Behavior of transplanted bone marrow-derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation.

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.     

Apical dominance

Apical dominance is the control exerted by the apical portions of the shoot over the outgrowth of the lateral buds. The classical explanations for correlative inhibition have focused on hormone/nutrient hypotheses. The remarkable progress that has been made in the technology of endogenous hormone quantification in plant tissue has not been accompanied by comparable progress in the elucidation of mechanisms of hormone action in apical dominance. Evidence from hormonal studies suggests that apically produced auxin indirectly suppresses axillary bud outgrowth that is promoted by cytokinin originating from roots/shoots. Significant involvement with other hormones, although less likely, has not been ruled out. Possible changes in tissue sensitivity to hormones should not be overlooked. Auxin-induced oligosaccharide signals originating from the cell walls of shoot tips or polyamines may function as secondary inhibitors to bud growth. Alternatively, apically produced auxin may suppress lateral bud growth by inhibiting auxin export from these buds. Support for a critical role for nutrients in apical dominance keeps resurfacing, especially for auxin-directed nutrient transport and for water as a possible inducing signal for bud outgrowth. Histological and biochemical analyses of lateral buds recently released from apical dominance are urgently needed. The feasibility of manipulating endogenous auxin/cytokinin content in plant tissue by gene insertion and modulation opens the door to exciting approaches as does the use of hormone insensitive/resistant mutants. There is also need to recognize the existence of variability of apical dominance mechanisms among different plant types. The aesthetic and economic implications of understanding apical dominance for the modification of plant structure and form are extremely significant.     

Apical ectodermal ridge induction by the transplantation of En-1-overexpressing ectoderm in chick limb bud

In the early chick embryo, the dorsal–ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Here it is reported that Engrailed-1 ( En-1 ), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1 -overexpressing ectoderm induces ectopic AER at the boundary of En-1 -positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud.     

Presence of anti-cystatin C-positive dendritic cells or macrophages and localization of cysteine proteases in the apical bud of the enamel organ in the rat incisor.

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C-positive. Anti-cystatin C-labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C-positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.     

The Effect of the Apical Bud on the Growth of Lateral Buds on Subterranean Shoots

The influence of the apical bud on the growth of the lateral buds on subterranean shoots was studied in Stachys sieboldiiMig. and Helianthus rigidus(Gass.) Desv. Removing and damaging the apical parts of subterranean shoots or their treatment with 2% chlorocholine chloride shoot enhanced shoot branching. The response to light of the apical bud was invariably negative: the stolons, which came out or were extracted from the soil, grew back into the ground (negative phototropism). The response to light of lateral buds was autonomous and depended on the conditions of their initiation. The lateral buds developed in darkness manifested negative phototropism when withdrawn from the soil and exposed to the light, whereas the buds developed in the light showed positive phototropism. The author concludes that the concept of apical dominance, thoroughly studied in aboveground shoots, is also valid for subterranean shoots. However, in contrast to the former, in the latter case, the apical bud does not control the growth orientation of the lateral buds.     

Tillering in Tussock Grasses in Relation to Defoliation and Apical Bud Removal

Experiments with five caespitose grass species from temperateand tropical environments showed that the number of lateralshoots (tillers) which emerged following defoliation was notincreased by leaving a greater residual leaf area. Increasedavailability of photosynthate (and perhaps other resources)was effective, however, in increasing the rate of growth anddegree of flowering of new lateral shoots in one tropical species,Panicum maximum. In two temperate Agropyron tussock grasses, decapitation (apicalbud removal) did not stimulate lateral shoot growth. This indicatedthat apical dominance was not a factor preventing growth oflateral buds just prior to inflorescence emergence on the parenttillers. However, defoliation, where both terminal buds andfoliage were removed from the parent tillers stimulated lateralbud growth. Hormones other than those produced by the apicalbud or light quality or intensity may control lateral bud growthin these species. In contrast to the temperate species, lateralbud growth was stimulated by both decapitation and defoliationin the three tropical species. This response is consistent withthe model of correlative inhibition by apical dominance. Agropyron desertorum, Agropyron spicatum, Heteropogon contortus, Panicum maximum, Themeda triandra, crested wheatgrass, bluebunch wheatgrass, black speargrass, green panic grass kangaroo grass, apical dominance, tillering, regrowth, grazing, tussock grasses     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

Intracellular Localization of 3H-IAA in the Apical Bud of Lycopersicon esculentum

Driss-Ecole, D. and Perbal, G. 1987. Intracellular localizationof ³H-IAA in the apical bud of Lycopersicon esculentum.—J.exp. Bot. 38: 1362–1372. High resolution autoradiography of ³H-IAA was performed on ultra-thinsections of the apical bud of Lycopersicon esculentum treatedby DCC or glutaraldehyde. A quantitative study of the localizationof the labelling in the compartments (cell wall, cytoplasm,vacuoles and nucleus) of four types of cells (cells of the lateralzone of the apex, cells of the pith meristem, proximal and distalcells of the upper part of the pith) was made. The statisticalanalysis of the results has proved that the variability of thelabelling of the cell compartments in the four cell types wassimilar after treatment by DCC or glutaraldehyde. The densityof labelling is higher in each compartment of the meristematiccells compared with those of the differentiating cells. Thecells of the pith meristem can be distinguished from the meristematiccells of the lateral zone of the apex by a greater density oflabelling in the cytoplasm and the nucleus. These two cellulartypes contain a high density of radioactivity in vacuoles. Byconsidering the percentage of labelling of each compartmentrelative to the total labelling in the cell, it can be shownthat the meristematic cells are characterized by a high percentagein the nucleus whereas the vacuoles of the differentiating cellscontain the highest percentage of radioactivity. Key words: ³H-IAA, tomato shoot     

Preliminary Study on Transformation of Wheat Apical Point

Apical points of young seedlings of wheat (Triticum aestivum) cultivar "Jing 411” and somatic calli of cultivar "FK8” were transformed with plasmids pBI121 and (or) pBIAH-A+ by using microprojectile bombardment. Histochemical assay of GUS activity showed positive reaction on some of the transformation processed apical points and calli. This demonstrated that foreign genes were introduced into the apical meristematic cells as well as the callus cells. The plantlets of cv. "Jing 411” survived after apical point transformation with pBIAH-A+ were transplanted into the field and the progenies were screened with kanamycin. 4 % of the screened seeds germinated into green seedlings with kanamycin resistance. Dot hybridization of total DNA from kanamycin resistant plants showed the existence of foreign DNA in some of the detected plants.     

Formation of supernumerary structures by the embryonic chick wing depends on the position and orientation of a graft in a host limb bud

The relationship between the position transplanted in a host limb bud, the orientation of a graft in a host limb bud, and the extra limb structures formed was studied by juxtaposing normally nonadjacent embryonic chick wing bud tissue. In one series of transplantation operations, two different wedges (ectoderm and mesoderm) of stage 21 right donor posterior wing bud tissue were transplanted to the middle of a host stage 20 to 22 right wing bud such that the dorsal-ventral polarity of the graft and host were the same or reversed. The results of these transplantation operations show that the formation of supernumerary limb structures depends on the position of origin of the donor tissue, the anterior-posterior position transplanted in a host limb bud, and the orientation of the graft in the host limb bud. In a second series of transplantation operations, the relationship between the proximodistal position where posterior donor tissue is transplanted in an anterior host site and the extra structures formed was studied. A wedge of posterior stage 21 right wing bud tissue was transplanted to an anterior proximal or anterior distal site of a stage 22 to 24 host right wing bud. The results of these transplantation operations show that when the donor tissue is transplanted to an anterior proximal position in a host wing bud, then limbs with only a duplicated humerus result, whereas, when transplanted to an anterior distal position, then limbs with a duplicated forearm element and extra digits result.     

Axillary Bud Formation in Two Isogenic Lines of Tomato Showing Different Degrees of Apical Dominance

Grafting experiments have been carried out in which rootstocksof the cultivar Craigella were paired with scions of an isogenicline Craigella Lateral Suppressor (ls ls) and vice versa, andthe levels of hormones in the roots and shoots of the graftedplants examined. The roots of Craigella plants differed from those of LateralSuppressor in that they contained a higher proportion of a cytokininthat co-chromatographed with N⁶ - (²—isopentenyl) adenosine.Reciprocal grafts did not lead to any qualitative or quantitativechanges in the cytokinins in the roots of either line. GraftingLateral Suppressor scions on Craigella rootstocks led to anincrease in the IAA content of the apical region and the ABAcontent of the stem tissue immediately below it, but when Craigellascions were grafted on Lateral Suppressor rootstocks there wereno changes in the level of either hormone. Cytokinins applied to the leaf axils of Lateral Suppressor plantsresulted in lateral bud initiation in the axils above the pointof treatment but not if the plants were also given a short periodof far-red light at the end of the photoperiod. Cytokinins wereineffective in initiating lateral buds in grafted Lateral Suppressorscions. It is suggested that root-produced cytokinins influence lateralbud outgrowth indirectly by way of their effect on the levelsof IAA and ABA in the shoot. Lycopersicon esculentum Mill., tomato, apical dominance, growth regulation, indol-3yl acetic acid, abscisic acid, cytokinins     

Tissue engineered hybrid tooth-bone constructs

Proper rehabilitation of craniofacial defects is challenging because of the complexity of the anatomy and the component tissue types. The ability to simultaneously coordinate the regeneration of multiple tissues would make reconstruction more efficient and might reduce morbidity and improve outcomes. The craniofacial complex is unique because of the presence of teeth, in addition to skin, bone, cartilage, muscle, vascular, and neural tissues since teeth naturally grow in coordination with the craniofacial skeleton, our group developed an autologous, tooth-bone hybrid model to facilitate repair of mandibular defects in the Yucatan minipig. The hybrid tooth-bone construct was prepared by combining tooth bud cell-seeded scaffolds with autologous iliac crest bone marrow derived stem cell-seeded scaffolds, which were transplanted back into surgically created mandibular defects in the same minipig. The constructs were harvested after 12 and 20 weeks of growth. The resulting bone/tooth constructs were evaluated by X-ray, ultra high-resolution volume computed tomography (VCT), histological, immunohistochemical analyses, and transmission electron microscopy (TEM). The observed formation of small tooth-like structures consisting of organized dentin, enamel, pulp, cementum, periodontal ligament, and surrounded by regenerated alveolar bone, suggests the feasibility for regeneration of teeth and associated alveolar bone, in a single procedure. This model provides an accessible method for future clinical applications in humans.     

Fate of amnion-derived stem cells transplanted to the fetal rat brain: migration, survival and differentiation

We have recently characterized a stem cell population isolated from the rodent amniotic membrane termed amnion-derived stem cells (ADSCs). In vitro ADSCs differentiate into cell types representing all three embryonic layers, including neural cells. In this study we evaluated the neuroectodermal potential of ADSCs in vivo after in utero transplantation into the developing rat brain. A clonal line of green fluorescent protein-expressing ADSCs were infused into the telencephalic ventricles of the developing embryonic day 15.5 rat brain. At E17.5 donor cells existed primarily as spheres in the ventricles with subsets fused to the ventricular walls, suggesting a mode of entry into the brain parenchyma. By E21.5 green fluorescent protein (GFP) ADSCs migrated to a number of brain regions. Examination at postnatal time points revealed that donor ADSCs expressed vimentin and nestin. Subsets of transplanted ADSCs attained neuronal morphologies, although there was no immunohistochemical evidence of neural or glial differentiation. Some donor cells migrated around blood vessels and differentiated into putative endothelial cells. Donor ADSCs transplanted in utero were present in recipients into adulthood with no evidence of immunological rejection or tumour formation. Long-term survival may suggest utility in the treatment of disorders where differentiation to a neural cell type is not required for clinical benefit.     

杨树顶芽细胞内质网与其他膜系统的结构联系及其在休眠过程中的变化

杨树（Ｐｏｐｕｌｕｓ ｄｅｌｔｏｉｄｅｓ Ｂａｒｔｒ．ｅｘ Ｍａｒｓｈ）顶芽分生组织细胞经一种改良的高锰酸钾固定法固定后,显示出一种十分清晰的内膜结构,尤其展现了内质网与其他膜系统存在一种结构上的密切联系。一些与核膜相连接的内质网伸展到细胞质中与线粒体、质体及高尔基体发生联系,可延伸到质膜。还有些内质网的一端与一个细胞的核膜相连结,其另一端穿过胞间连丝与邻近的另一个细胞的核膜相连结,在两个相邻的细     

Control of Lateral Bud Growth inPhaseolus vulgarisL. by Ethylene in the Apical Shoot

Elongation growth of the first trifoliate leaf axillary budwas induced by physical restriction of the apical growth orby treating the apical shoot with ethylene or ethephon. Thereis evidence to suggest that the promotion of axillary bud developmentby ethylene action on the apical shoot was associated with theavailability of freely diffusible ethylene in the tissues ofthe treated shoot. Loss of apical dominance was not, apparently,directly dependent on either the internal ethylene concentration(i.e. concentration in the vacuum – extracted gases) oron the rate of ethylene emanation. The effects of aminoethoxyvinylglycine (AVG) and silver nitrate on ethylene production wereexamined. Although treatment of the shoot with tri – iodobenzoicacid (TIBA) induced various morphological responses in the plant,including axillary bud outgrowth, which appeared similar tothe responses to ethylene, the initial effect of TIBA is notthought to be ethylene – mediated.     

Fetal myocardium in the kidney capsule: an in vivo model of repopulation of myocytes by bone marrow cells

Debate surrounds the question of whether the heart is a post-mitotic organ in part due to the lack of an in vivo model in which myocytes are able to actively regenerate. The current study describes the first such mouse model--a fetal myocardial environment grafted into the adult kidney capsule. Here it is used to test whether cells descended from bone marrow can regenerate cardiac myocytes. One week after receiving the fetal heart grafts, recipients were lethally irradiated and transplanted with marrow from green fluorescent protein (GFP)-expressing C57Bl/6J (B6) donors using normal B6 recipients and fetal donors. Levels of myocyte regeneration from GFP marrow within both fetal myocardium and adult hearts of recipients were evaluated histologically. Fetal myocardium transplants had rich neovascularization and beat regularly after 2 weeks, continuing at checkpoints of 1, 2, 4, 6, 8 and12 months after transplantation. At each time point, GFP-expressing rod-shaped myocytes were found in the fetal myocardium, but only a few were found in the adult hearts. The average count of repopulated myocardium with green rod-shaped myocytes was 996.8 cells per gram of fetal myocardial tissue, and 28.7 cells per adult heart tissue, representing a thirty-five fold increase in fetal myocardium compared to the adult heart at 12 months (when numbers of green rod-shaped myocytes were normalized to per gram of myocardial tissue). Thus, bone marrow cells can differentiate to myocytes in the fetal myocardial environment. The novel in vivo model of fetal myocardium in the kidney capsule appears to be valuable for testing repopulating abilities of potential cardiac progenitors.     

An immunohistochemical study on hard tissue formation in a subcutaneously transplanted rat molar

While dental pulp undergoes calcification following tooth replantation or transplantation, we actually know little about these mechanisms. We therefore conducted histological and immunohistochemical evaluations of mineralized tissue that formed in the pulp of rat maxillary molar transplanted into abdominal subcutaneous tissue. One, 2, 3, and 4 weeks post-transplantation, the teeth were investigated immunohistochemically using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue non-specific alkaline phosphatase (TNAP). In the 1st week after transplantation, cell-rich hard tissue was formed at the root apex. At 2 weeks, formations of hard tissue, with few cells in the root canals and bone-like tissue in the coronal pulp chamber, were noted. After 3 and 4 weeks, the amounts of these hard tissues were increased. The immunolocalization of OCN, OPN, and BSP was seen strongly in coronal and apical hard tissues, but weakly in the root hard tissue. Conversely, DSP localized in the root hard tissue, but not in other newly formed hard tissues. At 1 week, TNAP localized along the periphery of the apical hard tissue and the lower surfaces of root predentin. These results demonstrate that the newly formed hard tissues in the pulp cavity of subcutaneously transplanted molars could be classified into three types, suggesting that these might be formed by type-specific cells.     

Application of dedifferentiated fat cells for periodontal tissue regeneration

Periodontal diseases result from inflammation by bacterial infection in plaques, leading to tooth loss. However, regenerative approaches with periodontal tissue regeneration by guided tissue regeneration and enamel matrix derivative are not yet well established. Tissue regeneration requires three factors: cells, scaffold, and growth factors. Dedifferentiated fat cells (DFATs) are pluripotent with the same differentiation capacities as mesenchymal stem cells (MSCs). Access to MSCs is limited, whereas donor cells for DFATs are abundant in adipose tissues and can be non-invasively obtained. Therefore, we tested DFATs as a new source for periodontal tissue regeneration in an experimental periodontal tissue loss model in rats by transplanting DFATs on an atelocollagen scaffold using DFATs isolated from Sprague–Dawley (SD) rats expressing green fluorescent protein (GFP). GFP–DFAT cells were transplanted on the palatal side of the upper left first molar in SD rats and detected by H&E staining, GFP, and proliferating cell nuclear antigen (PCNA) expression. DFAT differentiation was also evaluated in three-dimensional cultures. GFP positive cells were detected in the regenerated tissue by the DFATs/scaffold mixture at 4 weeks after transplantation, and PCNA-positive cells were significantly increased in the periodontal ligament along the new bone in the DFATs/scaffold group more than in the scaffold-only group, suggesting that DFATs differentiate in the same manner as MSCs and regenerate in the defective areas. Consistent with previous reports, DFATs differentiation was slower than that with stem cells. The present study demonstrates that DFATs are pluripotent and an effective new source of cells for periodontal tissue regeneration.     

Construction of glomerular epithelial cells expressing both immune tolerance and GFP genes and application to cell therapy by cell transplantation

Cell therapy applied to wound healing or tissue regeneration presents a revolutionary realm to which principles of gene engineering and delivery may be applied. One promising application is the transplantation of cells into the wounded tissue to help the tissue repair. However, when cells are transplanted fromin vitro toin vivo, immune rejection occurs due to the immune response triggered by the activation of T-cell, and the transplanted cells are destroyed by the attack of activated T-cell and lose their function. Immune suppressant such as FK506 is commonly used to suppress immune rejection during transplantation. However, such kind of immune suppressants not only suppresses immune rejection in the periphery of transplanted cells but also suppresses whole immune response system against pathogenic infection. In order to solve this problem, we developed a method to protect the desired cells from immune rejection without impairing whole immune system during cell transplantation. Previously, we reported the success of constructing glomerular epithelial cells for removal of immune complex, in which complement receptor of type 1 (CR1) was over-expressed on the membrane of renal glomerular epithelial cells and could bind immune complex of DNA/anti-DNA-antibody to remove immune complex through phagocytosis [1]. Attempting to apply the CR1-expressing cells to cell therapy and evade immune rejection during cell transplantation, we constructed three plasmids containing genes encoding a soluble fusion protein of cytolytic T lymphocyte associated antigen-4 (CTLA4Ig) and an enhanced green fluorescent protein (EGFP). The plasmids were transfected to the above-mentioned glomerular epithelial cells to express both genes simultaneously. Using the clone cells for cell transplantation showed that mice with autoimmune disease prolonged their life significantly as compared with the control mice, and two injections of the cells at the beginning of two weeks resulted in remarkable survivability, whereas it requires half a year and 50 administrations of proteins purified from the same amount of cells to achieve the same effect.     

Application of autologous periosteal cells for the regeneration of class III furcation defects in Beagle dogs

The aim of this study was to evaluate the healing of class III furcation defects following transplantation of autogenous periosteal cells combined with β-tricalcium phosphate (β-TCP). Periosteal cells obtained from Beagle dogs’ periosteum explant cultures, were inoculated onto the surface of β-TCP. Class III furcation defects were created in the mandibular premolars. Three experimental groups were used to test the defects’ healing: group A, β-TCP seeded with periosteal cells were transplanted into the defects; group B, β-TCP alone was used for defect filling; and group C, the defect was without filling materials. Twelve weeks post surgery, the tissue samples were collected for histology, immunohistology and X-ray examination. It was found that both the length of newly formed periodontal ligament and the area of newly formed alveolar bone in group A, were significantly increased compared with both group B and C. Furthermore, both the proportion of newly formed periodontal ligament and newly formed alveolar bone in group A were much higher than those of group B and C. The quantity of cementum and its percentage in the defects (group A) were also significantly higher than those of group C. These results indicate that autogenous periosteal cells combined with β-TCP application can improve periodontal tissue regeneration in class III furcation defects.