High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development

Summary To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.     

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N-cadherin and its associated proteins

Retinoic acid (RA) is a well-known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal-regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N-cadherin expression. However, RA treatment caused sustained expression of N-cadherin and its associated proteins including alpha- and beta-catenin suggesting that modulation of expression of these molecules is associated with RA-induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA-induced sustained expression of cell adhesion molecules and overcomes RA-induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell-to-cell interactions at the post-precartilage condensation stage.     

Chondrogenesis and myogenesis in micromass cultures of mesenchyme from mouse facial primordia

Summary The face develops from small buds of tissue positioned around the primitive mouth. The chondrogenic and myogenic cell populations contained within these facial primordia in mouse embryos have been investigated in short-term micromass culture. Chondrogenesis occurred in frontonasal mass mesenchyme from E11-E13 embryos, in maxillary mesenchyme from E12.5 embryos and was absent in mandibular mesenchyme. Myogenesis was greatest in mandibular mesenchyme, moderate in maxillary mesenchyme and low in the frontonasal mass. When compared with chick embryos the mouse facial primordia have lower chondrogenic potential, which in the case of the frontonasal mass may be related to the relative outgrowth of the primordia in the two species. Chondrogenesis in the mouse mandibular mesenchyme may be affected by the presence of a large population of odontogenic mesenchyme. The behavior of myogenic cell populations is related to the pattern of the musculature of the face, as the mandible contains the most muscle, the maxilla some, and the frontonasal mass none. However, the presence of myoblasts in early mesenchyme of all primordia may indicate that, as with chick, facial primordia are initially seeded with muscle cells and that the size of the cell population is subsequently controlled according to the development of the musculature within the primordia.     

NuSerum,a synthetic serum replacement,supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities (1, 5, 10, or 20 × 10⁶ cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [³H]leucine and [³H]thymidine. Chondrogenesis was determined by the incorporation of [³⁵S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 10⁶ cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 × 10⁶ cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [³⁵S]sulfate (2.6-fold), [³H]leucine (1.4-fold), and [³H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 10⁶ cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors, including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro.     

Evaluation of Bupropion Hydrochloride Developmental Cardiotoxic Effects in Chick Cardiomyocyte Micromass Culture and stem cell derived Cardiomyocyte Systems

The use of antidepressant drug bupropion hydrochloride (BPN) during pregnancy results in increased cardiovascular anomalies. In this study, BPN developmental cardiotoxic effects in in vitro system were evaluated using chick cardiomyocyte micromass (MM) culture system and mouse embryonic stem cell derived cardiomyocyte (ESDC) system. In MM system, the cardiomyocyte contractile activity significantly decreased only at BPN 200 μM, while in ESDC system BPN concentration above 75 μM resulted in decreased contractile activity. The increase in drug concentration also affected the cardiomyocyte viability and total cellular protein content in both systems, but in ESDC system the cell viability failed to attain significant difference. The drug failed to induce reactive oxygen species production in both systems, but has affected the cardiac connexin43 expression especially in MM system. We observed that BPN showed developmental cardiotoxic effects irrespective of the stage of cardiac development in both in vitro systems     

Analysis of cartilage maturation using micromass cultures of primary chondrocytes

A micromass culture (MM-C) system of primary immature chondrocytes for functional analysis of soluble factors involved in the maturation step of cartilage was previously developed. Ectopically expressed BMP-2 was shown to induce the expression of the Ihh and Noggin genes. Here it is demonstrated that, upon longer culture, secreted bone morphogenetic protein-2 (BMP-2) further promotes the maturation step as judged by the induction of type X collagen and BMP-6 expression, which are known to be detectable in the later phase of cartilage maturation. Induction of all of these genes by secreted BMP-2 was not inhibited by ectopic expression of parathyroid hormone-related peptide (PTHrP) induced by retrovirus vector infection, although the same virus vector showed strong inhibitory effects on the expression of type X collagen gene or alkaline phosphatase activity in mature chondrocytes. These results suggest that the maturation-promoting activity exhibited by BMP-2 is dominant over the suppressive effect of PTHrP in immature chondrocytes. When the BMP-6 gene was introduced into the same virus vector as that used for BMP-2, it induced the same sets of genes (Ihh, Noggin, type X collagen and endogenous BMP-6) as BMP-2 did. These results also suggest that BMP-6 would autonomously maintain and/or promote a later stage of chondrocytic maturation.