Production of heparan sulphate proteoglycans by human bone marrow stromal cells

Haemopoietic progenitors from human bone marrow bind strongly to human marrow stromal cell cultures but the interaction only occurs if the stromal cells are maintained in methyl prednisolone. Heparan sulphate has been implicated in this interaction and in the binding of haemopoietic cell growth factors. In the present study we have compared the molecular structures of the heparan sulphate proteoglycans, metabolically labelled with [35S]sulphate, produced by methyl prednisolone-treated and untreated human marrow stromal cells in vitro. [35S]proteoglycans were examined in the cell layers (extracted with 1% (v/v) Triton X-100 in 6M urea) and in the culture medium. Fractionation of proteoglycans by ionexchange chromatography indicated that the heparan sulphate produced by the treated cultures eluted at a higher NaCl concentration than the counterpart from untreated cells. The heparan sulphate appeared to be mainly expressed on the cell surface, since it was efficiently extracted by treatment with dilute trypsin (50 μg ml−1 for 10 min). All cultures contained two heparan sulphate proteoglycan species, the major component eluted from a Sepharose CL-4B column with a median Kav of 0.33 and apparently contained an average of only one heparan sulphate chain. Small quantities of a larger proteolgycan, which was eluted in the void volume from the CL-4B column, was also detected, mainly in the cell layer extracts. The molecular structure of the heparan sulphate chains was analysed by oligosaccharide mapping, following specific enzymic depolymerisation, and separation of breakdown products by gradient PAGE. The maps revealed significant differences in overall enzyme susceptibilities and sulphation patterns of polysaccharides produced by methyl prednisolone-treated and untreated cultures. The heparan sulphate from the methyl predinsolone-treated cultures was particularly sensitive to the enzyme heparinase (Hep I), which cleaves highly sulphated disaccharides of structure GlcNSO3 (±6S) α (l,4)IdUA (2S). The relatively high frequency of these disaccharides may account for the stronger polyanionic properties of these heparan sulphates indicated by their more retarded elution from ionexchange columns. The changes in molecular structure of heparan sulphate expressed on the surfaces of methyl-prednisolone-treated stromal cells may contribute to their ability to promote binding of immature haemopoietic cells and could enhance the capacity of these cultures to present essential growth factors to the bound cells.