WO2012174467A2 - Cord blood-derived neurons by expression of sox2 - Google Patents

Abstract

Provided herein are induced neural progenitors cells generated from cord blood (CB-iNPCs). CB-iNPCs are generated by ectopic expression of SOX2, optionally in combination with c-MYC. CB-iNPCs differentiate into functional, mature neurons in vitro and in vivo. The mature CB-derived neurons form functional synapses, generate action potentials, and produce neurons when grafted in vivo. Thus, further provided herein are cell-base therapies for neurodegenerative diseases and traumatic injuries to the nervous system.

Description

CORD BLOOD-DERIVED NEURONS BY EXPRESSION OF SOX2

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/497,341 filed Jun 15, 2011, which is hereby incorporated in its entirety and for all purposes.

BACKGROUND OF THE INVENTION

[0002] During normal development or under physiological conditions, differentiated cells maintain their fate throughout life. Nevertheless, it has been demonstrated that the identity of terminally differentiated cells can be reprogrammed by experimental manipulation. The generation of inducible pluripotent stem cells (iPSCs) represents the latest and most dramatic evidence that the epigenomes of differentiated cells are remarkably plastic (Takahashi & Yamanaka, Cell 126, 663 (2006)). CB CD133+ cells are considered more amenable to reprogramming than other adult somatic cells, and only two transcription factors, OCT4 and SOX2, are required to generate CB-iPSCs (Giorgetti et al., Cell stem cell 5, 353 (2009)). Thus, stem cell populations may be more responsive to different stimuli than other adult cells, depending on their pre-existing transcriptional or epigenetic states (Kim et al., Nature 461, 649 (2009)).

[0003] The present results show that expression of SOX2 alone is sufficient to convert CB CD 133+ cells into induced neural progenitor cells (CB-iNPCs). The presence of c-MYC enhances the efficiency of this conversion. The CB-iNPCs express neural progenitor markers and differentiate into functional, mature neurons both in vitro and in vivo.

BRIEF SUMMARY OF THE INVENTION

[0004] In one aspect, an induced neuroprogenitor cell (iNPC) generated by a method including expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC is provided.

[0005] In another aspect, an induced neuroprogenitor cell (iNPC) generated by a method including increasing a level of endogenous SOX2 in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC is provided.

[0006] In one aspect, an induced neuronal cell generated by a method including expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC and differentiating the iNPC into a neuron is provided.

[0007] In another aspect, an induced neuronal cell generated by a method including increasing a level of endogenous SOX2 in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC and differentiating the iNPC into a neuron is provided.

[0008] In one aspect, a method of generating an induced neuroprogenitor cell (iNPC) is provided. The method includes expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC.

[0009] In another aspect, a method of generating an induced neuroprogenitor cell (iNPC) is provided. The method includes increasing a level of endogenous SOX2 in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC.

[0010] In one aspect, a method of generating an induced neuronal cell is provided. The method includes expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell, culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC and differentiating the iNPC into a mature neuron, thereby generating an induced neuronal cell.

[0011] In another aspect, a method of generating an induced neuronal cell is provided. The method includes increasing a level of endogenous SOX2 in a cord blood (CB) cell, culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC and differentiating the iNPC into a mature neuron, thereby generating an induced neuronal cell.

[0012] In one aspect, a method of treating a neurodegenerative disorder or neuronal injury in an individual in need of such treatment is provided. The method incldues administering to the individual an effective amount of an iNPC described herein including embodiments thereof, or an effective amount of an induced neuronal cell described herein including embodiments thereof, thereby treating the neurodegenerative disorder or neuronal injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. Representative dot-plot for CD133 cells purity. Quantification of total CD133+ cells and double positive CD133+/CD45+ cells after immuno-selection, by

cytofluorimetric analysis. The cells showed 93,69% of viability and were 94,93% double positive for CD133/CD45.

[0014] Figure 2: Quantitative Real-time PCR. Analysis for neural markers MAP2, OLIG02, PAX6, TBRI, OTX2 and GFAP in CD133+ cells, isolated from 3 independent CB units. HES-derived neurons were analysed, together with CB CD133+, as positive control. Error bars indicate the SD generated from triplicate.

[0015] Figure 3. CB-iNPC derivation and characterization. A, Timeline of CB stem cells conversion into neural progenitor cells. B,C, Representative phase contrast images of passage 0 and passage 2 CB-iNPCs generated with only SOX2. D, Alkaline phosphatase (AP) staining. E,F, Images of immunocytochemistry for neural and pluripotency markers such as OCT4, NANOG, Tuj-1, GFAP, MAP2 and NF of S CB-iNPC clone. Nuclei were stained with Dapi (scale bar, 100 um). G,H_> Representative phase contrast images of passage 0 and passage 2 CB- iNPCs generated with SOX2 and c-MYC. I, AP staining of SM CB-iNPCs . L-S,

Immunofluorescence analysis of SM CB-iNPC lines for pluripotency and neural markers. The colonies contained cells with neural morphologies and expressed Tuj-1, GFAP, MAP2, PAX6, OLIG02 and NF, but were negative for TH and pluripotency markers OCT4, NANOG, (scale bar, 50,100 and 250 μιη). T-Z, For the immunofluorescence, a CBiPS line was used as negative control. The colonies were positive for OCT4 but did not express the neural markers Tuj-1, MAP2 and NF (scale bar,100μm).

[0016] Figure 4. Quantitative Real-time PCR. Analysis for neural markers MAP2, GFAP, TBRI, PAX6, and OLIG02. HES-derived neurons were analysed, together with (SM) and (S) CB-iNPCs clones, as positive control. Error bars indicate the SD generated from triplicate.

[0017] Figure 5. CB-iNPCs lost hematopoietic phenotype. Flow Cytometry analysis of SM and S CB-iNPCs cultured in hES conditions. Cells were analysed for the haematopoietic markers CD45, CD34, CD38 and CD133, and for neural marker Synapsin. The analysis confirmed that CB-iNPCs have lost haematopoietic phenotype and start to acquire a neural mature phenotype.

[0018] Figure 6. Karyotyping. Representative high-resolution, G-banded karyotype indicating a normal, diploid, female chromosomal content in SM CB-iNPCs cells analysed after passage 15. [0019] Figure 7. Characterization and gene expression profile of CB-derived neurons. A,

CB-iNPCs after 6 weeks of differentiation on top of human astrocytes acquired a more mature phenotype highlighted by the expression of the excitatory markers VGLUT1 and the dendritic marker MAP2 (scale bar, 30μιη). B, CB-derived neurons were positive for inhibitory marker such as GABA, (scale, bar 80μηι). C, Synaptic buttons on CB-derived neurons were highlighted by the expression of Synapsin puncta on Tuj-1 positive cells (scale bar, 10 μηι). D, Hierarchical clustering of genome-wide expression profile of CB CD133+ cells, CB-iNPCs (t2), CB-derived mature neurons (t3) and neurons derived from human ES cells, HUES6 (as control). E, Average global gene expression patterns were compared between CB CD 133+ (3 replicates) CB-iNPCs (3 replicates), CB-derived mature neurons (3 replicates), and HUES6 (2 replicates). We highlighted some neural specific genes in the plots. F, Correlation coefficients of genome-wide

transcriptional profiles for all pairwise comparisons of CB-iNPCs, CB-derived mature neurons and positive controls.

[0020] Figure 8: Activity-dependent calcium transients in CB-iNPC-derived neurons. A, Representative example of Syn: :DsRed cultures of CB-iNPC-derived neurons used for calcium signal traces (scale bar, 50 μιη). B, Red traces correspond to the calcium rise phase detected by the algorithm used (see supplementary methods). Example of fluorescence intensity changes reflecting intracellular calcium fluctuations in CB-iNPC-derived neurons before and after glutamate receptor antagonist (CNQX/APV) treatment. Each number on the left corresponds to the tracing of a different neuron on the plate. C, Effects of TTX ( 1 μΜ) and CNQX/APV ( 1 ΟμΜ /20 μΜ) on intracellular calcium transient frequency of individual neurons analyzed

simultaneously by calcium imaging. D, Analysis of spontaneous intracellular calcium transients in CB-iNPC-derived neurons after 4 weeks of differentiation. CB104-13 and CB75-12, CB- iNPC-differentiated neurons derived from 2 distinct CB units showed similar prevalence of calcium signaling as well as similar transient frequency within the neuronal population. Data shown as mean ± s.e.m.

[0021] Figure 9: Electrophysiology and in vivo grafting of CB-iNPC-derived neurons. A,

Electrophysiological properties of CB-derived neurons: Representative fluorescence micrograph of CB-derived neurons expressing Synapsin: :DsRed, from which data shown in (B-D) were obtained (scale bar, 10 μηι). B, Transient Na+ currents and sustained K+ currents in response to voltage step (cell voltage-clamped at -70 mV command voltage from -55 to 0 mV, 5 mV step). C, Action potentials evoked by somatic current injections (cell current-clamped at around -70 mV, currents from 50 to 150 pA, 50 pA step). D, Spontaneous action potentials when the cell was current-clamped at -60 mV. E, Transplantation and integration of CB-iNPC-derived neurons in vivo at 2 weeks: An example of transplanted neurons integrating in the dorsal blade of the dentate gyrus (DG) of the hippocampus. Grafted neurons were positive for NeuN and extended Tujl+ processes around the granule neurons of the host tissue (arrow). F, grafted neurons also extended Tuj-1 processes along the mossy fiber track to contact endogenous pyramidal neurons in the CA2/CA3 regions (arrows). G, Interestingly, neurons not grafted to the hippocampus also showed the ability to integrate into the corpus callosum (CC), sending Tujl+ processes contra- laterally (arrow). H, CD 133+ cells grafted in the hippocampus, as controls did not integrate into the host tissue and did not express the neuronal markers Tuj 1 (arrow). I, Representative image of CB-derived neurons 4 weeks post transplantation showing increase in co-localization with mature neuronal marker, NeuN (arrow). Scale bars = 50 μπι. J, Quantification of percentage of CB-derived neurons positive for Tuj-1 and NeuN, 4 months after transplantation.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction [0022] The present results show that SOX2 is sufficient to direct neural differentiation of cord blood (CB) CD133+ cells, thereby forming CB-derived induced neural progenitor cells (CB- iNPCs). Additional expression of c-MYC, which at physiological levels regulates the self- renewal of neural progenitor cells (NPCs) (Kerosuo et al., Journal of cell science 121, 3941 (2008)), enhances the generation of CB-iNPCs. Two or one-factor CB-iNPCs express neural progenitor markers and differentiate in vitro into mature neurons. CB-derived neurons express neural genes, highlighted by up-regulation of specific mature neural genes such as MAP2, VGLUT-1 and SYN1. CB-derived neurons are also functional, generating calcium transients and action potentials. Upon in vivo transplantation, CB-derived neurons integrate into the neural network. Untreated CD 133+ cells did not differentiate into neurons either in vitro or in vivo. [0023] The biological characteristics and availability of CB CD133+ cells offer logistic advantages over other adult somatic cell types (Rocha et al., NEJM 351, 2276 (2004)). More than 450,000 immunologically characterized CB units are available worldwide through a network of CB banks, representing the most comprehensive collection of cells with diverse, well characterized human leukocyte antigen (HLA) types available (Wagner & Gluckman, Semin Hematol 47, 3 (2010)). Until the present disclosure, however, evidence suggesting that CB stem cells can adopt new fates has been unconvincing, and there are few data demonstrating functionality of CB derived cells. [0024] The present results show that one or two factors (SOX2, optionally in combination with c-MYC) can induce human CB CD 133+ cells to become functional neurons. The generation of functional CB-derived neurons, HLA matched for any given patient, provides a therapeutic option for treating neuronal injury and neurodegenerative diseases. II. Definitions

[0025] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed, J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0026] A "neuroprogenitor cell" or "neural progenitor cell" is a cell that can give rise to a number of different types of neuronal cells (e.g., motor neurons, sensory neurons). While the neuroprogenitor cell can divide, and is not terminally differentiated, it has more limited self- renewal potential than a stem cell. An "induced neural progenitor cell" or "iNPC" is an artificially generated neuroprogenitor cell, e.g., as described herein. The iNPCs described herein are produced by expressing SOX2, and optionally c-MYC, in CD133+ cord blood cells at a higher than normal level (i.e., at a higher level than in unmanipulated CD133+ cord blood cells). Neuroprogenitor cells can be identified as described herein using characteristic cell surface markers, gene expression, and/ or morphology.

[0027] A neuron or neuronal cell is a cell capable of electrical and chemical signaling, e.g., as described herein. Morphologically, a typical neuron has dendrites, an axon, a soma (cell body), synapses, etc., and can by associated with glial cells or myelin. An "induced neuron" or

"induced neuronal cell" is a cell generated by further differentiation of an iNPC as described herein. The iNPC is further differentiated using neurotrophic factors and/ or feeder cells that support growth and differentiation of neuronal cells. As with neuroprogenitor cells, neurons can be identified using characteristic cell surface markers, gene expression, and/ or morphology.

[0028] The term "feeder cell" is known in the art, and in the context of the present invention, includes all cells used to support the propagation of progenitor cells and neuronal cells, e.g., during differentiation. Feeder cells can be irradiated prior to being co-cultured with stem or progenitor cells in order to avoid the feeder cells outgrowing the stem or progenitor cells. Feeder cells provide a layer physical support for attachment, and produce growth factors and extracellular matrix proteins that support cells. Examples of feeder cells include fibroblasts (e.g., embryonic fibroblasts, foreskin fibroblasts), glial cells (e.g., astrocytes), endothelial cells, and macrophages.

[0029] A "stem cell" is a cell characterized by the ability of self-renewal through mitotic cell division and the potential to differentiate into a tissue or an organ. Among mammalian stem cells, embryonic and somatic stem cells can be distinguished. Embryonic stem cells reside in the blastocyst and give rise to embryonic tissues, whereas somatic stem cells reside in adult tissues for the purpose of tissue regeneration and repair.

[0030] "Self renewal" refers to the ability of a cell to divide and generate at least one daughter cell with the self-renewing characteristics of the parent cell. The second daughter cell may commit to a particular differentiation pathway. For example, a self-renewing hematopoietic stem cell can divide and form one daughter stem cell and another daughter cell committed to differentiation in the myeloid or lymphoid pathway. A committed progenitor cell has typically lost the self-renewal capacity, and upon cell division produces two daughter cells that display a more differentiated (i.e., restricted) phenotype. Non-self renewing cells refers to cells that undergo cell division to produce daughter cells, neither of which have the differentiation potential of the parent cell type, but instead generates differentiated daughter cells.

[0031] The term "pluripotent" or "pluripotency" refers to cells with the ability to give rise to progeny that can undergo differentiation, under appropriate conditions, into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to tissues of a prenatal, postnatal or adult organism. A standard art-accepted test, such as the ability to form a teratoma in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population. However, identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells. Expression or non-expression of certain combinations of molecular markers are examples of characteristics of pluripotent stem cells. More specifically, human pluripotent stem cells may express at least some, and optionally all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rexl, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics.

[0032] An adult stem cell is an undifferentiated cell found in an individual after embryonic development. Adult stem cells multiply by cell division to replenish dying cells and regenerate damaged tissue. An adult stem cell has the ability to divide and create another cell like itself or to create a more differentiated cell. Even though adult stem cells are associated with the expression of pluripotency markers such as Rexl, Nanog, Oct4 or Sox2, they do not have the ability of pluripotent stem cells to differentiate into the cell types of all three germ layers. Adult stem cells have a limited ability to self renew and generate progeny of distinct cell types. Adult stem cells can include hematopoietic stem cell, a cord blood stem cell, a mesenchymal stem cell, an epithelial stem cell, a skin stem cell or a neural stem cell. A tissue specific progenitor refers to a cell devoid of self-renewal potential that is committed to differentiate into a specific organ or tissue. A primary cell includes any cell of an adult or fetal organism apart from egg cells, sperm cells and stem cells. Examples of useful primary cells include, but are not limited to, skin cells, bone cells, blood cells, cells of internal organs and cells of connective tissue. A secondary cell is derived from a primary cell and has been immortalized for long-lived in vitro cell culture.

[0033] A "somatic cell" is a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germ cells.

[0034] "Allogeneic" refers to deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated but genetically similar. An "allogeneic transplant" refers to transfer of cells or organs from a donor to a recipient, where the recipient is the same species as the donor.

[0035] "Autologous" refers to deriving from or originating in the same subject or patient. An "autologous transplant" refers to collection and retransplant of a subject's own cells or organs. [0036] "Graft-versus-host response" or "GVH" or "GVHD" refers to a cellular response that occurs when lymphocytes of a different MHC (major histocompatibility complex) class are introduced into a host, resulting in the reaction of the lymphocytes against the host. The HLA (human leukocyte antigens) are a subset of antigen presenting MHC proteins found in humans {see, e.g., Bodner et al. (1992) Human Immunol. 34:4).

[0037] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, and 2- O-methyl ribonucleotides.

[0038] The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

[0039] The terms "protein", "peptide", and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0040] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0041] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0042] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0043] The terms "identical" or percent "identity," in the context of two or more nucleic acids or proteins, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Identity typically exists over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0044] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0045] The word "expression" or "expressed" as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0046] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0047] The term "heterologous" when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature {e.g., a fusion protein).

[0048] The term "exogenous" refers to a molecule or substance {e.g., nucleic acid or protein) that originates from outside a given cell or organism. Conversely, the term "endogenous" refers to a molecule or substance that is native to, or originates within, a given cell or organism. [0049] A "vector" is a nucleic acid that is capable of transporting another nucleic acid into a cell. A vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0050] A "viral vector" is a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors.

[0051] A "cell culture" is an in vitro population of cells residing outside of an organism. The cell culture can be established from primary cells isolated from a cell bank or animal, or secondary cells that are derived from one of these sources and immortalized for long-term in vitro cultures.

[0052] The terms "culture," "culturing," "grow," "growing," "maintain," "maintaining," "expand," "expanding," etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body {e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. The term does not imply that all cells in the culture survive or grow or divide, as some may naturally senesce, etc. Cells are typically cultured in media, which can be changed during the course of the culture.

[0053] The terms "media" and "culture solution" refer to the cell culture milieu. Media is typically an isotonic solution, and can be liquid, gelatinous, or semi-solid, e.g., to provide a matrix for cell adhesion or support. Media, as used herein, can include the components for nutritional, chemical, and structural support necessary for culturing a cell.

[0054] The term "derived from," when referring to cells or a biological sample, indicates that the cell or sample was obtained from the stated source at some point in time. For example, a cell derived from an individual can represent a primary cell obtained directly from the individual (i.e., unmodified), or can be modified, e.g., by introduction of a recombinant vector, by culturing under particular conditions, or immortalization. In some cases, a cell derived from a given source will undergo cell division and/ or differentiation such that the original cell is no longer exists, but the continuing cells will be understood to derive from the same source. [0055] The term "transfection" or "transfecting" is defined as a process of introducing a nucleic acid molecule to a cell using non-viral or viral-based methods. The nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof.

Typically, a nucleic acid vector, comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.). Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. For viral- based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno- associated viral vectors. In some aspects, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art.

[0056] Expression of a transfected gene can occur transiently or stably in a host cell. During "transient expression" the transfected nucleic acid is not integrated into the host cell genome, and is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. Expression of a transfected gene can further be accomplished by transposon-mediated insertion into to the host genome. During transposon- mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow insertion into the host genome as well as subsequent excision.

[0057] The term "transduction" as used herein refers to introducing protein into a cell from the external environment. Typically, transduction relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0058] The term "Yamanaka factors" refers to Oct3/4, Sox2, Klf4, and c-Myc, which factors are highly expressed in embryonic stem (ES) cells. Yamanaka factors can induce pluripotency in somatic cells from a variety of species, e.g., mouse and human somatic cells. See e.g.,

Yamanaka, 2009, Cell 137: 13-17. [0059] A "SOX2 protein" as referred to herein includes any of the naturally-occurring forms of the SOX2 transcription factor, or variants thereof that maintain SOX2 transcription factor activity (e.g. at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Sox2). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion, e.g., the DNA-binding region) compared to a naturally occurring Sox2 polypeptide (e.g. SEQ ID NO: l). In some aspects, the SOX2 protein is the protein as identified by SEQ ID NO: 1 or a variant having substantial identity to SEQ ID NO: l . [0060] A "c-MYC protein" refers to any of the naturally-occurring forms of the c-Myc transcription factor, or variants thereof that maintain c-Myc transcription factor activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to cMyc). In some aspects, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring c-Myc polypeptide (e.g. SEQ ID NO:6). In other aspects, the c-Myc protein is the protein as identified in SEQ ID NO:2, or a variant having substantial identity to SEQ ID NO:2.

[0061] The terms "agonist," "activator," "upregulator," etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or activity. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more higher than the expression or activity in the absence of the agonist.

[0062] A "SOX2 agonist" or "SOX2 signaling agonist" is a substance that increases the expression or activity of SOX2 in a cell. SOX2 expression can be increased, e.g., by introducing a nucleic acid encoding SOX2 protein into a cell under conditions permitting expression, or by addition or activation of a positive regulatory factor upstream of SOX2 expression. SOX2 activity can be increased, e.g., by transduction of a SOX2 protein into a cell, or addition or activation of a positive regulatory factor upstream of SOX2 activity. In some aspects, the SOX2 agonist is an inhibitor of an agent that represses SOX2 expression or activity.

[0063] The terms "inhibitor," "repressor" or "antagonist" or "downregulator" interchangeably refer to a substance that results in a detectably lower expression or activity level as compared to a control. The inhibited expression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the inhibition is 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control.

[0064] A "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life or engraftment potential) or therapeutic measures (e.g., comparison of side effects). Controls can be designed for in vitro applications, e.g., testing the activity of various SOX2 signaling agonists. One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0065] The terms "therapy," "treatment," and "amelioration" refer to any reduction in the severity of symptoms, e.g., of a neurodegenerative disorder or neuronal injury. As used herein, the terms "treat" and "prevent" are not intended to be absolute terms. Treatment can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, improved cognitive function or coordination, increase in survival time or rate, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.

[0066] The term "therapeutically effective amount," as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate a given disorder or symptoms. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0067] "Subject," "patient," "individual in need of treatment" and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.

[0068] In the context of the present invention, i.e., methods for generating iNPCs and induced neuronal cells, a subject in need of treatment can refer to an individual that is deficient in one or more neuronal cell population. The deficiency can be due to a genetic defect, injury, or pathogenic infection.

[0069] A "transplant," as used herein, refers to cells, e.g., hematopoietic cells, introduced into a subject. The source of the transplanted material can be cultured cells, cells from another individual, or cells from the same individual (e.g., after the cells are cultured in vitro).

III. Cells

[0070] In some embodiments, the starting cell population for generating induced

neuroprogenitor cells or induced neuronal cells are CD 133+ cord blood cells (CB cells). Such cells retain pluripotent characteristics, both in gene expression and morphology (see, e.g.,

Example 1 below and Pessina et al. (2010) Cell Biol. Int. 34:783). The cord blood cells can be obtained from any of a number of cord blood cell banks, or from a designated source, e.g., the individual's own cord blood. CD133+, and optionally CD45+ cells can be obtained, e.g., using cell surface markers and FACS sorting or beads, as known in the art. Typically, the cells are human, e.g., for use in human therapy or study of xenographic models, but cord blood cells can also be obtained from experimental animal models (e.g., mice, rats, non-human primates, or rabbits), livestock (bovine, equine, ovine, etc.), or pets (dogs, cats, etc.).

[0071] In one aspect, an induced neuroprogenitor cell (iNPC) generated by a method including expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC is provided. The culturing of the CB cell in conditions permitting differentiation of the CB cell into an iNPC thereby results in generating the iNPC. In some embodiments, the CB cell is a CD133+ CB cell. The expressing may include expressing an exogenous, recombinant nucleic acid encoding SOX2. In other embodiments, the method further includes expressing a recombinant c-MYC polypeptide in the CD133+ CB cell. In some embodiments, the expressing recombinant c-MYC polypeptide includes expressing an exogenous, recombinant nucleic acid encoding c-MYC. In some embodiments, the method further includes increasing a level of endogenous cMyc in the

CD133+ CB cell. In other embodiments, the CB cell is cultured in embryonic stem cell media. In some embodiments, the CB cell is cultured with feeder cells.

[0072] In another aspect, an induced neuroprogenitor cell (iNPC) generated by a method including increasing a level of endogenous SOX2 in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC is provided. The culturing of the CB cell in conditions permitting differentiation of the CB cell into an iNPC thereby results in generating the iNPC. In some embodiments, the CB cell is a CD133+ CB cell. In other embodiments, the method further includes expressing a recombinant c-MYC

polypeptide in the CD133+ CB cell. In some embodiments, the expressing recombinant c-MYC polypeptide includes expressing an exogenous, recombinant nucleic acid encoding c-MYC. In some embodiments, the method further includes increasing a level of endogenous cMyc in the CD 133+ CB cell. In other embodiments, the CB cell is cultured in embryonic stem cell media. In some embodiments, the CB cell is cultured with feeder cells.

[0073] The invention involves recombinant methods, e.g., for construction of vectors encoding SOX2 protein, c-MYC protein, or an antisense construct, e.g., specific for a SOX2 or c-MYC inhibiting factor. Standard recombinant methods are used for cloning, DNA and RNA isolation, amplification and purification. Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007 with updated through 2010) Current Protocols in Molecular Biology, among others known in the art. Standard transfection methods can be used to produce mammalian cells that express a selected protein. Transformation of eukaryotic can be performed according to standard techniques (see, e.g., Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu e/ o/., eds, 1983).

[0074] Recombinant expression vectors contain a coding sequence, e.g., for SOX2 or c-MYC, or an inhibitory sequence operably linked to suitable transcriptional or translational regulatory elements derived from mammalian or viral genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. An origin of replication and a selectable marker to facilitate recognition of transformants can be incorporated. The coding and regulatory sequences utilized in the present recombinant expression vectors can be on different expression vectors, e.g., such that the gene products are on at least two different vectors, or on the same vector. There may be reasons to divide up the gene products such as size limitations for insertions.

[0075] In some embodiments, an exogenous regulatory element is introduced to the cell to increase expression of the endogenous SOX2 or c-MYC gene, e.g., instead of or in combination with introducing an exogenous SOX2 and/or c-MYC encoding sequence to the CD 133+ cord blood cell. For example, a constitutive of inducible promoter can be targeted to operate on SOX2 or c-MYC.

[0076] The transcriptional and translational control sequences in expression vectors can be provided by non-viral or viral sources. Commonly used promoters and enhancers are derived, e.g., from beta actin, adenovirus, simian virus (SV40), and human cytomegalovirus (CMV). For example, vectors allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, transducer promoter, or other promoters shown effective for expression in mammalian cells are suitable. Further viral genomic promoter, control and/or signal sequences may be used, provided such control sequences are compatible with the host cell chosen.

[0077] Expression of proteins from eukaryotic vectors can also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.

[0078] In some aspects, a nucleotide sequence that specifically interferes with expression of a SOX2 inhibiting factor, at the transcriptional or translational level can be used. This approach may utilize, for example, siRNA and/or antisense oligonucleotides to block transcription or translation of a specific mRNA, either by inducing degradation of the mRNA with a siRNA or by masking the mRNA with an antisense nucleic acid. [0079] In some aspects, amplification of known sequences may be desirable, e.g., for cloning into appropriate expression vectors, or for detection of expression, e.g., using Q-PCR Such methods of amplification are well known to those of skill in the art. Detailed protocols for PCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). The known nucleic acid sequences for the genes listed herein is sufficient to enable one of skill to routinely select primers to amplify any portion of the gene.

[0080] Expression of SOX2 and/or c-MYC in CB cells induces the cells to generate induced neuroprogenitor cells (iNPCs). iNPCs are characterized by loss of pluripotent characteristics, such as OCT4, Nanog, CD133, CD34, and/ or CD38 expression, and ability to give rise to teratomas when injected into SCID mice. iNPCs are positively characterized by the ability to give rise to mature neurons (induced neuronal cells), and expression of neuronal lineage genes, such as Tuj l, GFAP, NF, PAX6, NESTIN, OLIG02, and/ or MAP2. In addition, as described herein, iNPCs can engraft, mature, and form part of a functional neuronal system upon administration in vivo.

[0081] iNPCs can also give rise to further differentiated neuronal cells in vitro. Such induced neuronal cells are functional (as evidenced by their in vivo functionality), and can generate calcium transients and action potientials. Induced neuronal cells are also positively characterized by neuronal gene expression (MAP2, PAX6, NF, NeuroDl, N-CAM1, VGLUT1, MAP1B and SYN1).

[0082] In one aspect, an induced neuronal cell generated by a method including expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC and differentiating the iNPC into a neuron is provided. The permitting differentiation of the CB cell into an iNPC and differentiating the iNPC into a neuron thereby results in generating the neuronal cell. In some embodiments, the CB cell is a CD 133+ CB cell. In other embodiments, the method further includes expressing a recombinant c-MYC polypeptide in the CD133+ CB cell. In some embodiments, the method further includes increasing a level of endogenous cMyc in the CD133+ CB cell. In another embodiment, the CB cell is cultured in embryonic stem cell media. In some embodiments, the CB cell is cultured with feeder cells. In other embodiments, the differentiating includes culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

[0083] In another aspect, an induced neuronal cell generated by a method including increasing a level of endogenous SOX2 in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC and differentiating the iNPC into a neuron is provided. The permitting differentiation of the CB cell into an iNPC and differentiating the iNPC into a neuron thereby results in generating the neuronal cell. In some embodiments, the CB cell is a CD 133+ CB cell. In other embodiments, the method further includes expressing a recombinant c-MYC polypeptide in the CD133+ CB cell. In some embodiments, the expressing recombinant c-MYC polypeptide includes expressing an exogenous, recombinant nucleic acid encoding c-MYC. In some embodiments, the method further includes increasing a level of endogenous cMyc in the CD133+ CB cell. In another embodiment, the CB cell is cultured in embryonic stem cell media. In some embodiments, the CB cell is cultured with feeder cells. In other embodiments, the differentiating includes culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

[0084] In some embodiments, the iNPCs and/ or induced neuronal cells generated as described herein can be compared to negative controls (pluripotent cells such as CB cells or non-neuronal cells) or positive controls (e.g., known NPCs or neurons). [0085] In one aspect, an induced neuroprogenitor cell (iNPC) including an exogenous, recombinant nucleic acid encoding SOX2 is provided. In some embodiments, the iNPC further includes an exogenous, recombinant nucleic acid encoding c-MYC. In other embodiments, the iNPC is derived from a CD133+cord blood (CB) cell.

IV. Culturing methods [0086] Suitable culture conditions are described herein, and can include standard tissue culture conditions. For example, CB cells, iNPCs, or neuronal cells can be cultured in a buffered media that includes amino acids, nutrients, growth factors, etc, as will be understood in the art. In some aspects, the culture includes feeder cells (e.g., fibroblasts), while in others, the culture is devoid of feeder cells. Cell culture conditions are described in more detail, e.g., in Picot, Human Cell Culture Protocols (Methods in Molecular Medicine) 2010 ed. and Davis, Basic Cell Culture 2002 ed.

[0087] In some aspects, the CB cells, iNPCs, or neuronal cells are cultured and allowed to divide. Stem cells and other less differentiated cells can give rise to additional pluripotent daughter cells, or to more differentiated cells (e.g., neurons). Cell division can be determined according to methods known in the art, e.g., detecting incorporation of labeled nucleic acids or amino acids. [0088] Culture conditions that support differentiation of neuronal cells from progenitors are described herein. For example, glial {e.g., astrocytes) or fibroblast feeder cells can be included in the culture to promote neuronal differentiation. Growth factors (e.g., neurotrophins) can also be included in the culture to promote neuronal differentiation, e.g., retinoic acid, brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurotropins 3 and/or 4, etc. (see, e.g., Ibanez and Ernfors (2007) Neuron 54:673).

[0089] In one aspect, a method of generating an induced neuroprogenitor cell (iNPC) is provided. The method includes expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC. In some embodiments, the CB cell is a CD133+ CB cell. In other embodiments, the expressing includes expressing an exogenous, recombinant nucleic acid encoding SOX2. In some embodiments, the method further includes expressing a recombinant c-MYC polypeptide in the CD133+ CB cell. In some embodiments, the expressing recombinant c-MYC polypeptide includes expressing an exogenous, recombinant nucleic acid encoding c-MYC. In other embodiments, the method further includes increasing a level of endogenous c-MYC in the CD 133+ CB cell. In some embodiments, the CB cell is cultured in embryonic stem cell media. In other embodiments, the CB cell is cultured with feeder cells. In some embodiments, the method further includes differentiating the iNPC into a neuron. In some further embodiments, the differentiating includes culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

[0090] In another aspect, a method of generating an induced neuroprogenitor cell (iNPC) is provided. The method includes increasing a level of endogenous SOX2 in a cord blood (CB) cell and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC. In some embodiments, the CB cell is a CD 133+ CB cell. In some embodiments, the method further includes expressing a recombinant c-MYC polypeptide in the CD 133+ CB cell. In some embodiments, the expressing recombinant c-MYC polypeptide includes expressing an exogenous, recombinant nucleic acid encoding c-MYC. In other embodiments, the method further includes increasing a level of endogenous c-MYC in the CD133+ CB cell. In some embodiments, the CB cell is cultured in embryonic stem cell media. In other embodiments, the CB cell is cultured with feeder cells. In some embodiments, the method further includes differentiating the iNPC into a neuron. In some further embodiments, the differentiating includes culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin. [0091] In one aspect, a method of generating an induced neuronal cell is provided. The method includes expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell, culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC and differentiating the iNPC into a mature neuron, thereby generating an induced neuronal cell. In some embodiments, the CB cell is a CD133+ CB cell. In other embodiments, the method further includes expressing a recombinant c-MYC

polypeptide in the CD 133+ CB cell. In some embodiments, the differentiating includes culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

[0092] In another aspect, a method of generating an induced neuronal cell is provided. The method includes increasing a level of endogenous SOX2 in a cord blood (CB) cell, culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC and differentiating the iNPC into a mature neuron, thereby generating an induced neuronal cell. In some embodiments, the CB cell is a CD 133+ CB cell. In other embodiments, the method further includes expressing a recombinant c-MYC polypeptide in the CD 133+ CB cell. In other embodiments, the method further includes increasing a level of endogenous c- MYC in the CD 133+ CB cell. In some embodiments, the differentiating includes culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

V. Therapeutic applications

[0093] The ability to induce neuronal cell development in cord blood cells that are readily available and HLA-characterized provides an exciting new option for cell based therapy. The induced neuronal cells can be used to replace cells lost or damaged in neurodegenerative disorders and neuronal injuries.

[0094] Thus, in some embodiments, the iNPCs or induced neuronal cells described herein are administered to an individual having a neurodegenerative disorder, a neurological disorder, or a neuronal injury to treat the disorder or injury.

[0095] In one aspect, a method of treating a neurodegenerative disorder or neuronal injury in an individual in need of such treatment is provided. The method incldues administering to the individual an effective amount of an iNPC described herein including embodiments thereof, or an effective amount of an induced neuronal cell described herein including embodiments thereof, thereby treating the neurodegenerative disorder or neuronal injury. In some embodiments, the iNPC or induced neuronal cell is administered to the site of the neurodegeneration or neuronal injury in the individual. In some embodiments, the neurodegenerative disorder is selected from the group consisting of Alzheimer's Disease (AD), Huntington's Disease (HD), Parkinson's Disease (PD) Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS) and Cerebral Palsy (CP). In some embodiments, the disorder is selected from the group consisting of: Alzheimer's Disease (AD), Huntington's Disease (HD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Cerebral Palsy (CP), Dentatorubro-pallidoluysian Atrophy (DRPLA), Neuronal Intranuclear Hyaline Inclusion Disease (NIHID), dementia with Lewy bodies, Down's Syndrome, Hallervorden-Spatz disease, prion diseases, argyrophilic grain dementia, cortocobasal degeneration, dementia pugilistica, diffuse neurofibrillary tangles, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Jakob-Creutzfeldt disease, Niemann-Pick disease type 3, progressive supranuclear palsy, subacute sclerosing

panencephalitis, Spinocerebellar Ataxias, Pick's disease, and dentatorubral-pallidoluysian atrophy. In some embodiments, the disorder is AD. In some embodiments, the disorder is selected from HD, PD, ALS, MS, and CP.

[0096] In some embodiments, the neuronal injury is selected from the group consisting of traumatic brain injury, stroke, and chemically induced brain injury. Neuronal injuries can result from any number of traumatic incidents, e.g., obtained in sport, accident, or combat. Neuronal injuries include concussion, ischemia (stroke), hemorrhage, or contusion resulting in damage to the neurons in an individual, or significant loss of neuronal tissue in drastic cases. Also included are neuronal injuries and loss caused by pathogenic infection, or chemically induced brain injury, e.g., due to medication, environmental factors, or substance abuse.

[0097] Methods for diagnosing neurodegenerative disorders, neurological disorders, and neuronal injuries are known in the art (see, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV-TR), American Psychiatric Assoc. 2000). Generally, a physician or neurologist will consider a number of factors in making a diagnosis in a particular individual or patient. For example, family history is often indicative of a risk of AD, HD, PD, and other neurodegenerative disorders. Doctors will also carry out chemical tests to check for normal blood count, thyroid function, liver function, glucose levels. Spinal fluid is often analyzed as part of this testing.

[0098] A physician can also use neuropsychological tests to assess memory, problem-solving, decision making, attention, vision-motor coordination and abstract thinking (see, e.g.,

Gleichgerrcht et al. (2010) Nat Rev Neurol. 6:611; Kovacs & Budka (2010) Clin Neuropathol. 29:271). These include spatial exercises and simple calculations. The Mini-Mental State Examination is also common.

[0099] CAT scans and MRIs can also be used to rule out tumors, and can provide clues as to degraded areas of the brain. Non-invasive medical imaging techniques such as Positron Emisson Tomography (PET) or single photon emission computerized tomography (SPECT) imaging are particularly useful for the detection of brain disease. PET and SPECT imaging shows the chemical functioning of organs and tissues, while other imaging techniques, such as X-ray, CT and MRJ, show structure. The use of PET and SPECT imaging has become increasingly useful for qualifying and monitoring the development of brain diseases. In some instances, the use of PET or SPECT imaging allows a neurodegenerative disorder to be detected several years earlier than the onset of symptoms.

[0100] Once an individual has been diagnosed as having a deficiency in neuronal cells, e.g., resulting from neurodegeneration or injury, the individual can be considered for treatment with the cell based therapies described herein. [0101] Methods of administering cells to neuronal tissues such as the brain or spinal cord are described herein and in, e.g., Blurton-Jones et al. (2009) Proc. Natl. Acad. Sci. 106:13594; Jin et al, (2009) J. Cereb. Blood Flow Metab. 30:534; and Lundberg et al, (2009) Neuroradiology 51 :661. Geron and SanBio are among the companies carrying out clinical trials for cell-based therapies of neuronal disorders in humans. Such administration of cells to neuronal tissues is also described, e.g., in J. Neurosci. (2005) 25; GLIA (2005) 49; and at the clinical trials website at clinicaltrials.gov (e.g., clinicaltrials.gov/ct2/show/NCT01287936).

[0102] The invention provides methods of treating, preventing, and/or ameliorating neurodegenerative or neurological disorders or neuronal injuries in a subject in need thereof (individuals having a neuronal cell deficiency). As with any therapy, the course of treatment is best determined on an individual basis depending on the particular characteristics of the subject and the type of treatment selected. The treatment can be administered to the subject one time, on a periodic basis (e.g., bi-weekly, monthly) or any applicable basis that is therapeutically effective. The treatment can be administered alone or in combination with another therapeutic agent, e.g., an agent that reduces pain, or an agent that encourages neuronal function or growth. The additional therapeutic agent can be administered simultaneously with the iNPCs or induced neuronal cells described herein, at a different time, or on an entirely different therapeutic schedule (e.g., the iNPCs or induced neuronal cells can be administered as needed, while the additional therapeutic agent is administered daily or weekly).

[0103] The dosage of iNPCs or induced neuronal cells administered to a patient will vary depending on a wide range of factors. For example, it would be necessary to provide substantially larger doses to humans than to smaller animals. The dosage will depend upon the size, age, sex, weight, medical history and condition of the patient, use of other therapies, and the frequency of administration.

[0104] Having indicated that there is variability in terms of dosing, it is believed that those skilled in the art can determine appropriate dosing, e.g., by initial animal testing, or by administering relatively small amounts and monitoring the patient for therapeutic effect. If necessary, incremental increases in the dose can be made until the desired results are obtained. Generally, treatment is initiated with smaller dosages which may be less than the optimum dose of the therapeutic agent. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For example, a dose of 10⁴- 10⁵ cells can be initially administered, and the subject monitored for effect (e.g., engraftment of the cells, improved neural function, increased neuronal density in an affected area). The dose of iNPCs or induced neuronal cells can be in the range of 10³-10⁷, 10⁴-10⁷, 10⁵-10⁸, 10⁶-10⁹, or 10⁶-10⁸. Again, the exact initial dosage is best determined by a medical professional depending on the characteristics of the individual to be treated. [0105] The pharmaceutical preparation comprising iNPCs or induced neuronal cells can be packaged or prepared in unit dosage form. The cells can be lyophilized and/ or frozen for increased shelf life, and resuspended prior to administration. In such form, the cellular preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., according to the dose of the therapeutic agent. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation. The composition can, if desired, also contain other compatible therapeutic agents.

[0106] The iNPCsor induced neuronal cell compositions disclosed herein can be administered by any appropriate method. Typically, the differentiated cells of the invention are injected (either bolus or infusion) or otherwise applied to the affected site. For example, administration can be intralesionally, intracranial ly, via a spinal injection, etc. Additional routes of

administration include intraocular and intramuscular (in the case of peripheral neurons). [0107] For parenteral administration in an aqueous solution, the solution should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution.

[0108] Sterile injectable solutions can be prepared by sterile filtration of the media or injection vehicle prior to incorporating the cells for injection. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium. Vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients, can be used to prepare sterile powders for reconstitution of sterile injectable solutions. The preparation of more concentrated solutions for direct injection is also contemplated. DMSO can be used as solvent for rapid penetration, delivering high concentrations of the active agents to a small area.

[0109] The pharmaceutical compositions of the invention can optionally comprise growth factors or cell matrix components to support growth of the iNPC or induced neuronal cell infusion. For example, the cells can be administered in a matrix solution {e.g., matrigel), optionally comprising neuronal growth factors.

[0110] The following discussion of the invention is for the purposes of illustration and description, and is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g. , as may be within the skill and knowledge of those in the art, after understanding the present disclosure. All publications, patents, patent applications, Genbank numbers, and websites cited herein are hereby incorporated by reference in their entireties for all purposes.

VI. Examples A. Methods

[0111] CD133+ cell purification, transduction, and CB-iNPC culture. Umbilical CB samples were obtained from the Banc de Sang i Teixits, Hospital Duran i Reynals, Barcelona. Mononuclear cells (MNC) were isolated from CB using Lympholyte-H (Cederlane, Ontario, CA) density gradient centrifugation. CD 133+ cells were positively selected using Mini-Macs immunomagnetic separation system (Miltenyi Biotec, Bergisch Gladbach, Germany).

Purification efficiency was verified by flow cytometric analysis staining with CD133- phycoerythrin (PE; Miltenyi Biotec, Bergisch Gladbach, Germany) and CD45 (APC, Becton Dickinson) antibodies. The CB CD 133+ cells were infected with a viral-based expression vector and cultured as previously described (Zhou & Melton, Cell stem cell 3, 382 (2008)).

[0112] Purification of Total RNA and Quantitative RT-PCR. Isolation of total RNA from CB CD133+ stem cells, CB-iNPCs and HUES6 cells was performed using either RNeasy Mini Kit (Qiagen) or RNAqueous-Micro kit (Ambion) based on the cell number available. All samples were treated with TURBO DNase inhibitor (Ambion) to remove any residual genomic DNA and 1 ug of RNA was used to synthesize cDNA using the Invitrogen Superscript II Reverse Transcriptase kit (Invitrogene). 25ng of cDNA were used to quantify gene expression by Quantitative RT-PCR using the following primers:

TBR-I F- 5' - CAA CTC AGT CAA CAG GAA GGC- 3' (SEQ ID NO:3)

R- 5' - AAA GAT GAT CTC CAG CAC AGC- 3' (SEQ ID NO:4)

GFAP: F 5 '-CCGACAGCAGGTCCATGT-3 ' (SEQ ID NO:5)

R 5 '-GTTGCTGGACGCCATTG-3 ' (SEQ ID NO:6)

MAP2: F 5 '-TTGGTGCCGAGTGAGAAGA-3 ' (SEQ ID NO:7)

R 5 '-GTCTGGC AGTGGTTGGTTAA-3 ' (SEQ ID NO:8)

F: 5 '-AATC AGAGAAG AC AGGCC A-3 ' (SEQ ID NO:9)

R: 5 '-GTGTAGGTATCATAACTC-3 ' (SEQ ID NO:10)

olig2: F: 5'-CAG AAG CGC TGA TGG TCA T-3' (SEQ ID NO: 11)

R: 5'-CG GCA GTT TTG GGT TAT TC-3' (SEQ ID NO: 12)

Otx2: F: 5 '-GAC CAC TTC GGG TAT GGA CT-3 ' (SEQ ID NO: 13)

R: 5'-TGG ACA AGG GAT CTG ACA GT-3' (SEQ ID NO: 14)

[0113] GeneChip Microarray Analysis. The GeneChip microarray processing was performed by the core facility Microarray Analysis Service (SAM) from IMIM-Hospital del Mar (Barcelona, Spain). Amplification, labeling and hybridization were performed according to the Ambion and Affymetrix protocol. Briefly, 200 ng of total RNA were amplified using the Ambion® WT Expression Kit (Ambion/Applied Biosystems, Foster city, CA, USA), labeled using the WT Terminal Labeling Kit (Affymetrix Inc., Santa Clara, CA, USA), and then hybridized to Human Gene 1.0 ST Array in a GeneChip® Hybridization Oven 640.

[0114] Washing and scanning were performed using the Hybridization Wash and Stain Kit and the GeneChip® System of Affymetrix (GeneChip® Fluidics Station 450 and GeneChip® Scanner 3000 7G). The data extraction was done by the Command Console software. [0115] The raw CEL files were normalized with the RMA algorithm using ArrayStar4. We considered a probset as differently expressed between samples if the fold change was at least 2 with a p-value lower than 0.05. The hierarchical clustering of samples was performed using correlation metric and the average linkage method with Cluster software and visualized by Tree View software. The correlation plots were generated using R (found at the R project website at r-project.org).

[0116] Immunofluoresence analysis and Alkaline Phosphatase (AP) analyses. CB-iNPCs were grown on plastic cover-slide chambers in the presence of HFFl feeder for 1 week and fixed with 4% PFA. The following antibodies were used: Tuj-1 (l :500;Covance), a-fetoprotein (1 :400; Dako), a-actin (1 :100; Sigma), OCT4 (1 : 100, SantaCruz), NANOG (1:100, Everest Biotech), smooth muscle actin (1:400, Sigma), FoxA2 (1 :50 R&D System), GFAP (1 :1000, Dako), MAP2 (1:100, Sigma), Pax6 (1 : 100, Dako), 01igo2 (l: 10,Dako), Neurofilament-200 (1 :80, Sigma), Nestin (1 :200;Chemicon). Images were taken using a Leica SP5 confocal microscope. Direct AP activity was analyzed using an Alkaline Phosphatase Blue/Red

Membrane substrate solution kit (Sigma) according to the manufacturer's guidelines.

[0117] After differentiation in co-culture with human astrocytes for 6 weeks the cells were fixed with 4% PFA and then permeabilized with 0.5% triton X-100 in PBS. The antibodies used to stain CB-derived neurons were: Tuj-1 (1 :500, Covance); Map2 (1 :100, Sigma); VGLUT1 (1 :200, Synaptic Systems); GABA (1 :100, Sigma); Synapsin (1 :400, Calbiochem) EGFP (1:200, Molecular Probes, Invitrogene).

[0118] CB-iNPC differentiation into mature neurons. CB-iNPCs were dissociated by trypsin and 10,000 single cells were plated on human astrocyte feeder layer on

Polyornithine/Laminin-coated plates in the presence of DMEM/F12 +N2 and B27 Supplements, RA (retinoic acid at ΙμΜ), BDNF, GDNF (both at 20ng/ml), Lam (1 μg/ml) and 0.5% FBS, for 4 weeks. The HUES6 line (Harvard) was differentiated in mature neurons as previously described (Marchetto et al. Cell 143, 527 (2010)).

[0119] Calcium imaging. Neuronal networks derived from CB-iNPCs were previously infected with the lentiviral vector carrying the Syn:DsRed reporter construct. Cell cultures were washed twice with sterile Krebs HEPES Buffer (KHB, 10 mM HEPES, 4.2 mM NaHC0₃, 10 mM dextrose, 1.18 mM MgS0₄'2H₂0, 1.18 mM KH₂P0₄, 4.69 mM KC1, 118 mM NaCl, 1.29 mM CaCl₂; pH 7.3) and incubated with 2-5 μΜ Fluo-4AM (Molecular Probes/Invitrogen, Carlsbad, CA) in KHB for 40 minutes at room temperature. Excess dye was removed by washing twice with KHB and an additional 20 minutes incubation was done to equilibrate intracellular dye concentration and allow de-esterification. Time-lapse image sequences (100X magnification) of 5000 frames were acquired at 28 Hz with a region of 336 x 256 pixels, using a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics K.K., Japan) with a 488 nm (FITC) filter on an Olympus 1X81 inverted fluorescence confocal microscope (Olympus Optical, Japan). Images were acquired with MetaMorph 7.7 (MDS Analytical Technologies, Sunnyvale, CA). Images were subsequently processed using ImageJ (http://rsbweb.nih.gov/ij/) and custom written routines in Matlab 7.2 (Mathworks, Natick, MA).

[0120] Quantification of calcium transients. For quantification of calcium transients, ImageJ, an NIH-funded open source, JAVA-based morphometric application, was used to allow manual selection of individual neurons on the Syn::DsRed image that correspond to each calcium movie using circular regions of interest (ROI) of 4 pixels (~5μπι) in diameter. Each cell was considered as an individual ROI and the average fluorescence intensity was calculated for each ROI through the entire acquired image sequence. Quantitative signal analysis and processing were done with custom-written Matlab routines. Individual temporal fluorescence intensity signals indicative of intracellular calcium fluctuations were filtered using power spectrum calculated from Fourier transforms to reduce noise. Amplitude of signals was presented as relative fluorescence changes (AF/F) after background subtraction. A first- derivative filter was used to identify regions of increase in calcium signal and a calcium event was identified by a positive derivative value of >2 SD above background and with a rise phase that persisted a minimum of 5 consecutive frames (~70ms). To assess changes in calcium signaling in response to perturbation of neuronal activity, 1 μΜ tetrodotoxin (TTX) or the glutamate receptor antagonists CNQX/APV (6-cyano-7-nitroquinoxaline-2,3-dione at 10 μΜ / (2R)-amino-5-phosphonovaleric acid; (2R)-amino-5-phosphonopentanoate at 20 μΜ, respectively) were applied by bath application.

[0121] Electrophysiology. Whole-cell patch clamp recordings were performed from cell? co- cultured with astrocytes after 6 weeks of differentiation. The bath was constantly perfused with fresh HEPES-buffered saline (115 mM NaCl, 2 mM KCI, 10 mM HEPES, 3 mM CaC12, 10 mM glucose and 1.5 mM MgCl₂ (pH 7.4). The recording micropipettes (tip resistance 3-6 ΜΩ) were filled with internal solution containing: 140 mM K-gluconate, 5 mM KCI, 2 mM MgC12, 10 mM HEPES and 0.2 mM EGTA, 2.5 mM Na-ATP, 0.5 mM Na-GTP, 10 mM Na₂-phosphocreatine (pH 7.4). Recordings were made using Axopatch 200B amplifier (Axon Instruments). Signals were filtered at 2 kHz and sampled at 5 kHz. The whole-cell capacitance was fully compensated. The series resistance was uncompensated but monitored during the experiment by the amplitude of the capacitive current in response to a 10-mV pulse. For measurement of voltage-gated Na+ currents, cells were clamped at -70 mV and stimulated by step depolarizations of 300 ms (command voltage from -55 to 0 mV in 5 mV step). Cells were current clamped at -70 mV to measure the spiking activities in response to somatic current injections (duration 300 ms, currents starting from 50 pA in 50 pA increment). All recordings were performed at room temperature and chemicals were purchased from Sigma.

[0122] In vivo assay. For the engrafting assay, CB-iNPCs were dissociated by trypsin and plated directly on Polyornithine/Laminin-coated plates in the presence of DMEM/F12 +N2 and B27 Supplements, RA (retinoic acid at 1 μΜ), BDNF, GDNF (both at 20ng/ml), Lam (1 μg/ml) and 0.5% FBS, for 4 weeks. Five days prior to engrafting, the cells were infected with lentiviral vector expressing EGFP. On the day of in vivo injection, CB-derived neurons were dissociated with Accutase and resuspended in PBS-glucose + ROK inhibitor, BDNF and GDNF (50,000 cells/μΐ). P14 mouse pups were anaesthetized using ketamine/xylazine (lOOmg/kg, lOmg/kg). For transplantation Ιμΐ of cell suspension (-50,000 cells) was delivered to the dentate gyrus of the mouse hippocampus in the right hemisphere through stereotaxic surgery. The injection site was determined using the difference between bregma and lambda (d), using the position of the bregma as reference: anterior/porterior, -(1/2) x d mm; lateral, -1.6mm (if d < 1.6) or -1.7mm; ventral, -1.9mm (from dura). CB CD133+ cells infected with Lenti-GFP were engrafted as negative controls.

[0123] In vivo assay sample preparation for morphological analysis and

immunohistochemistry. Animals 2 or 4 weeks post transplantation were anesthetized with ketamine/xylazine (100 mg/kg, 10 mg/kg) and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brain samples were post-fixed with 4% paraformaldehyde and equilibrated in 30% sucrose. Coronal sections of 40 μπι were prepared with a sliding microtome. Brain sections of one-in-four series were selected for immunostaining. The following antibodies were used: chicken anti-GFP (gift from Aves Lab), mouse anti-neuronal-specific nuclear protein (NeuN) (Chemicon, Temecula, CA), rabbit anti-Tuj 1 (Chemicon, Temecula, CA), FITC- conjugated donkey anti-chicken, cyanine 3-conjugated donkey anti-mouse antibodies, and cyanine 5 -conjugated donkey anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA) 4'6'-Diamidino-2-phenylindole (DAPI) was used to reveal nuclei. B. Example 2: Generationof CB-iNPCs from CD133+ cells

[0124] SOX2 is highly expressed in adult neural stem cells (NSCs) (Graham et ah, Neuron 39, 749 (2003); Pevny & Nicolis, Intl. J. biochem. cell bioh 42, 421 (2010)). To determine if SOX2 could induce the conversion of CB CD133+ cells into neurons, we isolated CB CD133+ cells using standard immuno-magnetic selection, obtaining a purity range of 92-97% (Fig. 1). Freshly isolated CB CD133+ cells do not express or have a very low level of expression of neural lineage genes (Zangiacomi et al., Stem cells dev. 17, 1005 (2008) and Fig. 2).

[0125] First, we attempted to reprogram CD133+ cells into neurons by the overexpression of SOX2 (S) and by culturing the cells directly in neural condition cultures. Four weeks post- infection, no neural cells were detected and flow cytometry analysis showed that the cells were in apoptotic. The culture conditions were thus changed, briefly, three days post-transduction, the cells were plated onto irradiated human foreskin fibroblast (HFF-1) feeder cells and cultured in human embryonic stem cell (hES) medium, as described in Giorgetti et al. (2009) Cell Stem Cell 5:353 (Fig. 3A).

[0126] Using the retroviral approach, we obtained an infection efficiency of approximately 28% as monitored by a constitutive GFP reporter retrovirus (Giorgetti et al., Nature Prot. 5, 811 (2010)). Three weeks after infection, on average lxlO⁵ CD133+ cells infected with SOX2 gave rise to 1-2 colonies (efficiency 0.003-0.005%) (Fig. 3B).

[0127] After two passages, the colonies acquired a more complex structure (Fig. 3C). The absence of alkaline phosphatase activity (Fig. 3D), combined with the negativity for the pluripotency markers OCT4 and NANOG (Fig. 3E-F) ruled out the possibility that these colonies could contain pluripotent cells. In addition, upon injection into immuno-compromised SCID beige mice, the generation of complex intra-testicular teratomas were not observed after 8 weeks. In contrast, staining for Tuj-1 (β-ΙΙΙ Tubulin), GFAP (glial fibrillary acidic protein), NF

(neurofilament) and MAP2 (microtubule-associated protein 2) revealed that the colonies contained cells with typical neural morphologies and were positive for neural progenitor markers (Fig. 3E-F). Thus, the overexpression of SOX2 alone was sufficient to induce the conversion of CB CD133+ cells into CB-iNPCs.

[0128] To improve the efficiency of neural fate conversion, we combined the expression of SOX2 with that of c-MYC. The following data show that c-MYC is not required for the reprogramming process, but rather enhances its efficiency. c-MYC has a role in controlling the self-renewal and proliferation of neural progenitor cells (Kerosuo et al., Differentiation 80, 68 (2010); Kerosuo et al., Journal of cell science 121, 3941 (2008); Wey et al., Cerebellum 9, 537 (2004)).

[0129] CB CD133+ cells were infected using a mix (1:1) of SOX2 and c-MYC (SM) and cultured them in hES conditions. Around 20 days post-infection, small colonies started to appear (Fig. 3G-I) and the presence of c-MYC increased the frequency of colonies by 15- to 25-fold (efficiency 0,05%-0,09%). SM CB-iNPCs expressed homogeneously multiple neural progenitor markers such as NESTIN, Tuj-1, GFAP, MAP2, PAX6, OLIG02 (oligodendrocyte lineage transcription factor 2), and NF as showed by immunofluorescence and gene expression analysis (Fig. 3H-S, Fig. 4). In addition, S and SM CB-iNPCs lost the hematopoietic phenotype, as exemplified by flow cytometry analysis for markers such as CD133, CD45, CD34 and CD38 (Fig. 5). Other Yamanaka factors (KLF-4 and OCT4) were used to reprogram CB CD 133+ cells into neurons without success. Likewise, unlike the case of CB CD 133+ cells, human fibroblasts were not induced to form iNPC colonies with S or SM.

[0130] To rule out the possibility that CB-iNPCs could be the result of direct differentiation from a subpopulation of CB cells, CD133+ cells were maintained under neural culture conditions for a long period of time. After three weeks of culture, CD133+ cells did not give rise to neuronal progenitor cells. Flow cytometry analysis revealed an apoptotic phenotype.

[0131] Cytogenetic analysis showed that the CB-iNPCs lines maintained a normal 46XY or 46 XX karyotype after more than 15 passages and could be maintained in hES condition culture in the presence of FGF2 for at least 30 passages (Fig. 6). PCR fingerprinting confirmed the CB- iNPCs derived from respective CB cells.

[0132] The results were repeated with ten independent CB units using the two conditions (S and SM) to generate 40 CB-iNPC lines. Eighteen lines were expanded and characterized for expression of neural markers. Seven independent CB-iNPC lines (6 SM and 1 S CB-iNPC lines) were further differentiated into mature neurons and explored for neural activity in vitro and in vivo.

C. Example 2: CB-iNPCs differentiate into mature neurons

[0133] To obtain mature neurons, the CB-iNPCs were disaggregated and re-plated as single cells, either onto polyornithine/laminin coated plates or in co-culture with human astrocytes, in the presence of neural differentiation medium for 4-6 weeks. Although CB-iNPCs under both culture conditions acquired the typical morphology of mature neurons, further differentiation and expression of proteins involved in synaptic transmission were observed in the presence of astrocyte feeders. Under these culture conditions, VGLUT1 (vesicular glutamate transporter- 1) puncta were detected along MAP2 -positive dendrites (Fig. 7A) after one month of

differentiation. Moreover, CB-derived neurons expressed the inhibitory GABAergic marker (GAB A) (Fig. 7B). The expression of Synapsin puncta on Tuj-1 -positive cells highlighted the presence of mature synaptic buttons on CB-derived neurons (Fig. 7C). The presence of

Synapsin-positive terminals and the expression of both excitatory (VGLUT1) and inhibitory (GABA) markers suggested that the CB-derived neurons had the protein machinery necessary to fire action potentials.

D. Example 3: Gene expression of CB derived neurons resembles that of human neurons

[0134] The global gene expression analysis of CB CD133+ cells was compared to that of CB- iNPCs and CB-derived mature neurons. The results show that CB-derived neurons have a similar neural transcription profile to neurons derived from human ES (HUES6) (Marchetto et al., Cell 143, 527 (2010)) (Fig.7D-F). A set of neural specific-genes (MAP2, Pax6, NF, NeuroDl, N-CAM1, VGLUT1, MAP1B and SYN1) is significantly up-regulated (fold change 2, pval <0.05) in CB-derived neurons in comparison with the starting population CB CD 133+ cells (Fig. 7E).

[0135] On the other hand, the down-regulation of hematopoietic-specific genes (CD34, CD38, CD31, CD48, FLT3 and GATA2) in CB-derived neurons indicated that they lose the hematopoietic phenotype. Thus, CB-iNPCs and in particular CB-derived mature neurons, have a gene expression profile comparable to HUES6. The reprogramming is broadly reflected in gene expression changes. E. Example 4: CB derived neurons generate calcium currents

[0136] Early in neural development, spontaneous electrical activity leads to increases in intracellular calcium levels and activation of signaling pathways that are important for the regulation of neuronal processes (Spitzer et al, Trends in neurosciences 27, 415 (2004)). The cells were preloaded with the calcium indicator fluo-4AM, and neurons highlighted using the Synapsin: :DsRed vector reporter, to test if CB-derived neuronal networks were functionally capable of generating calcium transients (Fig. 8A). Cultures with similar cell density and numbers of DsRed-positive neurons were used. Spontaneous calcium transients were detected in neuronal networks from CB-derived neurons that were generated from 2 distinct CB units (Fig. 8B-D). Calcium transients detected in CB-derived neurons were blocked after the addition of glutamate receptor antagonists (CNQX: AMPA receptor antagonist and APV: NMDA receptor antagonist) (Fig. 8B) and after the addition of TTX (tetrodotoxin) (Fig. 8C), indicating neuronal signaling dependence on local synaptic connections.

F. Example 5: CB derived neurons generate action potentials

[0137] To determine if CB-derived neurons could form functional electrophysiological synapses, whole-cell recording was performed on cells that had differentiated 6-8 weeks in co- culture with human astrocytes. Neurons were visualized by infection with the Synapsin::DsRED lentiviral vector (Fig. 9A). CB neurons derived from distinct CB units (CB75-C12 and CB104- C13) were able to fire action potentials after current injection (Fig. 9B-D). Together with the calcium imaging data, the electrophysiological recordings demonstrate that CB-derived neurons can form functional networks in vitro.

G. Example 6: CB derived neurons engraft into the hippocampus

[0138] The results show that CB-derived neurons have the phenotypic and electrophysiological characteristics of mature neurons in vitro, thus we sought to determine if CB-derived neurons could integrate in an endogenous neural environment in vivo. CB-iNPCs differentiated for 4 weeks were infected with an EGFP-expressing lentivirus and injected into the hippocampus of 14-day-old NOD-SCID mice.

[0139] The presence of EGFP+ cells 2 weeks post transplantation was determined. As shown in Figure 9 (E-H), transplanted cells expressed mature neuronal markers (Tuj-1 and NeuN) and integrated with the host tissue, extending processes to endogenous granule neurons of the dentate gyrus, as well as along the mossy fiber path to pyramidal neurons in the CA2/CA3 regions (Fig. 9 E and F). Additionally, grafted neurons were able to integrate along the corpus callosum, sending extensive Tuj-1+ processes to the contra-lateral hemisphere (Fig. 9G).

[0140] As a negative control, CB CD 133+ cells were infected with a constitutive EGFP lentivirus and transplanted into the hippocampus of 14-day-old mice. Two weeks after grafting, CD133+ EGFP+ cells were found in the mouse hippocampus but were not differentiated into neurons. The CD 133+ EGFP+ cells exhibited a round morphology with no processes and were negative for the typical neuronal markers Tuj-1 and NeuN (Fig. 9H). [0141] To determine whether CB-derived neurons were stably integrated into the endogenous neural network, the presence of EGFP+ cells was determined one month post-injection. EGFP+ cells were still present in the hippocampus. At one month post-transplantation, 90% of the CB- derived neurons transplanted were positive for pan-neuronal marker, Tuj-1, and 48% were positive for the mature neuronal marker, NeuN (Fig. 9 I, J). In contrast, almost no EGFP+ CB CD 133+ cells were found in the transplanted animals four weeks post-injection. The lack of engraftment is likely due to their inability to integrate into the neurogenic environment of the host tissue, further confirming that CB CD 133+ cells do not have direct neurogenic potential. CB-derived neurons generated as described above, however, were fully capable of engrafting and integrating into the host tissue after in vivo transplantation.

H. Informal Sequence Listing: Human SOX2 polypeptide sequence (SEQ ID NO:l; NP_003097.1; 317 amino acids)

MYNM ETELKPPGPQQTSGGGGGNSTAAAAGGNQK SPDRVKRPMNAFMVWSRGQRRK AQENPK HNSEI SKRLGA E KLLSETEKRPFI DEAKRLRALHMKEHPDY YRPRRKTKTLMKKDKYTLPGGLLAPGGNS ASGVGVGAGLGAGVN QRMDSYAH NGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQG TPGMALGS GSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMI SMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAI NGTLPLSH

Human c-MYC polypeptide sequence (SEQ ID NO:2; NP_002448.2; 454 amino acids) MDFFRVVENQQPPAT PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLS PSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLE VTELLGGD VNQSFICDPDDETFIKNI I IQDC WSGFS AAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECI DPSVVFPYPLNDSSS PKSCASQDSSAF S PSS DSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEI DVVSVEKRQAPGKRSESGSPSAGGHSKPPHS PLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQI SNNRKCTSPRSSDTEENVKRRTHNVLERQRRNEL KRS FFALRDQ I PELENNEKAPKVVI LKKATAY I LS VQAEEQKL I SEEDLLRKRREQLKHKLEQLRNSCA

VII. Embodiments

[0142] Embodiment 1. An induced neuroprogenitor cell (iNPC) generated by a method comprising: expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell; and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC. [0143] Embodiment 2. The iNPC of embodiment 1, wherein said CB cell is a CD133+ CB cell.

[0144] Embodiment 3. The iNPC of embodiment 2, wherein the expressing comprises expressing an exogenous, recombinant nucleic acid encoding SOX2.

[0145] Embodiment 4. The iNPC of embodiment 2, wherein the method further comprises expressing a recombinant c-MYC polypeptide in the CD 133+ CB cell.

[0146] Embodiment 5. The iNPC of embodiment 4, wherein the expressing recombinant c- MYC polypeptide comprises expressing an exogenous, recombinant nucleic acid encoding c- MYC. [0147] Embodiment 6. The iNPC of embodiment 1, wherein the CB cell is cultured in embryonic stem cell media.

[0148] Embodiment 7. The iNPC of embodiment 1, wherein the CB cell is cultured with feeder cells.

[0149] Embodiment 8. An induced neuronal cell generated by a method comprising expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell; culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC; and differentiating the iNPC into a neuron.

[0150] Embodiment 9. The induced neuronal cell of embodiment 8, wherein the CB cell is a CD 133+ CB cell.

[0151] Embodiment 10. The induced neuronal cell of embodiment 9, wherein the method further comprises expressing a recombinant c-MYC polypeptide in the CD133+ CB cell.

[0152] Embodiment 11. The induced neuronal cell of embodiment 8, wherein the CB cell is cultured in embryonic stem cell media.

[0153] Embodiment 12. The induced neuronal cell of embodiment 8, wherein the CB cell is cultured with feeder cells.

[0154] Embodiment 13. The induced neuronal cell of embodiment 8, wherein the differentiating comprises culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin. [0155] Embodiment 14. A method of generating an induced neuroprogenitor cell (iNPC) comprising: expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell; and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC.

[0156] Embodiment 15. The method of embodiment 14, wherein the CB cell is a CD133+ CB cell.

[0157] Embodiment 16. The method of embodiment 15, wherein the expressing comprises expressing an exogenous, recombinant nucleic acid encoding SOX2.

[0158] Embodiment 17. The method of embodiment 15, further comprising expressing a recombinant c-MYC polypeptide in the CD133+ CB cell.

[0159] Embodiment 18. The method of embodiment 17, wherein the expressing recombinant c-MYC polypeptide comprises expressing an exogenous, recombinant nucleic acid encoding c- MYC.

[0160] Embodiment 19. The method of embodiment 14, wherein the CB cell is cultured in embryonic stem cell media.

[0161] Embodiment 20. The method of embodiment 14, wherein the CB cell is cultured with feeder cells.

[0162] Embodiment 21. The method of embodiment 14, further comprising differentiating the iNPC into a neuron.

[0163] Embodiment 22. The method of embodiment 21, wherein the differentiating comprises culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

[0164] Embodiment 23. A method of generating an induced neuronal cell comprising:

expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell; culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC; and differentiating the iNPC into a mature neuron, thereby generating an induced neuronal cell.

[0165] Embodiment 24. The method of embodiment 23, wherein the CB cell is a CD 133+ CB cell.

[0166] Embodiment 25. The method of embodiment 24, further comprising expressing a recombinant c-MYC polypeptide in the CD133+ CB cell. [0167] Embodiment 26. The method of embodiment 23, wherein the differentiating comprises culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

[0168] Embodiment 27. A method of treating a neurodegenerative disorder or neuronal injury in an individual in need of such treatment, said method comprising administering to the individual an effective amount of an iNPC as in one of embodiments 1 to 7, or an effective amount of an induced neuronal cell as in one of embodiments 8 to 13, thereby treating the neurodegenerative disorder or neuronal injury.

[0169] Embodiment 28. The method of embodiment 27, wherein the iNPC or induced neuronal cell is administered to the site of the neurodegeneration or neuronal injury in the individual.

[0170] Embodiment 29. The method of embodiment 27, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's Disease (AD), Huntington's Disease (HD), Parkinson's Disease (PD) Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS) and Cerebral Palsy (CP).

[0171] Embodiment 30. The method of embodiment 27, wherein the neuronal injury is selected from the group consisting of traumatic brain injury, stroke, and chemically induced brain injury.

[0172] Embodiment 31. An induced neuroprogenitor cell (iNPC) comprising an exogenous, recombinant nucleic acid encoding SOX2.

[0173] Embodiment 32. The iNPC of embodiment 31, wherein the iNPC further comprises an exogenous, recombinant nucleic acid encoding c-MYC.

[0174] Embodiment 33. The iNPC of embodiment 31, wherein the iNPC is derived from a CD133+cord blood (CB) cell.

Claims

WHAT IS CLAIMED IS: 1. An induced neuroprogenitor cell (iNPC) generated by a method comprising:

expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell; and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC.

2. The iNPC of claim 1, wherein said CB cell is a CD133+ CB cell.

3. The iNPC of claim 2, wherein the expressing comprises expressing an exogenous, recombinant nucleic acid encoding SOX2.

4. The iNPC of claim 2, wherein the method further comprises expressing a recombinant c-MYC polypeptide in the CD133+ CB cell.

5. The iNPC of claim 4, wherein the expressing recombinant c-MYC polypeptide comprises expressing an exogenous, recombinant nucleic acid encoding c-MYC.

6. The iNPC of claim 1, wherein the CB cell is cultured in embryonic stem cell media.

7. The iNPC of claim 1, wherein the CB cell is cultured with feeder cells.

8. An induced neuronal cell generated by a method comprising expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell;

culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC; and

differentiating the iNPC into a neuron,

9. The induced neuronal cell of claim 8, wherein the CB cell is a CD 133+ CB cell.

10. The induced neuronal cell of claim 9, wherein the method further comprises expressing a recombinant c-MYC polypeptide in the CD133+ CB cell.

1 1. The induced neuronal cell of claim 8, wherein the CB cell is cultured in embryonic stem cell media.

12. The induced neuronal cell of claim 8, wherein the CB cell is cultured with feeder cells.

13. The induced neuronal cell of claim 8, wherein the differentiating comprises culturing the iNPC on a surface coated with feeder cells or polyomithine/ laminin.

14. A method of generating an induced neuroprogenitor cell (iNPC) comprising:

expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell; and culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC.

15. The method of claim 14, wherein the CB cell is a CD133+ CB cell.

16. The method of claim 15, wherein the expressing comprises expressing an exogenous, recombinant nucleic acid encoding SOX2.

17. The method of claim 15, further comprising expressing a recombinant c- MYC polypeptide in the CD133+ CB cell.

18. The method of claim 17, wherein the expressing recombinant c-MYC polypeptide comprises expressing an exogenous, recombinant nucleic acid encoding c-MYC.

19. The method of claim 14, wherein the CB cell is cultured in embryonic stem cell media.

20. The method of claim 14, wherein the CB cell is cultured with feeder cells.

21. The method of claim 14, further comprising differentiating the iNPC into a neuron.

22. The method of claim 21, wherein the differentiating comprises culturing the iNPC on a surface coated with feeder cells or polyomithine/ laminin.

23. A method of generating an induced neuronal cell comprising: expressing a recombinant SOX2 polypeptide in a cord blood (CB) cell;

culturing the CB cell in conditions permitting differentiation of the CB cell into an iNPC, thereby generating an iNPC; and differentiating the iNPC into a mature neuron, thereby generating an induced neuronal cell.

24. The method of claim 23, wherein the CB cell is a CD133+ CB cell.

25. The method of claim 24, further comprising expressing a recombinant c- MYC polypeptide in the CD133+ CB cell.

26. The method of claim 23, wherein the differentiating comprises culturing the iNPC on a surface coated with feeder cells or polyornithine/ laminin.

27. A method of treating a neurodegenerative disorder or neuronal injury in an individual in need of such treatment, said method comprising administering to the individual an effective amount of an iNPC as in one of claims 1 to 7, or an effective amount of an induced neuronal cell as in one of claims 8 to 13, thereby treating the neurodegenerative disorder or neuronal injury.

28. The method of claim 27, wherein the iNPC or induced neuronal cell is administered to the site of the neurodegeneration or neuronal injury in the individual.

29. The method of claim 27, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's Disease (AD), Huntington's Disease (HD), Parkinson's Disease (PD) Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS) and Cerebral Palsy (CP).

30. The method of claim 27, wherein the neuronal injury is selected from the group consisting of traumatic brain injury, stroke, and chemically induced brain injury.

31. An induced neuroprogenitor cell (iNPC) comprising an exogenous, recombinant nucleic acid encoding SOX2.

32. The iNPC of claim 31, wherein the iNPC further comprises an exogenous, recombinant nucleic acid encoding c-MYC.

33. The iNPC of claim 31, wherein the iNPC is derived from a CD133+cord blood (CB) cell.