinct groups of cell cycle genes. First, housekeeping genes enriched for known cell cycle functions; second, cell type-specific genes enriched for HaCaT-specific functions; and third, Polycomb-regulated genes. These Polycomb-regulated genes are specifically upregulated during DNA replication, and consistent with being epigenetically silenced in other cell cycle phases, these genes have lower expression than other cell cycle genes. We also find similar patterns in foreskin fibroblasts, indicating that replication-dependent expression of Polycomb-silenced genes is a prevalent but unrecognized regulatory mechanism.     

Muscle cell culture as a tool in animal growth research

Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.     

Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis

The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell division, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell division protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation-division cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell division and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to division sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.     

A method for prolonged survival of primary cell lines

Summary We have established a means for prolonged survival of primary cell cultures and establishment of continuous cell lines without genetic manipulations. Primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. Earlier attempts to circumvent this limitation have included transformation with oncogenes, spontaneous immortalization of primary cultures, and chemical carcinogenesis. We have found that addition of a complex of growth-promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. This same supplement allows the prolonged survival of primary cell cultures derived from adult rat ovaries. The rat ovary primary cultures consisted of mixed phenotypes, including epithelial, neuron-like, and mesenchymal cell types. Numerous cells stain positive for alkaline phosphatase in these cultures. Other primary cell lines were established from embryonic rat liver and from adult rat lungs, using the same supplement. The survival effect is reversible because cells degenerate when the supplement is removed. Therefore, the cell lines have neither acquired properties of a tumor cell line nor have they been immortalized by a virus infection. We expect that our approach will open the door to prolonged survival of other primary cell types.     

Defining the immune mechanism with monoclonal antibodies

Summary It has long been realized that only the study of homogeneous antibodies or cell populations could enable a definitive understanding of much of the immune mechanism. Hybridoma technology has greatly facilitated such approaches. Hybridoma antibodies have been used to delineate both B cell and T cell subpopulations. T cell studies per se have been accomplished by the use of T cell hybridoma cell lines producing a variety of factors. Anti-idiotypes against B cell hybridoma antibodies have been used to characterize T cell receptors and factors. B cell studies have been facilitated by hybridomas that have made available the immunoglobulin of pre-B cells or defective B cell lines. Hybridoma antibodies have also been used to dissect closely related antibody families and the potential for responsiveness against a variety of antigenic determinants. Finally, hybridomas have provided a primary source of material for protein and DNA sequence analysis. In our laboratories hybridoma antibodies derived against the murine H-2 locus have demonstrated the ability of B cell antibodies to discriminate amongst H2 mutants—a capacity previously attributed only to T cell specificities. Hybridoma antibodies have also been generated by fusions with antigen stimulated neonatal B cells to provide homogeneous antibodies reflective of the earliest developmental immunoglobulin readout. Such probes should increase our understanding of the processes involved in the generation of both the T and B cell repertoires. Presented in the symposium on the Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association. Washington, D.C., June 7–11, 1981. This work was supported by United States Public Health Service Grants AI 15797 and AI 15710. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

A proteomics approach to identifying fish cell lines

Fish cell lines are relatively easy to culture and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred. Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2-DE and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. Similar concordance was seen in cells derived from the same tissues than from other tissues within the same species. These profiles have been saved in an electronic databank and could be made available to be used for discerning the origins of the various cell lines evaluated. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines.     

Proteome and phosphoproteome dynamic change during cell dedifferentiation in Arabidopsis

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, we have established the proteome map of Arabidopsis cotyledons and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, we have investigated the cotyledon phosphoproteome using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. These studies have provided significant new insight into protein and phosphoprotein differential expression during cell dedifferentiation in plants.     

Stem cell plasticity, cell fusion, and transdifferentiation

One of the most contentious issues in biology today concerns the existence of stem cell plasticity. The term "plasticity" refers to the capacity of tissue-derived stem cells to exhibit a phenotypic potential that extends beyond the differentiated cell phenotypes of their resident tissue. Although evidence of stem cell plasticity has been reported by multiple laboratories, other scientists have not found the data persuasive and have remained skeptical about these new findings. This review will provide an overview of the stem cell plasticity controversy. We will examine many of the major objections that have been made to challenge the stem cell plasticity data. This controversy will be placed in the context of the traditional view of stem cell potential and cell phenotypic diversification. What the implications of cell plasticity are, and how its existence may modulate our present understanding of stem cell biology, will be explored. In addition, we will examine a topic that is usually not included within a discussion of stem cell biology--the direct conversion of one differentiated cell type into another. We believe that these observations on the transdifferentiation of differentiated cells have direct bearing on the issue of stem cell plasticity, and may provide insights into how cell phenotypic diversification is realized in the adult and into the origin of cell phenotypes during evolution.     

Cell cycle regulation in the developing lens

Regulation of cell proliferation is a critical aspect of the development of multicellular organisms. The ocular lens is an excellent model system in which to unravel the mechanisms controlling cell proliferation during development. In recent years, several cell cycle regulators have been shown to be essential for maintaining normal patterns of lens cell proliferation. Additionally, many growth factor signaling pathways and cell adhesion factors have been shown to have the capacity to regulate lens cell proliferation. Given this complexity, understanding the cross talk between these many signaling pathways and how they are coordinated are important directions for the future.     

Fibroblast and epidermal growth factors: their uses in vivo and in vitro in studies on cell functions and cell transplantation

Summary The events which led to the isolation of the fibroblast growth factor are described. Its impact on the development of new cell models which have been used for studies as diverse as the control of cell proliferation, cell differentiation, and cell transplantation have been reviewed.     

Smoothened, thickveins and the genetic control of cell cycle and cell fate in the developing Drosophila eye

The Hedgehog and Decapentaplegic pathways have several well-characterized functions in the developing Drosophila compound eye, including initiation and progression of the morphogenetic furrow. Other functions involve control of cell cycle and cell survival as well as cell type specification. Here we have used the mosaic clone analysis of null mutations of the smoothened and thickveins genes (which encode the receptors for these two signals) both alone and in combination, to study cell cycle and cell fate in the developing eye. We conclude that both pathways have several, but differing roles in furrow induction and cell fate and survival, but that neither directly affects cell type specification.     

Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK)

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.     

Morphology, ontogeny and histochemistry of secretory trichomes of Geranium robertianum (Geraniaceae)

Geranium robertianum bears three types of glandular uniseriate trichomes which originate from a single protodermal cell and develop through periclinal divisions. Type I trichomes are procumbent and have an oval apical cell, two stalk cells and a basal cell. Type II trichomes are erect and have a pear shaped apical cell, two stalk cells and a basal cell. Type III trichomes are much longer than the other two types and have an elongated apical cell, five long stalk cells and a basal cell. Type I and type II trichomes are common on leaves while III trichomes are more abundant on flower structures.
Type I and type II trichomes secrete terpenoids and phenols. Type III trichomes are characterized by the accumulation of anthocyanins in the apical cell and secrete flavonoids.     

讲座细胞药物的制备工艺——细胞药物连载之二

细胞药物制备的质量直接关系到细胞治疗的效果。由于细胞治疗所用细胞是具有生物学效应的,细胞药物的制备技术和应用方案具有多样性、复杂性和特殊性,不像一般生物药物那样有统一的制作标准。细胞药物的制备过程主要包括供者筛查、供者检测、采集、加工、分离纯化、储存等,以造血干细胞、间充质干细胞、肝细胞及树突状细胞为例对其进行简要介绍。     

Genome-wide annotation and expression profiling of cell cycle regulatory genes in Chlamydomonas reinhardtii

Eukaryotic cell cycles are driven by a set of regulators that have undergone lineage-specific gene loss, duplication, or divergence in different taxa. It is not known to what extent these genomic processes contribute to differences in cell cycle regulatory programs and cell division mechanisms among different taxonomic groups. We have undertaken a genome-wide characterization of the cell cycle genes encoded by Chlamydomonas reinhardtii, a unicellular eukaryote that is part of the green algal/land plant clade. Although Chlamydomonas cells divide by a noncanonical mechanism termed multiple fission, the cell cycle regulatory proteins from Chlamydomonas are remarkably similar to those found in higher plants and metazoans, including the proteins of the RB-E2F pathway that are absent in the fungal kingdom. Unlike in higher plants and vertebrates where cell cycle regulatory genes have undergone extensive duplication, most of the cell cycle regulators in Chlamydomonas have not. The relatively small number of cell cycle genes and growing molecular genetic toolkit position Chlamydomonas to become an important model for higher plant and metazoan cell cycles.     

Quantum dots do not affect the behaviour of mouse embryonic stem cells and kidney stem cells and are suitable for short-term tracking

Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.     

Asymmetric cell division: recent developments and their implications for tumour biology

The ability of cells to divide asymmetrically is essential for generating diverse cell types during development. The past 10 years have seen tremendous progress in our understanding of this important biological process. We have learned that localized phosphorylation events are responsible for the asymmetric segregation of cell fate determinants in mitosis and that centrosomes and microtubules play important parts in this process. The relevance of asymmetric cell division for stem cell biology has added a new dimension to the field, and exciting connections between asymmetric cell division and tumorigenesis have begun to emerge.     

Mutations of the secondary cell wall

It has not been possible to isolate a number of crucial enzymes involved in plant cell wall synthesis. Recent progress in identifying some of these steps has been overcome by the isolation of mutants defective in various aspects of cell wall synthesis and the use of these mutants to identify the corresponding genes. Secondary cell walls offer numerous advantages for genetic analysis of plant cell walls. It is possible to recover very severe mutants since the plants remain viable. In addition, although variation in secondary cell wall composition occurs between different species and between different cell types, the composition of the walls is relatively simple compared to primary cell walls. Despite these advantages, relatively few secondary cell wall mutations have been described to date. The only secondary cell wall mutations characterised to date, in which the basis of the abnormality is known, have defects in either the control of secondary cell wall deposition or secondary cell wall cellulose or lignin biosynthesis. These mutants have, however, provided essential information on secondary cell wall biosynthesis.     

A hypothesis for the mechanism of mycoplasma evolution

Mycoplasmas are wall-less prokaryotes which have small genomes and are known to have evolved from ancestors of Gram-positive bacteria. A model is proposed to explain how mycoplasmas may have evolved from these ancestors which had cell walls and large genomes. It is proposed that the initial step in this process was loss of the cell wall and conversion of the ancestral bacterium to an L-form. Fusion of L-forms would have resulted in a single cell that contained two or more complete genomes. It is thought that this bringing together of multiple genomes by cell fusion resulted in genetic recombination between genomes and loss of DNA segments from the cell. Data from bacterial systems are cited in support of this model.