Assessment of Differentiation Aspects by the Morphological Classification of Embryoid Bodies Derived from Human Embryonic Stem Cells
Assessment of Differentiation Aspects by the Morphological Classification of Embryoid Bodies Derived from Human Embryonic Stem Cells
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DOI：
10.1089/scd.2010.0476
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发表时间：
2011-11-01
影响因子：
4
通讯作者：
Chung, Hyung-Min
中科院分区：
文献类型：
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作者：
Kim, Jung Mo;Moon, Sung-Hwan;Chung, Hyung-Min
一般来说，胚状体（EBs）的形成是体外初始诱导人胚胎干细胞（hESCs）形成其衍生物的一种常见方法。尽管EBs具有模拟发育过程的能力，但EBs的规范和分类尚未广为人知。由于EBs表现出不同的分化潜能，这取决于聚集细胞的大小和形态，因此很难确定。在这里，我们试图通过形态学对EBs的分化潜力进行分类，从而使人们能够高效地控制特定谱系从hESCs的分化。为了诱导EB的分化，我们在无碱性成纤维细胞生长因子的hESC培养皿中建立了未分化hESC的漂浮培养。细胞首先聚集成球；与悬浮培养后10天类似，出现了一些不同类型的EB形态，我们将其分类为囊型、亮腔型和暗腔型EB。接下来，我们通过多重聚合酶链反应（PCR）、实时PCR和免疫细胞化学分析了每种类型EB分化为3种胚层的能力。结果表明，囊性EB内大部分细胞由内胚层系群组成，两种类型的EB均有3个胚层细胞。然而，明亮腔体的EBs分化能力比暗腔体快。因此，与其他类型的脑电波相比，本研究的亮腔脑电波具有相同的分化倾向，可作为脑电波体外工程的标准。这些结果表明，EB形态的分类可以估计EB的分化状态，并可能允许描述EB分化为特定细胞类型所需的条件子集。
In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; similar to 10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.