Microtiter micromass cultures of limb-bud mesenchymal cells

Abstract

A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (microT microM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This microT microM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d microT microM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the microT microM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation.

Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes.

“…Insulin and IGF-I have been used as supplements in defined medium for culture of chondrocytes from limb bud, growth plate, and articular cartilage to support chondrocyte phenotype and chondrogenesis (5,(56)(57)(58)(59)(60). It is possible that one function that is lost during immortalization is the production of a chondrocyte-specific growth factor such as IGF-I or expression of IGF-I receptors.…”

Mathematically guided approaches to distinguish models of periodic patterning

“…Although challenging, making broad perturbations may be feasible using in vitro models of patterning (Miura and Shiota, 2000a,b;Paulsen and Solursh, 1988;Yamanaka and Kondo, 2014), together with careful tuning of the extracellular environment. One way to control the external environment is to use hydrogels (Cushing and Anseth, 2007;Elisseeff, 2008), in which one can independently modulate the diffusion constants of secreted molecules, the motility of different cell types and the material properties of the underlying ECM (Forget et al, 2013;Kyburz and Anseth, 2013;Lin and Metters, 2006;Weber et al, 2009).…”

“…By contrast, a model based on cell movement will be affected by perturbing cell motility, and a mechanical model by perturbing the stiffness of the surrounding ECM. A particularly attractive system in which to perform these experiments is an in vitro model of chondrogenesis (Miura and Shiota, 2000a;Paulsen and Solursh, 1988;Raspopovic et al, 2014), where, as described above, there are methods for independently perturbing extracellular diffusion, cell motility and the mechanical properties of the ECM.…”

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

“…To determine the effect of phospho-p38 on limb differentiation, we added the p38 inhibitor SB203580 (Barančík et al, 2001;Chen et al, 2016;Engel et al, 2005;Hirose et al, 2003;Kim et al, 2016;Tong et al, 1997;Wang et al, 2012) to micromass cultures. After 48 h, control cells had formed nodules, an early step in chondrogenesis that precedes Alcian Blue staining (Barna and Niswander, 2007;Paulsen and Solursh, 1988), whereas the inhibitor-treated cells failed to do so (n=4/4; Fig. S10).…”