Chondrogenesis In Micromass Cultures Research Articles

Tbx4 function during hindlimb development reveals a novel mechanism to explain the origins of proximal limb defects

ABSTRACTWe dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects.

Deer thymosin beta 10 functions as a novel factor for angiogenesis and chondrogenesis during antler growth and regeneration

BackgroundDeer antlers are the only known mammalian organ with vascularized cartilage that can completely regenerate. Antlers are of real significance as a model of mammalian stem cell-based regeneration with particular relevance to the fields of chondrogenesis, angiogenesis, and regenerative medicine. Recent research found that thymosin beta 10 (TMSB10) is highly expressed in the growth centers of growing antlers. The present study reports here the expression, functions, and molecular interactions of deer TMSB10.MethodsThe TMSB10 expression level in both tissue and cells in the antler growth center was measured. The effects of both exogenous (synthetic protein) and endogenous deer TMSB10 (lentivirus-based overexpression) on antlerogenic periosteal cells (APCs; nonactivated antler stem cells with no basal expression of TMSB10) and human umbilical vein endothelial cells (HUVECs; endothelial cells with no basal expression of TMSB10) were evaluated to determine whether TMSB10 functions on chondrogenesis and angiogenesis. Differences in deer and human TMSB10 in angiogenesis and molecular structure were determined using animal models and molecular dynamics simulation, respectively. The molecular mechanisms underlying deer TMSB10 in promoting angiogenesis were also explored.ResultsDeer TMSB10 was identified as a novel proangiogenic factor both in vitro and in vivo. Immunohistochemistry revealed that TMSB10 was widely expressed in the antler growth center in situ, with the highest expression in the reserve mesenchyme, precartilage, and transitional zones. Western blot analysis using deer cell lines further supports this result. Both exogenous and endogenous deer TMSB10 significantly decreased proliferation of APCs (P < 0.05), while increasing the proliferation of HUVECs (P < 0.05). Moreover, deer TMSB10 enhanced chondrogenesis in micromass cultures and nerve growth as assessed using a dorsal root ganglion model (P < 0.05). Deer TMSB10 was proangiogenic using models of chicken chorioallantoic membrane, tube formation, and aortic arch assay. At the molecular level, endogenous deer TMSB10 elevated the expression of vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C, and VEGF-D, and VEGFR2 and VEGFR3 in HUVECs (P < 0.05).ConclusionsDeer TMSB10, in contrast to its human counterpart, was identified as a novel stimulating factor for angiogenesis, cartilage formation, and nerve growth, which is understandable given that deer antlers (as the arguably fastest mammalian growing tissue) may require this extra boost during a period of rapid growth and regeneration.

Rac1 promotes chondrogenesis by regulating STAT3 signaling pathway.

The small GTPase protein Rac1 is involved in a wide range of biological processes including cell differentiation. Previously, Rac1 was shown to promote chondrogenesis in micromass cultures of limb mesenchyme. However, the pathways mediating Rac1's role in chondrogenesis are not fully understood. This study aimed to explore the molecular mechanisms by which Rac1 regulates chondrogenic differentiation. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased as chondrogenesis proceeded in micromass cultures of chick wing bud mesenchyme. Inhibition of Rac1 with NSC23766, janus kinase 2 (JAK2) with AG490, or STAT3 with stattic inhibited chondrogenesis and reduced phosphorylation of STAT3. Conversely, overexpression of constitutively active Rac1 (Rac L61) increased phosphorylation of STAT3. Rac L61 expression resulted in increased expression of interleukin 6 (IL-6), and treatment with IL-6 increased phosphorylation of STAT3. NSC23766, AG490, and stattic prohibited cell aggregation, whereas expression of Rac L61 increased cell aggregation, which was reduced by stattic treatment. Our studies indicate that Rac1 induces STAT3 activation through expression and action of IL-6. Overexpression of Rac L61 increased expression of bone morphogenic protein 4 (BMP4). BMP4 promoted chondrogenesis, which was inhibited by K02288, an activin receptor-like kinase-2 inhibitor, and increased phosphorylation of p38 MAP kinase. Overexpression of Rac L61 also increased phosphorylation of p38 MAPK, which was reduced by K02288. These results suggest that Rac1 activates STAT3 by expression of IL-6, which in turn increases expression and activity of BMP4, leading to the promotion of chondrogenesis.

Role of c-Myb in chondrogenesis

The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5–E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation.

Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis

Wnt factors are involved in the regulation of all steps of cartilage development. The activity of Wnt factors is generally regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus and Wnt inhibitory factor 1 (Wif-1). Here we report that Wif-1 is highly expressed at cartilage-mesenchyme interfaces of the early developing skeleton. In fetal and postnatal skeletal development, Wif-1 is expressed in a sharply restricted zone in the upper hyaline layer of epiphyseal and articular cartilage and in trabecular bone. Coimmunoprecipitation and pull-down assays using recombinant Wif-1 and Wnt factors show specific binding of Wif-1 to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11. Moreover, Wif-1 was able to block Wnt3a-mediated activation of the canonical Wnt signalling pathway. Consequently, Wif-1 impaired growth of mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of chondrogenesis in micromass cultures of embryonic chick limb-bud cells. These results identify Wif-1 as a novel extracellular Wnt modulator in cartilage biology.

Wnt-5a is involved in TGF-β3-stimulated chondrogenic differentiation of chick wing bud mesenchymal cells

In this study we investigated whether signalling by TGF-β3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-β3 (TGF-β3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-β3. Overexpression of Wnt-5a or treatment with TGF-β3 stimulated the activation of protein kinase C-α (PKC-α) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-α and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-β3 and Wnt-5a. Similarly, partial reduction in TGF-β3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-α and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-β3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-β3 through upregulation of PKC-α and p38MAPK signaling.

Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro

Cartilage formation in the chick limb follows rapid proliferation, condensation and differentiation of limb mesenchyme. The control of these early events is poorly understood. Platelet-derived growth factor receptor alpha (PDGFR- α) is present throughout the mesenchyme of early chick limb buds, while its ligand, PDGF-A, is expressed in the surrounding epithelium. PDGFR- α is down-regulated in areas that will not give rise to cartilage and is then lost from cartilage forming areas after they begin to differentiate. PDGF-A increases chondrogenesis in micromass cultures of stage-20–24 limb buds, but not stage 25, where it inhibits chondrogenesis. Ectopic PDGF-A in the chick wing can lead to either a localized increase in cartilage formation, or an inhibition. Inhibition of PDGF signalling in the chick limb results in the loss of cartilage. These data demonstrate that PDGF-A functions to promote chondrogenesis at early stages of limb development and suggest that it inhibits chondrogenesis at later stages.

Sox9 Expression during Chondrogenesis in Micromass Cultures of Embryonic Limb Mesenchyme

Sox9 plays a crucial role in chondrogenesis. It encodes an HMG-domain transcription factor that activates an enhancer in the gene for type II collagen (Col2a1), a principal cartilage matrix protein. We have characterized the temporal pattern of Sox9 RNA expression in micromass culture, a widely used in vitro model for the analysis of embryonic cartilage differentiation. Cultures were prepared from distal subridge mesenchyme of the stage 24/25 chick embryo wing bud, which undergoes uniform chondrogenic differentiation in vitro. The early “prechondrogenic” phase of culture was characterized by the activation of Sox9 RNA expression, which preceded detectable upregulation of Col2a1 transcription. Sox9 RNA levels peaked between 20 and 65 h of culture, a phase of progressive Col2a1 transcript accumulation, then declined in the mature cartilage of 120-h cultures. Staurosporine treatment enhanced chondrogenesis in micromass culture by inducing a rapid quantitative increase in Sox9 transcript levels. However, PMA, a phorbol ester that inhibits Col2a1 expression and chondrocyte differentiation, had an unexpectedly modest effect on Sox9 RNA accumulation.

Changes in adenylate cyclase activity during chondrogenesis in cultured limb mesenchyme

An in vitro assay to examine the effects of forskolin on adenylate cyclase (AC) activity and chondrogenesis in micromass cultures of mesenchymal cells derived from the distal tip of stage 25 chick limb buds is described. Forskolin activation of AC increased 6 fold in prechondrogenic cells cultured for 48 hours relative to uncultured cells at the time of cell dissociation (Day 0) and remained elevated and unchanged in differentiated cells during the remaining four days of culture. Equivalent increases occurred in cyclic AMP (cAMP) concentrations over the six day period examined. Forskolin also accelerated chondrogenesis in these cultures. Quantitative analysis of Alcian green binding to cartilage matrix showed approximately 2 fold increases in forskolin-treated cultures relative to controls on each of days 2–4 of culture. These results demonstrate that dramatic increases in the total pool of AC activity occur in prechondrogenic mesenchyme prior to overt chondrogenesis which, through increases in cAMP, may play an important role in cartilage differentiation.

Characterization of chondrogenesis in cells isolated from limb buds in mouse

Micromass cultures of mesenchymal cells isolated from limb buds of 11.5-day-old mouse fetuses were used to study chondrogenesis. After 3 days of culture, dense cell aggregates were observed. They then were converted into macroscopically visible cartilage foci during the following 2-4 days. Comparison of 2-, 4- and 7-day-old cultures has shown that the cells first expressed collagen type I, then switched to collagen type II expression as shown by immunohistochemistry and in situ hybridization. At day 7, proteoglycans were synthesized centrally in the foci. At the same time, most cells expressed collagen type II, with the highest expression in the periphery of the aggregates. The oncogene c-fos and homeodomain protein FS-1 were found in the cells expressing collagen type II, indicating that these transcription factors may be involved in the regulation of cell differentiation. The expression of alkaline phosphatase was detected first in mature cartilage foci (day 4) and increased during culture. Early in culture, DNA-replicating cells were uniformly distributed. With differentiation, the proliferating cells were present predominantly between the aggregates and their total number became significantly reduced. Our results indicate that the process of chondrogenesis in micromass cultures of mesenchymal cells mimics the differentiation process occurring during fetal development in vivo and can be directly studied by in situ hybridization, immunohistochemical and histochemical methods.

Factors modulating chondrogenesis in micromass cultures from developing chick limbs

Factors modulating chondrogenesis in micromass cultures from developing chick limbs

Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis.

Cell adhesion molecules have been shown to be important mediators of morphogenesis and pattern formation. In this study, we have shown that N-cadherin is expressed in a specific spatiotemporal manner in the developing limb bud during chondrogenesis in vivo and in cultured limb mesenchyme in vitro. The time period of maximal expression of N-cadherin corresponds to the period of active cellular condensation, an event believed to be a necessary prerequisite for chondrogenic differentiation. To directly assess the functional involvement of N-cadherin in cellular condensation, we have examined the effects of perturbing N-cadherin activity on both cell aggregation and chondrogenesis using NCD-2, a rat monoclonal antibody directed against the binding region of N-cadherin. Non-immune rat IgG was used as a control. Our results show that functional N-cadherin is necessary for chondrogenesis to proceed both in vivo and in vitro. Limb mesenchymal cells exhibited characteristic Ca(2+)-dependent cell aggregation in suspension, which was inhibited in the presence of exogenous NCD-2. In micromass cultures of limb mesenchymal cells, NCD-2 inhibited overt chondrogenesis in a dose-dependent manner. Furthermore, NCD-2 inhibition of chondrogenesis in micromass cultures was time-dependent, suggesting that N-cadherin is crucially involved during the latter half of the first 24 hours of culture, a time period most likely corresponding to active cellular condensation. NCD-2 also significantly influenced limb development when injected into embryonic limb buds in vivo. In addition to significant inhibition of chondrogenesis and developmental delays, gross developmental deformities and perturbation of overall pattern formation were also observed. Taken together, these results demonstrate that N-cadherin is functionally required in mediating the cell-cell interactions among mesenchymal cells important for chondrogenesis in micromass culture in vitro and in the intact limb bud in vivo.

Ectodermal inhibition of cartilage differentiation in micromass culture of chick limb bud mesenchyme in relation to gene expression and cell shape.

Ectoderm inhibits the formation of cartilage by chick wing bud mesenchyme in micromass culture. This suggests that the pattern of cartilage formation in the limb bud may result from a restriction of cartilage cell differentiation to the limb bud core as cells leave the progress zone. We have used in situ hybridization to investigate whether ectodermal inhibition in micromass culture occurs at the level of gene transcription. We found that ectoderm completely inhibited the accumulation of cartilage-specific type II collagen transcripts in the mesenchyme cells, whilst the level of type I collagen transcripts was unaffected. Morphometric analysis of electron micrographs revealed that inhibition of chondrogenesis in micromass culture was not preceded by cell flattening. In fact, a rounded cell shape was found not to be a prerequisite for cartilage cell differentiation in micromass.

Limb bud cell culture for in vitro teratogen screening: Validation of an improved assessment method using 51 compounds

Rat embryo limb bud cells multiply and undergo chondrogenesis in micromass culture. Teratogenic agents are identified from their inhibition of chondrogenesis, which is quantified by determination of cartilaginous foci number or proteoglycan production. In other in vitro systems, the detection is based on their ability to affect cell proliferation. So far, these methods have failed to distinguish among true inhibition of differentiation, inhibition of cell proliferation, and nonspecific cytotoxicity. The improved technique involves simultaneous measurement of cartilage synthesis and cell multiplication. Differentiation was evaluated by measurement, using an Artek Counter, of nodule areas after Alcian blue staining and proliferation by spectrophotometric quantification of Crystal-Violet bound to micromass cells. Using this method, retinoic acid was shown to inhibit chondrogenesis without affecting cell multiplication, whereas 6-aminonicotinamide preferentially inhibited cell multiplication without affecting nodule size. Doxylamine (succinate), a known nonteratogen, induced inhibition of chondrogenesis, but with a parallel inhibition of cell multiplication, reflecting a nonspecific toxic effect. This improvement increases the specificity of the micromass culture test. Validation was performed using 51 compounds. Compounds were classified according to their inhibitory activity and their active concentration. The sensitivity of the test was 61%; the specificity, 100%; and the final accuracy, 75%. The method is fully miniaturised, automated, and computerised, allowing numerous compounds to be rapidly tested at very low cost.

Regulation of chondrogenesis by heparan sulfate and structurally related glycosaminoglycans

To test the possible involvement of heparan sulfate proteoglycans in chondrogenesis, we have studied the effect of their major glycosaminoglycan (GAG) component, heparan sulfate, and other structurally related GAGs on chondrogenesis in micromass cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23 24 ). Heparan sulfate and several of its analogs (heparin, dermatan sulfate, and dextran sulfate) were found to significantly stimulate cartilage nodule formation; in addition, heparan sulfate and heparin also promoted nodule growth. Chondroitin sulfate did not stimulate chondrogenesis. The activity of the GAGs was dependent on their dose, molecular size, charge, and chemical structure. Similar effects were also observed in micromass cultures derived from stage 25 embryonic wing-tip subridge mesoderm, an enriched population of chondroprogenitor cells. The time of action of the GAGs during culture suggested their involvement in post-cell aggregation events of chondrogenesis, such as the initial expression of the chondrocyte phenotype or the growth of cartilage nodules.

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two-to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.

The patterns of chondrogenesis of cells from facial primordia of chick embryos in micromass culture

Chondrogenesis of mesenchymal cells from the frontonasal mass, mandibles, and maxillae of stage-24 chick embryos has been investigated in micromass (high-density) cultures. Distinct differences in the amount and pattern of cartilage differentiation are found. In cultures of frontonasal mass cells, a central sheet of cartilage develops; in cultures of mandible cells, less cartilage differentiates and nodules form; while in cultures of maxillae cells, virtually no chondrogenesis takes place. The same patterns of cartilage are found in cultures established from stage-20 embryos. At stage 28, frontonasal mass cultures form cartilage nodules and the number of nodules in mandible cultures is markedly decreased. There are striking parallels between the chondrogenic patterns of cells from the face and limb buds in micromass culture. The frontonasal mass cell cultures of stage-20 and -24 chick embryos resemble those established from the progress zone of limb buds. The progress zone is an undifferentiated region of the limb in which positional cues operate. Cultures established from the frontonasal mass of stage-28 chick embryos and from the mandibles of all stages resemble cultures of whole limb buds. These contain a mixture of committed and uncommitted cells. Ectoderm from facial primordia locally inhibits chondrogenesis in micromass cultures and this could provide a positional cue. The differences in chondrogenic potential of cells from facial primordia may underlie the specific retinoid effects on the frontonasal mass.

The independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds

Micromass cultures prepared from stage 23, 24, or 25 chick wing buds and cultured under identical conditions produce similar numbers of myoblasts. After treatment with the DNA synthesis inhibitor cytosine-1-β- d-arabinofuranoside, [ 3H]thymidine labeling and autoradiography of the cultures show that the increase in myoblast number during the first 48 hr of culture is due primarily to cell division. Micromass cultures prepared from proximal and distal portions of stage 23 or 24 wing buds have very different chondrogenic potentials in vitro ( B. J. Swalla, E. M. Owens, T. F. Linsenmayer, and M. Solursh (1983). Dev. Biol. 97, 59–69) but a similar myogenic potential under these culture conditions. Medium supplements that significantly enhance chondrogenesis by proximal cell cultures, such as low serum or 1 m M db cyclic AMP, do not affect the number of myoblasts per unit area of culture during the first 3 days. Muscle cells are eventually reduced in number in whole limb micromass cultures, yet persist as long as 6 days in proximal and distal cultures. These results suggest that myogenic cells are already committed in the early limbs but are inhibited from differentiation in situ until a later time. Myogenesis and chondrogenesis occur independently in culture, consistent with the idea that these two differentiated cells are derived from two separate cell populations. Furthermore, treatments which enhance chondrogenesis do not act indirectly by killing the myoblast population in these cultures.

Prostaglandin synthesis during the course of limb cartilage differentiation in vitro

In the present study we have used radiometric thin layer chromatography (TLC) and radioimmunoassay (RIA) to examine the synthesis of various prostaglandins (PGs) during the progressive chondrogenic differentiation limb mesenchymal cells undergo in micromass culture. Throughout the 3-day culture period, [3H]arachidonic acid (AA) is metabolized to compounds which comigrate with authentic PGE2, PGF2 alpha, 6-keto-PGF1 alpha, TxB2, and PGD2. In micromass cultures prepared from the cells of whole stage-23/24 wing buds, all 3H-AA metabolites are produced in relatively small amounts during the initial period of culture, i.e. prior to the formation of extensive prechondrogenic cellular aggregates. Concomitant with maximum aggregate formation and the initiation of cartilage differentiation, there is a striking and progressive increase in the production of all the major classes of PGs from 3H-AA. PG production from 3H-AA is also at a maximum during the onset of chondrogenesis in micromass cultures prepared from the distal subridge mesenchymal cells of stage-25 wing buds in which more rapid, extensive, and homogeneous cartilage differentiation occurs. To complement these TLC studies, RIA has been used to examine the amount of various PGs synthesized from endogenous substrates by micromass culture homogenates at various times during in vitro chondrogenesis. These RIA studies also indicate that PG production is highest during periods of culture which coincide with the onset of overt chondrogenesis in both stage-23/24 whole limb and stage-25 subridge mesoderm micromass cultures. RIA indicates that PGE2 is the predominant PG produced from endogenous substrates during 1h incubations at the onset of chondrogenesis, while radiometric TLC indicates compounds which comigrate with PGF2 alpha are the major class of 3H-AA metabolites which accumulate during that time. This qualitative difference very likely reflects metabolism of parent PG compounds during the long (12h) labelling and postlabelling incubations utilized in the TLC analyses. The temporal correlation between PG production and the initiation of chondrogenesis in vitro is consistent with previous studies implicating PGs in the regulation of limb cartilage differentiation.

Uniform cartilage differentiation in micromass cultures prepared from a relatively homogeneous population of chondrogenic progenitor cells of the chick limb bud: effect of prostaglandins.

AbstractEven at very early stages of development (i.e., stages 22–24), the embryonic chick limb bud consists of a heterogeneous mixture of chondrogenic, myogenic, and fibrogenic progenitor cells in various stages of differentiation. In contrast, the distal subridge region of a stage 25 wing bud appears to be a relatively homogeneous population of undifferentiated chondrogenic progenitor cells. In the present study, we confirm the results of a number of investigators that when the heterogeneous cells of whole stage 23/24 limb buds are subjected to high density cell culture (micromass culture) the cells in various discrete regions of the culture aggregate with one another and differentiate into cartilage nodules. The cartilage nodules in such cultures, however, are separated from one another by a considerable amount of nonchondrogenic tissue. In fact, nonchondrogenic tissue constitutes a relatively high proportion of the micromass cultures of whole stage 23/24 limb bud cells. In contrast, when stage 25 subridge mesenchymal cells are subjected to micromass culture, a virtually uniform sheet of cartilage is formed throughout the culture, and little, if any, nonchondrogenic tissue is detectable. The micromass culture of stage 25 subridge mesenchymal cells thus provides an excellent system in which a relatively homogeneous group of chondrogenic progenitor cells progressively and uniformly undergo chondrogenesis in a readily manipulable culture situation. Such a system is ideally suited for studies on the molecular regulation of cartilage differentiation.In the present study, we have also demonstrated that exogenous prostaglandin E2 (PGE2) elicits a dose‐dependent stimulation of chondrogenesis in micromass cultures of both stage 25 subridge mesenchymal cells and the cells of whole stage 23/24 limb buds. The stimulatory effect of PGE2 is greatly potentiated by the phosphodiesterase inhibitor theophylline, suggesting that its influence on chondrogenesis is mediated by its ability to increase cAMP levels. PGE1 is just as effective as PGE2 in stimulating chondrogenesis, whereas PGF1α, 6‐keto PGF1α and thromboxane B2 have no effect. On the basis of these observations and previous studies, the possibility that endogenous prostaglandins might regulate limb cartilage differentiation by acting as local mediators of cAMP accumulation is discussed.