WO2013011093A1

WO2013011093A1 - Novel method for generation of neural progenitor cells

Info

Publication number: WO2013011093A1
Application number: PCT/EP2012/064191
Authority: WO
Inventors: Cathérine VERFAILLIE; Jeroen DECLERCQ; Anujith KUMAR
Original assignee: Katholieke Universiteit Leuven
Priority date: 2011-07-20
Filing date: 2012-07-19
Publication date: 2013-01-24
Also published as: GB201112433D0

Abstract

The present invention relates to methods for reprogramming cells to neural progenitor cells. The methods generally involve the modulation of pluripotency factors in combination with the modulation of ZIC3. The methods of this invention generally comprise a step of increasing the expression and/or activity of these pluripotency factors and/or ZIC3 or substrates, cofactors, and/or downstream effectors of any of these factors, including ZIC3.

Description

NOVEL METHOD FOR GENERATION OF NEURAL PROGENITOR CELLS FIELD OF THE INVENTION

The present invention relates to methods for reprogramming cells to neural progenitor cells. The methods generally involve the modulation of pluripotency factors in combination with the modulation of ZIC3. The methods of this invention generally comprise a step of increasing the expression and/or activity of these pluripotency factors and/or ZIC3 or substrates, cofactors, and/or downstream effectors of any of these factors, including ZIC3. BACKGROUND OF THE INVENTION

In medicine, more and more attention is focussed on the use of pluripotent stem cells and/or tissue-specific progenitor cells for the treatment of diseases or injuries. The ability of said cells to self-renew and give rise to subsequent generations with variable degrees of differentiation capacities, offers significant potential for generation of tissues that can potentially replace diseased or damaged areas in the body, with minimal risk of rejection and side-effects. Whereas lots of research has been focussed on the use of embryonic stem cells, from an ethical point of view, it is of course more desirable to use non- embyronic cells, provided that they have or are able to gain the ability to differentiate into the desired types of tissue. Therefore, in the last couple of years, different research programs have focussed on the reprogramming of non-embyonic, cells, such as (human) somatic cells, into induced pluripotent stem cells (iPSC).

For example, induced pluripotent stem cells (iPSC) could be obtained by ectopic expression of OCT4, SOX2, KLF4 and c-MYC in mouse fibroblasts; or OCT4, SOX2, NANOG and LIN28 in primary human fibroblasts (Yu et al., Science, 318:1917-1920 (2007)). Yang et al., Biotechnology and Bioengineering, 104:1047-1058 (2009) have further used these findings for developing a protein-based approach for iPSC generation, instead of viral constructs, thereby avoiding DNA integration of the ectopically expressed genes. These findings have opened the door to the generation of patient-specific cells for regenerative medicine and disease modeling starting from non-embryonic cells. The transcription factors (TF) OCT4, NANOG, and SOX2 have been found to be the core regulatory players in maintaining pluripotency, i.e. the embryonic stem cell (ESC) fate (Jaenisch et al., Cell, 132:567-582 (2008)). Chromatin immunoprecipitation (ChIP) experiments have demonstrated that KLF4, SOX2 and OCT4 together with other ESC- specific transcription factors such as NANOG often co-occupy target genes, including their own promoters, hence cooperating in regulatory feedback loops to maintain self-renewal and pluripotency. Recent advances in ESC biology have highlighted the role of additional TF in the maintenance of pluripotency, including the orphan nuclear receptor, DAX1 , the zinc finger transcription factor, ZFP206 and the zinc finger transcription factor in cerebellum-3, ZIC3 (Lim et al., 2010; Lim et al., 2007).

ZIC3, a member of the GLI superfamily of transcription factors, encodes five tandem C2H2 zinc finger domains that are highly conserved across species. In both human and mouse, Zic3 is located on the X chromosome. ZIC3 has been implicated in human congenital anomalies and is a marker for brain tumors (medulloblastoma and meningioma). During mouse development, Zic3 is expressed in both posterior ectoderm and mesoderm at the mid to late primitive streak stage and early neural fold stages, and in the hindbrain regions at later stages. Zic3 is also expressed in the dorsal neural tube, somites, and the segmenting trunk. Zic3 has been found to be required for the maintenance of pluripotency in mouse (m) and human (h)ESC by directly controlling expression of Nanog (Lim et al., 2010; Lim et al., 2007).

Although the versatility of iPSC's is very interesting, the use of tissue-specific progenitor cells, may have beneficial effects over the use of more pluripotent stem cells in treatment, since tissue-specific progenitor cells are more likely to differentiate into cells specific for said particular tissue, whereas, pluripotent stem cells, have a higher risk of differentiating into an undesired cell type. For example, in the treatment of brain diseases, it would be highly beneficial to use neural progenitor cells, which are only capable of differentiating into brain cell lineages, rather than pluripotent stem cells, which could give rise to the growth of non-brain cells in the brain. Furthermore, the process of making appropriate cells via induced pluripotent stem cells requires reprogramming of somatic cells and subsequent redifferentiation. Therefore, a method for directly converting somatic cells into lineage-specific stem/progenitor cells in one step, thereby bypassing the intermediate pluripotent stage, would significantly shortening the reprogramming process.

Kim et al., 201 1 (WO201 1 159726) showed that transient transduction of Oct4, Sox2, Klf4 and c-Myc can efficiently transdifferentiate mouse fibroblasts into functional neural stem/progenitor cells by using appropriate signalling inputs. However, as described by Yu et al., Science, 318:1917-1920 (2007), the use of c-Myc in transdifferentiating human somatic cells, is undesirable given the observation that c-Myc expression causes death and differentiation of human ES cells. Therefore, the method as described by Kim et al., 201 1 is likely not suitable for reprogramming human somatic cells into functional neural progenitor cells. Furthermore, to the best of our knowledge, there are currently no methods available for reprogramming human somatic cells into functional neural progenitor cells, and therefore it was an object of the present invention to provide such methods. As evident from the examples, that follow hereinafter, we have surprisingly found that Zic3 is essential for reprogramming cells to neural progenitor cells. Zic 3 has earlier been found to be required for maintenance of pluripotency in mouse and human embryonic stem cells (Lim et al., 2007) and is thus considered to be a pluripotency- maintaining factor (Aruga et al., Neurochemical Research, 36:1286-1292 (201 1 )). Even in neural progenitor cells, Zic3 has been shown to be such a pluripotency-maintaining factor of such neural progenitor cell (NPC) fate by preventing neuronal differentiation (Inoue et al., 2007). Furthermore, Zic3 was found to be an essential factor in neural development in zebrafish (Lee et al., Animal Cells and Systems, 12:23-33 (2008)) and Xenopus (Kitagushi et al., Society for Neuroscience Abstracts, 26:abstract 22.1 (2000)). However, notwithstanding this earlier identification that Zic3 as a pluripotency-maintaining factor, there have been no previous indications that Zic3 could reverse differentiation of somatic cells to a less differentiated state neural state as provided herein.

We have found that co-transduction of Zic3 together with other transcription factors such as OCT4, SOX2 and KLF4 creates stable hNPC lines from human fibroblasts. We further demonstrated that combined transduction of OCT4, SOX2 and KLF4 with Zic3 in human fibroblasts, yields within 15 days stable cell lines that have NPC characteristics, as demonstrated by transcriptome analysis, in vitro and in vivo differentiation studies.

Given the earlier identification of Zic3 as a direct transcriptional activator of the Nanog promotor in embryonic stem cells (Lim et al., 2010), this is even more surprising as it was to be expected that induction of Zic3, in non-embryonic cells, through the subsequent activation of Nanog, would give rise to iPSC's, as is the case for Oct4/Lin28/Sox2/Nanog induced non-embyonic cells (Yu et al., Science, 318:1917-1920 (2007)). However, surprisingly we have found that modulation of Zic3 in non-embryonic cells, does not give rise to iPSC's, but rather results in the reprogramming of cells into neural progenitor cells.

SUMMARY OF THE INVENTION

The present invention is based on the finding that ZIC3 is a factor that plays a role in reprogramming cells into neural progenitor cells (NPC). We demonstrate that by transducing human fibroblasts with Zic3/OCT4/SOX2/KLF4, we can create stable NPC. The present invention relates to the finding that addition of Zic3 during iPSC generation, allows for the derivation of stable, human NPC lines.

A first aspect of the present invention provides a method for reprogramming a cell to a neural progenitor cell comprising the step of inducing neural differentiation by modulation of the expression and/or activity of at least one, two or three pluripotency factors in combination with ZIC3, or a substrate, cofactor or downstream effector of any of these factors. In certain embodiments of the present invention said pluripotency factors are selected from the list containing: Sox2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, ESRRB, SOX1 , PAX6, ZIC1 and ZIC2. In more specific embodiments of the present invention said pluripotency factors are selected from the list containing: Sox2, c-Myc, Oct3/4, Klf4, Lin28 and Nanog. In particular embodiments of the present invention, said pluripotency factors are Oct4, Klf4 and Sox2.

In certain embodiments of the present invention said method for reprogramming a cell to a neural progenitor cell starts from cells that endogenously express one or more of said pluripotency factors. The starting cell to be reprogrammed to a neural progenitor cell can be any type of cell, such as an adult cell, an adult stem cell, an adult bone marrow cell, a non)adult cell, a cord cell, a placenta cell, a cord blood cell and a fibroblast.

In certain embodiments of this invention, said starting cell to be reprogrammed to a neural progenitor cell is of human origin.

Certain embodiments of the present invention relate to a method for reprogramming a cell to a neural progenitor cell comprising the step of inducing neural differentiation by modulation of the expression and/or activity of ZIC3, or a substrate, cofactor or downstream effector thereof, in combination with an Induced pluripotent stem cell (IPS) generation procedure. IPS generation procedures are known to a person skilled in the art and examples are modifications of a procedure originally discovered in 2006 (Yamanaka, S. et al., Cell Stem Cell, 1 :39-49 (2007)), such as the insertion of the four genes: Oct4, Sox2, Lif4 and c-myc. In certain embodiments of the present invention some factors are omitted in the IPS generation procedures, an example of such an omitted factor is c-myc. Certain embodiments of the present invention relate to the modulation of the expression and/or activity of some factors, in which said modulation comprises a step of transiently transfecting at least one of said factors. In other embodiments of the present invention, said modulation comprises a step of viral transduction of at least one of said factors. In other embodiments of the present invention, said modulation comprises a step of microinjection of at least one of said factors. Said factors include any pluripotency factor, and ZIC3.

In certain embodiments of the present invention, the step of treating the population of cells to induce neural differentiation comprises a step of contacting the population of cells with a small molecule, protein, peptide, sugar, or other compound, agent or treatment that effects modulation of at least one of said factors, or a substrate, cofactor or downstream effector thereof. In certain embodiments of the present invention, the step of treating the population of cells to induce neural differentiation comprises a step of increasing the expression of at least one of said factors, or a substrate, cofactor or downstream effector thereof.

Certain embodiments of the present invention relate to the monitoring of the neural progenitor phenotype, said monitoring can be performed by monitoring or measuring at least one cellular marker known by a skilled person in the art, said cellular marker can be selected from the list containing PAX6, OTX1 , FOXG1 , GLAST, P75, SOX1 , and OLIG2. Another aspect of the present invention relates to the method of the present invention, further comprising an amplification step of the neural progenitor cells that are produced by the method and/or comprising a differentiation step. Said differentiation step can be differentiation from neural progenitor cells into astrocytes, neural progenitor cells into oligodendrocytes, neural progenitor cells into motor neurons, or neural progenitor cells into dopaminergic neurons.

Another aspect of the present invention relates to the cells produced by any of the methods of this invention.

This invention further relates to a pharmaceutical composition containing the cells produced by any of the methods of this invention. Preferably, said pharmaceutical composition further comprises a physiologically compatible solution including, for example phosphate-buffered saline.

Another aspect of the present invention relates to a method for treating a neurodegenerative disease (e.g. Parkinson's disease) in a patient by administering to the brain of said patient any of the foregoing pharmaceutical compositions.

Another aspect of the present invention relates to the use of the cells produced by any of the methods of this invention, for pharmacological compound screening in neural disorders or toxicity effects.

Some embodiments of the invention are set forth in claim format directly below:

1 . A method for reprogramming a cell to a neural progenitor cell comprising the steps of:

(a) providing a population of cells;

(b) treating the population of cells to induce neural differentiation by modulating the expression and/or activity of at least three pluripotency factors selected from Sox2, c- Myc, Oct3/4, Klf4, Lin28, Nanog, ESRRB, SOX1 , PAX6, ZIC1 and ZIC2 together with the factor ZIC3, or a substrate, cofactor or downstream effector of any of these factors; and

(c) selecting for cells having a neural progenitor phenotype. 2. The method of embodiment 1 , wherein the cells endogenously express one or more of the pluripotency factors selected from Sox2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, ESRRB, SOX1 , PAX6, ZIC1 and ZIC2 or a substrate, cofactor or downstream effector thereof.

3. The method of embodiment 1 or 2, wherein the 3 pluripotency factors of step (b) are Oct4, Klf4 and Sox2.

4. The method of any of embodiments 1 to 3, wherein the population of cells comprises adult cells.

5. The method of any of embodiments 1 to 3, wherein the population of cells comprises adult stem cells.

6. The method of any of embodiments 1 to 3, wherein the population of cells comprises adult bone marrow cells.

7. The method of any of embodiments 1 to 3, wherein the population of cells comprises non-adult cells.

8. The method of any of embodiments 1 to 3, wherein the population of cells comprises fibroblasts.

9. The method of any of embodiments 1 to 8, wherein the population of cells are of human origin.

10. The method of any of embodiments 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of transiently transfecting at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

1 1. The method of any of embodiments 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of viral transduction of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

12. The method of any of embodiments 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of microinjection of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

13. The method of any of embodiments 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of contacting the population of cells with a small molecule, protein, peptide, sugar, or other compound, agent or treatment that effects modulation of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

14. The method of any of embodiments 1 to 13, wherein the step of treating the population of cells to induce neural differentiation comprises a step of increasing the expression of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

15. The method of any of embodiments 1 to 14, wherein the step of selecting for cells having a neural progenitor phenotype comprises monitoring at least one cellular marker selected from PAX6, OTX1 , FOXG1 , GLAST, P75, SOX1 , and OLIG2.

16. The method of any of embodiments 1 to 15, wherein the method further comprises amplifying the neural progenitor cells selected in step (c).

17. The method of any of embodiments 1 to 16, further comprising inducing differentiation of the neural progenitor cells into astrocytes.

18. The method of any of embodiments 1 to 16, further comprising inducing differentiation of the neural progenitor cells into oligodendrocytes.

19. The method of any of embodiments 1 to 16, further comprising inducing differentiation of the neural progenitor cells into motor neurons.

20. The method of any of embodiments 1 to 16, further comprising inducing differentiation of the neural progenitor cells into dopaminergic neurons.

21. A population of cells produced by the method of any of embodiments 1 to 20.

22. A pharmaceutical composition comprising a population of cells according to embodiment 21.

23. Use of a pharmaceutical composition according to embodiment 22 in human or animal medicine.

24. Use of a pharmaceutical composition according to embodiment 22 for the treatment of disorders of the brain.

25. Use according to embodiment 24, wherein the disorders of the brain are neurodegenerative disorders such as Alzheimer's Disease or Parkinson's Disease.

DESCRIPTION

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

Figure 1. Transduction of Zic3 together with OCT4, SOX2 and KLF4 in human fibroblasts yields SSEA1/AP positive colonies (neural progenitor like cells) within 8-10 days. A) Quantification of the number of colonies 8 (white bar) and 14 (black bar) days after retroviral transduction of BJ1 fibroblasts with OSK and OSKZ retroviruses (n=3, p=0.02). B) 250,000 mESC, ZiNPCI , ZiNPC2 and hESC were plated on MEF and cell numbers were measured using a nucleocounter as function of time after plating. N=3; p<0.0001 for mESC versus ZiNPCI and ZiNPC2 and; p<0.0001 for hESC versus ZiNPCI and ZiNPC2. Figure 2. A) Light microscopy of, from left to right, a ZiNPC colony, a mESC colony and a hESC colony. B) Alkaline phosphatase staining of ZiNPCs and of BJ 1 -fibroblasts transduced with OSK retroviruses.

Figure 3. A) Gene expression profile for the transgenes and endogenous ZIC3 in hESC, hiPSC, ZiNPCI and ZiNPC2 as determined by qRT-PCR. Data are shown as ACT values relative to GAPDH (n=3). B) The expression of Zic3 was silenced in ZiN PC by using two different shRNA constructs and the expression of neuronal genes was analyzed by qRT- PCR. Data are shown as relative expression levels as compared to the non transduced ZiNPCs (fold induction) (n=2).

Figure 4. Characterization of the expression profile of ZiNPC. A) Transcript levels for the endogenous pluripotency TF in hESC, hiPSC, ZiNPCI and ZiNPC2 as determined by RT- qPCR. Data are shown as ACT values relative to GAPDH. B) Transcript levels of endodermal (FOXA2, SOX17), mesodermal (BRACHYURY, GSC) and ectodermal (PAX6) genes in hESC, hiPSC, ZiNPCI and ZiNPC2 as determined by RT-qPCR. Data are shown as ACT values relative to GAPDH. C+D) Transcript levels of ectodermal genes in hESC, hiPSC, ZiN PC and neurospheres derived from those cells as determined by RT- qPCR. Data are shown as ACT values relative to GAPDH. All RT-qPCR data are presented as the mean ± SEM from triplicate experiments in representative cell lines. E) Venn diagram of genes significantly higher expressed in NSC derived from hESC and/or ZiNPC compared to hESC. Significance was assigned with a corrected p-value<0.05.

Figure 5. Assessment of the differentation potential of ZiNPC by embryoid body formation. ZiNPC and hESC were cultured in EB conditions and transcript levels of ectodermal (PAX6, SOX1), endodermal (AFP) and mesodermal (FLK1) genes analyzed by RT-qPCR. Data are shown as ACT values relative to GAPDH (n=2).

Figure 6. A) Gene expression profile for astrocyte-specific transcripts in ZiNPC and ZiNPC differentiated towards astrocyte lineage. Data are shown as ACT values relative to GAPDH (n=3). B) Gene expression profile for oligodendrocyte-specific transcripts in ZiNPC and ZiNPC differentiated towards oligodendrocyte lineage. Data are shown as ACT values relative to GAPDH (n=3). C) Gene expression profile for motor neuron and matured neuron-specific transcripts in ZiNPC and ZiN PC differentiated towards motor neuron lineage. Data are shown as ACT values relative to GAPDH (n=3). DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel methods to generate neural progenitor cells (NPC). The present invention provides cells and cell populations made by the methods of this invention, including NPCs, oligodendrocytes, astrocytes, motor neurons and dopaminergic neurons. The method of the present invention starts from a starting population of cells which can be any type of cell, such as adult cells and typical suitable cells are mesenchymal cell types such as fibroblasts (eg skin fibroblasts). Such starting population of cells may be derived from essentially any suitable source, and may be heterogeneous or homogeneous. In certain embodiments, the cells to be treated according to the invention are adult cells, including essentially any accessible adult cell types. In other embodiments, the cells used according to the invention are adult stem cell populations, including bone marrow, or non-adult cells such as cord and placenta cells and cord blood cells. In still other embodiments, the cells treated according to the invention include any type of adult cell that can give rise to a cancer or which can be transformed in cell culture. The cells generated in the present invention can be used, potentially in a cellular composition, as screening tools for compound screening, which compounds are potentially useful for the treatment of neural disorders, or for toxicity screenings, including toxicity analyses for said potential pharmaceutical compounds.

The cells generated in the present invention can be used, potentially in a pharmaceutical composition to treat brain disorders such as neurodegenerative diseases and can be used in cell replacement/transplantation therapies.

In a specific embodiment, fibroblasts are reprogrammed using the 3 transcription factors Oct4, SOX2 and Klf4 in combination with ZIC3. The reprogramming of the cells with these four factors reveals stable NPC called ZiNPC. Thus our method uses the classical pluripotency factors in combination with ZIC3 to generate NPC. Many combinations of pluripotency factors are known by the person skilled in the art for the generation of IPS cells.

"Induced pluripotent stem cells (IPS, IPSC or IPS cells)" are somatic cells that have been reprogrammed, for example, by introducing exogenous genes that confer on the somatic cell a less differentiated phenotype. These cells can then be induced to differentiate into less differentiated progeny. IPS cells have been derived using modifications of an approach originally discovered in 2006 (Yamanaka, S., Cell Stem Cell, 1 :39-49 (2007)). For example, in one instance, to create IPS cells, scientists started with skin cells that were then modified by a standard laboratory technique using retroviruses to insert genes into the cellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4, and c- myc, known to act together as natural regulators to keep cells in an embryonic stem celllike state. These cells have been described in the literature. See, for example, Wernig et al., PNAS, 105:5856-5861 (2008); Jaenisch et al., Cell, 132:567-582 (2008); Hanna et al., Cell, 133:250-264 (2008); and Brambrink et al., Cell Stem Cell, 2:151-159 (2008). These references are incorporated by reference for teaching IPS and methods for producing them. It is also possible that such cells can be created by specific culture conditions (exposure to specific agents).

In certain embodiments, it will be preferred to deliver one or more pluripotency factors including ZIC3 to a cell or a population of cells using a viral vector or other in vivo polynucleotide delivery technique. This may be achieved using any of a variety of well - known approaches including adenoviral vector based methods, retroviral technology, and adeno-associated viral (AAV-)technology.

Pluripotency factors, including ZIC3, or substrates, cofactors, or downstream effectors of any of these factors, can also be introduced into cells using, for example transient methods, e.g. protein transduction, microinjection, non-integrating gene delivery, mRNA transduction or any other suitable technique. Alternatively, or in addition, pluripotency factors, including ZIC3, can be exogenous molecules contacted with or otherwise introduced into cells (e.g., small molecules, proteins, peptides, sugars, etc) which modulate the factors themselves and/or the signaling pathways within which the pluripotency factors act.

Certain embodiments of the present invention relate to methods which comprise a selection step in which NPC are selected or isolated. Such selection step comprise methods of depletion of undesirable cell types and/or active selection of desired cell types. Markers to be actively selected for can contain at least one neural or NPC marker known in the art. Such marker can be selected from the list containing: PAX6, OTX1 , FOXG1 , GLAST, P75, SOX1 , and OLIG2.

Methods for Depleting Cell Populations of Undesirable Cell Types:

In certain embodiments of the present invention, it is desirable to deplete cell populations of undesirable cell types that contribute to deleterious effects or otherwise possess undesirable properties. Alternatively, cell populations may be depleted of cells which themselves do not possess undesirable properties, but are merely unwanted for the particular end-use of the final cell preparation. Methods for in vitro depletion and/or enrichment of cell populations to select for/against cells expressing certain markers (e.g., cell surface proteins such as SSEA-4) are well known. Active selection of desired cell types can be performed by methods well known in the art, including but not limited to: FACS, Immunomagnetic Cell Separations and cellular panning.

"Cell Sorting": Fluorescence-activated cell sorting (FACS) is a commonly used cell sorting technique. Cells are sorted based on the ability of fluorescently-labeled antibodies or other markers to bind to the cells of interest. Cells are separated by flow cytometry and sorted into different containers based on their fluorescent characteristics.

"Immunomagnetic Cell Separations": Immunomagnetic cell separations involve attaching antibodies directed to cell surface markers (e.g., proteins) to small paramagnetic beads. See, for example, Kruger et al., Transfusion 40: 1489-1493, 2000. When the antibody- coated beads are mixed with the cell sample, the antibodies attach to the cells expressing the marker of interest. The cell sample is then placed in a strong magnetic field, causing the paramagnetic beads (and the bound cells) to pellet to one side. Depending upon the marker of interest, the captured cells may represent either a desirably enriched cell population, with the unbound cells being discarded, or the unbound cells representing the enriched cell population with the unwanted cells removed.

"Cellular Panning": For this cellular separation technique, an antibody to the cell type in question is allowed to adhere to a surface, such as the surface of a plastic Petri dish. When the cell mixture is layered on top of the antibody-coated surface, the targeted cells tightly adhere. Non-adherent cells are rinsed off the surface, thereby effecting a cell separation. Cells that express a cell surface protein recognized by the antibody are retained on the plastic surface whereas other cell types are not. This technique is useful for capturing rare cells in a population, but the antibody-bound surface may become saturated and target cells lost in samples having relatively large numbers of target cells. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

The singular and plural forms are also interchangeable in this application where a cell or cells or cell lines and population of cells are used.

A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

The invention is further illustrated by way of the illustrative embodiments described below.

Transduction of human fibroblasts with a combination of Zic3 with OCT4, SOX2 and KLF4 generates colonies with mESC characteristics.

To investigate the consequence of Zic3 during the reprogramming of human fibroblasts to iPSC, human BJ1 fibroblasts were retrovirally transduced with OCT4 (O), SOX2 (S), KLF4 (K) with and without Zic3 (Z). Colonies started to appear in the culture plates transduced with OSKZ after 8 days. In contrast, iPSC like colonies were not detected in BJ1 fibroblasts transduced with OSK even till 30 days post transduction (Figure 1A). The colonies obtained (termed Zic3-induced neural progenitor cells or ZiNPC) after retroviral transduction with OSKZ displayed morphological features more typical for bright, tightly packed, dome-shaped mESC than the flattened two-dimensional colony morphology of hESC (Figure 2A). ZiNPC stained positive for alkaline phosphatase (Figure 2B) and were SSEA1 positive, typical for mESC, but negative for SSEA4 and TRA160, typical for undifferentiated hESC (Data not shown). Unlike hESC, ZiNPC could be propagated by trypsin digestion and passaged as single cells. Finally, ZiNPC proliferated significantly faster than hESC, but still slower than mESC (Figure 1 B).

ZiNPC show many characteristics of early neuronal progenitor cells.

The four transgenes were integrated in the ZiNPC, as demonstrated by genomic DNA PCR using transgene specific primers. The OCT4, SOX2 and KLF4 transgenes were silenced whereas the Zic3 transgene remained expressed (Figure 3A). We could, in contrast to OSKM induced hiPSC or hESC lines, only detect very low levels of endogenous OCT4 and NANOG mRNA. Although endogenous SOX2 was expressed, the expression was lower compared to hESC or hiPSC, whereas endogenous KLF4 was expressed at levels similar to those in hESC or hiPSC (Figure 4A). Endogenous ZIC3 was expressed at lower levels in ZiNPC compared to hESC and hiPSC (Figure 3A). However, the ectodermal TF PAX6 was expressed at much higher levels than in hESC and hiPSC, whereas the endodermal TF, FOXA2 and SOX17, and the mesodermal TF, BRACHYURY and GSC, were not expressed in ZiNPC (Figure 4B). As these results suggested that fibroblasts might be reprogrammed to an NPC fate and not to a pluripotent state, we next compared the expression levels of several neurectodermal TF in ZiNPC with those in hiPSC or hESC-derived neural stem cells (NSC), generated by dual SMAD inhibition with noggin and SB431542 as described in the prior art. The neuroectodermal genes, PAX6, OTX1 , FOXG1 , GLAST, P75, SOX1 , and OLIG2 were upregulated in hESC and hiPSC only upon differentiation towards NSC, whereas their expression levels were already high in ZiNPC, indicating an NPC-fate (Figure 4C+D). Near homogenous expression of PAX6, OTX1 and FOXG1 was confirmed at the protein level by immunostaining (Data not shown).

To investigate whether the continuous expression of the Zic3 transgene is important for the maintenance of the NPC fate, we silenced the expression of Zic3 by two independent shRNA's. Induction of Zic3-shRNA expression in ZiNPC induced a 82->90% decrease in Zic3 expression. This was associated with a 60-80% decrease in expression levels for PAX6, OTX1 , OTX2 (Figure 3B) without concomitant increase in endodermal or mesodermal gene expression.

To further ascertain the NPC like fate following OSKZ transduction of fibroblasts, the global gene expression profile of ZiNPC and hESC-derived NSC was compared by microarray analysis. Compared with undifferentiated ESC, 1617 genes were more highly expressed in hESC-derived NSC and 1876 genes in ZiNPC, whereas 1550 genes and 1977 genes, respectively, were expressed at lower levels in hESC-derived NSC and ZiNPC compared with undifferentiated hESC. Among the more highly expressed genes in ESC-NSC and ZiNPC, 563 were common in both cell types (Figure 4E). Gene ontology analysis (http://david.abcc.ncifcrf.gov/) demonstrated that many of these genes are involved in neural differentiation and development. Differentiation of ZiNPC is limited to the ectodermal lineage

To investigate the differentiation capacity of ZiNPC, embryoid body (EB) formation was performed. ZiNPC formed clusters of cells morphologically similar to EBs. Although clusters were formed, these represented neurospheres rather than EBs as only neuroectoderm specific genes were highly expressed, as demonstrated by RT-qPCR analysis (Figure 5). This suggested that the differentiation capacity of ZiNPC is limited to the ectodermal lineage. To further confirm this observation in vivo, one million cells dissolved in 50% matrigel were injected subcutaneously in immunodeficient mice. The mice developed large tumors, which were harvested 1 month post injection and evaluated by H&E staining as well as immunostaining. In contrast to teratomas generated by hESC and hiPSC, ZiNPC progeny did not form cells of the three germ layers and progeny had characteristic features of neuro-endocrine tumors. In addition, tumors were largely positive for 33-tubulin and synaptophysin (Data not shown). This further substantiated the notion that the differentiation capacity of ZiNPC is restricted to the (neur)ectodermal lineage.

ZiNPC can differentiate in vitro to astrocytes, oligodendrocytes and motor neurons We next tested if ZiNPC can be directly differentiated into mature neural progeny in vitro, including astrocytes, oligodendrocytes and motor neurons. By culturing in the presence of neural differentiation medium and 1 % fetal calf serum, ZiNPC could be committed to the astrocyte lineage, as shown by expression of the astrocyte-specific transcript, GFAP, by RT-qPCR (Figure 6A) and the astrocyte-specific proteins, S1003 and GFAP by immunostaining (Data not shown). Culture of ZiNPC under oligodendrocyte-inducing conditions resulted in the induction of the oligodendrocyte-specific transcripts OLIG2 and Myelin oligodendrocyte glycoprotein (MOG) (Figure 6B) and the 04-protein (Data not shown). Expression of the Zic3 transgene was decreased by 4 and 9.5 fold following differentiation to the astrocyte and oligodendrocyte lineage, respectively (Figure 6A and 6B). ZiNPC could also be committed to motor neuron-like cells, and this without initial SMAD inhibition. Resulting cells expressed the motor neuron-specific HB9, ISI1 , NF200 and MAP2 transcripts (Figure 6C), as well as proteins (Data not shown). Expression of the Zic3 transgene was decreased by 35 fold following differentiation to the motor neuron lineage (Figure 6C).

We finally assessed the in vivo differentiation potential of ZiNPCs by orthotopic injection of 150,000 GFP transduced ZiNPCs in the Striatum of 10-12 week old NODSCID yC KO mice. Analysis of the graft 4-5 weeks after transplantation revealed few cells expressing the mature neuronal markers, MAP2, 200 kDA NF, NeuN and also neural progenitor marker OTX1/2 , the oligodendrocyte marker 04 and the astrocyte marker GFAP (Data not shown). This indicates that in vivo ZiNPCs can mature further to neuronal, oligodendrocyte and astrocyte lineages under appropriate stimulation from the in vivo environment.

There are many strategies to reprogram adult cells to pluripotent cells or to transdifferentiate them to particular tissue types, which is in most instances achieved by transduction of either pluripotency TF or lineage specific TF. Aside from pluripotent iPSC generation from adult cells, reprogramming of fibroblasts to a different mature cell type has been described. For instance, forced combinatorial expression of the neural-lineage- specific transcription factors, AscH , Brn2 (also called Pou3f2) and Mytl l can rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro (Vierbuchen et al., 2010). Similar studies were performed to derive cardiomyocytes from mouse fibroblast by overexpression of the TF Gata4, Mef2c and Tbx5. Others have used TF modulation to fate ESC to a specific lineage. Extraembryonic endoderm differentiation can be induced in mESC by ectopic expression of Gata4 or Gata6 and in hESC by forced expression of SOX7, while forced expression of SOX17 in hESC specifies ESC to mesendodermal specific cell lines. By contrast, forced expression of Zfp521 is essential and sufficient for committing mESC to the neural lineage (Kamiya et al., 201 1 ). Since the original description of iPSC generation, many studies have assessed if additional TF known to play a role in pluripotency of ESC can aid in the generation of iPSC. Zic3 is another TF that partakes in the maintenance of ESC pluripotency (Lim et al., 2007). Like Oct4, Sox2 and Nanog, Zic3 is rapidly downregulated in differentiating mESC (Lim et al., 2007). Zic3 binds strongly to the Nanog promoter and functions as a transcriptional activator of Nanog (Lim et al., 2010). siRNA knock-down studies demonstrated that loss of Zic3 leads to reduced levels of Nanog, and to a lesser extent Sox2 and Oct4, with acquisition of endodermal lineage specific gene expression, such as Sox17, Foxa2, Gata4 and Gata6 (Lim et al., 2010). Forced expression of Zic3 in mESC induced sustained expression of Nanog following LIF withdrawal, but could not prevent that Oct4 and Sox2 transcripts decreased to a similar extent as in control mESC, consistent with the finding that the Zic3-expressing ESC colonies differentiated upon serial passage (Lim et al., 2010). Zic3 is important in controlling mesoderm development and is required for primitive ectoderm and neuroectoderm specification in vivo (Lim et al., 2007) as well as the maintenance of NPC fate by preventing neuronal differentiation (Inoue et al., 2007). We hypothesized that co-transduction of human fibroblasts with the OCT4, SOX2 and KLF4 together with Zic3 would either enhance reprogramming of fibroblasts to a pluripotent state, or might reprogram fibroblasts to an early neural fate.

We demonstrate that although transduction of human fibroblasts with OSKZ induced within 8-10 days colonies with morphological and phenotypical features of mESC cells, the established cell lines had many properties of NPC. Although the colonies were AP and SSEA1 positive, they did not express OCT4 or NANOG. Differentiating hESC are, however, SSEA1 but not SSEA4 positive as well as OCT4 and NANOG negative, and NSC express tissue nonspecific alkaline phosphatase. This suggested that the fibroblasts might be reprogrammed to a neuroectodermal progenitor fate. Consistent with this, ZiNPC expressed SOX2, PAX6, OTX1 , FOXG1 and OLIG2, and transcriptome analysis further demonstrated similarities between ZiNPC and NSC generated from hESC and hiPSC. The NPC fate was dependent on persistent transgenic expression of Zic3, as knock-down studies demonstrated that a >80% reduction in Zic3 mRNA levels was associated with a >60% decrease in N PC-specific transcripts. This was, however, not associated with the acquisition of a pluripotent state, consistent with the fact that endogenous OCT4 and NANOG were not induced following the combinatorial TF transduction. Suppression of Zic3 levels was also not associated with induction of endodermal or mesodermal transcripts. In vitro and in vivo studies demonstrated that ZiNPC by default differentiate to neuroectoderm only, and could be specified to mature neural lineages, without initial SMAD inhibition, as is needed for hESC or hiPSC.

In conclusion, we here demonstrate the very fast generation of neuroprogenitor cells from human fibroblasts by OSKZ transduction. The kinetics of generating ZiNPC are fast, as typical alkaline phospatase positive colonies were found as early as 8-10 days post transduction. ZiNPC can be propagated by trypsin digestion, can be easily grown clonally, and maintain a stable phenotype through many passages. As ZiNPC are committed to the neural lineage, they can be further specified to mature neural cell phenotypes without SMAD inhibition, required for hESC commitment to neural cells. These features of ZiNPC make them a very useful tool in pharmacological compound screening in neural disorders or for toxicity effects.

MATERIALS AND METHODS Cell culture

hESC (H9; purchased from WiCell, Madison, Wl, passages 36-65) and ZiNPC (ZiNPCI , ZiNPC2 and ZiNPC3: passages 12-38 and OSKM-induced hiPSC were cultured on mitomycin treated mouse embryonic fibroblasts (Globalstem, Rockville, MD). Cells were grown in DMEM/F12 (Invitrogen, Carlsbad, CA), 20% knockout serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), 6 ng/ml FGF-2 (Invitrogen), NEAA (Invitrogen), Sodium Pyruvate (Invitrogen) and Pen-strep (Invitrogen). Cells were passaged using 2mg/ml of collagenase IV (Invitrogen), washed and replated at a dilution of 1 :4 to 1 :10 every 3-4 days.

OSKM-induced hiPSC were generated by transduction of BJ1 fibroblasts with OCT4, SOX2, KLF4 and c-MYC retroviral vectors (Addgene, Cambridge, MA) and characterized by RT-qPCR for endogenous pluripotency gene expression and transgene expression, immunostaining for SSEA4, TRA160, OCT4 and NANOG, EB formation, teratoma formation, microarray analysis and targeted differentiation.

Plasmid construction, retroviral transduction

The coding region of the mouse Zic3 gene was amplified by RT-PCR with primers listed in Table 1 , and was cloned in pMIG-IRES-GFP plasmid (Addgene). The pMXs plasmids encoding for OCT4, SOX2 and KLF4 were purchased from Addgene. pMXs and pMIG- based retroviral vectors were transfected individually along with the viral packaging genes gag-pol (Addgene) and VSVG (Addgene) into 293T cells (ATCC) using Fugene HD reagent (Roche, Basel, Switzerland) according to the manufacturer's directions.

Generation of reprogrammed cells was performed as previously described (Okita et al., 2007) with some modifications. Briefly, human BJ1 cells (Lonza BioWhittaker, Basel, Switzerland) were seeded at 1.5 * 105 cells/well in 6-well plates (Sigma-Aldrich, St Louis, MO) without feeders. Cells were transduced with Zic3, KLF4, OCT4 and SOX2 retroviral vector containing supernatants mixed equally. To achieve maximum transduction efficiency, cells were transduced twice. Four days after transduction, the cells were reseeded at 1.5 ^χ 105 cells per 100-mm dish containing MEF and cultured in hESC medium. ZiNPC were picked 15-20 days post transduction, and expanded further.

Short Hairpin RNA (ShRNA) experiments

Two independent Zic3 shRNA sequences (V3THS_304236, V3THS_304235) obtained from Open Biosystems (Huntsville, AL) were cloned in pTRIPZ doxycycline inducible lentiviral vector (Open Biosystems) by digesting with EcoRI and Xhol (Roche). A universal non-silencing construct (Open Biosystems) was used as control. Constructs were sequenced and transfected into 293T cells using the viral packaging plasmids pMD2.G and psPAX2 (Addgene). Supernatants were collected after 48h and 72 h, and used to transduce ZiNPC. To select for the transduced cells, puromycin (1 mg/ml) (Sigma) selection was performed 1 d after transduction. Expression of Zic3 specific shRNA was induced by adding doxycycline (1 mg/ml) (Sigma) to the media.

Immunostaining

Paraffin sections (5 pm thick) were rehydratated using standard procedures or cells were fixed using 10% Neutral Buffered Formalin (NBF) for 15 minutes at room temperature, rinsed twice in PBS (Invitrogen). Permeabilization was performed for 15 minutes using PBS containing 0.2% Triton X-100 (PBST) (Acros Organics, New Jersey). Nonspecific blocking was carried out with 10% serum corresponding to the animal source of secondary antibody (Dako, Glostrup, Denmark) for 30 minutes. Primary antibodies at the dilutions described in supplementary Table 2 were diluted with Dako antibody diluent and the cells were incubated overnight at 4°C followed by incubation with secondary antibodies conjugated with Alexa dyes for 1 hr at room temperature (Invitrogen). For nuclear staining, 1 ug/ml Hoechst 33258 (Sigma-Aldrich) was added along with secondary antibody incubation. Differentiation studies:

Astrocyte differentiation: cells were cultured on gelatin-coated plates in neural stem cell medium (DMEM F12, N2 Supplement, MEM NEAA, Heparin 2 mg/ ml; Invitrogen) with 1 % FCS (Invitrogen) for 14 days (Hong et al., 2008).

Oligodendrocyte differentiation: ZiNPC were cultured in neural differentiation medium in the presence of 20ng/ml platelet-derived growth factor (R&D, Minneapolis, MN), 20 ng/ml bFGF (Invitrogen) and 10ng/ml EGF (Sigma) for 7 days, after which cells were maintained for an additional 14 days in medium alone (Hong et al., 2008).

Motor neuron differentiation: ZiNPC were differentiated towards the motor neuron lineage using previously described protocols (Chambers et al., 2009; Hu and Zhang, 2009) but without the initial SMAD inhibition step. ZiNPC were seeded on Poly-L-ornithine (Sigma) and Laminin (Sigma) coated plates with neural differentiation medium containing SHH (200ng/ml) (R&D) and RA (0.1 uM) (Sigma). On day 4, the medium was changed to medium containing BDNF (10ng/ml), GDNF (10ng/ml), IGF1 (10ng/ml) (R & D Systems), cAMP (1 uM) and Ascorbic acid (200ng/ml) (Sigma), for another 10 days.

Genomic DNA PCR

Genomic DNA was isolated as per manufacturers's instructions (Qiagen DNeasy kit). Using transgene specific primers (Table 1 ), the target genes were amplified and loaded on a 1 % Agarose (Sigma-Aldrich) gel and visualized to detect the presence of the integrated transgenes.

Real time Quantitative PCR

RNA was obtained from cells using the RNeasy microkit (Qiagen, Hilden, Germany). 1 mg of DNase treated RNA was reverse transcribed using a superscript III first strand cDNA synthesis kit (Invitrogen). cDNA was further diluted to 100 μΙ and 2 μΙ of cDNA was used for quantitative PCR using the Sybergreen PCR kit (Invitrogen). All primers are listed in Table 1. Embryoid body formation:

hESC and ZiNPC were plated on low adherent plates (Elscolab, Kruibeke, Belgium) in embryoid body (EB) medium (IMDM medium, 15% FBS, 2 mM L-glut, 1 % NEAA, 1 mM Sodium pyruvate, 100 U penicillin/streptomycin, 200 pg/ml Iron-saturated-transferrin, 10 μΜ β-mercaptoethanol, 50 pg/ml ascorbic acid (Sigma) for 8 days. Subsequently, the cells were cultured for another 8 days on plates coated with 0.1 % gelatin (Chemicon, Freiburg, Germany) in EB medium. In vivo tumor formation assay

106 ZiNPC were resuspended in 50% of hESC-qualified matrigel (VWR, Radnor, PA) and injected subcutaneously in 6-8 week old Rag2 c-l- mice. After 4 weeks, mice were sacrificed, the tumors were removed, fixed with formalin and embedded in paraffin. Paraffin blocks were sectioned, and sections stained with H&E or with antibodies against beta-tubulin-3 and synaptophysin. All experiments with mice were approved by the institutional review board of the KULeuven.

Generation of Neurospheres from hESC

hESC cultures were disaggregated using accutase (Sigma-Aldrich) for 10 min and plated on Matrigel-coated dishes in mTESR medium (Stem Cell Technologies, Vancouver, Canada). hESC were allowed to expand for 3 d or until they were nearly confluent. The differentiation towards neural progenitors was performed in hESC media lacking bFGF (R&D) with 10 μΜ TGF-β inhibitor (Tocris) and 500 ng/ml of Noggin (R&D). On day 5, the TGF-β inhibitor was withdrawn while maintaining 500 ng/ml of Noggin and increasing amounts of N2 media (25%, 50%, 75%) was added for an aditional 6 days with changing media every 2 days.

Microarray method

RNA was extracted from hESC, ZiNPC, and hESC-derived neurospheres. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND- 1000 (Nanodrop Technologies, Wilmington, DE) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent, Santa Clara, CA). 100 ng total RNA spiked with bacterial poly- A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA). The sample was converted and amplified to antisense cRNA in an in vitro transcription reaction, which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit. A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip Human Gene 1 .1 ST Arrays followed by staining and washing in the GeneTitan® Instrument (Affymetrix) according to the manufacturer's procedures. To assess the raw probe signal intensities, chips were scanned using the GeneTitan® HT Array Plate Scanner (Affymetrix). Analysis of the microarray data was performed in the R programming environment, in conjunction with the packages developed within the Bioconductor project ((Gentleman et al., 2004)). The analysis was based on the Robust Multichip Average (RMA) expression levels of the probe sets that had at least once a present detection above background (DABG) detection call. Differential expression was assessed via the moderated t-statistic (Smyth, 2004). To control the false discovery rate, multiple testing correction was performed and probes with a corrected p-value below 0.05 were selected.

The microarray data are deposited in the NCBI's Gene Expression Omnibus. Statistics

Where appropriate, results were expressed as means ± SEM. Statistical analysis was performed by unpaired Student's t test, where P < 0.05 was considered significant.

In vivo differentiation assay

Stereotactic surgery

10-12 week old Balb/cA RAG2 KO mice were anesthetized with 75mg/kg ketamine (Ketamine 1000, CEVA, Sante Animale) and 1 mg/kg medetomidin (Domitor, Orion Pharma) intraperitoneally and positioned in a stereotactic head frame (Stoelting) for stereotactic injection in the caudate putamen (striatum) using bregma as a reference point. Stereotactic coordinates starting from the dura were the following: anteroposterior 0.5mm; mediolateral= -1.7mm; dorsoventral= 2.5-1.5mm. Using a 26S Hamilton syringe (VWR international) 3μΙ of ZiNPCs, transduced with GFP, at 33.10³cells/pl suspended in PBS were injected at a rate of 0.5pl/min. After injection the needle was left in place for an additional 4 min and anesthesia was reversed with 0.5mg/kg atipamezole (antisedan, Orion Pharma) i.p.

Tissue processing and immunohistochemistry

Animals were sacrificed 4 weeks following stereotactic surgery with an i.p. overdose of pentobarbital (Nembutal, CEVA, Sante Animale) and transcardially perfused with 4% (w/v) PFA in PBS. Brains were removed and postfixed overnight in 4% PFA at 4°C. Serial 50pm coronal sections were made using a vibratome. Free floating sections were washed three times for 5 min in PBS/Triton 0.1 % X-100 (v/v) and immunostaining for GFP together with primary antibodies against Map2, GFAP, NeuN, OTX1/2, Sox2, 200 kDA NF, and 04 was performed.

A number of brains were paraffin embedded and 5pm coronal sections were made. Following deparaffinisation and rehydratation, antigen retrieval was performed by boiling the sections in Dako target retrieval solution (S1699) for 20 min. using a pressure cooker. After cooling down for 20 min to RT the sections were rinsed in AD for 5 min and washed twice in PBS/Triton 0.1 % for 5 min. Further staining was performed using the same protocol as described for processing vibratome sections followed by dehydratation and mounting in DPX before microscopy analysis.

Table 1 : Primer sequences

AFP Forward 5' AAATGCGTTTCTCGTTGCTT 3' 27 Reverse: 5' ACAAACTATTGGCCTGTGGC 3' 28

Flkl Forward 5' ACAACCAGACGGACAGTGGT 3' 29

Reverse: 5' AGCCTTCAGATGCCACAGAC 3' 30

FoxGl Forward 5' CCCTCCCATTTCTGTACGTTT 3' 31

Reverse: 5' CTGGCGGCTCTTAGAGAT 3' 32

Pax6 Forward 5' GGGCAATCGGTGGTAGTAAA 3' 33

Reverse: 5' CTAGCCAGGTTGCGAAGAAC 3' 34

Nestin Forward 5' AGACTTCCCTCAGCTTTCAG 3' 35

Reverse: 5' CTTGGTTCTTAAGAAAGGCTGG 3' 36

Mixll Forward 5' GGATCCAGGTATGGTTCCAG 3' 37

Reverse: 5' CATGAGTCCAGCTTTGAACC 3' 38

Otx2 Forward 5' AGAGGAGGTGGCACTGAAAA 3' 39

Reverse: 5' ATTGGCCACTTGTTCCACTC 3' 40

Otxl Forward 5' CCAAGACTCGCTACCCTGAC 3' 41

Reverse: 5' GAGCTAGAGGACGAGGC AGA 3' 42

GLAST Forward 5' CTCACAGTCACCGCTGTCAT 3' 43

Reverse: 5' CCATCTTCCCTGATGCCTTA 3' 44

Soxl Forward 5' GCAAGATGGCCCAGGAGAA 3' 45

Reverse: 5' CCTCGGACATGACCTTCCA 3' 46 p75 Forward 5' GC CT AC GGCT ACT ACC AGG A 3' 47

Reverse: 5' CACACGGTGTTCTGCTTGTC 3' 48

01ig2 Forward 5' CAGAAGCGCTGATGGTCATA 3 ' 49

Reverse: 5' TCGGCAGTTTTGGGTTATTC 3' 50

Map2 Forward 5' ACTGCAGCTCTGCCTTTAGC 3' 51

Reverse: 5' ATCGTGGAACTCCATCTTCG 3' 52

200kDA Forward 5' GAGGAGTGGTTCCGAGTGAG 3' 53 NF Reverse 5' GAGGAGTGGTTCCGAGTGAG 3' 54

MOG Forward 5' GATCTTCCCTTGGGCTTTTC 3' 55

Reverse 5' TATTCAGGTGCCTGGTCTCC 3' 56 Table 2: Antibodies

REFERENCES

Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and

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Hong, S., Kang, U.J., Isacson, O., and Kim, K.S. (2008). Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.

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Hu, B.Y., and Zhang, S.C. (2009). Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc 4, 1295-1304.

Inoue, T., Ota, M., Ogawa, M., Mikoshiba, K., and Aruga, J. (2007). Zic1 and Zic3 regulate medial forebrain development through expansion of neuronal progenitors. J Neurosci

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Kamiya, D., Banno, S., Sasai, N., Ohgushi, M., Inomata, H., Watanabe, K., Kawada, M., Yakura, R., Kiyonari, H., Nakao, K., et al. (201 1 ). Intrinsic transition of embryonic stem- cell differentiation into neural progenitors. Nature 470, 503-509.

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Claims

(a) providing a population of cells;

(c) selecting for cells having a neural progenitor phenotype.

2. The method of claim 1 , wherein the cells endogenously express one or more of the pluripotency factors selected from Sox2, c-Myc, Oct3/4, Klf4, Lin28, Nanog, ESRRB, SOX1 , PAX6, ZIC1 and ZIC2 or a substrate, cofactor or downstream effector thereof.

3. The method of claim 1 or 2, wherein the 3 pluripotency factors of step (b) are Oct4, Klf4 and Sox2.

4. The method of any of claims 1 to 3, wherein the population of cells comprises adult cells.

5. The method of any of claims 1 to 3, wherein the population of cells comprises adult stem cells.

6. The method of any of claims 1 to 3, wherein the population of cells comprises adult bone marrow cells.

7. The method of any of claims 1 to 3, wherein the population of cells comprises non-adult cells.

8. The method of any of claims 1 to 3, wherein the population of cells comprises fibroblasts.

9. The method of any of claims 1 to 8, wherein the population of cells are of human origin.

10. The method of any of claims 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of transiently transfecting at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

1 1. The method of any of claims 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of viral transduction of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

12. The method of any of claims 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of microinjection of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

13. The method of any of claims 1 to 9, wherein the step of treating the population of cells to induce neural differentiation comprises a step of contacting the population of cells with a small molecule, protein, peptide, sugar, or other compound, agent or treatment that effects modulation of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

14. The method of any of claims 1 to 13, wherein the step of treating the population of cells to induce neural differentiation comprises a step of increasing the expression of at least one of said factors of step (b), or a substrate, cofactor or downstream effector thereof.

15. The method of any of claims 1 to 14, wherein the step of selecting for cells having a neural progenitor phenotype comprises monitoring at least one cellular marker selected from PAX6, OTX1 , FOXG1 , GLAST, P75, SOX1 , and OLIG2.

16. The method of any of claims 1 to 15, wherein the method further comprises amplifying the neural progenitor cells selected in step (c).

17. The method of any of claims 1 to 16, further comprising inducing differentiation of the neural progenitor cells into astrocytes.

18. The method of any of claims 1 to 16, further comprising inducing differentiation of the neural progenitor cells into oligodendrocytes.

19. The method of any of claims 1 to 16, further comprising inducing differentiation of the neural progenitor cells into motor neurons.

20. The method of any of claims 1 to 16, further comprising inducing differentiation of the neural progenitor cells into dopaminergic neurons.

21. A population of cells produced by the method of any of claims 1 to 20.

22. A pharmaceutical composition comprising a population of cells according to claim

23. Use of a pharmaceutical composition according to claim 22 in human or animal medicine.

24. Use of a pharmaceutical composition according to claim 22 for the treatment of disorders of the brain.

25. Use according to claim 24, wherein the disorders of the brain are neurodegenerative disorders such as Alzheimer's Disease or Parkinson's Disease.