Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study

Abstract

The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

FIG. 1.

TEM of BALB/c ESCs. (A) n, nucleus; nu, nucleolus; rer, dilated rER; ly, lysosomes; apv, autophagocytic vacuole. (B) mv, microvilli; rb, residual bodies; m, juvenile mitochondria; cp, cell process. (C) m1, poorly developed mitochondria; m2, well-developed mitochondria; ld, lipid droplets; dv, digestive vacuole. (E) m, reticular form of mitochondria. (F) m, large poorly developed mitochondria, stalks of rER (arrows).

FIG. 2.

TEM of C57BL/6 ES cells. (B) pv, phagocytic vacuole; (arrowheads) stalks of RER. (C) m, large poorly developed mitochondria. (D) (*) Elongated mitochondria; m, juvenile mitochondria; (arrowheads) lipid droplets. (E) dj, desmosomal-like junction; pv, pinocytotic vacuole. (F) m, small poorly developed mitochondria.

FIG. 3.

TEM of 129 W9.5 ES cells. (A) m, elongated mitochondria. (B) m, large poorly developed mitochondria, reticular form of mitochondria (*). (C) m, juvenile mitochondria lipid droplets (arrowheads). (E) (arrows) Stalks of RER; g, Golgi apparatus. (E) m, small poorly developed mitochondria.

FIG. 4.

TEM of cells in BALB/c EBs. (A) m, poorly developed mitochondria; microvilli (arrows); (arrowheads) tight junction. (B) m, poorly developed mitochondria; (arrows) microvilli; (arrowheads) tight junction. (C) m, well-developed mitochondria; (arrow) basement-like membrane. (D) (arrowheads) Desmosomal-like junction (E) (arrowhead) Tight junction; m, well-developed mitochondria; gj, gap junction. (G) m, well-developed mitochondria; (*) lipid droplets. (I) (Arrowheads) Desmosomal-like junction; m, well-developed mitochondria.

FIG. 5.

TEM of cells in C57BL/6 EBs. (A) m, well-developed mitochondria. (B) (Arrowheads) Desmosomal-like junction. (C) (Arrowheads) Desmosomal-like junction; m, poorly developed mitochondria. (D) m1, juvenile mitochondria; m2, elongated mitochondria; (arrowheads) tight junction. (E) m, well-developed mitochondria.

FIG. 6.

TEM analysis of the terminal and peripheral cells from 129 W9.5 EBs. (A) m, juvenile mitochondria, basement-like structure (*). (B) tj, tight junction; m, poorly developed mitochondria. (D) m, elongated mitochondria. (F) m, well-developed mitochondria. (G) m, poorly developed mitochondria; desmosomal like junction (arrowheads). (H) Lipid plaques (*).

FIG. 7.

TEM analysis of the middle cells in 129 W9.5 EBs. (A) m, well-developed mitochondria. (B) Dividing cell (*). (C) Indication of cell necrosis (*).