Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

Acute exposure to triphenyl phosphate inhibits the proliferation and cardiac differentiation of mouse embryonic stem cells and zebrafish embryos

Attention has recently paid to the interaction of triphenyl phosphate (TPHP) and body tissues, particularly within the reproductive and development systems, due to its endocrine-disrupting properties. However, the acute effects of TPHP on early embryonic development remain unclear. Here, we used mouse embryonic stem cells (mESC) and zebrafish embryos to investigate whether TPHP is an embryo toxicant. First, we found that continuous exposure of TPHP decreased the proliferation and increased the apoptotic populations of mESCs in a concentration-dependent manner. Results of mass spectrometry showed that the intracellular concentration of TPHP reached 39.45 ± 7.72 µg/g w/w after 3 hr of acute exposure with TPHP (38.35 μM) but gradually decreased from 3 hr to 48 hr. Additionally, DNA damage was detected in mESCs after a short-term treatment with TPHP, which in turn, activated DNA damage responses, leading to cell cycle arrest by changing the expression levels of p53, proliferating cell nuclear antigen, and Y15-phosphorylated Cdk I. Furthermore, our results revealed that short-term treatment with TPHP disturbed cardiac differentiation by decreasing the expression levels of Oct4, Sox2, and Nanog and transiently reduced the glycolysis capacity in mESCs. In zebrafish embryos, exposure to TPHP resulted in broad, concentration-dependent developmental defects and coupled with heart malformation and reduced heart rate. In conclusion, the two models demonstrate that acute exposure to TPHP affects early embryonic development and disturbs the cardiomyogenic differentiation.     

Down-regulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells: effects on cellular proliferation, differentiation, and melanogenesis

The composition of the gangliosides of hamster melanoma cells is closely related to their cellular growth and degree of differentiation, with slow-growing, highly differentiated melanotic melanoma MI cells expressing GM3 and fast-growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O-acetyl-GD3. To study the putative function of O-acetyl-GD3, we established stably transfected AbC-1 amelanotic hamster melanoma cells with O-acetylesterase gene from influenza C virus to hydrolyze the O-acetyl group from O-acetyl-GD3. The content of O-acetyl-GD3 in the transfected cells expressing O-acetylesterase gene was reduced by >90%. These O-acetyl-GD3-depleted cells differed from the parental ones in their cellular morphology, growth behavior, and melanogenesis activity. The absence of O-acetyl-GD3 in the transfected cells was accompanied by increased thick dendrite formation with an enlarged cell body, which is in striking contrast to the control cells, which were rounded and flattened, with few processes. Their growth was significantly slower than that of the control cells. They also demonstrated significantly lower tyrosinase activity and melanogenic potential. We suggest that the enhanced expression of melanoma-associated O-acetyl-GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation but not melanogenesis.     

The different actions of normal and supranormal calcium concentrations on the proliferation of BALB/c 3T3 mouse cells

Summary In the presence of a normal (1.25 to 1.80 mM) calcium concentration, addition of fresh bovine calf serum or completely changing the medium induces proliferatively quiescent BALB/c 3T3 mouse cells in dense cultures to start a growth division cycle and initiate DNA synthesis about 12 hr later. Fresh, low-calcium (0.02 mM physiologically available) medium also causes cells to start a growth-division cycle. However, the development of such stimulated, calcium-deprived cells stops just before the expected time of initiation of DNA synthesis, which can then be rapidly induced by restoration of the normal calcium concentration. Simply raising the calcium concentration to nonphysiologically high levels (without otherwise altering the medium) can mimic the action of fresh serum or fresh whole medium by inducing some of the cells in proliferatively quiescent confluent cultures to start a growth-division cycle and initiate DNA synthesis 22 hr later. Issued as NRCC No. 15371.     

The effect of epidermal growth factor and transforming growth factor β1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The effects of two growth factors, EGF and TGFβ₁, on growth and differentiation of different populations of urothelial cells in explant cultures of mouse urinary bladder have been studied by electron microscopy and lectin analysis. In an explant culture 10 days after the implantation three different populations of urothelial cells can be distinguished. Original urothelial cells, which are integral part of the explant, new urothelial cells, which cover the baso-lateral sides of the explant and are organized in a multilayer epithelium, and new urothelial cells, which are not any more in direct contact with the explant and grow over the membrane in epithelium-like structure. Exogenously added EGF or TGFβ₁ did not affect either the formation or the thickness of multilayered urothelium, so cells were proliferating on the free surfaces of stroma as well as on the epithelium expanding over the membrane. In the absence of growth factors in medium, the newly formed baso-lateral multilayered epithelium bordering the stroma shows ultrastructural signs of terminal differentiation suggesting that for cell proliferation and differentiation the action of stroma is of crucial importance. On the other hand, the differentiation of the epithelium spreading over the membrane, but not its thickness, is affected by exogenously added TGFβ₁. Solely in TGFβ₁-treated cultures a differentiation sirnilar to that in vivo takes place. The apoptosis of the urothelial cells was not increased by TGFβ₁. The lectin analysis by WGA and ConA conjugated with ferritin revealed that ConA-ferritin combines only with the surface cells which grow over the membrane in the absence of TGFβ₁.