Expression of hyaluronan and the hyaluronan-binding proteoglycans neurocan, aggrecan, and versican by neural stem cells and neural cells derived from embryonic stem cells

Abstract

We have examined the expression and localization patterns of hyaluronan and hyaluronan-binding chondroitin sulfate proteoglycans in neural stem cells and differentiated neural cells derived from mouse embryonic stem cells. Expression of proteoglycans and hyaluronan was weak in the SSEA1-positive embryonic stem cells but increased noticeably after retinoic acid induction to nestin-positive neural stem cells. After subsequent plating, the hyaluronan-binding chondroitin sulfate proteoglycans aggrecan, neurocan, and versican are expressed by cells in both the astrocytic and neuronal lineages. During the time period that hyaluronan was present, it co-localized with each of the hyaluronan-binding proteoglycans studied and was found to be clearly associated with beta-III tubulin-expressing neurons and oligodendrocytes expressing the O4 sulfatide marker. Although proteoglycan expression levels increased to varying degrees following neural differentiation, they did not change noticably during the following 2 weeks in culture, but there was a significant decrease in hyaluronan expression. Our studies therefore demonstrate the expression by neural stem cells and neural cells derived from them of hyaluronan and its associated proteoglycans, thereby providing a necessary foundation for integrating their specific properties into developing strategies for therapeutic applications.

Fig. 1

Expression of aggrecan in mouse E14T embryonic stem cells (day 0, A-C), and in neural stem cells on the fourth day of retinoic acid treatment during the neural induction period (D-F). Embryonic and neural stem cells are identified by their expression of SSEA-1 and nestin, respectively. Expression of aggrecan by neural cells derived from embryonic stem cells at differentiated day 4 is shown in panels G-J and K-M. Astrocytes and neurons are identified by their expression of GFAP and β-III tubulin. (The punctate labeling occasionally seen in areas where cells are absent represents proteoglycan that is shed or secreted from the cells and adheres to the coverslip.) Nuclear staining with TO-PRO-3 is shown in blue. Magnification: 160×.

Fig. 2

A-C and D-F: Expression of neurocan by neural cells derived from embryonic stem cells at differentiated day 4. Astrocytes and neurons are identified by their expression of GFAP and β-III tubulin. Expression of hyaluronan by mouse embryonic stem cells one day after differentiation to oligodendrocytes expressing the O4 sulfatide marker (G-J), and to β-III tubulin-expressing neurons (K-M). Panels A-C show the feeder-independent E14T cell line. Nuclear staining with TO-PRO-3 is shown in blue. Magnification: 160×.

Fig. 3

Expression of α-versican and β-versican by neural cells derived from embryonic stem cells at differentiated day 7. Neurons and astrocytes are identified by their expression of β-III tubulin and GFAP. Nuclear staining with TO-PRO-3 is shown in blue. Magnification: 160×.

Fig. 4

Higher magnification images (250×) showing the co-expression of aggrecan, α-versican, β-versican and neurocan with neuronal and astrocytic markers (β-III tubulin and GFAP, respectively). Panels A-C, G-J and K-M show differentiated day 4 cells, and panels D-F show differentiated day 7 cells. Nuclear staining with TO-PRO-3 is shown in blue.