AND DIFFERENTIATION

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of the following U.S. Provisional Application

No.:61/602,399, filed February 23, 2012, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Injuries involving the central and/or peripheral nervous system (CNS) often result in lifelong disabilities due to loss of neural (e.g., motor and sensory) function. Recovery from such injuries is typically poor because the injured CNS is a highly inhibitory environment for axon regeneration that severely limits functional recovery, and because neurons lost due to injury do not typically regenerate. Transplantation of bioscaffold encased human embryonic stem cells (hESCs) has been proposed as a possible clinical therapy for various neurological lesions and disorders. Artificially synthesized carbon-based biomaterials such as carbon nanotube (CNT) and graphene have demonstrated feasibility in supporting stem cell attachment and differentiation. However, the applicability is significantly hampered by evidence of nanotoxic effects on multiple cell types. There is an urgent need for a carbonaceous biomaterial that would support neuronal growth, promote neuronal

differentiation and enhance axon outgrowth. Such a biomaterial would be useful for treating injuries, conditions and disorders affecting the nervous system. SUMMARY OF THE INVENTION

As described below, the present invention features microscale carbonaceous compositions (e.g., activated charcoal) comprising extracellular matrix components, embryonic stem cells or other neuronal progenitor cells and in vitro and in vivo methods of using such compositions to promote neuronal growth and differentiation.

In one aspect, the invention generally features method for generating a differentiated mammalian cell, the method involving culturing a mammalian stem cell on a carbonaceous substrate containing microscale carbon particles under conditions that promote cellular differentiation, thereby generating a differentiated mammalian cell.

In another aspect, the invention features a method for generating a differentiated neuronal cell, the method involving culturing a stem cell or other neuronal precursor on a carbonaceous substrate containing microscale carbon particles under conditions that promote neuronal differentiation, thereby generating a differentiated neuronal cell.

In still another aspect, the invention features a method for generating a differentiated neuronal cell, the method involving culturing an embryonic stem cell on an activated charcoal substrate under conditions that promote neuronal differentiation, thereby generating a differentiated neuronal cell.

In still another aspect, the invention features a carbonaceous substrate that supports the growth of a cell (e.g., a neuron), the substrate containing microscale carbon particles, and at least one extracellular matrix component.

In still another aspect, the invention features a carbonaceous scaffold for implantation into a subject, the scaffold containing a carbonaceous substrate containing microscale carbon particles and a mammalian cell.

In still another aspect, the invention features a method of ameliorating cell or tissue loss in a subject in need thereof, the method involving delivering to the subject an effective amount of a carbonaceous scaffold of any of the above aspects or otherwise delineated herein. In one embodiment, the cell or tissue loss or damage is associated with a condition that is any one or more of a central nervous system injury, spinal cord injury, peripheral nervous system injury, ischemic injury, stroke, or myocardial infarction. In another embodiment, the scaffold is delivered surgically or by injection.

In still another aspect, the invention features a pharmaceutical composition containing a carbonaceous scaffold of any of the above aspects in a pharmaceutically acceptable excipient.

In still another aspect, the invention features a culture system comprising a

carbonaceous substrate suitable for cell growth and directions for the use of the culture system to promote cell growth or differentiation according to a method of any of the above aspects or otherwise delineated herein. In one embodiment, the carbonaceous substrate is fixed to a cover slip, culture flask or culture plate.

In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the stem cell is a human embryonic stem cell, induced pluripotent stem cell, neuronal stem cell, or other neuronal progenitor. In various

embodiments of any of the above aspects, the differentiated cell is a neuron, motor neuron, sensory neuron, oligodendrocyte, or astrocyte. In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the carbonaceous substrate or scaffold contains activated charcoal. In certain embodiments, the substrate or scaffold contains activated charcoal and an extracellular matrix (ECM) component (e.g., collagen, chitosan or a collagen-chitosan combination). In particular embodiments, the carbonaceous substrate or scaffold contains one or more biopolymers (e.g., collagen, gelatin, hyaluronan, chitosan, and alginate. In other embodiments, the carbonaceous substrate or scaffold contains one or more growth factor selected from the group consisting of nerve growth factor, angiopoietin, acidic fibroblast growth factors (FGF), basic FGF, bone morphogenic protein, vascular endothelial growth factor, epidermal growth factor, transforming growth factor a, transforming growth factor β, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor a, hepatocyte growth factor, insulin like growth factor, erythropoietin, colony stimulating factor, macrophage-CSF, granulocyte/macrophage CSF and nitric oxide synthase. In other embodiments, a carbonaceous scaffold or substrate contains a differentiated mammalian cell that is a neuron, motor neuron, sensory neuron, oligodendrocyte, or astrocyte. .

The invention provides implantable carbonaceous compositions comprising embryonic stem cells, neuronal stem cells, or other cell types capable of generating neuronal cells, and methods of using such compositions in in vitro culture methods, or as implantable scaffolds to ameliorate an injury or other condition associated with neuronal cell death, or a decrease in neural function or activity. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10%, 25%, 40%, 50% or greater change.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical

modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

By "carbonaceous composition" is meant a material comprising 3-dimensional microscale carbon particles that support cell adhesion, growth, and/or differentiation, alone or in the presence of other materials. In preferred embodiments, a carbonaceous composition comprises activated charcoal and one or more extracellular matrix materials or artificial polymers. In one embodiment, the carbonaceous composition does not comprise or is substantially free of carbon nanotubes and/or graphene. If desired, the carbonaceous composition is functionalized with one or more proteins or other agents to enhance cell growth and differentiation. The carbonaceous composition of the invention are substantially free of carbon nanotubes. The carbonaceous composition of the invention are also substantially free of graphene. In one embodiment, carbon nanotubes are specifically excluded from compositions of the invention. In another embodiment, graphene is specifically excluded from the invention.

By "cell substrate" is meant the material that is in contact with a cell. A cell substrate of the invention comprises carbonaceous composition (e.g., activated charcoal) an

extracellular matrix component, and, if desired, polypeptides, peptides, or other molecular components.

By "central nervous system" (CNS) is meant the brain or spinal cord, and cellular or molecular components thereof, including the extracellular materials and fluids.

By "central nervous system disease or injury" is meant any disease, disorder, or trauma that disrupts the normal function or connectivity of the brain or spinal cord.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

By "effective amount" is meant the amount of a composition of the invention required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of a cellular composition used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

"Engraft" refers to the process of cellular contact and incorporation into an existing tissue of interest (e.g., neural) in vivo

By "enhancing axonal outgrowth" is meant increasing the number of axons or the distance of extension of axons relative to a control condition. Preferably the increase is by at least 2-fold, 2.5-fold, 3-fold or more.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a

polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "microscale" is meant between 1 μπι and 999 μπι in size. A particle that is microscale is larger in size than a nanotube.

By "neuron" is meant any cell that i) expresses one or more markers characteristic of a neuronal cell type; ii) has one or more functional characteristics of a neuron; and/or iii) has a neuronal morphology.

By "neural progenitor" is meant any cell capable of giving rise to a neuron under suitable conditions.

By "peripheral nerve graft" is meant any cellular or non-cellular material derived from the peripheral nervous system that is implanted into a heterologous environment. In one approach, the peripheral nerve graft generally comprises an acellular matrix that supports axonal extension.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

By "reference" is meant a standard or control condition.

By "restorative nervous system surgery" is meant any procedure carried out on the nervous system to enhance neurological function. An exemplary restorative surgery is a peripheral nerve graft.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By "embryonic stem cell" is meant a multipotent cell of embryonic origin.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 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, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and IB are images of activated charcoal-extracellular matrix (AC- ECM) composite substrate. Figure 1 A depicts brightfield images of varying

concentrations (0-50 g/L; inset) of AC-collagen composite substrates on cover slips. Figure IB depicts a scanning electron microscope (SEM) micrograph of AC-collagen composite substrate.

Figures 2A-2L show axonal growth of differentiated neurons on AC-ECM composite substrates. Figures 2A-2D depict representative images of βΙΙΙ tubulin staining of axonal development from differentiated neurons on AC-collagen substrate at different concentrations (0, 10, 25, and 50 g/L, respectively). Figures 2E-2H depict representative images of βΙΙΙ tubulin staining of axonal development from differentiated neurons on AC-chitosan substrate at different concentrations (0, 10, 25, and 50 g/L, respectively). Figures 2I-2L depict representative images of βΙΙΙ tubulin staining of axonal development from differentiated neurons on AC-collagen-chitosan substrate at different concentrations (0, 10, 25, and 50 g/L, respectively).

Figures 3A-3L show axonal growth of differentiated neurons on AC-ECM composite substrates. Figure 3A is a graph showing that ratios of increasd in axonal length as the concentration of activated charcoal increased on AC-collagen substrate, as examined by βΙΙΙ tubulin staining (n > 10, **p < 0.005). Figure 3B is a graph showing that ratios of increasd in axonal density as the concentration of activated charcoal increased on AC-collagen substrate, as examined by βΙΙΙ tubulin staining (n > 10, **p < 0.005). Figure 3C is a graph showing that ratios of increasd in axonal length as the concentration of activated charcoal increased on AC-collagen substrate, as examined by peripherin staining (n > 10, **p < 0.005). Figure 3D is a graph showing that ratios of increasd in axonal density as the concentration of activated charcoal increased on AC- collagen substrate, as examined by peripherin staining (n > 10, **p < 0.005). Figure 3E is a graph showing that ratios of increasd in axonal length as the concentration of activated charcoal increased on AC-chitosan substrate, as examined by βΙΙΙ tubulin staining (n > 10, **p < 0.005). Figure 3F is a graph showing that ratios of increasd in axonal density as the concentration of activated charcoal increased on AC-chitosan substrate, as examined by βΙΙΙ tubulin staining (n > 10, **p < 0.005). Figure 3G is a graph showing that ratios of increasd in axonal length as the concentration of activated charcoal increased on AC-chitosan substrate, as examined by peripherin staining (n > 10, **p < 0.005). Figure 3H is a graph showing that ratios of increasd in axonal density as the concentration of activated charcoal increased on AC-chitosan substrate, as examined by peripherin staining (n > 10, **p < 0.005). Figure 31 is a graph showing that ratios of increasd in axonal length as the concentration of activated charcoal increased on AC- collagen-chitosan, as examined by βΙΙΙ tubulin staining (n > 10, **p < 0.005). Figure 3J is a graph showing that ratios of increasd in axonal density as the concentration of activated charcoal increased on AC-collagen-chitosan, as examined by βΙΙΙ tubulin staining (n > 10, **p < 0.005). Figure 3K is a graph showing that ratios of increasd in axonal length as the concentration of activated charcoal increased on AC-collagen- chitosan substrate, as examined by peripherin staining (n > 10, **p < 0.005). Figure 3L is a graph showing that ratios of increasd in axonal density as the concentration of activated charcoal increased on AC-collagen-chitosan substrate, as examined by peripherin staining (n > 10, **p < 0.005). Figures 4A-4D are graphs showing comparison of human embryonic stem cell (hESC) neuronal differentiation between AC-collagen and AC-CNT substrates. Figure 4A is a graph depicting differences in the increasing ratios of axonal length between collagen control, AC-collagen and AC-CNT (n > 10, **p < 0.005). Figure 4B is a graph depicting differences in the increasing ratios of axonal density between collagen control, AC-collagen and AC-CNT (n > 10, **p < 0.005). Figure 4C is a graph providing a direct comparison between the AC-collagen and AC-CNT data shown in Figure 4A (n > 10, **p < 0.005). Figure 4D is a graph providing a direct comparison between the AC- collagen and AC-CNT data shown in Figure 4B (n > 10, **p < 0.005).

Figures 5A-5D depict cellular viability and morphology of differentiated cells on

AC-collagen substrate. Figure 5A is a graph depicting the results of a cellular viability assay, which are expressed as percentages of live and dead cells (n > 10, **p < 0.005). Figure 5B is an SEM image depicting the morphological features of differentiated neuronal-like cells on collagen control and AC-collagen matrix. Figures 5C and 5D are SEM images depicting the morphological features of differentiated neuronal-like cells on AC-collagen matrix. Figure 5D clearly shows thick axonal extension across the matrix.

Figures 6A-6F are micrographs showing immunofluorescent staining of differentiated neuronal cells with neuronal lineage markers. Figure 6A depicts Pax-6 staining of hESCs differentiated on AC-collagen 50 g/L substrates. Figure 6B depicts Nestin staining of hESCs differentiated on AC-collagen 50 g/L substrates. Figure 6C depicts Map-2 staining of hESCs differentiated on AC-collagen 50 g/L substrates.

Figure 6D depicts Neurofilament staining of hESCs differentiated on AC-collagen 50 g/L substrates. Figure 6E depicts Myelin basic protein staining of hESCs differentiated on AC-collagen 50 g/L substrates. Figure 6F depicts Olig-2 staining of hESCs differentiated on AC-collagen 50 g/L substrates.

Figures 7A-7B depict the results of functional assays showing depolarization- dependent cytosolic Ca ²⁺ signaling and synaptic vesicle recycling. Figure 7A is a graph depicting high K ⁺ induced depolarization-dependent calcium influx as rhod-2

fluorescence increased in cytosol in differentiated neuronal cells on AC-collagen 50 g/L substrates (n > 4, **p < 0.005). Figure 7B is a graph depicting reduced the fluorescence of FMl-43 dye loaded differentiated neurons by high K ⁺ stimulation, which suggests the presence of active synaptic vesicles recycling (n > 4, **p < 0.005).

Figures 8A-8B show that AC and AC-collagen substrate enhanced protein adsorption. Figure 8A is a graph depicting that AC-collagen composite substrate increased adsorption of proteins (BSA) in solution over time (n > 5, **p < 0.005).

Figure 8B is a graph depicting that AC particles increased adsorption of proteins (BSA) in solution over time (n > 5, **p < 0.005). The adsorption was AC concentration dependent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features carbonaceous compositions (e.g., activated charcoal), comprising embryonic stem cells, induced pluripotent stem cells, or other neural progenitors; and in vitro and in vivo methods of using such compositions to ameliorate an injury or condition (e.g., spinal cord injury) associated with a reduction in cell number, function, or activity.

The invention is based, at least in part, on the discovery that a natural bamboo-based activated charcoal (AC) composite biosubstrate can support and promote neuronal differentiation from human embryonic stem cells. Strikingly, the biocompatible activated charcoal composite biomatrix yielded more matured neuronal-like cells. Both axonal length and density were at least twice more abundant than control groups. Functional assays demonstrated that the derived neuronal-like cells responded to depolarization-dependent calcium signaling and possibly contains active synapses. In addition, the activated charcoal composite substrate concentrates growth factors and cell adhesion proteins, further encouraging attachment and differentiation. The results indicate that neuronal differentiation of hESCs can be markedly enhanced with the inclusion of nontoxic and economically manufactured activated charcoal. Moreover, the activated charcoal composite biomaterial can potentially be constructed into implantable 3D bioscaffolds, increasing the potential of regenerating injured neural and other tissues.

Carbonaceous Scaffolds

Plant derived activated charcoal (AC) is a unique, well established quasi-graphitic biomaterial (Thomas (2008) Biotechnol Adv 26: 618-631). Various physical characteristics of activated charcoal, such as dimension of particle and size, shape or density of pores can be easily controlled by modulating the synthesis process (Dastgheib et al., (2001) Carbon 39: 1849-1855). Activated charcoal is relatively hydrophilic and can be functionalized with specific charges or conjugated with bioactive materials. It is produced when pulverized organic substances, such as bamboo, sawdust, peat, coconut shell or other vegetation, are heated to 600-900°C (Olson et al., (2010) J Med Toxicol 6: 190-198). The activation process erodes the internal surfaces of the material with steam or hot air thereby creating a fine network of micro-pores (Olson et al., (2010) J Med Toxicol 6: 190-198). Large adsorptive surface area and volume of activated charcoal favors industrial applications such as gas separation, water filtration, waste decontamination and catalyst support.

Activated charcoal is also an effective semiconductor, routinely incorporated in the manufacturing of electrodes for battery and fuel cells. Besides the industrial relevance, activated charcoal has pervasive biological applications. Activated charcoal can enhance the growth of plant tissue culture by the sequestration of inhibitory substances in culture media, removal of phenolic oxidation, and continuous release of adsorbed nutrients in a controlled manner (Thomas (2008) Biotechnol Adv 26: 618-631). The biomedical utilizations of activated charcoal range from trapping bacteria and bacterial spores from solution to removing fetid odor from malignant fungating wounds. Furthermore, gastrointestinal decontamination with activated charcoal remains the clinically approved treatment of choice when removing ingested toxin or medication poisoning. Despite these conventional roles whether activated charcoal can serve as a novel biomaterial to support human embryonic stem cell (hESC) growth has never, heretofore, been explored. As reported in the Examples below, activated charcoal -extracellular matrix composite biomaterial was useful in promoting human embryonic stem cell differentiation towards neuronal lineages.

Methods for Constructing Carbonaceous Scaffolds

Carbonaceous scaffolds of the invention may be formed from virtually any carbon source. In particular embodiments, carbonaceous scaffolds are generated from charcoal (e.g., activated charcoal) generated from plant or other organic substances (e.g., bamboo, sawdust, peat, coconut shell) heated to 600-900°C as described, for example, in Olson et al., (2010) J Med Toxicol 6: 190-198. The physical properties of the activated charcoal can be modified to optimize the charcoal for use in cell culture as a substrate or for implantation into a subject. In particular, the size, shape or density of pores can be modified according to the methods described, for example, by Dastgheib et al., (2001) Carbon 39: 1849-1855. In other embodiments, the activated charcoal is functionalized with specific charges or conjugated with bioactive materials. The activation process erodes the internal surfaces of the material with steam or hot air thereby creating a fine network of micro-pores to enhance diffusion within the scaffold. See, for example, Olson et al., (2010) J Med Toxicol 6: 190-198. In various embodiments, a carbonaceous scaffold further comprises an ECM component. ECM components include structural proteins, such as collagen and elastin;

proteins having specialized functions, such as fibrillin, fibronectin, and laminin; and proteoglycans that include long chains of repeating disaccharide units composed of glycosaminoglycans (e.g., hyaluronan, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, aggrecan).

In other embodiments, a carbonaceous substrates comprises one or more of the following: collagen, chitosan, poly(D,L-lactide-co-glycolide (PLGA) fiber matrices, polyglactin fibers, calcium alginate, polyglycolic acid (PGA), poly-(L-lactic acid) (PLLA) and polyanhydrides. Carbonaceous substrates can include materials that are nonbiodegradable or biodegradable. Desirably, biodegradable materials will degrade over a time period of less than a year, more preferably less than six months.

The carbonaceous substrate may form a hydrogel. A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. Examples of materials that can be used to form a hydrogel include polysaccharides (e.g., alginate), polyphosphazenes, and polyacrylates (e.g., hydroxyethyl methacrylate). Other materials that can be used include proteins (e.g., fibrin, collagen, fibronectin) and polymers (e.g., polyvinylpyrrolidone), and hyaluronic acid. In general, these polymers are at least partially soluble in aqueous solutions, (e.g., water) buffered salt solutions, or aqueous alcohol solutions, which have charged side groups, or monovalent ionic salts thereof. Examples of polymers with acidic side groups that can be reacted with cations are poly(phosphazenes), poly( acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly( vinyl acetate), and sulfonated polymers (e.g., sulfonated polystyrene). Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.

Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted with anions are poly( vinyl amines), poly( vinyl pyridine), poly( vinyl imidazole), and some imino substituted polyphosphazenes. The ammonium or quaternary salt of the polymers can also be formed from the backbone nitrogens or pendant imino groups. Examples of basic side groups are amino and imino groups. Alginate can be ionically cross-linked with divalent cations in water at room temperature to form a hydrogel matrix. Additional methods for the synthesis of the other polymers described above are known to those skilled in the art (see, for example, Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, E. Goethals, editor, Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such as poly(acrylic acid), are commercially available.

Synthetic polymers can also be used to form a matrix, and are preferred for reproducibility and controlled release kinetics. Synthetic polymers that can be used include bioerodible polymers such as poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes, and non-erodible polymers such as polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly( vinyl imidazole), chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon™, and nylon. Non-degradable materials can also be used to form the matrix. Preferred polymers for use in the matrix have mechanical and biochemical properties that enhance viability and proliferation of transplanted cells, tissues, or organs.

Synthetic degradable polymer matrices have been proposed as a means of tissue reconstruction and repair. The matrix serves as both a physical support and an adhesive substrate for isolated cells during in vitro culturing and subsequent in vivo implantation. Matrices are used to deliver cells to desired sites in the body, to define a potential space for engineered tissue, and to guide the process of tissue development. Cell transplantations on substrates are useful for the regeneration of tissues and organs (e.g., skin, nerve, liver, pancreas, cartilage and bone tissue) using various biological and synthetic materials.

Carbonaceous scaffolds of the invention may be functionalized with one or more biologically active agents that support cell growth, proliferation, or differentiation. Such biologically active agents include cell adhesion molecules and growth factors. Cell adhesion molecules suitable for use in the invention include components of the extracellular matrix that promote cell spreading or extension or fragments thereof. Preferably, fragments of a cell adhesion molecule include the adhesion molecule binding domain. Exemplary cell adhesion molecules include cadherin (e.g., E-cadherin, N-cadherin), cell adhesion molecule (CAM) (e.g., neuronal cell adhesion molecule (NCAM), vascular cell adhesion molecule (VCAM)-l and intracellular adhesion molecule (ICAM)-l,), fibronectin, integrin (e.g., β-integrin), laminin, and selectin.

Growth factors are typically polypeptides or fragments thereof that support the survival, growth, or differentiation of a cell (e.g., a neuron or neural progenitor).

Carbonaceous scaffolds can comprise or be conjugated to virtually any growth factor known in the art. Such growth factors include nerve growth factor, angiopoietin, acidic fibroblast growth factors (aFGF) (GenBank Accession No. NP_149127) and basic FGF (GenBank Accession No. AAA52448), bone morphogenic protein (GenBank Accession No.

BAD92827), vascular endothelial growth factor (VEGF) (GenBank Accession No.

AAA35789 or NP_001020539), epidermal growth factor (EGF)(GenBank Accession No. NP_001954), transforming growth factor a (TGF-a) (GenBank Accession No. NP_003227) and transforming growth factor β (TFG-β) (GenBank Accession No. 1109243 A), platelet- derived endothelial cell growth factor (PD-ECGF)(GenBank Accession No. NP_001944), platelet-derived growth factor (PDGF)( GenBank Accession No. 1109245 A), tumor necrosis factor a (TNF- a) (GenBank Accession No. CAA26669), hepatocyte growth factor

(HGF)(GenBank Accession No. BAA14348), insulin like growth factor (IGF)(GenBank Accession No. P08833), erythropoietin (GenBank Accession No. P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF)(GenBank Accession No. AAB59527), granulocyte/macrophage CSF (GM-CSF) (GenBank Accession No. NP_000749) and nitric oxide synthase (NOS)(GenBank Accession No. AAA36365).

Cells of the Invention

Carbonaceous scaffolds contain a carbonaceous substrate, an ECM component, and a cell of the invention. Stem cells of the present invention include embryonic stem cells. The embryonic stem (ES) cell has unlimited self-renewal and pluripotent differentiation potential (Thomson, J. et al. 1995; Thomson, J.A. et al. 1998; Shamblott, M. et al. 1998; Williams, R.L. et al. 1988; Orkin, S. 1998; Reubinoff, B.E., et al. 2000). These cells are derived, for example, from the inner cell mass (ICM) of a pre-implantation blastocyst (Thomson, J. et al. 1995; Thomson, J.A. et al. 1998; Martin, G.R. 1981), or can be derived from the primordial germ cells from a post-implantation embryo (embryonal germ cells or EG cells). ES have been derived from multiple species, including mouse, rat, rabbit, sheep, goat, pig and more recently from human and human and non-human primates (U.S. Patent Nos. 5,843,780 and 6,200,806). Embryonic stem cells are well known in the art. For example, U.S. Patent Nos.

6,200,806 and 5,843,780 refer to primate, including human, embryonic stem cells. U.S.

Patent Applications Nos. 20010024825 and 20030008392 describe human embryonic stem cells. U.S. Patent Application No. 20030073234 describes a clonal human embryonic stem cell line. U.S. Patent No. 6,090,625 and U.S. Patent Application No. 20030166272 describe an undifferentiated cell that is stated to be pluripotent. U.S. Patent Application No.

20020081724 describes what are stated to be embryonic stem cell derived cell cultures. An exemplary embryonic cell line is H9 from Wicell (Madison, WI).

In one embodiment, a stem cell is present in a mixed population of cells, which can be purified to a degree sufficient to produce a desired effect. Those skilled in the art can readily determine the percentage of hematopoietic stem cells or their progenitors in a population using various well-known methods, such as fluorescence activated cell sorting (FACS).

Purity of the stem cells can be determined according to the genetic marker profile within a population. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).

Cell Isolation

One aspect of the invention pertains to the use of a carbonaceous scaffold comprising cells of interest (e.g., embryonic stem cells, induced pluripotent stem cells, neuronal progenitors, neuronal cells, and other differentiated cell types). In certain embodiments, the scaffold may comprises other cell types, such as glial cells, fibroblasts. The cells present in the scaffold may be derived from the recipient's own tissue, derived from a different individual of the same species, or derived from a mammalian species that is different from the recipient (e.g., pig or primate). Cells can be isolated from a number of sources, for example, from biopsies or autopsies using standard methods. The isolated cells are preferably autologous cells obtained by biopsy from the subject. The cells from biopsy can be expanded in culture. Cells from relatives or other donors of the same species can also be used with appropriate immunosuppression. Methods for the isolation and culture of cells are discussed in Fauza et al. (J. Ped. Surg. 33, 7-12, 1998)

Cells are isolated using techniques known to those skilled in the art. For example, an embryonic tissue can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with digestive enzymes (e.g., trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase, and dispase). Mechanical disruption can be accomplished by scraping the surface of the organ, the use of grinders, blenders, sieves, homogenizers, pressure cells, or sonicators. For a review of tissue disruption techniques, see Freshney, (Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, Ch. 9, pp. 107-126, 1987)

Preferred cell types include, without limitation, human embryonic stem cells, induced pluripotent stem cells, neuronal stem cells, or other neural progenitor. Once the tissue has been reduced to a suspension of individual cells, the suspension can be fractionated into subpopulations. This may be accomplished using standard techniques (e.g., cloning and positive selection of specific cell types or negative selection, i.e., the destruction of unwanted cells). Selection techniques include separation based upon differential cell agglutination in a mixed cell population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell sorting. For a review of clonal selection and cell separation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, Ch. 11 and 12, pp. 137-168, 1987).

Cell fractionation may be useful for the selection of cells capable of giving rise to differentiated neurons. Isolated cells can be cultured in vitro to increase the number of cells available for transplantation. The use of allogenic cells or autologous cells is useful\ to prevent tissue rejection. However, if an immunological response does occur in the subject after implantation of the neuronal scaffold, the subject may be treated with

immunosuppressive agents, such as cyclosporin or FK506, to reduce the likelihood of rejection.

Isolated cells may be transfected. Useful genetic material may be, for example, genetic sequences that are capable of enhancing neuronal growth or differentiation. For example, neurons or other cell types present in the neuronal scaffold may carry genetic information useful in supporting the long-term survival of neurons present in the scaffold. In other embodiments, the cells comprise sequences useful for detecting or monitoring the implanted cells. In one example, the neuronal cell or cells present in the scaffold are genetically modified to express a bioactive molecule that promotes neuronal growth or survival. In another example, the cell or cells of the microvascular scaffold are genetically modified to expresses a fluorescent protein marker. Exemplary markers include GFP, EGFP, BFP, CFP, YFP, and RFP. Transfection may be used for transient gene expression or stable gene expression by incorporation of the gene into the host cell.

Isolated cells can be normal or genetically-engineered to provide additional or normal function. Methods for genetically engineering cells with viral vectors such as retroviral vectors or other methods known to those skilled in the art can be used. These include using expression vectors which transport and express nucleic acid molecules in the cells (see, for example, Goeddel et al., (Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990).

Vector DNA is introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

Methods for Making Cell Suspensions

Isolated neuronal stem cells are mixed with a matrix and injected directly, or cultured for a time with a suitable matrix polymer. The polymer is dissolved in solution to a concentration capable of forming a polymeric hydrogel. The isolated cells, including neuronal cells or neural progenitors may be suspended in the polymer solution to a concentration of between 0.5 and 500 million cells/ml, preferably between 1 and 50 million cells/ml, and most preferably between 5 and 10 million cells/ml.

Cells are cultured on or embedded in a carbonaceous composition or injected into a carbonaceous scaffold already implanted at the desired site. Desirably, the matrix is a nontoxic, injectable carbonaceous scaffold that allows for neuronal growth. The carbonaceous scaffold should be shaped to maximize surface area, to allow adequate diffusion of nutrients and growth factors to the cells and to allow the engraftment of the neurons into a target tissue. The carbonaceous scaffold configuration is dependent on the tissue which is to be treated, repaired, or produced.

Therapeutic and Prophylactic Applications

The present invention provides a ready supply of carbonaceous scaffolds comprising cells of the invention (e.g., embryonic stem cells, induced pluripotent stem cells, neuronal progenitors, neuronal cells, and other differentiated cell types). The invention also provides methods of using these cells to ameliorate conditions associated with neuronal cell loss or injury. Cells of the invention are administered (e.g., directly or indirectly) to a damaged or diseased tissue or organ where they engraft and establish functional connections with a target tissue. For example, a carbonaceous scaffold of the invention can be used to establish functional neural connections within an injured spinal cord, or between the central or peripheral nervous system and one or more target tissues (e.g., muscle, skin). In one embodiment, an implanted scaffold comprising differentiated neuronal cells enhances neural connectivity. Methods for repairing damaged tissue or organs may be carried out either in vitro, in vivo, or ex vivo.

Administration

Cells of the invention include human embryonic stem cells that have been induced to express one or more markers characteristic of a differentiated neuronal cell (e.g., Nestin, Map2, Neurofilament, MBP). Such cells can be provided directly to a tissue or organ of interest (e.g., by direct injection or surgical implantation). If desired, the cells are delivered to a portion of the spinal cord that innervates a tissue or organ, where connectivity between the spinal cord and the target have been lost due to trauma or disease.

Advantageously, cells (e.g., embryonic stem cells, induced pluripotent stem cells, neuronal progenitors, neuronal cells, and other differentiated cell types) of the invention engraft within the central or peripheral nervous system and establish connectivity with a target tissue or organ. If desired, expansion and differentiation agents can be provided prior to, during or after administration of the cells to increase, maintain, or enhance production or differentiation of the cells in vivo. Compositions of the invention include pharmaceutical compositions comprising a carbonaceous substrate, cells (e.g., embryonic stem cells, induced pluripotent stem cells, neuronal progenitors, neuronal cells, and other differentiated cell types) and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, cells obtained from one subject, can be administered to the same subject or a different, compatible subject. Methods for administering cells are known in the art, and include, but are not limited to, catheter administration, systemic injection, localized injection, intravenous injection, intramuscular, intracardiac injection or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). Formulations

Cellular compositions of the invention can be conveniently provided as sterile preparations. In one embodiment, a composition of the invention is provided as a liquid, liquid suspension, gel, viscous composition, or solid composition. Liquid, gel, and viscous compositions are somewhat more convenient to administer, especially by injection. Viscous compositions can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid

polyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells (e.g., embryonic stem cells, neuronal progenitors, differentiated neurons) as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water,

physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid- filled form).

A method to potentially increase cell survival when introducing the cells into a subject is to incorporate cells or their progeny (e.g., in vivo, ex vivo or in vitro derived cells) of interest into a biopolymer or synthetic polymer. Depending on the subject's condition, the site of injection might prove inhospitable for cell seeding and growth because of scarring or other impediments. Examples of biopolymer include, but are not limited to, cells mixed with a carbonaceous scaffold comprising fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. This could be constructed with or without included expansion or

differentiation factors. Additionally, these could be in suspension, but residence time at sites subjected to flow would be nominal. Another alternative is a three-dimensional gel comprising the carbonaceous scaffold. Again, expansion or differentiation factors could be included with the cells. These could be deployed by injection via various routes described herein.

Exemplary agents that may be delivered together with a cell of the invention include, but are not limited to, any one or more of activin A, adrenomedullin, acidic FGF, basic fibroblast growth factor, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, angiostatin, angiotropin, angiotensin-2, bone morphogenic protein 1, 2, or 3, cadherin, collagen, colony stimulating factor (CSF), endothelial cell-derived growth factor, endoglin, endothelin, endostatin, endothelial cell growth inhibitor, endothelial cell-viability maintaining factor, ephrins, erythropoietin, hepatocyte growth factor, human growth hormone, TNF-alpha, TGF-beta, platelet derived endothelial cell growth factor (PD-ECGF), platelet derived endothelial growth factor (PDGF), insulin-like growth factor-1 or -2 (IGF), interleukin (IL)-l or 8, FGF-5, fibronectin, granulocyte macrophage colony stimulating factor (GM-CSF), heart derived inhibitor of vascular cell proliferation, IFN-gamma, IFN-gamma, integrin receptor, LIF, leiomyoma-derived growth factor, MCP-1, macrophage-derived growth factor, monocyte-derived growth factor, MMP 2, MMP3, MMP9, neuropilin, neurothelin, nitric oxide donors, nitric oxide synthase (NOS), stem cell factor (SCF), VEGF- A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, and VEGF 164. Other agents that may be delivered together with a cell of the invention include one or more of LIF, BMP-2, retinoic acid, trans-retinoic acid, dexamethasone, insulin, indomethacin, fibronectin and/or 10% fetal bovine serum, or a derivative thereof.

Those skilled in the art will recognize that the polymeric components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the cell as described in the present invention. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue

experimentation), from this disclosure and the documents cited herein. Dosages

One consideration concerning the therapeutic use of cells of the invention is the quantity of cells necessary to achieve an optimal effect. In general, doses ranging from 1 to 4 x 10' cells may be used. However, different scenarios may require optimization of the amount of cells injected into a tissue of interest. Thus, the quantity of cells to be

administered will vary for the subject being treated. In a preferred embodiment, between 10 ⁴ to 10 8 , more preferably 105 to 107 , and still more preferably, 1, 2, 3, 4, 5, 6, 7 x 107 stem cells of the invention can be administered to a human subject.

Fewer cells can be administered directly a tissue where an increase in cell number is desirable. Preferably, between 10 ² to 10 ⁶ , more preferably 10 ³ to 10 ⁵ , and still more preferably, 10 ⁴ neuronal progenitor cells can be administered to a human subject. However, the precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. As few as 100-1000 cells can be administered for certain desired applications among selected patients. Therefore, dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

Cells of the invention can comprise a purified population of cells expressing one or more neuronal cell markers (e.g., Nestin, Map2, Neurofilament). As described herein, cells of the invention are identified by the expression of markers, by cellular morphology, by the ability to form a particular cell type (e.g., neuronal cell), or by biological function (e.g., synaptic vesicle recycling, depolarization-dependent calcium signaling). Those skilled in the art can readily determine the percentage of cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Preferable ranges of purity in populations comprising neuronal progenitors or differentiated neuronal cells are about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%. More preferably the purity is about 70 to about 75%, about 75 to about 80%, about 80 to about 85%; and still more preferably the purity is about 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Purity of a cell population can be determined according to the marker profile within a population. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). Other cells, i.e., cells other than a cell of interest, that may be present in scaffolds of the invention include, but are not limited to, fibroblasts, oligodendrocytes, astrocytes, and endothelial cells.

The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions to be administered in methods of the invention. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD ₅₀ in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.

If desired, cells of the invention (e.g., embryonic stem cells, induced pluripotent stem cells, neuronal progenitors, neuronal cells, and other differentiated cell types) are delivered in combination with (prior to, concurrent with, or following the delivery of) agents that increase survival, increase proliferation, enhance differentiation, and/or promote maintenance of a differentiated cellular phenotype. Expansion agents include growth factors that are known in the art to increase proliferation or survival of neuronal progenitors or differentiated neuronal cells. In vitro and ex vivo applications of the invention involve the culture of a neuronal progenitor cell with a selected agent or combination of agents to achieve a desired result. If desired, neuronal progenitor cells or differentiated neural cells are delivered in combination with other factors that promote cell survival, differentiation, or engraftment.

Delivery Methods

Compositions of the invention (e.g., scaffolds comprising cells) can be provided directly to a tissue or organ of interest, such as a tissue having a deficiency in neural cell number or neural cell function as a result of injury or disease (e.g., by administration into the central or peripheral nervous system). Compositions can be administered to subjects in need thereof by a variety of administration routes. Methods of administration, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include surgical engraftment or injection (e.g., intramuscular, intra-cardiac, intraocular, intracerebro ventricular). In one approach, cells present in a carbonaceous scaffold are implanted into a host. The

transplantation can be autologous, such that the donor of the cells is the recipient of the transplanted cells; or the transplantation can be heterologous, such that the donor of the cells is not the recipient of the transplanted cells. Once transferred into a host, neuronal cells are engrafted, such that they assume the function and architecture of the native host tissue. In another embodiment, once transferred into a host, neuronal progenitor cells undergo differentiation. The cells are then engrafted, such that they assume the function and architecture of the native host tissue.

Neural progenitor cells can be cultured, treated with agents and/or administered as part of a carbonaceous scaffold. If desired, agents described herein are incorporated into a carbonaceous scaffold to promote cell survival, proliferation, or enhance maintenance of a cellular phenotype. Carbonaceous scaffolds are designed to optimize gas, nutrient, and waste exchange by diffusion. The carbonaceous scaffold can be shaped to maximize surface area, to allow adequate diffusion of nutrients and growth factors to the cells. Taking these parameters into consideration, one of skill in the art could configure a carbonaceous scaffold having sufficient surface area for the cells to be nourished by diffusion.

Expression of Recombinant Proteins

In another approach, neuronal progenitor cells of the invention may be engineered to express a gene of interest whose expression promotes cell survival, proliferation,

differentiation, maintenance of a cellular phenotype, or otherwise enhances the engraftment of the cell. The gene of interest may be constitutively expressed or its expression may be regulated by an inducible promoter or other control mechanism where conditions necessitate highly controlled regulation or timing of the expression of a protein, enzyme, or other cell product. Such neuronal progenitor cells, when transplanted into a subject produce high levels of the protein to confer a therapeutic benefit. For example, w cell of the invention, its progenitor or its in vzYro-derived progeny, can contain heterologous DNA encoding genes to be expressed, for example, in gene therapy. Insertion of one or more pre-selected DNA sequences can be accomplished by homologous recombination or by viral integration into the host cell genome. The desired gene sequence can also be incorporated into the cell, particularly into its nucleus, using a plasmid expression vector and a nuclear localization sequence. Methods for directing polynucleotides to the nucleus have been described in the art. The genetic material can be introduced using promoters that will allow for the gene of interest to be positively or negatively induced using certain chemicals/drugs, to be eliminated following administration of a given drug/chemical, or can be tagged to allow induction by chemicals, or expression in specific cell compartments.

Calcium phosphate transfection can be used to introduce plasmid DNA containing a target gene or polynucleotide into a neuronal progenitor cell and is a standard method of DNA transfer to those of skill in the art. DEAE-dextran transfection, which is also known to those of skill in the art, may be preferred over calcium phosphate transfection where transient transfection is desired, as it is often more efficient. Since the cells of the present invention are isolated cells, microinjection can be particularly effective for transferring genetic material into the cells. This method is advantageous because it provides delivery of the desired genetic material directly to the nucleus, avoiding both cytoplasmic and lysosomal degradation of the injected polynucleotide. Cells of the present invention can also be genetically modified using electroporation.

Liposomal delivery of DNA or RNA to genetically modify the cells can be performed using cationic liposomes, which form a stable complex with the polynucleotide. For stabilization of the liposome complex, dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPQ) can be added. Commercially available reagents for liposomal transfer include Lipofectin (Life Technologies). Lipofectin, for example, is a mixture of the cationic lipid N-[l-(2, 3-dioleyloxy)propyl]-N-N-N- trimethyl ammonia chloride and DOPE. Liposomes can carry larger pieces of DNA, can generally protect the polynucleotide from degradation, and can be targeted to specific cells or tissues. Cationic lipid- mediated gene transfer efficiency can be enhanced by incorporating purified viral or cellular envelope components, such as the purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G). Gene transfer techniques which have been shown effective for delivery of DNA into primary and established mammalian cell lines using lipopolyamine- coated DNA can be used to introduce target DNA into the neuronal progenitor cells described herein.

Naked plasmid DNA can be injected directly into a tissue comprising cells of the invention (e.g., neuronal progenitor cells). This technique has been shown to be effective in transferring plasmid DNA to skeletal muscle tissue, where expression in mouse skeletal muscle has been observed for more than 19 months following a single intramuscular injection. More rapidly dividing cells take up naked plasmid DNA more efficiently.

Therefore, it is advantageous to stimulate cell division prior to treatment with plasmid DNA. Microprojectile gene transfer can also be used to transfer genes into cells either in vitro or in vivo. The basic procedure for microprojectile gene transfer was described by J. Wolff in Gene Therapeutics (1994), page 195. Similarly, microparticle injection techniques have been described previously, and methods are known to those of skill in the art. Signal peptides can be also attached to plasmid DNA to direct the DNA to the nucleus for more efficient expression.

Viral vectors are used to genetically alter cells of the present invention and their progeny. Viral vectors are used, as are the physical methods previously described, to deliver one or more target genes, polynucleotides, antisense molecules, or ribozyme sequences, for example, into the cells. Viral vectors and methods for using them to deliver DNA to cells are well known to those of skill in the art. Examples of viral vectors that can be used to genetically alter the cells of the present invention include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), alphaviral vectors (e. g., Sindbis vectors), and herpes virus vectors.

Kits

Carbonaceous scaffolds comprising cells of the invention may be supplied along with additional reagents in a kit. The kits can include instructions for the treatment regime, reagents, and equipment (test tubes, reaction vessels, needles, syringes, etc.). The instructions provided in a kit according to the invention may be directed to suitable operational parameters in the form of a label or a separate insert.

In one embodiment, cells generated using the culture methods of the invention are useful for the treatment or prevention of central or peripheral nervous system injury or disease. The present invention provides compositions and methods of treating diseases and/or disorders or symptoms thereof characterized by the loss of neuronal cells or of neural function or activity. The methods of the invention comprise administering a therapeutically effective amount of a cellular composition described herein to a subject (e.g., a mammal, such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder characterized by neuronal cell loss or damage. The method includes the step of administering to the mammal a therapeutic amount of a cellular composition herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated. The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a cellular composition described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compositions herein, such as a cellular composition described herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis,

1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

Example 1. AC composite substrate morphology.

Activated charcoal (AC) composite matrices were formed by thorough mixing of AC and ECMs followed by drying on the glass cover slips. Representative matrix made of AC and collagen were examined by microscopy using brightfield and SEM resolution (Figures 1A and IB). Increasing the AC concentration from 0-50 g/L increased the coverage of AC on the cover slip, as indicated by a decrease in the amount of light available for penetration (Figure 1 A). Example 2. AC composite substrates promoted neuronal differentiation.

Biomatrices incorporated with ECM have been shown to enhance nerve regeneration. To test whether the AC composite substrates promoted neuronal differentiation, three different types of ECMs were blended with AC to produce AC- collagen, AC-chitosan or AC-collagen-chitosan substrates. The ratios of axonal growth and density increased in an AC concentration dependent manner as demonstrated by the staining of axonal markers βΙΙΙ tubulin (Figures 2A-2L) and peripherin. Fluorescent staining of βΙΙΙ tubulin confirmed the markedly longer and denser axons on AC-collagen matrix than collagen only control (Figures 2A-2D).

Neuronal differentiation efficiency on AC substrates was further assessed by measuring the increases in ratios of axonal length and density over control (AC free). Increases in axonal extension were approximately 1.8, 2.2, and 2.5 times higher than control in 10, 25, and 50 g/L of AC-collagen treatments, respectively (Figure 3A). At the same time, axonal density also increased by approximately 1.2, 1.5 and 1.8 fold under the same AC conditions (Figure 3B). For AC-chitosan and AC-collagen-chitosan matrices, increase in axonal length at the 50 g/L was approximately 2 and -1.8 fold higher, respectively, than that of the corresponding controls (AC free) when evaluated by staining of βΙΙΙ tubulin (Figures 3E and 31) and peripherin (Figures 3G and 3K). Meanwhile, enhancement in axonal density at 50 g/L of AC-chitosan and AC-collagen- chitosan was about ~2 and 1.4 fold more, respectively, than corresponding controls when assessed by staining of βΙΙΙ tubulin (Figures 3F and 3J) and peripherin (Figures 3H and 3L).

Thus, neuronal differentiation occurred significantly faster on all three AC-ECM substrates when compared to the corresponding controls (Figures 3A-3L). AC-collagen matrix was most effective in differentiating neuronal-like cells since the axonal length and density ratios were the highest among AC-chitosan and AC-chitosan-collagen matrices (Figures 3A-3L).

Example 3. AC composite substrate is more effective at promoting hESC neuronal differentiation compared to CNT composite substrate.

To further understand neuronal differentiation efficacy among different carbon- based substrates, a comparison between AC-collagen and CNT-collagen substrates were made. Because AC-collagen composite substrate was effective in promoting neuronal differentiation, AC-collagen was further examined by comparing its effects to that of CNT-collagen composite material. AC-collagen substrate significantly increased the axonal length and density compared to CNT-collagen substrate (Figures 4A-4D). AC- collagen matrix significantly improved neuronal differentiation over CNT-collagen matrix by at least 1.7 fold (Figures 4C and 4D). Without being bound to a particular theory, this result was attributed to the nanotoxic effects exerted by CNT. Thus, the data indicated that AC-collagen is more effective than CNT-collagen substrate at promoting neuronal differentiation from hESC.

In addition, the hESC differentiation rate was AC concentration dependent.

Without being bound to a particular theory, such phenomenon could be due to the increase in AC surface area and roughness created during AC processing by the generation of micropores. Similar physical characteristics have been found in CNTs which aid cell adhesion, growth and differentiation (Baker et al. (2009) Rev Med Devices 6: 515-532).

AC-collagen composite yielded the best differentiation. On the other hand, less neuronal differentiation on AC-chitosan may be due to reduced cell-matrix adhesion as suggested by a number of studies (Huang et al. (2005) Biomaterials 26: 7616-7627).

Without being bound to a particular theory, (i) biophysical properties of collagen and (ii) change in composite matrix stiffness contribute to differentiation on AC-collagen composite.

Collagen is the most abundant ECM existed in natural mammalian tissues (Ruoslahti (1997) Stretching is good for a cell. Science 276: 1345-1346; Flanagan et al. (2006) J Neurosci Res 83: 845-856) and critically regulates neuronal development in nervous system (Hubert et al. (2009) Cell Mol Life Sci 66: 1223-1238). Type 1 collagen, which was used in the studies, contains specific motifs, particularly favoring the growth of many neuronal cultures (Lin et al. (2004) Brain Res Dev Brain Res 153: 163-173; Yang et al. (2004) Biomaterials 25: 1891-1900) and the neural differentiation of hESCs (Ma et al. (2008) BMC Dev Biol 8: 90; Sridharan et al. (2009) Biochem Biophys Res Commun 381: 508-512). Its flexible fibrous networks provide a firm anchorage and thereby support the growth of neural precursors (Ma et al. (2008) Tissue Eng Part A 14: 1673-1686). Because hESCs and neuron-like cells are anchorage dependent, the AC-collagen substrate may render the most suitable environment necessary for differentiation into neuronal circuits. The data are also consistent with the finding that decellularized brain matrix enriched with collagen I-VI cultivates neuronal differentiation from induced pluripotent stem cells (Dequach et al. (2011) Tissue Eng Part A). Corroborating data further unveils that the storage modulus values of collagen gels ranges between 1.5-1.7 kPa (Lee et al. (2011) J R Soc Interface 8: 998-1010) which are within the modulus ranges reported for native nerve tissues, such as brain and spinal cord (Gupta et al. (2006) Fast-gelling injectable blend of hyaluronan and

methylcellulose for intrathecal, localized delivery to the injured spinal cord.

Biomaterials 27: 2370-2379; Gefen et al. (2004) J Biomech 37: 1339-1352).

Without being bound to a particular theory, the synergistic effect on neuronal differentiation presented by AC-collagen matrix when compared with collagen only control is due to their distinctions in material stiffness. Stiffer composite substrates exert higher tensional forces on cellular cytoskeleton (Sridharan et al. (2009) Biochem Biophys Res Commun 381: 508-512). Without being bound to a particular theory, these forces, mediated by integrins and cadherins adhesion molecules, transduce to

downstream signals activating Fak-Mek/Erk pathway, regulating neurite outgrowth and neuronal stem cell differentiation (Engler et al. (2006) Cell 126: 677-689; Chen et al. (2010) Biomaterials 31: 5575-5587; Mruthyunjaya et al. (2010) Biochem Biophys Res Commun 391: 43- 48). Thus, changes in tensional forces offered by AC-collagen substrate may modulate neuronal differentiation. Therefore, AC-collagen composite provides an appropriate microphysical environment for hESC to recognize and differentiate into neuronal lineage. Example 4. Survival and integration of differentiated cells on AC-collagen composite substrate.

A cellular viability assay was used to assess the biocompatibility between AC- collagen substrate, hESC, and differentiated cells. After a 7 day differentiation period, the ratios of live to dead cells were calculated for both control (collagen only) and AC- collagen conditions. More than 99% of cells were alive in both AC-collagen and collagen only conditions (Figure 5A). The analysis confirmed that AC-collagen substrate was biocompatible and non-toxic to differentiated cells and hESCs for sustaining long term propagation needed for transplantation. .

The direct integration of differentiated cells with AC-collagen substrates was further demonstrated. SEM images showed that the neuronal-like cells adhered and propagated well on AC-collagen matrices displaying typical cell body morphology and size (Figures 5C and 5D). Neuronal growth was observed, accompanied by large number of long neurite extensions on AC-collagen substrate. Extensions with lengths at least two times longer that the cell body indicated expansion of axons/neurites (Figures 5C and 5D). The cells also displayed a more three-dimensional structure when compared with those observed for neuronal-like cells grown on control (collagen only) (Figure 5B).

On the ground that 50 g/L AC-collagen substrate provided best differentiation, we then assessed its biocompatibility by testing cell viability. Figure 5A showed that

>99% of differentiated neuronal like cells were alive at the end of the 7 day period. The data indicated that the AC-collagen interface is highly bio-friendly to neuronal cells and sustains long term propagation needed for transplantation. A bio-friendly interface is important for the differentiation of neuronal lineages. SEM images showed that hESCs firmly attached on AC-collagen substrates and the differentiation of neuronal like cells was strikingly efficient.

Example 5. AC-collagen susbstrate enhanced neuronal protein expression.

To determine the sort of mature cell types derived from AC-collagen substrate after 7 days of culturing and differentiation, immunostaining was performed using 6 different markers that detect both early ectodermal and late neural development: Pax6 (early neurons), Nestin (neurons), Map2 (neuronal axons), Myelin Basic Protein (MBP, neuronal axons), Neurofilament (neuronal axons) and Olig-2 (oligodendrocytes).

Especially for neurofilament, this marker appears in more developed neurons and is expressed exclusively by central and peripheral nervous systems (Prabhakaran et al. (2009) Biomaterials 30: 4996-5003.).

Immunohistochemistry confirmed the presence of the mature markers expressed by differentiated neuronal like cells on AC-collagen substrate after a week. Pax6 staining was localized to the cytoplasm, which displays a clear neuronal phenotype (Figure 6A). Fluorescence staining of Nestin, Map2 and Neurofilament concentrated on the axonal protrusion (Figures 6B-6D). Axons were also positively stained with MBP which targets the myelin sheath wrapping the axons (Figure 6E). The use of Olig-2 confirmed the presence of oligodendrocytes in the culture (Figure 6F). Thus, hESC culture on AC-collagen susbstrates gave rise to various neuronal cell types.

Example 6. Neuronal cells derived from hESCs differentiated on AC-collagen have properties of functional neuron-specific synapses.

The maturity of the differentiated neuronal cells was further investigated by characterizing their neuronal properties. Depolarization-dependent Ca ²⁺ influx and release of synaptic vesicles are inherent properties of developing and mature neurons.

To determine whether differentiated neuronal like cells undergo depolarization- dependent calcium influx, intracellular Ca ²⁺ levels were measured by loading Rhod-2 dye. The cells were depolarized with high K ⁺ , and the fluorescence was measured for a total of 14 minutes. Differentiated cells on AC-collagen substrate were more responsive to depolarization as higher cytosolic [Ca ²⁺ ] (-1.4 times higher) was observed suggesting a faster influx (Figure 7A). The control showed minimal response.

The recycling of synaptic vesicles at the nerve terminal is another property of functional neurons. Recycling of synaptic vesicles was examined to authenticate the presence of functional neuronal like cells from differentiated hESC on AC-collagen substrate. FM1-43 marker was implemented to investigate synaptic vesicle recycling (exocytotic and endocytotic activities) at living axonal terminals. Vesicular release and recycling in neuronal networks are mostly linked with rising cytosolic [Ca ²⁺ ] and are labeled with FM1-43 dye. Under prolonged exposure to depolarization buffer, high K ⁺ lead to a reduction in fluorescence intensity (-50%) in AC-collagen substrate

differentiated cells (Figure 7B). The decreased FM1-43 fluorescence indicated that the cells were experiencing depolarization-dependent synaptic vesicle recycling at active neuronal circuits and further indicated that these neuronal cells have properties of functional neuron- specific synapses. Thus, AC-collagen matrix was able to promote the maturity of the differentiated cells.

Example 7. AC and AC-collagen substrates enhanced protein adsorption

Protein adsorption on substrate is an important factor for promoting cell adhesion and differentiation. A direct correlation exists between adsorption capacity of the substrate for growth factors or cell adhesive proteins with cellular proliferation. Thus, it was postulated that the large surface area of AC substrates renders high adsorption capacity. Similarly, the substrate enhances adsorption of growth factors in N2 media and cell adhesion proteins such as laminin during substrate coating. Previous studies have shown that neurons increase differentiation, higher expansion and neurite outgrowth on laminin-rich substrates in a dose-dependent manner (Ma et al. (2008) BMC Dev Biol 8: 90). Alternatively, the enhanced adsorption of serum proteins on graphene selectively promotes MSC anchorage and osteogenic differentiation (Lee et al. (2011) ACS Nano 5: 7334-7341). Thus, AC substrate serves as a concentrating platform for neuronal inducers by (1) enhancing EB adherence to the ECM and (2) exposing hESC to surface densely coated with growth factors favoring neuronal differentiation.

To demonstrate the adsorption characteristics of AC substrates, BSA was used as a representative protein. The amount of available free BSA was investigated by soaking AC-collagen substrate in BSA solution. The measured free BSA ratio correlated with the amount of BSA adsorbed by AC-collagen matrix. AC-collagen matrix was able to reduce the amount of free BSA by 50% after 3 days of incubation in a concentration- and time-dependent manner (Figure 8A). AC alone can adsorb protein in solution without the collagen matrix (Figure 8B). Thus, the results demonstrated that protein adsorption was enhanced in AC and AC-collagen substrates.

The results reported herein were obtained using the following methods and materials.

AC-ECM substrate manufacturing

AC was weighed out, autoclaved and mixed with type 1 collagen, chitosan, or collagen-chitosan at 10 g/L, 25g/L, and 50g/L to allow adsorption. Excess collagen and chitosan was then washed out. The mixtures were briefly sonicated. AC-ECM

composite materials were coated onto glass cover slips, allowed to dry overnight and soaked in DPBS supplemented with penicillin-streptomycin. The matrices were subsequently coated with Poly-L- Ornithine and laminin before seeding EBs on top.

Carbon nanotube tube (CNT)-collagen substrate manufacturing

CNTs were weighed out, autoclaved, and mixed with collagen at 50g/L. This mixture was briefly sonicated. CNT-collagen composite material was coated onto glass cover slips, allowed to dry overnight and soaked in DPBS supplemented with penicillin- streptomycin. The matrices were subsequently coated with poly-L-Ornithine and laminin before seeding embryoid (Ebs) on top. hESC Culture

hESC lines H9 from Wicell (Madison, WI) (passage 30-50) were cultured in 20% knockout serum replacement medium on mitomycin C (Sigma-Aldrich) treated mouse embryonic fibroblasts (MEFs) feeder layers. The standard 20% knockout serum replacement medium contained 20% KO serum replacement (Invitrogen), 1%

nonessential amino acids (Invitrogen), 1 mM L-glutamine (Invitrogen), Dulbecco's modified Eagle' s medium (DMEM/F12) (Invitrogen), 0.1 mM β-mercapto ethanol

(Sigma-Aldrich), and 4 ng/ml FGF-2 (Sigma-Aldrich). The medium was changed every day and hESCs were passed every 7 days.

Embryoid body (EBs) Formation and differentiation

hESC colonies were treated with dispase (0.5 mg/ml, Invitrogen) to remove colonies from MEF feeder layer. The colonies were cultured in ultra-low attachment dishes (Costar) for 5 days in 20% Knockout serum replacement medium without FGF-2 to form embryonic bodies (Ebs). To induce differentiation, EBs were transferred to AC composite material with poly-L- ornithine (PLO) and laminin coated and treated with N2 medium supplement consisting of DMEM/F12, nonessential amino acids, sodium pyruvate (Invitrogen), N2 supplement (Invitrogen), 2ug/ml of Heparin, O. lmM β- mercaptonethanol (Sigma-Aldrich) and FGF-2 (8 ng/ml). Cells were allowed to culture for a maximum of 7 days. Immunocytochemistry

Adherent cells were rinsed with HANKS and fixed with 4% paraformaldehyde for 20 minutes. Cells were treated with 0.1% Triton X-100 and 1% BSA for 30 minutes before they were incubated at 4°C overnight in primary antibodies: βΙΙΙ tubulin (1:500, Millipore), Peripherin (1:200, SantaCruz), Pax6 (1:200, Millipore), Map-2 (1:500, Millipore), Myelin Basic Protein (1:200, Millipore), Nestin (1:200, Millipore),

Neurofilament (1: 1000, Millipore), Olig-2 (1:500, Millipore). Secondary fluorescent antibodies were used at 1:500 for 1 hour at room temperature (AlexaFluor 488,

Invitrogen). Cell imaging was obtained using same exposure time for image acquisitions. Images were acquired by Nikon Eclipse TE2000-U fluorescent microscope (Nikon Eclipse TE2000-U, Tokyo, Japan).

Functional Assays

Depolarization-dependent calcium signaling

After differentiation for 7 days on the composite materials, cells were rinsed lx with DPBS and loaded with Rhod-2 AM dye (1 μΜ) (λΕχ = 552 nm and lEm = 581) (Invitrogen, CA, USA) for 45 minutes in Hanks' buffer. The dye was thoroughly rinsed with DPBS and replaced with normal Hanks' solution. Depolarizing solution (high K ⁺ ) (in mM: 20.9 NaCl, 100 KC1, 1.2 MgCb, 1.2 NaH ₂ P04, 1.2 Na ₂ S04, 2.5 CaCb, 25 NaHC03 and 10 glucose, pH 7.3) was then added to induce intracellular Ca ²⁺ influx (Williams et al. (2008) Nat Protoc 3: 835-839). Calcium signaling experiments were carried out on a Nikon microscope (Nikon Eclipse TE2000-U, Tokyo, Japan). Depolarization-dependent synaptic vesicle recycling

After differentiation for 7 seven days on the composite materials, cells were rinsed lx with DPBS. For staining recycling vesicles, cells were loaded with FM1-43 dye (2 μΜ) (λΕχ = 510 nm and λΕπι = 626) (Invitrogen, CA, USA) and incubated for 5 minutes at 37° C. Extracellular FM dyes were carefully rinsed with Tyrode solution (pH 7.3) twice. Exocytosis of synaptic vesicles were triggered before adding depolarizing solution (high K ⁺ ) (in mM: 20.9 NaCl, 100 KC1, 1.2 MgCb, 1.2 NaH ₂ P04, 1.2 Na ₂ S04, 2.5 CaCb, 25 NaHC03 and 10 glucose, pH 7.3) to induce synaptic vesicles exocytosis (Williams et al. (2008) Nat Protoc 3: 835-839). Synaptic vesicle recycling experiment were carried out on a Nikon Microscope (Nikon Eclipse TE2000-U, Tokyo, Japan).

Scanning Electron Microscope

Adherent cells were washed with cacodylate buffer and fixed with

glutaraldehyde for 20 minutes. The samples were washed again and dehydrated in 25%, 50%, 75%, 90%, 95%, and 100% EtOH for 5 minutes each. The samples were then inserted into a critical point dryer for 1 hour before being examined with FEI Quanta 200 ESEM (North America NanoPOrt, OR, USA). Statistical Analysis

Data is presented as means ± SD. Each experiment was performed independently at least three times. Statistical significance was determined using a Student's t-test analysis with p values of <0.005 (Microsoft Excel and GraphPad Prism 4.0, GraphPad Software, Inc., San Diego, CA, USA).

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.