Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment.
Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment.
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DOI：
10.1007/s11626-016-0013-5
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发表时间：
2016-05
期刊：
影响因子：
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通讯作者：
Ko MS
中科院分区：
文献类型：
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作者：
Sharova LV;Sharov AA;Piao Y;Stagg CA;Amano T;Qian Y;Dudekula D;Schlessinger D;Ko MS
视黄酸（RA）是小鼠胚胎干细胞（ESC）分化最有效的诱导剂之一。然而，之前的研究表明，对在白血病抑制因子（LIF）存在下培养的细胞进行 RA 处理也会导致 Zscan4 基因的上调，该基因的瞬时表达是未分化 ESC 的标志。我们探索了 RA 这两种看似对立的作用之间的平衡。 RA 处理后，ESC 确实在 LIF 存在的情况下分化，但未分化的 ESC 集落最终从这些分化的细胞中出现——即使在 RA 存在的情况下也是如此。这些集落被称为次级集落，由三种细胞类型组成：表达多能性基因（如 Pou5f1、Sox2 和 Nanog）的典型未分化 ESC；以及表达多能性基因的典型未分化 ESC。表达 Zscan4 的细胞；和位于集落外围的内胚层样细胞。确认了所有八个测试的 ESC 系形成次生集落的能力。来自次级集落的细胞在转移到标准 ESC 培养基后保留了多能性，根据其强碱性磷酸酶 (ALP) 染色、典型的集落形态、基因表达谱、稳定的核型、在胚状体形成测定中分化为所有三个胚层的能力以及注射到囊胚后成功形成嵌合体来判断。基于流式细胞术分析（FACS），次级集落中Zscan4阳性细胞的比例高于标准ESC集落，这可能解释了ESCs抵抗RA分化作用并形成未分化ESCs次级集落的能力。这一假设得到了细胞谱系追踪分析的支持，该分析表明次级集落中的大多数细胞是瞬时表达 Zscan4 的细胞的后代。
Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells — even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies — after transfer to the standard ESC medium — retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.