Induced pluripotent stem cells and senescence: learning the biology to improve the technology

The discovery that adult somatic cells can be reprogrammed into pluripotent cells by expressing a combination of factors associated with pluripotency holds immense promise for a wide range of biotechnological and therapeutic applications. However, some hurdles—such as improving the low reprogramming efficiencies and ensuring the pluripotent potential, genomic integrity and safety of the resulting cells—must be overcome before induced pluripotent stem cells (iPSCs) can be used for clinical purposes. Several groups have recently shown that key tumour suppressors—such as members of the p53 and p16^INK4a/retinoblastoma networks—control the efficiency of iPSC generation by activating cell‐intrinsic programmes such as senescence. Here, we discuss the implications of these discoveries for improving the safety and efficiency of iPSC generation, and for increasing our understanding of different aspects of basic biology—such as the control of pluripotency or the mechanisms involved in the generation of cancer stem cells.     

Q&A: Mesenchymal stem cells — where do they come from and is it important?

Mesenchymal stem — or stromal — cells (MSCs) have been administered in hundreds of clinical trials for multiple indications, making them some of the most commonly used selected regenerative cells. Paradoxically, MSCs have also long remained the least characterized stem cells regarding native identity and natural function, being isolated retrospectively in long-term culture. Recent years have seen progress in our understanding of the natural history of these cells, and candidate native MSCs have been identified within fetal and adult organs. Beyond basic knowledge, deciphering the biology of innate MSCs may have important positive consequences for the therapeutic use of these cells.     

The pro-metastatic role of bone marrow-derived cells: a focus on MSCs and regulatory T cells

Several bone marrow-derived cells have been shown to promote tumour growth and progression. These cells can home to the primary tumour and become active components of the tumour microenvironment. Recent studies have also identified bone marrow-derived cells—such as mesenchymal stem cells and regulatory T cells—as contributors to cancer metastasis. The innate versatility of these cells provides diverse functional aid to promote malignancy, ranging from structural support to signal-mediated suppression of the host immune response. Here, we review the role of mesenchymal stem cells and regulatory T cells in cancer metastasis. A better understanding of the bipolar nature of these bone marrow-derived cells in physiological and malignant contexts could pave the way for new therapeutics against metastatic disease.     

Mise en évidence des cellules inflammatoires dans le sperme

A high level of round cells in semen decreases the fertility capacity. These cells can be either immature germinal cells from testicular disorders characterized by teratospermia, or polymorphonuclear cells if they are peroxidase positive. However it often happens that neither teratospermia nor peroxidase reaction can explain the high proportion of round cells. For these semen we have used the nonspecific esterase staining to search for macrophages, plasma-cells and lymphocytes. These three types of cells were detected in certain non-leucospermic semen which reflects a chronic infection in development. Also these cells, macrophages, plasma-cells and lymphocytes were found in some leucospermic semen samples. If we considere the populations leucospermic and non leucospermic together, macrophages are correlated — with plasma-cells (r=0,65 — p=0,01) and with lymphocytes (r=0,56 — p=0,05). The correlation between plasma-cells and lymphocytes was more important (r=0,91 — p=0,001).     

Therapeutic targets in cancer cell metabolism and autophagy

The metabolism of cancer cells is reprogrammed both by oncogene signaling and by dysregulation of metabolic enzymes. The resulting altered metabolism supports cellular proliferation and survival but leaves cancer cells dependent on a continuous supply of nutrients. Thus, many metabolic enzymes have become targets for new cancer therapies. Recently, two processes—expression of specific isoforms of metabolic enzymes and autophagy—have been shown to be crucial for the adaptation of tumor cells to changes in nutrient availability. An increasing number of approved and experimental therapeutics target these two processes. A better understanding of the molecular basis of cancer-associated metabolic changes may lead to improved cancer therapies.     

Anti-Müllerian hormone: Hormone or growth factor?

Anti-Müllerian hormone (AMH) is a dimeric glycoprotein member of the TGF-β family. It is synthesized by immature Sertoli cells, and, to a lesser degree, by adult Sertoli and granulosa cells. AMH is responsible for the regression of Müllerian ducts in the male fetus; it also has deleterious effects on the female fetal reproductive tract, destroying Müllerian primordia and germ cells, and masculinizing the fetal ovary on the rare occasions female fetuses become exposed to its effects. All other suggested actions for AMH—retardation of oocyte meiosis, inhibition of EGF receptor autophosphorylation, anti-cancer activity—have been reported with crude hormone preparations, and have not been confirmed using pure AMH. Its relatively limited sphere of action—the fetal genital tract—and the fact that it is secreted into the general circulation and can act at long range, imply that AMH is more like a hormone than a growth factor, but the complex interaction between hormones and growth factors make a formal distinction impossible.     

Implications of stochastic inheritance

Imperfect coordination between an autonomous genetic element, or plasmid, and a host bacterium reproducing by binary fission is shown to have two effects. The proportion of cells which have lost the plasmid completely tends to a constant for populations growing under constant selective pressure. At the same time, the average number of copies of the plasmid per cell—averaged over those cells which inherit one or more—tends to a constant, which is determined essentially by the ratio of two characteristic probabilities: that of failure of the plasmid to duplicate before a cell divides, and that of failure—of any two copies of the plasmid resulting from the same act of duplication—to separate to opposite halves of a dividing cell.The theory given predicts the proportion of cells which fail to inherit plasmid-like particles after any number of generations of bacterial growth. The manner in which the mean number of particles per particle-containing cell approaches its final constant value is also predicted.The effect of non-random inheritance on cells containing non-replicating particles is determined.Application of the theory is illustrated by experimental data from the literature.     

Reaction of human lymphocytes with aggregated IgG columns. Effect on antibody-dependent cytotoxicity and EAC rosette formation.

The origin and life span of long-lived small lymphocytes in the bone marrow of mice have been evaluated by the use of radioautography, scintillation counting, and anti-theta serum. Thymus-deprived BALB/C mice and nude mice had a smaller percentage of long-lived lymphocytes in bone marrow and in thoracic duct lymph than sham-operated or normal littermates. Furthermore, the long-lived lymphocytes in the marrow of nudes have more varied—but generally shorter—life spans than long-lived lymphocytes from mice having a thymus. In thoracic duct lymph of nude mice a more homogeneous long-lived population—according to life span—was found.It was concluded that both long-lived T cells and long-lived B cells are normal residents in the bone marrow. Furthermore, it was concluded that cells of variable life spans comprise the B lymphocyte population: short-lived cells with life spans of 3–5 days and long-lived lymphocytes with life spans of weeks to months.     

Induction and axial patterning of the neural plate: Planar and vertical signals

In this review I summarize recent findings on the contributions of different cell groups to the formation of the basic plan of the nervous system of vertebrate embryos. Midline cells of the mesoderm—the organizer, notochord, and prechordal plate—and midline cells of the neural ectoderm—the notoplate and floor plate—appear to have a fundamental role in the induction and patterning of the neural plate. Vertical signals acting across tissue layers and planar signals acting through the neural epithelium have distinct roles and cooperate in induction and pattern formation. Whereas the prechordal plate and notochord have distinct vertical signaling properties, the initial anteroposterior (A-P) pattern of the neural plate may be induced by planar signals originating from the organizer region. Planar signals from the notoplate may also contribute to the mediolateral (M-L) patterning of the neural plate. These and other findings suggest a general view of neural induction and axial patterning. © 1993 John Wiley & Sons, Inc.     

Studies on a mammalian cell protein (P8) with affinity for DNA in vitro

A protein (P8) found in cultured mammalian cells has been highly purified by DNA—cellulose chromatography alone. The protein is quite abundant, especially in human diploid fibroblasts, is readily extractable at low-salt concentration, and has an isoelectric pH close to neutrality. Its synthesis is not coupled to that of DNA, but it accounts for a greater proportion of the total soluble protein synthesis in growing than in resting cells. Among the proteins identified after DNA—cellulose chromatography, only P8 has affinity for single-stranded DNA but not for double-stranded DNA. It appears to have properties different from those of other DNA-binding proteins that have been described.     

EVOLUTION OF DEVELOPMENTAL PROGRAMS IN VOLVOX (CHLOROPHYTA)1

The volvocine green algal genus Volvox includes ～20 species with diverse sizes (in terms of both diameter and cell number), morphologies, and developmental programs. Two suites of characters are shared among distantly related lineages within Volvox. The traits characteristic of all species of Volvox—large (>500) numbers of small somatic cells, much smaller numbers of reproductive cells, and oogamy in sexual reproduction—have three or possibly four separate origins. In addition, some species have evolved a suite of developmental characters that differs from the ancestral developmental program. Most multicellular volvocine algae, including some species of Volvox, share an unusual pattern of cell division known as palintomy or multiple fission. Asexual reproductive cells (gonidia) grow up to many times their initial size and then divide several times in rapid succession, with little or no growth between divisions. Three separate Volvox lineages have evolved a reduced form of palintomy in which reproductive cells are small and grow between cell divisions. In each case, these changes are accompanied by a reduction in the rate of cell division and by a requirement of light for cell division to occur. Thus, two suites of characters—those characteristic of all Volvox species and those related to reduced palintomy—have each evolved convergently or in parallel in lineages that diverged at least 175 million years ago (mya).     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

Fluorescence techniques in biotechnology

The high specificity and sensitivity of fluorescence techniques have made them important analytical tools in medicine and biotechnology. Besides monitoring and quantitative detection of biomolecules these methods can be used for controlling bacterial activities or for measuring physiological states of cells or tissues. Three topics of importance in biotechnology — immunoassays, photosynthesis and fermentation — are treated in detail.     

Induction of Oxidative Stress in Tumor Cells: A New Strategy for Drug Therapy of Malignant Tumors

Biophysics - Abstract—Tumor cells have a higher basal ROS level than normal cells. This phenomenon may provide grounds for the development of novel antitumor drugs that are capable of...     

Isolation and Purification of Viable Sperm Cells from Stored Bicellular Pollen of Lilium davidii Duch.

A successful mass isolation of viable sperm cells from stored bicellular pollen of Lilium davidii Duch. was reported. When fresh pollen was cultured in BKS 15 medium, 87 % germinated in which the generative cells of the fresh pollen underwent mitosis and formed sperm cells within 28 hours. For pollen stored at -20℃ and -70℃ for 6 months, only less than 20 % germinated; but the germination percentage rose to 80 % after they have been hydrated and gradually warmed. Pollen grains of L. davidii which have been stored at -70 ℃ for 6 months, after being thawed were firstly germinated in a 15% sucrose medium for 28 hours, and then osmotically shocked with 10 % sucrose Solution. The solution was later adjusted to a final sucrose concentration of 15%. After density gradient centrifugation, 4 mL suspension of purified sperm cells with a density of 6 × 106 cells/mL were obtained at the interphase of 5%—15% percoll, with a 12% yield of viable sperm cells. The purified sperm cells had a diameter of 13—15 μm and reacted positive to fluorochrome, indicating that they have intact plasma membrane.     

ONTOGENY AND STRUCTURE OF CONIFEROUS SIEVE CELLS

Studies of the secondary phloem of 6 species of conifers revealed that mature sieve-cell protoplasts contain internal strands which are derived ontogenetically from slime bodies of immature cells. These strands, each measuring about 0.3 μ in diameter, traverse the cell and run from cell to cell through sieve-area pores. Coniferous sieve cells have much in common—both ontogenetically and structurally—with dicotyledonous sieve-tube members.     

Mass-flowering crops enhance wild bee abundance

Although agricultural habitats can provide enormous amounts of food resources for pollinator species, links between agricultural and (semi-)natural habitats through dispersal and foraging movements have hardly been studied. In 67 study sites, we assessed the interactions between mass-flowering oilseed rape fields and semi-natural grasslands at different spatial scales, and their effects on the number of brood cells of a solitary cavity-nesting bee. The probability that the bee Osmia bicornis colonized trap nests in oilseed rape fields increased from 12 to 59 % when grassland was nearby, compared to fields isolated from grassland. In grasslands, the number of brood cells of O. bicornis in trap nests was 55 % higher when adjacent to oilseed rape compared to isolated grasslands. The percentage of oilseed rape pollen in the larval food was higher in oilseed rape fields and grasslands adjacent to oilseed rape than in isolated grasslands. In both oilseed rape fields and grasslands, the number of brood cells was positively correlated with the percentage of oilseed rape pollen in the larval food. We show that mass-flowering agricultural habitats—even when they are intensively managed—can strongly enhance the abundance of a solitary bee species nesting in nearby semi-natural habitats. Our results suggest that positive effects of agricultural habitats have been underestimated and might be very common (at least) for generalist species in landscapes consisting of a mixture of agricultural and semi-natural habitats. These effects might also have—so far overlooked—implications for interspecific competition and mutualistic interactions in semi-natural habitats.     

Initiation, Growth and Nuclear Characteristics of Tissue Cultures of Paeonia suffruticosa

Tissue cultures of the garden paeony, Paeonia suffruticosa have been established using explants of etiolated stems. Callus formation was induced on agar-solidified media containing ammonium ions or amino acids together with the hormones 2,4-dichlorophenoxyacetic acid and kinetin, but not on media lacking the reduced nitrogen component. Attempts to induce callus from explants from green plants were completely unsuccessful and were characterized by the production of intense brown colorations, both of the explant and the medium. Subcultured tissue without the added hormones produced roots, both on solid and liquid media. Growth was tested on a range of liquid media, SH/2, SH, SH × 2 and SH—M, containing 1250, 2500, 5000 and 2500 mg/l potassium nitrate. The SH—M medium also contained 1650 mg/l ammonium nitrate. Growth measured as increased fresh weight was best in the SH/2, SH and SH—M media and was curtailed in the SH × 2 medium. Soluble protein content was highest at the lowest nitrogen concentration. A histochemical comparison of tissue grown on the SH/2, SH—M and SH—M lacking hormones showed that the cells in all the cultures remained diploid while differences were established in total nuclear protein, measured using the ninhydrin-Schiff procedure. Nuclei from SH—M grown cells have a higher protein content than those from the SH/2 medium while cells from the SH—M medium lacking hormones show a further increase in nuclear protein. This raises the question whether this change in nuclear protein is related to the morphogenesis of roots which occurs in this medium.     

Effects of synthetic defensin fragments on aggregation and adhesion of epitheliolike cells

Normal course of processes of regeneration and epithelization of damaged tissues has been shown to be based on the capability of cells participating in these processes for selective adhesion. In the case of the complete or partial absence of this capability in the cells-participants of the wound healing process, the so-called non-healing wounds appear. In this connection, it remains actual to search for natural agents promoting healing of chronic non-healing wounds. In the present work, we studied effects of synthetic fragments of leukocytic antimicrobial peptides defensines—GER, FGER, and GERA—on aggregation and adhesion of epitheliolike cells of the CHO-K1 line. These peptides have been established to have aggregate-stimulating properties; besides, they enhance adhesion of the cells to the untreated plastic and inhibit fibronectinmediated cell adhesion. Possible pathways of regulation by peptides of processes of intercellular and cell-matrix interaction are discussed as well as ways of release of these compounds in an organism and their functional role in an organism.     

DISRUPTION OF CELL—CELL ADHESION IN THE PRESENCE OF SODIUM BUTYRATE ACTIVATES EXPRESSION OF THE 92kDa TYPE IV COLLAGENASE IN MDCK CELLS

We have investigated the effects of altered cell shape on the regulation of the 92kDa type IV collagenase. In MDCK cells, anti-E-cadherin antibodies alter cell shape by disrupting normal cell—cell contacts, while sodium butyrate causes a marked flattening and spreading of cells. The disruption of cell—cell contacts led to a faint expression of the 92kDa collagenase. This effect was enhanced by sodium butyrate, which by itself did not induce collagenase expression. In contrast, stromelysin expression was not regulated in these conditions. Although mRNA expression was enhanced, the secreted collagenase activity was not altered in these conditions in either cell line. Examination of cytoskeletal and extracellular matrix proteins and cell—cell and cell—matrix adhesion proteins by immunofluorescence and Western blot revealed a disruption of the actin network, tight junctions, and fibronectin deposition by anti E-cadherin antibodies, and alterations in actin, cytokeratin 8, cytokeratin 14, laminin and β1 integrin induced by sodium butyrate. Thus, the induction of collagenase expression in epithelial cells by disrupted cell—cell adhesion and sodium butyrate is associated with changes in cell shape and structure.