Agents for the prevention and/or treatment of central nervous system damage

Abstract

The present invention relates to the use of agents (including heparin derivatives) for the prevention and/or treatment of CNS damage.

Claims

1. A method of transplanting cells into central nervous system (CNS) tissue in a patient in need of such treatment, said method comprising administering said cells into said CNS tissue and treating said patient with a therapeutically effective amount of an agent selected from the group consisting of: (i) a heparin derivative which is: substantially 6-O desulphated and 2-N desulphated; substantially 2-O desulphated and 6-O desulphated; and/or substantially 2-O desulphated, 6-O desulphated and 2-N desulphated, wherein a heparin derivative that is substantially 6-O desulphated has 50% to 100% of the 6-O atoms of glucosamine moieties desulphated; and (ii) a selective FGF-1 and/or FGF-9 inhibitor, wherein the inhibitor is an antibody; or a pharmaceutically acceptable salt or solvate of said agent, either prior to, during or following cell transplantation into said CNS tissue.

2. The method according to claim 1, wherein said agent is the heparin derivative.

3. The method according to claim 1, wherein the heparin derivative exhibits less than 5% of the Anti-Factor Xa activity of unmodified porcine intestinal mucosal heparin.

4. The method according to claim 1, wherein 50 to 100% of the 6-O atoms of the glucosamine moieties of the heparin derivative are substituted with hydrogen.

5. The method according to claim 1, wherein the heparin derivative has the general structural formula I shown below: ##STR00005## wherein: R.sup.1 and R.sup.2 are selected from hydrogen or sulphate; n is 1 to 30; R.sup.3 is selected from sulphate, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted acyl, substituted or unsubstituted amido and phosphate; R.sup.4, R.sup.5 and R.sup.6 are each separately selected from the group consisting of hydrogen, sulphate, phosphate, substituted or unsubstituted (1-6C)alkyl, substituted or unsubstituted (1-6C)alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted acyl, and substituted or unsubstituted amido; and R.sup.7 and R.sup.8 are each separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted acyl, a terminal monosaccharide group, a terminal disaccharide group and/or fragments or derivatives thereof; or a pharmaceutically acceptable salt or solvate thereof; with the proviso that: (i) between 50% to 100% of the R.sup.2 groups present are hydrogen and between 50% to 100% of the R.sup.3 groups present are hydrogen or a substituent group other than sulphate; (ii) between 50% to 100% of the R.sup.1 and R.sup.2 groups present are hydrogen; or between 50% to 100% of the R.sup.1 and R.sup.2 groups present are hydrogen; and between 50% to 100% of the R.sup.3 groups present are hydrogen or a substituent group other than sulphate.

6. The method according to claim 5, wherein in the heparin derivative of formula I: (i) between 80-100% of all R.sup.2 groups present are hydrogen and 80-100% of the R.sup.3 groups present are hydrogen or a substituent group other than sulphate (e.g. acetyl) and greater than 75% of the R.sup.1 groups are sulphate; (ii) between 80-100% of all R.sup.2 and R.sup.1 groups present are hydrogen and greater than 75% of the R.sup.3 groups are sulphate; and/or (iii) between 80-100% of all R.sup.2 and R.sup.1 groups present are hydrogen and 80-100% of the R.sup.3 groups present are hydrogen or a substituent group other than sulphate.

7. The method according to claim 5, wherein R.sup.4, R.sup.5 and R.sup.6 are each independently selected from hydrogen or sulphate.

8. The method according to claim 1, wherein the heparin derivative has an average molecular weight of from 500 Da to 30 kDa.

9. The method according to claim 1, wherein the degree of polymerisation of the heparin derivative ranges from 2 monomer units to 60 monomer units.

10. The method according to claim 1, wherein the cells are a glial cells.

11. The method according to claim 1, wherein the cells are selected from Schwann cells and olfactory ensheathing cells.

12. The method according to claim 1, wherein a heparin derivative that is substantially 6-O desulphated has 75% to 100% of the 6-0 atoms of glucosamine moieties desulphated.

13. The method according to claim 1, wherein a heparin derivative that is substantially 6-O desulphated has 90% to 100% of the 6-0 atoms of glucosamine moieties desulphated.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. HS from a variety of tissue sources can induce a boundary in OEC:astrocyte confrontation assays. 30 g/ml HS purified from brain (BHS, B), liver (LHS, C) and porcine intestinal mucosa (PMHS, D), were added to confrontation assays of OECs and astrocytes and compared to an untreated control (A). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75.sub.NTR (green). The numbers of OECs mingling with astrocytes across a 300 m line were counted (E). HS from all 3 tissue sources prevented OECs from crossing into the astrocyte monolayer, resulting in the formation of a boundary. Error bars indicate SEM. Scale bar 50 m. * p<0.05, *** p<0.001 versus control.

(2) FIG. 2. Both HS and protein components of SCM are required for boundary formation. Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); heparinase treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75.sub.NTR (green). The number of cells mingling with astrocytes was counted across a 300 m line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate SEM. Scale bar 50 m. ** p<0.01 versus control.

(3) FIG. 3. HS sulfation is critical for boundary formation. Confrontation assays of OECs and astrocytes were carried out in the presence of 10 g/ml modified heparins (A-J). The disaccharide structures of the heparins are indicated. After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75.sub.NTR (green). Assays were scored using a graded scoring system to assess the ability of each modified heparin to promote boundary formation, i.e., mingling (), partial boundary (+/) or complete boundary (++) (K). Control heparin effect was scored as ++. Modified heparins with the highest levels of sulfation (Hep1, Hep2, Hep3, Hep4 and Hep9) induced boundaries between OECs and astrocytes, whereas those with lower levels of sulfation (Hep5, Hep6, Hep7 and Hep8) did not. The images are representative of at least 3 independent experiments. Scale bar 50 m.

(4) FIG. 4. SAX-HPLC analysis of fluorescently labelled HS disaccharides purified from OCM and SCM indicate that SCs and OECs secrete distinct HS structures. HS purified from OCM (A) and SCM (B) was digested with heparinase enzymes to generate HS disaccharides, which were fluorescently labelled with BODIPY and separated by SAX-HPLC over a 45 minute, 0-1.5 M NaCl gradient, (A and B, black lines). HS disaccharide standards were separated over the same gradient as a reference guide (A and B, dashed lines). Relative abundances of the eight HS standard disaccharides in each conditioned medium sample were calculated as a percentage of total HS (C). A summary table detailing the composition of OCM HS and SCM HS is also presented (D). SCM HS was found to be more highly sulphated than OCM HS, with an average of 1.02 sulfates per disaccharide compared to 0.75 sulfates per disaccharide in OCM HS (D). SCM HS contained a higher proportion of di- and tri-sulphated disaccharides (disaccharides 4, 5, 8 and 6) and the singly 6-O-sulphated disaccharide (disaccharide 2) compared to OCM HS (C), which contained a higher proportion of the unsulphated disaccharide (disaccharide 1) (C, D). (UA) uronic acid, (GlcN) glucosamine, (NAc)N-acetyl, (NS)N-sulfate, (6OS) 6-O-sulfate, (2OS) 2-O-sulfate.

(5) FIG. 5. Expression profile of HS biosynthetic enzymes and HSPGs in OECs and SCs. Using qRT-PCR, the expression of HS biosynthetic enzymes and HSPG core proteins by OECs and SCs were compared. Data are represented as relative quantification (RQ) of HS biosynthetic enzymes (A-C) and HSPGs (D-E) in SCs, normalised to the expression in OECs. HS6ST2, Sulf1 and Sulf 2 were more highly expressed by OECs. HS3ST6 was expressed uniquely by SCs, whereas HS6ST3 and HS3ST3a1 were expressed uniquely by OECs. HS3ST2, GPC2 and GPC5 were not expressed by either cell type. Error bars indicate SEM. * p<0.05, *** p<0.001 versus control.

(6) FIG. 6. Knockdown of Sulf expression in OECs promotes boundary formation with astrocytes. Confrontation assays were performed using astrocytes and either OECs transfected with nontargeting control siRNA (A) or OECs transfected with siRNAs targeted to Sulf 1 and Sulf 2 (B). After 2 days, cells were fixed and stained for GFAP (red) and p75.sub.NTR (green). OECs depleted of Sulf 1 and Sulf 2 expression formed a boundary on contact with astrocytes, whereas astrocytes and control treated OECs mingled. The extent of gene knockdown was assessed by qPCR using RNA purified from siRNA treated cells and Sulf 1 and Sulf 2 specific primers and was found to be 70% and 63% reduced respectively (C). Error bars indicate SEM. Scale bar 50 m. *p<0.05, ** p<0.01 versus control.

(7) FIG. 7. Interference of endogenous SC HS function using HS mimetics to inhibit boundary formation. Confrontation assays of SCs and astrocytes were carried out in the absence (A) or presence (B-D) of 10 g/ml selectively chemically modified heparins (Hep 6, 7, 8; see FIG. 3 for structural details) with low levels of sulfation. After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75.sub.NTR(green) and the numbers of SCs mingling with astrocytes across a 300 m line were counted (E). The addition of low sulphated modified heparins (HSmimetics) to confrontation assays resulted in an increased number of SCs mingling with astrocytes (B-D) compared to in control cultures (A). Error bars indicate SEM. Scale bar 50 m. * p<0.05 versus control.

(8) FIG. 8. Neutralisation of FGF1 and FGF9 disrupts boundary formation between SCs and astrocytes. SC and astrocyte confrontation assays were treated with 1 g/ml FGF1 (B), FGF2 (C) or FGF9 (D) blocking antibodies for 2 days after the cell populations had met. Cells were fixed and stained for GFAP (red) and p75.sub.NTR(green) and the numbers of SCs mingling with astrocytes across a 300 m line were counted (E). Neutralisation of FGF1 and FGF9, but not FGF2, resulted in an increase in mingling between SCs and astrocytes compared to control cultures (A). Error bars indicate SEM. Scale bar 50 m. * p<0.05, *** p<0.001 versus control.

(9) FIG. 9. A model of HS function in SC/astrocyte boundary formation. SCs secrete a highly sulphated HS (green chains), due to low level expression of Sulf enzymes. This HS promotes FGF1 and/or FGF9 ligand (black circles) binding to and/or activation of FGFR3-IIIb receptor (blue) on astrocytes, activating intracellular signalling pathways and resulting in altered cell phenotype (blocking of mingling) and the formation of a boundary. OECs secrete a lower sulphated HS (red chains), due to higher expression of Sulf enzymes (yellow stars), which selectively remove 6-O-sulfates. These lower sulphated HS chains cannot promote FGF1 or FGF9 binding to and/or activation of FGFR3-IIIb on astrocytes, and so fail to activate the required signalling pathways that lead to boundary formation, thus, enabling OECs to mingle with astrocytes.

(10) FIG. 10. Table showing the disaccharide composition of the heparin derivatives prepared in Example 1.

EXAMPLES

(11) The invention will now be illustrated in the following Examples.

Example 1

Preparation of Modified Heparin Derivatives

(12) Chemically modified heparin compounds (A) to (I) were prepared by the following combinations of reactions (a) to (g) set out below:

(13) HEP1 PIMH starting material (Celsus Labs, Cincinnati, Ohio);

(14) HEP2 N-acetyl heparin (d) (f);

(15) HEP3 Ido 2-de-O-sulphated heparin (a);

(16) HEP4 6-O-desulphated heparin (b) (e);

(17) HEP5 Ido 2-de-O-sulphated, N-acetylated heparin (a) (d) (f);

(18) HEP6 6-O-desulphated, N-acetylated heparin (b) (f);

(19) HEP7 6-O-desulphated, 2-O-desulphated heparin (c) (e);

(20) HEP8 6-O-desulphated, 2-O-desulphated, N-acetylated heparin (c) (f); and

(21) HEP9 Per-sulphated heparin (g) (e).

(22) Compounds were characterized by .sup.1H and .sup.13C NMR as previously described. (Yates et al., Carbohydrate Research 1996, 294, 15-27.) Compounds were desalted, lyophilized and re-suspended in the appropriate buffer prior to assay.

(23) Chemical Reactions

(24) (a) Selective removal of iduronate 2-O-sulphate was achieved as described by Jaseja and Perlin. (Jaseja, M.; Rej, R. N.; Sauriol, F.; Perlin, A. S. Can. J. Chem. 1989, 67, 1449-1456.) Note that there is concomitant modification in the small number of N- and 3-O-sulphated glucosamine units. (Santini, F.; Bisio, A.; Guerrini, M.; Yates, E. A. Carbohydrate Research 1997, 302, 103-108.) (b) Selective removal of glucosamine 6-O-sulphate was carried out according to a modification (Yates, E. A. et al. supra.) of the method described. (Inoue, S.; Miyawaki, M. Analytical Biochemistry 1975, 65, 164-174.) (c) Complete removal of O- and N-sulphates was achieved using solvolytic de-sulphation by the method described. (Inoue, S.; Miyawaki, M. supra.) (d) Selective de-N-sulphation was carried out employing controlled solvolytic de-sulphation under kinetic control as described. (Inoue, Y.; Nagasawa, K. Carbohydrate Research 1976, 46, 87-95.) (e) Re N-sulphation was achieved by use of trimethylamine.sulfur trioxide complex as described. (Lloyd, A. G.; Embery, G.; Fowler, L. J. Biochemical Pharmacology 1971, 20, 637-648.) (f) Re N-acetylation employed acetic anhydride in saturated sodium bicarbonate. (Yates, E. A. et al. supra.) (g) Complete O-sulphation of all available hydroxyl groups was carried out using excess sulfur trioxide pyridine complex on the tetrabutylammonium salt of heparin in pyridine as described (Yates, E. A.; Santini, F.; De Cristofano, B.; Payre, N.; Cosentino, C. et al. Carbohydrate Research 2000, 329, 239-247.) followed by re-N-sulphation (Lloyd, A. G. et al. supra.) taking precautions to avoid formation of an unusual N-sulfoaziridine modification. (Yates, E. A.; Santini, F.; Bisio, A.; Cosentino, C. Carbohydrate Research 1997, 298, 335-340).
Compound Purity

(25) The starting material for all chemical modifications was PIMH (Celsus Labs, Cincinnati, Ohio, USA; lot PH-42800 with anticoagulant activity 201 IU/mg).

(26) Each polysaccharide HEP1-8 was subjected to purification by size-exclusion chromatography (Sephadex G-25, recovering only the exclusion limit; M.sub.w>5 KDa) and treated with ion exchange resin (Dowex, W-50, Na.sup.+ form) prior to NMR and activity testing.

(27) Size-exclusion chromatography analysis of the 8 polysaccharides was also conducted with a TSK gel G2000SW.sub.XL column (7.8 mm30 cm with 0.5 m particle size; Supelco) eluting with water at 1 ml/min and detecting at 190 nm. All the samples exhibited a single major peak, with very similar retention times (mean, 6.07 minutes; .sub.n-1=0.05). In all cases, there were no contaminants greater than 5%.

(28) Polysaccharides HEP1-8 were also exhaustively digested with a mixture of heparinases I, II and III to their constituent disaccharides (here denoted D1 to D8) determined. Disaccharides were separated by strong-anion exchange HPLC (Propac PA-1 column, Dionex UK; [ref 2]) and quantified (A.sub.232) with reference to authentic standards (Dextra Labs, Reading, UK) and showed the following composition (%). In all cases, unidentified peaks were <5% of the total area of the constituents. The data is shown in FIG. 10 and conforms exactly to the compositions predicted from the modifications performed, and are in agreement with the NMR structure data (see below).

(29) NMR Spectroscopy

(30) The polysascharides were characterised by .sup.1H and .sup.13C NMR to confirm their structure [Yates, E. A.; Santini, F.; Bisio, A.; Cosentino, C. Carbohydrate Research 1997, 298, 335-340]. NMR spectra were recorded in D.sub.2O at 40 C. on a 400 MHz instrument. Assignment was by a combination of COSY, TOCSY, HMBC two-dimensional spectra. .sup.13C spectra were recorded on 150 mg samples of the polysaccharide. Chemical shift values were recorded relative to trimethylsilyl propionate as reference standard at 40 C.

(31) TABLE-US-00001 Table of .sup.1H and .sup.13C NMR chemical shift values for heparin derived polysaccharides HEP1-9 Glucosamine Iduronate Polysaccharide A-1 A-2 A-3 A-4 A-5 A-6 I-1 I-2 I-3 I-4 I-5 HEP1 99.5 60.7 72.5 78.8 72.0 69.2 102.1 78.9 72.1 79.0 72.3 5.42 3.31 3.69 3.79 4.05 4.30-4.42 5.23 4.37 4.22 4.14 4.82 HEP2 96.6 56.2 73.0 79.3 72.3 69.6 102.2 76.8 67.3 74.2 70.8 5.15 4.03 3.76 3.78 4.04 4.31-4.37 5.20 4.37 4.31 4.08 4.91 HEP3 98.1 60.3 72.4 80.1 71.5 68.7 104.6 71.1 70.4 77.2 71.2 5.34 3.24 3.65 3.71 4.02 4.36 4.23 5.04 3.78 4.12 4.08 4.84 HEP4 100.0 60.8 72.4 80.5 73.8 62.6 102.0 77.6 70.7 78.7 71.4 5.31 3.27 3.71 3.70 3.89 3.86-3.88 5.26 4.35 4.25 4.06 4.84 HEP5 97.1 56.2 72.5 79.6 71.8 68.8 104.6 72.0 71.4 77.0 71.9 5.18 4.00 3.78 3.79 4.08 4.37-4.26 5.01 3.75 3.42 4.10 4.78 HEP6 96.8 56.6 72.9 80.6 74.2 62.9 102.3 76.6 67.1 74.1 70.6 5.14 4.03 3.79 3.76 3.91 3.87-3.92 5.26 4.37 4.28 4.07 4.91 HEP7 98.2 60.5 72.5 80.2 73.5 62.4 104.3 72.2 71.5 77.8 72.2 5.39 3.26 3.67 3.72 3.87 3.84-3.88 4.95 3.74 4.11 4.08 4.77 HEP8 97.1 56.2 72.3 79.6 73.7 62.3 104.3 72.5 72.2 77.3 72.6 5.18 3.97 3.76 3.74 3.89 3.85-3.88 4.92 3.69 3.89 4.07 4.73 HEP9 99.6 59.3 82.9 76.8 72.1 68.7 100.8 73.6 72.9 73.3 69.8 5.32 3.50 4.48 4.04 4.05 4.27-4.41 5.32 4.55 4.72 4.39 5.05

(32) The .sup.1H chemical shift values quoted for position-6 of glucosamine residues (A-6) are intervals. Signals from the carbonyl group of iduronate and acetyl CH.sub.3 groups of N-acetylated glucosamine derivatives are not shown.

Example 2

The Anticoagulant Activity of the Heparin Derivatives Prepared in Example 1

(33) Experimental Protocol:

(34) The anti-factor Xa activity was measured against a PIMH standard of known activity using a diagnostic grade Coatest Heparin test kit (Chromogenix, MA), adapted to a 96-well plate format, reading A405.

(35) Results:

(36) The Table below shows the anticoagulant activity (anti-Xa activity) of the heparin derivatives described in Example 1 herein. Seven of the heparin-derivatives (HEP 2 to H) 50 including the six O-de-sulphated heparins of the present invention (HEP to HEP8) show <1.5% anti-Xa activity compared with that of the standard heparin (HEP1).

(37) Anticoagulant Activity Data.sup.16

(38) TABLE-US-00002 Anti- coagulant Compound R.sub.1 R.sub.2 R.sub.3 activity HEP1 PMIH SO.sub.3 SO.sub.3 SO.sub.3 100% HEP2 N-acetyl SO.sub.3 SO.sub.3 COCH.sub.3 0.03% HEP3 UA-2-OH H SO.sub.3 SO.sub.3 0.4% HEP4 GlcN-6-OH SO.sub.3 H SO.sub.3 0.5% HEP5 UA-2-OH, N-Acetyl H SO.sub.3 COCH.sub.3 0.03% HEP6 GlcN-6-OH, N-acetyl SO.sub.3 H COCH.sub.3 0.03% HEP7 UA-2-OH, GlcN-6-OH H H SO.sub.3 0.03% HEP8 UA-2-OH, GlcN-6-OH, H H COCH.sub.3 0.03% N-acetyl HEP9 Per-sulphated SO.sub.3 SO.sub.3 SO.sub.3 35.0%

Example 3

Assessments of Anti-Coagulant Activity of the Heparin Derivatives

(39) Factor Xa and Factor II activity was measured using a colorimetric substrate assay as previously described.sup.17. Briefly, in a 96 well ELISA plate, antithrombin III (American Diagnostica, 30 mIU/ml final concentration) was incubated with heparin or polysaccharide fractions in 0.9% NaCl at 37 C. for 2 minutes. Factor IIa (Sigma, 15 mU/ml final concentration) or Factor Xa (Thermo Scientific, 15 mU/ml final concentration) was added and the samples incubated for a further 1 minute at 37 C. Factor Xa Chromogenic substrate (Sigma, 240 uM final concentration) was added to the samples and incubated for 10 minutes at 37 C. The reaction was stopped with glacial acetic acid (Sigma, 25% final concentration). Colour change of the substrate was measured at 405 nm.

(40) APTT and PT assays were performed using a Axis Shield Thrombotrack 1 instrument using normal human plasma, Pathrombin SL reagent and Thromborel S (all from Axis Shield) reagents according to manufacturers instructions.

(41) For all assays, heparins and polysaccharides were tested up to 100 ug/ml. Heparin was an anticoagulant in all assays with IC50 values of 0.9, 1.0, 1.2 and 41.4 ug/ml for the Factor Xa, Factor II, APTT and PT assays respectively.

(42) Results and Conclusion:

(43) No polysaccharide fraction had anticoagulant activity (<1% of heparin activity).

(44) Table Showing Anticoagulant Activity of Heparin and HEP5-8

(45) TABLE-US-00003 Com- IC50 IC50 IC50 IC50 pound Structure Factor Xa Factor IIa APTT PT Heparin 0.9295 1.0326 1.2461 41.4005 Hep5 I2OH, NAc NI NI NI NI Hep6 A6OH, NAc NI NI NI NI Hep7 I2OH, A6OH NI NI NI NI Hep8 I2OH, A6OH, NAc NI NI NI NI IC50s in ug/ml NI: No inhibition of coagulation up to 100 ug/ml of compound

Example 4

Biological Evaluation

(46) Materials and Methods

(47) Generation of Purified Glial Cells

(48) All primary neural cultures were generated from Sprague-Dawley rat pups of either sex. As described previously.sup.10,37, purified type 1 astrocytes were prepared by digesting cortices (dissected from 1-day old Sprague Dawley (SD) rats) in 1.33% (w/v) collagenase (Sigma-Aldrich, Gillingham, UK), seeding (2107 cells per 75 cm.sup.2 flask) and culturing the cells in poly-L-lysine (PLL, 13.3 g/ml, Sigma-Aldrich, Gillingham, UK) coated 75 cm2 flasks for 10-12 days. The cells were maintained in DMEM (Invitrogen, Paisley, Scotland) supplemented with 10% (v/v) foetal bovine serum (FBS) (Sigma-Aldrich, Gillingham, UK) and L-glutamine (2 mM, Sigma-Aldrich, Gillingham, UK) (DMEM-FBS). Confluent flasks were shaken on a rotary platform overnight at 37 C. to remove contaminating oligodendrocyte precursor cells. The remaining cells were 85-95% type 1 astrocytes as identified by labelling for glial fibrillary acidic protein (GFAP), an astrocyte cell specific marker.

(49) OECs were isolated from the olfactory bulb of 7 day old SD rat pups and purified using magnetic nanoparticles (STEMCELL Technologies, UK) pre-bound with the p75.sup.NTR antibody (mouse IgG1, Abcam, Cambridge, UK).sup.30. The cells were grown in low glucose DMEM with 5% (v/v) FBS and 2 mM L-glutamine and further supplemented with DMEM-BS (Bottenstein, 1979), FGF2 (25 ng/ml, Peprotech, London UK), heregulin -1 (50 ng/ml, R&D Systems, Oxon, UK), forskolin (510-7 M Sigma-Aldrich, Gillingham, UK) and astrocyte conditioned media (ACM) (1:5, fresh serum-free media taken after incubation with a confluent astrocyte monolayer for 48 h).sup.18, 37.

(50) SCs were purified using a modification of the method described by Brockes and colleagues.sup.20. This modification involved treating the cultures with cytosine arabinoside (AraC, 10.sup.5 M, Sigma-Aldrich, Gillingham, UK), followed by incubation with anti-Thy1.1 antibody (1:1 supernatant, Sigma-Aldrich, Gillingham, UK) and rabbit complement (1:6, Harlan Laboratories Ltd., UK) to reduce contamination by fibroblasts.sup.10. All cell cultures were grown in PLL coated tissue culture flasks.

(51) Collection of SC Conditioned Medium (SCM), OEC Conditioned Medium (OCM) and Astrocyte Conditioned Medium (ACM)

(52) Confluent cultures of purified SCs or OECs in 75 cm2 flasks (maintained in vitro for 2-6 weeks) were rinsed twice with phosphate buffered saline (PBS), pH 7.4 and 7 ml of DMEM-BS without growth factors added. Cultures were maintained for a further 2 days before medium collection. Collected medium was centrifuged to remove cellular debris and filtered through a 0.2 m filter (Millipore, Hertfordshire, UK). The same procedure was used for generating ACM, except that confluent astrocyte cultures were maintained in 11 ml of DMEM-BS. Conditioned media was added to cell cultures at a 1:1 ratio with DMEM-FBS.

(53) Confrontation Assays

(54) Confrontation assays were performed as described by Wilby et al. (1999) and Lakatos et al. (2000) with some modifications.sup.10,48. Briefly, 70 l containing 10,000 OECs or SCs were seeded into one well of a silicon Ibidi culture insert on a PLL-coated glass coverslip (Ibidi GmbH, Munich, Germany). Into the opposing, parallel well, 10,000 astrocytes were seeded. Cells were allowed to attach for 1 h before careful removal of the insert followed by a wash with DMEM-FBS to remove unattached cells. Cultures were maintained in DMEM-FBS and allowed to grow towards each other over a period of 5-7 days, allowing time for cells to make contact and interact.sup.10. In some experiments, modified heparins or blocking antibodies were added to the cultures after the cells had contacted each other. Cultures were then immunolabelled using anti-GFAP for astrocytes (1:500; anti-rabbit (Dako, Ely, UK)) and anti-p75.sub.NTR for OECs and SCs (1:1; IgG1; hybridoma supernatant.sup.49. Fluorescent images were captured using an Olympus BX51 fluorescent microscope and Image-Pro software. Using Adobe Photoshop Elements 7.0, a 300 m line was drawn along the interface between astrocytes and either OECs or SCs. The numbers of OECs or SCs crossing the cell:cell boundary were counted and averaged over five randomly chosen fields. Experiments were repeated at least three times.

(55) Treatments:

(56) Modified Heparins

(57) Modified heparins were produced semisynthetically by chemical modification (selective desulfation) of heparin as described in Example 1. These structurally distinct, model HS-mimetic polysaccharides.sup.50 are useful tools for investigating structure-activity relationships of HS.sup.15-17,31. Heparins were added to confrontation assays at 10 g/ml at the stage when cells made contact (day 0) and treatment was repeated on day 2. Cultures were fixed and stained as described above on day 3.

(58) HS from Various Tissue Sources

(59) Porcine mucosal HS (PMHS) was a gift from Organon (Oss, Netherlands), porcine liver and rat brain HS were purified using previously described methods (Lyon and Gallagher, 1991; Esko, 2001). Confrontation assays were treated with polysaccharides for 2 days (day 0 and day 2) at a final concentration of 30 g/ml.

(60) Heparinase and Trypsin Treatment of Conditioned Medium

(61) Proteins in SCM were digested by the addition of trypsin at a final ratio of 1:50 trypsin:protein and incubated for 12 h at 37 C. (amount of protein was estimated by measuring the UV absorbance at 280 nm). Trypsin was inactivated by addition of soybean trypsin inhibitor ( 1/10 of final trypsin concentration). HS was digested by the addition of 10 mU each of heparinase I (EC 4.2.2.7), II (EC number not assigned) and III (EC 4.2.2.8) (Ibex Technologies, Montreal, Canada) to 4 ml SCM, followed by incubation at 37 C. for 6 h. A further 10 mU of each heparinase enzyme was added to SCM and the reaction incubated overnight at 37 C. Treatment of confrontation assays was carried out by replacing half of the media with untreated SCM, heparinase-treated SCM, trypsin-treated SCM or 50:50 heparinase:trypsin treated SCM. Confrontation assays were treated for 2 days.

(62) FGF Inhibition

(63) Anti-FGF2 (Clone bFM-1) (Millipore, Hertfordshire, UK), anti-FGF9 (Clone 36912) and anti-FGF1 (both R&D Systems, Oxon, UK) neutralising antibodies were added to confrontation assays at a final concentration of 1 g/mL, at the stage when cells made contact. Treatment was repeated for 2 days and then cultures were fixed and stained as described above on day 3.

(64) HPLC Analysis of HS Disaccharides in SCM and OCM

(65) SCM and OCM were collected from 75 cm2 flasks of confluent OECs or SCs, frozen and stored at 20 C. until enough material was collected for detection (180 mL OCM and 90 mL SCM). OCM and SCM were rotated with 0.1 ml DEAE-sephacel (Sigma-Aldrich, Gillingham, UK) per 10 mL, overnight at 4 C., and then centrifuged at 3382g for 5 min and unbound material in the supernatant removed. DEAE beads were washed three times with 10 column volumes of PBS, and then washed twice with 10 column volumes of 0.25 M NaCl in PBS. Bound material containing HS proteoglycans (HSPGs) was eluted with 10 column volumes of 2 M NaCl in PBS and desalted over two, in-line 5 mL Hi Trap desalting columns (GE Healthcare UK Ltd, Buckinghamshire, UK) using an AKTA-FPLC system. Desalted material was then freeze-dried.

(66) Heparinase Digestion

(67) Lyophilised material was taken up in heparinase buffer (100 mM sodium acetate, 0.1 mM calcium acetate, pH 7) and 2.5 mU each of heparinase I, II and III were added and incubated for 3 h at 37|C. After this time, a further 2.5 mU of heparinase I, II and III were added and the reaction incubated overnight at 37|C. Another 2.5 mU of heparinase I, II and III were then added and incubated for a further 3 h. As a control, 100 g heparin was digested in the same way. Digested samples were then incubated at 95 C. for 5 min to stop the reaction and taken up in 800 l HPLC-grade water.

(68) C18 HPLC

(69) HS disaccharides were separated out from samples using a Discovery C18 HPLC column (Supelco, Sigma-Aldrich, Gillingham, UK) (25 cm4.6 mm, 5 m) on a Simadzu SPD 10 A HPLC system. Buffer A was HPLC-grade water and Buffer B was 80% (v/v) methanol. Elution profiles were monitored by UV absorbance at 232 nm. Samples were injected in buffer A and the void volume containing HS disaccharides was collected and freeze-dried for BODIPY labelling. Bound material (containing hydrophobic material, including HSPG core proteins), was eluted using a linear gradient of 0-50% buffer B over 30 min at a flow rate of 1 ml min.sup.1.

(70) BODIPY Labelling of HS Disaccharides

(71) Freeze-dried HS disaccharides were labelled with BODIPY FL hydrazide (5 mg/ml; 4,4-di fluoro-5,7-di methyl-4-bora-3a,4a-diaza-S-indacene-3-propionic acid hydrazide; Molecular Probes) as previously described.sup.52-54. Digested heparin control and HS disaccharide standards (Iduron, Manchester, UK) were labelled in the same way. Labelled samples were applied to silica gel thin layer chromatography (TLC) aluminium plates and free BODIPY tag separated from labelled disaccharides with butanol. Labelled HS disaccharides were removed from the TLC plates and solubilised in 800 l HPLC-grade water.

(72) SAX-HPLC

(73) SAX separations were performed on a Propac PA1 column (25 cm9 mm, 5 m) using a Simadzu SPD 10A HPLC system. Elution profiles were monitored by UV absorbance at 232 nm and fluorescent detection using a Shimadzu RF10AXL spectrofluorometer. Buffer A was 150 mM NaOH and Buffer B was 150 mM NaOH, 2 M NaCl. Elution profiles were monitored by UV absorbance at 232 nm and fluorescent detection at .sub.ex=488 nm .sub.em=520 nm. Samples were injected and the flow held at 2 ml min.sup.1 in buffer A until all remaining free tag had been eluted. Fluorescently labelled disaccharides were then eluted using a linear gradient of 0-50% buffer B over 45 min at 2 ml min.sup.1. The column was then washed with a 10 min elution in 300 mM NaOH, 2 M NaCl, before returning to 150 mM NaOH.

(74) Quantitative Real-Time PCR

(75) RNA from monocultures of OECs and SCs was extracted using a Qiagen RNeasy Mini Kit (Qiagen, West Sussex, UK) following manufacturer's instructions and RNA quality and integrity were checked using the Nanodrop 1000 (Thermo Fisher Scientific Inc, IL, USA). Following RNA extraction, cDNA was synthesised from 1 g RNA using the Quantitect Reverse Transcription kit (Qiagen, West Sussex, UK). Real-time PCR was performed with 100 ng cDNA in a 20 l reaction volume using QuantiTect primer assays and the Quantifast SYBRgreen PCR kit (Qiagen, West Sussex, UK). Experiments were performed in triplicate for each sample in 96-well plates using the Applied Biosystems 7500 real-time PCR system. PCR cycle settings were 95 C. for 5 min, followed by 40 cycles of 95 C. for 10 s, then 60 C. for 30 s. Cycle threshold was calculated based on GAPDH (endogenous control), which was confirmed to be comparable in both cell types. Expression of all genes were expressed relative to GAPDH in each sample, derived using the comparative delta delta threshold change method (relative quantification, RQ). Three independent cell preparations were analysed.

(76) Quantitative Real-Time PCR

(77) RNA from monocultures of OECs and SCs was extracted using a Qiagen RNeasy Mini Kit (Qiagen, West Sussex, UK) following manufacturer's instructions and RNA quality and integrity were checked using the Nanodrop 1000 (Thermo Fisher Scientific Inc, IL, USA). Following RNA extraction, cDNA was synthesised from 1 g RNA using the Quantitect Reverse Transcription kit (Qiagen, West Sussex, UK). Real-time PCR was performed with 100 ng cDNA in a 20 l reaction volume using QuantiTect primer assays and the Quantifast SYBRgreen PCR kit (Qiagen, West Sussex, UK). Experiments were performed in triplicate for each sample in 96-well plates using the Applied Biosystems 7500 real-time PCR system. PCR cycle settings were 95 C. for 5 min, followed by 40 cycles of 95 C. for 10 s, then 60 C. for 30 s. Cycle threshold was calculated based on GAPDH (endogenous control), which was confirmed to be comparable in both cell types. Expression of all genes were expressed relative to GAPDH in each sample, derived using the comparative delta delta threshold change method (relative quantification, RQ). Three independent cell preparations were analysed.

(78) siRNA Transfections

(79) Purified OECs were seeded at a density of 5000 cells/100 l into one chamber of an Ibidi culture insert (Ibidi GmbH, Munich, Germany) sealed onto a PLL coated coverslip in a well of a 24-well plate. Cells were cultured in defined OEC medium for 24 h, after which, the medium was replaced with low serum (2% (v/v) FBS) OEC medium containing 1 M siRNA. siRNA sequences were obtained from Dharmacon/Thermo Scientific (Sulf 1: E-093746-00-0005; Sulf2: E-093673-00-0005; non-targeting: D-001910-01-05; Thermo Fisher Scientific Inc, IL, USA). Sulf 1 and Sulf 2 siRNAs were added in combination. After 72 h, astrocytes were seeded into the opposing chamber of the culture insert and confrontation assays performed as previously described. The extent of gene knockdown was assessed by qPCR using RNA purified from siRNA treated cells and Sulf 1 and Sulf 2 specific primers.

(80) Results

(81) The induction of astrocyte hypertrophy by SCs and the resulting formation of a cellular boundary remains a barrier to their use in cell transplantation therapies for the repair of spinal cord injury. Previous work has shown that SC induced boundary formation involves HS and FGFRs, since digestion of HS or chemical blockage of FGFR inhibits boundary formation and promotes cell mingling.sup.11. Improved knowledge of the biological factors and signalling pathways underlying SC-induced boundary formation will aid the development of strategies to improve the incorporation of transplanted SCs into host CNS tissue and prevent activation of an astrocytic stress response by invading host SCs, to facilitate injury repair.

(82) HS from Different Sources Induces OEC/Astrocyte Boundaries

(83) Heparin has been shown to induce a boundary between OECs and astrocytes.sup.11. To determine if this activating effect of heparin on boundary formation could be induced by more physiologically relevant HS species, confrontation assays of OECs and astrocytes were treated with HS purified from different tissues. Whilst OECs and astrocytes mingled freely in control assays (FIG. 1A), strong boundaries were formed upon addition of HS purified from rat brain (FIG. 1B), rat liver (FIG. 1C) or porcine intestinal mucosa (FIG. 1D). Quantification of the extent of boundary formation, assessed by the number of OECs crossing into the astrocyte monolayer, showed that HS from all 3 sources had similar activity (FIG. 1E).

(84) Induction of an OEC/Astrocyte Boundary by SCM Requires Both a Protein and HS Component

(85) Initially, the relative requirements for protein and HS components in SCM for boundary formation were investigated. The active role of SCM HS in the induction of an OEC:astrocyte boundary was confirmed, since heparinase treatment of SCM negates the boundary forming effect (FIG. 2C). SCM treated separately with either heparinase or trypsin did not induce boundary formation (FIG. 2C, D, F), whilst combining the separately treated SCM samples reconstituted its biological activity and induced boundary formation (FIG. 2E, F). These data demonstrate that the activity of SCM in inducing boundary formation between cells requires both an HS and protein component, and that individually, these components are insufficient to induce a boundary.

(86) HS Sulfation Determines the Induction of OEC/Astrocyte Boundaries

(87) In order to establish the effects of sulfation pattern on the inductive activity of HS, a panel of structurally defined, chemically modified (selectively desulphated) heparins were used to investigate the structural specificity of HS activity on boundary formation. After treatment with modified heparins (10 g/ml) for 2 days, confrontation assays were stained for p75.sup.NTR and GFAP to visualise OECs and astrocytes, respectively. Results indicated that the most highly sulphated heparins (normal heparin (Hep1) or oversulphated heparin (Hep 9)) induced the strongest boundaries between OECs and astrocytes (FIG. 3 A, I), whilst lower sulphated heparin variants allowed OECs and astrocytes to mingle (FIG. 3 E-H). Interestingly, boundary formation was particularly dependent on O-sulfation, since N-acetylated heparin (in which N-sulfates are replaced with N-acetyl groups) induced significant boundary formation (Hep 2, FIG. 3 B), whereas either de-2-O-sulphated or de-6-O-sulphated heparins demonstrated lower boundary forming activity (Hep 3 and Hep 4, FIG. 3 C, D).

(88) SCs Secrete More Highly Sulfated HS than OECs

(89) Since it has been have shown that HS in SCM plays an active role in boundary formation, the structure of HS synthesised by SCs and OECs and shed into their surrounding environment was directly analysed. The disaccharide composition of SCM and OCM HS was, therefore, analysed via separation by strong anion exchange (SAX)-HPLC (FIG. 4A, B) and the relative abundance of each of the eight standard HS disaccharides were calculated as a percentage of total HS (FIG. 4C). SCM contains approximately two fold more HS than OCM, and in addition, SCM HS is more highly sulfated, with an average of 1.02 sulfates per disaccharide, compared to 0.75 sulfates per disaccharide in OCM HS (FIG. 4D). SCM HS consists of a higher proportion of di and tri-sulfated disaccharides (disaccharides 4, 5, 8 and 6) compared to OCM HS, and also a higher proportion of the singly 6-O-sulfated disaccharide (disaccharide 2). OCM HS, in contrast, contains a higher proportion of the unsulfated disaccharide (disaccharide 1). The proportion of singly N-sulfated (disaccharide 3) and singly 2-O-sulfated (disaccharide 7) disaccharides are similar in HS from both conditioned media.

(90) SCs and OECs Express Different Levels of HS Biosynthetic Enzymes

(91) To determine if differences in the expression of HS biosynthetic enzymes could account for the higher sulfation of SCM HS, quantitative PCR was carried out using cDNA generated from monocultures of OECs and SCs. Whilst there appeared to be a trend towards differences in expression of several enzymes by SCs compared to OECs, these were not significant due to variability in biological replicates. For example, there were no significant differences in the expression of N-deacetylase/N-sulfotransferase (NDST1-4) enzymes or many of the sulfotransferase enzymes. However, HS6-O-sulfotransferase 2 (HS6ST2) was expressed at a significantly higher level by OECs compared to SCs (FIG. 5B). In addition, HS6-O sulfotransferase 3 (HS6ST3) and HS3-O-sulfotransferase 3a1 (HS3ST3a1) were found to be uniquely expressed by OECs and HS3-O-sulfotransferase 6 (HS3ST6) was uniquely expressed by SCs (FIG. 5A). Interestingly, OECs express significantly higher levels of HS6-O-sulfatase enzymes compared to SCs (Sulf 1 and Sulf 2; 8-fold and 4-fold respectively) (FIG. 5C), which would be expected to reduce 6-O-sulfation of OECHS. These differences in HS biosynthetic and modification enzymes provide a potential explanation for the structural differences in HS synthesised and secreted by the two glial cell types. SCs and OECs express similar levels of the most common HSPG core proteins (FIG. 5D-E).

(92) Reduction of Sulf 1 and Sulf 2 Expression in OECs Using RNAi Promotes Boundary Formation with Astrocytes

(93) Consistent with increased levels of 6-O-sulphated HS in SCDM, qPCR data indicated that SCs express lower levels of Sulf 1 and Sulf 2 6-O-endosulfatase enzymes compared to OECs. To determine if this was important for the ability of SCs to induce a boundary with astrocytes, OECs were transfected with siRNA targeted to Sulf 1 and Sulf 2 to see if the reduction in Sulf activity converted them to a more SC-like phenotype in confrontation assays. Prior to the addition of astrocytes, OECs were transfected with siRNA for 72 hours and the reduction of Sulf 1 and Sulf2 mRNA was confirmed by qPCR (73% and 63% knockdown respectively). Once the cells had met in confrontation assays, the numbers of OECs mingling with astrocytes across a 300 m line were counted. Significantly less Sulf siRNA-treated OECs crossed into the astrocyte monolayer than control siRNA-treated OECs (4.61.3 Sulf siRNA cells compared with 151.8 control SiRNA OECs. p<0.01) and a clear boundary was observed, whereas control OECs and astrocytes mingled freely (FIG. 6A-B). This suggests that an increase in HS 6-O-sulfation in OECs, via reduced Sulf expression, induces SC-like behaviour. The complimentary experiment to over-express Sulf 1 and Sulf 2 in SCs to determine if they adopted an OEC-like phenotype was attempted using pcDNA3.1/Myc-His()-MSulf-1 and pcDNA3.1/Myc-His()-MSulf-2.sup.36. However, it was not possible to gain conclusive data using transfected cells in confrontation assays due to the low transfection efficiency of primary SCs (<3%, data not shown), which has also been reported by others.sup.55. An alternative experiment based on the hypothesis that it may be possible to interfere competitively with endogenous highly sulphated HS in SC/astrocyte boundaries using an excess of low sulphated modified heparins that do not promote boundary formation (eg., Hep 6, 7 and 8 in FIG. 3). Confrontation assays of SC and astrocytes treated with each of the low sulphated modified heparins demonstrated significantly increased cell mingling (FIG. 7), confirming the hypothesis that these HS mimetics can inhibit boundary formation, presumably by competing for endogenous FGF ligands and reducing activation of astrocyte FGFR.

(94) Inhibition of FGF1 or FGF9 Disrupts SC Boundary Formation with Astrocytes

(95) It has long been established that HS is required for the proper function of FGF by supporting the binding of all members of the FGF family to their cognate FGFRs.sup.38,44,51. The differential sulfation of HS is also known to regulate FGF activity.sup.28,41. Previously, it has been shown that FGFR inhibition disrupts SCM-induced boundary formation in OEC/astrocyte cultures.sup.11, suggesting that a target of HS regulation of boundary formation is a member of the FGF family. We, therefore, investigated the role of particular FGF ligands in boundary formation. The FGFR inhibitor used in the aforementioned study (SU5402) was previously thought to specifically inhibit FGFR1, however, it has also been shown to effectively inhibit FGFR3.sup.27,39. Astrocytes, but not OECs or SCs, express FGFR3-IIIb.sup.11, suggesting that the response observed with the SU5402 inhibitor in confrontation assays may be due to inhibition of this receptor on astrocytes. FGF1 and FGF9 are the only known ligands for FGFR3-IIIb.sup.21,29,45, therefore, the effect of blocking FGF1 or FGF9 using neutralising antibodies was investigated. Since FGFR3-IIIb does not bind FGF2.sup.21, a neutralising antibody against FGF2 was used as a negative control. Inhibition of FGF1 or FGF9 in SC/astrocyte confrontation assays resulted in reduced boundary formation and increased cell mingling, whereas inhibition of FGF2 had no effect (FIG. 8).

DISCUSSION

(96) Glial cell transplantation is a promising strategy for the repair of damaged CNS following injury or disease, with OECs and SCs as potential candidates. OECs may be the preferred candidate due to their ability to evoke less of a stress response in astrocytes.sup.10,11,23. However, since SCs often invade the CNS after injury when the blood brain barrier is breached.sup.14, it is important to understand the mechanisms by which they induce an astrocytic stress response, in order to devise strategies to prevent it. Previously, it has been shown that addition of heparin to OEC/astrocyte co-cultures induces boundary formation and removal of endogenous HS or inhibition of HS sulfation in SC/astrocyte cultures results in cell mingling.sup.11. In this study, we have demonstrated that the sulfation of HS synthesised by SCs and OECs is a crucial feature of their molecular phenotype influencing their activity on contacting astrocytes. The ability of HS to induce a boundary between SCs and astrocytes is shown to be dependent upon the level and pattern of HS sulfation, is modulated by the extracellular sulfatases, Sulf 1 and Sulf 2, and is likely to be mediated via FGF1 and/or FGF9 activation of astrocyte FGFR3-IIIb signalling. This information provides new targets for the development of strategies to enhance post-transplantation integration of SCs into CNS tissue.

(97) Using chemically modified heparins, which are model HS compounds with different levels of sulfation, it was possible to correlate HS sulfation with the extent of mingling/boundary formation induced in OEC/astrocyte confrontation assays. The more highly sulphated structures induced a stronger OEC:astrocyte boundary compared to the less sulphated structures, with a particular dependence on O-sulfation (FIG. 3). In support of this, HS isolated from SCM, which induces a boundary in OEC/astrocyte cultures, was 27% more sulphated than OCM HS, with a particular enhancement of 6-O-sulphated and 2-O-sulphated disaccharides (FIG. 4). Differences in the structure of HS synthesised by the glial cells will reflect a difference in biological activity and function, since HS:protein interactions are mediated by specific HS structures.

(98) Further evidence for a role of Sulf modified HS in boundary formation was obtained by siRNA mediated knock down of Sulf 1 and Sulf 2 expression in OECs (FIG. 6). Cell behaviour was altered dramatically, resulting in a phenotypic switch towards a SC-like response on contact with astrocytes and formation of a boundary. This strongly suggests that Sulf enzymes expressed by OECs play a vital role in modifying HS fine structure to determine their biological function.

(99) Removal of specific 6-O-sulfates from HS by Sulf enzymes will affect HS:protein interactions and, therefore, subsequent signalling pathways involved in boundary formation, allowing the cells to mingle with astrocytes. Reduction in Sulf enzyme expression diminishes this extracellular control of HS 6-O-sulfation, resulting in the synthesis of OEC HS that is more highly sulphated and SC-like and, thus, able to activate the signalling cascades that underlie boundary formation. Conversely, by saturating SC/astrocyte confrontation assays with low sulphated HS mimetics, we demonstrated that it is possible to inhibit SC-induced boundary formation (FIG. 7), potentially by out-competing SC HS for the activating FGF ligands. This is consistent with the notion that highly sulphated HS produced by SCs is essential for activation of boundary forming signalling pathways.

(100) In addition to the requirement for an HS component in SCM to induce an OEC:astrocyte boundary, a protein factor in SCM is also needed, since treatment of SCM with trypsin abolishes SCM activity. Reconstitution of separately heparinase- or trypsin-treated SCM samples restores SCM activity, further demonstrating the necessity of both an HS and protein component for activity (FIG. 2). Previously, FGFs were implicated, since inhibition of FGFR in SC:astrocyte cultures and OEC:astrocyte cultures treated with SCM reduced astrocyte hypertrophy and boundary formation.sup.11. Notably, in the same study, the expression of FGFRs in SCs, OECs and cortical astrocytes were analysed, and FGFR3-IIIb was found to be uniquely expressed by astrocytes. FGF1 and FGF9 are the only known ligands for FGFR3-IIIb.sup.21,29,45. In the current study, inhibition of FGF1 and FGF9 activity in confrontation assays resulted in mingling of SCs and astrocytes, whilst inhibition of FGF2 had no effect (FIG. 8). Interestingly, FGF9, also known as glial activating factor.sup.45, has previously been reported to stimulate GFAP expression in human glioma cells.sup.35, a process that also occurs during boundary formation.sup.10. This data is consistent with the notion that signalling through FGFR3-IIIb is crucial for astrocyte boundary formation. FIG. 9 presents a model in which FGF1 and/or FGF9 can only bind to and activate FGFR3-IIIb if a more highly sulphated HS is present in the conditioned medium via direct secretion of an extracellular matrix HSPG such as perlecan or by shedding of cell surface HSPGs such as syndecans.sup.19,43 or glypicans.sup.32,47. This leads to activation of astrocytes and subsequent boundary formation with SCs.

(101) Lower sulphated HS synthesised by OECs, formed as a result of higher Sulf expression, cannot support FGF induced FGFR3-IIIb signalling, resulting in mingling between astrocytes and OECs.

(102) The findings of this study demonstrate the prospect for significant advancements in combinatorial approaches for CNS repair after injury and in neurodegenerative diseases in which astrocytes become reactive. For example, transiently modifying HS structure by modulating the expression of HS sulfatase enzymes in transplanted SCs to enhance engraftment is a viable option.

(103) It may also be possible to manipulate HS sulfation levels at the CNS injury site, possibly by direct addition of Sulf enzymes into the lesion, or alternatively, to interfere with endogenous HS activities using HS mimetics.

REFERENCES

(104) 1. Sofroniew M, Vinters H V (2010). Acta Neuropathol 119:7-35. 2. Eng L et al., (1971) Brain Res 8:351-4. 3. Maragakis N J, Rothstein J D (2006) Nat Clin Pract Neurol 2:679-89. 4. Eddleston M, Mucke L (1993) Neurosci 54:15-36. 5. Liberrto et al., (2004) J Neurochem 89:1092-100. 6. Nash B, et al., (2010) Astrocyte phenotypes and their relationship to myelination. J Anat 219:44-52. 7. Barnett S C, Riddell J S. (2007) Nat Clin Pract Neurol 3:152-61. 8. Reier P J (2004) NeuroRx 1:424-51. 9. Franklin R J et al., (2008) Nat Rev Neurosci 9:839-55. 10. Lakatos A et al., (2000) Glia 32:214-25. 11. Santos-Silva A et al., (2007) J Neurosci 27:7154-67. 12. Fairless R et al., (2005) Mol Cell Neurosci. 28:253-63. 13. Franklin R J M, Blakemore W F (1993) Int J Dev Neurosci 11:641-49. 14. Bruce et al., (2000) J Neurotrauma 17:781-8. 15. Yates E A et al., (2004) J Med Chem 47:277-280. 16. Patey S J et al., (2006) J Med Chem 49, 6129-6132. 17. Guimond S E et al., (2006) Macromol Biosci. 6; 681-686. 18. Alexander C L et al., (2002) Glia 37:349-364. 19. Asundi V K et al., (2003) J Neurosci Res 73:593-602. 20. Brockes J P et al., (1979) Brain Res 165:105-118. 21. Chellaiah A T et al., (1994) J Biol Chem 269:11620-11627. 22. Chuah M I, West A K (2002) Microsc Res Tech 58:216-227. 23. Fairless R, Barnett S C (2005) Int J Biochem Cell Biol 37:693-699. 24. Fawcett J W, Asher R A (1999) Brain Res Bull 49:377-391. 25. Franklin R J, Barnett S C (2000) Neuron 28:15-18. 26. Franssen E H et al., (2007) Brain Res Rev 56:236-258. 27. Grand E K et al., (2004) Leukemia 18:962-966. 28. Guimond S et al., (1993) J Biol Chem 268:23906-23914. 29. Hecht D et al., (1995) Growth Factors 12:223-233. 30. Higginson J R, Barnett S C (2010) Exp Neurol 229:2-9. 31. Irie A et al., (2002) Development 129:61-70. 32. Kreuger J et al., (2004) Dev Cell 7:503-512. 33. Lakatos A et al., (2003) Exp Neurol 184:237-246. 34. Li Y et al., (2005) Glia 52:245-251. 35. Miyagi N et al., (1999) Oncol Rep 6:87-92. 36. Morimoto-Tomita M et al., (2002) J Biol Chem 277:49175-49185. 37. Noble M, Murray K (1984) EMBO J 3:2243-2247. 38. Omitz D M et al., (1992) Mol Cell Biol 12:240-247. 39. Paterson J L et al., (2004) Br J Haematol 124:595-603. 40. Pekny M, Nilsson M (2005) Glia 50:427-434. 41. Pye D A et al., (1998) J Biol Chem 273:22936-22942. 42. Raisman G (1985) Neuroscience 14:237-254. 43. Ramani V C et al., (2012) J Biol Chem 287:9952-61. 44. Rapraeger A C et al., (1991) Science 252:1705-1708. 45. Santos-Ocampo S et al., (1996) J Biol Chem 271:1726-1731. 46. Silver J, Miller J H (2004) Nat Rev Neurosci 5:146-156. 47. Traister A et al., (2008) Biochem J 410:503-511. 48. Wilby M J et al., (1999) Mol Cell Neurosci 14:66-84. 49. Yan Q, Johnson E M, Jr. (1988) J Neurosci 8:3481-3498. 50. Yates E A et al., (1996) Carbohydr Res 294:15-27. 51. Yayon A et al., (1991) Cell 64:841-848. 52. Skidmore M A et al., (2009) Methods Mol Biol 534:157-169. 53. Skidmore M A et al., (2010) Nat Protoc 5:1983-1992. 54. Skidmore M A et al., (2006) J Chromatogr A 1135:52-56. 55. Kraus A, et al., Neuron Glia Biol 6:225-230.