Heparan sulphate in embryonic stem cells
Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass/epiblast of pre-implantation embryos. Differentiation or the retention of pluripotency requires a variety of growth factors/morphogens, many of which are known to be regulated by HS. During neural differentiation, which is dependent on FGF4, the expression and sulphation levels of cellular HS increase; in the mesodermal lineage, other HS-dependent factors (eg VEGF) play a critical role (Figure 3).
Figure 3: Schematic model of ES cell differentiation
The maintenance of ES cell pluripotency, and differentiation to distinct cell lineages, requires the co-ordinated involvement of a number of different growth factors, many of which can be regulated by HS, eg BMP, FGFs and VEGF. Defined neural and mesodermal differentiation protocols can be used to detail the evolution of cell-type specific HS.
In addition, the availability of HS-mutant ES lines, such as the HS-sulphotransferase mutant (Hs2st-/-), are useful for further understanding the role of HS in maintenance of pluripotency and early differentiation.
The group are interested in correlating changes in HS structure with:
- The expression patterns of the many enzymes involved in HS biosynthesis/modification
- The requirements for specific HS-dependent factors
These approaches should help us to understand how the appearance of inductive sequence motifs in HS is regulated.
Mice that harbour mutations in various HS biosynthetic/modification enzymes present developmental defects ranging from a failure to gastrulate through to limited, tissue-specific effects such as renal agenesis. We have established ES cells from various mutant embryos and are studying their differentiation potentials in vitro, with a view to identifying the signaling pathways that are disrupted. In parallel we are also exploiting an inducible RNAi system to allow us to specifically modulate the HS biosynthetic machinery in real-time, and look for consequent developmental effects.