Targeted deletion of either of the HS polymerases EXT1 or EXT2 results in early embryonic lethality (38
), as does lack of both NDST1 and NDST2 (31
ES cells have previously been derived and studied (40
). These cells do not express any HS and fail to transit to differentiation upon leukemia inhibitory factor withdrawal (41
) and are not able to differentiate in vitro
into neural cells (40
ES cells synthesize HS lacking N
-sulfate groups but with a low degree of 6-O
). Lack of both NDST1 and NDST2 has been shown to result in an inability of the cells to respond to VEGF, preventing angiogenic sprouting (32
). In a recent study by Lanner et al.
), it was suggested that NDST1−/−NDST2−/−
ES cells display a general failure to differentiate upon embryoid body formation. However, the methods used by us for in vitro
differentiation are designed to yield specific cell fates, including stepwise formation of intermediate progenitors, enabling primitive cells of ectodermal and endodermal type to appear. Using this approach, we can show that NDST1−/−NDST2−/−
mouse ES cells can take on a mesodermal fate and differentiate into osteoblasts, albeit with a lower efficiency (). In addition, the expression of FGF5 in NDST1−/−NDST2−/−
ES cells cultured under neural inducing conditions () indicates that the cells can enter a primitive ectoderm-like state (37
). FGF5 expression was recently demonstrated also for the EXT1−/−
ES cells (42
). Thus, HS appears not to be necessary for the formation of primitive ectoderm.
BMP signaling is essential for bone formation (43
). Our finding that functional BMP pathways exist in the double knock-out cells () may thus explain why osteoblasts can form. Adipocyte differentiation from ES cells also involves BMP-4 (44
), but contrary to calcified bone, fat would not form in the NDST1−/−NDST2−/−
ES cell cultures (). It is currently unknown where the differentiation block toward adipocytes lies, but terminal markers of adipogenesis are lacking. HS is implied in transport of fatty acids over the plasma membrane (45
), and a possible explanation for the failure to stain for Oil Red O could be a defect in lipid accumulation.
The vast majority (more than 90%) of the mutant ES cells were unable to differentiate into neural progenitors; nor did they show neural specific nestin expression (). In combination with the reduced proliferation in FGF-containing medium and inability to down-regulate markers of pluripotency, our findings highlight the need for functional HS in neural commitment. In the embryo, FGF5 marks the transition from inner cell mass to epiblast (46
), and when its expression declines, neural cells can form. Because NDST1−/−NDST2−/−
ES cells show expression of FGF5 during neural inducing conditions, we suggest that they become blocked at the stage of primitive ectoderm and cannot proceed to neural differentiation. Similarly, expression of GATA4, a key marker of early endoderm (47
) in mutant ES cells undergoing differentiation, shows an ability of the cells to form primitive endoderm. However, the inability of NDST1−/−NDST2−/−
embryoid bodies to differentiate into endothelial cells shows that this capacity is limited (32
). As previously demonstrated for WT ES cells (40
), NDST3 and in particular NDST4 became up-regulated during neural differentiation (). In mutant cells, a less pronounced increase in NDST4 expression was noted (). However, no N
-sulfotransferase activity was detected in mutant cells, suggesting that HS structure in these cells was unaltered after 6 days in neural differentiation medium.
Inhibition of BMP signaling is needed for neural differentiation (48
), and we therefore investigated if the failure of mutant ES cells to form neural progenitors was due to an inability to properly respond to BMP inhibitors. However, the addition of BMP4 to NDST1−/−NDST2−/−
ES cells induced a non-neural fate that in turn could be blocked by concomitant noggin addition, excluding a defective BMP pathway (). There are many examples in the literature of cell surface HS proteoglycans as critical determinants of the biological activity of BMPs and their endogenous antagonists in vivo
and in vitro
). For example, chordin binding in mouse embryonic tissues was shown to be dependent upon its interaction with cell surface HSPG (50
). Our finding that functional HS is not needed to modulate the signaling efficiency of BMP was therefore somewhat unexpected but was in line with a recent study where HSPG were found to play no role in BMP signaling in the early Drosophila
Undifferentiated ES cells are known to produce FGF4 (52
), also evident in our cells where both WT and mutant cells expressed the growth factor (). According to the literature, exposure to FGF signaling via the ERK1/MAPK pathway is required before the cells can respond to the anti-neural action of BMP (37
). Hence, it is possible that the low level of FGF4 signaling taking place in mutant cells is sufficient to make the cells sensitive to BMP but not for differentiation into neural progenitors.
Neural differentiation of the mutant ES cells could be rescued by the addition of conditioned medium from WT cells (), thus showing that soluble factors were enough to restore the capacity to form neural progenitors. WT ES cells readily formed neural progenitors (), despite a quite modest ERK phosphorylation in response to added FGF4 (). When instead FGF was added together with heparin, massive ERK phosphorylation was seen, in particular in the mutant ES cells (). Somewhat surprisingly, the addition of heparin alone had a similar effect on ERK phosphorylation in both WT and mutant cells (). It has been reported that heparin, in its role as co-factor for FGF receptors, can activate FGFR-4 in the absence of ligand (54
). This effect was enhanced in cells lacking heparan sulfate, which goes well together with our finding that ERK activation was stronger in KO cells than in WT ES cells.
We found that heparin could either cause rescue or cell death depending on the dose. The high degree of sulfation of heparin compared with that of endogenous heparan sulfate may explain the lethal effect of heparin at high concentration. The deleterious effect may be related to the recently recognized ability of heparin to induce apoptosis in cancer cells (55
). The mechanism has not been elucidated, but it may be related to the capacity of the highly negatively charged polysaccharide to interfere with transcription factor activity. Another possibility is that heparin in the extracellular space captures survival factors produced by non-neural cells in the cultures (57
). Adding FGF4 may balance the potentially negative effects of heparin and thus enable differentiation.
Heparin at lower concentrations, on the other hand, was not lethal but induced neural differentiation in NDST1−/−NDST2−/− ES cells. These cells produce FGF4 (), but the concentration of growth factor may be too low to induce differentiation. The rescuing effect of low doses of heparin may thus be to enhance FGF signaling to levels sufficient for differentiation. Taken together, our data thus suggest that the molar ratio of HS to FGF4 is essential for neural differentiation.
The addition of heparin to ES cells lacking the HS polymerase EXT1 results in a partial rescue of neural differentiation also in the absence of FGF (40
), and a restoration of FGF2 induced ERK phosphorylation (41
), but it was not studied if this was the case also for phosphorylation in response to FGF4. In experiments performed by Lanner et al.
), FGF2 induced ERK phosphorylation in NDST1−/−NDST2−/−
ES cells without the addition of heparin, whereas FGF4 did not have this effect. It is possible that factors in addition to FGF4, stimulated by the added heparin, were responsible for the rescue of the neural differentiation of the NDST1−/−NDST2−/−
ES cells (). However, Pickford et al.
) could recently show that heparin cannot support neural differentiation of EXT1−/−
ES cells when FGF signaling is inhibited, demonstrating the importance of FGF in this process.
In summary, we show that NDST1−/−NDST2−/− ES cells, which synthesize HS with a very low sulfate content, can take on a mesodermal fate and differentiate into primitive ectodermal cells. The cells express FGF4, and autocrine FGF4 signaling in these cells appears to be sufficient to render the cells sensitive to BMP signaling. However, to undergo neural differentiation, the ratio between heparin (or HSPG) and FGF appears to be the crucial factor determining if the cells will die (too much heparin/HSPG), survive but not differentiate (too little of either heparin/HSPG or FGF), or differentiate into neural cells (optimal ratio between heparin/HSPG and FGF).