An insight into spore dispersal of <Emphasis Type="Italic">Ganoderma boninense</Emphasis> on oil palm

The disease of oil palm caused by Ganoderma boninense, although universally referred to as Ganoderma basal stem rot, occurs in three very distinct phases, with basal stem rot only part of the disease cycle. G. boninense also causes a seedling disease and an upper stem rot. An understanding of spore dispersal provides an insight into where spores of G. boninense have a role in the infection process. This role will be discussed in relation to each of these three infection phases. This understanding is a critical component of developing a successful disease control strategy.     

In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells

The purpose of this study has been to investigate the possible effects of the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs). Autologous bone marrow was aspirated from the iliac crest of male sheep. MSCs were purified, expanded, and labeled with the fluorescent dye PKH26. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The cells labeled with red fluorescent PKH26 in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs.This work was supported by the National Natural Science Foundation of China (nos. 39900036, 20174006, and 20221402), the National Advanced Technology Programs of China (nos. 2003AA744051, 2003AA205041), the Award Foundation for Young Teachers from the Ministry of Education, 973 project (no. G1999054306-03), and the 248 key innovative project of Beijing (no. H010210190123).     

Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis. S. Ichinose and I. Sekiya contributed equally to this study     

A comparative histological study between normal and fasciated shoots of <Emphasis Type="Italic">Prunus avium</Emphasis> generated <Emphasis Type="Italic">in vitro</Emphasis>

A combination of Murashige and Skoogs medium and N⁶–benzyladenine (BA) at various concentrations (0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg l^–1) was supplied to shoot tips from root cuttings of a 50-year-old wild-cherry tree (Prunus avium). The concentration of BA in the growing medium was a determining factor with respect to the number of proliferated shoots per explant in vitro.Normal and fasciated shoots were generated when BA was present at 0.5, 0.75, 1.0 and 1.25 mg l^–1 in the medium and the mean numbers of normal shoots per explant were 3.63, 5.37, 8.93 and 7.30 respectively, and those of the fasciated shoots per explant were 0.03, 0.1, 0.47 and 0.4 respectively. Anatomical analysis by confocal microscopy of sections of paraffin-embedded specimens revealed that the cell structure and organization of the cortex and vascular cylinder in the fasciated shoots was similar to that in normal shoots. However, the cross-sectional area of stem of the fasciations was apparently greater than that of the normal shoots. In particular, the volume of vascular tissues, of pith and of some individual parenchyma cells in the cortex and pith was apparently greater in fasciated shoots than in normal shoots. Increases in cytokinesis and morphogenetic activity, such as the development of callus-like regions and the formation of adventitious shoots, were observed in the cortex and pith throughout the fasciations. The fasciated shoots had numerous buds and initiating new shoots at their apices while normal shoots had a single dominant axial bud.     

Myocardial regeneration with bone-marrow-derived stem cells

Despite significant therapeutic advances, heart failure remains the predominant cause of mortality in the Western world. Ischaemic cardiomyopathy and myocardial infarction are typified by the irreversible loss of cardiac muscle (cardiomyocytes) and vasculature composed of endothelial cells and smooth muscle cells, which are essential for maintaining cardiac integrity and function. The recent identification of adult and embryonic stem cells has triggered attempts to directly repopulate these tissues by stem cell transplantation as a novel therapeutic option. Reports describing provocative and hopeful examples of myocardial regeneration with adult bone-marrow-derived stem and progenitor cells have increased the enthusiasm for the use of these cells, yet many questions remain regarding their therapeutic potential and the mechanisms responsible for the observed therapeutic effects. In this review article we discuss the current preclinical and clinical advances in bone-marrow-derived stem or progenitor cell therapies for regeneration or repair of the ischaemic myocardium and their multiple related mechanisms involved in myocardial repair and regeneration.     

In search of markers for the stem cells of the corneal epithelium

The anterior one-fifth of the human eye is called the cornea. It consists of several specialized cell types that work together to give the cornea its unique optical properties. As a result of its smooth surface and clarity, light entering the cornea focuses on the neural retina allowing images to come into focus in the optical centres of the brain. When the cornea is not smooth or clear, vision is impaired. The surface of the cornea consists of a stratified squamous epithelium that must be continuously renewed. The cells that make up this outer covering come from an adult stem cell population located at the corneal periphery at a site called the corneal limbus. While engaging in the search for surface markers for corneal epithelial stem cells, vision scientists have obtained a better understanding of the healthy ocular surface. In this review, we summarize the current state of knowledge of the ocular surface and its adult stem cells, and analyse data as they now exist regarding putative corneal epithelial stem cell markers.     

Rotenone-induced caspase 9/3-independent and -dependent cell death in undifferentiated and differentiated human neural stem cells

We used human neural stem cells (hNSCs) and their differentiated cultures as a model system to evaluate the mechanism(s) involved in rotenone (RO)- and camptothecin (CA)-induced cytotoxicity. Results from ultrastructural damage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining indicated that RO-induced cytotoxicity resembled CA-induced apoptosis more than H(2)O(2)-induced necrosis. However, unlike CA-induced, caspase 9/3-dependent apoptosis, there was no increased activity in caspase 9, caspase 3 or poly (ADP-ribose) polymerase (PARP) cleavage in RO-induced cytotoxicity, in spite of time-dependent release of cytochrome c and apoptosis-inducing factor (AIF) following mitochondrial membrane depolarization and a significant increase in reactive oxygen species generation. Equal doses of RO and CA used in hNSCs induced caspase 9/3-dependent apoptosis in differentiated cultures. Time-dependent ATP depletion occurred earlier and to a greater extent in RO-treated hNSCs than in CA-treated hNSCs, or differentiated cultures treated with RO or CA. In conclusion, these results represent a unique ultrastructural and molecular characterization of RO- and CA-induced cytotoxicity in hNSCs and their differentiated cultures. Intracellular ATP levels may play an important role in determining whether neural progenitors or their differentiated cells follow a caspase 9/3-dependent or -independent pathway in response to acute insults from neuronal toxicants.     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.     

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.     

Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.