Heparan sulphates
10266612 ยท 2019-04-23
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61L2430/02
HUMAN NECESSITIES
C08B37/0075
CHEMISTRY; METALLURGY
C12N5/0663
CHEMISTRY; METALLURGY
C12N2501/115
CHEMISTRY; METALLURGY
A61P19/08
HUMAN NECESSITIES
A61K31/737
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
International classification
A61K31/737
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
A novel class of structurally and functionally related isolated Heparan sulphate is disclosed. The novel class of Heparan sulphates has been found to bind FGF2 and enhance the proliferation of stem cells while maintaining their pluripotency/multipotency.
Claims
1. A method of culturing stem cells in vitro, the method comprising contacting a stem cell culture with isolated or purified heparan sulphate HS8, wherein: the heparan sulphate HS8 is capable of specifically binding a peptide consisting of the amino acid sequence GHFKDPKRLYCKNGGF (SEQ ID NO:1); and the proportion of cells in the culture that are multipotent or pluripotent increases as compared to a control culture of stem cells that differ only by lack of the presence of exogenous heparan sulphate HS8.
2. The method according to claim 1, wherein following digestion with heparin lyases I, II and III and then subjecting the resulting disaccharide fragments to capillary electrophoresis analysis the heparan sulphate HS8 has a disaccharide composition comprising: TABLE-US-00005 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.7 3.0 UA,2S-GlcNS 7.2 2.0 UA-GlcNS,6S 15.5 3.0 UA,2S-GlcNAc,6S 6.5 2.0 UA-GlcNS 15.7 3.0 UA,2S-GlcNAc 1.0 0.5 UA-GlcNAc,6S 8.9 3.0 UA-GlcNAc 32.5 3.0.
3. The method according to claim 1, wherein following digestion with heparin lyases I, II and III and then subjecting the resulting disaccharide fragments to capillary electrophoresis analysis the heparan sulphate HS8 has a disaccharide composition comprising: TABLE-US-00006 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.7 1.0 UA,2S-GlcNS 7.2 0.4 UA-GlcNS,6S 15.5 1.0 UA,2S-GlcNAc,6S 6.5 0.6 UA-GlcNS 15.7 3.0 UA,2S-GlcNAc 1.0 0.4 UA-GlcNAc,6S 8.9 1.0 UA-GlcNAc 32.5 1.6.
4. The method according to claim 1, wherein the HS8 is obtained by a method comprising: (i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide comprises a heparin-binding domain having the amino acid sequence YCKNGGF (SEQ ID NO:2); (ii) contacting the polypeptide molecules with a mixture comprising glycosaminoglycans such that polypeptide-glycosaminoglycan complexes are allowed to form; (iii) partitioning polypeptide-glycosaminoglycan complexes from the remainder of the mixture; (iv) dissociating glycosaminoglycans from the polypeptide-glycosaminoglycan complexes; (v) collecting the dissociated glycosaminoglycans.
5. The method according to claim 4 wherein the polypeptide comprises the amino acid sequence selected from GHFKDPKRLYCKNGGF (SEQ ID NO:1).
6. The method according to claim 4 wherein the mixture comprising glycosaminoglycans is a heparan sulphate preparation obtained from porcine intestinal mucosa.
7. The method according to claim 1, wherein the heparan sulphate is formulated as a pharmaceutical composition or medicament.
8. The method according to claim 7 wherein the pharmaceutical composition or medicament further comprises FGF2 protein and/or mesenchymal stem cells.
9. The method according to claim 7, wherein the pharmaceutical composition or medicament further comprises stem cells.
10. The method according to claim 7, wherein the pharmaceutical composition or medicament further comprises a pharmaceutically acceptable carrier, adjuvant or diluent.
11. The method according to claim 7, wherein the pharmaceutical composition or medicament is in the form of a biomaterial suitable for implantation in a tissue or administration to a subject.
12. The method according to claim 11, wherein the biomaterial is selected from a hydrogel, a fibrin web or mesh, or a collagen sponge.
13. The method according to claim 11, wherein the biomaterial further comprises at least one biologically active molecule selected from the group consisting of BMP-2, BMP-4, OP-1, FGF-1, FGF-2, TGF-1, TGF-2, TGF-3, VEGF, collagen, laminin, fibronectin and vitronectin.
14. The method according to claim 7, wherein the pharmaceutical composition or medicament is formulated in fluid or liquid form for injection.
15. A method of enriching for colony forming units (CFU-F) in a culture of mesenchymal stem cells (MSC), the method comprising culturing MSCs in vitro in contact with isolated or purified heparan sulphate HS8, wherein: the heparan sulphate HS8 is capable of specifically binding a peptide consisting of the amino acid sequence GHFKDPKRLYCKNGGF (SEQ ID NO:1); and the proportion of cells in the culture that are multipotent or pluripotent increases as compared to a control culture of stem cells that differ only by lack of the presence of exogenous heparan sulphate HS8.
16. The method according to claim 15, wherein the MSCs are cultured such that cultured cells proliferate and the population of MSCs expands, and wherein the expanded MSC population is characterised in that: 2% of the MSC population express any of CD45, CD34, CD14, CD19, HLA-DR; and 95% of the MSC population express CD105, CD73 and CD90; and 40% of the MSC population express CD49a and/or 50% of the MSC population express SSEA-4 and/or 20% of the MSC population express STRO-1.
17. The method according to claim 15 further comprising passaging the MSCs, wherein after one or more passages the MSC population is characterised in that: 2% of the MSC population express any of CD45, CD34, CD14, CD19, HLA-DR; and 95% of the MSC population express CD105, CD73 and CD90; and 40% of the MSC population express CD49a and/or 50% of the MSC population express SSEA-4 and/or 20% of the MSC population express STRO-1.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
(2)
(3)
(4)
(5)
(6)
(7) TABLE-US-00004 Numbered row of the table Corresponding in FIG. 5 SEQ ID NO(s) 1 SEQ ID NOs: 3 and 4 2 SEQ ID NO: 5 3 SEQ ID NO: 6 5 SEQ ID NO: 7 6 SEQ ID NO: 8 8 SEQ ID NO: 9 9 SEQ ID NO: 10 10 SEQ ID NO: 11 11 SEQ ID NO: 12 13 SEQ ID NO: 13 14 SEQ ID NO: 7 15 SEQ ID NO: 14
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
(50)
(51)
(52)
(53)
(54)
DETAILED DESCRIPTION OF THE INVENTION
(55) The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.
EXAMPLES
Example 1
(56) We investigated the purification of a new FGF2 binding HS from commercially available Porcine Celsus Heparan sulphate sources suitable for scale up of heparan sulphate (HS) preparations that can be readily used in the clinic.
(57) The Heparin binding domain (HBD) peptide sequence GHFKDPKRLYCKNGGF [SEQ ID NO:1] from FGF2 was selected (The structure of glycosaminoglycans and their interactions with proteins; Gandhi N S and Mancera R L., Chem Biol Drug Des. 2008 December; 72(6):455-82) and used to purify specific HS species binding to FGF2.
(58) Upon synthesizing the peptide, it was subjected to the .sup.3H Heparin assay where specific binding of .sup.3H Heparin to the peptide soaked to a nitrocellulose membrane in a dose dependent manner was compared to the total counts of the .sup.3H Heparin. Once the specific binding of .sup.3H Heparin to the FGF2-HBD peptide was shown the peptide was used to pull down a specific HS from Porcine Celsus HS which binds to FGF2 by affinity chromatography. This new HS species was named as HS8 (and was given the variant name HS8G).
(59) HS8 was analysed for its specificity in binding with FGF2 with glycosaminoglycan (GAG) binding plates where the specific binding of HS8 to FGF2 was measured in comparison to Heparin, Porcine Celsus HS and HS8 negative fraction.
(60) The GAGs were plated on GAG binding plate (5 g/ml) overnight and later incubated with recombinant human FGF2 (0-100 ng/ml) and an ELISA method was used to check the specificity of binding of GAGs to FGF2.
(61) The results clearly showed that HS8 has more binding to FGF2 compared to other GAG species (
(62) HS8 was also subjected to in vitro cell proliferation assay with STRO1 human mesenchymal stem cells (hMSCs) to determine the bioactivity of HS8. We used HS8 as a stand-alone media supplement with different doses (50 ng/ml, 100 ng/ml, 500 ng/ml, 1000 ng/ml, 5000 ng/ml and 10000 ng/ml) in short term growth of hMSCs compared to the controls. Without any addition of exogenous growth factors we observed a dose dependent growth of hMSCs with HS8 (
Example 2
(63) Mesenchymal Stem Cells (MSCs)
(64) MSCs are widely defined as plastic-adherent cells which can be directed to differentiate in vitro into osteogenic, chondrogenic, adipogenic, myogenic, and other lineages and recently, the name multipotent mesenchymal stromal cells was also coined to MSCs by the International Society for Cytotherapy (Zulma et al, 2011). MSCs are been found in bone marrow, adipose tissue, dermal tissue, intervertebral disc, amniotic fluid, various dental tissues, human placenta and cord blood (Si et al, 2011 and Zulma et al, 2011). The therapeutic potential of MSCs have been recognized and been used in many clinical applications such as bone tissue regeneration and non skeletal tissue regenerations. In recent years the immunosuppressive and anti-inflammatory effects of MSCs were described. This is due to MSCs weak immunogenicity by expressing low levels of major histocompatibility complex-I molecules (MHC-1) on their cell surface, ability to suppress the activation and proliferation of both T and B lymphocytes and modifying the microenvironment of injured tissues while protecting damaged tissues (Si et al, 2011 and Zulma et al, 2011). This MSC-mediated immunosuppression which can be effectively used to treat GVHD has a species variation in mechanism (Ren et al. 2009 and Shi et al, 2010). The cytokine-primed mouse MSCs is mediated by nitric oxide (NO) and cytokine-primed human MSCs is executed through indoleamine 2, 3-dioxygenase (IDO).
(65) Heparin and Heparan Sulfate Glycosaminoglycans (HSGAGs)
(66) Heparin is produced and stored in mast cells and in comparison, HSGAGs are found in all animal tissues and they can occur as a proteoglycan where HS chains are bound to cell surface or ECM proteins. HS affects metabolism, transport, information transfer, cell adhesion, cell growth and differentiation, and support in all organ systems (Bishop et al, 2007 and Gandhi et al, 2008). Heparin and HS are linear polysaccharides consisting of repeating uronic acid-(1.fwdarw.4)-D-glucosamine disaccharide subunits. Uronic acid can either be D-glucouronic acid or L-iduronic acid. In addition, modifications at specific places give rise to different N-sulfated, 0-sulfated and N-acetylated complex sequences [Ori et al 2008]. The most abundant disaccharide in heparin is IdoA(2S)-(1.fwdarw.4)-GlcNS(6S) therefore giving rise to highly negative charge throughout the chain length, which makes heparin less or no selectivity in binding to proteins. On the other hand, HS has the unsulfated GlcA-(1.fwdarw.4)-GlcNA disaccharide as the most common form which giving rise to segregated blocks of unsulfated NA domains and blocks of highly sulfated, heparin-like IdoA-(1.fwdarw.4)-GlcNS disaccharides (NS domain). The NA and NS domain is separated by NA/NS transition domains. This diversity of HS structure is responsible for wide range of biological functions.
(67) Fibroblast Growth Factor (FGF) Proteins and Heparin Binding Domains
(68) Fibroblast growth factors (FGFs) are large family of polypeptide growth factors which comprise of 22 members in humans. They play a major role in development, differentiation, cell proliferation, angiogenesis and wound healing by binding and activating a subfamily of FGF cell surface receptor tyrosine kinases known as FGF receptors (FGFR) (Ornitz et al 1996). Furthermore, the FGFs are among the best-studied heparin-binding proteins, and HSGAGs regulate FGF signaling by direct molecular association with FGFRs (Pellegrini, 2001). In addition, FGF2 signaling through FGFR1 is important for MSC expansion (Gronthos et al, 1999).
(69) Interactions of Heparin/HS with FGF2
(70) Various studies have recognized common structural features in the heparin/HS binding sites of proteins (Gandhi et al, 2008; Hileman et al, 1998 and Ori et al, 2008). Cardin and Weintraub in 1989 made a first attempt to determine the heparin binding domain (HBD) after analyzing 21 heparin-binding proteins and proposed that typical heparin-binding sites may have the sequence XBBXBX or XBBBXXBX, where B is a positively charged amino acid (arginine, lysine and rarely histidine) and X is a hydropathic residue. The next consensus sequence TXXBXXTBXXXTBB, was introduced by Hileman et al in 1998 after Comparing X-ray and NMR of several proteins. In this sequence T defines a turn, B a basic amino acid (arginine or lysine) and X a hydropathic residue.
(71) Strong ionic interactions are expected between GAGs and proteins with positively charged basic amino acids form ionic bonds with negatively charged sulphate or carbon/late groups on heparin chains. Their role is determinant for the interaction with heparin and, possibly, with the highly sulfated regions within HS like NS domains (Fromm et al, 1997 and Ori et al, 2008). In addition, there are other types of bonds namely van der Waals forces, hydrogen bonds and hydrophobic interactions. These bonds will come in to play for the interactions with the more heterogeneous HS, where neutral amino acids are also required (Fromm et al, 1997 and Ori et al, 2008). In considering FGF2, Glutamine and asparagine amino acids play an important role for the interaction with HS by forming hydrogen bonds with the hydroxyl groups of the sugar in addition to the ionic bonds (Thompson et al, 1994).
(72) According to the numerous published studies so far, there are different peptide sequences as the heparin binding domain of FGF2 and those have been compiled in the table 1. Here we have adopted a numbering system where the amino acids are numbered according to the full FGF2 sequence (288aa).
(73) Graft Versus Host Disease (GVHD)
(74) Haemopoietic-cell transplantation (HCT) is an intensive therapy used to treat haematological malignant diseases where allogeneic HCT procedures are increasing annually (Ferrara et al, 2009). The major complication of HCT is GVHD, an immunological disorder that affects mainly gastrointestinal tract, liver, skin, and lungs. According to Billingham, 1966-67 three requirements should be fulfilled if GVHD to occur namely, 1) the graft must contain immunologically competent cells which are T lymphocytes, 2) the recipient must express tissue antigens that are not present in the transplant donor, and 3) the patient must be incapable of mounting an effective response to abolish the transplanted cells. GVHD pathophysiology starts when myeloablative conditioning regimes are used to remove the host defective bone marrow. The host's antigen presenting cells get activated because of the cytokines (TNF, IL1, LPS) produced by the damaged tissues. Once the allogeneic HCT has been performed at this stage donor T cells get activated which thereby produce more cytokines leading to cellular and inflammatory reactions resulting in GVHD. Non-haemopoietic stem cells; MSCs, can reduce allogeneic T-cell responses due to their potent immunosuppressive and ameliorate GVHD (Le Blanc et al, 2008; Meuleman et al, 2009 and Toubai et al, 2009).
(75) Need to Scale Up of hMSC for Therapeutic Purposes
(76) A major drawback of hMSCs usage in cell based therapies is that the difficulty in achieving sufficient cell numbers, though they are already been used in the clinics. The low numbers of hMSCs, where it can be as low as 0.01% to 0.0001% of bone marrow mononuclear cells hinders the widespread usage of it. According to Caplan, 2009 where they obtained bone marrow from different aged donors, dispersed, placed on culture flasks, later counted the CFU-Fs and shown by decade of age versus the MSCs per nucleated marrow cells. A remarkable decrease in MSCs per nucleated marrow cells was observed, with a 10-fold decrease from birth to teens and another 10-fold decrease from teens to the elderly. Clearly, with age the number of MSCs in marrow decreased. In addition, Caplan pointed out that these decreases paralleled the observed fracture healing rates of young and adults. In comparison, the titres of haematopoietic stem cells in marrow which were around one per 10.sup.4 nucleated marrow cells, remained constant throughout the age of the individual.
(77) Current Expansion Methods of hMSCs
(78) Researchers have thought that, if they can mimic the bone marrow microenvironment in culturing hMSCs they can achieve therapeutic numbers of hMSCs for clinical use. Basically, mimicking can be achieved by two broad ways namely growing hMSCs with ECM and with exogenous growth factor supplementation. When ECM substrates were used, increased hMSC attachment and cumulative cell number were observed (Grnert et al, 2007 and Matsubara et al, 2004) but, the expanded cells were lacking stemness (Cool et al, 2005). In addition, FGF2 was commonly used as exogenous growth factor supplementation which also showed marked amplification of cell number (Ling et al, 2006 and Sotiropoulou et al, 2006) compared to the controls with standard culture media. In line with the cells grown with ECM substrates the cells grown with FGF2 also had increased amounts of differentiated progenitors compared to multipotant hMSCs in controls (Gronthos et al, 1999 and Walsh et al, 2000). Hence, identification of a molecule which prompts the proliferation of hMSCs where we can achieve therapeutic numbers of hMSCs without adversely affecting the stemness shows a great promise in clinical use of hMSCs for bone regeneration and bone marrow transplantations to alleviate GVHD.
(79) HS GAGs Improve the Growth of hMSCs without Affecting the Stemness of Cells
(80) Nurcombe et al in 1993 have shown that, activity of FGF on murine neural precursor cells regulated by HS GAGs and this interaction is a requirement for the binding of FGF2 to their receptors. In addition, there was a significant difference in binding of HSGAGs to FGFs where at day 9 HS GAGs produced by these cells preferentially bound to FGF2 and by day 11, HSGAGs binding shifted to FGF1. Furthermore, these unique heparan sulfates mediate the binding of FGF2 to specific receptors via interacting with cell-surface receptors on neural precursor cells (Brickman et al, 1995). In 1998, Brickman et al further supported these findings by isolation and characterization of two separate HS pools from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells. One pool was derived from cells in log growth phase, which increased the activity of FGF-2, and the other pool from cells undergoing contact-inhibition and differentiation, which had preference to FGF1. As described previously by our lab, an embryonic HS GAG preparation named HS2 increased the hMSCs growth without significant loss of multipotentiality and lead to increased bone formation in mice when transplanted in vivo. This evidence suggests that ECM component HS GAGs improve the growth of the hMSCs without adversely affecting the multipotentiality. Hence, there is a specific need for a HS variant that is having high binding affinity to FGF2 and potentiates its activity on cell growth which can be readily scalable to be used in clinical settings compared to HS2.
(81) Results
(82) Isolation of the Heparan Sulfate with Higher Binding Affinity to FGF-2 by Column Chromatography (HS8)
(83) In line with the strategy of purifying the FGF2 binding HS2, we seek the possibility of purifying another FGF2 binding HS from commercially available Porcine Celsus heparan sulphate sources (Celsus Laboratories, USA) in order to scale up the HS preparation which could be readily used in the clinics. Out of these peptides sequences which are presented in the table 1 .sup.157GHFKDPKRLYCKNGGF.sup.172 (SEQ ID NO:1) (Gandhi et al, 2008) which was named FGF2-Gandhi-HBD was used.
(84) [.sup.3H] Heparin Assay
(85) Upon synthesizing the peptides, they were subjected to .sup.3H Heparin assay where the capability of the FGF2-HBD-peptides binding to heparin was tested. Known amounts of peptides or saturating amounts of peptides were dried onto identical nitrocellulose membranes which were first air dried and then further dried for 45 min in a vacuum oven at 80 C. Then membranes were washes with 1 phosphate buffered saline (PBS) and incubated in counting vials for 16 hr with 0.1 Ci of [.sup.3H] heparin (Perkin Elmer, Boston, USA) in 4% (w/v) bovine serum albumin (BSA)/PBS. After that membranes were washed and the radioactivity was determined by Perkin Elmer Tri-Carb 2800 TCR Liquid Scintillation Analyzer.
(86) When known amounts of peptide (SEQ ID NO:1) was used they were showing increasing CPM dose dependently, where BMP2-HBD was used as a positive control [
(87) Characterization of HS8
(88) GAG Binding Affinity Assays
(89) HS8 was subjected to its affinity in binding to FGF2 and other proteins (R&D Systems) with 96 well GAG binding plates (Iduron, UK) where the specific binding of HS8+ to FGF2 measured in comparison to heparin (Sigma), Porcine Celsus HS (Celsus Laboratories, USA) and HS8 negative fraction. The GAGS were plated on GAG binding plate (2.5-10 g/ml) overnight and blocked with 0.2% Fish Gelatin (Sigma) in standard assay buffer (SAB) for 1 hour at 37 C.
(90) Then incubated with 200 l/well of 0-100 ng/ml of recombinant human FGF2 for 2 hrs at 37 C. and later incubated with 200 l/well of 250 ng/ml primary biotinylated antibody (R&D Systems) for 1 hour in 37 C. In the next step plate was incubated with 200 l/well of 220 ng/ml ExtrAvidin-AP (Sigma) for 30 min at 37 C. From overnight incubation up to this step plate was washed 3 times with SAB in between each step. Finally, incubated with 200 l/well SigmaFAST p-Nitrophenyl phosphate (Sigma) for 40 min and absorbance was read at 405 nm by Victor.sup.3 1420 multi-label counter, PerkinElmer.
(91) Binding of all the GAGs in all 3 concentrations tested (2.5, 5 and 10 g/ml) to FGF2 increased similarly with increasing amounts of FGF2 and reached a saturation at 100 ng/ml FGF2 (
(92) Then we tested the ability of HS8+ and HS8 () fractions binding to different proteins (
(93) Ability of different GAGs competes with heparin with FGF2 tested in this assay, modified from Ono et al, 1999. A known concentration of FGF2 (R&D Systems) with differing concentrations of GAGs was mixed for 30 min at room temperature (RT) in a microtube.
(94) To this 40 l of beads solution [20 l of heparin-agarose beads. (Type I, Sigma) and polyacrylamide gel (Bio-Gel P-30, Bio-Rad)] were added and mixed for 30 min at RT. The heparin beads were washed 3 times by centrifugation (2000 rpm for 1 minute) with BSA-PBS (1% BSA in PBS) and 3 times with PBST (PBS containing 0.02% Tween) and to each tube, 100 l of 1:500 biotinylated anti-FGF2 (R&D Systems) added and incubated at RT for 1 hr. After washing as above, 100 l of 1:10 TMB substrate (R&D Systems) was added and mixed for 30 min at RT. Stop solution (50 l of 2N H.sub.2SO.sub.4) was added and 100 l of the supernatant after centrifugation was transferred to a 96 well plate. The absorbance was read at 450 nm by Victor.sup.3 1420 multi label counter, Perkin Elmer.
(95) Firstly, the amount of FGF2 needed for binding with the amount of heparin beads added was measured by the FGF2 optimization and the FGF2 dose 20 ng/ml were chosen for the next set of experiments [
(96) Then in order to get the range of GAGs to be used in the competition assay we initially used different amount of heparin. With the addition 50 g of heparin was almost sufficient enough to compete with the internal heparin attached to the beads [
(97) Proliferation Assays
(98) Two varieties of hMSCs were used in this assays from a 21 year old Hispanic male donor, where STRO1 positive cells isolated by magnetic activated cell sorting (passage 5-7) and HM21 cells isolated by conventional plastic adherence (passage 5). Cells were seeded 3000 cells/cm.sup.2 seeding density and allowed to attach to the plate for 24 hrs. Then the different concentrations of HS 8+ used as stand-alone media supplement ranging from 50-10000 ng/ml and as a positive control 2.5 ng/ml human recombinant FGF2 (R&D Systems) was added to media. Media change was performed either at 2 or 3 days. In STRO 1 cells, where the media change was done every 2 days, with HS8+ with increasing concentration increased the viable cell count and by day 6 compared to the controls, 5000 ng/ml was giving the highest counts (
(99) Because of the significant difference observed in the viable cell counts with the two cell types we conducted a proliferation assay with Passage 5 STRO1 and HM21 cells for 6 days and compared to the controls where media change was done in every 3 days. STRO 1 was showed slight higher proliferation counts compared to the HM21 cells but in both cell types, controls and the treated cells were giving almost the same cell counts [(
(100) Summary
(101) We used the sequence .sup.157GHFKDPKRLYCKNGGF.sup.172 (SEQ ID NO:1) to prepare higher affinity binding HS (HS8) to FGF2 from Porcine Celsus HS by affinity chromatography.
(102) In the glycosaminoglycan (GAG) binding assay results, which clearly showed that HS8+ had the highest affinity of binding to FGF2 compared to other GAG species. In addition the fold difference of Celsus HS: HS8+ at 100 ng/mIFGF2 point, the ratio was 1; 1.51. In heparin beads completion assays considering the percentage of competition, heparin was the most competitive where by adding 50 g it reached around 13%, followed by HS8+ 43%, Celsus HS 50% and HS8() 63%. STRO1+hMSCs isolated by magnetic activated cell sorting and HM21 hMSCs isolated by conventional plastic adherence were used in cell proliferation assays, where higher cell counts were obtained when HS8+ used as a standalone media supplement at a concentration of 5 g/ml and when the media change done in every 2 days. In conclusion, we now have successfully isolated higher binding affinity heparan sulfate (HS8) to FGF2 from a pool of commercially available heparan sulfate source which possess higher binding affinity to FGF2 and increase the ability to proliferate hMSCs.
(103) In conclusion, we now have successfully isolated higher binding affinity heparan sulfate (HS8) to FGF2 from a pool of commercially available heparan sulfate source and shown that it has higher binding capacity compared with other GAGs including heparin. In addition, HS8+ when used as stand-alone media supplement increases the cell proliferation when media change done in every 2 days. Accordingly, we believe we have addressed the need for high quality ex vivo expanded MSCs by culturing these cells in a heparan sulphate (HS8) engineered to have high affinity for FGF2.
(104) Additional Studies
(105) Isolation of Specific HS (HS8) to FGF2
(106) Although we have successfully achieved in isolating HS8, a higher binding affinity HS to FGF2 we would be further testing the other FGF2 HBD peptides sequences (table 1) in the means of [.sup.3H] Heparin Assay, GAG binding assays and cell attachment assays according to Lee et al, 2007.
(107) Binding Affinity Assays
(108) The binding affinity of HS8 has already confirmed by GAG binding plates and will be further validated by dot blot assays and kinetic binding with BIAcore T100 (Cain et al, 2005).
(109) Competition Assays
(110) The results from ELISA method will be further confirmed by western blot method.
(111) Proliferation Assays
(112) Results of proliferation assays will be further validated by using more hMSC lines and also with lower passage of cells. In addition, short term proliferation assays will be carried out by using BRDU (Roche) and WST-1 (Roche) reagents.
(113) Disaccharide Analysis
(114) Disaccharide analysis of HS8 will be carried out using anion exchange chromatography according to Murali et al, 2009 and the composition of the HS8 can be revealed.
(115) Stability of FGF2
(116) Stability assays will be carried out as SYPRO assays and FGF2 quantikine assays. In the SYPRO assay, interactions of FGF2 protein with GAGs will be measured as denaturing temperatures of proteins by a specific Sypro Orange dye (Uniewicz et al, 2010). The FGF2 quantikine assays will be carried out as with manufacturer's recommendations (R&D Systems Quantikine ELISA Cat No. DFB50) in order to measure FGF2 concentrations in cell cultures. Results are shown in
(117) Check the Biological Activity of hMSC Grown with HS8 In-Vitro
(118) Multipotentiality will be checked for plastic adherence, differentiating to osteogenic, adipogenic and chrondogenic tissues and FACS for surface markers (Dominici et al 2006). The CFU-Fs assays will be performed from bone marrow aspirates and expanded hMSCs with or without HS8 (Cawthon, 2002 and Guillot et al, 2007). Immunomodulatory activity of hMSCs will be assessed by mixed T lymphocyte assays.
(119) Check the Biological Activity of hMSC Grown with HS8 In-Vivo
(120) The cells isolated and grown in the presence of HS8 will be used in mouse bone regeneration models (Zannettino et al, 2010) and also will be used in xenogenic human NOD-SCID mice model of GVHD (Tiasto et al, 2007 and Toubai et al, 2009)
Example 3
(121) The binding capacity of different GAGs for FGF2 was assessed using GAG-binding plates (Iduron). The binding capacity of different GAGs for the heparin-binding growth factors (HBGFs) BMP-2, FGF1, FGF2, FGF7, PDGF-BB and VEGF was also assessed using GAG-binding plates (Iduron). The materials and methodology used are described below.
(122) HS8 was found to bind FGF-2 almost as well as heparin, and certainly better than the raw starting Celsus HS and the HS8 flow through fraction (
(123) HS8 (HS8+) preferentially binds FGF2 over all the other HBGFs tested and has a higher binding capacity for FGF2 than heparin, i.e. HS8 exhibits specific binding to FGF2. HS8- and raw starting Celsus HS displayed little preference for any of the HBGFs tested (
(124) Materials 1. Standard Assay Buffer (SAB)100 mM NaCl, 50 mM sodium acetate, 0.2% v/v tween 20, pH 7.2 2. Blocking buffer0.4% Fish gelatin (Sigma Cat No. 67041)+SAB 3. GAG binding Plate (Iduron, UK) 4. Proteins from R& D Systems: BMP2Cat No. 355 BM, FGF 1Cat No. 231 BC, FGF 2233 FB, FGF7Cat No, 251 KG, PDGF BBCat No. 220 BB, VEGFCat No. 293 VE 5. Antibodies from R & D Systems: BMP2Cat No. BAM 3552, FGF 1Cat No. BAF232, FGF 2BAM233, FGF7Cat No. BAF251, PDGF BBCat No. BAF220, VEGFCat No. BAF 293 6. ExtraAvidin-AP (Sigma Cat No. E2636) 7. Sigma FAST p-Nitrophenyl phosphate (Sigma, N2770)
(125) Method 1. Dissolve GAG in SAB (5 g/ml) 2. Add 200 l of GAG solution/well into GAG binding plate and incubate overnight at RT protected from light 3. Wash plate carefully 3 with 250 l/well with SAB 4. Incubate plate with 250 l/well blocking buffer for 1 hour at 37 C. protected from light 5. Wash plate carefully 3 with 250 l/well with SAB 6. Dissolve proteins with blocking buffer and perform serial dilution: 0, 0.781, 1.56, 3.125 nM 7. Dispense 200 l/well of diluted protein to GAG coated plate and incubate for 2 hours at 37 C. 8. Wash plate carefully 3 with 250 l/well with SAB 9. Add 200 l/well of 250 ng/ml of biotinylated primary antibody in blocking solution and incubate for 1 hour at 37 C. 10. Wash plate carefully 3 with 250 l/well with SAB 11. Add 200 l/well of 220 ng/ml of ExtraAvidin-AP in blocking solution and incubate for 30 min at 37 C. 12. Wash plate carefully 3 with 250 l/well with SAB 13. Add 200 l/well of Development reagent: Sigma FAST p-Nitrophenyl phosphate in DI water and incubate for 40 min at RT 14. Read absorbance at 405 nm
Example 4
(126) A BrdU incorporation proliferation assay was conducted to establish the effect of HS8 on hMSC proliferation (protocol described below).
(127) Dose-responses of human mesenchymal stem cells to HS8 (HS8+) were monitored by BrdU incorporation over 36 hours. FGF2 was used as a dosing positive control. HS8+ was found to enhance hMSC proliferation and provide significantly more stimulus than the other GAGs (
(128) Protocol (Cell Proliferation ELISA, BrdU (Colorimetric) Roche) 1. Cell Seeding5000 cells in 190 l of media/well (96 well plate) 2. MediaDMEM with 1000 mg/L+10% Fetal calf Serum (FCS)+1% 2 mM L-gluatamine+1% Penicillin and Streptomycin 3. Incubate for 6 hours in 37 C. and 5% CO.sub.2 4. Add different doses of treatments in 10 l of media for designated wells as in the layout after 6 hours of incubation 5. FGF2 (ng/ml) and GAGs (g/ml)10, 5, 2.5, 1.25, 0.625, 0.3125 6. Incubate for 36 hours with treatments in 37 C. and 5% CO.sub.2 7. Add BrdU into each well. 8. Label the cells with BrdU for 2 hours in 37 C. and 5% CO.sub.2 (Add 20 l of BrdU labeling solution/well) 9. Remove labeling medium by tapping off the plate 10. Add 200 l/well FixDenat to the cells and incubate for 30 min at 15-25 C. 11. Remove FixDenat solution thoroughly by flicking and tapping 12. Add 100 l/well anti-BrdU-POD working solution and incubate for 90 min at 15-25 C. 13. Remove antibody conjugate by flicking off and rinse wells three times with 250 l/well washing solution (lx PBS) 14. Remove washing solution by tapping. 15. Add 100 l/well substrate solution and incubate for 30 min at 15-25 C. 16. Measure the absorbance at 370 nm (reference wave length: 492 nm)
Example 5
(129) A FACS based cell proliferation assay was conducted to establish the effect of HS8 on hMSC proliferation (protocol described below).
(130) Dose-responses of human mesenchymal stem cells to HS8 (HS8+) were monitored by Guava ViaCount (FACS-based) method over 6 days. FGF2 was used as a dosing positive control. HS8 was found to enhance hMSC proliferation and provide a significant stimulus.
(131) Cell Proliferation Protocol
(132) Materials 1. HM20 hMSCMale Hispanic 20 year old donor (purchased from Lonza) 2. FGF 2 (R & D systems. Cat No. 233-FB-025) 3. Maintenance media: DMEM (10 mg/I glucose), 10% FCS, 1% Pen/Strp, 2 mM L-glutamine 4. HS8(+) Batch 2, HS8() Batch 2, Porcine mucosal heparan sulfate (Celsus laboratories, USA), Heparin (Sigma Cat No. H3149) 5. Guava Flex reagent (Millipore)
(133) Methods 1. HM20 cells are plated on 24-well plates at 3000 cells/cm.sup.2 in 500 l/well media (Day 0) 2. Day 1Media changed with increasing concentrations of FGF2 (ng/ml) and GAGs (g/ml)10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156 in 500 l of fresh media. 3. Media change every 2 days 4. Cells are harvested at designated time points (Day 2, Day 4 and Day 6)with 100 l of trypsin and neutralized with 100 l of media (t=Day 2) or 300 l of media (Day 4 and 6) 5. Cells were counted in Guava machine (Guava flex reagent: cell suspension is 1:200)
Example 6Human Mesenchymal Stem Cell Isolation
(134) Preparation of Human Bone Marrow (BM) Mononuclear Cells
(135) Collection of Human Bone Marrow (BM) and Preparation of BM Mononuclear Cells by Density Gradient Separation
(136) 1. Following informed consent, approx 40 mL of human bone marrow (BM) is collected from healthy young volunteers (18-40 y) by aspiration from the posterior iliac crest (hip bone). BM is placed immediately into a preservative-free, sodium heparin-containing 50-mL tube (10,000 units/tube).
(137) 2. A 10-L aliquot is removed and diluted 1:20 into White Cell Fluid (WCF) and nucleated cell content enumerated with a hemocytometer.
(138) 3. An equal volume of blocking buffer is then added to the BM aspirate, mixed well, then strained through a 70-m Falcon cell strainer to remove any small clots and bone fragments.
(139) 4. Then 3 mL of Ficoll-Hypaque (Lymphoprep) solution is dispensed into the bottom of approx. 12 round bottom 14-mL polystyrene Falcon tubes and carefully overlayed with 7.5 mL of diluted BM.
(140) 5. Tubes are centrifuged at 400 g for 30 min at room temperature.
(141) 6. Using a disposable plastic Pasteur pipette, the leucocyte band is recovered from all tubes and pooled into 414 mL polypropylene tubes.
(142) 7. Cells are diluted with HHF wash buffer and the BMMNC pelleted by centrifugation of the sample at 400 g for 10 min at 4 C.
(143) 8. The buffer is aspirated and cells are pooled into one tube.
(144) Isolation of MSCs by Adherence
(145) 1. BMNC fractions are seeded into 15 cm dishes in maintenance media (DMEM, 1 g/l glucose, 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin) and cells allowed to adhere for 3 days before the first media change.
(146) 2. The cells are cultured in maintenance medium with a media change every 3-4 days and routinely passaged upon 85% confluence using 0.125% trypsin. On re-plating, cells are seeded at 3,000/cm.sup.2. All cultures are maintained in a humidified incubator at 37 C. with 5% CO.sub.2.
(147) 3. Cells are removed from culture using a non-enzymatic cell dissociation solution (CellStripper, Mediatech, USA) and washed once in PBS before counting. 110.sup.5 cells are then aliquotted into a 96-well plate and cells pelleted at 450g for 5 min. Pre-diluted immunophenotyping antibody solutions in 2% FCS/PBS are subsequently added and cells incubated on ice for 20 min. Cells are then washed twice in 2% FCS/PBS before resuspension in 2% FCS/PBS and analysed on a GUAVA PCA-96 bench-top flow cytometer (Guava Technologies Inc., USA). All samples are measured in triplicate.
(148) Magnetic Activated Cell Sorting (MACS) of STRO-1 Positive BMSSC
(149) The use of MACS allows for partial purification of the BMSSC population and the processing of large numbers of BMMNC without compromising high losses in overall stem cells yield. Following density gradient centrifugation, approx 1-210.sup.8 mononuclear cells are recovered from a BM aspirate of 40 mL. Before immunolabeling, BMMNC are resuspended in 0.5 mL blocking buffer and incubated on ice for approx 30 min to reduce the possibility of Fc receptor-mediated binding of antibodies.
(150) Isolation of STRO-1+ BMSSC Using Magnetic Activated Cell Sorting (MACS)
(151) 1. BMMNC are pelleted by centrifugation at 400 g at 4 C. for 10 min and resuspended in 500 l of STRO-1 supernatant per 510.sup.7 BMMNC and incubated on ice for 60 min with occasional, gentle mixing.
(152) 2. BMMNC are washed twice in HHF wash buffer and then resuspended in 0.5 mL of HHF containing biotinylated goat anti-mouse IgM (-chain specific) at a 1/50 dilution and incubated at 4 C. for 45 min.
(153) 3. The BMMNC are washed three times in MACS buffer and resuspended in 450 L of MACS buffer to which 50 L of streptavidin microbeads are added (10 L of microbeads/10.sup.7 cells in 90 L MACS buffer). The mixture is incubated on ice for 15 min.
(154) 4. After one wash in ice-cold MACS buffer, a small aliquot of cells is removed for flow cytometric analysis (pre sample). The remaining cells are then placed onto a mini MACS column (column capacity of 10.sup.8 cells, Miltenyi Biotec, MS column). The STRO-1 cells (negative fraction) are not retained within the column and pass through into a fresh 2 mL polypropylene tube, under gravity into the effluent, while the STRO-1+ cells remain attached to the magnetised matrix.
(155) 5. The column is washed 3 times with 0.5 mL MACs buffer to remove any nonspecifically bound STRO-1 cells, which are collected in a fresh 2 mL polypropylene tube.
(156) 6. The STRO-1+ cells (positive fraction) are recovered by flushing the column with MACS buffer into a fresh 2-mL polypropylene tube after withdrawing the column from the magnetic field. The STRO-1+ cells are then counted and processed for two-color FACS.
(157) 7. Small samples (0.5-1.010.sup.5 cells) from each of the pre-MACS, STRO-1-, and STRO-1+ fractions are removed into separate 2 mL polypropylene tubes containing 0.2 mL of streptavidin-FITC conjugate (1/50). The cell samples are then incubated for an additional 5 min on ice to enable assessment of the enrichment procedure. A sample of (1.010.sup.5 cells) unlabeled pre-MACS cells serves as a negative control.
(158) 8. These samples are washed twice in HHF, fixed in FACS Fix solution and subsequently analysed by flow cytometry to assess purity and recovery.
(159) 9. At this point, the partially purified STRO-1+ BMSSC can be culture expanded or further purified by two-color FACS.
(160) Assessment of Bone Marrow Quality by Colony-Efficiency Assay
(161) The expected incidence of CFU-F colony in human bone marrow aspirates is approx 5-10 CFU-F per 10.sup.5 cells plated.
(162) 1. The BMMNC are seeded into 6-well culture plates at 0.3, 1.0, and 3.010.sup.5 cells per well in -MEM supplemented with 20% (v/v) FBS, 2 mM l-glutamine, 100 M l-ascorbate-2-phosphate, 50 U/mL penicillin, 50 mg/mL streptomycin, and -mercaptoethanol (510.sup.5 M). Cultures are set up in triplicate and incubated at 37 C. in 5% CO2 and >90% humidity for 12 days.
(163) 2. Day 12 cultures are washed twice with PBS and then fixed for 20 min in 1% (w/v) paraformaldehyde in PBS.
(164) 3. The fixed cultures are then stained with 0.1% (w/v) toluidine blue (in 1% paraformaldehyde solution) for 1 h then rinsed with tap water and allowed to dry. Aggregates of greater than 50 cells are scored as CFU-F.
(165) Fluorescence Activated Cell Sorting of Highly Purified BMSSC
(166) While all measurable CFU-F are contained within the STRO-1+BMMNC fraction, BMSSC represent only less than 2% of the total STRO-1+ population. The majority of the STRO-1+ cells are glycophorin-A+ nucleated red cells and some CD19+ B-cells. Therefore, the selection of BMSSC based on STRO-1 expression alone results in only a partial enrichment of CFU-F (approx 10-fold). Clonogenic BMSSC are all contained within the STRO-1.sup.bright cell fraction that can be further discriminated by dual-color FACS based on the expression of markers that are absent on nucleated red cells and lymphocytes, particularly CD106 and CD146. The methods described below enable the isolation of a minor subpopulation of the total STRO-1+ cell fraction, STRO-1.sup.bright/CD106+ BMMSC (1.4%0.3; n=20), in which 1 in every 2-3 cells plated have the capacity to form a CFU-F. This level of enrichment is almost 5,000-fold higher than the average incidence of CFU-F observed with unfractionated BMMNC (1 CFU-F per 10,000 cells plated).
(167) Isolation of STRO-1Bright/CD106+ BMSSC Using Flow Cytometric Cell Sorting (FACS)
(168) 1. Before immunolabeling, the MACS-isolated STRO-1+ cell BMMNC (routinely 2-510.sup.6 cells-from 110.sup.8 BMMNC) are resuspended in 0.5 mL HHF in preparation for 2-color immunofluorescence and FACS.
(169) 2. Approx 3-510.sup.5 MACS-isolated STRO-1+ cell are dispensed into 3 appropriately labeled tubes, to which the following are added: (i) No primary antibody (double negative control), kept on ice. (ii) Streptavidin-FITC conjugate (1/100 dilution in HFF) incubated on ice for 30 min (FITC control). The cells are then washed twice in HHF. (iii) 0.5 mL of murine IgG anti-human CD106 (VCAM-1) diluted to 20 g/mL in HFF. The STRO-1+ cells are incubated on ice for 30 min, washed twice in HHF and resuspended in 0.2 mL of PE-conjugated goat anti-mouse IgG (-chain specific), (PE control). The sample is incubated and washed then resuspended in HFF. (iv) The remaining 1-210.sup.6 MACS-isolated STRO-1+ cells are resuspended in 0.5 mL murine IgG anti-human CD106 (VCAM-1) and incubated as above, washed twice in HHF and resuspended in 0.2 mL of PE-conjugated goat anti-mouse IgG (-chain specific) and Streptavidin-FITC conjugate (1/100 dilution in HHF), then incubated on ice for 30 min (sorting sample). The cells are then washed as before then resuspended in HHF.
(170) 3. The samples are resuspended at a concentration of 110.sup.7 cells per mL in HHF before sorting on any sorter fitted with a 250 MW argon laser emitting light at a wavelength of 488 nm able to simultaneously detect FITC and PE. Samples (i-iii) are used to establish compensation for both FITC and PE. 5. Sorted STRO-1.sup.bright/VCAM-1+ cells from sample (iv) are collected in tubes containing appropriate growth media and mixed. 6. A cell count is performed as described above. The sorted cells are then cultured.
(171) Ex Vivo Culture of Human BMSSC
(172) Serum Replete Medium
(173) 1. The STRO-1.sup.bright/CD106+ isolated BMSSC populations (at 1-310.sup.4 per cm.sup.2) are cultured in tissue culture flasks or plates containing -modification of Eagle's Medium (-MEM) supplemented with 20% foetal bovine serum, 100 M l-ascorbate-2-phosphate, 2 mM l-glutamine, 50 U/mL penicillin and 50 g/mL streptomycin at 37 C. in 4% CO2 at relative humidity of >90% for 2 wk. Primary BMSSC populations are passaged when the cultures achieve 80-90% confluency.
(174) 2. Adherent cultures are washed 1 with serum free HBSS and the cells liberated by enzymatic digestion by the addition of 2 mL of 0.5% Trypsin/EDTA solution per T75 flask for 5-10 min at 37 C.
(175) 3. Cell viability is assessed by preparing a 1:5 dilution of single cell suspension in 0.4% trypan blue/PBS, and the number of viable cells determined using a haemocytometer.
(176) 4. BMMSC single cell suspensions are pooled and re-seeded at 0.5-1.010.sup.4 per cm.sup.2 in -MEM growth medium supplemented with 10% FBS, 100 M l-ascorbate-2-phosphate, 2 mM 1-glutamine, 50 U/mL penicillin and 50 g/mL streptomycin and incubated at 37 C. in 5% CO.sub.2 at relative humidity of >90%. Cultures are fed twice weekly by aspirating out the medium and replacing with an equal volume of freshly prepared medium warmed to 37 C.
(177) Serum Deprived Medium
(178) This method is a modification of the serum deprived medium (SDM) developed initially for the growth of hematopoietic progenitor cells.
(179) 1. Fibronectin-coated plates or flasks are prepared by precoating with 5 g per cm.sup.2 fibronectin solution for 90 min at room temperature. After this, the fibronectin solution is aspirated off and the culture vessels washed once with sterile PBS before seeding with cells.
(180) 2. The STRO-1.sup.bright/CD106+ isolated BMSSC populations (at 1-310.sup.4 per cm.sup.2) are cultured in the fibronectin-coated tissue culture flasks or plates suspended in media containing -MEM supplemented with 2% (w/v) bovine serum albumin (Cohn fraction V), 10 g/mL recombinant human insulin, human low density lipoprotein, 200 g/mL iron saturated human transferrin, 2 mM l-glutamine, dexamethasone sodium phosphate (10.sup.8 M), 100 M l-ascorbic acid-2-phosphate, -mercaptoethanol (510.sup.5 M), 10 ng/mL platelet derived growth factor-BB, 50 U/mL penicillin and 50 g/mL streptomycin.
(181) 3. The cultures are then incubated at 37 C. in 4% CO.sub.2 at relative humidity of >90% for 2 wk. Primary BMSSC populations are passaged when the cultures achieve 80-90% confluency.
(182) Cryopreservation of Ex Vivo Expanded MSCs
(183) 1. Routinely, single cell suspensions of culture expanded MPC are prepared by trypsin/EDTA digest as described above. The cells are then diluted and washed in cold HFF.
(184) 2. The cell pellet is resuspended at a concentration of 110.sup.7 cells per mL in FBS and maintained on ice. An equal volume of freeze mix (20% DMSO in cold FBS) is then added gradually while gently mixing the cells to give a final concentration of 510.sup.6 cells per mL in a 10% DMSO/FBS. One-milliliter aliquots are then distributed into 1.8-mL cryovials precooled on ice, then frozen at a rate of 1 C. per minute using a rate control freezer.
(185) 3. The frozen vials are then transferred to liquid nitrogen for long-term storage. Recovery of the frozen stocks is achieved by rapid thawing the cells in a 37 C. water bath. The cells are then resuspended in cold HFF and spun at 280 g for 10 min.
(186) 4. To assess viability of the cells, a 1:5 dilution is prepared in 0.4% trypan blue/PBS, and the number of cells determined using a haemocytometer. Typically this procedure gives viabilities between 80-90%.
Example 7Colony-Forming Units-Fibroblastic (CFU-F) Assay
(187) The CFU-F property of hMSC was assessed using the methodology described below. hMSCs were grown in one of unsupplemented control media for 4 passages and then grown in one of unsupplemented control media, or control media plus one of Heparin (1.25 g/ml), Celsus HS (1.25 g/ml), HS8 (1.25 g/ml), HS8 (HS8+) (1.25 g/ml) or HS8 (HS8+) (2.5 g/ml). Results are shown in
(188) Materials
(189) 1. hMSC(purchased from Lonza).
(190) 2. Maintenance media: DMEM (1000 mg/L glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine.
(191) 3. Crystal Violet 0.5% in 100% Methanol.
(192) Methods
(193) 1. MSCs are plated in triplicate in 10015 mm petri dishes at 150 cells/cm.sup.2 with 10 ml/dish maintenance media.
(194) 2. Cells were cultured for 14 days with media change at day 7.
(195) 3. At Day 14 the plates were stained with crystal violet (0.5% in 100% Methanol) as below: a. Remove media and wash twice with PBS b. Add 10 ml/dish of crystal violet and incubate for 30 minutes c. Wash once with PBS and once with H.sub.2O and dry the plates d. Colonies with more than 50 cells that were not in contact with other colonies were counted
Example 8Multipotent Characteristic of MSCs
(196) Maintenance of the multipotent character of MSCs was tested by assaying for ability of the MSCs to differentiate into bone (osteogenesis) and fat (adipogenesis), according to the methodology described below. Results are shown in
(197) Cells
(198) Passage 4 cells (P4)hMSCs cultured in normal maintenance media
(199) Passage 7 cells (P7)hMSCs cultured from P4 to P7 in normal maintenance media containing the one of the following treatments: HS8 (HS8(+)) 2.5 ug/mL HS8() 1.25 ug/mL Celsus HS 1.25 ug/mL Heparin 1.25 ug/mL FGF2 1.25 ug/mL
(200) Osteogenic Differentiation
(201) Materials 1. hMSC(purchased from Lonza) 2. Maintenance media: DMEM (1000 mg/L glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine 3. Treatment maintenance media: DMEM (1000 mg/L glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine, 10 nM dexamethazone, 10 mM -glycerol-phosphate and 25 g/mL L-ascorbate-2-phosphate 4. Paraformaldehyde 4% in PBS 5. Alizarin Red Solution: 1.37 g in 100 mL H.sub.2O; pH 4.1-4.3
(202) Methods 1. Cells were seeded (3,000 cells/cm.sup.2) in 6-well plates for 24 h 2. Changed media for control wells with maintenance media 3. Changed media for treated wells with maintenance media containing 10 nM dexamethazone, 10 mM -glycerol-phosphate and 25 g/mL L-ascorbate-2-phosphate 4. All cells then cultured for 28 days with media change in every 3 days 5. Cells were then stained with Alizarin Red: a. Wash three times with PBS b. Fix cells with 4% Paraformaldehyde for 10 min c. Wash three times with ddH.sub.2O d. Add Alizarin Red solution to the cells and incubate for 30 min, slowly shaking e. Wash three times with ddH.sub.2O f. Air dry the stained cells
(203) Adipogenic Differentiation
(204) Materials 1. hMSC(purchased from Lonza) 2. Adipocyte maintenance media: DMEM (4500 mg/L glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine 3. Adipocyte treatment media: DMEM (4500 mg/L glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine, 1 M dexamethazone, 10 M insulin, 20 M indomethazine and 115 g/mL 3-isobutyl-1-methylxanthine 4. Paraformaldehyde 4% in PBS 5. Oil Red O Solution: 0.36% in 60% isopropanol
(205) Methods 1. Cells were seeded (18,000/cm.sup.2) in 6-well plates in triplicates 2. Cells were cultured to confluence 3. Changed media for control wells with adipocyte maintenance media (4500 mg/L glucose) 4. Changed media for treated wells with adipocyte treatment media containing 1 M dexamethazone, 10 M insulin, 20 M indomethazine and 115 g/mL 3-isobutyl-1-methylxanthine 5. Subsequently, cultured for 28 days with media change in every 3 days 6. Cells were then stained with Oil-Red O: a. Wash three times with PBS b. Fix cells with 4% Paraformaldehyde for 60 min c. Wash once with ddH.sub.2O d. Add Oil Red O Solution to the cells and incubate for 1 h with slow shaking e. Wash two times in 60% isopropanol f. Wash three to five times with ddH.sub.2O g. Leave ddH.sub.2O on the plate or air dry the stained cells
Example 9
(206) The effect of HS8 on FGF-2 mediated growth hMSC was investigated using the methodology described below. HS8 was found to enhance FGF-2 mediated MSC growth (
(207) Cells
(208) Passage 4 cellshMSCs cultured in normal maintenance media with and without one of the following treatments: FGF2 0.156 ng/mL only FGF2 0.156 ng/mL with varying doses of HS8(+)
(209) Cell Proliferation Protocol
(210) Materials
(211) 1. hMSC(purchased from Lonza).
(212) 2. FGF 2 (R & D systems. Cat No. 233-FB-025).
(213) 3. Maintenance media: DMEM (1000 mg/I glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine.
(214) 4. HS8 (HS8(+)), HS8(), Porcine mucosal heparan sulfate (Celsus laboratories, USA), Heparin (Sigma Cat No. H3149).
(215) 5. Guava Flex reagent (Millipore).
(216) Methods
(217) 1. Cells are plated on 24-well plates at 3000 cells/cm.sup.2 in 500 l/well media (Day 0).
(218) 2. Day 1Media changed to contain maintenance media plus FGF2 (0.156 ng/mL) alone or FGF2 (0.156 ng/mL) with various concentrations of HS8(+): 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156 (g/ml) in 500 l of fresh media.
(219) 3. Media change every 2 days.
(220) 4. Cells are harvested at Day 4 with 100 l of trypsin and neutralized with 300 l of media.
(221) 5. Cells were counted in GUAVA machine (Guava flex reagent: cell suspension is 1:200).
Example 10
(222) The effect of HS8 on FGF-2 signaling via the ERK pathway was investigated using the methodology described below. HS8 was found to enhance/sustain FGF2 mediated signalling of the ERK pathway, as measured by phosphorylation of ERK1/2 and FRS2a (
(223) Cells
(224) P4hMSCs were cultured in normal maintenance media with and without the following treatments: FGF2 0.312 ng/mL only HS8 (HS8(+)) 2.54/mL
(225) Western Blot
(226) Materials 1. hMSC(purchased from Lonza) 2. FGF2 (R & D systems. Cat No. 233-FB-025) 3. Maintenance media: DMEM (1000 mg/L glucose), 10% FCS, 1% Pen/Strep, 2 mM L-glutamine 4. Serum-free media: DMEM (1000 mg/L glucose), 0.2% FCS, 1% Pen/Strep, 2 mM L-glutamine 5. Antibody against phospho-FRS2a (Cell Signaling. Cat No. 3861) 1:1000 in 5% BSA in TBST 6. Antibody against phospho-ERK1/2 (Cell Signaling. Cat No. 9106L) 1:2000 in 5% BSA in TBST 7. Antibody against total ERK1/2 (Cell Signaling. Cat No. 9102L) 1:1000 in 5% BSA in TBST 8. Antibody against actin (Millipore Chemicon. Cat No. MAB1501R) 1:8000 in 5% BSA in TBST
(227) Methods 1. Cells are plated at 10,000/cm.sup.2 on 6-well plates in maintenance media 2. Day 1: Change media to serum-free media at 2 mL/well 3. Day 3: Add treatment to well. Required amount of HS8(+) and/or FGF2 is dosed in serum-free media and added at 10 L/well 4. Cells are harvested in 100 L/well with 1.5 laemmli buffer at different time points (30 min and 24 h) 5. Lysates were heated for 5 min at 95 C. and stored at 20 C. 6. Samples are freeze-thawed only once 7. 20 L/well of sample is loaded into each lane of Novex 4-12% Bis-Tris SDS PAGE gel, 10 well (Invitrogen. Cat No. NP0335BOX) 8. Gel was run at 180V with 1MOPS Buffer for 50 min 9. Resolved protein bands were then transferred to nitrocellulose membrane at 100V in 1 Transfer Buffer for 1 h 30 min 10. Nitrocellulose membrane was stained with Ponceau S solution and cut into strips according to the size of the protein of interest 11. Membranes were then blocked in either 5% BSA or 5% Non-fat Milk in TBST for 30 min to 1 h at room temperature with slow shaking 12. Primary antibodies of recommended dilutions were then incubated overnight at 4 C. with slow shaking 13. Blots were then washed three times with TBST for 5 min each 14. Secondary antibodies of recommended dilutions were then incubated for 1 h to 2 h at room temperature with slow shaking 15. Blots were washed three times with TBST for 5 min each 16. Incubate blots with Chemiluminescence reagents and proceed to dark room to develop x-ray films for band visualization.
Example 11NMR Analysis of HS8
(228) A sample of HS8 was stored at 20 C. prior to analysis. NMR analysis was completed by dissolution in D2O (600 uL) that contained the internal standard tBuOH (200 L, 1.24 ppm) that is used for chemical shift comparison and quantitation. Celsus HS was weighed accurately in 1, 4 and 7 mg amounts, made up in the working D2O/tBuOH solution and analysed in the same run as HS8. Line fitting of the standard solutions gave regression of 0.995 or better for integration of the acetyl methyl region, the region 3.15-3.25 ppm and the lowest field portion of the anomeric region 5.15-5.65 ppm compared to the internal standard.
(229) Due to the small sample size which results in low signal to noise only the acetyl region data was used to calculate the amount of HS8 delivering a value of 0.7 mg. A second experiment was completed comparing signal to noise of the acetyl peak and a value of 0.5 mg was recorded. This is an absolute value not related to the internal standard. After three freeze-dry steps to remove the tBuOH prior to further analysis the mass recorded was 1.2 mg. Of note is the SEC HPLC data can be integrated to give an approximate purity value and it also recorded 58% suggesting 0.7 mg of HS-GAG present in the material. As this weight discrepancy is not a new phenomenon in small GAG samples the assumption is made that varying humidity and a proportion of salt must be affecting the recorded mass.
(230) The 1H NMR spectrum of HS8, Celsus HS and HS3* is displayed in
(231) Closer examination of the methine and methylene regions of the HS8 1H NMR showed differences compared to Celsus HS and HS3 (
(232) [*HS3 is an isolated heparan sulphate having specific and high binding affinity for a heparan binding domain of BMP-2. HS3 is described in WO2010/030244]
Example 12HPLC-SEC-RI of HS8 and Other HS Preparations
(233) Heparan sulfate preparations (approximately 1 mg, weighed accurately) were made up to 2 mg/mL in water. Heparin lyase I, II and III digests of these preparations were 2 mg/mL in water. The solutions were centrifuged (14 000 g, 2 min) and 200 L aliquots were taken for analysis.
(234) The SEC-RI system consists of a Waters 2690 Alliance separations module and a Water 2410 refractive index monitor (range 64). The do/dc for quantitation from the RI chromatograms was set at 0.129 (ref). Samples were injected (50 L) and eluted with 50 mM ammonium acetate with a flow rate of 0.5 mL/min from two Superdex Peptide 10/300 GL columns in series (30010 mm GE Healthcare, Buckinghamshire, UK). Data was collected and analysed using ASTRA software (Version 4.73.04, Wyatt Technology Corp).
(235) The size-exclusion chromatography of the whole HS8 preparation displayed a distinct size-exclusion profile. The Celsus HS starting material shows a voiding signal at 15 mL with additional material of a range of sizes eluting to approximately 23 mL of eluent. As shown in
(236) This is distinct again from the size profile of the HS3 preparations, showing an intermediate size profile between the HS8 and Celsus HS profiles (
(237)
(238) The size profile of the heparin lyase digest of HS8 (
Example 13[3H] Heparin Assay
(239) The heparin binding ability of SEQ ID NO:1 derived from the amino acid sequence of FGF2 was assessed using the protocol described below. Results are shown in
(240) Materials
(241) (1) Peptides:
(242) Gandhi et al (HS8)Manufactured by Nanyang Technological University GHFKDPKRLYCKNGGF-Ahx-(K)Biotin
(243) (2) 3H Heparin 0.1 Ci (Perkin Elmer, Boston, USA)
(244) (3) Nitrocellulose Membrane (Bio-Rad, USA)
(245) (4) Bovine Serum Albumin 4% (w/v) in PBS
(246) (5) Vacuum Oven (Thermo Fisher Scientific, USA)
(247) (6) Tri-Carb 2800 TCR Liquid Scintillation Analyzer (Perkin Elmer, Boston, USA)
(248) Methods
(249) (1) Make up FGF2-HBD-peptides to desired concentrations (4.6610-9, 9.3210-9, 1.8610-8, 3.73108 moles) with PBS
(250) (2) Soak identical nitrocellulose membranes in duplicates with known concentrations of peptides
(251) (3) Air dry the membranes for 1 h
(252) (4) Further drying in vacuum oven at 800 C for 45 mins
(253) (5) Wash membranes 3 times with PBS
(254) (6) Add 3H Heparin 0.1 Ci to the membranes and incubate for 16 h in scintillation counting vials
(255) (7) Wash membranes 4 times with PBS
(256) (8) Determine radioactivity with Tri-Carb 2800 TCR Liquid Scintillation Analyzer (Perkin Elmer, Boston, USA)
Example 14
(257) The ability of heparin binding domain peptide SEQ ID NO:1 to bind immobilized heparin was assessed using the protocol described below. Results are shown in
(258) Materials
(259) 1. Standard Assay Buffer (SAB)100 mM NaCl, 50 mM sodium acetate, 0.2% v/v tween 20, pH 7.2
(260) 2. Blocking buffer0.4% Fish gelatin (Sigma Cat No. 67041)+SAB
(261) 3. GAG binding Plate (Iduron, UK)
(262) 4. Peptides:
(263) Gandhi et al (HS8)Manufactured by Nanyang Technological University GHFKDPKRLYCKNGGF-Ahx-(K)Biotin
(264) 5. ExtraAvidin-AP (Sigma Cat No. E2636)
(265) 6. Sigma FAST p-Nitrophenyl phosphate (Sigma, N2770)
(266) Method 1. Dissolve Heparin in SAB (5 g/ml) 2. Add 200 l of Heparin solution/well into GAG binding plate and incubate overnight at RT protected from light 3. Wash plate carefully 3 with 250 l/well with SAB 4. Incubate plate with 250 l/well blocking buffer for 1 hour at 370 C protected from light 5. Wash plate carefully 3 with 250 l/well with SAB 6. Dissolve peptides in blocking buffer and perform serial dilution: 0, 50, 100, 200 nM 7. Dispense 200 l/well of diluted protein to GAG coated plate and incubate for 2 hours at 370 C 8. Wash plate carefully 3 with 250 l/well with SAB 9. Add 200 l/well of 220 ng/ml of ExtraAvidin-AP in blocking solution and incubate for 30 min at 370 C 10. Wash plate carefully 3 with 250 l/well with SAB 11. Add 200 l/well of Development reagent: Sigma FAST p-Nitrophenyl phosphate in DI water and incubate for 40 min at RT 12. Read absorbance at 405 nm
Example 15
(267) FGF-2 was assessed for its ability to bind HS8. This was compared to binding with the raw starting HS (HS-PM porcine mucosa), or no sugar. Results are shown in
(268) Materials
(269) 1. Standard Assay Buffer (SAB)100 mM NaCl, 50 mM sodium acetate, 0.2% v/v tween 20, pH 7.2
(270) 2. Blocking buffer0.4% Fish gelatin (Sigma Cat No. 67041)+SAB
(271) 3. GAG binding Plate (Iduron, UK)
(272) 4. Proteins from R& D Systems: FGF 2233 FB
(273) 5. Antibodies from R & D Systems: FGF 2BAM233
(274) 6. ExtraAvidin-AP (Sigma Cat No. E2636)
(275) 7. Sigma FAST p-Nitrophenyl phosphate (Sigma, N2770)
(276) Method
(277) 1. Dissolve GAG in SAB (5 g/ml)
(278) 2. Add 200 l of GAG solution/well into GAG binding plate and incubate overnight at RT protected from light
(279) 3. Wash plate carefully 3 with 250 l/well with SAB
(280) 4. Incubate plate with 250 l/well blocking buffer for 1 hour at 370 C protected from light
(281) 5. Wash plate carefully 3 with 250 l/well with SAB
(282) 6. Dissolve proteins with blocking buffer and perform serial dilution: 0, 0.781, 1.56, 3.125 nM
(283) 7. Dispense 200 l/well of diluted protein to GAG coated plate and incubate for 2 hours at 370 C
(284) 8. Wash plate carefully 3 with 250 l/well with SAB
(285) 9. Add 200 l/well of 250 ng/ml of biotinylated primary antibody in blocking solution and incubate for 1 hour at 370 C
(286) 10. Wash plate carefully 3 with 250 l/well with SAB
(287) 11. Add 200 l/well of 220 ng/ml of ExtraAvidin-AP in blocking solution and incubate for 30 min at 370 C
(288) 12. Wash plate carefully 3 with 250 l/well with SAB
(289) 13. Add 200 l/well of Development reagent: Sigma FAST p-Nitrophenyl phosphate in DI water and incubate for 40 min at RT
(290) 14. Read absorbance at 405 nm
Example 16
(291) Proliferation of plastic adherent mesenchymal stem cells over 6 days in the presence of HS8 was analysed. Results are shown in
(292) Cell Proliferation Protocol
(293) Materials
(294) 1. HM20 hMSCMale Hispanic 20 year old donor (purchased from Lonza)
(295) 2. FGF 2 (R & D systems. Cat No. 233-FB-025)
(296) 3. Maintenance media: DMEM (1000 mg/I glucose), 10% FCS, 1% Pen/Strp, 2 mM L-glutamine
(297) 4. HS8
(298) 5. Guava Flex reagent (Millipore)
(299) Methods
(300) 1. HM20 cells are plated on 24-well plates at 3000 cells/cm2 in 500 l/well media (Day 0)
(301) 2. Day 1Media changed GAGs (g/ml)2.5 and 0.5
(302) 3. Media change every 2 days
(303) 4. Cells are harvested at designated time points (Day 6)with 100 l of trypsin and neutralized with 300 l of media
(304) 5. Cells were counted in Guava machine (Guava flex reagent: cell suspension is 1:200)
Example 17
(305) Proliferation of STRO-1-isolated mesenchymal stem cells over 36 hours in the presence of isolated HS8, as compared to the raw Celsus starting HS (HS-PM), or the non-binding HS flow-through (HS8-) was measured by BrDU incorporation as described below. Results are shown in
(306) Protocol (Cell Proliferation ELISA, BrdU (Colorimetric), Roche)
(307) 1. Cell Seeding5000 cells in 190 l of media/well (96 well plate)
(308) 2. MediaAlpha MEM+10% Fetal calf Serum (FCS)+1% L-gluatamine+1% Penicillin and Streptomycin+100 nM L-glutamate
(309) 3. Incubate for 6 hours in 370 C and 5% CO2
(310) 4. Add different doses of treatments in 10 l of media for designated wells as in the layout after 6 hours of incubation
(311) 5. GAGs (g/ml)10, 5, 2.5, 1.25, 0.625, 0.3125
(312) 6. Incubate for 36 hours with treatments in 37 C. and 5% CO2
(313) 7. Add BrdU into each well.
(314) 8. Label the cells with BrdU for 2 hours in 37 C. and 5% CO2 (Add 20 l of BrdU labeling solution/well)
(315) 9. Remove labeling medium by tapping off the plate
(316) 10. Add 200 l/well FixDenat to the cells and incubate for 30 min at 15-25 C.
(317) 11. Remove FixDenat solution thoroughly by flicking and tapping
(318) 12. Add 100 l/well anti-BrdU-POD working solution and incubate for 90 min at 15-25 C.
(319) 13. Remove antibody conjugate by flicking off and rinse wells three times with 250 l/well washing solution (1PBS)
(320) 14. Remove washing solution by tapping.
(321) 15. Add 100 l/well substrate solution and incubate for 30 min at 15-250 C
(322) 16. Measure the absorbance at 370 nm (reference wave length: 492 nm)
Example 18Capillary Electrophoresis (CE) Analysis of Disaccharides
(323) Heparan sulfate (HS) was from Celsus Laboratories Inc. (HO-03103, Lot #HO-10697). Disaccharide standards (UA,2S-GlcNS,6S; UA,2S-GlcNS, UA,2S-GlcNAc,6S, UA-GlcNS,6S, UA-GlcNS, UA-GlcNAc, UA,2S-GlcNAc, UA-GlcNAc,6S, UA,2S-GlcN, UA,2S-GlcN,6S, UA-GlcN,6S, UA-GlcN Cat No. HD001 to HD013, Iduron Ltd, Manchester, UK), derived from the digestion of high-grade porcine heparin by bacterial heparinases, were purchased from Iduron Ltd, Manchester, UK. A synthetic derivative of a not naturally occurring disulfated disaccharide (UA,2S-GlcNCOEt,6S), was also purchased from Iduron for use as an internal standard. Heparin Oligosaccharides (dp4, dp6, dp8, dp10, dp12 (Cat. No. HO04, HO06, HO08, HO10, HO12)) and selectively desulfated heparin standards (2-O, 6-O and N-desulfated heparin) (Cat No. DSH001/2, DSH002/6, DSH003/N, Iduron Ltd, Manchester, UK) were also purchased from Iduron Ltd, Manchester, UK.
(324) Heparin lyase I (Heparitinase, EC 4.2.2.8, also known as heparitinase I), heparin lyase II (heparitinase II, no EC number assigned) and heparin lyase III (heparinase, EC 4.2.2.7, also known as heparitinase III) were obtained from Seikagaku Corporation, Japan. The enzymes, supplied as lyophilised powders (0.1 U/vial), were dissolved in 0.1% BSA to give solutions containing 0.5 mU/L. Aliquots (5 L; 2.5 mU) were frozen (80 C.) until needed.
(325) Digestion of HS Preparations with Heparin Lyase Enzymes
(326) HS preparations (1 mg) were each dissolved in 500 L of sodium acetate buffer (100 mM containing 10 mM calcium acetate, pH 7.0) and 2.5 mU each of the three enzymes was added. The samples were incubated at 37 C. overnight (24 h) with gentle inversion (9 rpm) of the tubes. A further 2.5 mU each of the three enzymes was added to the samples which were incubated at 37 C. for a further 48 h with gentle inversion (9 rpm) of the tubes. Digests were halted by heating (100 C., 5 min) and then lyophilized. The digests were resuspended in 500 L water and an aliquot (50 L) was taken for analysis by CE.
(327) Capillary Electrophoresis (CE)
(328) The capillary electrophoresis operating buffer was made by adding an aqueous solution of 20 mM H.sub.3PO.sub.4 to a solution of 20 mM Na.sub.2HPO.sub.4.12H.sub.2O to give pH 3.5. The column wash was 100 mM NaOH (diluted from 50% w/w NaOH). The operating buffer and column wash were both filtered using a Millipore filter unit fitted with 0.2 m cellulose acetate membrane filters (47 mm ; Schleicher and Schuell, Dassel, Germany). Stock solutions of the 12 disaccharide standards were prepared by dissolving the disaccharides in water (1 mg/mL). To determine the calibration curves for the standards, a mix containing all twelve standards was prepared. The stock solution of the 12 standard mix contained 10 g/100 L of each disaccharide and a dilution series containing 10, 5, 2.5, 1.25, 0.625, 0.3125 g/100 L was prepared; including 2.5 g of internal standard (UA,2S-GlcNCOEt,6S). The digests of HS were diluted (50 L/mL) with water and the same internal standard was added (2.5 g) to each sample. The solutions were freeze-dried and re-suspended in water (1 mL). The samples were filtered using PTFE hydrophilic disposable syringe filter units (0.2 m; 13 mm ; Advantec, Toyo Roshi Kaisha, Ltd., Japan).
(329) The analyses were performed using an Agilent.sup.3DCE (Agilent Technologies, Waldbronn, Germany) instrument on an uncoated fused silica capillary tube (75 m ID, 64.5 cm total and 56 cm effective length, Polymicro Technologies, Phoenix, Ariz., Part Number TSP075375) at 25 C. using 20 mM operating buffer with a capillary voltage of 30 kV. The samples were introduced to the capillary tube using hydrodynamic injection (50 mbar12 sec) at the cathodic (reverse polarity) end.
(330) Before each run, the capillary was flushed with 100 mM NaOH (2 min), with water (2 min) and pre-conditioned with operating buffer (5 min). A buffer replenishment system replaced the buffer in the inlet and outlet tubes to ensure consistent volumes, pH and ionic strength were maintained. Water only blanks were run at both the beginning, middle and end of the sample sequence. Absorbance was monitored at 232 nm. All data was stored in a ChemStore database and was subsequently retrieved and re-processed using ChemStation software.
(331) Eleven of the 12 heparin disaccharides in the standard mix were separated using conditions detailed above. The 12th disaccharide, UA-GlcN, does not migrate under the conditions used for these experiments. However, this disaccharide has not been reported to occur in heparan sulfates. The R2 values for the standard calibration curves ranged from 0.9949 to 1.0.
(332) The heparin lyase I, II and III digest of the HS preparations was done in duplicate and each duplicate was injected twice in the CE. Therefore, the normalized percentage of the disaccharides in the HS digest is the mean average of the results for the analyses. Of the 11 disaccharides separated in the standard mixes, only eight of these are detected in the HS digests. Other small signals are seen on the baseline of the electrophoretograms of the digests and these may correspond to oligosaccharides >2 dp. As mentioned above, the larger oligosaccharides will have less UV absorbance compared with the disaccharides.
(333) Duplicate analyses were completed on a sample of Celsus HS (Lot #10697) and compared to a previous set of analyses on the same sample: these results are displayed in
(334) The disaccharide composition for HS8 is comparable to that of HS3 (an HS isolated from Celsus HS through affinity for a heparin binding domain from BMP2, as described in WO2010/030244) in that a more sulfated (charged) fraction has in general been prepared from the Celsus HS. However; there is a striking difference in that there is a greater proportion of UA-GlcNS,6s and a lesser proportion of US-GlcNS for HS8 in comparison to HS3.
(335) Raw Celsus HS from which HS8 was derived has an average molecular weight of 20-25 kDa (compared with 15 kDa for heparin), and the process of identifying HS8 by affinity chromatography did not result in a substantial change in the observed molecular weight of HS chains. Each disaccharide unit is expected to have a molecular weight in the range 430 to 650 KDa. Using a rough average of 500 daltons per disaccharide (the average disaccharide in heparin is 650 daltons, for example), indicates (as a basic approximation) a chain length for HS8 of about 44 rings per average (22 kDa) HS8.
Example 19
(336) Having identified that HS8 preferentially binds to FGF2 and increases the growth rate of hMSCs, we further explored the mechanism of HS8 activity.
(337) Either FGF2 neutralizing antibody or FGFR1 inhibitors (both kinase inhibitor and neutralizing antibody) is able to reduce the proliferative effect of HS8 in hMSCs (
(338) We have shown (above) that HS8 enhances hMSC self-renewal while maintaining multipotency. To test if HS8 supplementation in hMSC routine culture can expand the culture faster, we grew hMSCs from three individual donors separately in HS8 supplemented medium. We noted that hMSCs exposed to HS8 were able to form more colonies (
(339) We also found that these cells are able to readily differentiate into all three mesenchymal stem cell lineages (including bone, as measured by Alizarin red, Von Kossa, Oil-Red-O and Alcian Blue staining) and have enhanced osteogenesis, suggesting this strategy may be effective for orthopedic trauma therapy. HS8 did not adversely affect the ability of MSCs to differentiate into bone.
(340) Therefore, we applied HS8 to a calvarial defect model in rats (
REFERENCES
(341) 1. Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004). Characterization of Growth Factor-binding Structures in Heparin/Heparan Sulfate Using an Octasaccharide Library. The Journal of Biological Chemistry 279(1)3, 12346-12354 2. Baird, A., Schubert, D., Ling, N., and Guillemin, R. (1988). Receptor- and heparin-binding domains of basic fibroblast growth factor. Proc. Natl. Acad. Sci. USA 85, 2324-2328. 3. Billingham, R. E. (1966-67). The biology of graft-versus-host reactions. Harvey Lect 62, 21-78. 4. Bishop, J. R., Schuksz, M., and Esko, J. D. (2007). Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030-1037 5. Brickman, Y. G., Ford M. D., Small, D. H., Bartletti, P., F., and Nurcombe, V. (1995). Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. J Biolo. Chem. 270(42), 24941-24948. 6. Brickman, Y. G., Nurcombe, V., Ford, M. D., Gallagher, J. T., Bartlett, P. F. and Turnbull, J. E. (1998). Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line. Glycobiology 8(5), 463-471. 7. Cain, S. A., Baldock, C., Gallagher, j., Morgan, A., Bax D. V., Weiss, A. S., Shuttleworth, A. C., and Kielty C. M. (2005). Fibrillin-1 Interactions with heparin-Implications for microfibril and elastic fibre assembly. The Journal of biological Chemistry 280(34), 30526-30537. 8. Caplan, A. I. (2009). Why are MSCs therapeutic? New data: new insight. J Pathol 217, 318-324 9. Cardin, A. D., and Weintraub, H. J. (1989), Molecular modeling of protein-glycosaminoglycan interactions. Arterioscler. Thromb. Vasc. Biol. 9, 21-32 10. Cawthon, R. M. (2002) Telomere measurement by quantitative PCR. Nucleic Acids Res 30, e47. 11. Cool, S. M., and Nurcombe, V. (2005) Substrate Induction of Osteogenesis from Marrow-Derived Mesenchymal Precursors. Stem Cells Dev. 14(6), 632-642. 12. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F. C., Krause, D. S., Deans R. J., Keating, A., Prockop, D. J., and Horwitz, E. M. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4), 315-317 13. Esch, F., Baird, A., Ling, N., et al. (1985) Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci USA; 82:6507-6511 14. Faham, S., Hileman, R. E., Fromm, J. R., Linhardt, R. J., and Rees, D. C. (1996) Heparin structure and interactions with basic fibroblast growth factor. Science 271, 1116-1120 15. Ferrara, J. L. M., Levine, J. E., Reddy, P., and Holler, E. (2009). Graft-versus-host disease. Lancet 373, 1550-61 16. Fromm, J. R., Hileman, R. E., Caldwell, E. E., Weiler, J. M., and Linhardt, R. J. (1997). Pattern and spacing of basic amino acids in heparin binding sites. Arch. Biochem. Biophys. 343, 92-100 17. Gandhi, N. S., and Mancera, R. L. (2008). The Structure of glycosaminoglycans and their interactions with proteins. Chem. Biol. Drug. Des. (2008) 72, 455-482 18. Gronthos, S., Zannettino, A. C. W., Graves, S. E., Ohta, S., Hay, S. J., and Simmons, P. J. (1999). Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J. Bone Min. Res. 14(1), 47-56. 19. Grnert, M., Dombrowski, C., Sadasivam, M., Manton, K., Cool, S. M., and Nurcombe, V. (2007). Isolation of a native osteoblast matrix with a specific affinity for BMP2. J. Mol. Histo 38, 393-404. 20. Guillot, P. V., Gotherstrom, C., Chan, J., Kurata, H. and Fisk, N. M. (2007) Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells 25, 646-654. 21. Hileman, R. E., Fromm, J. R., Weiler, J. M., and Linhardt, R. J. (1998). Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins. Bioessays 20(2), 156-67 22. Kinsella, L., Chen, H., Smith, J. A., Rudland, P. S., and Fernig D. G. (1998). Interactions of putative heparin-binding domains of basic fibroblast growth factor and its receptor, FGFR-1, with heparin using synthetic peptides. Glycoconjugate Journal 15, 419-422 23. Le Blanc, K., Frassoni, F., Ball, L., Locatelli, F, Roelofs, H., Lewis, I., Lanino, E., Sundberg, B., Bernardo, M. E., Remberger, M., Dini, G., Egeler, R. M., Bacigalupo, A., and Fibbe, W., Ringdn, O. (2008). Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371, 1579-1586 24. Lee, J. Y., Choo, J. E., Choi, Y. S., Lee, K. Y., Min, D. S., Pi, S. H., Seol, Y. J., Lee, S. J., Jo, I. H., Chung, C. P., and Park, Y. J. (2007). Characterization of the surface immobilized synthetic heparin binding domain derived from human fibroblast growth factor-2 and its effect on osteoblast differentiation. J Biomed Mater Res A 15; 83(4), 970-9. 25. Matsubara, T., Tsutsumi. S., Pan, H., Hiraoka, H., Oda, R., Nishimura, M., Kawaguchi, H., Nakamura, K., and Kato, Y. (2004). A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix. Biochemical and Biophysical Research Communications 313(3),503-508 26. Meuleman, N., Tondreau, T., Ahmad, I., Kwan, J., Crokaert, F., Delforge, A., Dorval, C., Martiat, P., Lewalle, P., Lagneaux, L., and Bron D. (2009). Infusion of mesenchymal stromal cells can aid hematopoietic recovery following allogeneic hematopoietic stem cell myeloablative transplant: a pilot study. Stem Cells Dev. 18(9),1247-52. 27. Murali, S., Manton, K. J., Tjong, V., Su, X., Haupt, L. M, Cool, S. M., and Nurcombe, V. (2009) Purification and characterization of heparan sulfate from human primary osteoblasts. Journal of cellular biochemistry 108, 1132-1142 (2009) 28. Nurcombe, V., Ford, M. D., Wildschut, J. A., and Bartlett, P. F. (1993). Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science, 260(5104), 103-106. 29. Ono, K., Hattori, H., Takeshita, S., Kurita, A., and Ishihara, M. (1999). Structural features in heparin that interact with VEGF165 and modulate its biological activity. Glycobiology. 9(7), 705-11. 30. Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulieri, F., Gao, G., and Goldfarb, M. (1996). Receptor Specificity of the Fibroblast Growth Factor Family. J Biol. Chem. 271(25), 15292-15297 31. Ori, A., Free, P., Courty, J., Wilkinson, M. C., and Fernig D. G. (2009). Identification of Heparin-binding Sites in Proteins by Selective Labeling. Molecular & Cellular Proteomics 8, 2256-2265 32. Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008). The heparanome and regulation of cell function: structures, functions and challenges. Front Biosci. 1(13), 4309-38. 33. Pellegrini, L. (2001). Role of heparan sulfate in fibroblast growth factor signalling: a structural view. Curr. Opin. Struc. Biol. 11, 629-634 34. Ren, G. S, J., Zhang, L., zhao, X., Ling, W., L'huillie, A., Zhang, J., Lu, Y., Roberts, A. I., Ji, W., Zhang, H., Rabson, A. B., and Shi, Y. (2009). Species Variation in the Mechanisms of Mesenchymal Stem Cell-Mediated Immunosuppression. Stem cells 27, 1954-1962 35. Shi, Y., Hu, G., Su, J., Li, W., Chen, C., Shou, P. Xu, C, Chen, X., Huang, Y., Zhu, Z., Huang, X., Han, X., Xie, N., and Ren, G. (2010). Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Research 20, 510-518. 36. Si, Y. L., Zhao, Y. L., Hao, H. J., Fu, X. B., and Han, W. D. (2011). MSCs: Biological characteristics, clinical applications and their outstanding concerns. Ageing Research Reviews 10, 93-103 37. Sotiropoulou, P. A., Perez, S. A., Salagianni, M., Baxevanis, C. N, and Papamichail, M. (2006). Characterization of the Optimal Culture Conditions for Clinical Scale Production of Human Mesenchymal Stem Cells. Stem cells 24, 462-471 38. Tisato, V., Naresh, K., Girdlestone, J., Navarrete, C., and Dazzi, F. (2007). Mesenchymal stem cells of cord blood origin are effective at preventing but not treating graft-versus-host disease. Leukemia 21, 1992-9. 39. Toubai, T., Paczesny, S., Shono, Y., Tanaka, J., Lowler, K. P., Malter, C. T., Kasai, M., and Imamura, M. (2009). Mesenchymal stem cells for treatment and prevention of graft-versus-host disease after allogeneic hematopoietic cell transplantation. Curr. Stem. Cell. Res. Ther. 4(4), 252-9. 40. Thompson, L. D., Pantoliano, M. W., and Springer B. A. (1994). Energetic characterization of the basic fibroblast growth factor-heparin interaction: identification of the heparin binding domain. Biochemistry 33, 3831-3840 41. Uniewicz, K. A., Ori, A., Xu, R., Ahmed, Y., Wilkinson, M. C., Fernig, D. G., and Yates, E. A. (2010). Differential Scanning Fluorimetry Measurement of Protein Stability Changes upon Binding to Glycosaminoglycans: A Screening Test for Binding Specificity. Anal. Chem., 82 (9), 3796-3802 42. Walsh, S., Jefferiss, C., Stewart, K., Jordan, G. R., Screen, J., and Beresford, J. N. (2000). Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1-4. Bone 27(2). 185-195. 43. Zannettino, A. C., Paton, S., Itescu, S., and Gronthos, S. (2010). Comparative assessment of the osteoconductive properties of different biomaterials in vivo seeded with human or ovine mesenchymal stem/stromal cells. Tissue Eng Part A. 16(12), 3579-87. 44. Zulma Gazit, Z., Pelled, G. Sheyn, D., Kimelman, N. and Gazit, N. (2011).
(342) Mesenchymal Stem Cells. Principles of Regenerative Medicine Chapter 17, 285-304 45. Friedenstein A J, Piatetzky S II, Petrakova K V (1966) Osteogenesis intransplants of bone marrow cells. J Embryol Exp Morphol 16:381-390 46. Gimble J, Guilak F (2003) Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5:362-369 47. Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W, Katz A J, Benhaim P, Lorenz H P, Hedrick M H (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211-228 48. Bieback K, Kern S, Kluter H, Eichler H (2004) Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625-634 49. Erices A, Conget P, Minguell J J (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109:235-242 50. Goodwin H S, Bicknese A R, Chien S N, Bogucki B D, Quinn C O, Wall D A (2001) Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 7:581-588 51. Kogler G, Sensken S, Airey J A, Trapp T, Muschen M, Feldhahn N, Liedtke S, Sorg R V, Fischer J, Rosenbaum C et al (2004) A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 200:123-135 52. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W, Ho A D (2005) Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 33:1402-1416 53. Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie C M (2002) Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 30:896-904