Paulsen D F, Chen W D, Pang L, Johnson B, Okello D
Department of Anatomy, Morehouse School of Medicine, Atlanta, Georgia 30310.
Dev Dyn. 1994 May;200(1):39-52. doi: 10.1002/aja.1002000105.
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 alloys 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. Four-day 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 stage- and 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.
在发育过程中，肢芽间充质细胞进行复杂的时空生长和分化模式。组织和器官培养有助于分析环境对这些细胞行为的影响，从而将其部分解析为外源性和内源性成分。使这种体外分析复杂化的两个因素是培养细胞的异质性和对培养基成分的不精确了解。肢体间充质由异质性细胞群体组成，细胞类型存在重要的区域差异。将肢体划分为子区域有助于限制细胞异质性，并且使用化学成分明确的无血清培养基可消除对培养基成分的担忧。在本研究中，来自第21 - 22阶段和第23 - 24阶段鸡翼芽前后轴和近远轴不同区域的间充质在高密度微孔培养中，分别在化学成分明确的培养基和含血清的培养基中生长。对各个区域的四天培养物在培养形态、阿尔新蓝阳性软骨基质和DNA积累方面进行了比较。结果表明这些细胞在体外生长、分化和反应性方面存在阶段和区域依赖性差异。例如，翼芽远端前部区域的间充质比其他区域表现出更低的内在软骨生成能力和对血清更高的反应性。体外软骨生成模式还表明，在所研究的阶段，远端翼芽间充质可能比人们认为的更不均匀。提出了无血清培养基适用于未来鸡肢体芽间充质体外研究的观点。结合肢体发育过程和模式相关基因的区域表达对结果进行了讨论。
