Characterization of mesenchymal progenitor cells in crown and root pulp from human mesiodentes.

OBJECTIVE: Mesiodentes are usually found in the central position of the upper or lower jaw as supernumerary teeth. Here, we obtained 10 mesiodentes and three permanent teeth (PT) and separated the dental pulp (DP) from these into crown and root portions. We then characterized and compared the isolated crown portion-derived cells (crown cells) with root portion-derived cells (root cells) using a range of in vitro assays. MATERIALS AND METHODS: Crown cells and root cells were examined for cell surface marker expression, colony-forming unit-fibroblast (CFU-F), cell proliferation, cell cycle characteristics and markers, and osteogenic and adipogenic differentiation. RESULTS: The proportion of CD105-positive cells (CD105(+) cells) in the crown cells vs the root cells varied among the mesiodentes, but not among the PT. When there were more CD105(+) cells in the root cells than in the crown cells, the root cells showed higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In contrast, when the crown cells contained more CD105(+) cells than the root cells, the crown cells showed the higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In addition, the sorted CD105(+) cells showed higher CFU-F and proliferation capacity than the sorted CD105(-) cells. CONCLUSION: These results indicated that proportion of CD105(+) cells is an effective means of characterizing DP-derived cells in mesiodentes.

Development of collagen fibres and lysyl oxidase expression in the presumptive dermis of chick limb bud.

Lysyl oxidase (LOX) plays a critical role in the formation of cross-linkages in extracellular matrix molecules. Thus, it is essential for the biogenesis and homeostasis of the connective tissue matrix. During development, collagen fibres and elastic system fibres emerge and accumulate in a temporospatial manner in the presumptive dermis of chicks. In this study, we investigated LOX mRNA expression by laser capture microdissection and RT-qPCR and LOX protein localization by immunohistochemistry. The picrosirius polarization method was used to investigate a relation between collagen accumulation and LOX expression. PCR analysis showed that the expression of LOX mRNA in the presumptive dermis became apparent at embryonic day 13 and increased considerably by ED17. Immunohistochemical staining for LOX in the dermis was very low at all stages of development. Accumulation of collagen fibres was seen in the dermis on ED10, and higher wavelengths of birefringence became evident by ED13. Our findings suggest that the temporal pattern of LOX mRNA expression correlates with collagen fibre accumulation in the dermis of the developing chick limb bud, whereas LOX expression was relatively constant at the protein level.

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification.

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Possible participation of isolated epicardial cell clusters in the formation of chick embryonic epicardium.

The embryonic epicardium is formed by the spreading of cells derived from the extracardiac proepicardial organ over the myocardial surface after transfer to the dorsal side of the myocardium via a bridge of villous projections. Using whole-heart immunostaining for keratin, we found that the chronology and pattern of epicardial formation in the chick was basically identical to that reported previously in the quail. However, discrete epicardial islands were observed on the ventrolateral surface of the atrioventricular canal as well as in two previously reported areas. Closer examination by scanning electron microscopy demonstrated the presence of isolated, sparsely distributed epicardial cell clusters on both the dorsal and ventral surfaces of the myocardium. These cells showed a surface morphology similar to that of the epicardial cells at the advancing edge of the spreading epicardial sheet and possessed numerous well-developed filopodia, suggesting active motility. These clusters are probably seeded onto the myocardium by vesicular transport from proepicardial villi, and our findings suggest that the resulting small, localised patches of epicardial cells might accelerate, supplement and tune the epicardial formation mediated by radial spreading of the epicardial sheet in the chick embryonic heart.

Observation of the abluminal surface of the atrioventricular endothelium which undergoes transition into cardiac cushion mesenchymal cells in the chick embryo.

In the developing chick heart, endothelial cells in the atrioventricular canal (AV) undergo a series of morphological changes and transform into cushion mesenchymal cells. In the present scanning electron microscopic study, we examined the abluminal surface features of the AV endothelium through an artificial window in the myocardial wall. The AV endothelial cell at stages 12 or earlier had a smooth, flattened basal surface with only a few blebs. In the successive stages, the abluminal surface exhibited remarkable changes; 1) the number of blebs increased, 2) elongated microvillous projections emerged, and 3) a thick filopodium, or a migratory appendage developed. It appeared, however, that these changes do not occur synchronously within the entire AV endothelium but were initially observed mostly in the proximity of the endothelial "crease" which was a limited invagination of the endothelial sheet towards the underlying acellular matrix. In addition, even in the proximity of the crease, endothelial cells with flattened basal surfaces were also observed next to endothelial cells that showed apparent morphological indications of transition into mesenchymal cells. These findings suggest that AV endothelial cells are possibly heterogeneous in the competency of transformation into mesenchymal cells and such heterogeneity would be important for maintaining the continuity of the AV endothelium.

Endothelial outgrowth and the subsequent cell transition in the culture of embryonic atrioventricular canal placed in an inverted polarity.

Endothelial cells in the atrioventricular (AV) segment of the developing chicken heart undergo a transition into mesenchymal cells. When the AV segment is explanted onto a hydrated collagen gel, endothelial cells grow out and reproduce in vivo cell transition regardless of the precise orientation of the explant on a gel. Our results showed that when the luminal side of an explant was placed towards a gel surface, the inverted polarity of endothelium was not adjusted by direct reorganization of polarity, but that the endothelium crawled down so as to settle on a gel surface in the correct original cell polarity. Subsequently, endothelial cells showed cytoplasmic hypertrophy, formed microvillous projections and then extended filopodial migratory appendages. These cellular changes were quite similar to those in vivo. However, the continuity of the endothelial layer was specifically disrupted in AV explant cultures. Such disruption was never observed in ventricle explant cultures in which endothelial-mesenchymal cell transition did not occur. The disintegration of AV endothelial outgrowth must be closely related to its capability to transform into mesenchymal cells and mitotic activity to keep a depository of endothelial cells.

Visualization of sulfated glycoconjugates in the chicken embryonic heart by whole-mount alcian blue staining.

In the atrioventricular canal (AVC) and outflow tract (OT) of the developing heart, endothelial cells transform specifically to mesenchymal cells. The mesenchymal cells migrate into the underlying acellular matrix termed cardiac jelly and form endocardial cushion tissue. It is believed the that the highly hydrated nature of cardiac jelly is ascribed to sulfated glycoconjugates in the components of jelly matrix. In the present study, we have visualized the distribution and its temporal changes of sulfated glycoconjugates in the embryonic heart from stage 12 to 26 using whole mount alcian blue (AB) histochemistry. Atrial matrix was AB-negative in all the stages examined. Cardiac jelly in the AVC and OT were positive and the staining intensity increased as heart development proceeded, while AB-positive staining in the matrix of the ventricle became negative by stage 19. At stages later than 19, AB-positive matrix was localized in only the AVC and OT where endothelially-derived mesenchymal cells populated.

Immunohistochemical characterization of monoclonal antibodies (PDs) as markers of the periderm in the developing chicken embryo.

The outermost surface cell layer of the developing embryo, the periderm, arises from the initial single layer of ectoderm and is eventually exfoliated from the stratified epidermis, which has the same ectodermal origin. In this study, monoclonal antibodies against chicken limb bud ectoderm were generated and screened for those which stained the periderm. Four separate antibodies termed PD2, 3, 7 and 9 were obtained from 180 mixed hybridomas. These PD antibodies stained the periderm selectively at all stages examined (stage 20-42). By correlating the results of immunohistochemistry with observations made by transmission electron microscopy, it was revealed that PD antibodies stained both the squamous periderm at an early stage and rounded bulging peridermal cells just before exfoliation. Therefore we feel that PD antibodies may be useful in further systematic investigations of the development and function of the chicken embryonic periderm.

The influence of scattering on temporo-regional proliferation in the cultured Madin-Darby canine kidney (MDCK) cells.

An epithelial Madin-Darby canine kidney (MDCK) cell line was grown in two kinds of standard media and a medium conditioned by MRC-5 embryonic lung cells, and temporo-regional differences in cell proliferation were examined by immunohistochemical detection of incorporated bromodeoxyuridine (BrdU). MRC-5-conditioned medium is known to contain scatter factor, which induces scattering of MDCK cells. In a proliferation assay, the BrdU labeling index (percentage ratio of BrdU-positive to total nuclei) was highest in day 2 cultures in the standard media, but on day 3 in the conditioned medium. MDCK cells in the standard media formed multiple epithelial sheets by day 2. In the conditioned medium, however, epithelial sheet formation was retarded and observed only after day 3. Once epithelial sheets had formed, cells close to the edge of the sheet were more proliferative than those distant from the edge in both standard and conditioned media. These findings suggest that proliferation of MDCK cells is suppressed during the scattering induced by the conditioned medium, and that their DNA synthesis becomes most active when cell scattering has ceased and epithelial sheets have begun to form.

Transformation of cardiac endothelium into cushion mesenchyme is dependent on ES/130: temporal, spatial, and functional studies in the early chick embryo.

ES/130 is a novel 130-kDa protein that has been linked previously to the transformation of endocardial endothelium into cushion mesenchyme. In the present study we report the localization of protein and mRNA for ES/130 in stages 7-plus through 20 chick embryos and present functional data related to a potential mechanism for ES/130. The temporal and spatial regulation of ES/130 expression suggests that this epithelial-to-mesenchymal transformation is a result of homogenetic induction. Functional studies indicate that myocardially derived ES/130 elicits expression of this protein by target AV endothelial cells, which is linked to a signal transduction cascade. The localization of ES/130 to other sites of inductive interactions (e.g., limb bud ectoderm, gut, and notochord) implies that this protein may have a more widespread importance to embryogenesis beyond its involvement in cardiac cushion tissue formation.

Production and characterization of an anti-chicken transferrin.

Several lines of recent evidence have suggested that transferrin plays a significant role in tissue interaction or morphogenesis at early stages of embryo development. In the present study, an anti-chicken transferrin antibody was produced and its basic characteristics were clarified as a basis for use in further studies. An antiserum termed Toraji 3 was raised against chicken transferrin and purified into IgG and ligand-affinity-purified fractions. These three preparations of the antibody gave an intense immunohistochemical signal in visceral yolk sac and developing liver, both of which are known to be major producers of transferrin in early development. In immunoblot analysis, these three preparations detected 70-kDa transferrin, whereas the ligand-affinity-purified preparation showed higher specificity. It was also demonstrated by enzyme-linked immunosorbent assay and immunoblotting that Toraji 3 antibody bound preferentially to chicken transferrin and showed a negligible binding to human transferrin.

Identification of transferrin as one of multiple EDTA-extractable extracellular proteins involved in early chick heart morphogenesis.

It was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial-mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive interactions, e.g., developing heart, limb buds, and neural crest forming region [Krug et al., 1987, Dev Biol 120:348-355; Mjaatvedt et al., 1991, Dev Biol 145:219-230). In the present study, by using an antiserum (ES3) to a similar immunogen, we affinity purified four major EDTA-soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS-PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): 1) amino acid sequencing showed that eight N-terminal residues were identical to those of chicken transferrin, 2) acid hydrolysates of both proteins had nearly identical compositions, 3) the protein co-migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and 4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin demonstrated that anti-transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial-mesenchymal formation. However, both cardiac endothelium and migrating mesenchymal cells became immunoreactive with anti-transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial-mesenchymal transformation.

Identification of an extracellular 130-kDa protein involved in early cardiac morphogenesis.

Previous studies indicate that the transformation of cardiac endothelium into mesenchyme is dependent upon a developmentally regulated signal expressed by its associated myocardium. This process can be mimicked in culture by substituting a non-cytolytic EDTA extract of embryonic heart tissue for the presence of myocardium. Polyclonal antibodies (ES1) generated against the EDTA-extractable proteins both localized to the cardiac extracellular matrix preceding the transformation of endothelium and blocked this process in culture. Based on these observations, we hypothesized that ES1 antigens participate in the formation of cardiac mesenchyme. The present study was undertaken to prepare cDNA and antibody probes for individual ES1 antigens to better characterize their involvement in this important morphogenetic event. An expression library was constructed in Uni-ZAP using poly(A+) RNA from embryonic cardiocyte cultures that had been shown previously to secrete proteins that engender the formation of cardiac mesenchyme. Screening of this expression library with ES1 antibodies resulted in several clones, one of which ("ES1-2.1a") is described in this report. ES1-2.1a has a 2.6-kilobase pair insert, the sequence of which exhibits no apparent homology to those in data banks. A fragment (852 base pairs) from the 5' region of ES1-2.1a cDNA was subcloned into the expression vector pGEX-2T, and a 20-kDa fragment of the resulting protein used to prepare affinity-purified antibodies. Immunoblotting detected a 130-kDa protein ("ES/130") in two preparations that elicit mesenchyme formation, i.e. EDTA extracts of embryonic hearts and conditioned medium of cardiocyte cultures. Functional studies showed that antibodies to ES/130 inhibited the epithelial-mesenchymal transformation of cardiac endothelium in culture. Immunohistochemistry of cardiocyte cultures localized ES/130 protein to the vacuolar system and secretory granules. By polymerase chain reaction analysis, the message for ES/130 was detected in the developing heart just prior to and during mesenchyme formation. These results are consistent with ES/130 being involved at a critical step in the initiation of the epithelial-mesenchymal transformation of cardiac endothelium.

Leg bud mesoderm retains morphogenetic potential to express limb-like characteristics ("limbness") in collagen gel culture.

Recent in situ hybridization studies have correlated expression of potential regulatory genes with pattern formation in limb bud mesoderm (Tabin: Cell 66:199-217, 1991); however, the mechanism(s) controlling their expression in mesoderm and their relevance to the establishment of a limb morphogenetic pattern remain unknown. One likely candidate for regulating patterning events in limb mesoderm is the apical ectodermal ridge, as its removal in ovo results in a graded truncation of limb skeletal elements in the proximal-distal axis dependent upon the time of excision (Rowe and Fallon: J Embryol Exp Morph 68:1-7, 1982). In the present study, we investigate whether the hypothetical imprint of ridge ectoderm is retained in cultured mesoderm. Specifically, we sought to determine if a subpopulation of limb mesoderm that forms in collagen gel culture (Markwald et al: Anat Rec 226:91-107, 1990), retains any expression of "limbness" in the absence of limb ectoderm as characterized by the formation of a predictable number and distribution of limb-like chondrogenic elements in comparison to the temporal and spatial relationships of the in situ proximal, hindlimb skeletal structures. Accordingly, explants of undissociated mesoderm from stage 18-22 chicken leg buds were cultured without ectoderm on collagen gel lattices and the central subpopulation of mesoderm was examined morphologically. We show that this central subset of mesoderm will form chondrogenic cells which were not expressed uniformly throughout the subset, but rather distinct nodules or elements of cartilage were elaborated. Moreover, the number of elements expressed by the central subset increased with the age of the mesoderm at the time of explantation; spatially and temporally, the sequence of elements that formed always proceeded from the proximal, anterior margin of the subset to its distal, posterior border. The shapes of the initial elements (designated I and II) resembled the forms of in situ proximal skeletal structures (girdle and femur-like), whereas more distal elements (III-V) were often fused and without structural similarity to in situ skeletal structures. When cultures were established from the posterior mesoderm of stage 19/20 or 21 mesoblasts, the frequency of element I formation was reduced approximately one-half, whereas formation of more distal elements was unaffected. Conversely, element formation from the central subset established from isolated anterior mesoderm was virtually identical to intact mesoblasts, indicating a capacity to regulate for the loss of mesoderm as occurs in situ (Hampé: Archs Anat Microsc Morph Exp 48:345-378, 1959).(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochemical detection of disulfide and sulfhydryl groups in lamprey aortic microfibrils.

A cytochemical study was performed on the lamprey ventral aorta with special reference to disulfide and sulfhydryl groups of microfibrils, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method combined with several other types of treatment. The HID-TCH-SP staining observed was classified into three categories: 1) weak staining in the periphery of collagen fibrils, 2) moderate staining in the boundaries of collagen fibrils, microfibrils and smooth muscle cells, and 3) intense staining of microfibrils. The first and second categories of staining were considered to represent chondroitin and/or heparan sulfate because of sensitivity of the staining to chondroitinase ABC (ChABC) and its specific localization. By contrast, the third category of staining was considered to represent disulfide and sulfhydryl groups of microfibrillar glycoprotein, because it was disclosed only after Oxone oxidation or thiosulfation and was not removed by ChABC digestion. Although this staining reactivity was not apparently altered by SH blockade prior to oxidation or thiosulfation, it was markedly diminished or completely inhibited by S-S reduction followed by SH blockade. These results indicate that lamprey aortic microfibrils contain more S-S groups than SH groups.

Ultrastructural and cytochemical study of elastic fibers in the ventral aorta of a teleost, Anguilla japonica.

Previous studies have revealed that amorphous elastin and microfibrils are structural entities of mammalian elastic fibers. Elastin shows a wide phylogenetic distribution, but the presence of elastin-associated microfibrils has not been demonstrated in teleost aorta. Thus, we have ultrastructurally and cytochemically examined elastic fibers in the ventral aorta of eel, a teleost, by utilizing routine uranyl acetate and lead double staining, the tannic acid (pH 7.0)-uranyl acetate (TA-UA) method, elastase en bloc digestion, Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, and the horseradish-peroxidase-labeled concanavalin A (Con A) method. In the ventral aorta of eel, a little ultrastructural difference between elastic fibers in the intima and media and those in the adventitia was noticed, but in either tunic each elastic fiber was basically composed of a "fibrillar core" and surrounding microfibrils. The fibrillar core was a collection of fibrils which showed a tendency to coalesce with each other, and these constituent fibrils were TA-UA positive and elastase-sensitive, representing their nature of elastin. By contrast, microfibrils associated with the fibrillar core were TA-UA negative and elastase-resistant, and their glycoproteinaceous nature was demonstrated by PA-TCH-SP and Con A methods. Thus, this study provides evidence for the presence of elastin-associated microfibrils in teleost aorta. These results are discussed in relation to the topographical difference of elastic fibers in eel aortic wall.

Ultrastructural cytochemistry of aortic microfibrils in the Arctic lamprey, Lampetra japonica.

In the ventral aorta of lamprey, microfibrils are major components of the extracellular matrix. With special reference to these microfibrils, we have cytochemically examined the lamprey ventral aorta, utilizing the tannic acid (pH 7.0)-uranyl acetate (TA-UA) method, elastase en bloc digestion, Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, and ferritin- or horseradish peroxidase-labeled concanavalin A (Con A) methods. The lamprey microfibrils were strongly stained with PA-TCH-SP and both Con A methods, but did not show TA-UA staining nor elastase sensitivity. These cytochemical properties of lamprey microfibrils are identical with those of mammalian elastin-associated microfibrils. On the other hand, in spite of extensive examination, TA-UA positive and elastase-sensitive extracellular components were not found, so that lamprey ventral aorta does not appear to contain elastin. These results indicate that lamprey aortic connective tissue contains microfibrils as elastic components, but deposition of amorphous elastin does not occur.

Ultrastructural cytochemistry of trout arterial fibrils as elastic components.

The trout arterial wall contains numerous extracellular fibrils that are presumed to be elastic. However, the cytochemical properties of the arterial fibrils have not been studied. Thus, we have ultrastructurally and cytochemically examined these fibrils, utilizing routine uranyl acetate and lead (UA-Pb) double staining, the tannic acid (pH 7.0)-uranyl acetate (TA-UA) method as an electron-dense staining for elastin, and Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method to localize vicinal glycol-containing complex carbohydrates. The arterial fibrils, about 23 nm in thickness, were interwoven at random but frequently showed the circular alignment to the long axis of the aorta. Occasionally they appeared to coalesce side by side, and the coalesced portion tended to lose its affinity for UA-Pb stains. The TA-UA method stained the fibrils moderately to intensely and stained the coalesced parts of the fibrils more intensely. All of those TA-UA positive fibrils were completely removed after elastase en bloc digestion. The PA-TCH-SP method did not stain the arterial fibrils but stained another kind of much thinner interfibrillar filamentous structure. These results suggest that the fibrils in the wall of trout ventral aorta are elastin in nature and do not contain vicinal glycols, although the fibrils usually exist in a fibrillar form, which is unlike mammalian amorphous elastin.