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The key enzyme of the sialic acid metabolism is involved in embryoid body formation and expression of marker genes of germ layer formation.

Weidemann W, Hering J, Bennmann D, Thate A, Horstkorte R - Int J Mol Sci (2013)

Bottom Line: Sialic acids are negatively charged nine carbon amino sugars and are found on most glycoproteins and many glycolipids in terminal positions, where they are involved in a variety of biological important molecular interactions.As a result, GNE-deficient embryonic stem cells fail to form proper embryoid bodies (EB) within the first day of culture.In contrast, when culturing these cells in sialic acid reduced medium, GNE-deficient embryonic stem cells proliferate faster and form larger EBs without any change in the expression of markers of the germ layers.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie, Martin-Luther-Universität Halle-Wittenberg, Hollystr.1, Halle D-06114, Germany. ruediger.horstkorte@medizin.uni-halle.de.

ABSTRACT
The bi-functional enzyme UDP-N-acetyl-2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the sialic acid biosynthesis. Sialic acids are negatively charged nine carbon amino sugars and are found on most glycoproteins and many glycolipids in terminal positions, where they are involved in a variety of biological important molecular interactions. Inactivation of the GNE by homologous recombination results in early embryonic lethality in mice. Here, we report that GNE-deficient embryonic stem cells express less differentiation markers compared to wild-type embryonic stem cells. As a result, GNE-deficient embryonic stem cells fail to form proper embryoid bodies (EB) within the first day of culture. However, when culturing these cells in the presence of sialic acids for three days, also GNE-deficient embryonic stem cells form normal EBs. In contrast, when culturing these cells in sialic acid reduced medium, GNE-deficient embryonic stem cells proliferate faster and form larger EBs without any change in the expression of markers of the germ layers.

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Embryoid body formation and qRT-PCR analysis in the absence of sialic acids. Wild-type (wt) and GNE-deficient (GNE-deficient (−/−)) embryonic stem cells were cultured in hanging drops in sialic acid free serum replacement medium for 72 h. (A) Representative micrographs were shown. Bar = 100 μm; (B) Bars represent fold change of expression of GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells. Each experiment was performed twice in triplicates.
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f3-ijms-14-20555: Embryoid body formation and qRT-PCR analysis in the absence of sialic acids. Wild-type (wt) and GNE-deficient (GNE-deficient (−/−)) embryonic stem cells were cultured in hanging drops in sialic acid free serum replacement medium for 72 h. (A) Representative micrographs were shown. Bar = 100 μm; (B) Bars represent fold change of expression of GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells. Each experiment was performed twice in triplicates.

Mentions: Recently, we found that GNE-deficient embryonic stem cells proliferate much faster in Sia-free or Sia-reduced culture media. Therefore, we analyzed the embryonic body formation in Sia-reduced SR (=serum replacement) culture medium (note that full FCS-containing culture medium is very enriched in Sia; see: [12]). Whereas wildtype embryonic stem cells do not distinguish between full Sia FCS containing medium and Sia-reduced SR medium, GNE-deficient embryonic stem cells form much larger embryoid bodies after three days of culture in Sia-reduced medium (Figure 3a). When quantifying the expression of the differentiation marker genes, we found an 10-fold up-regulation of Nkx2,5 and an 6-fold up-regulation of Cdx-2 in GNE-deficient embryoid bodies (Figure 3B). Expression of Nodal, Nestin, Sialoadhesion and beta-1 integrin is not different between wildtype or GNE-deficient embryoid bodies (data not shown). In summary, we were able to culture embryoid bodies in serum replacement medium. This medium contains only very low concentration of Sia compared to FCS-containing medium [12]. In the same study [12] we already demonstrated that the Sia content of the medium is involved in proliferation of embryonic stem cells. GNE-deficient embryonic stem cells proliferate much faster compared to wildtype embryonic stem cells in the absence of Sia. In addition, the presence of Sia-precursors interferes with proliferation. In the present study we made a similar observation: embryonic bodies of GNE-deficient embryonic stem cells grow faster in the absence of Sia. Unfortunately, long-term culture (longer than two days) of embryonic bodies in the absence of Sia is not possible because cells undergo apoptosis in complete serum-free medium (data not shown). Our understanding is that embryoid body formation is more comparable to early embryogenesis than to later development processes. In all in vitro experiments such as that described here, there is one very important point to mention: the placenta. The placenta is responsible for the supply of the embryo, whereas under all in vitro conditions the cells of the embryoid bodies are forced to use all supplement direct from the medium and no cellular mechanism can supplement missing components, such as Sia. Here, it is noteworthy that the placenta expresses high quantities of GNE [6]. Therefore, Sia might be responsible for the proper function of the placenta by itself or Sia synthesized by the placenta are used for generation of highly sialylated proteins provided for the embryo.


The key enzyme of the sialic acid metabolism is involved in embryoid body formation and expression of marker genes of germ layer formation.

Weidemann W, Hering J, Bennmann D, Thate A, Horstkorte R - Int J Mol Sci (2013)

Embryoid body formation and qRT-PCR analysis in the absence of sialic acids. Wild-type (wt) and GNE-deficient (GNE-deficient (−/−)) embryonic stem cells were cultured in hanging drops in sialic acid free serum replacement medium for 72 h. (A) Representative micrographs were shown. Bar = 100 μm; (B) Bars represent fold change of expression of GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells. Each experiment was performed twice in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821630&req=5

f3-ijms-14-20555: Embryoid body formation and qRT-PCR analysis in the absence of sialic acids. Wild-type (wt) and GNE-deficient (GNE-deficient (−/−)) embryonic stem cells were cultured in hanging drops in sialic acid free serum replacement medium for 72 h. (A) Representative micrographs were shown. Bar = 100 μm; (B) Bars represent fold change of expression of GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells. Each experiment was performed twice in triplicates.
Mentions: Recently, we found that GNE-deficient embryonic stem cells proliferate much faster in Sia-free or Sia-reduced culture media. Therefore, we analyzed the embryonic body formation in Sia-reduced SR (=serum replacement) culture medium (note that full FCS-containing culture medium is very enriched in Sia; see: [12]). Whereas wildtype embryonic stem cells do not distinguish between full Sia FCS containing medium and Sia-reduced SR medium, GNE-deficient embryonic stem cells form much larger embryoid bodies after three days of culture in Sia-reduced medium (Figure 3a). When quantifying the expression of the differentiation marker genes, we found an 10-fold up-regulation of Nkx2,5 and an 6-fold up-regulation of Cdx-2 in GNE-deficient embryoid bodies (Figure 3B). Expression of Nodal, Nestin, Sialoadhesion and beta-1 integrin is not different between wildtype or GNE-deficient embryoid bodies (data not shown). In summary, we were able to culture embryoid bodies in serum replacement medium. This medium contains only very low concentration of Sia compared to FCS-containing medium [12]. In the same study [12] we already demonstrated that the Sia content of the medium is involved in proliferation of embryonic stem cells. GNE-deficient embryonic stem cells proliferate much faster compared to wildtype embryonic stem cells in the absence of Sia. In addition, the presence of Sia-precursors interferes with proliferation. In the present study we made a similar observation: embryonic bodies of GNE-deficient embryonic stem cells grow faster in the absence of Sia. Unfortunately, long-term culture (longer than two days) of embryonic bodies in the absence of Sia is not possible because cells undergo apoptosis in complete serum-free medium (data not shown). Our understanding is that embryoid body formation is more comparable to early embryogenesis than to later development processes. In all in vitro experiments such as that described here, there is one very important point to mention: the placenta. The placenta is responsible for the supply of the embryo, whereas under all in vitro conditions the cells of the embryoid bodies are forced to use all supplement direct from the medium and no cellular mechanism can supplement missing components, such as Sia. Here, it is noteworthy that the placenta expresses high quantities of GNE [6]. Therefore, Sia might be responsible for the proper function of the placenta by itself or Sia synthesized by the placenta are used for generation of highly sialylated proteins provided for the embryo.

Bottom Line: Sialic acids are negatively charged nine carbon amino sugars and are found on most glycoproteins and many glycolipids in terminal positions, where they are involved in a variety of biological important molecular interactions.As a result, GNE-deficient embryonic stem cells fail to form proper embryoid bodies (EB) within the first day of culture.In contrast, when culturing these cells in sialic acid reduced medium, GNE-deficient embryonic stem cells proliferate faster and form larger EBs without any change in the expression of markers of the germ layers.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie, Martin-Luther-Universität Halle-Wittenberg, Hollystr.1, Halle D-06114, Germany. ruediger.horstkorte@medizin.uni-halle.de.

ABSTRACT
The bi-functional enzyme UDP-N-acetyl-2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the sialic acid biosynthesis. Sialic acids are negatively charged nine carbon amino sugars and are found on most glycoproteins and many glycolipids in terminal positions, where they are involved in a variety of biological important molecular interactions. Inactivation of the GNE by homologous recombination results in early embryonic lethality in mice. Here, we report that GNE-deficient embryonic stem cells express less differentiation markers compared to wild-type embryonic stem cells. As a result, GNE-deficient embryonic stem cells fail to form proper embryoid bodies (EB) within the first day of culture. However, when culturing these cells in the presence of sialic acids for three days, also GNE-deficient embryonic stem cells form normal EBs. In contrast, when culturing these cells in sialic acid reduced medium, GNE-deficient embryonic stem cells proliferate faster and form larger EBs without any change in the expression of markers of the germ layers.

Show MeSH
Related in: MedlinePlus