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. 2016 May;52(5):616-24.
doi: 10.1007/s11626-016-0013-5. Epub 2016 Apr 29.

Emergence of Undifferentiated Colonies From Mouse Embryonic Stem Cells Undergoing Differentiation by Retinoic Acid Treatment

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Free PMC article

Emergence of Undifferentiated Colonies From Mouse Embryonic Stem Cells Undergoing Differentiation by Retinoic Acid Treatment

Lioudmila V Sharova et al. In Vitro Cell Dev Biol Anim. .
Free PMC article

Abstract

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.

Keywords: Mouse embryonic stem cells; Pluripotency; Retinoic acid; Zscan4.

Figures

Figure 1
Figure 1
Emergence of undifferentiated ESC colonies in retinoic acid conditions. Left panel: the experiment design, red cells are ALP-positive. Right panel: MC1-ZE-3 cells with Emerald-Zscan4 reporter (green) plated at clonal density show the emergence of secondary colonies.
Figure 2
Figure 2
Expression of genes in secondary colonies. Expression of (A) pluripotency related genes, (B) genes associated with Zscan4 expression in ESCs, and (C) differentiation-related genes in manually picked secondary colonies (MC1 cells in 50 nM retinoic acid on day 7) and in descendants of these cells cultured in standard ESC conditions for 10 passages (derived cells), based on microarray data; expression is normalized to undifferentiated standard ESCs (line = 1). Heterogeneity of gene expression: (D-G) expression of Nanog (immunostaining), Zscan4 (Emerald reporter), and Gata4 (immunostaining). Expression of Zscan4 in early secondary colonies (day 3, Emerald reporter) (H, I) and cumulative expression in late secondary colonies (day 7, lacZ staining in Zscan4-CreERT2) (J, K) combined with immunostaining for pluripotency-related factors Pou5f1 (H, J) and Nanog (I, K); lacZ staining in Zscan4-CreERT2 cells shows cells that previously expressed Zscan4.
Figure 3
Figure 3
Contribution to chimeras and genome integrity of cells exposed to retinoic acid (RA) (i.e., from secondary colonies) and then cultured in standard conditions for 10 passages, as compared to control cells (i.e., parental ESCs that underwent the same number of passages). (A) Proportion of chimeric mice obtained from cell injection into blastocysts (number of pups shown); (B) telomere length assessed with qPCR; (C) karyotype of cells exposed to RA and in control cells; asterisk (*) indicates significant difference (p < 0.05).

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