Gap junctional communication during limb cartilage differentiation

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.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

The differentiation of normal and muscle-free distal chick limb bud mesenchyme in micromass culture

Distal chick wing bud mesenchyme from stages 19 to 27 embryos has been grown in micromass culture. The behavior of cultures comprising mesenchyme located within 350 microns of the apical ectodermal ridge (distal zone mesenchyme) was compared to that of cultures of the immediately proximal mesenchyme (subdistal zone cultures). In cultures of the distal mesenchyme from stages 21-24 limbs, all of the cells stained immunocytochemically for type II collagen within 3 days, indicating ubiquitous chondrogenic differentiation. At stage 19 and 20, this behavior was only observed in cultures of the distal most 50-100 microns of the limb bud mesenchyme. Between stages 25 and 27, distal zone cultures failed to become entirely chondrogenic. At all stages, subdistal zone cultures always contained substantial areas of nonchondrogenic cells. The different behavior observed between distal zone and corresponding subdistal zone cultures appears to be a consequence of the presence of somite-derived presumptive muscle cells in the latter, since no such difference was observed in analagous cultures prepared from muscle-free wing buds. The high capacity of the distal zone for cartilage differentiation supports a view of pattern formation in which inhibition of cartilage is an important component. However, its consistent behavior in vitro indicates that micromass cultures do not reflect the in vivo differences between the distal zones at different stages. The subdistal region retains a high capacity of cartilage differentiation and the observed behavior in micromass reflects interactions with a different cell population.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Abstract. Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism^H ( bp ^H ) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bp ^H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

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-beta-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 mM 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.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

The patterns on 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.     

Position-related capacity for differentiation of limb mesenchyme in cell culture.

A quantitative comparison (i.e., number of cartilage nodules) of cartilage differentiation was made between micromass cell cultures prepared with cells from different locations (core vs periphery) within prechondrogenic chick wing buds. Wing bud core cells in micromass culture exhibit a greater developmental bias toward cartilage differentiation than periphery cells from the same limbs. In addition, myogenic cells appear more frequently in cultures prepared from wing bud periphery than in those prepared from core tissue. Therefore a stage 23–24 wing bud is not a homogeneous population of multipotential mesenchymal cells. Instead, a stage 23–24 wing bud contains two classes of cells, each characterized by a bias for either cartilage or muscle differentiation, and a third class of uncharacterized mesenchymal cells.     

Cell sorting and chondrogenic aggregate formation in micromass culture

A fundamental feature of cartilage differentiation in the developing limb is the formation of a prechondrogenic cell condensation. An apparently similar process of prechondrogenic cell aggregation occurs in micromass cultures of limb bud mesenchyme with the formation of cellular aggregates which often differentiate into cartilage nodules. We have investigated the process of aggregate formation in micromass culture using chimaeric mixtures of potentially chondrogenic and nonchondrogenic cell types. Two systems were studied: mixtures of distal and proximal limb mesenchyme cells and mixtures of distal limb cells with avian tendon fibroblasts. In both cases cultures of varying proportions of each cell type have been prepared. The results demonstrate that aggregate formation in vitro is the consequence of a cell sorting process which can involve prechondrogenic cells of widely different spatial origins within the developing limb. This contrasts with in vivo prechondrogenic condensation in which there is no evidence of cell sorting (Searls, R.L. (1967), J. Exp. Zool. 166, 39-50). However, our findings do indicate that cell surface differences occur in apparently undifferentiated limb mesenchyme. The results also suggest that mesenchymal cell aggregates must achieve a threshold size before chondrogenesis can proceed. In addition, the results show that under some culture conditions nonchondrogenic cells will form aggregates.     

Inhibition of limb chondrogenesis by fibronectin

Abstract. This study compares the chondrogenic capacity of high density cultures prepared from either the develop-mentally younger, distal region or more advanced proximal region of stage 23/24 limb mesenchyme in high density cultures. Distal cultures undergo extensive chondrogenesis whether in F₁₂ medium supplemented with 10% fetal calf serum, 5% fetal calf serum, or fibronectin. On the other hand, proximal cultures fail to undergo chondrogenesis in medium containing 10% fetal calf serum or fibronectin, but do form cartilage in medium containing a decreased serum concentration or no serum. Furthermore, if the cells are cultured at low densities in native type I collagen gels, proximal cells have a reduced chondrogenic capacity in the presence of fibronectin, while chondrogenesis by distal cells is unaffected by the addition of fibronectin. The results demonstrate that proximal and distal cells respond differentially to serum and to fibronectin, and they suggest that the response of the cell to prevalent components of the extracellular matrix might change with development.     

Evaluation of the sensitive step of inhibition of chondrogenesis by retinoids in limb mesenchymal cells in vitro

The sensitive step of inhibition of chondrogenesis in vitro by retinoids was investigated in modified micromass cultures of limb bud mesenchymal cells from mouse embryos of day 11 and 12. Evaluation of chondrogenesis was performed after alcian blue staining, using a simple random hit counting of cartilage nodules. All-trans-retinoic acid, 13-cis-retinoic acid, and a newly developed arotinoid, RO 13-6298, were tested for their ability to inhibit chondrogenesis. We found that inhibition of chondrogenesis depended on the dosage and the duration of treatment with the different retinoids. Further analysis showed that chondrogenesis in limb bud mesenchymal cells from the proximal part was irreversibly inhibited after one hour of treatment, whereas distal cells showed a reduction of cartilage development only after a treatment period of 12 and more hours. In respect to the doses of the retinoids, proximal cells were about one magnitude more vulnerable than distal cells. These proximo-distal differences were obtained with 13-cis-retinoic acid at 10 micrograms/ml, with all-trans-retinoic acid at 1 microgram/ml and with arotinoid RO 13-6298 with 10 ng/ml. It is supposed that the late blastemal stage of chondrogenic differentiation before the onset of matrix synthesis is the step which is most vulnerable to retinoid treatment.     

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.     

Enhanced chondrocytic differentiation in chick limb bud cell cultures by inhibitors of poly(ADP-ribose) synthetase

Inhibitors of poly(ADP-ribose) synthetase, namely nicotinamide, benzamide, m-methoxybenzamide and 3-aminobenzamide, augmented chondrocytic differentiation chick embryo limb bud mesenchymal cells, in culture. These inhibitors stimulated early appearance and massive formation of cartilage nodules in micromass cultures stage 23-24 chick embryos. They also induced nodule formation in micromass and cartilage colonies at micromass plating densities from stage 18-19 embryo Benzamide, however, did not prevent differentiated chondrocytes from undergoing a pleiotypic change in cell type. These results are compatible with the putative regulatory function of poly(ADP-ribose) on cell differentiation.     

MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK cascade, has been shown to regulate cartilage differentiation in embryonic limb mesoderm and several chondrogenic cell lines. In the present study, we employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene. Transfection of frontonasal mesenchyme cells with dominant negative ERK increased collagen II enhancer activation, whereas transfection of constitutively active MEK decreased its activity. Thus, MEK-ERK signaling inhibits chondrogenesis in stage 24/25 frontonasal mesenchyme. Conversely, MEK-ERK signaling enhanced chondrogenic differentiation in mesenchyme of the stage 24/25 mandibular arch. In mandibular mesenchyme cultures, pharmacological MEK inhibition decreased cartilage matrix deposition, cartilage-specific RNA levels, and collagen II enhancer activity. Expression of constitutively active MEK increased collagen II enhancer activation in mandibular mesenchyme, while dominant negative ERK had the opposite effect. Interestingly, MEK-ERK modulation had no significant effects on cultures of maxillary or hyoid process mesenchyme cells. Moreover, we observed a striking shift in the response of frontonasal mesenchyme to MEK-ERK modulation by stage 28/29 of development.     

Evidence for histogenic interactions during in vitro limb chondrogenesis

The requirement for homotypic cell interaction was studied by making chimeric micromass cultures containing various proportions of chick and quail limb mesenchyme. Cultures made from limb mesenchyme from embryos of Hamburger and Hamilton stages 23–24 produce large clumps of cartilage cells, identified by the accumulation of an extracellular matrix which stains with alcian blue at pH 1 and by the ability of cells to take up ³⁵SO₄ rapidly, as demonstrated autoradiographically. Dissociated mesenchyme from stage 19 embryos did not produce cartilage in micromass cultures, but only precartilage cell aggregates. Micromass cultures prepared from mixtures of mesenchyme cells obtained from stage 19 and stages 23–24 embryos contained decreasing numbers of cartilage nodules as the proportion of stage 19-derived mesenchyme increased. At the same time the number of aggregates was not affected. When the ratio of stage 19- to stage 24-derived cells was 3:1 or greater, no nodules were detected. The actual number of cells from each stage was verified by using mixtures of quail and chick cells, which are microscopically distinguishable. Additional evidence suggests that the stage 19-derived mesenchyme inhibits chondrogenesis by passively preventing stage 24-derived cells from interacting. The results presented are consistent with the suggestions that (1) homotypic cell interaction plays a role in limb chondrogenesis and (2) the capacity to interact in the required manner is acquired after the embryos have reached stage 19. These phenomena might be involved in the normal histogenesis of cartilage tissue.     

Ethanol Exposure Stimulates Cartilage Differentiation by Embryonic Limb Mesenchyme Cells

Studies of neural, hepatic, and other cells have demonstrated thatin vitroethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformitiesin vivo,prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23–25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant ofin vitrochondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in³⁵SO₄incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol onin vitrochondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.     

Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody

We have addressed the problem of the segregation of cell lineages during the development of cartilage and muscle in the chick limb bud. The following experiments demonstrate that early limb buds consist of at least two independent subpopulations of committed precursor cells-- those in (a) the myogenic and (b) the chondrogenic lineage--which can be physically separated. Cells obtained from stage 20, 21, and 22 limb buds were cultured for 5 h in the presence of a monoclonal antibody that was originally isolated for its ability to detach preferentially myogenic cells from extracellular matrices. The detached limb bud cells were collected and replated in normal medium. Within 2 d nearly all of the replated cells had differentiated into myoblasts and myotubes; no chondroblasts differentiated in these cultures. In contrast, the original adherent population that remained after the antibody-induced detachment of the myogenic cells differentiated largely into cartilage and was devoid of muscle. Rearing the antibody-detached cells (i.e., replicating myogenic precursors and postmitotic myoblasts) in medium known to promote chondrogenesis did not induce these cells to chondrify. Conversely, rearing the attached precursor cells (i.e., chondrogenic precursors) in medium known to promote myogenesis did not induce these cells to undergo myogenesis. The definitive mononucleated myoblasts and multinucleated myotubes were identified by muscle- specific antibodies against light meromyosin or desmin, whereas the definitive chondroblasts were identified by a monoclonal antibody against the keratan sulfate chains of the cartilage-specific sulfated proteoglycan. These findings are interpreted as supporting the lineage hypothesis in which the differentiation program of a cell is determined by means of transit through compartments of a lineage.     

Chondrogenesis in chick limb mesenchyme in vitro derived from distal limb bud tips: changes in cyclic AMP and in prostaglandin responsiveness

Chondrogenesis was monitored in micromass cultures of mesenchymal cells derived from the distal tip of stage-25 chick limb buds over a 6-day period. Alcian green staining and immunofluorescent localization of cartilage-specific proteoglycans revealed the appearance of cartilage matrix by day 3 of cell culture. By day 6, cultures contained a uniform and homogeneous population of fully differentiated chondrocytes throughout the cell layer, with only a narrow rim of nonchondrogenic cells around the extreme periphery of the culture. Synthesis of sulfated glycosaminoglycans also progressively increased between days 3 and 6, being 8-fold higher at day 6 than at day 1 of culture. Both adenylate cyclase (AC) activity and cAMP concentrations increased dramatically during the first 2 days of culture, reaching maximal levels by day 2, which remained elevated and stable throughout the remaining chondrogenic period (days 3-6). Responsiveness of both AC and cAMP concentrations of the cells to PGE2 was maximal by day 1 of culture and was increased over control cells by 12-fold and 8-fold respectively. Both responses, however, were dramatically reduced by day 3, at which time the initiation of cartilage formation was apparent. Responsiveness of cells during the prechondrogenic period to PGE2 was relatively specific in that no effects could be demonstrated with equivalent concentrations of PGF2 alpha or 6-keto-PGF1 alpha, although PGl2 did produce increases in cAMP concentrations of about 50% of those of PGE2. These results indicate that previously reported changes in the cAMP system in heterogeneous cell cultures derived from whole limb buds reflect changes occurring in the chondrogenic cell type and indicate further that peak responsiveness of the cAMP system of these cells to prostaglandins is restricted to prechondrogenic developmental periods.     

Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage

Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self‐repair. Because of their unlimited capacity for self‐renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair. To this end, we have subjected undifferentiated pluripotent hESCs to the high density micromass culture conditions we have extensively used to direct the differentiation of embryonic limb bud mesenchymal cells into chondrocytes. We report that micromass cultures of pluripotent hESCs undergo direct, rapid, progressive, and substantially uniform chondrogenic differentiation in the presence of BMP2 or a combination of BMP2 and TGF‐β1, signaling molecules that act in concert to regulate chondrogenesis in the developing limb. The gene expression profiles of hESC‐derived cultures harvested at various times during the progression of their differentiation has enabled us to identify cultures comprising cells in different phases of the chondrogenic lineage ranging from cultures just entering the lineage to well differentiated chondrocytes. Thus, we are poised to compare the abilities of hESC‐derived progenitors in different phases of the chondrogenic lineage for cartilage repair. J. Cell. Physiol. 224: 664–671, 2010. © 2010 Wiley‐Liss, Inc.