Additional Supporting Information may be found in the online version of this article.

ana23788-sup-0001-SuppTables.docx12KSupplementary Tables
ana23788-sup-0001-SuppFig1.tif5995KSupplementary Figure 1: Characterization of mouse OPC cultures. (A-C) Phase contrast photomicrographs of OPCs derived from neurospheres (A); pre-OLs grown in the presence of T3 and NAC for 24 hours to promote differentiation into pre-OLs (B); and OPCs grown in the presence of T3 and NAC for 72 hours to generate mature OLs. (D) Expression of PDGFR-α (red) in OPC cultures; (E) pre-OLs generated after culturing OPCs for 24 hours with T3 and NAC showing that most of the cells are O4+ (red) with occasional O4+/GalC+ (yellow) cells, characteristic of early OLs; (F) GalC (green) and MBP (red) expression in OLs that differentiated in OPC cultures after 72 hours in the presence of T3 and NAC.
ana23788-sup-0002-SuppFig2.tif6266KSupplementary Figure 2: Accumulation of HA in proliferating OPCs (A-C) and OPCs cultured under pro-differentiation conditions (D-E). OPCs were plated in serum-free media and fixed (A) 1, (B) 3, or (C) 12 hours later, then labeled with an anti-PDGFR-α antibody (green) and HABP (red). OPCs were grown in the presence of T3 and NAC for (D) 24 and (E) 72 hours, then labeled with either an anti-PDGFR-α (D, green) or an anti-MBP (E, green) antibody and HABP (red). Note that the area highlighted in the upper left portion of panel E is shown in the lower right corner of the panel with HABP alone, demonstrating that HA is degraded an MBP+ (*) cell. Cells were counterstained with DAPI (blue) to label cell nuclei. (F) RT-PCR of Has gene expression in OPC cultures.
ana23788-sup-0003-SuppFig3.tif2961KSupplementary Figure 3: Example of a lysolecithin-induced demyelinating lesion labeled with DAPI (A, blue), PDGFR-α (B, green), and CC1 (C, red). (D) A merged image of PDGFR-α and CC1 labeling. To quantify the proportion of cells labeled for PDGFR-α (to visualize OPCs) and CC1 (to visualize the cell bodies of mature OLs), counts were performed in specific zones in and around each lesion as defined by DAPI labeling (see Methods). Zone 1 is the lesion itself excluding the lesion border, seen as an area of increased cell density in panel A. Zone 2 is the border of the lesion, which includes the lesion edge and the area adjacent to the lesion edge where DAPI labeling is decreased. Zone 3 is adjacent unaffected white matter. Note that compared with unaffected white matter, there are elevated numbers of PDGFR-α immunolabeled OPCs (B) and areas of depleted CC1-immunolabeled OLs (arrows, C) whereas at the lesion border there is a modest increase in PDGFR-α labeling (zone 2, B) but a substantial increase in CC1 labeling (see Fig. 3F-G for quantification of three separate experiments).
ana23788-sup-0004-SuppFig4.tif5520KSupplementary Figure 4: Additional examples of PH20 immunolabeling in chronic MS patient lesions. (A) a chronic lesion, showing the border of the lesion (MBP, red) and elevated PH20 expression (green) mostly within the lesion. (B) A high power image of the lesion in A, showing that the majority of the PH20-immunolabeled cells are within the lesion. Cell nuclei are stain with DAPI (blue). (C, D) Lesions from two other cases, showing PH20 expression at varying degrees within lesions and at lesion borders.
ana23788-sup-0005-SuppFig5.tif2571KSupplementary Figure 5: Example of HABP labeling (red, A) in a chronic demyelinated MS patient lesion in areas where PH20 expression (B, green, arrows) is elevated. (C) A merged image, demonstrating that HA is reduced in areas where PH20 labeling is highest.
ana23788-sup-0006-SuppFig6.tif1770KSupplementary Figure 6: Inhibitory activity of increasing concentrations of the pan hyaluronidase inhibitor VCPAL (6-O-Palmitoyl-L-ascorbic acid) on BTH/PH20 as determined using a turbidimetric assay. The final BTH/PH20 concentration was 100 U/ml. Each point represents the mean?SEM of 4 replicate experiments. The VCPAL IC50 was approximately 33 μM.

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