Ultrastructural comparison of porcine putative embryonic stem cells derived by in vitro fertilization and somatic cell nuclear transfer

Abstract

The ultrastructure of porcine putative embryonic stem cells and porcine fetal fibroblasts (PFFs) was analyzed by transmission electron microscopy. The aim of this study was to compare the features of organelles in in vitro fertilization (IVF) derived porcine embryonic stem cells (IVF-pESCs) and somatic cell nuclear transfer (SCNT) derived pESCs (SCNT-pESCs). Also, the features of organelles in high-passage IVF-pESCs were compared with those in low-passage cells. The ultrastructure of PFFs showed rare microvilli on the cell surfaces, polygonal or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, elongated mitochondria, rich lysosomes and rich phagocytic vacuoles. IVF-pESCs showed rare microvilli on the cell surfaces, round or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, long stacks of rough endoplasmic reticulum, elongated mitochondria, rare lysosomes and rare autophagic vacuoles. By contrast, SCNT-pESCs showed rich microvilli with various lengths and frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, round mitochondria, rich lysosomes and rich phagocytic vacuoles with clear intercellular junctions. Furthermore, high-passage IVF-pESCs showed irregularly shaped colonies, pyknosis and numerous lysosomes associated with autophagic vacuoles showing signs of apoptosis. In conclusion, this study confirms that the ultrastructural characteristics of pESCs differ depending on their origin. These ultrastructural characteristics might be useful in biomedical research using pESCs, leading to new insights regarding regenerative medicine and tissue repair.

Fig. 1.

Transmission electron micrographs of the cell surface. (A) Porcine fetal fibroblasts, magnification × 600. (B) In vitro fertilization-derived IVF 0227 pESCs, magnification × 1500. (C) pESC lines co-cultured with mouse embryonic fibroblasts (MEFs), magnification × 3000. n, nucleus; nu, nucleolus; ne, nuclear envelope; pv, phagocytic vacuole; rer, rough endoplasmic reticulum. (D) Transgenic pESCs derived by SCNT, magnification × 8000. mv, microvilli.

Fig. 2.

Transmission electron micrographs of the nucleus. (A, B) Porcine fetal fibroblasts, magnification × 5000. (C) In vitro fertilization-derived IVF 0227 pESCs, magnification × 4000. (D) Transgenic pESCs derived by SCNT, magnification × 10000. n, nucleus; nu, nucleolus; ne, nuclear envelope.

Fig. 3.

Transmission electron micrographs of protein-synthesis-associated organelles. (A) Porcine fetal fibroblasts, magnification × 5000. (B) In vitro fertilization-derived IVF 0227 pESCs, magnification × 4000. (C) In vitro fertilization-derived IVF 0227 pESCs, magnification × 6000. (D) Transgenic pESCs derived by SCNT, magnification × 10000. r, ribosome; rer, rough endoplasmic reticulum; g, Golgi apparatus.

Fig. 4.

Transmission electron micrographs of intracellular digestion-associated organelles. (A, B) Porcine fetal fibroblasts, magnification × 5000. (C) In vitro fertilization-derived IVF 0227 pESCs, magnification × 4000. (D) Transgenic pESCs derived by SCNT, magnification × 10000. pv, phagocytic vacuole; apv, autophagic vacuole; ly, lysosome.

Fig. 5.

Transmission electron micrographs of mitochondria. (A) Porcine fetal fibroblasts, magnification × 5000. (B) Porcine fetal fibroblasts, magnification × 6000. (C) In vitro fertilization-derived IVF 0227 pESCs, magnification × 6000. (D) Transgenic pESCs derived by SCNT, magnification × 10000. m, mitochondrion.

Fig. 6.

Transmission electron micrographs of intercellular junctions. (A) Porcine fetal fibroblasts, magnification × 1500. (B) Transgenic pESCs derived by SCNT, magnification × 10000. gj, gap junction; dj, desmosome-like junction.

Fig. 7.

Transmission electron micrographs of in vitro fertilization-derived IVF0214 pESCs (high passage and cultured in vitro after 37 passages). (A) Ultrastructure of a nucleus-associated organelle, magnification × 1500. (B) Ultrastructure of a protein-synthesis-associated organelle, magnification × 2000. (C) Ultrastructure of a nucleus-associated organelle, magnification × 3000. (D) Ultrastructure of an intracellular digestion-associated organelle, magnification × 3000. (E) Ultrastructure of a mitochondrion-associated organelle, magnification × 5000. (F) Ultrastructure of a nucleus-associated organelle, magnification × 5000. n, nucleus; nu, nucleolus; ne, nuclear envelope; m, mitochondrion; pv, phagocytic vacuole; apv, autophagic vacuole; ly, lysosome; v, vesicle; ld, lipid droplets; rer, rough endoplasmic reticulum; mv, microvilli.