Stage‐ and region‐dependent chondrogenesis and growth of chick wing‐bud mesenchyme in serum‐containing and defined tissue culture media

Abstract

During development, limb-bud mesenchymal cells carry out complex spatiotemporal patterns of growth and differentiation. Tissue and organ culture facilitate analysis of environmental influences on these cell behaviors, allowing their partial dissection into exogenous and endogenous components. Two factors that complicate such in vitro analyses are the heterogeneity of the cultured cells and imprecise knowledge of culture medium composition. Limb mesenchyme comprises a heterogenous cell population with important regional differences in cell type. Dividing the limb into subregions helps limit the cellular heterogeneity and using chemically defined, serum-free medium allays concerns about medium composition. In the present study, mesenchyme from different regions along the anteroposterior and proximodistal axes of stage 21-22 and stage 23-24 chick wing buds was grown in high-density microtiter cultures in chemically defined and in serum-containing medium. Fourday cultures of the various regions were compared in terms of culture morphology and the accumulation of Alcian blue-positive cartilage matrix and DNA. The results demonstrate stageand region-dependent differences in the in vitro growth, differentiation, and responsiveness of these cells. For example, mesenchyme from the distal anterior region of the wing bud exhibited lower intrinsic chondrogenic capacity and greater responsiveness to serum than other regions. Patterns of in vitro chondrogenesis also suggest that, at the stages examined, distal wing-bud mesenchyme may be less homogeneous than has been believed. A case is made for the suitability of serum-free medium for future in vitro studies of chick limb-bud mesenchyme. The results are considered in relation to the process of limb development and regional expression of pattern-related genes. 0 1994 Wiley-Liss, Inc.

Changes in the expression of fibroblast growth factor receptors mark distinct stages of chondrogenesis in vitro and during chick limb skeletal patterning

“…The course of chondrogenic differentiation in wing bud mesenchymal cell micromass cultures is well described (Paulsen et al, 1994).…”

“…Eggs were incubated at 37-38°C in a humidified egg incubator, windowed on the third day of incubation, and embryos were staged in ovo. We followed the procedures described by Ahrens et al (1977) and Paulsen et al (1994) to establish micromass cultures. Stage 23-24 embryos were collected in cold Earle's Balanced Salt Solution (EBSS) (Gibco, Grand Island, NY), and the distal two-thirds of wing buds were cut off, The wing bud pieces were incubated in 0.5% trypsin in Dulbecco's Ca' + and Mg' + free medium on ice for 1-2 hr, and then transferred to cold 2% fetal bovine serum (FBS) in EBSS.…”

Stage‐ and region‐dependent responses of chick wing‐bud mesenchymal cells to retinoic acid in serum‐free microcultures

“…The results indicate that under these conditions, chick wing-bud mesenchyme does indeed exhibit detectable anteroposterior differences in its responsiveness to exogenous RA. The results further indicate that the distal anterior wingbud mesenchymal cells exhibit a unique responsiveness to exogenous RA, as they do to the inclusion of serum in the culture medium (Paulsen et al, 199413).…”

“…High density microcultures of the mesenchyme from each region were established in half-well microtiter culture plates (Corning, Corning, NY) as previously described in detail (Paulsen et al, 199413). The control cultures were grown in defined medium (DM) consisting of DMEM/F12 (l:l, Media Tech, Herndon, VA) supplemented with 5 pg/ml bovine insulin (Collaborative Research, Waltham, MA), 5pg/ml chicken transferrin (conalbumin), 100 nM hydrocortisone, 50 pg/ml L-ascorbic acid (all Sigma), and antibiotics (GIBCO, Grand Island, NY).…”

Retroviral Gene Transfer in Chondrogenic Limb Bud Micromass Cultures

“…These studies have been facilitated by the development of serum-free media (33,34). Progress has also been made in dissecting out some of the signaling pathways involved in chondrogenesis (23,25,41).…”