Engineering human cell spheroids to model embryonic tissue fusion in vitro.
Engineering human cell spheroids to model embryonic tissue fusion in vitro.
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DOI：
10.1371/journal.pone.0184155
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Abbott BD
中科院分区：
文献类型：
--
作者：
Belair DG;Wolf CJ;Wood C;Ren H;Grindstaff R;Padgett W;Swank A;MacMillan D;Fisher A;Winnik W;Abbott BD
上皮-间充质相互作用在发育过程中驱动胚胎融合事件，这些相互作用的扰动可能导致出生缺陷。腭裂和神经管缺陷可能是由发育过程中的遗传缺陷或环境暴露引起的，但由于缺乏相关和可靠的人类体外发育融合行为分析，人们对化学物质暴露对人类发育过程中融合事件的影响知之甚少。鉴于腭裂的病因和流行率，以及胚胎腭部相对简单的结构和组成，我们试图开发一种模拟胚胎腭部的三维培养系统，并可用于研究使用人类细胞的体外融合行为。我们设计了大小可控的人Wharton‘s Jelly stromal cell(HWJSC)球体，并证实在成骨分化培养液中培养7天足以促进与胚胎腭间充质一致的成骨表型。HWJSC球体可能通过间充质球体沉积I型和IV型胶原、纤维连接蛋白和层粘连蛋白，支持人表皮角质形成细胞前体细胞(HPEKp)在球体外表面的附着。包被HPEKp细胞的HWJSC球体在培养中表现出融合行为，从球体之间的缝隙中去除上皮细胞表明，这依赖于表皮生长因子信号和成纤维细胞生长因子信号，这与腭部融合文献一致。这里描述的方法可以广泛地应用于三维上皮-间充质共培养的产生，以符合预测毒理学应用的形式来研究发育融合事件。
Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.
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