In vitro reconstruction of full thickness human skin on a composite collagen material

Rapid progress of in vitro techniques in the lastyears enabled the creation of organotypic skin cultures offering newpossibilities in wound treatment. Rebuilding of graft is one of the keyelementsof successful outcome of the procedure.In search for the best scaffold for organotypic skin culture, the novelcomposite xenogenic collagen based material with unique properties has beencreated and used to reconstitute full thickness human skin invitro. Based on our long established technology used for theproduction of collagen dressings for the treatment of burns, this novel,composite material offers excellent growth support of highly biodegradablespongy layer, combined with mechanical strength of collagen membrane. Themodulation of collagen properties was accomplished by consecutive treatmentwithhigh temperature and gamma irradiation. The use of the substrate enabled toobtain organotypic culture that resembles full thickness skin with fibroblastslayer and well-developed multilayer epithelium. Our new material offers easyhandling of obtained graft during surgery along with accelerated cell growth andcontrolled biodegradation of the culture support.     

Neuronal differentiation from postmitotic precursors in the ciliary ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment.     

Feasibility of ex vivo gene therapy for neurological disorders using the new retroviral vector GCDNsap packaged in the vesicular stomatitis virus G protein

Neuronal progenitor cells (NPC) are particularly suited as the target population for genetic and cellular therapy of neurological disorders such as Parkinson's disease or stroke. However, genetic modification of these cells using retroviral vectors remains a great challenge because of the low transduction rate and the need for fetal calf serum (FCS) during the transduction process that induces the cell differentiation to mature neurons. To overcome these problems, we developed a new retrovirus production system in which the simplified retroviral vector GCDNsap engineered to be resistant to denovo methylation was packaged in the vesicular stomatitis virus G protein (VSV-G), concentrated by centrifugation, and resuspended in serum-free medium (StemPro-34 SFM). In transduction experiments using enhanced green fluorescent protein (EGFP) as a marker, the concentrated FCS-free virus supernatant infected NPC at a high rate, while maintaining the ability of these cells to self-renew and differentiate in vitro. When such cells were grafted into mouse brains, EGFP-expressing NPC were detected in the region around the injection site at 8 weeks post transplantation. These findings suggest that the gene transfer system described here may provide a useful tool to genetically modify NPC for treatments of neurological disorders.     

Effect of epidermal growth factor on intestinal adaptation after allogeneic small bowel transplantation in rats

We reported that epidermal growth factor (EGF) stimulated graft adaptation in a rat model of syngeneic small bowel transplantation. However, graft rejection is a severe problem with clinical small bowel transplantation, because small intestinal wall contains large amounts of lymphoid tissue. Studies were performed to investigate the effect of EGF on allogeneic graft adaptation after small bowel transplantation in rats treated with an immunosuppressant FK506. The transplanted animals received intraperitoneally EGF or saline (untreated) after surgery and were examined for analysis one week later. EGF-treated group markedly enhanced the water absorption and induction of sodium glucose cotransporter (SGLTI) as compared with EGF-untreated group. EGF-treated group also increased the mucosal crypt depth and its cell proliferating rate, although there was no significant difference in the mucosal villus height between the two groups. These results indicate that EGF accelerates intestinal allograft adaptation in part by the recovery of mucosal structure and function after small bowel transplantation in rats. EGF may have relevance to promote graft function in clinical small intestinal transplantation.     

Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum

Microtubule dynamics in Paramecium caudatum were investigated with an anti-alpha-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-alpha-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.     

Autotetraploid tangor plant regeneration from in vitro Citrus somatic embryogenic callus treated with colchicine

In vitro chromosome doubling of embryogenic callus lines of the Citrus cultivars Umatilla and Dweet tangors (Citrus reticulata Blanco×C. sinensis [L.] Osb.), Caffin clementine (C. clementina Hort. ex Tan.) and Wheeny grapefruit (C. paradisi Macf) was carried out in the presence of either 0.05 or 0.1% colchicine, or 0.01, 0.05 or 0.1% oryzalin. Embryogenic callus development was partly suppressed in the presence of colchicine, and completely suppressed by oryzalin at all concentrations tested. No plants were regenerated from any of the oryzalin treatments. Ploidy level of plants regenerated from the colchicine treatments was determined using flow cytometry and chromosome squashes. Three desirable non-chimeric, autotetraploid plants of the mono-embryonic cultivar Umatilla were produced using 0.05% colchicine and one from 0.1% colchicine. One mixoploid Dweet plant was produced using 0.1% colchicine.     

The role of heme oxygenase signaling in various disorders

Modern methods of cell and molecular biology, augmented by molecular technology, have great potential for helping to unravel the complex mechanisms of various diseases. They also have the potential to help us try to dissect the events which follow the altered physiological conditions. Thus, there is every reason to believe that some of the potential mechanisms will be translated sooner or later into the clinic. Heme oxygenase (HO)-related mechanisms play an important role in several aspects of different diseases. In the past several years, significant progress has been made in our understanding of the function and regulation of HO. The objective of this article is to review current knowledge relating to the importance of HO mechanism in various diseases including myocardial ischemia/reperfusion, hypertension, cardiomyopathy, organ transplantation, endotoxemia, lung diseases, and immunosuppression. The morbidity and mortality of these diseases remain high even with optimal medical management. Furthermore, in this review, we consider various factors influencing the HO system and finally assess current pharmacological approaches to their control.     

Factors Influencing the Differentiation of Dopaminergic Traits in Transplanted Neural Stem Cells

1. Our previous studies demonstrated that when neural stem cells (NSCs) of the C17.2 clonal line are transplanted into the intact or 6-hydroxydopamine (6-OHDA) lesioned rat striatum, in most, but not all grafts, cells spontaneously express the dopamine (DA) biosynthetic enzymes, tyrosine hydroxylase (TH), and aromatic L-amino acid decarboxylase (Yang, M., Stull, N. D., Snyder, E. Y., Berk, M. A., and Iacovitti, L. (2002). Exp. Neurol.).2. These results suggested that there were certain conditions which were more conducive to the development of DA traits in NSCs and possibly other neurotransmitter phenotypes.3. In the present study, we modified a number of variables in vitro (i.e. passage number, confluence) and/or in vivo (degree, type, and site of injury) before assessing the survival, migration, and differentiation of engrafted NSCs.4. We found that low confluence cultures were comprised exclusively of flattened polygonal cells, which when transplanted, migrated widely in the brain but did not express TH.5. In contrast, high confluence cultures contained both polygonal cells and an overlying bed of fusiform cells.6. When these NSCs were maintained for 12–20 passages and then transplanted, virtually all engrafted cells in 65% of the grafts expressed TH but not markers of other neurotransmitter systems.7. Importantly, all TH⁺ grafts were accompanied by significant physical damage to the brain while TH^– grafts were not, suggesting that local injury-related factors were also important.8. Of no apparent influence on TH expression, regardless of how cells were grown prior to implantation, was the site of transplantation (cortex or striatum) or the degree of chemical lesion (intact, partial or full).9. We conclude that transplanted NSCs can express traits specifically associated with DA neurons but only when cells are grown under certain conditions in vitro and then transplanted in proximity to injury-induced factors present in vivo.     

The golden anniversary of cloning: a celebratory essay

May 2002 marked the golden anniversary of the first cloned tadpoles. We celebrate this anniversary, as nuclear transplantation of frog cells into enucleated eggs became the prototype for cloning insects, fish, and mammals. We briefly review the salient results from amphibian cloning. Extension of these studies to mammalian species led to cloning adult cells, important advances in understanding nuclear reprogramming, and the construction of transgenic clones for biomedical applications. In addition, murine cloning clarified two problems unresolved in frog cloning: the unequivocal demonstration that nuclei of fully differentiated cells can direct the formation of fertile adults, and that abnormal expression of genes was responsible for the endoderm and neural syndromes in Rana clones.