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1.
Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25-26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20-21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro. 相似文献
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R E Seegmiller 《Developmental biology》1977,58(1):164-173
Exposure of the chick embryo to the nicotinamide analog, 6-aminonicotinamide (6-AN), causes specific changes in chondrogenic cells that result in limb deformity. Autoradiography has further delineated these changes and relates them to altered utilization of molecular precursors of cartilage matrix and DNA. With 35SO4 to monitor synthesis of glycosaminoglycan, it was shown that, at 6 hr and persisting until 24 hr after treatment, 6-AN inhibited utilization of sulfate by cells in the chondrogenic core while having no detectable effect on cells in the chondrogenic periphery. Similarly, 6-AN suppressed incorporation of [3H]thymidine into core cells while having no effect to a slight enhancement effect on chondrogenic and nonchondrogenic cells surrounding the core. These observations support the view that, in response to 6-AN-inhibited NAD(P)-dependent reactions, limb chondrogenic cells (CORE) cease to produce matrix glycosaminoglycan, cease to synthesize DNA, and ultimately succumb. Conversely, presumably as a result of more efficient energy production because they lie closer to a vascular supply of oxygen, cells in the chondrogenic periphery withstand the teratogenic insult and continue proliferating to become the source where subsequent partial repair of the limb occurs. 相似文献
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When limb bud mesodermal cells of stages 23–24 chick embryos were plated at low cell density (2 × 105 cells/cm2) and cultured in medium containing 10% fetal calf serum (FCS) (serum-rich medium), all cells became fibroblastic and no chondrocyte differentiation occurred in the culture. However, when cells of the same origin were cultured in a medium containing only 0.1% FCS (serum-poor medium), almost all the cells formed aggregates which developed further to form cartilage nodules. The loss of chondrogenic activity in serum-rich medium culture was irreversible: cultivation of the limb bud cells in serum-rich medium for 12 h abolished chondrogenic activity completely and these cells could not resume activity on re-cultivation in serum-poor medium. Calf, horse and chick serum at a concentration of 10% also induced the loss of chondrogenic activity in low cell density culture. Failure of chondrogenesis in serum-rich medium culture seemed to be due to the commitment of bipotential limb bud mesodermal cells to fibroblastic cells rather than to selective detachment of pre-committed chondroblasts. 相似文献
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The relationship of nicotinamide adenine dinucleotide to the chondrogenic differentiation of limb mesenchymal cells 总被引:1,自引:0,他引:1
The nicotinamide adenine dinucleotide (NAD) content of mesenchymal cells from the embryonic chick limb has been hypothesized to control the differentiation of these cells by modulation of ADP-ribosylations. To test this hypothesis, [35S]sulfate incorporation into proteoglycans was monitored as an estimate of the chondrogenic expression of cultured limb mesenchymal cells treated with nicotinamide and nicotinic acid to elevate cellular NAD levels or with nicotinamide and benzamide compounds to inhibit ADP-ribosylations. The results of this study indicated that serum component(s) modulate the interactions between these chemical agents and limb mesenchymal cells and, thus, complicate the interpretations of experiments performed in the presence of serum. With a chemically defined medium that promotes limb mesenchymal cell proliferation and differentiation in vitro, it was demonstrated that: (1) no clear correlation exists between cellular NAD content and the chondrogenic expression of cultured limb mesenchymal cells, (2) nicotinamide and benzamide compounds reduce cell proliferation and, at the higher doses tested, considerably reduce chondrogenesis in limb mesenchymal cell cultures, and (3) limb mesenchymal cells exhibit an enhanced susceptibility to benzamide compounds at a time very early in the culture period which temporally coincides with a transient increase in cellular ADP-ribosylation activity and initial chondrogenic differentiation. These results suggest that NAD does not control the differentiation of limb mesenchymal cells and that ADP-ribosylations are an integral, though not controlling, component of limb mesenchyme cytodifferentiation. A model is presented which proposes a role for ADP-ribosylations during the differentiation of limb mesenchymal cells. 相似文献
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Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures 总被引:13,自引:0,他引:13
When chick limb bud mesenchyme cells from stage 23 to 24 embryos are plated at high density, they rapidly divide and a large proportion initiate chondrogenic expression during the first 2 to 3 days in culture. Between Days 4 and 8, the emergent chondrocytes mature and elaborate a cartilaginous matrix. The proteoglycans synthesized by the newly emergent Day 3 to 4 chondrocytes differ from those synthesized by either the prechondrogenic mesenchyme cells or the mature Day 8 chondrocytes. Cultures were grown from initial plating (Day 0) or from Day 2 in the continuous presence of 1 mM 4-methyl umbelliferyl-beta-D-xyloside, which acts intracellularly as a competitive acceptor with the endogenous core protein of proteoglycans for chondroitin sulfate synthesis. The proteoglycans synthesized by Day 8 cultures which had been maintained on xyloside or to which xyloside was added only 1 h prior to labeling were essentially identical. They were able to form aggregates, and they contained the same number of keratan sulfate chains, but only about 40% as many chondroitin sulfate chains, as normal. Additionally, both the chondroitin sulfate and keratan sulfate chains were 25% shorter than in the normal proteoglycans. The proteoglycans synthesized by cells in a culture maintained on xyloside until Day 8, and then switched to medium with no xyloside 1 h prior to labeling, were characteristic of those synthesized by normal mature Day 8 chondrocytes. These data suggest that stage 23 to 24 mesenchyme cells undergo normal chondrogenic maturation in culture in the presence of xylosides even though (a) most of the polysaccharides are synthesized onto the exogenously supplied xyloside substrate and released into the medium, (b) the proteoglycans that are synthesized are greatly reduced in polysaccharide content, and (c) the extracellular matrix as a consequence is greatly depleted in chondroitin sulfate content and, therefore, is abnormal in general morphology. 相似文献
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Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations. 相似文献
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Nicotinamide adenine dinucleotide levels in cells of developing chick limbs: possible control of muscle and cartilage development 总被引:2,自引:0,他引:2
The studies reported here show that NAD+ levels are low in chick limbs which have not yet attained the stage of cellular commitment, that these low levels persist during a time period when major chondrogenic commitment and expression occur, that beyond this stage the NAD+ levels in chick limbs rise dramatically and continuously, corresponding to the period of major myogenic development, and that developing cultures of stage 24 mesodermal cells seem to mimic these in vivo events in that myogenic cells are observed when NAD+ levels are high and chondrogenic cells are observed when NAD+ levels are low. These observations are consistent with the hypothesis that pyridine nucleotides may play some role in the control of muscle and cartilage development in embryonic chick limbs. 相似文献
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The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation. 相似文献
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Demineralized adult bone contains factors which stimulate nonskeletal mesenchymal cells to undergo a developmental progression resulting in de novo endochondral ossification. In this study, isolated embryonic stage 24 chick limb bud mesenchymal cells maintained in culture were utilized as an in vitro assay system for detection of specific bioactive components solubilized from adult chicken bone matrix. Guanidinium chloride extracts (4 M) of demineralized-defatted bone were fractionated and tested in limb mesenchymal cell cultures for possible effects upon growth and chondrogenesis. Two low-molecular-weight fractions were found to be active in these cultures. A cold water-insoluble, but warm Trisbuffered saline-soluble fraction provoked a dose-dependent increase in the amount of cartilage formed after 7 days of continuous exposure as evidenced by an increased number of chondrocytes observed in living cultures, elevated cell-layer-associated 35S incorporation per microgram DNA, and greater numbers of toluidine blue-staining foci (i.e., cartilage nodules). Growth inhibitory substances were detected in a low-molecular-weight, water-soluble fraction; 7 days of continuous exposure to this material resulted in less cartilage formation and reduced cell numbers (accumulated DNA) on each plate. These observations demonstrate the usefulness of stage 24 chick limb bud cell cultures for identifying bioactive factors extracted from adult bone matrix. In addition, the action of these factors on mesenchymal cells may now be studied in a cell culture system. 相似文献
11.
Previous studies showed that cultures of chick limb bud mesenchymal cells plated at high density, to maximize chondrogenic expression, had a much reduced extracellular matrix around chondrocytes when exposed to 4-methyl-, umbelliferyl-β-d-xyloside. The majority of newly synthesized chondroitin sulfate chains were found in the culture medium presumably bound to the xyloside as opposed to their normal deposition on the core protein of proteoglycan. The question remained open as to whether the development of an abnormal matrix affected the synthesis of extracellular deposition of other cartilage-specific macromolecules. We have analyzed, both morphologically and biochemically, the synthesis and deposition of Type I and Type II collagen by β-d-xyloside-treated cultures of limb mesenchymal cells. While the rate of collagen synthesis per plate and its extracellular accumulation after 8 days in culture were reduced to some extent, the ratios of Type II to Type I collagen and the morphological distribution of these macromolecules were not affected by exposure to β-d-xyloside. We conclude that the expression of the cartilage-specific Type II collagen during chondrogenic differentiation is, although reduced, qualitatively not dependent on the amount of extracellular chondroitin sulfate chains attached to matrix-associated proteoglycan core protein. However, prolonged exposure of limb bud cells to xylosides leads to the formation of a chondroitin sulfate- and collagen-deficient matrix which, in turn, reduces the capacity of limb bud cells to synthesize Types I and II collagen. 相似文献
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An in vitro Analogue of Early Chick Limb Bud Outgrowth 总被引:2,自引:0,他引:2
Our culture system appears to represent an in vitro analogue of early chick limb morphogenesis. Organized mesodermal cell accumulations resembling limb buds were derived from a monolayer of limb mesoderm cells when covered by limb ectoderm which included the apical ectodermal ridge (AER). The ridge retained its normal configuration when grown over a limb mesoderm monolayer and the mesoderm cells accumulated under the ridge to form a multilayered structure (10–25 cells in thickness) with the characteristic shape of a limb bud. Ectoderm which did not include the ridge failed to promote the formation of limb-like mesodermal accumulations thus the action of the ridge appears to be specific. The AER-elicited expression of mesodermal cell behaviour leading to early limb outgrowth is discussed in terms of possible morphogenetic mechanisms involved i.e. differential mitosis, cell migration, changes in cell shape and especially the adhesive properties of the cells. 相似文献
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Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction
of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells
differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation
events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up
to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic
stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For
example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated
expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular
therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the
transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective
enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These
cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes.
However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating
that chondrocytes derived from ES cells posses a distinct differentiation plasticity.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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AMDM, a form of osteochondrodysplasia, is due to the loss-of-function mutations in NPR-B gene. This study investigated the functional involvement of CNP-3, chick homolog of human CNP, and its receptor NPR-B in chondrogenesis utilizing the micromass culture of the chick limb mesenchymal cells. Results revealed CNP-3 and NPR-B expression in the chick limb bud making stage-specific peak levels first at Hamburger-Hamilton stage 23-24, and second at stage 30-31, corresponding to pre-chondrogenic mesenchymal condensation and initiation of chondrogenic maturation-hypertrophy in vivo, respectively. CNP-3 and NPR-B expression in vitro increased parallel to collagen type X expression, but not to that of collagen type II. Treatment of cultures with CNP significantly increased N-cadherin, and collagen type X expression, glycosaminoglycan synthesis and chondrogenesis. Collagen type II expression was not significantly affected. Thus, results implicated CNP-3/NPR-B signaling in pre-chondrogenic mesenchymal condensation, glycosaminoglycan synthesis and late differentiation of chondrocytes in the process of endochondral ossification. 相似文献
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Hidetoshi Sakurai Yasuko Sakaguchi Emi Shoji Tokiko Nishino Izumi Maki Hiroshi Sakai Kazunori Hanaoka Akira Kakizuka Atsuko Sehara-Fujisawa 《PloS one》2012,7(10)
Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α+/Flk-1− population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α+/KDR− population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α+/KDR− population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle. 相似文献
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Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes 总被引:7,自引:0,他引:7
M Solursh K L Jensen R S Reiter T M Schmid T F Linsenmayer 《Developmental biology》1986,117(1):90-101
We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Isolation and characterization of chondrocytes and non-chondrocytes from high-density chick limb bud cell cultures 总被引:1,自引:0,他引:1
D P Lennon P Osdoby D A Carrino B M Vertel A I Caplan 《Journal of craniofacial genetics and developmental biology》1983,3(3):235-251
This communication describes a replating technique for the separation of the chondrogenic and non-chondrogenic cells from stage-24 chick limb bud mesenchymal cell cultures by means of sequential digestion with collagenase. Four sub-populations of cells were obtained: The first consisted solely of non-chondrocytes and the next three were progressively enriched in chondrocytes. In addition to morphological differences, the four cell populations differed from each other in their rates of incorporation of sulfate into macromolecular material which were roughly proportional to the percentage of chondrocytes. The chondrocytes and non-chondrocytes no longer exhibited a density dependence of phenotype. In addition, the normal multilayered nodular morphology associated with cartilage development was not observed. These isolated cells have been used as starting material for detailed biochemical studies. Together, these studies indicate that the expressional program governing biosynthetic changes in chondrocytes is not controlled by the extracellular matrix. 相似文献