Patent Publication Number: US-8110400-B2

Title: Culture of mammalian pluripotent stem cells in the presence of hyaluronan induces differentiation into multi-lineage progenitor cells

Description:
FIELD OF INVENTION 
     The present invention relates to methods for the differentiation of pluripotent stem cells into mortal cells, which retain the potential to form adult cell lineages. 
     BACKGROUND OF THE INVENTION 
     A stem cell is defined as a self-renewing primitive cell that can divide indefinitely (i.e. it is immortal) and can develop into functional, differentiated cells. Differentiated cells are of value for the treatment of degenerative disease or injury by transplantation, or as in vitro model systems for pharmaceutical or toxicology screening. A limitation of differentiated cells however, is their limited capacity to proliferate in culture. 
     Stem cells can be derived from embryonic, fetal and adult tissue, with embryo stem cells exhibiting the greatest capacity for immortal self-renewal (i.e. expansion), and pluripotency to form differentiated cells. However, the immortal character of stem cells when undifferentiated can result in the formation of tumours if transplanted into a living organism. This is a critical problem for the use of stem cells in the treatment of degenerative diseases or injuries such as for example Parkinson&#39;s disease, diabetes, spinal cord repair, or bone repair, etc. Therefore what is needed are methods to control the immortality of cells without compromising their useful proliferative capacity and their lineage potential. That is, what is needed is to re-create a proliferative but mortal population of cells, similar to mesenchyme, which forms during embryo gastrulation, whose lineage potential is retained and controlled by local microenvironments. 
     Immortal stem cell self-renewal can be accomplished in vitro by growth of cells on either differentiated helper/feeder cells or under feeder-free conditions on extracellular matrix substrates by supplementation of media with defined growth factors or cytokines. For example, human embryo stem cells (hESCs) can be self-renewed by growth in feeder-free culture system consisting of growth on Matrigel™ (MG), a commercially available matrix derived from mouse tumours containing mostly laminin, collagen IV, and heparin sulfate proteoglycan) (Xu et al., 2001). Growth of hESCs on this matrix still requires prior conditioning of culture medium by feeder cells and supplementation with bFGF. Under these conditions laminin could also support hESC self-renewal, whereas other extracellular matrix (ECM) molecules such as Fibronectin and Collagen could not (Xu et al., 2001). However, hESC interactions with the ECM may be growth factor dependent since Fibronectin can support hESC self-renewal provided that the medium is further supplemented with transforming growth factor β1 (TGFβ1) and Leukemia inhibitory Factor (LIF) in addition to bFGF (Amit M et al., 2004). 
     The first ES cell lines derived from either non-human primates or humans were obtained using medium containing bovine fetal serum and mitotically inactivated mouse embryonic fibroblasts (MEF) as supporting feeder layers ( 1 ,  2 ). Prior to this, derivation of hES without feeders resulted in uncontrolled cell differentiation with successive passages ( 3 ). Recently, it has become possible to maintain hES in an undifferentiated state by relying instead on MEF conditioned medium and cultivation on the extra-cellular matrix (ECM) molecule laminin alone or with collagen, and heparan sulfate in a matrigel matrix ( 4 ). Medium conditioned by mouse embryonic or human adult fibroblasts or epithelial cells immortalized with hTERT cannot prevent hES from differentiating, despite growth on ECM substrates ( 4 ). Thus, one or more factors secreted by feeder cells, such as mouse embryo-derived fibroblasts, uniquely contribute to hES cell maintenance on ECM substrates. 
     Stem cell differentiation can be mediated in vitro by a variety of methods involving culture of cells that are adherant to a substrate or in suspension. For example:
         Indirect embryoid body mediated differentiation of cell representative of all germinal lineages (endoderm, ectoderm, mesoderm) (Keller, 1995; Itskovitz-Eldor et al., 2000; Conley et al., 2004)   Directed differentiation of neural cells by suspension culture of neurospheres (Zhang et al., 1998; Uchida et al., 2000; Schumacher et al., 2003)   Differentiation of cells grown on differentiated helper/feeder cells (Richards et al., 2002; Amit et al., 2003; Amit et al., 2004)   Differentiation of cells grown on specified extracellular matrix molecules (Xu et al., 2001; Rosler et al., 2004)       

     The problem with all of these strategies is that they favour either a heterogeneous or single population of terminally differentiated cell phenotypes whose proliferative capacity is lost. What is needed is a method for the controlled differentiation of an intermediate progenitor population with proliferative capacity that retains its potential to yield a single lineage in response to specific environmental cues. In animal development cells that conceptually possess such properties evolve during the process of gastrulation, which results in the formation of three primary germ layers, ectoderm, mesoderm, and endoderm. Of these layers, mesenchymal cells derived from the mesoderm layer have the unique capacity to proliferate and migrate to specific sites, in developing organisms referred to as limb buds, at which they become terminally differentiated and normally give rise to muscle, skeletal (i.e. bone), blood, vascular and urogenital systems and connective tissue, specifically osteoblasts, chondroblasts, adipocytes, fibroblasts, cardiomyoctes and skeletal myoblasts. 
     In the adult, mesenchymal cells can be recovered from bone marrow. These are referred to as mesenchymal stem cells because they can be cultured ex-vivo for a limited number of passages and be differentiated at the single cell level into mesodermal cell types as described. When introduced in vivo bone mesenchymal cells can differentiate into the same array of cell types, as well as cells with characteristics of cells outside the mesoderm, including endothelium, neuroectoderm, and endoderm (reviewed in Verfaillie et al. 2002). The formation of mesenchyme during gastrulation in the embryo, or in bone marrow during development or in the adult organism, is a complex process mediated by the interaction of a broad range of soluble and insoluble factors, which include amongst others glycosominoglycans. The complexity of this process is such however, that it is unlikely that a single factor could yield the unique properties of mesenchymal cells. 
     Glycosaminoglycans (GAGs) are unbranched chains composed of repeating disaccharide units. These disaccharide units always contain an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated, with the second sugar usually being a uronic acid (glucuronic or iduronic). GAGs are highly negatively charged because of the presence of carboxyl or sulfate groups on most of their sugar residues. As such they are strongly hydrophilic. GAGs tend to adopt highly extended conformations and form matrices that are space filling and resistant to compressive forces. Four main groups of GAGs have been distinguished by their sugar residues, the type of linkage between these residues, and the number and location of sulfate groups. They include: (1) hyaluronan, (2) chondroitin sulphate and dermatan sulfate, (3) heparan sulfate and heparin, and (4) keratan sulfate. 
     Hyaluronan (also called hyaluronic acid or hyaluronate) is the simplest of GAGs. It consists of a regular repeating sequence of non-sulfated disaccharide units, specifically N-acetylglucosamine and glucuronic acid. Its molecular weight can range from 400 daltons (the disaccharide) to over a million daltons. It is found in variable amounts in all tissues, such as the skin, cartilage, and eye, and in most if not all fluids in adult animals. It is especially abundant in early embryos. Space created by hyaluronan, and indeed GAGs in general, permit it to play a role in cell migration, cell attachment, during wound repair, organogenesis, immune cell adhesion, activation of intracellular signalling, as well as tumour metastasis. These roles are mediated by specific protein and proteoglycan binding to Hyaluronan. Thus, in articular cartilage, hyaluronan and aggrecan form large aggregates important for the function of cartilage (Hardingham and Muir, 1972). Furthermore, cell motility and immune cell adhesion is mediated by the cell surface receptor RHAMM (Receptor for Hyaluronan-Mediated Motility; Hardwick et al., 1992) and CD44 (Jalkenan et al., 1987; Miyake et al., 1990). 
     Hyaluronan is synthesized directly at the inner membrane of the cell surface with the growing polymer extruded through the membrane to the outside of the cell as it is being synthesized. Synthesis is mediated by a single protein enzyme, hyaluronan synthetase (HAS) whose gene family consists of at least 3 members. By contrast other GAGs are synthesized inside the cell in the Golgi apparatus, possibly in association with some core protein, and then released by exocytosis. Hyaluronan degradation in vertebrate tissues in vivo is mediated by hyaluronidase, and exoglycosidases that remove sugars sequentially Mammalian-type hyaluronidases have both hydrolytic and transglycosidase activities and can degrade hyaluronan and chondroitin as well as, to a small extent, dermatan sulfate. 
     Human ES cells will have several distinct roles in medicine, for which it is important that lines are available from a variety of different genotypes. Ideally it should be practicable to obtain ES cells with a new specific genotype by their derivation from embryos produced by nuclear transfer. In addition to their potential use in cell therapy, such lines will create new opportunities for studies of drug toxicology, for drug discovery and studies of diseases that have a genetic component, but not necessarily a result of inappropriate function of a single gene. Although human embryonic stem (hES) cell lines show great promise for the development of cell-based therapies to replace damaged tissues, the currently available lines may be of little utility for this purpose. This is because of their provenance. To be used a source of tissue for transplantation in the future, hES cells will have to be derived and maintained under far more stringent conditions of 1) informed consent from the donor and 2) bio-safety, than hitherto observed. Central to bio-safety issues is the capacity to derive and maintain hES cells or their derivatives under defined and pathogen free conditions. This requirement is severely constrained by the dependence of existing protocols on animal feeder cells and undefined factors provided by feeder cells, conditioned medium, or exogenously supplied serum. 
     It has now been found that Hyaluronan can surprisingly assist in the differentiation of pluripotent stem cells to derive a mortal multi-lineage progenitor cell population which resemble adult mesenchymal stem cells. 
     SUMMARY 
     According to a first aspect of the invention, there is provided a method for the differentiation of mammalian pluripotent stem (PS) cells into a mortal multi-lineage progenitor cell population, comprising culturing the pluripotent stem cells in the presence of Hyaluronan (HA). 
     Such in vitro methods of differentiation provide a mortal multi-lineage progenitor whose terminal differentiation may be dictated by local microenvironment more readily than embryo stem cells. The mortal multi-lineage progenitor cell population may be a population of mesenchymal stem cells. Suitably, the mortal multi-lineage progenitor cell population may have the capacity to form mesodermal lineages, including bone (i.e. osteoblasts), cartilage (i.e. chondroblasts), fat (i.e. adipocytes), connective tissue (i.e. fibroblasts), muscle (i.e. myoblasts or cardiomyocytes), as well as the plasticity to form ectoderm (i.e. epithelial cells, neurons) or endoderm (i.e. hepatocytes, pancreatic islet cells) lineages when cultured subsequently with at least one appropriate stem cell growth factor. 
     Mesenchymal cells may be characterised by expression of the following markers; CD71, CD90, GATA6, Nodal, BMP-2. Additional markers include, but are not restricted to BMPR-IA/ALK-3, BMPR-IB/ALK-6, BMPR-II, Endoglin/CD105, Nucleostemin, Sca-I, SCF R/c-kit, STRO-1, VCAM-1. 
     The process of differentiation of mesenchymal cells into adipocytes, chondrocytes, myocytes, and osteocytes may be characterised by expression of specific markers. Adipogenesis markers include PPARG, Adiponectin/Acrp30, FABP4, Glut4, Leptin. Chondrogenesis markers include SOX9, Collagen 2 (i.e. COL2), COL10, MMP13, Aggrecan, DSPG3. Myogenesis markers include FABP3, GATA-4, NKX2.5, Pax3, Pax7, delta-Sarcoglycan, alpha-Smooth Muscle Actin, Troponin T. Osteogenesis markers include Alkaline Phosphatase, CBFA1/RUNX2, Collagen I, Decorin, Fibronectin, Osteoadherin, Osteocalcin, SPARC, Thrombopoietin, PTHR1, PTHR2. 
     Mesenchymal stem cell growth factors include BMPs (Bone Morphogenetic Proteins), EGF Family, GDFs (Growth Differentiation Factors), IGF Family, VEGF/PDGF Family. The BMPs (Bone Morphogenetic Proteins) include BMP-2, BMP-3, BMP3b/GDF-10, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-15 and Decapentaplegic. 
     The IGF family includes IGF ligands such as IGF-I and IGF-II, the IGF-I Receptor (CD221), the IGF Binding Protein (IGFBP) Family which includes ALS, CTGF/CCN2, Endocan, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, IGFBP-rp1/IGFBP-7 and NOV/CCN3. 
     The EGF family includes the EGF ligands which include Amphiregulin, Betacellulin, EGF, Epigen, Epiregulin, HB-EGF, Neuregulin-3, NRG-isoform GGF2, NRG1-isoform SMDF, NRG1-alpha/HRG1-alpha, NRG1-beta1/HRG1-beta 1, TGF-alpha, TMEFF1/Tomoregulin-1, TMEFF2. The EGF family also includes the EGF R/ErB receptor family which includes EGF R, ErbB2, ErbB3 and ErbB4. 
     The VEGF/PDGF family includes Neuropilin-1, Neuropilin-2, PDGF, PDGF R alpha, PDGF R beta, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGF-AB, PIGF, VEGF R1/Flt-1, VEGF R2/Flk-1, VEGF R3/Flt-4, VEGF, VEGF-B, VEGF-C and VEGF-D. 
     The GDFs (Growth Differentiation Factors) family includes GFD-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-11, GDF-15. 
     Embryonic stem cell markers include ABCG2, Alkaline phosphatase, E-Cadherin, CCR4, CD9, Cripto, DPPA5/ESG1, FGF-4, FGF R4, FoxD3, FoxP3, GDF-3, Integrin alpha 6/CD49f, Integrin beta 1/CD29, Lefty, Nanog, Oct 3/4, Podocalyxin, SOX2, SPARC, SSEA-1, SSEA-3, SSEA-4, STAT3. 
     Early ectodermal lineage markers include Nestin, Otx2, TP63/TP73L, beta-III Tubulin, SHH, and PAX6. Ectoderm has the potential to form cell types such as neurons and early neuronal lineage markers include ACE, ALCAM, CD90/Thy1, GAD1/GAD67, Glut1, MAP2, NCAM-L1, Nectin-2/CD112, NeuroD1, NF-L, NF-M, ROBO3, gamma-Secretase, alpha-Secretase, beta-Secretase, beta-III tubulin, Tyrosine Hydroxylase. Neural stem cell markers include ABCG2, CXCR4, FGF R4, Frizzled-9, Musashi-1, Nestin, Noggin, Nucleostermin, Prominin 2, SOX2, Vimentin. 
     Neural stem cell growth factors include Activins, ALKs (Activin Receptor-like Kinases, BNPs, EGF family, FGF family, GDFs, Hedgehog family, IGF family, VEGF/PDGF family, Wnt-related family as described herein. 
     Early endodermal lineage markers include those for definitive endodemm such as FABP1, FABP2, GATA-4, HNF-3 beta, as well as those markers for primitive endoderm such as alpha-Fetoprotein, beta-Catenin, GATA-4, SOX17 and SOX7. 
     Early mesodermal cell markers include Brachyury, Cryptic and FABP4. 
     Markers for primordial germ cells include early primordial germ cell markers such as GCNF/NR6A1, Oct-3/4 and SSEA-1, as well as late primordial germ cell markers such as FABP9, Stella/Dppa3 and VASA. 
     Other markers are as described in the Examples contained herein. 
     Further differentiation into ectodermal cells can suitably be achieved by culturing progenitor cells in a culture medium comprising BMP/GDF-9. 
     Further differentiation into endodermal cells can suitably be achieved by culturing progenitor cells in a culture medium comprising wnt. 
     Further differentiation into mesodermal cells can suitably be achieved by culturing progenitor cells in a culture medium comprising Activin, Nodal and/or TGF-β. 
     Other differentiation protocols are as described in the Examples contained herein. 
     The invention applies to cells of an embryonic or adult origin in mammals, both human and non-human mammals, including but not limited to human and non-human primates, ungulates, ruminants and rodents. Ungulate species include, but are not limited to, cattle, sheep, goats, pigs, horses. Rodent species include, but are not limited to, rats and mice. The invention may also find application in other mammalian species such as rabbits, cats and dogs. 
     Examples of preferred pluripotent stem cell populations which can be differentiated according to the present invention include primate pluripotent stem (pPS) cells, such as human pluripotent stem (hPS) cells. Such cells include adult human mesenchymal cells. However, the invention may have its greatest application in the differentiation of cells derived from embryonic or fetal sources. 
     Cells derived from embryonic sources may include embryonic stem cells or stem cell lines obtained from a stem cell bank or other recognised depository institution. Other means of producing stem cell lines include the method of Chung et al (2006) which comprises taking a blastomere cell from an early state embryo prior to formation of the blastocyst (at round the 8-cell stage). The technique corresponds to the pre-implantation genetic diagnosis technique routinely practised in assisted reproduction clinics. The single blastomere cell is then co-cultured with established ES-cell lines and then separated from them to form fully competent ES cell lines. 
     Another procedure is that of Meissner &amp; Jaenisch (2006), in which the cdx2 gene is silenced in the donor nucleus during the process of nuclear transfer to prepare a reconstructed embryo from which a line of ES cells is derived. The cdx2 gene is turned back on in the isolated blastocyst cell taken from the embryo which is used to prepare the cell line. This is an example of, so-called, “Alternative Nuclear Transfer” where the embryo is not capable of implantation but the ES cell line derived therefrom is fully competent. 
     A pluripotent stem (PS) cell includes an embryonic stem cell. Pluripotent stem (PS) cells also include pluripotent cells which arise from the further division of totipotent cells that are capable of developing into any other cell type in the adult body but which do not have the potential to develop into an entire body. 
     A key advantage of the methods of the present invention is the fact that HA induces differentiation of a proliferative and mortal cell population of mesodermal derived cell-types whose potential includes (but is not limited to) the formation of osteoblasts, neurons, hepatocytes, germ cells, myocytes and cardiomyocytes, and which potential is retained for an extended period. This should benefit therapeutic strategies involving the transplantation of osteoblasts by minimising the potential for transmission of teratoma forming undifferentiated cells. 
     Hyaluronan is an extracellular matrix glycosoaminoglycan molecule that has not been considered to date in the context of hESC culture. HA (also called hyaluronic acid or sodium hyaluronate) is the simplest type of Glycosaminoglycan (GAG), consisting of up to 25,000 unbranched repeating non-sulfated disaccharide units (M r  401), specifically N-acetylglucosamine and glucuronic acid. Accordingly, it can exist in both low (M r  1500-5000) and high (M r &gt;10,000) molecular weight forms. In addition to being found in variable amounts in all adult tissues, such as the skin, cartilage, and eye, it is also a predominant feature of embryonic ECM, especially during epiblast formation and embryo outgrowth, and in vivo provides the necessary microenvironment for undifferentiated cell expansion (Brown and Papaioannou, 1993). References to Hyaluronan (HA) therefore include references to Hyaluronic acid, Sodium Hyaluronate, or Hyaluronate disaccharide, and may be used interchangeably unless the context clearly specifies otherwise. 
     As a large negatively charged macromolecule with viscoelastic properties, HA physically provides hydrated matrices that facilitate cell proliferation and migration. These processes are mediated by HA&#39;s interactions with a broad range of molecules from lipids and other GAGs, to proteoglycans and proteins. In culture, HA is believed to serve as a reservoir for growth factors, including bFGF, protecting them from tryptic digestion (Locci et al., 1995; Radomsky et al., 1998). Most significantly, HA can modulate growth factor signal transduction via its predominant receptor CD44, also known as the Homing-Cell Adhesion Molecule (H-CAM) for its involvement in mediating cell migration (reviewed in Isacke and Yarwood, 2002). This has been exemplified in a mouse embryonic heart explant model wherein HA augments the transformation of differentiated cardiac endothelial cells into heart-valve mesenchyme via CD44 interaction with ErB2-ErbB3 receptors, and Ras-GTPase signalling (Camenisch et al., 2000; ibid, 2002). 
     In one embodiment, it may be convenient to use Hyaluronate disaccharide in a range of from 5 μg/ml to 100 μg/ml. optionally of from 10 μg/ml to 50 μg/ml, suitably at 10 μg/ml. The formulation may be made in any convenient diluent, including water, isotonic water, and/or phosphate buffered saline (PBS). Additionally, or alternatively, it may be convenient to use Hyaluronic Acid in a range of from 50 μg/ml to 5 mg/ml, optionally of from 75 μg/ml to 500 μg/ml, suitably at 100 μg/ml. The formulation may be made in any convenient diluent, including water, isotonic water, and/or phosphate buffered saline (PBS). 
     Other growth factors, chemicals, nutrients may be present as desired in the medium. Such components may include, but are not limited to, vitamins, antibiotics, etc. 
     The present invention therefore also extends to the use of Hyaluronan as a medium for the differentiation of mammalian pluripotent stem (PS) cells into a mortal multi-lineage progenitor cell population. 
     Hyaluronan may be used in the form of Hyaluronate disaccharide (M r  401), specifically N-acetylglucosamine and glucuronic acid, or Sodium Hyaluronate and/or Hyaluronic Acid, in either or both of the low (M r  1500-5000) and high (M r &gt;10,000) molecular weight forms. 
     According to a second aspect of the invention, there is provided a population of a mortal multi-lineage progenitor cells differentiated from a population of mammalian pluripotent stem (PS) cells according to a method of the first aspect of the present invention. 
     According to a third aspect of the present invention, there is provided a system for the differentiation of a population of a mortal multi-lineage progenitor cells, said system comprising a population of mammalian pluripotent stem (PS) cells cultured in the presence of Hyaluronan and a population of mortal multi-lineage progenitor cells differentiated from said PS cells. 
     Such a system therefore comprises a composition comprising a population of mammalian pluripotent stem (PS) cells cultured in the presence of Hyaluronan and a population of mortal multi-lineage progenitor cells differentiated from said PS cells. 
     According to a fourth aspect of the present invention, there is provided a method for the treatment of a patient suffering from a degenerative disease or injury, the method comprising the transplantation into said patient of a population of mortal multi-lineage progenitor cells prepared according to a method of the present invention. This aspect of the invention, therefore also extends to such cells for use in transplantation. 
     Degenerative diseases or injuries that may be treated in accordance with this aspect of the invention, include but are not limited to bone and cartilage repair, Parkinson&#39;s disease, diabetes, nerve repair, including spinal cord repair, etc. Embodiments of this aspect of the invention, therefore extend to the use of cells prepared according the present invention in the preparation of a medicament for the treatment of a degenerative disease or injury. 
     In one embodiment of the invention, there is therefore provided a culture medium comprising Hyaluronan for the differentiation of mammalian pluripotent stem (PS) cells into a mortal multi-lineage progenitor cell population. 
     The culture medium may be provided in the form of a coating of Hyaluronan for cell attachment. The Hyaluronan may be present as Hylauronan disaccharide (M.Wt. 401.3 D), Sodium Hyaluronate and/or Hyaluronic Acid (M.Wt. 200 kD). The coatings may be applied at around 0.1 ml per cm 2  for 30 minutes at 4° C., before warming to room temperature, followed by aspiration of excess and transfer of cells. Hyaluronate disaccharide may be applied at 10 μg/ml in PBS and/or Hyaluronic acid at 100 μg/ml. Hyaluronic acid may be applied up to at least around 1 mg/ml if desired, or more if required. 
     The present invention therefore also extends to articles coated with Hyaluronan, such as tissue culture dishes, multi-well plates (e.g. 1, 2, 4, 8, 24, 48, 96-wells, etc), Petri-dishes, tissue culture flasks, fermentors, bioreactors etc for the differentiation of mammalian pluripotent stem (PS) cells into a mortal multi-lineage progenitor cell population. Such articles ma be composed of any generally suitable material, such as a plastics material, for example polypropylene, or other materials such as glass, metal etc. Suitable metals include mirror-polished metals, e.g. mirror-polished stainless steel. 
     Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. 
    
    
     
       The present invention will now be further described with reference to the following Examples which are present for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference in the Examples is made to a number of drawings in which: 
         FIG. 1  shows HA and HA receptor expression in hESCs and differentiation after growth on HA/HD or plastic alone. (I) Treatment of both H1 (shown) and H9, with a fluorescently-tagged HA-binding protein specifically stains both undifferentiated colony cells (A) and differentiating cells surrounding each colony (A′), indicative of endogenous HA expression. By immunofluorescence, both cell types in each line are also positive for the HA-binding proteoglycan syndecan, also known as CD138 (B), and the constitutive isoform of the CD44 receptor lacking variant exons, known as CD44s (C). hESCs treated with fluorescently conjugated secondary antibody alone and imaged digitally at the same settings used for HA-binding proteins exhibit negligible staining (D). Bars equal 10 microns. (II) Undifferentiated HESC colonies (see * in A, as shown for H1) can be maintained in mouse embryo fibroblast conditioned media supplemented with bFGF and KOSR if cells are on Matrigel™. However, plating on a substrate of low molecular weight HA (2000 kD; shown B) or Hyaluronan Disachamide induces a fibroblast morphology, comparable to plating on plastic alone (C). Bars equal 10 microns. 
         FIG. 2  shoes the loss of undifferentiated hESC markers following growth on Hyaluronan. (I) By Flow cytometry, undifferentiated H1 (shown) or H9 hESC on Matrigel™ are positive for SSEA4, Tra-1-60, Tra-1-81, and CD9, but negative for SSEA-1 (A). By contrast all cell surface markers, with the exception of CD9, are lost following growth on HA (B, @p11), HD (C, @p11), or untreated plastic (D, @p7). (II) By TAQMAN RT-PCR, the abundance of mRNA transcripts for genes associated with hESC pluripotency, namely Oct-4 and Cripto, are reduced following successive passage (p) on HA or HD, relative to growth on Matrigel™. Return to Matrigel™ does not result in a rise in the expression of these genes. The bar indicates the % of the total population of cells positive for the given marker. In all graphs, y-axis is “Events” from 0 to 100; x-axis in the graphs in (A), (B) and (D) is FL1-H, FL2-H, FL2-H, FL2-H, FL2-H from left to right; in (C) the x-axis is “Empty”; for all x-axes the scale reads in gradations of 10 0 , 10 1 , 10 2 , 10 3  and 10 4    
         FIG. 3  shows loss of Embryoid body formation/mediated differentiation post hESC growth on HA/HD. Suspension culture in DMEM+10% FBS of H1 &amp; H9 (shown) hESCs previously maintained in MEFCM/MG form compact aggregates within 2 days (A) and cystic Embryoid Bodies within 4 days (B). By contrast suspension culture of both hESC lines induced to differentiate by growth on HA or HD (shown H9 on HA for 11 passages) form smaller cell aggregates after 2 days (C), which coalesce to form larger dense aggregates that are not cystic after 4 days (D). Formation of dense cellular aggregates which predominates after 10 passages on HA, correlates with loss of potential to form cells which are positive for ectoderm (D-tubulin III), endoderm (α-fetoprotein) and mesoderm (muscle-specific actin) germinal lineage markers (data not shown). Bar equals 100 microns. 
         FIG. 4  shows similarities in surface marker expression between undifferentiated and HA-differentiated hESCs with human Mesenchymal Stem Cells (hMSCs) from bone marrow. By flow cytometry, undifferentiated H1 (shown) and H9 hESC grown on Matrigel™ (A), differentiated H1 (B) and H9 (C) grown on HA for 20 or 15 passages, respectively, or hMSC maintained in vitro for 11 passages, are positive for CD71 and 90 and negative for CD34 and 45. In the graphs, the y-axis is “Events” from 0 to 100 (except for the first two graphs in (B) and (D) from the left where the “Events” scale runs from 0 to 64; the x-axis in all the graphs is FL1-H and the scale reads in gradations of 10 0 , 10 1 , 10 2 , 10 3  and 10 4 . 
         FIG. 5  shows induction of mineralising osteoblasts from HA-differentiated cells. (I) Alizarin Red S staining was used to detect mineralised nodules in H9 embryoid body (EB) derived cells (+ve control) and H1 cells cultured on HA for 10 and 20 passages following 0, 3, 7 and 10 days of culture in the presence or absence of osteogenic supplement (±OS) as previously described (Sottile et al., 2003). In all treatment groups, staining was only observed after 7 days in the presence of OS, with no staining in its absence. (II) Time course over days in culture of calcium deposition in H9 EB (A) and H1-HA for 10 passages B) cells cultured±OS. Data are presented as mean±SEM (n=4) 
         FIG. 6  shows the effect of HA as an hESC substrate. The mRNA detection was by reverse transcription polymerase chain reaction (RT-PCR) of genetic markers for meso/endoderm (i.e. Gata-6), primitive and definitive endoderm (i.e. HNF-3β and HNF-4α), and ectoderm (i.e. PAX-6 and SHH) in H1 hESC cell line. (A) 100 base pair molecular weight marker with asterisk denoting 300 bp band. (B) H1 grown in culture conditions maintaining undifferentiated cell self-renewal involving mouse embryo fibroblast conditioned medium supplemented with 4 ng/ml bFGF on a substrate of Matrigel™ (Xu et al., 2001). (C-E) H1 hESC grown for 10, 15, and 20 passages in the media specified in (A) but on substrate of 100 μg/cm2 HA. (F) Four day suspension cultured embryoid bodies of differentiating H1 hESCs previously maintained in culture conditions specified in (A). These results suggest that despite HA induced differentiation cells still express gene products associated with mesodermal, endodermal and ectodermal lineages, and thus may still harbour the potential to form such lineages. 
         FIG. 7  shows in situ immunofluorescence staining for epitopes of CD44, a major receptor for HA, in H1 and H9 hESCs grown under self-renewing conditions as specified by Xu et al., 2001. Immunostaining suggests that multiple CD44 isoforms are conservatively expressed in different hESC lines. They include isoforms containing epitopes encoded by standard (s) and variant exons v3, 4, 5, 6, 7, and 10. Immunostaining for v3 was predominantly of undifferentiated colony cells that are known to have self-renewing capacity. 
         FIG. 8  shows detection by RT-PCR of CD44 isoforms in undifferentiated H1 hESCs grown in self-renewing conditions as described by Xu et al., (2001) using primers to exons 5 and 15 flanking variant extracellular exons. At an RNA level, the predominant isoforms appear to be those which either lack variant exons, or contain variant exons v10, v10 &amp; v9, or v10, v9 &amp; v3 
         FIG. 9  shows the effect of HA as an hESC substrate. mRNA detection by reverse transcription polymerase chain reaction (RT-PCR) of genetic markers of mesoderm and pluripotency. (a) expression of mesoderm markers Nodal (562 bp) and BMP-2 (586 bp). (b) expression of pluripotency markersOct-4 (126 bp), Nanog (178 bp) and Vasa (238 bp). In both a and b, from left to right: Lane 1 a portion of 100 base pair molecular weight marker showing markers above or below specified products Lane 2 H1 grown in culture conditions maintaining undifferentiated cell self-renewal involving mouse embryo fibroblast conditioned medium supplemented with 4 ng/ml bFGF on a substrate of Matrigel™ (Xu et al., Nat Biotech 2001; 19:971) (i.e. H-MG). Lane 3-8, H1 hESC grown for 7, 9, 11, 12, 13, 21 passages in the media specified in (A) but on substrate of 100 μg/cm2 HA. Lane 9, Water negative control. 
         FIG. 10  shows effect of HA as an hESC substrate. Expression of neuronal progenitor marker Nestin post neural induction protocol at p10 as described by Gerard et al., 2005). 
         FIG. 11  shows effect of HA as an hESC substrate on mRNA expression of mesoderm lineage markers by RT-PCR. From left to right: Lane 1, H1 hESC grown under conditions that maintain pluripotency (Xu et al., 2001). Lane 2 H1 hESC differentiated as embryoid bodies in suspension culture as per Xu et al., (2001). Lane 3 Adult human osteosarcoma cell line Saos2 treated with OS as per Sottile et al., 2003) for 18 days (superscript 1). Lane 4, H1 hESC grown on HA substrate at 100 μg/cm2 for 10 passages (H1HA-superscript 2). Lane 5-6, HLHA cells grown for 18 days without (lane5) or with (lane 6-superscript 3) OS. Expression patterns suggests that HA treatment causes induction of mesenchymal progenitor cell phenotype with capacity to support fetal endochondrial bone development Abbreviations: OS=Osteogenic supplement; ACTB—β-actin (control); PTIIR1/2—Parathyroid hormone receptor 1/2; ALPL—Alk.phosphatase L (Tissue non-spec.); COL1—Collagen 1 (Major comp. of mesenchyme); OPN—Osteopontin (Secreted Phosphoprotein); RunX2—(CBFA1)/Transcrip. Factor targeting osteocalcin; BSP—Bone Sialo protein (Mature Osteoblast); Sox9—(Transcription factor); Col2—Collagen 2 (Major component of Hyaline cartilage—Chondrocyte; Col10—Collagen 10 (Hypertrophic cartilage @transition to bone); MMP13—Matrix metaloprotein 13r; PPARG-2—peroxisome proliferator-activated receptor-gamma 
     
    
    
     MATERIALS AND METHODS 
     HESC Culture 
     Experiments were performed on both H1 and H9 hESC lines (Thomson et al., 1998). Treatments were replicated in 6 well plates (9.6 cm 2 /well) seeded at approximately 5×10 5 , and passaged/split 1:3 at confluence every 4-6 days, depending on the treatment condition. As a reference standard control cultures of hESC lines were maintained as described by Xu et al. (2001) in Mouse Embryo Fibroblast (MEF) Conditioned Medium (CM) composed of 80% KNOCKOUT-Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), 20% KNOCKOUT serum replacement (KOSR; Invitrogen), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng/ml human basic fibroblast growth factor (hbFGF). Cell cultures were normally grown in an atmosphere of 5% CO 2  in humidified air (20% O 2 ) at 37° C., and fed fresh MEF CM daily. Cultures were normally passaged by incubation in 200 units/ml Collagenase IV for 5-10 min at 37° C. Alternatively, hESCs were grown in an atmosphere of 5% CO 2 , 5% O 2 , and 90% N 2  (May 5, 1990), and/or passaged by incubation in Trypsin-EDTA (Invitrogen) for 5-10 min at 37° C. 
     Embryoid Body Mediated Differentiation 
     Human ES cells were dissociated into small clumps by treatment for 10 min in 200 Units/Collagenase IV in KO-DMEM at 37° C., and cultured for 4 days in suspension on low cluster plates in embryoid body differentiation (EBD) medium consisting of 80% KO-DMEM, 20% FBS (not heat inactivated), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids. The resulting embryoid bodies were transferred onto glass slide tissue culture chambers coated with 0.5% gelatin @ 0.1 ml/cm 2 , and cultured for an additional 11 days, feeding every second day. 
     Induction of Osteogenesis 
     Osteogenic differentiation was induced as previously described by Sottile et al., (2003). An osteogenic (OS) supplement consisting of 50 μM ascorbic acid phosphate (Wako, Neuss, Germany), 10 mM β-glycerophosphate (Sigma), and 100 nM dexamethasone (Sigma) was added to EBD medium. Cells derived from embryoid bodies (H9 control) or grown previously on HA (H1) for 10 or 20 passages were plated on 0.1% gelatin and treated with EBD medium±OS that was changed every 2 days over the indicated intervals. 
     HMSC Cell Culture 
     The hiMSC/c151 immortalised human bone marrow stromal cell line (Okamoto et al., 2002) kindly provided to the McWhir lab by Dr. J. Toguchida (Kyoto University, Japan)—referred to as hMSC) was cultured and expanded as previously described in DMEM containing 10% FCS, L-glutamine and nonessential amino acids. 
     Tissue Culture Plate Coating 
     Matrigel™ (Becton Dickinson) diluted 1:30 in cold KO-DMEM was applied as a coating of 0.1 ml per cm 2 , with coated plates used between 1-7 days of storage at 4° C. Hyaluronate Disaccharide (HD) at 10 μg/ml in PBS, and Hyaluronic acid (HA; 2000 kD purified from Streptococcus sp.) at 100 μg/ml in PBS, were applied as coatings of 0.1 ml per cm 2  for 30 min at 4° C., before warming to room temperature, aspiration of excess and transfer of cells. 
     Flow Cytometry 
     Single cell suspensions of were prepared by treatment with Trypsin EDTA, resuspended in KO DMEM and aliquoted for each probe to be evaluated. Cells were spun at (11000 g) for 5 min, and resuspended in 50 μl of Blocking Buffer consisting of 40% heat inactivated Rabbit serum (Jackson&#39;s Immunosystems, Stratech Scientific Ltd) in Staining Buffer (SB) composed of 2% FCS in PBS and 2 mM EDTA. Cells were blocked for 10-15 min on ice, followed by addition of primary antibody probes at the recommended concentration (see supplementary table 4) and incubation on ice for 30 min. To remove unbound probe, 3 mls of SB were added per group, and decanted after centrifugation at (1300 rpm) # g&#39;s for 5 min. When necessary exposure to the requisite dilution of fluorescent probe conjugated secondary antibody was in 100 μl of SB on ice for 30 min in the dark. Unbound probe was removed by addition of 3 ml of PBS, decanted following centrifugation at 1000 g&#39;s for 5 min. For immediate assessment on a Becton Dickinson FACSCAN, cells were resuspended in 0.5-11.0 ml of PBS. Otherwise cells were resuspended in 0.5-1 ml of 1% paraformaldehyde (PFA) in PBS, and stored in the dark at 4° C. for assessment within 24 h. 
     Immunocytochemistry 
     Cells were plated on either 4 well-glass chambers or microscope slides precoated with Matrigel™ as previously described, and cultured to desired confluence or differentiation. Cells were washed with PBS before fixation in 4% PFA in PBS for 20 min at room temperature. Fix was removed by washing 2×5 min with PBS, and immunostained immediately or after storage in PBS for up to 2 weeks. Cells were blocked with PBS containing 10% Normal Goat Serum and 0.01% Triton-X for 1 h at room temperature. Primary and secondary antibodies at optimised dilutions (see supplementary table) were prepared in PBS containing 1% NGS and 0.01% Triton-X. These were used at room temperature for 1-2 h, with unbound antibody removed by 3×5-10 min washes in PBS at room temperature. Slides were mounted in Vectashield containing Dapi (Cat. No., Supplier, City, Country), and stored at 4° C. in the dark prior to viewing on an Zeiss Axiovert S100 equipped for epifluorescence and digital imaging and analysis. 
     Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis 
     RNA was isolated using RNAzol and reverse transcribed using a First strand cDNA synthesis kit (Supplier, City, Country) according to manufacturers instructions. Gene-specific PCR was performed in accordance with gene requirements. To quantify the steady state level of mRNA for undifferentiated hESC markers (Oct-4, and Cripto) transcript abundance was measured by quantitative Real-Time RT-PCR using a TaqMan Universal PCR Master Mix protocol for an ABI Prism 7700 Sequence Detection System equipped with version 1.9.1 analysis software. Transcript abundance in experimental treatment groups was first normalised to amplification of 18S rRNA, and then expressed relative to normalised transcript abundance in control undifferentiated HESC culture on MG. Oligonucleotide primers and probes are summarised in supplementary table 2, with TAQMAN used according to manufacturers recommendations (ABI Biosystems: http://www.europe.appliedbiosystems.com). 
     Osteoblast Staining and Mineralisation Assays 
     Detection of mineralised nodules by Aliziarin-Red S (Sigma) staining and calcium deposition were as described previously by Sottile et al., 2003. Prior to staining cells were washed twice with calcium- and magnesium-free PBS, followed by fixation in 95% methanol. Fixed cells were incubated with a 1% Alizarin-Red S (Sigma) solution for 10 min, and then washed twice with water. Cell matrix-associated calcium deposition was determined in 96 well plates using the Sigma calcium assay kit (Sigma), in triplicate wells. 
     Example 1 
     Capacity of Hyaluronan to Serve as a Soluble Substrate for Human Embryo Stem Cells (hESC) Growth 
     Under culture conditions which maintain hESC self-renewal, hESCs secrete Hyaluronan and express receptors which can bind to it and mediate its effects, most notably Syndecan (CD138) and the Homing-Cell Adhesion Molecule (H-CAM, also known as CD44) ( FIG. 1 ). 
       FIG. 1(I)  shows the results of in situ fluorescence microscopy on undifferentiated hESC H1 (shown) and H9 with a fluorescently-tagged HA-binding protein stains undifferentiated colony (A) and differentiating non-colony cells (A′). Both hESC populations are also positive for the HA-binding proteoglycan syndecan, also known as CD138 (B), and the constitutive isoform of the CD44 receptor lacking variant exons, known as CD44s (C). hESCs treated with fluorescently conjugated secondary antibody alone and imaged digitally at the same settings used for HA-binding proteins exhibit negligible staining (D). Bars equal 10 microns. FIG.  1 (II) shows maintenance of H1 (shown) and H9 in MEFCM/MG as described in the methods preserves undifferentiated colony cells (see * in A) whereas plating of both lines on HA (shown) or HD in high (shown) or low oxygen atmosphere, and passaging with Collagenase (shown) or Trypsin, results in differentiation to a fibroblast-like-morphology which proliferate rapidly as a monolayer for over 25 passages. By contrast, growth on plastic alone (C) yields a similar morphology but growth arrest after 5-7 passages. Bars equal 10 microns. 
     With this insight, the capacity of Hyaluronan was investigated for its ability to serve as a soluble substrate for human embryo stem cells (hESC) growth in bFGF supplemented and feeder cell-conditioned medium that normally supports self-renewal. Culture of both H1 and H9 cell lines on Hyaluronan Disaccharide (HD) or low molecular weight HA (2000 kD) promotes differentiation into a fibroblast-like morphology within 5 passages of growth in a high or low oxygen atmosphere (i.e. 5% CO2 in air (20% O2), or 5% CO2, 5% O2, 90% N2 (May 5, 1990)), with cells dissociated using collagenase or trypsin ( FIG. 1 ). 
     Co-incident with their differentiation, HA induced cells lose most undifferentiated hESC markers, including SSEA-4, Tra-1-60, Tra-1-80, Oct-4, Cripto, but retain CD-9 ( FIG. 2 ). 
       FIG. 2(I)  shows results using Flow cytometry, undifferentiated H1 (shown) or H9 hESC on MEFCM/MG are +ve for SSEA4, Tra-1-60, Tra-1-81, and CD9, but −ve for SSEA-1 (A). By contrast all cell surface markers, with the exception of CD9, are lost following growth on HA (B, @p11), HD (C, @p11), or untreated plastic (D, @p7). FIG.  2 (II) shows results using TAQMAN RT-PCR, the abundance of mRNA transcripts for genes associated with hESC pluripotency, namely Oct-4 and Cripto, are reduced following successive passage (p) on HA or HD, relative to growth on Matrigel™. Return to Matrigel™ does not result in a rise in the expression of these genes. 
     Unlike hESCs plated on plastic alone that also become fibroblast-like and gradually cease to proliferate after 5-10 passages, HA induced cells grow rapidly as a monolayer for over 25 passages before their arrest. Their ability to form Embryoid bodies (EBs) and indirectly differentiate into adult cells representative of endodermal, mesodermal, and ectodermal lineages, specifically those expressing α-fetoprotein, muscle-specific actin, and β-tubulin III, respectively, is lost by 10 passages on HA or HD ( FIG. 3 ). Despite this HA induced cells retain the capacity to form mineralising osteoblasts, by direct differentiation protocol after a further 10 passages ( FIG. 5 ). In addition, RT-PCR for definitive markers of endoderm and lineages, specifically GATA-6 (meso/endoderm), HNF-3b and HNF-4-a (primitive and definitive endoderm), and PAX-6 and SHH (ectoderm), continued to be expressed at later passages near the time of loss of proliferative potential ( FIG. 6 ). These cells also retained expression of adult mesenchymal markers CD71 and 90 like embryo stem cells, but not CD34 and 45 expressed by hematopoietic cells ( FIG. 4 ). Thus, when provided as a matrix for hESC attachment under media conditions that normally maintain hESC self-renewal, HA induces differentiation of a proliferative and mortal cell population whose potential to form osteoblasts is retained for an extended period. This may benefit strategies for the therapeutic transplantation of osteoblasts by minimising the potential for transmission of teratoma forming undifferentiated cells. 
     Example 2 
     Expression of CD44 Isoforms on hESCs 
     The predominant receptor for HA is CD44, mediating most of its roles in cell attachment, migration, proliferation and differentiation. CD44 is encoded by a single gene expressed as multiple isoforms as a result of alternative splicing and post-translational modifications. Expression of these variant exons can both modulate and be modulated by growth factor ligands and receptors. We have also investigated the expression of CD44 variant (v) exons in human embryo stem cell lines at a protein and mRNA level in order to understand HA signalling pathways affecting stem cells, specifically hESCs. By indirect immunofluorescence microscopy both H1 and H9 hESCs exhibited significant staining above that observed with secondary antibody alone with all antibody probes evaluated that were directed against the standard isoform of CD44 (CD44s) as well as variant exons for v3, 4, 5, 6, 7 and 10. Immunostaining for CD44v3, was predominantly localised to undifferentiated colony cells. ( FIG. 7 ). Using RT-PCR primers to conserved exons which flanked variant extracellular domains for CD44 on H1 mRNA we amplified, subcloned and sequenced four major products of 300 bp [conserved exons 5, 15], 400 bp [exons 5, 14 (v10), 15], 500 bp (exons 5, 13 (v9), 14 (v10), 15) and 600 bp (5, 8 (v3), 13 (v9), 14 (v10), 15). These results indicate that multiple isoforms of CD44 are expressed by undifferentiated hESCs, the predominant of which are those containing the standard exons and no variant exons, or variant exons 10, 9 and 10, or 3, 9 and 10. ( FIG. 8 ). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Summary of FACS and cytochemical probes. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Targeted 
                   
                   
                   
                   
                   
               
               
                 Epitope 
                 Probe type-(Conjugate) 
                 Supplier 
                 Catalogue # 
                 Dilution 
                 Use 
               
               
                   
               
               
                 AFP 
                 Mouse monoclonal IgG2a 
                 Sigma-Aldrich 
                 A8452 
                  1:500 
                 FM 
               
               
                 β-Tubulin 
                 Mouse monoclonal IgG2b 
                 Sigma-Aldrich 
                 T8660 
                  1:1000 
                 FM 
               
               
                 CD44 
                 Mouse monoclonal IgG1 
                 Santa Cruz 
                 sc-7297 
                  1:200 
                 FM 
               
               
                 (HCAM)* 
               
               
                 Muscle Actin 
                 Mouse monoclonal IgG1 
                 DAKO 
                 M0635 
                 1:50 
                 FM 
               
               
                   
                 Kappa 
               
               
                 Syndecan 
                 Mouse monoclonal IgG1 
                 Cymbus 
                 CBL588 
                 1:50 
                 FM 
               
               
                 Non-specific 
                 Mouse monoclonal 
                 Sigma 
                 M3645 
                  1:100 
                 FACS 
               
               
                 serum ? 
                 IgG3 
               
               
                 Non-specific 
                 Mouse monoclonal IgM 
                 Sigma 
                 M3795 
                  1:100 
                 FACS 
               
               
                 serum ? 
               
               
                 SSEA1 
                 IgM 
                 DSHB 
                 MC- 
                 1:5  
                 FACS 
               
               
                   
                   
                   
                 480 
               
               
                 SSEA4 
                 IgG3 
                 DSHB 
                 MC-813- 
                 15:50  
                 FACS 
               
               
                   
                   
                   
                 70 
               
               
                 Tra-1-60 
                 Mouse monoclonal IgM 
                 Chemicon 
                 MAB 
                 1:10 
                 FACS 
               
               
                   
                   
                   
                 4360 
               
               
                 Tra-1-81 
                 Mouse monoclonal IgM 
                 Chemicon 
                 MAB 
                 1:20 
                 FACS 
               
               
                   
                   
                   
                 4381 
               
               
                 CD-9 
                 Mouse monoclonal IgG1- 
                 BD Pharmingen 
                 555372 
                 1:5  
                 FACS 
               
               
                   
                 (PE) 
               
               
                 Mouse Ig —   
                 Goat polyclonal IgG- 
                 Sigma-Aldrich 
                 F5262 
                 1/100 
                 FM 
               
               
                   
                 (FITC) 
               
               
                 Mouse Ig —   
                 Donkey polyclonal IgG- 
                 Jackson 
                 715-095- 
                 1/100 
                 FM 
               
               
                   
                 (Cy3) 
                   
                 150 
               
               
                 Rabbit Ig —   
                 Goat polyclonal IgG- 
                 Sigma-Aldrich 
                 F9887 
                 1/100 
                 FM 
               
               
                   
                 (FITC) 
               
               
                 Mouse Ig —   
                 Donkey polyclonal IgG- 
                 Jackson 
                 715-025- 
                 1/100 
                 FM 
               
               
                   
                 (TRITC) 
                   
                 150 
               
               
                 Mouse Ig —   
                 Goat polyclonal IgG- 
                 Southern 
                 1102-02 
                 1/100 
                 FACS 
               
               
                   
                 (FITC) 
                 Biotechnologies 
               
               
                 Mouse Ig —   
                 Goat polyclonal IgG-(PE) 
                 Southern 
                 1022-09. 
                 1/100 
                 FACS 
               
               
                   
                   
                 Biotechnologies 
               
               
                 Mouse IgG1 
                 Rat 
                 BD Pharmingen 
                 349073 
                 1/100 
                 FACS 
               
               
                 (Kappa) 
               
               
                 Non-specific 
                 IgG1-(PE) 
                 BD Pharmingen 
                 555749 
                 1/100 
                 FACS 
               
               
                 serum ? 
               
               
                 Hyaluronan 
                 HA Binding Protein- 
                 CalBiochem 
                 385906 
                 1/100 
                 FM 
               
               
                   
                 (FITC) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Oligonucleotide Primer Information 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Primer Seq 
                 Accession 
                 C DNA 
                 Gen DNA 
                 Spans 
                   
               
               
                 Name 
                 (5′-3′) 
                 No. 
                 posn. 
                 posn. 
                 Intron 
                 Chrom 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 GATA6 BP 
                 CCATGACTC 
                 &gt;gi|40288196| 
                 1803- 
                 18014909- 
                   
                   
               
               
                 for 
                 CAACTTCCA 
                 ref|NM_0052 
                 1822 
                 18014928 
                   
                   
               
               
                   
                 CC-3′ 
                 57.3| 
                   
                   
                   
                   
               
               
                   
               
               
                 GATA6 BP 
                 ACGGAGGAC 
                 &gt;gi|40288196| 
                 1998- 
                 18032718- 
                 YES 
                   
               
               
                 rev 
                 GTGACTTCG 
                 ref|NM_0052 
                 2016 
                 18032736 
                   
                   
               
               
                   
                 GGC′ 
                 57.3| 
                   
                   
                   
                   
               
               
                   
               
               
                 HNF-3β 
                 AGATGGAAG 
                 &gt;gi|5805393|g 
                 1621- 
                 22559865- 
                   
                 20 
               
               
                 for 
                 GGCACGAGC′ 
                 b|AF176110.1 
                 1638 
                 22559882 
                   
                   
               
               
                   
                   
                 |AF176110 
                   
                   
                   
                   
               
               
                   
               
               
                 HNF-3β 
                 CAGGCCGGC 
                 &gt;gi|5805393|g 
                 2729- 
                 22558757- 
                 No 
                   
               
               
                 rev 
                 GTTCATGTT 
                 b|AF176110.1 
                 2746 
                 22558774 
                   
                   
               
               
                   
                   
                 |AF176110 
                   
                   
                   
                   
               
               
                   
               
               
                 HNF-4α 
                 CTGCTCGGA 
                 &gt;gi|1217960|e 
                 878-901 
                 43732531- 
                   
                   
               
               
                 for 
                 GCCACCAAG 
                 mb|Z49825.1| 
                   
                 43732554 
                   
                   
               
               
                   
                 AGATCCTG 
                 HSHNF4A 
                   
                   
                   
                   
               
               
                   
               
               
                 HNE-4α 
                 ATCATCTGC 
                 &gt;gi|1217960|e 
                 1224- 
                 43738226- 
                 YES 
                   
               
               
                 rev 
                 CCACGTGAT 
                 mb|Z49825.1| 
                 1248 
                 43738249 
                 (2) 
                   
               
               
                   
                 GCTCTGCA′ 
                 HSHNF4A 
                   
                   
                   
                   
               
               
                   
               
               
                 HSHH for 
                 TTAGCCTAC 
                 &gt;gi|663156|gb 
                 275-296 
                 155021527- 
                   
                 7 
               
               
                   
                 AAGCAGTTT 
                 |L38518.1|HU 
                   
                 155021548 
                   
                   
               
               
                   
                 ATCC 
                 MSHH 
                   
                   
                   
                   
               
               
                   
               
               
                 HSHH rev 
                 ACTCGTAGT 
                 &gt;gi|663156|gb 
                 661-686 
                 155015972- 
                 YES 
                   
               
               
                   
                 ACACCCAGT 
                 |L38518.1|HU 
                   
                 155015997 
                   
                   
               
               
                   
                 CG 
                 MSHH 
                   
                   
                   
                   
               
               
                   
               
               
                 hPAX6 for 
                 AACAGACAC 
                 gi|189632|gb| 
                 1368- 
                 31779354- 
                   
                 11 
               
               
                   
                 AGCCCTCAC 
                 M93650.1|H 
                 1390 
                 31779376 
                   
                   
               
               
                   
                 AAACA 
                 UMPAX6AN 
                   
                   
                   
                   
               
               
                   
               
               
                 HPAX6 rev 
                 CGGGAACTT 
                 gi|189632|gb| 
                 1620- 
                 31775835- 
                 YES 
                   
               
               
                   
                 GAACTGGAA 
                 M93650.1|H 
                 1642 
                 31775957 
                 (2) 
                   
               
               
                   
                 CTGAC 
                 UMPAX6AN 
                   
                   
                   
                   
               
               
                   
               
               
                 β-actin 
                 TCACCACCA 
                 X00351 
                 640-658 
                   
                 YES 
                   
               
               
                 for 
                 CGGCCGAGC 
                   
                   
                   
                   
                   
               
               
                   
                 G 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 β-actin 
                 TCTCCTTCTG 
                 X00351 
                 990-971 
                   
                   
                   
               
               
                 rev 
                 CATCCTGTC 
                   
                   
                   
                   
                   
               
               
                   
                 G 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 hOct-4 F 
                 ACATCAAGC 
                 NA 
                   
                   
                   
                   
               
               
                   
                 TCTGCAGAA 
                   
                   
                   
                   
                   
               
               
                   
                 AGAAC 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 hOct-4-R 
                 CTGAATACC 
                 NA 
                   
                   
                   
                   
               
               
                   
                 TTCCCAAAT 
                   
                   
                   
                   
                   
               
               
                   
                 AGAACCC 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 hNanog-F 
                 CAGCTGTGT 
                 NA 
                   
                   
                   
                   
               
               
                   
                 GTACTCAAT 
                   
                   
                   
                   
                   
               
               
                   
                 GATAGATTT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-Nanog-R 
                 ACACCATTG 
                 NA 
                   
                   
                   
                   
               
               
                   
                 CTATTCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 CAGTTG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-Vasa-F 
                 AAGAGAGGC 
                 NA 
                   
                   
                   
                   
               
               
                   
                 GGCTATCGA 
                   
                   
                   
                   
                   
               
               
                   
                 GATGGA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-Vasa-R 
                 CGTTCACTTC 
                 NA 
                   
                   
                   
                   
               
               
                   
                 CACTGCCAC 
                   
                   
                   
                   
                   
               
               
                   
                 TTCTG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-Nodal-F 
                 CGACCAACC 
                 NA 
                   
                   
                   
                   
               
               
                   
                 ATGCATACA 
                   
                   
                   
                   
                   
               
               
                   
                 TCC 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-Nodal-R 
                 GTGACTTCA 
                 NA 
                   
                   
                   
                   
               
               
                   
                 TCCCACCTC 
                   
                   
                   
                   
                   
               
               
                   
                 CAA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-BMP2-F 
                 CTTCTAGCG 
                 NA 
                   
                   
                   
                   
               
               
                   
                 TTGCTGCTT 
                   
                   
                   
                   
                   
               
               
                   
                 CC 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 h-BMP2-R 
                 TGCTTGCAT 
                 NA 
                   
                   
                   
                   
               
               
                   
                 TCTGATTCA 
                   
                   
                   
                   
                   
               
               
                   
                 CC 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAQMAN 
                 GAA ACC 
                 gi|18088410|g 
                 557-578 
                 31239192- 
                   
                   
               
               
                 OCT for 
                 CAC ACT 
                 b|BC020712. 
                   
                 31239211 
                   
                   
               
               
                   
                 GCA GCA GA 
                 1| 
                   
                   
                   
                   
               
               
                   
               
               
                 TAQMAN 
                 CAC ATC CTT 
                 gi|18088410|g 
                 599-617 
                 31238889- 
                   
                   
               
               
                 OCT rev 
                 CTC GAG 
                 b|BC020712. 
                   
                 31239169 
                   
                   
               
               
                   
                 CCC A 
                 1| 
                   
                   
                   
                   
               
               
                   
               
               
                 TAQMAN 
                 CAG CCA 
                 gi|18088410|g 
                 578-597 
                 31239190- 
                   
                   
               
               
                 OCT Probe 
                 CAT CGC 
                 b|BC020712. 
                   
                 31239170 
                   
                   
               
               
                   
                 CCA GCA GC 
                 1| 
                   
                   
                   
                   
               
               
                   
               
               
                 TAQMAN 
                 TGA GCA 
                 &gt;gi|30220|em 
                 565-581 
                 46581915- 
                 Y 
                   
               
               
                 Cripto for 
                 CGA TGT 
                 b|X14253.1|H 
                   
                 46581931 
                   
                   
               
               
                   
                 GCG CA 
                 SCRTPTO 
                   
                   
                   
                   
               
               
                   
               
               
                 TAQMAN 
                 TTC TTG GGC 
                 &gt;gi|302200|em 
                 627-610 
                 46582057- 
                   
                   
               
               
                 Cripto 
                 AGC CAG 
                 b|X14253.1|H 
                   
                 46582074 
                   
                   
               
               
                 rev 
                 GTG 
                 SCRIPTO 
                   
                   
                   
                   
               
               
                   
               
               
                 TAQMAN 
                 AGA GAA 
                 &gt;gi|30220|em 
                 583-608 
                 46581933- 
                   
                   
               
               
                 Cripto 
                 CTG TGG GTC 
                 b|X14253.1|H 
                   
                 46582055 
                   
                   
               
               
                 Probe 
                 TGT GCC CCA 
                 SCRIPTO 
                   
                   
                   
                   
               
               
                   
                 TG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Primary and secondary antibodies for CD44 screening 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 DF1485 
                 Santa Cruz IgG1 Mouse monoclonal HCAM (CD44s) 
               
               
                 AB2081 
                 Chemicon IgG Rabbit polyclonal (CD44v3) 
               
               
                 MCA1728 
                 Serotec IgG1 Mouse monoclonal (CD44v4) 
               
               
                 MCA1729 
                 Serotec IgG1 Mouse monoclonal (CD44v5) 
               
               
                 MCA1730 
                 Serotec IgG1 Mouse monoclonal (CD44v6) 
               
               
                 MCA1731 
                 Serotec IgG1 Mouse monoclonal (CD44v7) 
               
               
                 MCA1733 
                 Serotec IgG1 Mouse monoclonal (CD44v10) 
               
               
                 555748 
                 Becton Dickinson Pharmingen IgG1 FITC (neg. control) 
               
               
                 F9887 
                 Sigma Anti-Rabbit FITC (for CD44v3 and its 
               
               
                   
                 negative control) 
               
               
                 F5262 
                 Sigma Anti-Mouse FITC (for the rest CD44 variants) 
               
               
                   
               
            
           
         
       
     
                     TABLE 4               Dilutions of antibodies                                        CD44s (HCAM)   1/100, 2ndary anti-mouse FITC 1/100       CD44v3   1/25, 2ndary anti-rabbit FITC 1/100       CD44v4   1/100, 2ndary anti-mouse FITC 1/100       CD44v5   1/100, 2ndary anti-mouse FITC 1/100       CD44v6   1/100, 2ndary anti-mouse FITC 1/100       CD44v7   1/100, 2ndary anti-mouse FITC 1/100       CD44v10   1/100, 2ndary anti-mouse FITC 1/100       Pre-conjugated IgG1   20 μl antibody + 100 μl NGS 1%, no secondary       negative control   antibody       Negative v3   no primary antibody (100 μl NGS 1%),           secondary anti-rabbit FITC 1/100                    
Summary of Results:
 
1. Characterisation of hESC growth on Low Molecular Wt HA or HD vs. Matrigel in MEF CM/low bFGF culture.
         a. Both H1 and H9 react with and express HA-binding proteins ( FIG. 1I ).
           i. Fluorescent HA-Binding protein staining reflects endogenous HA.   ii HA-binding proteoglycan (Syndecan, CD138) expressed by colony and differentiating cells.   iii Constitutive form of predominant HA receptor (CD44s) expressed by both colony and differentiating cells.   
           b. For both H1 &amp; H9, growth on HA/HD promotes differentiation to fibroblast-like morphology, which is irreversible after 5-7 passages ( FIG. 1  II).
           i Differentiation occurs regardless of whether in low oxygen or high oxygen (5% CO 2  in air) tension, and method of cell dissociation (collagenase vs. trypsin)—data not shown.   ii Unlike hESC plated on plastic alone that also differentiate into fibroblast morphology and cease to proliferate by p10, HA induced cells proliferate until approximately p27.   
           c. Karyotype of HA induced cells is preserved until end of proliferative life (data not shown).   d. Differentiation is characterised by loss of most undifferentiated surface markers by FACS (i.e. SSEA4, Tra-1-60, Tra-1-81, but not CD9) &amp; genetic markers by TAQMAN markers (i.e. Oct-4, Cripto, hTert, podocalyxin, GRP-R) ( FIG. 2 ).   e. HA induced cells lose ability to form embryoid bodies and indirect differentiation by approximately p10 ( FIG. 3 ).   f. HA induced fibroblasts and undifferentiated hESC resemble human bone marrow stromal cells in that they are +ve for CD71, CD90 &amp; CD44 and negative for CD34 and 45 ( FIG. 4 ).   g. After loss of EB forming capacity, HA induced cells respond to osteogenic factors to form mineralising osteoblasts ( FIG. 5 ).       

     Example 3 
     Expression of Lineage Markers in hESCs Cultured on HA 
     The effect of HA as an hESC substrate on expression of lineage markers can be seen as follows. Mesoderm markers Nodal and BMP-2 are shown in  FIG. 9(   a ) and pluripotency cell markers Oct-4, Nanog and Vasa in  FIG. 9(   b ). Further evidence of mesodermal marker expression is shown in  FIG. 10  where the neuronal progenitor cell marker Nestin is expressed at passage  10 , 
       FIG. 11  shows a more comprehensive survey of mesodermal marker expression in hESCs cultured on HA as a substrate. Expression suggests that HA induction of mesenchymal progenitor cells and fetal endochondrial bone development (vs intramem in adult). 
     The markers shown to be expressed include ACTB—β-actin (control); PTBR1/2—Parathyroid hormone receptor 1/2; ALPL—Alk.phosphatase L (Tissue non-spec.); COL1—Collagen 1 (Major comp. of mesenchyme); OPN—Osteopontin (Secreted Phosphoprotein); RunX2—(CBFA1)/Transcrip. Factor targeting osteocalcin; BSP—Bone Sialo protein (Mature Osteoblast); Sox9—(Transcription factor); Col2—Collagen 2 (Major component of Hyaline cartilage—Chondrocyte; Col10—Collagen 10 (Hypertrophic cartilage @transition to bone); MMP13—Matrix metaloprotein 13r; PPARG-2—peroxisome proliferator-activated receptor-gamma 
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