In vitro induction and transplantation of eye during early Xenopus development

A vertebrate eye was induced via a series of coordinated inductive interactions. Here, we describe a novel in vitro system to induce eye formation at high frequency using Xenopus early gastrulae. The eye formed in vitro is morphologically similar to the normal eye. When the in vitro eye was transplanted into a stage-33 tadpole, the optic nerve was seen extending from the grafted eye to the tectum of the host brain and the in vitro eye graft was retained after metamorphosis. In addition, we transplanted the eye formed in vitro into a tadpole with both eyes removed. The resultant juvenile frogs could perceive brightness using the grafted eye and thereby control their skin color, suggesting that the eye formed in vitro could function normally.     

Articular Cartilage Chondrocytes are more Advantageous for Generating Hyaline-like Cartilage than Mesenchymal Cells Isolated from Microfracture Repairs

Articular cartilage lacks self-repair capacity. Currently, two methods employing autologous cells are used to stimulate repair of articular cartilage. Micro-fracture induced repair induces autologous mesenchymal cell migration from bone marrow. Autologous chondrocytes' transplantation involves in vitro expansion of chondrocytes, and later implantation. In 15 patients de-differentiated chondrocytes obtained by cartilage biopsy were compared to cells derived from repair tissue induced by micro-fracture. These patients all underwent micro-fracture during the cartilage biopsy procedure. Autologous chondrocytes' transplantation was performed at least two months later then the biopsy. Tissue bits from articular cartilage and micro-fracture repair tissue were incubated in-vitro and explant cell cultures established. The cell cultures were assessed by immunohistochemistry and induced to differentiate. Differentiation into bone tissue was stimulated by addition of basic fibroblast growth factor, ascorbate and dexamethasone. High density (micro-mass) culture was used to stimulate chondrogenesis. Both cell cultures consist of mesenchymal progenitors as indicated by fibroblast growth factor receptor 3 expression and anti-CD-34+ antibodies. However, the micro-fracture generated repair tissue consists of osteocalcin-expressing cells destined to become bone. Collagen type II expression does not occur in these cells compared to autologous chondrocytes. Inducible nitric oxide synthase expression by microfracture cells is likely to damage surrounding articular cartilage in vivo. In conclusion, cells recruited by micro-fracture are inferior for cartilage regeneration purposes to those from cartilage biopsies.     

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.