LU92771B1 - Long-term self-renewing neural stem cells - Google Patents

Abstract

The present invention relates to methods for obtaining a neural stem cell (NSC). These NSCs can be further differentiated into neurons and glial cells. The invention further provides for NSCs, neurons and astrocytes obtainable by the methods of the present invention as well as preparations and pharmaceutical compositions comprising these cells. In addition the present invention relates to NSCs, neurons and astrocytes of the present invention for use in therapy.

Description

New patent application in Luxembourg Applicant: Université du Luxembourg our ref.: LUX15282LU Date: 10 July 2015

LONG-TERM SELF-RENEWING NEURAL STEM CELLS FIELD OF THE INVENTION

The present invention relates to methods for obtaining a neural stem cell (NSC). These NSCs can be further differentiated into neurons and giial cells. The invention further provides for NSCs, neurons and astrocytes obtainable by the methods of the present invention as well as preparations and pharmaceutical compositions comprising these cells. In addition, the present invention relates to NSCs, neurons and astrocytes of the present invention for use in therapy.

DESCRIPTION

[001] Recent advances in the field of somatic cell reprogramming have enormously furthered the use and optimization of pluripotent stem cells (iRSCs) since the seminal studies by Yamanaka and coworkers (Takahashi and Yamanaka (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-76; Takahashi et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell; 131(5):861-72). Several human iPSC lines derived from patients suffering from different diseases have been generated,: including Parkinson's disease, Alzheimer's disease and schizophrenia. Moreover, gene-editing approaches have been used to correct genetic mutations on PD patient derived-iPSC, resulting in the successful reversal of pathological phenotypes (Reinhardt et al. (2013) Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 8;e59252; Major et al. (2011) Transgene excision has no impact on in vivo integration of human iPS derived neural precursors. PLoS One;6(9):e24687).

[002] As iPSCs are derivable in a patient-specific manner, they are suitable for autologous engraftments (Morizane et al. (2013) Direct Comparison of Autologous and Allogeneic Transplantation of ÎPSC731 Derived Neural Cells in the Brain of a Nonhuman Primate. Stem Cell Reports 1:283-92) and for personalized disease modeling (Payne et al. (2015) Application of human induced pluripotent stem cells for modeling and treating neurodegenerative diseases. N Biotechnol. 32(1):212-28). iPSCs might also offer a powerful tool in preclinical research to test both toxicity and efficacy of new drug candidates. Basing preclinical tests directly on the target human cells rather than on surrogate cell models, often from other rodents, hold the promise of leading to more efficient drug screening (inoue et al. (2014) IPS cells: a game changer for future medicine. EMBO J 33:409-17). Notably, in the field of neurodegenerative diseases, hundreds of compounds have been successfully used in animal models to ameliorate induced-neuropathology or cognitive deficits. Yet, this has not translated into effective disease-modifying therapies for humans. Obviously, differences between rodent and human physiology impede translation of rodent results to humans (Geerts (2009) Of mice and men: bridging the translational disconnect in CNS drug discovery. CNS Drugs; 23(11):915-26). Thus, the possibility of testing drug candidates on patient-specific cells is of fundamental importance for diseases such as neurodegenerative disorders, which only affect humans.

[003] A second application of iPSC is replacement therapies (Barberi et al. (2003) Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 21:1200-7; Soundararajan et al. (2007) Easy and rapid differentiation of embryonic stem cells into functional motoneurons using sonic hedgehog-producing cells. Stem Cells 25:1697-706). The use of iPSCs overcomes the legal/ethical concerns as well as the risks of immune rejection of human embryonic stem cells (hESCs). Despite these advantages, iPSCs, as well as hESCs, have the inherent potential for teratoma formation (Liu et al. (2013) The tumourigenicity of iPS cells and their differentiated dérivâtes. J Cell Mol Med 17:782-91) most likely due to aberrant reprogramming. Despite the enormous progressions in the field of stem cell differentiation, the directed and homogenous differentiation of hiPSCs into a specific cell type remains technically challenging. The limited ability to generate pure neural cell populations still renders the clinical use of pluripotent-derived cells an obstacle.

[004] Thus, utilization of somatic fate restricted stem cells might represent an attractive alternative to achieve faster and more homogenous differentiation into the desired terminally differentiated cell types.

[005] The technical problem can thus be seen in the provision of an alternative method to obtain NSCs andOr an improved method to obtain neurons and/or astrocytes from iPSCs.

[006] The technical problem is solved by the embodiments reflected in the claims, described in the description, and illustrated in the Examples and Figures.

[007] The above being said, the present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem cells (iPSCs); b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and e) maintaining the cells obtained in d) in a medium comprising (i) a FGF signaling activator; (ii) an EGF signaling activator; and (iii) a LIF signaling activator, and thereby obtaining a NSC.

[008] The present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem cells (iPSCs); b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and d.2) further cultivating the cells obtained in d) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and (iv) an FGF signaling activator, thereby obtaining a NSC.

[009] In addition, the present invention relates to a NSC obtainable by a method of the present invention.

[010] Furthermore, the present invention relates to a neuron obtainable by a method as of the present invention.

[011] The present invention also relates to an astrocyte obtainable by a method of the present invention.

[012] The present invention further relates to a NSC of the present invention, neuron of the present invention or astrocyte of the present invention for use in therapy.

[013] The present invention also relates to a pharmaceutical composition comprising a NSC of the present invention, neuron of the present invention or astrocyte of the present invention.

[014] In addition, the present invention relates to a pharmaceutical composition of the present invention for use in therapy.

[015] The present invention further relates to a preparation obtainable by methods of the present invention.

[016] The present invention also relates to a preparation comprising a NSC of the present invention, neuron of the present invention or astrocyte of the present invention.

[017] Also, the present invention relates to an in vitro method or test system, wherein the method or test system comprises (i) NSCs of the present invention, (ii) neurons of the present invention or (iii) astrocytes of the present invention.

[018] The present invention also relates to a method of treating a disease, optionally a neurodegenerative disease, in a subject, comprising administering a therapeutically effective amount of a NSC of the present invention, neuron of the present invention or astrocyte of the present invention to said subject.

[019] The present invention also relates to a use of a NSC of the present invention, neuron of the present invention or astrocyte of the present invention for the preparation of a medicament.

[020] In addition, the present invention relates to a use of a NSC of the present invention, neuron of the present invention or astrocyte of the present invention in a method of treating a disease optionally a neurodegenerative disease.

[021] The figures show: [022] Figure 1. Generation of human neural stem cells (a) Schematic representation for directed differentiation of iPSC cells to hNSC. (b-g) Phase contrast of images of the generation of hNSC. (b) Feeder-free hiPSC. (c) Embryonic bodies after 3 days of differentiation, (d) Tube like structures after six days of differentiation, (e) Induction of neural rosette formation at day 10. (f) First passage of hNSCs at day 14 of hiPSC differentiation; (g) heterogeneous cell population becomes homogeneous after several passages. hNSC. (h-s) Immunofluorescence labeling of the neural stem cell markers Nestin, Sox2, Sox1 and Pax6 as well as of the cell cycle marker Ki67 of hNSCs at Passage 3 (h, k, n and q), Passage 6 (i, I, o and r) and Passage 27 (j, m, p ands). Scale bar 10 pm.

[023] Figure 2. Molecular Characterization of hNSCs (a) Multi-Dimensional Scale analysis based on the mRNA transcription profiles of hiPSCs (light grey), hNSCs (white) and hMLDCs (dark grey). All profiled cell-types were generated from the same individual, (b) mRNA scatter blot of hiPSCs and hNSC; hNSC specific genes, hiPSC specific genes, 561 genes observed in both cell types (black), (c) mRNA expression levels of OCT4, Nanog, Ki67, SOX2 and Nestin in hiPSC and hNSC quantified by RT-qPCR. (d) Gene Ontology analysis based on the 1428 genes specifically expressed in hNSCs when compared to hiPSCs and hMLDCs. (e) GO terms were subjected to a network analysis based on ReviGO (http://revigo.irb.hr/) and CytoScape (http://www.cytoscape.org/) softwares. The main GO sub-clusters are highlighted.

[024] Figure 3. Rapid generation of hNSC derived astrocytes

Representative confocal images illustrating the expression of astrocytic markers (a) S1008 after 50 days of differentiation, (b-d) GFAP-vimentin, (e) Tuj1-vimentin, (f-h) S100ß-Ki67. (i) mRNA expression levels of Nestin, KÎ67, GFAP in hNSC and astrocyte quantified by RT-qPCR.

[025] Figure 4. Astrocytes displayed mature functions

Representative confocal images illustrating the expression of astrocytic markers (a) AQP4 and (b) EAAT2 after 60 days of differentiation. Scale bars 50pm. (c) Glutamate up-take in astrocytes DIV 80-90 and HEK 293. Mean ± SEM; n=3 indipendent experiments.

[026] Figure 5. Stable isotope assisted metabolic profiling of hNSCs derived astrocytes (a) Atom transition model of uniformly labeled [U-13C]giucose. Glucose derived [U-13C]pyruvate can enter the TCA cycle via pyruvate dehydrogenase (PDH) dependent oxidation of pyruvate to acetyl-coA. In this case citrate molecules show a mass increase by two (M2 isotopologue). Subsequent TCA cycle metabolites are also M2 isotopologues. As an alternative way to enter the TCA cycle pyruvate can be carboxyiated by pyruvate carboxylase, resulting in oxaloacetate M3 isotopologues. The dotted line indicates the start and the end of the cycle. ALAT: alanine aminotransferase; LDH: lactate dehydrogenase; PC: pyruvate carboxylase; CS: citrate synthase; ACO: Aconitase; IDH: isocitrate dehydrogenase; GDH: glutamate dehydrogenase; AT: aminotransferase; SDH: succinate dehydrogenase; FH: fumarate hydratase; MDH: malate dehydrogenase, (b-f) Mass isotopomer distributions (MIDs) and carbon contributions. Ceils were labeled for 24h prior extraction of intracellular metabolites and analysis by GC/MS. M1-M6 indicates the number of 13C atoms incorporated into the metabolite, (b) MID of aspartate using [U-13C]glucose as a tracer, (c) MID of citrate using [U-13C]glucose as a tracer, (d) Calculated carbon contribution from gluocose, glutamine and other carbon sources (e.g. lipids, branched chain amino acids) to citrate (left) and glutamate (right), (e) MID of glutamate using [U-13C]glucose as a tracer, (f) MID of citrate using [U13C]glutamine as a tracer (f). Mean ±SEM, n = 3 independent experiments in triplicate; p-value < 0.05.

[027] Figure 6. Stable isotope assisted metabolic profiling reveals differences in serine biosynthesis in hNSCs derived astrocytes and hNSCs derived MLDC. (a) Cartoon illustrating possible atom transitions for serine in respect to the reversibility of the enzyme serine hydroxy methyl transferase (SHMT) and potential metabolic fates for serine. The hydroxymethylgroup of serine is transferred on tetrahydrofolate resulting in M2 glycine isotopologues. In the reversible reaction serine can be MO, M1, M2 or again M3 depending on the combination of labeled and unlabeled metabolite, (b) MID of serine using [U-13C]glucose as a tracer, (c) Carbon contribution of glucose to serine. Glutamine has no contribution to the serine pool (data not shown) (d) MID of glycine using [U-13C]glucose as a tracer, (e) MID of lactate using [U-13C]glucose as a tracer. Mean±SEM, n = 3 independent experiments in triplicate; p-value < 0.05.

[028] Figure 7. Transplantation of hNSC derived neurons and astrocytes in NOD/SCID mice a) Schematic representation of the protocol followed. hNSC were kept in maintenance medium for 3 days after splitting. Then the maintenance medium was switched to glial differentiation medium. Stereotactic injection into the subventricular zone of NOD/SCID mice was performed 7 days later. Representative images showing that clusters of GFAP positive cells formed by pre-differentiated hNSC (b-e).

[029] Figure 8. Multilinear differentiation of hNSCs

Multilinear differentiation is achieved by culturing hNSCs in the basic medium (ground medium), supplemented with 10% of FCS. NSCs differentiated into TuJ1 positive neurons (a) and GFAP positive astrocytes (b).

[030] Figure 9. Gene expression profiling of hNSCs (a) Growth curve of hNSCs maintained under proliferative conditions over 21 Passages, (b) Paired analysis of the microarray based expression intensities of hiPSC and hNSCs associated genes, (c-k) hNSC associated GO-terms organized by GO-sub-clusters.

[031] Figure 10. Derivation of functional neurons. (a-f) Representative confocal images illustrating the expression of neuronal markers Tuj1 (a), DCX (b), MAP2(c), GABA-Tuj1 (d), vGlut1-Tuj1 (e) and TH-Tuj1 (f). (g) Quantification of the number of neurons positive for MAP2, Tuj1, vGLUH, GABA, TH, GFAP. (h) mRNA expression levels of Nestin, Ki67, MAP2, TH, GABA and GLUT1 in hNSC and neurons quantified by RT-qPCR.

[032] Figure 11. hNSC derived neurons develop synapses and electrophysiological activity. (a-h) Representative confocal images illustrating the expression of neuronal markers synaptophysin (j), NCAM (k), PSD95 (n), Tuj1 (o). (i) Current-voltage protocol (-60 to +20 mV, 50 ms) shows fast inward current and slower outward currents, (j) The fast inward current is identified as Na+ 627 -current by its sensitivity to TTX (0.5 μΜ; red trace), (k) In current clamp recordings cells fired spontaneous action potentials (APs), when the membrane potential was set to -50 mV. (I) Current steps (5-15 pA, 0.3 s) activated overshooting APs, stronger injections elicited series of APs with moderately declining amplitudes, (m) Puff-application of glutamate (1 mM) from a gias pipette (15 pm tipp diameter) placed 50 - 80 pm away from the recorded ceil elicited an inward current when the cell was voltage clamped to -70 mV. (n) During current clamp, the cells depolarized in response to the glutamate puff.

[033] Figure 12, Transplantation of hNSC derived neurons in NOD/SCID mice, (a-h) Representative images of the subventricular zone showing that the predifferentiated hNSC differentiate into Tuj1 (c) and Doublecortin (OCX) (g) positive cells.

[034] A novel protocol for the generation of hNSCs from hiPSCs is described by the present invention (e.g. in Examples 2 and 3). This fate transition is achieved by the chronological administration of media with defined compositions. The here presented protocoi is very robust and independent of any sorting method. In contrast to other protocols for the generation of human neural precursor cells, no small molecules were needed for keeping hNSCs under self-renewing condition (Kim et al. 2012; Koch et al. 2009; U et al. 2011; Reinhardt et al. 2013). Differently to previous studies where neural progenitors grow and proliferate as neurospheres (Ebert et al. 2013) or neural rosettes (Elkabetz et al. 2008), hNSCs were homogeneously maintained in a two-dimensional adherent cell system. Spontaneous differentiation of hNSC into other cell types was negligible. hUF in the media blocked fate transition of hNSC into neuronal cells. Although hLIF has been reported to induce astrocytes differentiation in synergy with bone morphogenetic protein (BMP) 2 from mouse neuroepithelial cells (Nakashima K, Yanagisawa M, Arakawa H, Taga T. 1999. Astrocyte differentiation mediated by LIF in cooperation with BMP2. FEBS Letters 457:43-46), significant amounts of GFAP-positive ceils in the hNSC under maintenance condition were not detected.

[035] Cells (NSCs) obtained by the methods of the present invention conserved the two main characteristics of neural stem cells, i.e. self-renewing and multi-linear differentiation capacities. Since they grow in homogenous cultures, these cells are an attractive tool for expression profiling, disease modeling and high content screenings. Besides their ability to differentiate into functional neurons, hNSCs differentiated into glial ceils within a relatively short time period. A gene expression profile that distinguishes hNSC from less differentiated hiPSC and more differentiated neurons and astrocytes is shown in Example 3. Since these profiles were generated from cells from the same individual (starting iPSC line), the degree of comparability is very high and the derived signatures purely represent the differentiation status.

[036] Besides the generation of NSCs the present invention also provides for the preparation of neurons and glial cells from iPSCs via NSCs. Especially, the generation of astrocytes is of particular relevance. Several papers described the generation of human astrocytes from fetal or adult post-mortem central nervous system by the expansion of neuronal precursors (Haidet-Phillips et al. (2011) Astrocytes from familial and sporadic ALS patients are toxic to motor neurons Nature Biotechnology 29:824-828; Verwer et al. (2007) Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin. Brain: a journal of neurology 130:3321-3335). This approach required 6 months to generate a pure population of astrocytes (Krencik (2011) Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nature Biotechnology 29:528-534). Other recent papers described the differentiation of astrocytes from iPSC with protocols requiring from 35 days (Emdad et al. (2012) Efficient differentiation of human 682 embryonic and induced pluripotent stem cells into functional astrocytes. Stem Cells Dev 683 21:404-10) up to 4 months (Juopperi (2012) Astrocytes 702 generated from patient induced pluripotent stem cells recapitulate features of Huntington's disease patient cells. Mol Brain 5:17). Importantly, the obtained populations seem to represent astrocytes just in a reactive form as shown by the almost 100% immunoreactivity for GFAP. Therefore, these cultures might not be suitable to completely model mature astrocyte functions or to mirror patho- and physiological conditions (Roybon et al. (2013) Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Cell Rep 4:1035-48).

[037] The present invention now provides for an imporved method to generate astrocytes. The derivation of astrocytes by the methods of the present invention was achieved by a cost-efficient media composition, which ensures a highly pure culture as shown by the negligible contamination with Tuj1 positive cells. Unlike other protocols (Yuan et al. (2011) Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6:e17540), the described protocol is simple and does not require any antibody-based sorting step of glia or neuronal progenitors. Remarkably, by the methods of the present invention a population of mature astrocytes both in a quiescent state with a protoplasmic morphology (negative for GFAP) as well as in a reactive phenotype characterized by GFAP expression can be obtained. The expression of EAAT2 in ail the cells and the ability to uptake glutamate strongly supported the acquisition of mature functions. The importance of this feature was highlighted by the different effects of immature and mature astrocytes on axonal regeneration (Goldshmit et al. (2012) Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J Neurosci 32:7477-92; Tom et al. (2004) Studies on the development and behavior of the dystrophic growth cone, the hallmark of regeneration failure, in an in vitro model of the glial scar and after spinal cord injury. J Neurosci 24:6531-9).

[038] The high pyruvate carboxylase activity observed e.g. in the Examples described herein further confirmed the acquisition of metabolic specialization of hNSC-derived astrocytes as pyruvate carboxylation is an important anaplerotic reaction, specifically occurring in astrocytes (Shank et al. (1985) Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res 329:364-7; Amaral et al. (2011) A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose. Front Neuroenergetics. 3:5.). Moreover, the relative glucose flux into the TCA cycle was higher in astrocytes resulting in higher glucose derived carbon contribution to glutamate (Pardo (2011) Brain glutamine synthesis requires neuronal-bom aspartate as amino donor for glial glutamate formation. J Cereb Blood Flow Metab; 31(1):90-101).

[039] Notably, as shown in the Examples described herein in astrocytes as well as MLDCs (muitilineage differentiated cells) around 50% of the citrate carbons derived from other sources than glucose or glutamine. These other sources might be represented by lipid oxidation and/or degradation of amino acids such as branched chain amino acids.

[040] The present invention provides for the generation of astrocytes from iPSCs via NSCs. This approach provides for some advantages. For example, it has been shown in mice that primary neural stem cells (NSCs) bear the advantage of being expandable while maintaining their neurogenic and gliogenic differentiation potential (Conti and Cattaneo (2010) Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176-87; Conti (2005) Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS biology 3:e283). Therefore, the use of hiPSC derived hNSCs as source for glia cells and neurons represents a promising strategy (Wernig et al. (2008) Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci U S 779 A 105:5856-61). Stable cultures of hNSC avoid time and cost consuming maintenance of pluripotent stem cells as well as potential immune rejection and teratoma formation {Liu et al. (2013) The tumourigenicity of iPS cells and their differentiated dérivâtes. J Cell Mol Med 17:782-91). In addition, to fully mirror complex neurodegenerative mechanisms, multiple cell types might need to be generated from patient-specific iPSC. Particularly interesting is the possibility of generating human astrocytic cultures starting from the same precursor cells used to generate neurons.

[041] Traditionally, astrocytes were solely described as supporting elements for neurons. However, since decades it is known that astrocytes are essential players involved in brain information processing (Araque et al. (1999) Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci 22:208-15; Brockhaus and Deitmer (2002) Long-lasting modulation of synaptic input to Purkinje neurons by Bergmann glia stimulation in rat brain slices. J Physiol 545:581-93; Haydon and Carmignoto (2006) Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 86:1009-31; Ota et al. (2013) The role of astrocytes in the regulation of synaptic plasticity and memory formation. Neural Plast 2013:185463). Alteration of their functions has emerged as a clear factor in disease pathogenesis, especially for neurodegenerative diseases.

[042] Additionally, growing evidences support the ciritical role of astroglia in disease processes. As an example it has been shown that glia cells have a direct, non-cell autonomous effect on motor neuron survival in a mouse model of amyptrophic lateral sclerosis (ALS) (Di Giorgio et al. (2007) Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci;10(5):608-14).

[043] Despite these growing evidences for the importance of astrocytes, they are less investigated compared to neurons. The lack of an acknowledged and well-characterized set of human astroglial markers and the difficulty to obtain cultures of human astrocytes under defined conditions is partly accounting for that. Moreover, human astroglial culture purity is generally not high due to contamination of other cell types as microglia, which might affect astrocyte functional evaluation. The present invention now provides for the robust and rapid generation of hNSCs from hiPSCs. Furthermore, their maintenance and directed differentiation is also provided by the present invention.

[044] Importantly, the provision of astrocytes and especially the generation of autologous astrocytes from iPSCs are of important clinical relevance. Recently, the outstanding importance of astrocytes for neurological disease got into focus of several research approaches. As an example, it has been shown that astrocytes strongly contribute to the development of the Down syndrome (Chen et al. (2014) Role of astroglia in Down's syndrome revealed by patient-derived human663 induced pluripotent stem cells. Nat Commun 5:4430). Additionally, a recent study clearly demonstrated that transplantation of astrocytes was extremely beneficial in a rat model of Parkinson’s disease (Proschel et al. (2014). Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons. EMBO Mol Med 6:504-18).

[045] Thus, the availability of an effective method to generate mature astrocytes, as described herein, is of key importance for convincing disease-modelling studies and replacement therapy strategies. This is important, especially, for neurodegenerative diseases such as Parkinson's and Alzheimer's disease.

[046] The present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem cells (iPSCs), preferably from a fibroblast; b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and e) maintaining the cells obtained in d) in a medium comprising (i) a FGF signaling activator; (ii) an EGF signaling activator; and (tit) a LI F signaling activator, and thereby obtaining a NSC, [047] Additionally or alternatively the present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem cells (iPSCs); b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and d.2) further cultivating the cells obtained in d) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (Hi) an antioxidant; and (iv) an FGF signaling activator, thereby obtaining a NSC.

[048] The methods of the present invention are directed at the generation of a neural stem cell (NSC) also referred to as primary neural stem cells. Primary neural stem cells (NSCs), can generally be obtained, besides via methods of the present invention, from actual stem cells in several different stages of neural development and also from adult stem cells present in a subject. NSCs are somatic fate restricted. This means that these cells are multipotent. So, for example, primary neural stem cells have the capacity to differentiate further into multiple types of cells, such as neurons, astrocytes and other glial cells.

[049] Furthermore, NSCs are able to self-renew. Self-renewal is the ability to go through numerous cell cycles of cell division while maintaining the undifferentiated state. Methods for testing if a cell has the capacity to self-renew and if a cell is multipotent are known to the skilled artesian. Self-renewal may be tested by passaging the cells over more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more passages. Passaging includes splitting of the cells before re-plaiting them as a single cell suspension. Multipotency can be tested by differentiating said cells into different lineages such as astrocytes, oiigodendroctyes and neurons.

[050] Furthermore, a NSC obtained by the methods of the present invention can express markers such as PAX6, SOX2, Ki67, NESTIN and SOX1. Furthermore, a NSC obtained by the methods of the present invention can be characterized by a lack or reduced of expression of the markers OCT4 and/or NANOG compared to iPSCs from which they have been generated. Furthermore, a NSC obtained by the methods of the present invention can express markers such as SOX2, Ki67 and NESTIN to a higher extend compared to the iPSC cell from which it has been derived.

[051] It is further envisioned by the present invention that the NSC can be a mammalian NSC. It is also encompassed by the present invention that the NSC is a human NSC (hNSC). A NSC of the present invention is obtained by the methods of the present invention.

[052] For the generation of NSCs in accordance with the present invention, it is necessary that induced pluripotent stem cells (iPSCs) are obtained. "Induced pluripotent stem cells", as used herein, refers to adult somatic cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Thus, induced pluripotent stem cells derived can be from a non-pluripotent cell.

[053] Induced pluripotent stem cells are an important advancement in stem cell research, as they allow obtaining pluripotent stem cells without the use of embryos. Mouse iPSCs were first reported in 2006 (Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”. Cell 126 (4): 663-76), and human iPSCs (hiPSCs) were first reported in 2007 (Takahashi et al. (2007) “induction of pluripotent stem cells from adult human fibroblasts by defined factors.” Cell; 131(5):861-72). Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expression of stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. Such stem cell markers can include Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) as well as stem cell-specific antigen 3 and 4 (SSEA3/4). Also the chromatin méthylation patterns of iPSC are similar to that of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014) “Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014, Ser. B 90).

[054] In addition, iPSCs are able to self-renew in vitro and differentiate into all three germ layers. The pluripotency or the potential to differentiate into different cell types of iPSC can tested, e.g., by in vitro differentiation into neural or glia cells or the production of germline chimaeric animals through blastocyst injection.

[055] Methods for the generation of human induced pluripotent stem ceils are well known to the skilled person. Usually forced expression of Oct3/4, Sox2 and Klf4 (as well as OCT3/4, SOX2 and KLF4) is sufficient to generate an induced pluripotent stem cell out of an adult somatic cell, such as a fibroblast. However, also the combination of Oct3/4, Sox2, c-Myc and Klf4 (as well as OCT3/4, SOX2, C-MYC) and KLF4 is sufficient for the generation of a iPSC from an adult somatic cell. In addition also the combination of OCT3/4, SOX2, NANOG and LIN28 was efficient for reprogramming (Tanabe, Takahashi, Yamanaka (2014) “Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014, Ser. B 90). For this, these genes are usually cloned into a retroviral vector and transgene-expressing viral particles or vectors, with which the somatic cell is co-transduced. However, also other techniques known to the skilled artesian can be used for that purpose. Human skin fibroblasts can also be co-transduced with all four vectors e.g. via protein transduction or naked DNA.

[056] Further methods for obtaining iPSCs are also known to the skilled artesian and for example described in W02009115295, W02009144008 or EP2218778. Thus, the skilled artesian can obtain an iPSC by any method.

[057] In principle, induced pluripotent stem cells may be obtained from any adult somatic cell (of a subject). Exemplary somatic cells include peripheral blood Mononuclear Cells (PBMCs) from blood or fibroblasts, such as for example fibroblasts obtained from skin tissue biopsies.

[058] Therefore, it is envisioned by the present invention that the iPSC is produced or derived or obtained from any somatic cell of a subject. In addition, it is envisioned by the present invention that the iPSCs can be produced from somatic cells such as fibroblasts. Additionally or alternatively, the iPSC can be a human iPSC (hiPSC).

[059] It is further encompassed by the present invention that the somatic cells such as fibroblasts have been obtained from a subject. For example, the fibroblast can be obtained from a human.

[060] The term “subject” can mean human or an animal. The subject can be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, dogs, horses, mice and rats. A mammal can be a human, dog, cat, cow, pig, mouse, rat etc. Thus, in one the subject is a vertebrate. The subject can also be a human subject.

[061] The subject can also be a subject suffering from a neurodegenerative disease. The term “neurodegenerative disease" can concern a group of hereditary and sporadic conditions characterised by progressive dysfunction, degeneration and death of specific populations of neurons, which are often synapticaily interconnected.

[062] Examplary neurodegenerative diseases include, but are not limited to, Parkinson’s disease, progressive supranuclear palsy, corticobasal degeneration, multisystem atrophy with striatonigral degeneration, Huntington's disease, dentatorubropallidoiuysial atrophy, Friedrich’s ataxia, spinocerebellar degeneration,

Amyotrophic lateral sclerosis, spinobulbar muscle atrophy (Kennedy’s disease), spinal muscular atrophy, Alzheimer's disease, dementia with Lewy bodies, Frontotemporal lobar degeneration or Prion disease.

[063] The neurodegenerative disease can thus include Parkinson’s disease, Down syndrome, or Alzheimer’s disease. In particular, the subject may be a subject comprising the LRRK2-G2019S mutation, which is associated with familial Parkinson’s disease. The subject can also be a subject not suffering from a neurodegenerative disease as described herein or not suffering from Parkinson's disease, Down syndrome, or Alzheimer’s disease. Also encompassed by the present invention is that the subject is a healthy subject.

[064] The methods of the present invention further require that cells such as iPSCs and NSCs are cultivated. In general, the methods of the present invention can be carried out in any cell culture. Culture conditions may vary, but the artificial environment in which the cells are cultured often comprise a suitable vessel comprising one or more of the following: a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, gases (02, C02) and/or regulated physico-chemical environment (pH, osmotic pressure, temperature). Cell culture as described herein refers to the maintenance and growth of cells in a controlled laboratory environment. Such in vitro cell culture models are well-known in experimental ceil biological research. For example, cells can be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture). Cells can also be grown floating in the culture medium (suspension culture).

[065] It is also envisioned by the present invention that cells such as iPSCs can be maintained on another cell type such as mouse embryonic fibroblasts (MEFs). The maintaining on another cell type can be performed under feeder free conditions. Thus, the methods of the present invention also contemplate that method steps b), c) and d) (optionally also d.2) are performed on a feederlayer. The feeder layer can comprise or be consistent of mouse embryonic fibroblasts.

[066] In addition, cells can be cultured in a two-dimensional cell culture. This type of cell culture is well-known to the person skilled in the art. In two-dimensional (2D) cell culture cells are grown on flat plastic dishes such as Petri dish, flasks and multi-well plates. However, biologically derived matrices (e.g. fibrin, collagen, feeder layers as further described herein) and synthetic hydrogels (e.g. PAA, PEG and as further described herein) can be used to elicit specific cellular phenotypes that are not expressed on rigid surfaces.

[067] The methods of the present invention can also be carried out in a three dimensional cell culture. A “three-dimensional cell culture“ or “3D cell culture” as used herein means that cells are grown in an artificially-created environment in which cells are permitted to grow or interact with its surroundings in all three dimensions. This concept is known to the skilled artesian and for example described in Ravi et al. (2015) “3D cell culture systems: advantages and applications.” J Cell Physiol. 230(1):16-26 and Antoni et al. (2015) “Three-Dimensional Cell Culture: A Breakthrough in Vivo.” Int J Mol Sci. 16(3):5517-5527). To achieve the three dimensional property of the cell culture, cells are grown or differentiated in matrices or scaffolds. In principle, suitable matrices or scaffolds, which can be used in three dimensional cell cultures are known to the skilled artesian. Such matrices or scaffolds can therefore be any matrix or scaffold. For example, the matrix or scaffold can be an extracellular matrix comprising either natural molecules or synthetic polymers, a biological and synthetic hybrid, metals, ceramic and bioactive glass or carbon nanotubes.

[068] The methods of the present invention require that cells such as iPSCs are cultivated in different media. The media as used in the methods of the present invention can comprise a “basic/basal" culture medium. Such media are known to the skilled artesan and also publicly available. Exemplary “basic/basal” culture media include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM) (e.g. (ATCC® 30-2002™), DMEM-F12 medium (e.g. ATCC® 30-2006™), RPMI medium (e.g. RPMI-1640 Medium (ATCC® 30-2001 ™), Iscove's modified Dulbecco's medium (IMDM) (e.g. ATCC® 30-2005™), Eagle's Minimum Essential Medium (EMEM) (e.g. ATCC® 30-2003™), F-12K Medium (Kaighn's Modification of Ham's F-12 Medium e.g. ATCC® 30-2004™), Leibovitz's L-15 Medium (e.g. ATCC® 30-2008™), McCoy's 5A Medium (e.g. ATCC® 30-2007™) or Neurobasal medium (e.g. obtainable from Gibco).

[069] In addition to the basic culture medium, media as used in the methods of the present invention can comprise further additives/suppiements. Suitable additives/supplements are known to the person skilled in the art and also publicly avaiiale. For example supplements/additives can include signaling inhibitors/activators, reducing agents» growth factors, nutritiens, amino acids, sugars, B27, N2, G5, serum, antibiotics, antoxidants just to name a few.

[070] The media used in the methods of the present invention can comprise a signaling activator or a signaling inhibitor. The term "activator", as used herein, is defined as a compound/molecule enhancing or achieving the activity of a target molecule and/or signaling pathway. The activator may achieve this effect by enhancing or inducing the transcription of the gene encoding the protein to be activated and/or enhancing the translation of the mRNA encoding the protein to be activated. It can also be that the protein to be activated performs its biochemical function with enhanced efficiency in the presence of the activator or that the protein to be activated performs its cellular function with enhanced efficiency in the presence of the activator. Accordingly, the term "activator" encompasses both molecules/compounds that have a directly activating effect on the specific signaling pathway but also molecules that are indirectly activating, e.g. by interacting for example with molecules that negatively regulate (e.g. suppress) said pathway. The activator can also be an agonist of the signaling pathway (receptor) to be activated. Methods for testing if a compound/molecule is capable to induce or enhance the activity of a target molecule and/or pathway are known to the skilled artesian. For example an activator of a SHH, WNT or other activator as described herein can be tested by performing Western Blot analysis of the amount of e.g. pathway effector proteins such as Gli proteins, LEF1 orTCFI protein, respectively.

[071] The compound/molecule that can be used as an activator can be any compound/molecule, which can activate the respective pathway or which inhibits a suppressor of the pathway to be activated. Exemplary activators can include suitable binding proteins directed e.g. against suppressors of a certain pathway.

[072] For example, the binding protein can be an antibody or a divalent antibody fragment comprising two binding sites with different specificities. Non limiting examples of such divalent antibody fragments include a (Fab)2’-fragment, a divalent single-chain Fv fragment, a bsFc-1/2-dimer or a bsFc-CH3-1/2 dimer. Alternatively, the binding protein can also be a bivalent proteinaceous artificial binding molecule such as a lipocalin mutein that is also known as “duocalin”. The binding protein may also only have a single binding site, i.e., may be monovalent. Examples of monovalent binding proteins include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties.

Examples of monovalent antibody fragments include, but are not limited to a Fab fragment, a Fv fragment, a single-chain Fv fragment (scFv) or an scFv-Fc fragment.

[073] The binding protein can also be a proteinaceous binding molecule with antibody-like binding properties. Exemplary but non-limiting proteinaceous binding molecules include an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, an avimer or a (recombinant) receptor protein.

[074] The activator can also be a nucleic acid molecule, such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer. Such an aptamer is an oligonucleic acid that binds to a specific target molecule. These aptamers can be classified as: DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides. Also the nucleic acid molecules may be used to suppress a repressor of a pathway to be activated.

[075] It is also encompassed by the present invention that the activator is a small molecule or protein/polypeptide. Such a small molecule can have is a low molecular weight of less than 900 daltons (da), less than 800 da, less than 700 da, less than 600 da or less than 500 da. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. So for example an activator of the SHH pathway can be purmorphamine, which is a small-molecule agonist developed for the protein Smoothened. Thus, the activator can also be an agonist of the pathway to be activated.

[076] An activator may enhance or increase the pathway to be activated by 10%, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % or more when compared to the activity of the pathway without the addition of the activator.

[077] On the contrary to the activator or signaling/pathway activator as described herein an “inhibitor" as used herein is defined as a compound/molecule reducing or blocking the activity of a target molecule and/or signaling pathway. The inhibitor may achieve this effect by reducing or blocking the transcription of the gene encoding the protein to be inhibited and/or reducing/blocking the translation of the mRNA encoding the protein to be inhibited. It can also be that the protein to be inhibited performs its biochemical function with decreased efficiency in the presence of the inhibitor or that the protein to be inhibited performs its cellular function with reduced efficiency in the presence of the inhibitor. Accordingly, the term "inhibitor" encompasses both molecules/compounds that have a directly reducing/blocking effect on the specific signaling pathway but also molecules that are indirectly inhibiting, e.g. by interacting for example molecules that positively regulate (e.g. activate) said pathway. The inhibitor can also be an antagonist of the pathway (receptor) to be inhibited. Methods for testing if a compound/moiecule is capable to reduce or block the activity of a target molecule and/or signaling pathway are known to the skilled artesian. For example an inhibitor of BMP as described herein can be tested by performing Western Blot analysis of the amount of e.g. pathway effector proteins such as GFAP protein, respectively.

[078] The compound/moiecule that can be used as an inhibitor can be any compound/moiecule, which can reduce or block the respective pathway or which inhibits a activator of the signaling (pathway) to be inhibited. Exemplary inhibitors can include suitable binding proteins as described herein, which are directed e.g. against activators of a certain pathway.

[079] The inhibitor can also be a nucleic acid molecule, such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer as described herein. Also the nucleic acid molecules may be used to suppress an activator of a pathway to be inhibited.

[080] It is also encompassed by the present invention that the inhibitor is a small molecule or protein/polypeptide. As decribed herein such a small molecule can have is a low molecular weight of less than 900 daltons (da), less than 800 da, less than 700 da, less than 600 da or less than 500 da. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. So for example an inhibitor of the BMP pathway can be dorsomorphine, which is a small-molecule antagonist for bone morphogenetic protein (BMP) type I receptors (ALK2, ALK3 and ALK6). Thus, the inhibitor can also be an antagonist of the pathway/signaling patthway to be inhibited.

[081] An inhibitor may reduce or decrease the pathway to be inhibited by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more when compared to the activity of the pathway without the addition of the inhibtor. A block of the pathway to be inhibited is present when the pathway is inhibited by 100 % when compared to the activity of the pathway without the addition of the inhibitor.

[082] The media as used in the methods of the present invention can for example comprise an activin/TGF-ß inhibitor. For example, the medium used in step b) and step c) of the methods of the present invention comprises an activin/TGF-ß inhibitor.

[083] The activin/TGF-ß signaling pathway is known in the art and for example described in Heldin, Miyazono and ten Dijke (1997) “TGF-bold beta signaling from cell membrane to nucleus through SMAD proteins.” Nature 390, 465-471. In short, Receptor ligands, including, for example, TGFB1, TGFB2, TGFB3, ACTIVIN A, ACÏIVIN B, ACTIVIN AB and/or NODAL, bind to a heterotetrameric receptor complex comprising two type I receptor kinases, including, for example, TGFBR2, ACVR2A, and/or ACVR2B, and two type II receptor kinases, including, for example, TGFBR1 (ALK5), ACVR1B (ALK4) and/or ACVR1C (ALK7). This binding triggers phosphorylation and activation of a heteromeric complex consisting of an R-smad, including, for example, SMAD2, and/or SMAD3, and a Co-smad, including, for example, SMAD4. Accordingly, the term "activator of the activin/TGF-ß signaling pathway" refers to an activator of any one of the above recited molecules that form part of this signaling pathway, while the term "inhibitor of the activin/TGF-ß signaling pathway" refers to inhibitors of any one of the above recited molecules that form part of this signaling pathway. In addition, such an activator can be an agonist of the ACVR2A and/or ACVR1B (ALK4) receptor or an agonist of the TGFßRII receptor and/or ALK5 receptor. Such an inhibitor can be an antagonist of the ACVR2A and/or ACVR1B (ALK4) receptor or an antagonist of the TGFßRII receptor and/or ALK5 receptor. In principle such inhibitors/activators of the activin/TGF-ß signaling pathway are known to the skilled artesian and are commercially available.

[084] The invention contemplates that the activin/TGF-ß inhibitor is an inhibitor of the TGF-ß type I receptor activin receptor-like kinase(s). Further envisioned by the present invention is that the activin/TGF-ß inhibitor inhibits ALK5, ALK4 and/or ALK7. Exemplary but non-limiting exammples of an activin/TGF-ß inhibitor are A-83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1 H-pyrazole-1 -carbothioamide; CAS No.: 909910-43-6), D4476 (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1 H-imidazol-2-yl]benzamide; CAS No.: 301836-43-1), GW788388 (4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide; CAS No.: 452342-67-5), LY364947 ( 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline; CAS No.: 396129-53-6), R268712 (4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1 H-pyrazoie-1 -ethanol; CAS No.: 879487-87-3), SB-431542 (4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate; CAS No.: CAS Number 301836-41-9), SB-505124 (2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate; CAS No.: 694433-59-5), SD208 (2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine; CAS No.: 627536-09-8), SB-525334 (6-[2-tert-Butyl-5-(6-methyi-pyridin-2-yl)-1 H-imidazol-4-yl]- quinoxaline; CAS No.: 356559-20-1) and ALK5 inhibitor II (CAS: 446859-33-2). The activin/TGF-ß inhibitor can thus be SB-431542.

[085] The activin/TGF-ß inhibitor such as SB-431542 can be employed in a concentration of between about 0,01 μΜ and about 1 M, more preferably between about 5 μΜ and about 15 μΜ, and most preferably the amount is about 10 μΜ. For example, SB-431542 can be obtained from Ascent Scientific.

[086] The media as used in the methods of the present invention can additionally or alternatively comprise a canonical WNT-signaling activator. For example, the medium used in step b), step c), step d) of the methods of the present invention comprises a canonical WNT-signaling activator. Also media used in step d.1) and/or d.2) as described herein can comprise a WNT-signaling activator.

[087] The canonical Wnt signaling pathway is known to the skilled artesian and for example described in Logan and Nusse (Annu. Rev. Cell Dev. Biol. (2004) 20:781-810). In short, a Wnt ligand binds to Frizzled receptors, which triggers displacement of the multifunctional kinase GSK-3ß from a regulatory APC/Axin/GSK-3ß-complex. In the absence of Wnt-signal (Off-state), ß-catenin, is targeted by coordinated phosphorylation by CK1 and the APC/Axin/GSK-3ß-complex leading to its ubiquitination and proteasomal degradation through the ß-TrCP/SKP pathway. In the presence of Wnt ligand (On-state), the co-receptor LRP5/6 is brought in complex with Wnt-bound Frizzled. This leads to activation of Dishevelled (Dvl), which displaces GSK-3ß from APC/Axin. The transcriptional effects of Wnt ligand is mediated via Rad-dependent nuclear translocation of ß-catenin and the subsequent recruitment of LEF/ TCF DNA-binding factors as co-activators for transcription. Exemplary Wnt ligands include for example Wnt1, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt7b, and/or Wnt11.

[088] Accordingly, the term “canonical WNT-signaling activator” as described herein refera to an activator of any one of the above recited molecules that form part of this signaling pathway.

[089] Exemplary canonical WNT-signaling activators include Norrin, R-spondin 2 or WNT protein. However, the canonical WNT-signaling activator can also block Axin or APC. This can be achieved for example via siRNA or miRNA technology. It is also encompassed by the present invention that the canonical WNT-signaling activator is a GSK-3 inhibitor. Exemplary GSK-3 inhibitors include CH1R 99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CAS No.: 252917*06*9), SB-216763 (3-(2,4-Dichlorophenyl)-4-(1-methyl-1 H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS No.: 280744-09-4), 6- bromoindirubin-3'-oxime (CAS No.: CAS 667463-62-9), Tideglusib (4-Benzyl-2-(naphthalen-1 -yl)-1,2,4-thiadiazolidine-3,5-dione), GSK-3 inhibitor 1 (CAS No.: 603272-51-1), AZD1080 (CAS No.: 612487-72-6), TDZD-8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione; CAS No.: 327036-89-5), TWS119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol; CAS No.: 601514-19-6), CHIR-99021 (CAS No.: 252917-06-9), CHIR-98014 (N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-Pyridinediamine; CAS No.: 252935-94-7), SB 415286 (3-[(3-Chloro-4-hydroxyphenyl)-amino]-4-(2-nitrophenyl)-1 H-pyrrol-2,5-dione; CAS No.: 264218-23-7), LY2090314 (3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1 -hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1 H-pyrrole-2,5-dione; CAS No.: 603288-22-8), AR-A014418 (N-(4-Methoxybenzy!)-N’-(5-nitro-1,3-thiazol-2-yl)urea; CAS No.: 487021-52-3 and/or IM-12 (3-(4-

Fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yi)-1H-pyrrole-2,5-dione; CAS No.: 1129669-05-1). Thus, the GSK-3 inhibitor can also be CHIR 99021.

[090] The canonical WNT-signaling activator such as CHIR 99021 can be employed in a concentration of between about 0,01 μΜ and about 1 M, more preferably between about 0,1 μΜ and about 5 μΜ, and most preferably the amount is about 3 μΜ. CHIR 99021 can for example be obtained from Axon Medchem.

[091] The media as used in the methods of the present invention can additionally or alternatively comprise a BMP signaling inhibitor. For example, the medium in step b), step c) used in the methods of the present invention compises a BMP signaling inhibitor.

[092] The BMP signaling pathway is known to the skilled artesian and for example described in Jiwang Zhanga, Linheng Lia (2005) BMP signaling and stem cell regulation Developmental Biology Volume 284, Issue 1,1 August 2005, Pages 1-11.

[093] In short, BMP functions through receptor-mediated intracellular signaling and subsequently influences target gene transcription. Two types of receptors are required in this process, which are referred to as type I and type II. While there is only one type II BMP receptor (Bmprll), there are three type I receptors: Alk2, Alk3 (Bmprl a), and Alk6 (Bmprlb). BMP signal transduction can take place over at least two signaling pathways. The canonical BMP pathway is mediated by receptor I mediated phosphorylation of Smadl, Smad5, or Smad8 (R-Smad). Two phosphorylated R-Smads form a heterotrimeric complex coaggregate with a common Smad4 (co-Smad). The Smad heterotrimeric complex can translocate into the nucleus and can cooperate with other transcription factors to modulate target gene expression. A parallel pathway for the BMP signal is mediated by TGFßl activated tyrosine kinase. 1 (TAK1, a MAPKKK) and through mitogen activated protein kinase (MARK), which also involves cross-talk between the BMP and Wnt pathways.

[094] It is envisioned by the present invention that the inhibitors of BMP signaling can only block/reduce the canonical BMP pathway. Thus, the BMP signaling inhibitor can be a cancoical BMP signaling inhibitor. One such inhibitor selective for cannocial BMP signaling pathway is dorsomorphin. Exemplary, but non-limiting, examples of BMP signaling inhibitors include chordin, noggin, DMH1 (CAS 1206711-16-1), K 02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol; CAS No.: 1431985-92-0), dorsomorphin (6-[4-(2-Piperidin-1-yIethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine; CAS No.: 866405-64-3) and LDN 193189 (4-[6-[4-(1-Piperazinyl)phenyi]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride, CAS No.: 1062368-24-4). The BMP signaling inhibitor can also be dorsomorphin.

[095] The BMP signaling inhibitor such as dorsomorphin can be employed in a concentration of between about 0,01 μΜ and about 1 M, more preferably between about 0,1 μΜ and about 5 μΜ, and most preferably the amount is about 0,1 μΜ. Dorsomorphin can for example be obtained from Tocris.

[096] The media as used in the methods of the present invention can additionally or alternatively comprise a SHH-pathway activator. For example, the medium in step b), step c) and step d) of the methods of the present invention compise a SHH-pathway activator.

[097] The "Hedgehog signaling pathway" or “SHH pathway” is well known in the art and has been described, for example, in Choudhry et al. (2014) “Sonic hedgehog signalling pathway: a complex network.” Ann Neurosci. 21(1):28-31. Hedgehog ligands, including, for example, Sonic hedgehog, Indian hedgehog, and/or Desert hedgehog, bind to the receptor, including, for example, Patched or the patched-smoothened receptor complex, which induces a downstream signaling cascade. Downstream target genes of SHH signaling include GLU, GLI2 and/or GLI3. Accordingly, the term "activator of the Hedgehog signalling pathway" also refers to an activator of any one of the above recited molecules that form part of this signaling pathway.

[098] Exemplary activators of the Hedgehog signaling (SHH) include purmorphamine (PMA; 2-(1-Naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine 9- Cyclohexyl-N-[4-(4-morpholinyi)phenyl]-2-(1-naphthalenyloxy); CAS No.: 483367- 10- 8), SHH, smoothened agonist (SAG; 3-chioro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]-benzo[b]thiophene-2-carboxamide; CAS No.: 912545-86-9 ) and Hh-Ag 1.5 (3-chloro-4,7-difluoro-N-(4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide; CAS No.: 612542-14-0) as well as Gli-2. The SHH-pathway activator can also be selected from the group consisting of purmorphamine, SHH, SAG Analog and Gli-2. The SHH-pathway activator can therefore be purmorphamine. The SHH pathway activator can also be a recombinant or truncated form of SHH, which retains SHH pathway activating functions such as e.g. SHH C24II.

[099] The SHH signaling pathway activator such as purmorphamine can be employed in a concentration of between about 0,25 μΜ and about 1 M, more preferably between about 0,4 μΜ and about 0,5 μΜ, and most preferably the amount is about 0,5 μΜ.

[100] The SHH signaling pathway activator such as SHH can also be employed between about 50 and about 1000 ng/ml. The SHH signaling pathway activator such as SHH C24II can also be employed in a concentration of about 10 and about 500 ng/ml. The SHH signaling pathway activator such as SAG can be employed in a concentration of about 1 and about 100 nM. The SHH signaling pathway activator such as Hh-Ag1.5 can also be employed in a concentration of about 1 and about 50 nM.

[101] The medium used in step b) in the methods of the present invention is also referred to as “Embryoid Body (EB) Formation Medium” herein. The term “embryoid bodies" as used herein refers to aggregates of cells derived from pluripotent stem ceils (iPSCs). Embryoid bodies (embryoid body; EB) are generally comprised of a large variety of differentiated cell types. Cell aggregation can for example be imposed by hanging drop or other methods that prevent cells from adhering to a surface, thus allowing the embryoid bodies to form their typical colony growth. Upon aggregation, differentiation is typically initiated and the cells begin to a limited extent to recapitulate embryonic development.

[102] The EB medium comprises a “basic/basal” medium as described herein, which can compise supplements/additives as described herein. As basic medium the EB medium or the medium used in step b) of the methods of the present invention can for example be a DMEM medium. Thus, the medium used in step b) of the methods of the present invention can comprise DMEM medium. The DMEM medium can for example be a knockout DMEM medium. “Knockout DMEM” medium is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells. The knockout DMEM medium may contain no L-glutamine. The knockout DMEM medium can for example be the Knockout™ DMEM medium from Gibco.

[103] The medium used in step b) of the methods of the present invention can additionally or alternatively comprise a serum replacement. A serum replacement is known to a skilled artesian and commercially available. A “serum replacemenf is usually used to replace serum in a medium. For example, the serum replacement can be a knockout serum replacement. The “knockout serum replacemenf is a defined, serum-free formulation optimized to grow and maintain undifferentiated ES cells in culture. It can directly replace FBS in protocols/methods. The knockout serum replacement can be used in conjunction with knockout DMEM. The knockout serum replacement can for example be the Knockout™ SR from Gibco.

[104] The medium used in step b), c) and step d), d.1) and d2) and also the neuron differentiation medium and ground medium as described herein of the methods of the present invention can additionally or alternatively comprise one or more non-essential amino acid(s) (NEAA(s)). Also these are known to the skilled artesian and are commercially available e.g. as 100X MEM (minimum essential medium) Non-Essential Amino Acids Solution from Gibco. The medium can thus comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the non-essential amino acids described herein. In general, the non-essential amino acids can comprise at least one of arginine, asparagine, glutamine, glycine, alanine, cysteine, aspartic acid, glutamamic acid, proline, tyrosine and serine. The non-essential amino acids can also comprise at least one of alanine, aspartic acid, cysteine or glutamamic acid. For example the medium used in step b) of the present invention can comprises NEAA obtainable form Gibco. It is furthermore envisioned by the present invention that the one or more of the amino acids are present as L-stereoisomers.

[105] The medium used in step c) and step d) (d.1; d.2) and also the neuron differentiation and ground medium as described herein of the method of the present invention can for example comprise glutamine. The glutamine can for example be obtained from Invitrogen.

[106] The medium used in step b) of the methods of the present invention can additionally or alternatively comprise a reducing agent(s). A “reducing agent” is an element or compound that loses (or "donates") an electron to another chemical species in a redox chemical reaction. Since the reducing agent is losing electrons, it is said to have been oxidized. Exemplary reducing agents include but are not limited to ß-mercaptoethanol, DL-Dithiothreitol (DTT), 2-Mercaptoethylamine-HCI, TCEP, TCEP-HCI or Cysteine-HCl. The reducing agent can be ß-mercaptoethanol.

[107] The medium used in step b), c) and step d) (d.1; d.2) of the methods of the present invention as well as the neuron differentiation medium as well as the ground medium as described herein can additionally or alternatively comprise an antibiotic(s). Such an antibiotic can for example be a mix of penicillin and streptomycin. These antibiotics can be present at a concentration of 0.3 %, 0.5 %, 0.7 %, 1 %, 1.3 %, 1.5 %, 1.7 %, 2 %, 3 %, 4 %, 5 % or more. Thus, the antibiotic such as a mix of penicillin and streptomycin can be present in a total concentration of 1 % (including both penicillin and streptomycin).

[108] The medium used in step c) and d) (d.1; d.2) in the methods of the present invention and also in the neuron differentiation medium as described herein is also referred to as “N2B27 Medium”. This medium comprises a “basic/basai" medium as described herein which can compise supplements/additives as described herein. As basal medium for the N2B27 Medium or the medium used in steps c) and/or d) (d.1; d.2) of the methods of the present invention and also in the neuron differentiation medium as described herein can for example be a DMEM-F12 medium. Additionally or alternatively, the (basal) medium used in steps c) and/or d) of the methods of the present invention and also in the neuron differentiation medium as described herein can comprise neurobasal medium. It is further envisioned by the present invention that the medium used in step c) and/or step d) of the method of the present invention and also in the neuron differentiation medium as described herein comprises DMEM-F12 medium and neurobasal medium in a proportion of 50:50. Both, the DMEM-F12 medium and the neurobasal medium can for example be obtained from Gibco.

[109] The medium used in step c) and d) (d.1; d.2) in the methods of the present invention and also in the neuron differentiation medium as described herein or the N2B27 Medium can additionally or alternatively comprise N2 supplement. N2 supplement is known to the skilled artesian and commercially available. “N2 Supplement” is a chemically defined, serum-free supplement based on Bottenstein’s N-1 formulation. N2 Supplement can be used in connection with with Neurobasal® media. The N2 supplement can be added in a concentration of 1:10 (N2:medium), 1: 50, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500 or lower to the medium such as N2B27 medium. N2 can be added in a concentration of 1:200 to the medium.

[110] The medium used in step c) and d) (d.1; d.2) in the methods of the present invention and also in the neuron differentiation medium as described herein or the N2B27 Medium can additionally or alternatively comprise B27 lacking vitamin A. This supplement is also known to the skilled and commercially available. For example, comprise B27 lacking vitamin A can be obtained from Invitrogen. The B27 lacking vitamin A can be added in a concentration of 1:5 (B27 lacking vitamin A:medium), 1:10, 1:25, 1:50, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500 or lower. B27 lacking vitamin A can be added in a concentration of 1:100 to the medium.

[111] The medium used in step c) and d) (d.1, d.2) in the methods of the present invention and also in the neuron differentiation medium and ground medium as described herein or the N2B27 Medium can additionally or alternatively comprise glutamine. For example, the medium can further comprise L-glutamine. L-glutamine can be added at a concentration of 0.5 mM, 1mM, 1.5 mM, 2 mM, 2.5 mM 3 mM 3.5 mM, 4 mM or more. Thus, L-glutamine can be added at a concentration of 2 mM.

[112] The medium used in step d) (d.1, d.2) of the methods of the present invention can additionally or alternatively comprise an antioxidant. An “antioxidant” is a molecule that inhibits the oxidation of other molecules. The terms "oxidation" and “antioxidant” are well known in the art and have been described, for example, in Nordberg J, Amér ES. (2001) “Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.” Free Radie Biol Med. 31(11):1287-312. In short, oxidation is a chemical reaction involving the loss of electrons or an increase in oxidation state. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. Accordingly, an antioxidant refers to an inhibitor of a molecule involved in cellular oxidative processes.

[113] Exemplary antioxidants include ascorbic acid, superoxide dismutase 1, superoxide dismutase 2, superoxide dismutase 3, glutathione, lipoic acid, epigallocatechin gallate, curcumine, melatonin, hydroxytyrosol, ubiquinone, catalase, vitamin E or uric acid. The antioxidant can also be antioxidant, which is selected from the group consisting of ascorbic acid, glutathione, lipoic acid and uric acid. Thus, the antioxidant can be ascorbic acid.

[114] The antioxidant such as ascorbic acid can be utilized in an amount of about 50 μΜ to about 1 mM, or between about 100 μΜ and about 500 μΜ, or the amount can about 150 μΜ. The antioxidant such as superoxide dismutase 1, 2 or 3 can also be employed between about 10 and about 500 units/ml. The antioxidant such as glutathione can also be employed between about 1 and about 10 ng/μΙ. Lipoic acid can be employed between about 200 and about 1000 μΜ. The antioxidant such as epigallocatechin gallate can be employed between about 10 and about 100 pg/ml. The antioxidant such as curcumin can be employed between about 10 and about 100 μΜ. The antioxidant such as melatonin can be employed between about 10 and about 200 μΜ. The antioxidant such as hydroxytyrosol can be employed between about 10 and about 100 μΜ. The antioxidant such as ubiquinone can be employed between about 10 and about 50 μΜ. The antioxidant such as catalase can be employed between about 10 and about 500 units/ml. The antioxidant such as vitamin E can be employed between about 100 and about 1000 μΜ.

[115] Cells can be cultivated in the media used in the methods of the present invention for any period of time. For example, cells can be kept in the EB medium or medium of step b) used in the method of the present invention for 12, 24, 48, 60, 72, 96 or more hours. Thus, the cells can be cultivated for 48 hours in the medium used in step b) of the method of the present invention.

[116] Additionally or alternatively cells can be kept in the N2B25 medium or medium of step c) and/or d) (d.1, d.2) used in the method of the present invention for 12, 24, 48, 60, 72, 96, 108,120, 144, 168 or more hours. Thus, cells can be cultivated for 24 to 96 hours in the medium used in step c) and/or d) of the method of the present invention. For example, cells can be cultivated for 48 hours in the medium used in step c).

[117] Additionally or alternatively cells can be cultivated for 96 hours in the medium used in step d) of the method of the present invention. The medium used in step d) of the method of the present invention can also be changed once. This means that cells can be cultivated for 48 hours in the medium used in step d) of the method of the present invention and then be cultivated, after medium change, for another 48 hours in the medium used in step d) of the method of the present invention.

[118] It is further envisioned by the present invention that cells obtained after/in the cultivating step d) of the method of the present invention can plated on Matrigel coated plates. For example, cells can be plated after 12, 24, 48 or 72 or 96 hours of cultivating in a medium of step d) of the method of the present invention. Cells can be plated after 48 hours of cultivating in a medium of step d) of the method of the present invention. These Matrigel coated plates can for example be 12-well plates.

[119] This means that step d) of the method of the present invention can comprise d) further cultivating the cells obtained in c) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and d.1) further cultivating the cells obtained in d) in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant.

The step d.1) can further comprise, before performing cultivation of cells in the medium, one or more of 1) disintegration of neural tube like structures and/or 2) plating on Matrigel coated well plates and/or 3) medium change and/or 4) medium change after 48 hours.

[120] It is also contemplated by the present invention that step d) of the method of the present invention further comprises a step d.2) comprising culturing the cells obtained in step d) or d.1) in a medium comprising a FGF signaling activator. This means that step d) of the method of the present invention can comprise the step d.2) comprising further cultivating the cells obtained in d) or d.1) in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and (iv) an FGF signaling activator, 1121] An FGF receptor signaling activator activates FGF signaling. The FGF signaling pathway is well known to the skilled artesian and for example described in Coutu and Galipeau (2011) Roles of FGF signaling in stem ceil self-renewal, senescence and aging. Aging (Albany NY); 3(10):920-33. In short, FGF signaling is activated by binding of an FGFR agonist e.g. fibroblast growth factor (FGF) to the FGF receptor (FGFR), which induces FGFR dimerization, which juxtaposes the intracellular Tyr kinase domains of the receptors so that kinase activation by transphosphorylation can occur. Activated FGFR kinase in turn activates its intracellular substrates by phosphorylation. The signal can be further relayed through four distinct pathways: the Janus kinase/signal transducer and activator of transcription (Jak/Stat), phosphoinositide phospholipase C (PLCg), phosphatidylinositol 3-kinase (PI3K) and Erk pathways. In principle, any molecule that induces/increases FGF signaling can serve as activator of FGF signaling. The activator of FGF signaling can also be an FGF receptor agonist.

[122] Exemplary FGF signaling activators include but are not limited to FGF ligands, including, for example, FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11 , FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, and/or FGF23. The FGF signaling activator can also include FGF1, FGF2, FGF3, FGF4 and FGF8. The FGF signaling activator can also be FGF2 such as basic FGF2 (bFGF2).

[123] The FGF signaling activator as used in step d.2) of the method of the present invention can, for example, be added to the medium/cell culture in a concentration of 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/mi, 25 ng/ml, 30 ng/ml or more. The FGF receptor agonists as used in step d.2) of the method of the present invention can be added to the medium/cell culture in a concentration of 20 ng/ml. The FGF receptor agonist can, for example, be obtained from Peprotech.

[124] After cultivating cells in media used in step b), c) and d) of the methods of the present invention and optionally in media as described in step d.1), d.2), in principle already the desired NSCs are obtained. However, these cells can then be cultivated in the so called “human NSC maintenance medium" or a medium as used in step e) of the method of the present invention. This medium comprises an FGF signaling activator, an EGF signaling activator and LIF signaling activators. Furthermore, step e) of the methods of the present invention may be performed in matrigel coated dishessuch as 10 cm dishes.

[125] Before starting cultivating cells in a medium as disclosed in step e) of the methods of the present invention, cells may be detached e.g. by dispase.

[126] The FGF signaling activator as used in step e) of the methods of the present invention can be added to the medium/cell culture in a concentration of 50 ng/μΙ, 100 ng/μΙ, 150 ng/μΙ, 200 ng/μΙ, 250 ng/μΙ, 300 ng/μΙ, 350 ng/μΙ or more. The FGF receptor agonists as used in step e) of the method of the present invention can be added to the medium/cell culture in a concentration of 200 ng/μΙ. The FGF receptor agonist can be for example obtained from Peprotech.

[127] The medium used in step e) of the methods of the present invention comprises an FGF receptor agonist or an activator of FGF signaling as described herein.

[128] Furthermore, the medium used in step e) of the method of the present invention comprises an EG F receptor agonist or EGF signaling activator. The EGF signaling pathway is well known to the skilled artesian and for example described in Ayuso-Sacido et al. (2006) The duality of epidermal growth factor receptor (EGFR) signaling and neural stem cell phenotype: cell enhancer or cell transformer? Curr Stem Cell Res Ther; 1(3):387-94. In short, EGFR (EGF receptor) is a member of the receptor tyrosine kinase family that also includes erbB2, erbB3 and erbB4. After ligand (such as an EGF agonist e.g. EGF) binding, EGFR homo- or heterodimerizes and its intrinsic tyrosine kinase activity trans-phosphorylates specific Tyrosine (Tyr) residues located in the C-terminal cytoplasmic tail. Phosphorylated tyrosine residues function as docking sites for proteins with Src homology domain 2 (SH2) or phospho-tyrosine binding domains (PTB). These proteins are involved in the activation of different downstream pathways that regulate the phenotype and behavior of cells including migration, proliferation, differentiation, survival, and cell growth.

[129] In principle any molecule that induces/increases EGF signaling can serve as an activator of EGF signaling.

[130] Exemplary but non-limiting examples of EGF signaling activator(s) include epidermal growth factor (EGF), transforming growth factor a (TGFa), amphiregulin (AR), heparin-binding EGF-like growth factor (ΗΒ-EGF), betacellulin (BTC), epigen (EPG) and epiregulin (EPR). Notably, EGF, BTC and EPR bind to erbB4 while Neuregulins (NR) bind to either erbB3 or erbB4. In principle, however, any molecule that induces/increases EGF signaling can serve as an activator of EG F signaling. The EG F signaling activator can also be selected from the group consisting of EGF, TGF-α, Epigen, heparin-binding EGF-like growth factor (ΗΒ-EGF), betacellulin and amphiregulin (AR).

[131] The EGF signaling activator as used in step e) of the method of the present invention can be added to the medium/cell culture In a concentration of 50 ng/μΙ, 100 ng/pl, 150 ng/μΙ, 200 ng/pl, 250 ng/μΙ, 300 ng/μΙ, 350 ng/μΙ or more. The EGF receptor agonists as used in step e) of the methods of the present invention can be added to the medium/cell culture In a concentration of 200 ng/μΙ. The EGF signaling activator can, for example, be obtained from Peprotech.

[132] Furthermore, the medium used in step e) of the method of the present invention comprises a (Leukemia Inhibitory Factor) LI F receptor-α agonist or LI F receptor-α signaling activator. The LIF receptor-α signaling pathway is well known to the skilled artesian and for example described in Mathieuet al. (2012) LIF-Dependent Signaling: New Pieces in the Lego Stem Cell Rev.; 8(1): 1-15.

[133] In short, a LIF receptor agonist, such as LIF binds to the specific LIF receptor (LIFR-α) which forms a heterodimer with a specific subunit common to all members of that family of receptors, the GP130 signal transducing subunit. This leads to activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen activated protein kinase) cascades. In cell cultures, removal of LIF can push stem cells toward differentiation, but upon presence of LIF they may retain their proliferative potential or pluripotency. Thus, LIF is typically added to stem cell culture medium to reduce spontaneous differentiation.

[134] In principle, any molecule that induces/increases LIF signaling can serve as an agonist of the LIF a receptor or LIF a signaling activator. The LIF signaling activator in e) can be LIF. The LIF signaling activator in e) can be LIF human LIF.

[135] The present invention also relates to a method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem cells (iPSCs); b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and d.2) further cultivating the cells obtained in d) cells in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and (iv) an FGF signaling activator, thereby obtaining a NSC.

[136] The present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem cells (iPSCs); b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaiing activator; (Hi) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) cells in a medium comprising (i) a canonical WNT-signaiing activator; (ii) SHH-pathway activator; and (Hi) an antioxidant; and d.1) further cultivating the cells obtained in d) cells in a medium comprising (i) a canonical WNT-signaiing activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and d.2) further cultivating the cells obtained in d) cells in a medium comprising (i) a canonical WNT-signaiing activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and (iv) an FGF signaling activator, e) maintaining the cells obtained in d.2) in a medium comprising (i) a FGF signaling activator; (ii) an EGF signaling activator; and (iii) a LIF signaling activator, and thereby obtaining a NSC.

[137] After cultivation of ceils in step e) but also d), d.1), d.2) cells obtained in step e) but also d), d.1), d.2) can be further differentiated. Further differentiation can be achieved by switching the medium to a specific “differentiation medium". Before changing the medium, cells (NSCs) may be splitted. It is further encompassed by the present invention that cells (NSCs) are splitted at a confluency of 70-80% and then cultivated in a differentiation medium so in a further step f) in the method of the present invention.

[138] Thus the present invention can further comprise f) further cultivating the cells obtained in e), d), d.1) or d.2) (i) in a differentiation medium, optionally in a neuron differentiation medium or a ground medium.

[139] Thus, the the method of the present invention can further comprise differentiation of the cells (NSCs) obtained in step e), d), d.1), d.2) into neurons and glia cells. For example, cells may be differentiated into neurons and/or astrocytes.

[140] For example, the method of the present invention can comprise differentiation of the cell obtained in step e), d), d.1) or d.2) into a (i) Map2; (ii) TH; (ili) GABA; (iv) vGlut; (v) dcx; (vi) synaptophysin; (vii) postsynaptic density protein 95 (PSD 95); (viii) Tuj1; and/or

(ix) NCAM expressing cell. Such a cell can be characterized as an immature or mature neuron depending on the observed marker expression.

[141] In accordance with the present invention, "Map2" refers to Microtubule-associated protein 2, a protein that in humans is encoded by the MAP2 gene. Map2 belongs to the microtubule-associated protein family. The proteins of this family are thought to be involved in microtubule assembly, which is an essential step in neuritogenesis. Human MAP2 mRNA is represented by the NCBI reference NMJ501039538 (SEQ ID NO: 1) and the protein by Uniprot No. P11137 (SEQ ID NO: 2). The term Map2 embraces any Map2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[142] In accordance with the present invention, TH" refers to Tyrosine hydroxylase/tyrosine 3-monooxygenase/tyrosinase, a protein that in humans is encoded by the TH gene. TH is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). Human TH mRNA is represented by the NCBI reference NMJ300360 (SEQ ID NO: 3) and the protein by Uniprot No. P07101 (SEQ ID NO: 4). The term TH embraces any TH nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[143] In accordance with the present invention, "GABA“ refers to y-Aminobutyric acid, a protein that is an inhibitory neurotransmitter in the mammalian central nervous system. It is synthesized from glutamate using the enzyme L-glutamic acid decarboxylase (GAD) and pyridoxal phosphate (which is the active form of vitamin B6) as a cofactor. The term GABA embraces any polypeptide and can also comprise fragments or variants thereof.

[144] In accordance with the present invention, "vGlut" refers to vesicular glutamate transporter 1/BNPI/SLC17A7, a protein that in humans is encoded by the VGLUT gene. The protein encoded by this gene is a vesicle-bound, sodium-dependent phosphate transporter that is specifically expressed in the neuron-rich regions of the brain. Human vGlut mRNA is represented by the NCBI reference NM_020309 (SEQ ID NO: 5) and the protein by Uniprot No. Q9P2U7 (SEQ ID NO: 6). The term vGlut embraces any vGlut nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[145] In accordance with the present invention, "dcx" refers to doublecortin, also known as doublin or lissencephalin-X, a protein that in humans is encoded by the DCX gene. The protein is a microtubule-associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures. Human dcx mRNA is represented by the NCBI reference NMJ300555 (SEQ ID NO: 7) and the protein by Uniprot No. 043602 (SEQ ID NO: 8). The term dcx embraces any dcx nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[146] In accordance with the present invention, "Synaptophysin" is a protein that in humans is encoded by the SYN gene. The protein is a synaptic vesicle glycoprotein with four transmembrane domains weighing 38kDa. Human synaptophysin mRNA is represented by the NCBI reference NM_003179 (SEQ ID NO: 9). and the protein by Uniprot No. P08247 (SEQ ID NO: 10). The term synaptophysin embraces any synaptophaysin nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[147] In accordance with the present invention, "PSD95" refers to postsynaptic density protein 95 also known as Disks large homolog 4 that in humans is encoded by the DLG4 gene. The PSD95 is a protein dense specialization attached to the postsynaptic membrane. Human PSD95 mRNA is represented by the NCBI reference NM_001128827 (SEQ ID NO: 11) and the protein is represented by Uniprot No. P78352 (SEQ ID NO: 12). The term PSD95 embraces any PSD95 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[148] In accordance with the present invention, "Tuj1" also known as “ßlll Tubulin” is a protein that in humans is encoded by the TUBB3 gene. The protein pill Tubulin (TuJ1) is present in newly generated immature postmitotic neurons and differentiated neurons and in some mitotically active neuronal percursors. Human Tuj mRNA is represented by the NCBI reference NM_006086.3 (SEQ ID NO: 13) and the protein by Uniprot No. Q13509 (SEQ ID NO: 14). The term Tuj1 embraces any Tuj1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[149] In accordance with the present invention, “NCAM“ also known as “Neural cell adhesion molecule”, also called “CD56” is a protein that in humans is encoded by the NCAM gene. NCAM is a homophilic binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle and natural killer cells. Human NCAM mRNA is represented by the NCBI reference NM_000615 (SEQ ID NO: 67) and the protein by Uniprot No. P13591 (SEQ ID NO: 68). The term NCAM embraces any NCAM nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[150] The differentiation into neurons can for example comprise cultivating cells obtained in step e), d), d.1) or d.2) of the method of the present invention in a “neuron differentiation medium”. The neuron differentiation medium can be a N2B27 medium as described herein. For example the neuron differentiation medium can comprise one or more of (1) DMEM-F12 medium as described herein, (2) Neurobasal medium as described herein, (3) N2 supplement as described herein, (4) B27 supplement lacking vitamin A as described herein, (5) antibiotics as described herein, (6) glutamine as described herein. The neuron differentiation medium can also comprise all of (1 )-(6) listed above.

[151] The neuron differentiation medium can additionally or alternatively comprise (i) at least two different neurotrophins; and (ii) an antioxidant as described herein.

[152] The term "neurotrophins", as used herein, relates to a family of proteins that regulate the survival, development, and function of neurons. Family-members include for example nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) as well as the GDNF family of ligands and ciliary neurotrophic factor (CNTF).

[153] Accordingly, the term "at least two different neurotrophins" refers to two or more of the above recited molecules. Preferably, the at least two different neurotrophins are BDNF and GDNF (Gene Symbols: BDNF and GDNF, respectively; (Jiang et al. (Chin Med J (Engl) (2011 ) 124:1540-1544); Glavaski-Joksimovic et al. (J Neurosci res (2010) 88:2669-2681).

[154] Amounts of BDNF and GDNF, which can be employed, can be between about 0.5 and about 50 ng/ml each, more preferably between about 2 and about 20 ng/ml each, and most preferably the amount is about 10 ng/ml each. BDNF and GDNF may for example be obtained from Peprotech.

[155] The neuron differentiation medium can additionally or alternatively comprise a (iii) activin/transforming growth factor-ß (TGF-ß) signaling activator. TGF-ß signaling activators are elsewhere described herein. Exemplary activators of the activin/TGF-ß signaling pathway include TGFßl, TGFß2, TGFß3, activin A, activin B, activin AB or nodal. Thus, the activator of activin/TGF-ß signaling pathway can be TGFß3. The activator of the activin/TGF-ß signaling pathway such as TGFß3 can be utilized in an amount of 0.001 ng/ml to 10 ng/ml such as e.g. in an amount of 1 ng/ml. TGFß3 can be obtained from Peprotec, for example.

[156] The neuron differentiation medium can additionally or alternatively comprise a cAMP analogue. Such cAMP analogs are compounds that have similar physical, chemical, biochemical, or pharmacological properties as the cyclic adenosine monophosphate (cAMP). cAMP is known to the skilled artesian and described in e.g. Fimia GM, Sassone-Corsi P. (2001) “Cyclic AMP signalling.” J Cell Sci; 114(Pt 11):1971-2.

[157] Exemplary cAMP analogues include forskolin, 8-(4-chloro-phenylthio)-2’-0- methyladenosine-3’,5'-cyclic monophosphate (8CPT-2Me-cAMP), 8-Chloro-cAMP (8-CI-cAMP), Bucladesine, Rp-adenosine .3., 5.,-cyclic monophosphorothioate sodium salt (Rp-cAMPS), Sp-8-hydroxyadenosine .3., 5.,-cyclic monophosphorothioate sodium salt (Sp-80H-cAMPS) and Rp8-hydroxyadenosine .3., 5.,-cyclic monophosphorothioate sodium salt (Rp-80H-cAMPS) or dbcAMP. Thus, the cAMP analogue can be dbcAMP. The cAMP analogue such as dbcAMP can be utilized in an amount of 50 μΜ to 1000 μΜ such as e.g. in an amount of 500 μΜ. dbcAMP can be obtained from Sigma Aldrich, for example.

[158] The differentiation into neurons may take any period of time. Differentiation into neurons can take at least 14 days, 21 days, 28 days, 35 days, 42 days, 49 days or longer. For example, the process of differentiation into neurons can take at least 4 weeks.

[159] Differentiation into neurons in the neuon differentiation medium as described herein can result in the generation of GABAergic cells (cells positive for GABA). The percentage of GABA+ cells among the total of cells/the total of cells in the culture (in the neuron differentiation medium) can be about 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 % or higher. For example, about 36 % of the total of cells/the total of cells in the culture can differentiate into GABA ergic cells (cells positive for GABA).

[160] Additionally, or alternatively differentiation into neurons in the neuron differentiation medium as described herein can result in the generation of glutamatergic cells (vGlut+ cells). The percentage of glutamatergic cells (e.g.vGlut+ cells) among the total of cells/the total of cells in the culture (in the neuron differentiation medium) can be about 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 % or higher. For example, about 40 % of the total of cells/the total of ceils in the culture can differentiate into glutamatergic cells (cells positive for vGiut+).

[161] Additionally, or alternatively differentiation into neurons in the neuron differentiation medium as described herein can result in the generation of dopaminergic cells (e.g. TH+ cells). The percentage of dopaminergic ceils (e.g. TH+ cells) among the total of cells/the total of cells in the culture (in the neuron differentiation medium) can be about 3 %, 7 %, 10 %, 12 % or higher. For example, about 13 % of the total of cells/the total of cells in the culture can differentiate into dopaminergic cells (e.g. TH+ cells).

[162] The present invention further contemplates that when cells (NSCs) are differentiated into neurons, differentiation does virtually not result in the generation of astrocytes. For example, the culture (differentiated to neurons) can comprise less than 20 %, 15 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 % astrocytes.

[163] Additionally to the generation of neurons the present invention also provides for methods for the generation of astrocytes. Accordingly, the method of the present invention can also comprise differentiation of cells (NSCs) obtained in step e), d), d.1), d.2) into a (i) GFAP; (ii) S100b; (iii) vimentin; (iv) aquaporin 4; and/or (V) EAAT2 expressing cell.

[164] For example, after differentiation of 45-50 days astrocytes can express S100ß but can lack expression of GFAP. After a differentiation of 60 days astrocytes may express both S100ß and GFAP. Furthermore, after 60 days of differentiation astrocytes may additionally or alternatively express AQP4 and or EAAT2. After 80-90 days of differentiation astrocytes may be able to transport glutamate intracellularly. The person skilled in the art knwons how to measure such a glutamate transport, whih is also described in the Examples herein.

[165] in accordance with the present invention, "GFAP" also known as Glial fibrillary acidic protein is a protein that in humans is encoded by the GFAP gene. Gliai fibrillary acidic protein is an intermediate filament (IF) protein that is expressed by numerous cell types of the central nervous system (CNS) including astrocytes. Human GFAP mRNA is represented by the NCBI reference NM_001131019 (SEQ ID NO: 15) and the protein by Uniprot No. P14136 (SEQ ID NO: 16). The term GFAP embraces any GFAP nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[166] In accordance with the present invention, "S100b“ or “S100ß” also known as S100 calcium-binding protein B is a protein that in humans is encoded by the S100 gene. S100 proteins are localized in the cytoplasm and nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Human S100b mRNA is represented by the NCBI reference NM_006272 (SEQ ID NO: 18) and the protein by Uniprot No. P04271 (SEQ ID NO: 19). The term S100b embraces any S100b nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[167] In accordance with the present invention, "vimentin" is a protein that in humans is encoded by the VIM gene. Vimentin is a type III intermediate filament (IF) protein that is expressed in mesenchymal cells. Human vimentine mRNA is represented by the NCBI reference NM_003380 (SEQ ID NO: 19) and the protein by Uniprot No. P08670 (SEQ ID NO: 20). The term vimentin embraces any vimentin nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[168] In accordance with the present invention, "aquaporin 4" also termed “AQP4” is a protein that in humans is encoded by the AQP4 gene. AQP4 belongs to the aquaporin family of integral membrane proteins that conduct water through the cell membrane. Human aquaporine 4 mRNA is represented by the NCBI reference NM_0Q1650 (SEQ ID NO: 21) and the protein by Uniprot No. P55087 (SEQ ID NO: 22). The term aquaporin 4 embraces any aquaporin 4 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[169] In accordance with the present invention, ΈΑΑΤ2" Excitatory amino-acid transporter 2 (EAAT2) also known as solute carrier family 1 member 2 (SLC1A2) is a protein that in humans is encoded by the SLC1A2 gene. SLC1A2/EAAT2 is a member of a family of the solute carrier family of proteins. Human EEAT2 mRNA is represented by the NCBI reference NMJD01195728 (SEQ ID NO: 23) and the protein by Uniprot No. P43004 (SEQ ID NO: 24). The term EAAT2 embraces any EAAT2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[170] In addition to the differentiation into neurons, cells as obtained in step e), d), d.1), d.2) of the methods of the present invention can be differentiated into astrocytes or multilineage cells (MLDC; hMLDC for human MLDC). To achieve this cells obtained in step e), d), d.1), d.2) of the methods of the present invention can be differentiated in a basic medium described herein. The basic medium can for example be a “ground medium”. The ground medium can comprise a basic medium as described herein and can additionally comprise supplements/additives.

[171] For example the ground medium can comprise one or more of (1) DMEM-F12 medium as described herein, (2) an antibiotics as described herein, (3) glutamine as described herein. The ground medium can also comprise all of (1)-(3) listed above. In addition or alternatively the ground medium can comprise serum.

[172] “Serum” as used herein is known to the skilled artesian and different sera are commercially available. Commonly serum is used as growth supplement for cell culture media because of its high content of growth promoting factors. The serum can for example be fetal calf serum (FCS) and fetal bovine serum (FBS), The serum can be FCS. Serum such as for example FCS may be obtained from e.g. Gibco.

[173] The amount of serum to be utilized depends on the intention of the differentiation. If cells are to be differentiated into astrocytes serum can be employed at a concentration of about 0.1 %, 0.25 %, 0.5 %, 0.75 %, 1 %, 1.1 %, 1.25 %, 1.3 % to about 4 %. For the differentiation into astrocytes, serum can also be employed in a concentration of 1 % for example in the ground medium as described herein.

[174] When cells are to be differentiated into MLDCs, serum can be employed at a concentration of about 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 20 % or more. For the differentiation into MLDC serum can also be employed in a concentration of 10 % for example in the ground medium as described herein.

[175] The differentiation of cells (NSCs) in the ground medium to generate astrocytes or MLDCs can be performed for any period of time. For example, cells may be differentiated for 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105,110,115 or more days. Thus, the process of differentiation can take 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or more days. In accordance with the present invention, ceils can be differentiated in ground medium for 45-50 days, 60 days or 80-90 days. For example, the process of differentiation can take 45-50 days. The process of differentiation can also take 60 days. Alternatively, the process of differentiation can also take 80-90 days.

[176] It is further contemplated by the the present invention that when cells (NSCs) are differentiated into astrocytes, differentiation can result in the generation of S100ß positive ceils, with a percentage of 50 %, 60 % 70%, 75 %, 80 %, 85 %, 90 %, 95 %, 97 %, 98 %, 99% or higher of the total amount of cells. It is also envisioned by the methods of the present invention that 100% of the differentiated cells are s100b-positive.

[177] Alternatively or additionally, it is envisioned by the present invention that when cells (NSCs) are differentiated into astrocytes, differentiation can result in the generation of vimentine positive cells, with a percentage of 50 %, 60 % 70%, 75 %, 80 %, 85 %, 90 %, 95 % 97 %, 98 %, 99% or higher of the total amount of cells. It is also envisioned by the methods of the present invention that 100% of the differentiated cells are vimentine-positive.

[178] The present invention also relates to a NSC obtainable by a method of the present invention, it is envisioned by the present invention that the so obtained NSC can express PAX6 with a fold change of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 or more relative to the iPSC (cell or culture from which it has been generated). The NSC of the present invention can, for example, express PAX6 with a fold change of about 16 relative to the iPSC (cell or culture from which it has been generated).

[179] A “fold change” as used herein is a measure describing how much a quantity changes going from an initial to a final value. For example, an initial value of 30 and a final value of 60 corresponds to a fold change of 2, or in common terms, a two-fold increase. Fold change is calculated simply as the ratio of the final value to the initial value, i.e. if the initial value is A and final value is B, the fold change is B/A.

[180] It is also contemplated by the present invention that the NSC expresses SOX1 with a fold change of at least 2, 3, 4, 5, 6, 7, 8, 9 or more relative to the iPSC (cell or culture from which it has been generated). The so obtained NSC can for example express SOX1 with a fold change of about 6 relative to the iPSC (cell or culture from which it has been generated).

[181] It is further contemplated by the present invention that the so obtained NSC can express SOX2 with a fold change of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 or more relative to the iPSC (cell or culture from which it has been generated). The so obtained NSC can express SOX2 with a fold change of about 1.3 relative to the iPSC (cell or culture from which it has been generated).

[182] It is further contemplated by the present invention that the so obtained NSC can express Ki-67 with a fold change of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 or more relative to the iPSC (cell or culture from which it has been generated). The so obtained NSC can express Ki67 with a fold change of about 1.3 relative to the iPSC (cell or culture from which it has been generated).

[183] It is also envisioned by the present invention that the NSC expresses higher and amounts of Ki-67 than the iPSC (cell or culture from which it has been generated). It is also envisioned by the present invention that the NSC expresses higher amounts of SOX2 than the iPSC (cell or culture from which it has been generated). It is also envisioned by the present invention that the NSC expresses higher amounts of nestin than the iPSC (cell or culture from which it has been generated).

[184] It is also contemplated by the present invention that the NSC lacks expression of at least one of Nanog, Klf4, LIN28A, Oct4 or Myc. For example, the NSC can lack expression of of Nanog and/or Oct4.

[185] In accordance with the present invention, "PAX6" also referred to as Paired box protein Pax-6 also known as aniridia type II protein (AN2) or oculorhombin is a protein that in humans is encoded by the PAX6 gene. Pax6 is a transcription factor present during embryonic development. The encoded protein contains two different binding sites that are known to bind DNA and function as regulators of gene transcription. It is a key regulatory gene of eye and brain development. Human PAX6 mRNA is represented by the NCBI reference NM_000280 (SEQ ID NO: 25) and the protein by Uniprot No. P26367 (SEQ ID NO: 26). The term PAX6 embraces any PAX6 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[186] In accordance with the present invention, “SOX1” also referred to as “SOX1 Sex determining region Y-box 1” is a protein that in humans is encoded by the SOX1 gene. SOX1 (for Sex determining region Y-box 1) is a transcription factor in the Sox protein family. Human SOX1 mRNA is represented by the NCBI reference NM_005986.2 (SEQ ID NO: 27) and the protein by Uniprot No. 000570 (SEQ ID NO: 28). The term SOX1 embraces any SOX1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[187] in accordance with the present invention, ,,SOX2” also known as sex determining region Y)-box 2, is a protein that in humans is encoded by the SOX2 gene. SOX2 is a transcription factor that is essential for maintaining self-renewal, or pluripotency, of undifferentiated embryonic stem ceils. Sox2 has a critical role in maintenance of embryonic and neural stem cells. Human SOX2 mRNA is represented by the NCBI reference NM_003106 (SEQ ID NO: 29) and the protein by Uniprot No. P48431 (SEQ ID NO: 30). The term SOX2 embraces any SOX2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[188] In accordance with the present invention, "Nanog" is a protein that in humans is encoded by the NANOG gene. NANOG is a transcription factor critically involved in self-renewal of undifferentiated embryonic stem cells. Human Nanog mRNA is represented by the NCBI reference NM_001297698 (SEQ ID NO: 31) and the protein by Uniprot No. Q9H9S0 (SEQ ID NO: 32). The term Nanog embraces any Nanog nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[189] in accordance with the present invention, "Klf4" also known as Kruppei-like factor 4, is a protein that in humans is encoded by the KLF4 gene. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Human Klf4 mRNA is represented by the NCBI reference NM_004235.4 (SEQ ID NO: 33) and the protein by Uniprot No. 043474 (SEQ ID NO: 34). The term Klf4 embraces any Klf4 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[190] In accordance with the present invention, "LIN28A“ is a protein that in humans is encoded by the LIN28 gene. LIN28 is thought to regulate the self-renewal of stem cells. Human LIN28A mRNA is represented by the NCBI reference NM_024674.4 (SEQ ID NO: 35) and the protein by Uniprot No. Q9H9Z2 (SEQ ID NO: 36). The term LIN28A embraces any LIN28A nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[191] In accordance with the present invention, "Oct4" also known as octamer-binding transcription factor 4) also known as POU5F1 (POU domain, class 5, transcription factor 1) is a protein that in humans is encoded by the POU5F1 gene. Oct-4 is a homeodomain transcription factor of the POU family. This protein is critically involved in the self-renewal of undifferentiated embryonic stem cells. Human Oct4 mRNA is represented by the NCBI reference NM_001173531 (SEQ ID NO: 37) and the protein by Uniprot No. Q01860 (SEQ ID NO: 38). The term Oct4 embraces any Oct4 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[192] In accordance with the present invention, "Myc" also known as c-Myc is a protein that in humans is encoded by the MYC gene. This protein is a regulator gene that codes for a transcription factor. Human Myc mRNA is represented by the NCBI reference NM_002467 (SEQ ID NO: 39) and the protein by Uniprot No. P01106 (SEQ ID NO: 40). The term Myc embraces any Myc nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[193] In accordance with the present invention, “nestin" Nestin is a protein that in humans is encoded by the NES gene. Nestin is a type VI intermediate filament (IF) protein. Human nestin mRNA is represented by the NCBI reference NMJ306617 (SEQ ID NO: 69) and the protein by Uniprot No. P48681 (SEQ ID NO: 70). The term nestin embraces any nestin nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[194] It is further contemplated by the present invention that the NSCs obtained by methods of the present invention can be passaged for more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 23 or more times. Additionally or alternatively the NSCs obtained by a method of the present invention can undergo about 1 population doubling in one passage. To calculate population doublings, cells can passaged once they have a confluency of between 70% to 80%.

[195] The “population doubling level (PDL)” as used herein refers to the total number of times the cells in the population have doubled since their primary isolation in vitro. This is usually an estimate rounded off to the nearest whole number. A formula to use for the calculation of population doublings is as follows: n = 3.32 (log UCY - log I) + X, where n = the final PDL number at end of a given subculture, UCY = the cell yield at that point, I = the cell number used as inoculum to begin that subculture, and X = the doubling level of the inoculum used to initiate the subculture being quantitated. For example, the NSCs obtained by a method of the present invention can undergo about one population doubling in one passage. Thus, in 20 passages cells can undergo about 20 population doublings. This is also shown in Figure 9a of the present application.

[196] The present invention also relates to a neuron obtainable by methods of the present invention.

[197] In addition, the present invention relates to an astrocyte obtainable by a method of the present invention.

[198] The present invention also relates to a NSC of the present invention, a neuron of the present invention or an astrocyte of the present invention for use in therapy. Therapy may for example comprise transplantation of a NSC of the present invention, a neuron of the present invention or an astrocyte of the present invention into a subject. However, therapy may also comprise administering a ceil culture medium as described herein, in which cells have been grown/maintained/differentiated. This media are also referred to as conditioned media. For example, cells/media may be transplated/administered into the brain or areas of the peripheral ervous system of the subject.

[199] When cells/culture media/preparations/pharmaceutical compositions as disclosed herein are transplanted into a subject, it is further envisioned by the present invention that cells are pre-differentiated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 days in a differentiation medium as described herein (neuronal/gound mdium) before transplantation. For example, cells may be pre-differntiated for 7 days before transplantation. This means that cells to be transplantated do not need not to be fully differentiated into neurons and/or glial cells such as astrocytes at the time point of differentiation. Rather it is sufficient to initially push these cells ino a certain differentiation direction before transplantation.

[200] The present invention also relates to a NSC of the present invention, a neuron of the present invention or an astrocyte of the present invention for use in treating a disease. The disease can for example be a neurodegenerative disease as described herein.

[201] As such the term “treating” or “treatment” includes administration of cell and/or conditioned medium as described herein preferably in the form of a medicament, to a subject suffering from a diease comprising a need for the purpose of ameliorating or improving symptoms. Similarly included is the administration of a cell and/or conditioned medium as described herein preferably in the form of a medicament, to a subject suffering from a diease such as a neurodegenerative disase for the purpose of ameliorating or improving symptoms.

[202] The present invention also relates to a pharmaceutical composition comprising a NSC of the present invention, neuron of the present invention or astrocyte of the present invention.

[203] In addition, the present invention relates to a pharmaceutical composition of of the present invention, NSC of the present invention, neuron of the present invention or astrocyte of the present invention for use in therapy/treatment of a disease.

[204] The present invention further relates to a preparation obtainable by a method of the present invention.

[205] The present invention also relates to a preparation comprising a NSC of the present invention, neuron of the present invention or astrocyte of the present invention. The term "preparation” relates to a purification/isoiation/ recovery of a NSC of the present invention, neuron of the present invention or astrocyte of the present invention, from the medium and/or from a cell extract. The term preparation can also comprise a cell present in any of the method steps of the present invention. For example, a preparation can also comprise a ceil present in step d), d.1), d.2) of the present invention. The preparation can also contain culture medium such as any of the media and conditioned media as described herein.

[206] Also, the present invention relates to an in vitro method or test system, wherein the method or test system comprises (i) NSCs of of the present invention, (ii) neurons of the present invention or (iii) astrocytes of the present invention. ''"[207] The in vitro method or test system of the present invention can be for use in testing efficiency or toxicity of a molecule of interest in therapy, wherein the method or test system further comprises (iv) contacting the NSCs, neurons and or astrocytes with a molecule of interest. Moreover, the in vitro method or test system of the present invention can : further comprise (iv) correlating the result obtained to the efficiency or toxicity of a molecule of interest in therapy/treatment of disease.

[208] The compound of interest may for example be a binding protein such as an antibody-or antibody molecule,i'a small molecule, a chemical compound, siRNA, mRNA, miRNA or any other compound.

[209] It is also contemplated by the present invention that the in vitro method or test sÿstem of the present invention can be used in screening, expression profiling or disease modeling.

[210] The present invention also relates to a method of treating a disease, optionally a heurodegenerative disease, in a subject, comprising administering a therapeutically effective amount of a NSC of the present invention, neuron of the present invention or astrocyte of thè present invention to said subject.

[211] The “therapeutically effective amount’ for each cell type and the pharmaceutical composition can vary with factors including but not limited to the activity of the cells used, stability of the cells in the patient's body, the severity of the conditions to be alleviated, the age and sensitivity of the patient to be treated, adverse events, and the like, as will be apparent to a skilled artisan. The amount of administration can be adjusted as the various factors change over time.

[212] The present invention also relates to a use of a NSC of any one of the present invention, neuron of the present invention or astrocyte of the present invention for the preparation of a medicament.

[213] In addition, the present invention relates to a use of a NSC of the present invention, neuron of the present invention or astrocyte of the present invention in a method of treating a disease optionally a neurodegenerative disease.

[214] The skilled person is aware of suitable methods of determining whether one or more of the above recited markers/polypeptides/nucleic acid molecules are expressed by the cells obtained by the methods of the present invention. Exemplary methods are also described in the Examples herein. Such methods include, without being limiting, determining the expression of a marker on the amino acid (polypeptide) level as well as on the nucleic acid molecule level. The present invention also envisiones that nucleic acid molcules encoding proteins as described herein, as well as RNA and proteins as described herein can be detected by e.g. RNA and protein analysis. For example, the skilled artesian knows how to determine if a cell expresses a certain marker. For example, expression of a certain molecule/marker can be determined by RNA and/or protein analysis.

[215] Methods for the determination of expression levels of a marker on the amino acid level include but are not limited to immunohistochemical methods as described in the appended examples but also e.g. western blotting or polyacrylamide gel electrophoresis in conjunction with protein staining techniques such as Coomassie Brilliant blue or silver-staining. Also of use in protein quantification is the Agilent Bioanalyzer technique. Further methods of determination include, without being limiting, cell sorting approaches such as magnetic activated cell sorting (MACS) or flow cytometry activated cell sorting (FACS) or panning approaches using immobilised antibodiesm as described for example in Dainiak et al. (Adv Biochem Eng Biotechnol. 2007;106:1-18). Methods for determining the expression of a protein or the nucleic acid level include, but are not limited to, northern blotting, PCR, RT-PCR or real time PCR as well as techniques employing microarrays. All these methods are well known in the art and have been described in part in the appended examples.

[216] All of the definitions provided herein above for markers expressed by the cells in accordance with the invention, in particular the methods referred for determining the presence or absence of marker expression, apply mutatis mutandis to these markers that are lacking expression in the cells in accordance with the invention.

[217] It is further envisioned by the present invention that also variants and fragments of the markers as described herein can be detected.

[218] With regard to the term “nucleic acid molecule” when used herein encompasses any nucleic acid molecule having a nucleotide sequence of bases comprising purine- and pyrimidine bases which are comprised by said nucleic acid molecule, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences can include DNA, cDNA, genomic DNA, RNA, both sense and antisense strands. The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

[219] A variety of modifications can be made to DNA and RNA; thus, the term "nucleic acid molecules" can embrace chemically, enzymatically, or metabolically modified forms. “Modified" bases include, for example, trityiated bases and unusual bases such as inosine.

[220] The term “polypeptide” when used herein means a peptide, a protein, or a polypeptide, which is used interchangeable and which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. Also encompassed by the invention are amino acids other than the 20 gene-encoded amino acids, such as selenocysteine.

[221] The term polypeptide also refers to, and does not exclude, modifications of the polypeptide. Such modifications are known to the skilled artesian.

[222] A “variant” of a polypeptide when used herein encompasses a polypeptide wherein one or more amino acid residues are substituted. For example, the substitution can be a conservative substitution compared to said polypeptide or to a polypeptide as depicted in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 68, 70. The variant can however still have the same functional properties as any of the polypeptides of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 68, 70. Such variants include insertions, inversions, repeats, and substitutions selected according to general rules known in the art which have no effect on the activity of the polypeptide compared to e.g. a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 68, 70.

[223] A “variant” of a nucleic acid molecule of the present invention encompasses a nucleic acid molecule comprising a mutation. The mutation can be present with regard to any of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 67, 69. Such mutations can include one or more point mutations, such as 1, 2, 5, 10, 15, 20, 50 or more point mutations. A variant can also comprise insertions (addition of one or more nucleotides to the DNA/RNA), such as 1, 2, 3, 5, 6, or more insertions. Both, point mutations and insertions can be selected according to general rules known in the art, which can have no effect on the activity of the nucleic acid molecule compared to e.g. a nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21,23, 25, 27, 29, 31, 33, 35, 37, 39, 67, 69.

[224] Similarly, a “fragment” as used herein can be any nucleic acid molecule or polypeptide which comprises a deletion of 1,2, 3, 4, 5,10, 20, 30 or more amino acid residues of any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 68, 70. or a deletion of more than 1, 2, 3, 4,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or more nucleic acid bases compared to a nucleic acid molecule of any of SEQ ID NO: 1,3, 5, 7, 9,11,13,15,17,19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 67, 69. The fragment can however still have the same functional properties as any of the polypeptides of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 68, 70 or the nucleic acid molecules of SEQ ID NO: 1,3, 5, 7, 9,11,13,15,17,19, 21,23, 25, 27, 29, 31,33, 35, 37, 39, 67, 69.

[225] Given that also variants and fragments of the markers (polypeptides, nucleic acid molcules) as described herein are encompassed by the present invention, the present invention also encompasses detection of sequences which have a sequence identity of 80 %, 85 %, 90 %, 95 %, 97 %, 99 % or 100 % with any of the polypeptides/nucleic acid molecules of any of SEQ ID NO: 1-40, 67-70.

[226] In accordance with the present invention, the term “identical” or “percent identity" in the context of two or more nucleic acid molecules or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 95 %, 96 %, 97 %, 98 % or 99 % identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 80 % to 95 % or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

[227] Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word size (W) of 28, an expectation (E) of 10, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, and an expectation (E) of 10. Furthermore, the BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) can be used.

[228] For example, BLAST2.0, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments.

[229] In general, for the detection of DNA or RNA such as mRNA it may be useful to utilize one, two, three or more oligonucleotides (also called primers), which specifically hybridize to a marker as described herein or fragments or variants thereof. Such oligonucleotides can have a length of 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, 20, 22, 25, 30, 40 or more nucleic acid bases. Knowing the nucleic acid sequence of a marker as described herien (e.g. SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 67, 69) various oligonucleotide primers spanning the locus/RNA or HPV DNA or RNA may be designed e.g. in order to amplify the genetic material by Polymerase Chain Reaction (PCR). For example such a oligonucleotide can have a sequence of any of SEQ ID NO: 41-66.

[230] Exemplary means to detect a marker/protein as described herein can include suitable binding proteins as described herein.

[231] The present invention is further characterized by the following items: [232] 1. Method for obtaining a neural stem cell (NSC), the method comprising a) optionally obtaining/providing induced pluripotent stem ceils (iPSCs); b) cultivating said iPSCs in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; and (iii) an antioxidant; and e) maintaining the cells obtained in d) in a medium comprising (i) a FGF signaling activator; (ii) an EGF signaling activator; and (iii) a LIF signaling activator, and thereby obtaining a NSC.

[233] 2. The method of item 1, wherein the NSC is a human NSC (hNSC).

[234] 3. The method of item 1 or 2, wherein the ÎPSC is a human iPSC (hiPSC).

[235] 4. The method of any one of items 1-3, wherein the iPSC has been obtained from a fibroblast.

[236] 5. The method of any one of items 3, wherein the fibroblast has been obtained from a human.

[237] 6. The method of any one of items 1-5, wherein activin/TGF-ß inhibitor is an inhibitor of the TGF-ß type I receptor activin receptor-like kinase(s).

[238] 7. The method of claim 6, wherein the activin/TGF-ß inhibitor inhibits ALK5, ALK4 and/or ALK7.

[239] 8. The method of any one of items 1-7, wherein the activin/TGF-ß inhibitor is selected from the group consisting of A-83-01, D4476, GW788388, LY364947, R268712, SB-431542, SB-505124, SD208, SB-525334 and ALK5 Inhibitor II (CAS: 446859-33-2).

[240] 9. The method of item 8, wherein the activin/TGF-ß inhibitor is SB-431542.

[241] 10. The method of any one of items 1-9, wherein the canonical WNT-signaling activator is selected from the group consisting of Norrin, R-spondin 2 and Wnt protein.

[242] 11. The method of any one of items 1-10, wherein WNT-signaling is activated by blocking Axin or APC e.g. via siRNA.

[243] 12. The method of any one of items 1-11, wherein WNT-signaiing is activated by the addition of a GSK-3 inhibitor.

[244] 13. The method of item 11, wherein the GSK-3 inhibitor is selected from the group consisting of CHIR 99021, SB-216763, 6-bromoindirubin-3'-oxime, Tideglusib, GSK-3 inhibitor 1, AZD1080, TDZD-8, TWS119, CHIR-99021, CHIR-98014, SB 415286, SB 216763, LY2090314, AR-A014418 and IM-12, preferably CHIR 99021.

[245] 14. The method of item 12, wherein the GSK-3 inhibitor is CHIR 99021.

[246] 15. The method of any one of items 1-14, wherein the BMP signaling inhibitor is selected from the group consisting of chordin, noggin, DMH1 (CAS 1206711-16-1), K 02288, dorsomorphin and LDN 193189.

[247] 16. The method of item 15, wherein the BMP signaling inhibitor is dorsomorphin.

[248] 17. The method of any one of items 1-16, wherein the SHH-pathway activator is selected from the group consisting of purmorphamine, SHH, SAG Analog and Gli-2.

[249] 18. The method of item 17, wherein the SHH-pathway activator is purmorphamine.

[250] 19. The method of any one of items 1-18, wherein the medium in b) comprises DMEM medium.

[251] 20. The method of item 19, wherein the DMEM medium is a knockout DMEM medium.

[252] 21. The method of any one of items 1-20, wherein the medium in b) further comprises knockout serum replacement (knockout SR).

[253] 22. The method of any one of items 1-21, wherein the medium in b) further comprises non-essential amino acids (NEAA).

[254] 23. The method of item 22, wherein the non-essential amino acids comprise at least one of arginine, asparagine, glutamine, glycine, alanine, cysteine, aspartic acid, glutamamic acid, proline, tyrosine and serine.

[255] 24. The method of item 23, wherein one or more of the amino acids are present as L-stereoisomers.

[256] 25. The method of any one of items 1-24, wherein the medium in b) further comprises a reducing agent.

[257] 26. The method of any one of items 1-25, wherein the reducing agent is selected from the group consisting of ß-mercaptoethanol, DL-Dithiothreitol (DTT), 2-Mercaptoethylamine-HCI, TCEP, TCEP-HCI or cysteine-HCl.

[258] 27. The method of item 26, wherein the reducing agent is ß-mercaptoethanol.

[259] 28. The method of any one of items 1-27, wherein the medium in b) further comprises antibiotics.

[260] 29. The method of any one of items 1-28, wherein the medium in c) and/or d) is a N2B27 medium.

[261] 30. The method of any one of items 1-29, wherein the medium in c) and/or d) comprises DMEM-F12 medium.

[262] 31. The method of any one of items 1-30, wherein the medium in c) and/or d) comprises neurobasal medium.

[263] 32. The method of any one of items 1-31, wherein the medium in c) and/or d) comprises DMEM-F12 medium and neurobasal medium in a proportion of 50:50.

[264] 33. The method of any one of items 1-32, wherein the medium in c) and/or d) further comprises N2 supplement.

[265] 34. The method of any one of items 1-33, wherein the medium in c) and/or d) further comprises B27 lacking vitamin A.

[266] 35. The method of any one of items 1-34, wherein the medium in c) and/or d) further comprises glutamine.

[267] 36. The method of any one of items 1-35, wherein the medium in c) and/or d) further comprises antibiotic.

[268] 37. The method of any one of items 1-36, wherein the antioxidant is selected from the group consisting of ascorbic acid, glutathione, lipoic acid, superoxide dismutase 1, superoxide dismutase 2, superoxide dismutase 3, epigallocatechin gallate, curcumine, melatonin, hydroxytyrosol, ubiquinone, catalase, vitamin E and uric acid.

[269] 38. The method of item 36 or 37, wherein the antioxidant is ascorbic acid.

[270] 39. The method of any one of items 29 or 36, wherein said cells are cultivated in the medium for 48 hours.

[271] 40. The method of any one of items 1-39, wherein said cells are cultivated in the medium of step d) for 96 hours.

[272] 41. The method of item 40, wherein the medium is changed once.

[273] 42. The method of item 41, wherein the medium is changed after 48 hours.

[274] 43. The method of any one of items 40-43, wherein the cells are plated on

Matrigel coated plates, preferably 12-well plates, after 48 hours.

[275] 44. The method of any one of items 1-43, wherein step d) can further comprise a step d.2) comprising culturing the cells obtained in step d) in a medium comprising a FGF signaling activator.

[276] 45. The method of any one of items 1-44, wherein the FGF signaling activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4 and FGF8.

[277] 46. The method of item 45, wherein the FGF signaling activator is FGF2 such as basic FGF2.

[278] 47. The method of any one of items 1-46, wherein step d), further comprises d.2) cultivating said cells obtained in d) in a medium comprising (i) a canonical WNT-signaling activator; (ii) SHH-pathway activator; (iii) an antioxidant; and (iv) an FGF signaling activator.

[279] 48. The method of any one of items 1-47, wherein the EGF signaling activator is selected from the group consisting of epidermal growth factor (EGF), transforming growth factor a (TGFa), amphiregulin (AR), heparin-binding EGF-like growth factor (ΗΒ-EGF), betacellulin (BTC), epigen (EPG) and epiregulin (ERR).

[280] 49. The method of item 48, wherein the EGF signaling activator is EGF.

[281] 50. The method of any one of items 1-49, wherein the LI F signaling activator in step e) is LIF, preferably human LIF.

[282] 51. The method of any one of items 1-50, wherein the medium in e) comprises a medium as defined in any one of items 29-34, 36.

[283] 52. The method of any one of items 1-51, wherein the medium in e) further comprises B27 with vitamin A.

[284] 53. The method of any one of items 1-52, wherein the method further comprises differentiation of the cells obtained in step e) into neurons or glial cells, preferably astrocytes.

[285] 54. The method of any one of items 1-53, wherein the method further comprises differentiation of the cell obtained in step e) into a (i) Map2; (ii) TH; (iii) GABA; (iv) vGlut; (v) dcx; (vi) synaptophysin; (vii) postsynaptic density protein 95 (PSD 95); (viii) Tuj1 and/or

(ix) NCAM expressing cell.

[286] 55. The method of any one of items 1-53, wherein the method further comprises differentiation of the cell obtained in step e) into a (i) GFAP; (ii) S100b; (iii) vimentin; (iv) aquaporin 4; and/or (v) EAAT2 expressing cell.

[287] 56. The method of item 53 and 54, wherein the differentiation comprises culturing cells obtained in step e) in a medium as defined in any one of items 29-36.

[288] 57. The method of any one of items 53, 55 and -56, wherein the differentiation comprises culturing cells obtained in step e) in a medium comprising (i) at least two different neurotrophins; and (ii) an antioxidant.

[289] 58. The method of item 57, wherein the at least two different neurotrophins are selected from the group consisting of BDNF, NGF, GDNF, NT-3, NT-4, or CNTF, preferably GDNF and BDNF.

[290] 59. The method of any one of items 53, 54, 56-58, wherein the medium further comprises a (iii) activin/transforming growth factor-ß (TGF-ß) signaling activator.

[291] 60. The method of item 59, wherein the activin/TGF-ß signaling activator is TGF-ß 1, TGF-ß 2, TGF-ß 3, activin A, activin B, activin A/B or nodal, preferably TGF-ß 3- [292] 61. The method of any one of items 53, 54, 56-60, wherein the medium further comprises a cAMP analogue.

[293] 62. The method of claim 61, wherein the cAMP analogue is selected from the group consisting of forskoiin, 8-(4-ehloro-phenylthio)-2'-0-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), 8-Chloro-cAMP (8-CI-cAMP), Bucladesine, Rp-adenosine .3., 5.,-cyclic monophosphorothioate sodium salt (Rp-cAMPS), Sp-8-hydroxyadenosine .3., 5.,-cyclic monophosphorothioate sodium salt (Sp-80H-cAMPS) and Rp8-hydroxyadenosine .3., 5.,-cyclic monophosphorothioate sodium salt (Rp-80H-cAMPS) or dbcAMP, preferably the cAMP analogue is dbcAMP.

[294] 63. The method of any one of claims 53, 54, 56-62, wherein the process of differentiation takes at least 4 weeks.

[295] 64. The method of any one of items 53, 54, 56-63, wherein differentiation results in the generation of GABAergic cells, with a percentage of 10 %, 15 %, 20 %, 25 %, 30 %, 35 % or higher of the total amount of cells.

[296] 65. The method of any one of items 53, 54, 56-64, wherein differentiation results in the generation of glutamatergic cells, with a percentage of 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 % or higher of the total amount of cells.

[297] 66. The method of any one of items 53, 54, 56-65, wherein differentiation results in the generation of dopaminergic cells, with a percentage of 3 %, 7 %, 10 %, 12 % or higher of the total amount of cells.

[298] 67. The method of any one of items 53, 54, 56-66, wherein differentiation does virtually not result in the generation of astrocytes, wherein preferably the culture comprises less than 20 %, 15 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 % astrocytes of the total amount of cells.

[299] 68. The method of item 53 or 55, wherein the differentiation comprises culturing the cells obtained in step e) in a medium comprising (i) serum.

[300] 69. The method of item 68, wherein the serum is selected from the group consisting of fetal calf serum (FCS) and fetai bovine serum (FBS), preferably FCS.

[301] 70. The method of item 68 or 69, wherein the serum is present in a concentration between 1 %-9 %, preferably in a concentration of 1 %.

[302] 71. The method of any one of items 68-70, wherein the medium comprises DMEM medium.

[303] 72. The method of item 71, wherein the DMEM medium is a DMEM-F12 medium.

[304] 73. The method of any one of items 68-72, wherein the medium further comprises glutamine.

[305] 74. The method of any one of items 68-73, wherein the medium further comprises antibiotics.

[306] 75. The method of any one of items 53, 55, 68-74, wherein the process of differentiation takes 45 days.

[307] 76. The method of any one of items 53, 55, 68-75, wherein differentiation results in the generation of SlOOß positive cells, with a percentage of 50 %, 60 % 70%, 75 %, 80 %, 85 %, 90 %, 95 % or higher of the total amount of cells.

[308] 77. The method of any one of items 53, 55, 68-76, wherein differentiation results in the generation of vimentin positive cells, with a percentage of 50 %, 60 % 70%, 75 %, 80 %, 85 %, 90 %, 95 % or higher of the total amount of cells.

[309] 78. NSC obtainable by a method as defined in any one of items 1-52.

[310] 79. NSC of item 78, wherein the NSC expresses PAX6 with a fold change of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 or more relative to the iPSC.

[311] 80. NSC of item 78 or 79, wherein the NSC expresses SOX1 with a fold change of at least 2, 3, 4, 5, 6, 7 or more relative to the iPSC.

[312] 81. NSC of any one of items 71-74, wherein the NSC lacks expression of at least one of Nanog, Klf4, LIN28A, Oct4 or Myc.

[313] 82. NSC of any one of items 71-75, wherein the NSC expresses Ki67 with a fold change of at least 1.1 or more relative to the iPSC.

[314] 83. NSC of any one of items 71-76, wherein the NSC expresses S0X2 with a fold change of at least 1.1 or more relative to the iPSC.

[315] 84. NSC of any one of items 71-77, which can be passaged for more than 10, 11, 12, 13, 14,15,16,17,18,19,20, 21,22 or 23 or more times.

[316] 85. Neuron obtainable by a method as defined in any one of items 53, 54, 56-67.

[317] 86. Astrocyte obtainable by a method of any one of items 53, 55, 68-77.

[318] 87. Pharmaceutical composition comprising a NSC of any one of items 78- 84, neuron of item 85 or astrocyte of item 86.

[319] 88. Pharmaceutical composition of item 87, NSC of any one of items 78-84, neuron of item 85 or astrocyte of item 86 for use in therapy.

[320] 89. A preparation obtainable by a method of any of items 1-52.

[321] 90. A preparation comprising a NSC of any one of items 78-84, neuron of item 85 or astrocyte of item 86.

[322] 91. In vitro method or test system, wherein the method or test system comprises (i) NSCs as defined in any one of items 78-84, (ii) neurons as defined in item 85 or (Hi) astrocytes as defined in item 86.

[323] 92. In vitro method or test system of item 91, for use in testing efficiency or toxicity of a molecule of interest in therapy, wherein the method or test system further comprises (iv) contacting the NSCs, neurons and or astrocytes with the molecule of interest.

[324] 93. In vitro method or test system of item 92, wherein the method or test system further comprises (iv) correlating the result obtained to the efficiency or toxicity of the moiecuie of interest in therapy.

[325] 94. In vitro method or test system of any one of items 91-93, for use in screening, expression profiling or disease modeling.

[326] 95. A method of treating a disease, optionally a neurodegenerative disease, in a subject, comprising administering a therapeutically effective amount of a NSC of any one of items 78-84, neuron of item 85 or astrocyte of item 86 to said subject.

[327] 96. Use of a NSC of any one of items 78-84, neuron of item 85 or astrocyte of item 86 for the preparation of a medicament.

[328] 97. Use of a NSC of any one of items 78-84, neuron of item 85 or astrocyte of item 86 in a method of treating a disease, optionally a neurodegenerative disease.

[329] The different seuquences described herein are summarized in the table 1 below.

Table 1. Sequences as described herein.

EXAMPLES EXAMPLE 1: Materials and Methods [330] Cell culture iPSC were detached from plates using dispase (1mg/mL) for 30min at 37 °C with the help of a scraper in case of big colonies. Colonies were collected by sedimentation and resuspended in “EB Medium" (Knockout™ DMEM (Gibco), Knockout™ SR (Gibco), NEAA (Gibco), penicillin/streptomycin (Invitrogen) and ß-mercaptoethanol (Gibco)) supplemented with 10 pMSB-431542 (Ascent Scientific), 1 μΜ dorsomorphin (Tocris), 3 μΜ CHIR 99021 (Axon Medchem) and 0.5 μΜΡΜΑ (Alexis).

[331] 48 hours later, the medium was replaced by N2B27 medium (DMEM-F12 (Gibco)/Neurobasal (Gibco) 50:50 supplemented with 1:200 N2 supplement (Invitrogen), 1:100 B27 supplement lacking vitamin A (Invitrogen), penicillin/streptomycin and glutamine (Invitrogen) supplemented with 10 μΜ SB-431542, 1 μΜ dorsomorphin, 3 μΜ CHIR 99021 and 0.5 μΜ PMA.

[332] After additional 48 hours, the medium was replaced by N2B27 medium supplemented with 3 μΜ CHIR 99021, 0.5 μΜ PMA and 150 μΜ ascorbic acid (Sigma Aldrich).

[333] Two days later, neural tube like structures were disintegrated by pipetting and small pieces were plated on Matrigel (BD Biosciences) coated 12-well plates in N2B27 medium supplemented with 3 μΜ CHIR 99021, 0.5 μΜ PMA and 150 μΜ ascorbic acid.

[334] At day 8, the medium was exchanged by N2B27 medium supplemented with 3 μΜ CHIR 99021, 0.5 μΜ PMA, 150 μΜ ascorbic acid and 20 ng/ml basic (b) FGF2 (Peprotech).

[335] At day 12, cells were detached using dispase and cultivated in hNSC Maintenance Medium on Matrigel coated 10cm dishes. “Human NSC Maintenance Medium” was composed by DMEM HAM’S F12 medium (Gibco) supplemented with 200ng/pl EGF (Peprotech), 200ng/pl bFGF2, N2 supplement, B27 supplement (with vitamin A), glutamine, Penicillin/Streptomycin and hLIF (150U/ml).

[336] Human NSCs were splitted (1:2 ratio) at a confluence of 70-80% by using dispase. At a confluence of 70-80% the hNSC Maintenance Medium was switched to “Neuron differentiation medium” which was composed by N2B27 Medium supplemented with 10 ng/mL BDNF (Peprotech), 10 ng/mL GDNF (Peprotech), 1 ng/mL TGF-ß3 (Peprotech), 200 μΜ ascorbic acid and 500 μΜ dbcAMP (Sigma Aldrich). Cultures were tested/harvested after 4 weeks of neuronal differentiation.

[337] Astrocytic differentiation was induced by switching the hNSC Maintenance Medium to the basic medium/ground medium (DMEM HAM's F12 medium, glutamine, Penicillin/Streptomycin) supplemented with 1% FCS (Gibco) (Conti and Cattaneo (2010) Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176-87; Conti et al. (2005) Niche-independent symmetrical selfrenewal of a mammalian tissue stem cell. PLoS biology 3:e283).

[338] Finally, multilinear differentiation was achieved by replacing the maintenance medium by the basic medium (ground medium) containing 10% of FCS.

[339] Immunoeytochemistry

For immunohistochemical analysis, cells were fixed with 4% paraformaldehyde (PFA) in 120mM phosphate buffer (PBS), pH 7.4, permeabilized with 0.05% Triton X-100 in PBS, blocked with 10% goat serum in PBS and subjected to immunohistochemistry staining with primary and secondary antibodies diluted in the blocking solution.

For immunolabelling the following antibodies at indicated dilutions were used: anti-Nestin (1:400; BD Bioscience), anti-Sox1 (1:100; R&amp;D Systems), anti-Sox2 (1:200; Abeam), anti-Pax6 (1:200; DSHB), anti-Ki67 (1:200; Vector Labs), anti-TuJ1 (1:400; Covance), anti-Map2 (1:200; Millipore), anti-DCX (1:200; Miilipore), anti-GFAP (1:200; Millipore), anti-04 (1:50, Sigma-Aldrich), anti-GABA (1:200,Abeam), anti-vGlutl (1:200; Millipore), anti-TH (1:100, Millipore), anti-Synaptophysin (1:100; Millipore), anti-PSD95 (1:200; Invitrogen), anti-vimentin (1:5000; Abeam), anti-S100B (1:1000; Sigma-AIdrich), anti-aquaporin 4 (AQP4, 1:100; Santa Cruz Biotechnology) and anti-excitatory amino acid transporter 2 (EAAT2, 1:100; Santa Cruz Biotechnology), secondary Alexafluorophore-conjugated antibodies (1:1000, Invitrogen). DNA was stained using Hoechst 33258 (1:10000,invitrogen).

[340] Microarray mRNA was extracted from hiPSCs, hNSCs and derived cells using the RNAeasy kit (Quiagen) following manufacturer’s recommendations. mRNA quantity and purity were determined by using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Additional quality check was performed by the Agilent Bioanalyzer (Agilent). Gene expression profiles were generated using HumanGene 2.0ST arrays according to manufacturer’s recommendations (Affymetrix).

[341] Bioinformatics

Correspondence analysis (CoA) from the TIGR MeV (MultiExperimentViewer; http://www.tm4.org/mev/) expression analysis platform was performed on the whole transcriptome established by the HumanGene 2.0ST arrays after RMA normalization proposed by the Affymetrix [342] Expression Console

GeneOntolgy (GO) analysis was performed on the 1428 transcripts specifically differing hNSCs from 156 parental hiPSCs and from filial hMLDCs by a 2-fold difference. Here, we used the GO-biological process analyzer implemented in the DAVID analysis platform (.http://david.abcc.ncifcrf.gov/) (Huang, Sherman, Lempicki (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44-57). GO terms and associated p-values (0.05 or lower) were then introduced into the REVIGO Webserver (Supek et al. (2011). REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6:e21800) to establish network files. These files were than uploaded to the cytoscape software where the final GO-Network was built up (www.cytoscape.org) (Cline et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nat Protoc 2:2366-82).

[343] RT-qPCR

For quantification analysis, total RNA (mRNAs and miRNAs) was extracted from hiPSCs, hNSCs, human neurons (4 weeks/28 days of differentiation) and human astrocytes (6weeks/42 days of differentiation) by the RNAeasy kit (Quiagen) following manufacturer’s recommendations. Gene expression levels were evaluated by the SYBR-Green Jump Start Taq Ready Mix (Sigma-Aldrich) following manufacturer’s recommendations. Gene-related intensity levels were evaluated upon normalization with GAPDH levels. The following primers were used: GAPDH: GTGGACCTGACCTGCCGTCT (SEQ ID NO: 41), GGAGGAGTGGGTGTCGCTGT(SEQ ID NO: 42) OCT4: CCT C AGIT C ACTGC ACT GTA (SEQ ID NO: 43), CAGGTTTTCTTTCCCTAGCT (SEQ ID NO: 44) NANOG: T G AACCTCAGCTACA AAC AG (SEQ ID NO: 45), TGGTGGTAGGAAGAGTAAAG (SEQ ID NO: 46) SOX2: CCCAGCAGACTTCACAT GT (SEQ ID NO: 47), CCTCCCATTTCCCTCGTTTT (SEQ ID NO: 48) KÏ67: AT ACGT G AACAGG AGCC AG (SEQ ID NO: 49), CCTTGGAATCTTGAGCTTTCTG (SEQ ID NO: 50) SOX1: AATTTTATTTTCGGCGTTGC (SEQ ID NO: 51), TGGGCTCTGTCTCTTAAATTTGT (SEQ ID NO: 52) NESTIN: CTGGAGCAGGAGAAACAGG (SEQ ID NO: 53), TGGGAGCAAAGATCCAAGAC (SEQ ID NO: 54) PAX6: ATGTGTGAGTAAAATTCTGGGCA (SEQ ID NO: 55), GCTTACAACTTCTGGAGTCGCTA (SEQ ID NO: 56) GFAP: CTGCTCAATGTCAAGCTGG (SEQ ID NO: 57), AATGGTGATCCGGTTCTCC (SEQ ID NO: 58) MAP2: GG AG ACAG AG AT G AG AATT CCT (SEQ ID NO: 59), GAATTGGCTCTGACCTGGT (SEQ ID NO: 60) TH: GTGCTAAACCTGCTCTTCTC (SEQ ID NO: 61), TTCAAACGTCTCAAACACCT (SEQ ID NO: 62) SLC6A11 (GABA): CAACAACTGCTACAGGGAC (SEQ ID NO: 63), GAGAAGATGGCAAACCCAG (SEQ ID NO: 64) SLC17A7 (Glutamate transporter): COAT G ACT AAGC AC AAG ACT C (SEQ ID NO: 65), AGATGACACCTCCATAGTGC (SEQ ID NO: 66).

[344] Extraction of intracellular metabolites and Gas Chromatography-Mass Spectrometry

Astrocytes and MLD cultures were cultivated for 6 weeks in 12-well plates and washed with 1 ml of 0.9% NaCI and quenched with 0.2 ml -20 °C methanol. After adding an equal volume of 4 °C cold water, cells were collected with a ceil scraper and transferred in tubes containing 0.2 mi -20 °C chloroform. The extracts were shaken at 1400 rpm for 20 min at 4 °C (Thermomixer Eppendorf) and centrifuged at 16,000xg for 5 min at 4 °C. 0.2 ml of the upper aqueous phase was collected in specific glass vials with micro inserts and evaporated under vacuum at -4°C using a refrigerated CentriVap Concentrator (Labconco). Metabolite derivatization was performed using a Gerstel MRS. Dried polar metabolites were dissolved in 15 μΙ of 2% methoxyamine hydrochloride in pyridine at 40 °C under shaking. After 60 min an equal volume of MTBSTFA was added and held for 60 min at 40°C. 1 μΙ sample was injected into an SSL injector at 270°C in splitless mode. GC/MS analysis was performed using an Agilent 7890A GC equipped with a 30m DB-35MS + 5m Duraguard capillary column. Helium was used as carrier gas at a flow rate of 1.5 ml/min. The GC oven temperature was held at 100 °C for 2 min and increased to 300 °C at 10 °C/min. After 3 min, the temperature was increased to 325 °C. The GC was connected to an Agilent 5975C inert XL MSD, operating under electron ionization at 70 eV. The MS source was held at 230 °C and the quadrupole at 150 °C. The detector was operated in scan mode with mass range m/z 70-800. The total run time of one sample was 25.00 min. All GC/MS chromatograms were processed 203 by using MetaboliteDetector (Hiller et al., (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem; 81(9):3429-39). Mass isotopomer distributions (MIDs) were determined and corrected for natural isotope abundance using MetaboliteDetector (Hiller et al., (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem; 81(9):3429-39). For determination of MIDs the following ions were selected (Wegner et al., (2014) Fragment formula calculator (FFC): determination of chemical formulas for fragment ions in mass spectrometric data. Anal Chem; 86(4):2221-8): Asparatate_3TB D MS : 418-427; Citrate_4TBDMS: 591-601; Glutamate_3TBDMS: 432-442; SerineJTBDMS: 390-397; Glycine_2TBDMS: 246-252; Lactate_2TBDMS:261 -269.

[345] Electrophysiology

For electrophysiological recordings coverslips with hNSC derived neurons were transferred to a recording chamber mounted on an upright microscope (Zeiss, Oberkochen, Germany) and kept in a bath solution containing (in mM): NaC1130, KCI 3, NaHCOa 10, CaCI2 1.5, MgCI2 1, Glucose 11, HERES 10, pH 7.3 with NaOH. Patch pipettes with 2 -4 M were filled with (in mM) K-gluconate 125, KCI 20, EGTA 0.5, MgATP 4, MgCI2 4, NazGTP 0.3, HEPES 10, pH 7.4 with KOH. Measurements were done with an EPC10 amplifier and Patchmaster software (HEKA, Lambrecht, Germany). For puff-application of KCI or glutamate, a pipette with 15 pm tip diameter was placed 60 - 100 pm away from the recorded cell. Pressure ejection was controlled by a pico pump (PV830, WPI, Sarasota, FL).

[346] Measurement of Glutamate Uptake

Astrocytes DIV 80-90 and HEK 293 cells were incubated for 10 minutes at 37°C in basic media containing 50 ,pM of L-glutamic acid (Sigma). Glutamate uptake was measured using a colorimetric kit (abeam) according to manufacturer’s instructions. Absorbance measurements were normalized to total protein per culture well.

[347] Mice 12 week old male NOD/SCID mice were kept under standard conditions according to governmental rules and regulations. All experiments have been conducted according to the German Animal Welfare Act and have been approved by the responsible authorities (Landesamt fiir Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen). For each transplantation approach, 3 animals were used.

[348] hNSC transplantation

For stereotactic injections, mice were under deep anesthesia (intraperitoneal injection of 17 μΜ of 2.5% 230 Avertin per gram of body weight) and fixed into a stereotactic frame (Kopf). Three microlitres of cell suspension (in total: 1.105 -2.105 cells) were injected into the lateral ventricle over 5 minutes using a Hamilton 7005KH 5pl syringe. Following stereotactic coordinates in relation to bregma were used: anteroposterior: 1,4 mm, mediolateral: ±0,84 mm, dorsoventral: -2,5 below skull. In order to control the hNSC fate after transplantation, we introduced an in vitro predifferentiation step in our experimental set-up. At the first step, passage 6 or higher hNSCs were splitted in a 1:2 ratio. Three days later, the hNSC maintenance medium in one of the dishes was changed to neuronal differentiation medium, while the second dish was subjected to astroglial differentiation by replacing the maintenance medium by glial differentiation medium. After one week of pre-differentiation, 1.106 to 2.106 cells were transplanted into the brain of adult NOD/SCID mice. The cell fate was analyzed 6 additional weeks later.

[349] Perfusion, sectioning and immunohistochemistry

Mice under deep anesthesia were perfused with 50 ml PBS following 50 ml 4% PFA /1 PBS solution. After dissection, isolated brains were post-fixed in 4% PFA /1 PBS solution over night at 4°C. 40 μηη sagittal brain sections were cut using a Vibratom (Leica VT 1200 S). Free-floating sections were permeabilized in Tris-buffered saline solution with 0.1M Tris, 150mM NaCI, pH 7.4 / 0.5% Triton-X 100 / 0.1% Na-Azide / 0.1% Na-Citrate / 5% normal goat serum (TBS+/+/+) for at least 1 h. The primary antibodies anti-Hu Nuclei (1:200; Millipore), anti-DCX (1:400; Abeam), anti-TuJ1 (1:600; Covance) and anti-GFAP (1:100; Millipore) were diluted in TBS+/+/+ and incubated for 48 h on a shaker at 4°C. For immunofluorescence staining, secondary Alexa-fiuorophore conjugated antibodies (Invitrogen) and Hoechst 33258 (1:10000, Invitrogen) were used. Sections were analyzed with a Zeiss LSM 710 confocal microscope.

[350] Statistical analysis

Data presented are means ± SEM. Statistical significance was tested with Sigma Plot software. Results were denoted statistically significant when p values were <0.05; number (n) of samples/repeats are given in the Results and Figure legends [351] EXAMPLE 2 Newly generated hNSCs conserved seif-renewing characteristics

Human iPSCs were maintained on on mouse embryonic fibroblasts (MEFs) or under feeder free conditions, and treated them according to the scheme in Figure 1a. As described previously (Reinhardt et al. (2013) Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 8:e59252), the neural induction of embryonic bodies from hiPSC (Figure 1b, c) was achieved by inhibition of BMP and TGFß signaling (Chambers et al. (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275-80; Kim et al. (2010) Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev 6:270-81). Simultaneously, CHIR99021 and Purmorphamine were administered to stimulate the canonical WNT- and SHH-pathways (Sineva and Pospelov (2010) Inhibition of GSK3beta enhances both adhesive and signaling activities of beta-catenin in mouse embryonic stem cells. Biol Cell 102:549-60; Wu et al. (2004) Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem Biol 11:1229-38).

[352] After three days, these neural-induced embryonic bodies (Figure 1c) were cultivated under defined conditions for three more days until neural tube like structures appear (Figure 1d). Neural rosette-like structure formation was induced by supplementing the culture medium with bFGF2 (Conti and Cattaneo (2010) Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176-87) for four additional days (Figure 1e). After re-plating, the cells were cultured in presence of EGF, bFGF, N2, B27 and hLIF. Following the first passages, the initially heterogeneous cell clusters adopted a homogeneous morphology (Figure 1f and g). Induction of differentiation into either the neuronal or the glial lineage (details see below) induced further changes in morphology.

[353] One key characteristic of neural stem cells is their extensive self-renewal potential. This ability was evaluated by measuring the cell number over the first 21 passages following their generation. The resulting exponential growth curve showed stable proliferation rates over the 21 passages analyzed (Figure 9a).

[354] To confirm that generated hNSCs preserved self-renewing characteristics, the presence of the stem cell markers Nestin, Sox2, Sox1 and Pax6 at early (passage 3 and 6) and late (passage 27) passages (Figure 1 h-p) was evaluated. While Nestin, Sox1 and Sox2 showed very similar expression patterns, Pax6 displayed cytoplasmic labeling at lower passage numbers and more nuclear labeling at higher passages (Figure 1n-p), which is in agreement with data on brain development (Yan et al. (2010) Sumoylation activates the transcriptional activity of Pax-6, an important transcription factor for eye and brain development. Proc Natl Acad Sci U S A 107:21034-9).

[355] Finally, hNSCs maintained proliferation characteristics, as demonstrated by the positive labeling of the cell cycle marker Ki67 across passages (Figure 1q-s). These results demonstrate that generation and maintenance of hiPSC-denved hNSCs was achieved robustly, and that hNSCs maintained self-renewing characteristics over numerous passages.

[356] EXAMPLE 3 hNSCs revealed distinct gene expression profile

In order to further characterize the generated hNSCs, the mRNA transcriptome of hiPSCs, and hNSCs derived from these progenitors was compared (Figure 2). Additionally, differentiated cells, derived from these hNSCs were included in the analysis. Since the induced differentiation was undirected, multiple cell types were present in this population; consequently this was considered a multilineage differentiation (hMLDCs) (Figure 8). To minimize the interlineage differences existing between iPSC-lines generated from different individuals (Cahan and Daley (2013) Origins and implications of pluripotent stem cell variability and heterogeneity. Nat Rev Mol Cell Biol 14:357-68), the comparison was based on hiPSCs, hNSCs and hMLDCs derived from the same individual. The transcriptome-based correspondence analysis (CoA) of the three cell types demonstrated that hiPSCs, hNSCs and hMLDCs were characterized by distinct molecular expression signatures (Figure 2a).

[357] The paired comparison of hiPSCs and of hNSCs in a scatter blot highlights that neural-stem-cell-genes (Pax6, Sox1 and Nestin) were higher expressed in hNSCs when compared to their parental hiPSCs. Similarly, hNSCs showed a significant downregulation of the pluripotency genes Lin28a, Nanog and Oct4 (Figure 2b and Figure 9b). RT-qPCR analysis comparing the expression levels of the pluripotency genes Oct4 and Nanog as well as the neural-stem-cell-gene Nestin between hiPSCs and hNSCs confirmed these data (Figure 2c). Interestingly, the neural stem cell specific marker Nestin was found nearly absent in hiPSCs when compared to hNSCs. The marker Sox2, predicted to be strongly expressed within pluripotent (Takahashi and Yamanaka (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-76) and neural stem cells (Qu and Shi (2009) Neural stem cells in the developing and adult brains. J Cell Physiol 221:5-9), as well as the proliferation marker Ki67, exhibited similar expression levels within hiPSCs and hNSCs).

[358] Further 1428 transcripts that differed at least twofold between hNSCs, hiPSCs and hMLDCs were isolated. The resulting Gene Ontology (GO) analysis revealed that genes specifically expressed in hNSCs were mainly involved in the biological processes development, differentiation, neurogenesis and cell adhesion (Figure 2d), in order to better decipher key mechanisms important for hNSC maintenance, a network analysis on the “biological-process GO-terms” that were statistically associated with the 1428 enriched hNSCs genes was performed. Within the GO-Network, it was observed that the GO terms clustered as sub-networks that may be outlined as ‘Tissue development”, “Cell growth and differentiation”, “Neurogenesis and neuron development", “Regulation of catabolism and metabolism” and “Regulation of BMP signaling pathway” (Figure 2e). By this approach a unique expression signature that clearly distinguished hNSCs from less differentiated hiPSCs as well as from the more differentiated hMLDCs was defined.

[3593 Thus, a novel protocol for the generation of hNSCs from hiPSCs has been described in Examples 2 and 3. This fate transition is achieved by the chronological administration of media with defined compositions. The here presented protocol is very robust and independent of any sorting method. In contrast to other protocols for the generation of human neural precursor cells, no small molecules were needed for keeping hNSCs under self-renewing condition.

[360] The here described cells conserved the two main characteristics of neural stem cells, i.e. self-renewing and multi-linear differentiation capacities. Since they grow in homogenous cultures, these cells are an attractive tool for expression profiling, disease modeling and high content screenings. Besides their ability to differentiate into functional neurons, hNSCs differentiated into glial cells within a relatively short time period.

[361] A gene expression profile that distinguishes hNSC from less differentiated hiPSC and more differentiated neurons and astrocytes has also been shown in Example 3. Since these profiles were generated from cells from the same individual (starting iPSC line), the degree of comparability is very high and the derived signatures should purely represent the differentiation status.

[362] EXAMPLE 4 hNSCs differentiated into mature astrocytes

In the next step we wanted to demonstrate the ability of hNSCs to differentiate into astrocytes. The astrocytic differentiation medium consisted of the basic cultivation medium (ground medium) supplemented with 1%FCS. The most commonly used marker protein for astrocytic differentiation is GFAP. However, astrocytes positive for GFAP are considered to show a reactive phenotype while astrocytes negative for GFAP show a quiescent phenotype with protoplasmic morphology, as described previously (Roybon (2013) Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Cell Rep 4:1035-48). Therefore the additional markers S100B and vimentin were used to investigate astrocyte differentiation.

[363] After 45-50 days of differentiation, it was observed that nearly 100% of the cells were expressing S100B (Figure 3a). After 60 days, some astrocytes showed GFAP immunoreactivity while all the cells expressed vimentin (Figure 3b-d). S100B was expressed in 100% of the cells also at this time point (Figure 3f-h). The contamination with neurons was negligible as shown by the Tuj1 staining (Figure 5e).

[364] After 60 days of differentiation, some cells still expressed the proliferation marker Ki67 (Figure 5g), indicating that they are able to proliferate, a capacity shared with primary astrocytes (Bardehle et al. (2013) Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation. Nat Neurosci 16:580-6; Lundgaard et al. (2013) White matter astrocytes in health and disease. Neuroscience. 276:161-73). In support, loss of neural stem cell- and proliferation markers with simultaneous increase of astrocytic markers was also observed by RT-qPCR. Nestin and KÎ67 were significantly downregulated upon 45 days of glial differentiation and we observed a massive increase in the expression levels of GFAP gene (Figure 3i).

[365] Remarkably, after 60 days of differentiation all astrocytes also expressed a basal level of AQP4 (Figure 4a), a marker for the main water channel in the perivascular membranes, typically present in astrocytic endfeet in brain tissue. Additionally, they expressed the glutamate transporter EAAT2 (Figure 4b) that is used by astrocytes in situ to remove glutamate from the extracellular space. This is a key function of mature astrocytes (Huang and Bergles (2004) Glutamate transporters bring competition to the synapse. Curr Opin Neurobiol; 14(3):346-52) that was also confirmed by a glutamate uptake assay (Figure 4c). Astrocytes differentiated for 80-90 days were able to transport glutamate intracellularly. We assessed HEK 293 as a cell type with no specific glutamate intake ability (p=0,016,Student t test).

[366] The results demonstrate that by replacing the hNSC maintenance medium with the adequate differentiation medium it was possible to induce a robust and homogenous differentiation into astrocytes. The homogeneous immunoreactivity toward AQP4 and EAAT2 and the ability to transport glutamate suggest that hNSC-derived astrocytes reached a certain level of maturity and functionality.

[367] Astrocytes showed increased pyruvate carboxylase activity and reduced serine biosynthesis. A hallmark of astrocytes is the presence and the relative high activity of the metabolic enzyme pyruvate carboxylase (PC) (Gamberino et al. (1997) Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes. J Neurochem 69: 688 2312-2325). PC catalyzes the carboxylation of pyruvate to the tricarboxylic acid cycle (TCA) intermediate oxaloacetate. This reaction is important during anabolic processes to replenish the TCA cycle with oxaloacetate. To investigate the presence and the activity of pyruvate carboxylase in hNSCs derived astrocytes uniformly labeled [U-13 CJglucose was used to monitor the fate of glucose in astrocytes. Application of this stable isotope tracer results in isotopic enrichment in all metabolites downstream of the tracer (e.g. metabolites of the glycolysis and the TCA cycle). The isotopic enrichment can be measured using Gas Chromatography/Mass Spectrometry. In case pyruvate is oxidized via pyruvate dehydrogenase to acetyl-coA the succeeding citrate molecule shows a mass enrichment by two (M2 isotopologue). In case of pyruvate carboxylation by PC the succeeding citrate molecule shows a mass enrichment by three (M3 isotopologue). M5 citrate isotopologues result from both, pyruvate dehydrogenase and PC activity (Figure 5a).

[368] Using this approach, pure astrocyte cultures were compared with multi-lineage differentiation cultures (MLDCs) and increased PC activity in the pure astrocyte culture was observed (17 % enrichment vs. 4 % enrichment of M3 aspartate) (Figure 5b). Also increased M3 and M5 isotopologue abundance of citrate originating from M3 oxaloacetate as the precursor was observed (Figure 5c). To compare the amount of glucose contributing to the citrate pool, the total carbon contribution from glucose to citrate in both culture conditions was calculated (Figure 5d). Determined were 24% glucose carbon contribution to citrate in pure astrocyte cultures and only 15% glucose carbon contribution in MLDCs. This difference was also visible in glutamate (Figure 5d, e). In summary, these data show increased PC activity and glucose derived carbon contribution into the TCA cycle in astrocytes compared to MLDCs.

[369] Besides glucose, the amino acid giutamine represents the most important carbon source for cells. This is especially pronounced in proliferating cells or in any cell type with increased anabolic demands (Wise and Thompson (2010) Glutamine addiction: a new therapeutic target in cancer. Trends Biochem Sci 35:427-33). To evaluate the impact of glutamine to the TCA cycle of astrocytes we used uniformly labeled [U-13C]glutamine and investigated the isotopic enrichment in citrate. In the astrocyte cultures it was found that glutamine provided similar amounts of carbons for the synthesis of citrate as glucose (22%) (Figure 5d). However, in MLDCs (where the glucose contribution was lower as in astrocytes) we found an increased contribution from glutamine (34%), probably to compensate for the decreased glucose contribution.

[370] In line with increased glutamine derived carbon contribution also higher M4 and M5 istopologue abundances of citrate were found (Figure 5f). Furthermore, also unexpected features of astrocyte metabolism compared to MLDCs were identified. By using [U-13C]glucose clear differences in the MID of serine were found. This non-essential amino acid can be derived from 3-phosphoglycerate, an intermediate of the glycolysis {Figure 6a). As an alternative to de novo biosynthesis it can be taken up from the medium. Based on stable isotope experiments higher relative serine biosynthesis rates were found in MLDCs (24%) as in pure astrocyte cultures (5%) (Figure 6b, c) where most serine originated from the medium (MO isotopologue abundance).

[371] Interestingly, although at least 5% (astrocytes) and 24% (MLDCs) of relative glucose contribution to serine was found, there was very little labeling in glycine (Figure 6d). This indicated that the equilibrium of the SHMT catalyzed reaction is far on the side of serine and that most glycine is taken up from the medium rather than being synthesized from glucose.

[372] Similar to serine, also increased labeling in lactate (M3) was found in MLDCs (Figure 6e). Lactate has an important role in maintaing the cytosolic redox balance of NADH/NAD+. The reason for different lactate labeling patterns could be caused by two reasons: first, pure astrocyte cultures might consume more (unlabeled) pyruvate from the medium (present at 1mM concentration) resulting in higher MO abundance and second, pyruvate and thus lactate might be produced from serine via serine dehydratase.

[373] Several papers described the generation of human astrocytes from fetal or adult post-mortem central nervous system by the expansion of neuronal precursors (Haidet-Phillips et al. (2011 ) Astrocytes from familial and sporadic ALS patients are toxic to motor neurons Nature Biotechnology 29:824-828; Verwer et al. (2007) Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin. Brain: a journal of neurology 130:3321-3335). This approach required 6 months to generate a pure population of astrocytes (Krencik (2011 ) Specificationof transplantable astroglial subtypes from human pluripotent stem cells. Nature Biotechnology 29:528-534). Other recent papers described the differentiation of astrocytes from iPSC with protocols requiringfrom 35 days (Emdad et al. (2012) Efficient differentiation of human 682 embryonic and induced pluripotent stem cells into functional astrocytes. Stem Cells Dev 683 21:404-10) up to 4 months (Juopperi (2012) Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington's disease patient cells. Mol Brain 5:17). Importantly, the obtained populations seem to represent astrocytes just in a reactive form as shown by the almost 100% immunoreactivity for GFAP. Therefore, these cultures might not be suitable to completely model mature astrocyte functions or to mirror patho- and physiological conditions (Roybon et al. (2013) Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Cell Rep 4:1035-48).

[374] In the here presented Examples the derivation of astrocytes was achieved by a cost-efficient media composition, which ensures a highly pure culture as shown by the negligible contamination with Tuj1 positive cells. Unlike other protocols (Yuan et al. (2011) Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6:e17540), the described protocol is simple and does not require any antibody-based sorting step of glia or neuronal progenitors. Remarkably, we were able to obtain a population of mature astrocytes both in a quiescent state with a protoplasmic morphology (negative for GFAP) as well as in a reactive phenotype characterized by GFAP expression. The expression of EAAT2 in all the cells and the ability to uptake glutamate strongly supported the acquisition of mature functions. The importance of this feature was highlighted by the different effects of immature and mature astrocytes on axonal regeneration (Goldshmit Yet al. (2012) Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J Neurosci 32:7477-92; Tom et al. (2004) Studies on the development and behavior of the dystrophic growth cone, the hallmark of regeneration failure, in an in vitro model of the glial scar and after spinal cord injury. J Neurosci 24:6531-9).

[375] The high pyruvate carboxylase activity confirmed the acquisition of metabolic specialization of hNSC-derived astrocytes as pyruvate carboxylation is an important anaplerotic reaction, specifically occurring in astrocytes (Shank et al. (1985) Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res 329:364-7; Amaral et al. (2011) A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose. Front Neuroenergetics. 3:5.). Moreover, the relative glucose flux into the TCA cycle was higher in astrocytes resulting in higher glucose derived carbon contribution to glutamate (Pardo (2011) Brain glutamine synthesis requires neuronai-born aspartate as amino donor for glial glutamate formation. J Cereb Blood Flow Metab 2011 Jan;31(1):90-101).

[376] Interestingly, in astrocytes as well as MLDCs around 50% of the citrate carbons derived from other sources than glucose or glutamine. These other sources might be represented by lipid oxidation and/or degradation of amino acids such as branched chain amino acids. Compared to other cell lines 50% of carbons derived from alternative carbon sources is relatively high, at least at basic culture conditions and high oxygen tension (unpublished data). The reasons for that can be multiple [377] Identifying the relevance of alternative carbon sources for glial specific metabolism could help for a better understanding of cell survival and integrity. Particularly interesting is the role of serine as it represents an important metabolic intersection point to i) provide one-carbon units to the folate mediated one-carbon metabolism, to ii) serve as a precursor for the transsulfuration pathway to generate cysteine from serine and homocysteine and iii) as the precursor to produce the non-essential amino acid glycine. Serine and its connected metabolic pathways have been shown to be of special importance in neuronal oxidative stress conditions (Krug et al. (2014) Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+).Cell Death Dis 5:e1222). Astrocytes have an important supportive function in protecting neurons against oxidative stress by providing the antioxidant glutathione (Dringen (2000) Glutathione metabolism and oxidative stress in neurodegeneration. Eur J Biochem 267:4903). Glycine represents together with cysteine and glutamate one of the three amino acids that build the antioxidant glutathione.

[378] EXAMPLE 5 hNSCs differentiated into various neuronal subtypes

Also the ability to differentiate into neurons is a hallmark of NSCs and therefore needs to be evaluated for the characterization of NSCs. To induce neuronal differentiation in the hNSC culture system oft he present invention a treatment with the factors BDNF (brain-derived neurotrophic factor), GDNF (glial cell line-derived neurotrophic factor), TGFB-3 (transforming growth factor beta-3), dbcAMP (dibutyryi-cAMP) and ascorbic acid was used. After 4 weeks of neuronal differentiation, derived cells showed positive labeling for early neuronal markers such as TuJ1 and Doublecortin (Figure 10a and b) as well as for the advanced maturation marker MAP2 (Figure 10c). The neuronal differentiation protocol enabled hNSCs to differentiate into GABAergic (GABA, 36.21%), giutamatergic (vGlutl, 40.34%) and dopaminergic neurons (TH, 12.68%) (Figure 10d-f), whereas differentiation into GFAP-positive cells was low (Figure 10g).

[379] To test these results at the mRNA level, we used RT-qPCR to quantitate expression of the neural stem ceil marker Nestin, the proliferation marker Ki67, the neuronal marker MAP2, as well as the neuronal sub-type markers for GABAergic (GABA), glutamatergic (GLUT, vGlutl) and dopaminergic (TH) neurons. This analysis was performed in a comparison of hNSC under maintenance conditions and after 4 weeks of neuronal differentiation (Figure 1Gh). The results demonstrate that neural stem cell identity and proliferation capacities decreased with differentiation. At the same time, we observed increased expression levels of MAP2, GABA, vGlutl and TH (Figure 10h), indicating ongoing neuronal differentiation.

[380] To examine whether these neurons were functional, immunolabelling for synaptic markers was performed after 4 weeks of differentiation, the pre- and postsynaptic markers synaptophysin and PSD95 were reliably detected (Figure 11a-d and 11 e-h), consistent with the expression of a more mature neuronal phenotype.

[381] Next, whole-cell patch-clamp recordings were performed following standard procedures. Under voltage clamp conditions, neurons showed a fast transient inward current in response to depolarizing voltage steps (-1.9 ±0.4 nA, n = 30; Figure 11 i) that was identified as a sodium (Na+-) current by blocking its influx with 0.5 μΜ TTX (Figure 11 j). The transient Na+-current was followed by an outward current with partial inactivation, typical for a mixture of voltage activated potassium channels. This outward current included an A-current that was identified in 10 out of 12 cells tested with a pre-pulse protocol (data not shown). The input resistance of the induced neurons was 1.5 ±0.2 GQ (n = 33). The amount of sodium current as well as the input resistance are typical for mature neurons (Belinsky et al. (2011) Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol. Stem Cells Dev 20:1733-46; Pre et al. (2014) A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs). PLoS One 9:e103418).

[382] In current clamp recordings, cells showed spontaneous action potential (AP) firing, when depolarized to a membrane potential near -50 mV by current injection (Figure 11k). Depolarizing currents elicited action potentials (Aps) with +19 ±5 mV peak potential (n =15 cells; Figure 111) and a width at half-maximal amplitude of 3.8 ±0.1 ms (n = 10). While the generation of APs is an important aspect of the neuronal lineage, we observed no spontaneous postsynaptic currents in continuous voltage clamp recordings (>3 min per cell; n = 30).

[383] Similarly, evoked postsynaptic currents could not be elicited by moderate depolarization with KCI applied in bath solution (10 mM; n = 5) or by puffs with high KCI (150 mM; 1-5 s duration; n = 6) directly onto patched cells. In contrast, puff-application of glutamate (1 mM, 0.5 s) elicited inward currents (608 ± 362 pA; n = 4) under voltage clamp conditions (Figure 11m), or depolarizations to membrane potentials near 0 mV (n = 4) during current clamp (Figure 11 n).

[384] These data indicate the presence of typical sodium channels and glutamate receptors in differentiated hNSCs cells.

[385] EXAMPLE 6 Transplanted hNSCs survived and differentiated in vivo

Finally, hNSC derived cells were characterized in vivo. The use of hiPSC-derived cells mandates that firstly, transplanted cells are devoid of tumor formation potential, and secondly, the transplanted cells are able to survive in vivo. Since the potential of tumor formation is inversely correlated to the degree of differentiation, cells after 6 passages were selected for transplantation because nearly all hNSCs were Nestinpositive and Oct4-/Nanog-negative at this time point. Stereotactic injection of self-renewing hNSCs into the subventricular zone of NOD/SCID mice was never followed by any tumoral growth. In total, 1 *10s to 2*106 hNSCs were transplanted into the brain hemispheres of 9 mice. 3 mice were sacrificed after 6 weeks, 3 other mice were sacrificed after 3 month and the final 3 mice were sacrificed after 6 months. In none of these mice, we observed tumoral outgrowth.

[386] To control cells fate after transplantation, hNSC were subjected to a pre-differentiation step. The cell fate was analyzed 6 additional weeks later (Figure 7a). Since injected cells were of human origin, the transplant could be identified by immunofluorescence with a specific antibody directed against human nuclei (hNuc). Human NSCs that were neuronal pre-differentiated one week before transplantation, were able to survive and to differentiate into TuJ1 (Figure 12c) and Doublecortin (Figure 12g) positive neurons. hNSCs that were differentiated to astroglia for 1 week prior transplantation formed clusters of GFAP positive astrocytes (Figure 7d).

[387] These data demonstrate that transplanted pre-differentiated hNSCs have no tumorigenic potential. More importantly, it was revealed that pre-differentiation prior to transplantation can direct the fate of transplanted cells after grafting. The ability to define the fate of cells after transplantation is of outstanding importance for controlled cell replacement therapies.

[388] Recently, the outstanding importance of astrocytes for neurological disease got into focus of several research approaches. As an example, it has been shown that astrocytes strongly contribute to the development of the Down syndrome (Chen et al. (2014) Role of astroglia in Down's syndrome revealed by patient-derived human induced pluripotent stem cells. Nat Commun 5:4430). Additionally, a recent study clearly demonstrated that transplantation of astrocytes was extremely beneficial in a rat model of Parkinson’s disease (Proschel et al. (2014). Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons. EMBO Mol Med 6:504-18). Thus, the availability of an effective method to generate mature astrocytes, as described in this study, is of key importance for convincing disease-modelling studies and replacement therapy strategies. This is becoming a relevant field of investigation especially for neurodegenerative diseases such as Parkinson's and Alzheimer's disease.

[389] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent" includes one or more of such different reagents and reference to “the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[390] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[391] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[392] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise", and variations such as “comprises" and “comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising" can be substituted with the term “containing” or sometimes when used herein with the term “having”.

[393] When used herein “consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[394] In each instance herein any of the tenus "comprising”, "consisting essentially of and “consisting of may be replaced with either of the other two terms.

[395] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[396] When used herein, the term "about” is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value. For example, if a formulation comprises about. 5 mg/ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as “(from) X to Y” equates with an interval which is defined as “between X and Y”. Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of “5 mg/ml to 10 mg/ml” or “between 5 mg/ml and 10 mg/ml” includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value.

REFERENCES

Altschul, Nucl. Acids Res. 25 (1997), 3389-3402 Altschul, J. Mol. Evol. 36 (1993), 290-300 Altschul, J. Mol. Biol. 215 (1990), 403-410 Altschul Nucl. Acids Res. 25 (1977), 3389-3402

Amaral Al, Teixeira AP, Hàkonsen BI, Sonnewald U, Alves PM. 2011. A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose. Front Neuroenergetics. 3:5

Antoni et al. (2015) “Three-Dimensional Cell Culture: A Breakthrough in Vivo.” Int J Mol Sci. 16(3):5517-5527

Araque A, Parpura V, Sanzgiri RP, Haydon PG. 1999. Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci 22:208-15

Ayuso-Sacido A, Graham C, Greenfield JP, Boockvar JA. The duality of epidermal growth factor receptor (EGFR) signaling and neural stem cell phenotype: cell enhancer or ceil transformer? Curr Stem Cell Res Ther. 2006 Sep;1(3):387-94

Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H, Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N and others. 2003. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnoi 21:1200-7.

Bardehle S, Kruger M, Buggenthin F, Schwausch J, Ninkovic J, Oevers H, Snippert HJ, Theis FJ, Meyer-Luehmann M, Bechmann I and others. 2013. Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation. Nat Neurosci 16:580-6

Belinsky GS, Moore AR, Short SM, Rich MT, Antic SD. 2011. Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol. Stem Cells Dev 20:1733-46

Brockhaus J, Deitmer JW. 2002. Long-lasting modulation of synaptic input to Purkinje neurons by Bergmann glia stimulation in rat brain slices. J Physiol 545:581-93

Cahan P, Daley GQ. 2013. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat Rev Mol Cell Biol 14:357-68

Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275-80

Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC and others. 2014. Role of astroglia in Down's syndrome revealed by patient-derived human induced pluripotent stem cells. Nat Commun 5:4430

Choudhry et al. (2014) “Sonic hedgehog signalling pathway: a complex network.” Ann Neurosci. 21(1):28-31

Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas R, Avila-Campilo I, Creech M, Gross B and others. 2007. Integration of biological networks and gene expression data using Cytoscape. Nat Protoc 2:2366-82

Conti L, Cattaneo E. 2010. Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176-87

Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E and others. 2005. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS biology 3:e283

Dringen R. 2000. Glutathione metabolism and oxidative stress in neurodegeneration. Eur J Biochem 267:4903

Coutu DL, Galipeau J. Roles of FGF signaling in stem cell self-renewal, senescence and aging. Aging (Albany NY). 2011 Oct;3(10):920-33

Dainiak et al. (Adv Biochem Eng Biotechnol. 2007;106:1-18

Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K. Non-cel! autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci. 2007 May;10(5):608-14

Ebert AD, Shelley BC, Hurley AM, Onorati M, Castiglioni V, Patitucci TN, Svendsen SP, Mattis VB, McGivern JV, Schwab AJ and others. 2013. EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs. Stem Cell Res 10:417-27

Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L. 2008. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. .Genes Dev 22:152-65

Emdad L, D'Souza SL, Kothari HP, Qadeer ZA, Germano IM. 2012. Efficient differentiation of human embryonic and induced pluripotent stem cells into functional astrocytes. Stem Cells Dev 21:404-10

English K, Wood KJ. 2011. Immunogenicity of embryonic stem cell-derived progenitors after transplantation. Curr Opin Organ Transplant 16:90-5

Fimia GM, Sassone-Corsi P. (2001) “Cyclic AMP signalling.” J Cell Sci; 114(Pt 11):1971-2

Gamberino WC, Berkich DA, Lynch CJ, Xu B, La Noue KF. 1997. Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes. J Neurochem 69: 2312-2325

Geerts H. Of mice and men: bridging the translational disconnect in CNS drug discovery. CNS Drugs. 2009 Nov;23(11):915-26

Goldshmit Y, Sztal TE, Jusuf PR, Hail TE, Nguyen-Chi M, Currie PD. 2012. Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J Neurosci 32:7477-92

Haidet-Phillips AM, Hester M.E., Miranda, C.J., Meyer K., Braun L., Frakes A., Song S., Likhite S., Murtha M.J., Froust K.D., Rao M., Eagle A., Kammesheidt A., Christensen A., Mendell J.R., Burghes A.H., Kaspar B.K. 2011. Astrocytes from familial and sporadic ALS patients are toxic to motor neurons Nature Biotechnology 29:824-828

Haydon PG, Carmignoto G. 2006, Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 86:1009-31

Heldin, Miyazono and ten Dijke (1997) “TGF-bold beta signaling from cell membrane to nucleus through SMAD proteins.” Nature 390, 465-471

Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915

Hiller K, Hangebrauk J, Jager C, Spura J, Schreiber K, Schomburg D. MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem. 2009 May 1;81 (9):3429-39

Huang YH, Bergies DE. Glutamate transporters bring competition to the synapse. CurrOpin Neurobiol. 2004 Jun;14(3):346-52

Huang da W, Sherman BT, Lempicki RA. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44-57.

Inoue H, Nagata N, Kurokawa H, Yamanaka S. 2014. iPS cells: a game changer for future medicine. EMBO J 33:409-17

Jiang et al. (Chin Med J (Engl) (2011) 124:1540-1544); Glavaski-Joksimovic et al. (J Neurosci res (2010) 88:2669-2681

Jiwang Zhanga, Linheng Lia BMP signaling and stem cell regulation Developmental Biology Volume 284, Issue 1,1 August 2005, Pages 1-11

Juopperi TA, Kim WR, Chiang CH, Yu H, Margolis RL, Ross CA, Ming GL, Song H. 2012. Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington's disease patient cells. Mol Brain 5:17

Kim DS, Lee DR, Kim HS, Yoo JE, Jung SJ, Lim BY, Jang J, Kang HC, You S, Hwang DY and others. 2012. Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and ÎPSC706 derived neural rosettes. PLoS One 7:e39715

Kim DS, Lee JS, Leem JW, Huh YJ, Kim JY, Kim HS, Park IH, Daley GQ, Hwang DY, Kim DW. 2010. Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev 6:270-81

Kirkeby A, Grealish S, Wolf DA, Nelander J, Wood J, Lundblad M, Lindvall O, Parmar M. 2012. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep 1:703-14

Koch P, Opitz T, Steinbeck JA, Ladewig J, Brustle O. 2009. A rosette-type, self-renewing human ES cell derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Natl Acad Sci U S A 106:3225-30

Krencik R, Weick JP, Liu Y, Zhang ZJ, Zhang SC. 2011. Specificationof transplantable astroglial subtypes from human pluripotent stem cells. Nature Biotechnology 29:528-534

Krug AK, Gutbier S, Zhao L, Pöltl D, Kullmann C, Ivanova V, Forster S, Jagtap S, Meiser J, Leparc G, Schildknecht S, Adam M, Hiller K, Farhan H, Brunner T, Hartung T, Sachinidis A, Leist M. 2014. Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+).Cell Death Dis 5:e1222

Li W, Sun W, Zhang Y, Wei W, Ambasudhan R, Xia P, Talantova M, Lin T, Kim J, Wang X and others. 2011. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc Natl Acad Sci U S A 108:8299-304

Liu Z, Tang Y, Lu S, Zhou J, Du Z, Duan C, Li Z, Wang C. 2013. The tumourigenicity of iPS cells and their differentiated dérivâtes. J Cell Mol Med 17:782-91

Logan and Nusse (Annu. Rev. Cell Dev. Biol. (2004) 20:781-810)

Lundgaard I, Osorio MJ, Kress BT, Sanggaard S, Nedergaard M. 2013. White matter astrocytes in health and disease. Neuroscience. 2014 Sep 12;276:161-73

Major T, Menon J, Auyeung G, Soldner F, Hockemeyer D, Jaenisch R, Tabar V. Transgene excision has no impact on in vivo integration of human iPS derived neural precursors. PLoS One. 2011;6(9):e24687

Mathieu, Saucourt, Moumetas, Gauthereau, Thézé, Praloran, Thiébaud, and Bœuf (2012) LIF-Dependent Signaling: New Pieces in the Lego Stem Ceil Rev.; 8(1): 1-15

Morizane A, Doi D, Kikuchi T, Okita K, Hotta A, Kawasaki T, Hayashi T, Onoe H, Shiina T, Yamanaka S and others. 2013. Direct Comparison of Autologous and

Allogeneic Transplantation of iPSC Derived Neural Cells in the Brain of a Nonhuman Primate. Stem Cell Reports 1:283-92

Nakashima K, Yanagisawa M, Arakawa H, Taga T. 1999. Astrocyte differentiation mediated by LIF in cooperation with BMP2. FEBS Letters 457:43-46

Nordberg J, Arnér ES. (2001) “Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.” Free Radie Biol Med. 31(11):1287-312

Ota Y, Zanetti AT, Hallock RM. 2013. The role of astrocytes in the regulation of synaptic plasticity and memory formation. Neural Plast 2013:185463

Payne NL, Sylvain A, O'Brien C, Herszfeld D, Sun G, Bernard CC. 2014. Application of human induced pluripotent stem cells for modeling and treating neurodegenerative diseases. N Biotechnol.

Pardo B, Rodrigues TB, Contreras L, Garzón M, Liorente-Folch I, Kobayashi K, Saheki T, Cerdan S, Satrustegui J. 2011. Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation. J Cereb Blood Flow Metab 31:90-101

Pre D, Nestor MW, Sproul AA, Jacob S, Koppensteiner P, Chinchalongporn V, Zimmer M, Yamamoto A, Noggle SA, Arancio O. 2014. A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells 743 (iPSCs). PLoS One 9:e103418

Proschel C, Stripay JL, Shih CH, Munger JC, Noble MD. 2014. Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons. EMBO Mol Med 6:504-18 PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983), pgs. 1-12

Qu Q, Shi Y. 2009. Neural stem ceils in the developing and adult brains. J Cell

Physiol 221:5-9

Ravi et al. (2015) “3D cell culture systems: advantages and applications." J Cell Physiol. 230(1):16-26

Reinhardt P, Glatza M, Hemmer K, Tsytsyura Y, Thiel CS, Hoing S, Moritz S, Parga JA, Wagner L, Bruder JM and others. 2013. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 8:e59252,

Roybon L, Lamas NJ, Garcia-Diaz A, Yang EJ, Sattler R, Jackson-Lewis V, Kim YA, Kachel CA, Rothsteîn JD, Przedborski S and others. 2013. Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Ceil Rep 4:1035-48

Sineva GS, Pospelov VA. 2010. Inhibition of GSK3beta enhances both adhesive and signaling activities of beta-catenin in mouse embryonic stem cells. Biol Cell 102:549-60

Shank RP, Bennett GS, Freytag SO, Campbell GL. 1985. Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res 329:364-7.

Soundararajan P, Lindsey BW, Leopold C, Rafuse VF. 2007. Easy and rapid differentiation of embryonic stem cells into functional motoneurons using sonic hedgehog-producing cells. Stem Cells 25:1697-706.

Seifter, Meth. Enzymol. 182 (1990); 626-646, Rattan, Ann. NY Acad. Sci. 663 (1992); 48-62

Supek F, Bosnjak M, Skunca N, Smuc T. 2011. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6:e21800

Tabar V, Tomishima M, Panagiotakos G, Wakayama S, Menon J, Chan B, Mizutani E, Al-Shamy G, Ohta H, Wakayama T and others. 2008. Therapeutic cloning in individual parkinsonian mice. Nat Med 14:379-81

Tanabe, Takahashi, Yamanaka (2014) “Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014, Ser. B 90

Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem ceils from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-76

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov 30:131(5):861-72

Tom VJ, Steinmetz MP, Miller JH, Doller CM, Silver J. 2004. Studies on the development and behavior of the dystrophic growth cone, the hallmark of regeneration failure, in an in vitro model of the glial scar and after spinal cord injury. J Neurosci 24:6531-9

Thompson Nuci. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Verwer RW, Sluiter AA, Balesar RA, Baayen JC, Noske DP, Dirven CM, Wouda J, van Dam AM, Lucassen PJ, Swaab DF. 2007. Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin. Brain: a journal of neurology 130:3321 -3335

Wegner A, Weindl D, Jäger C, Sapcariu SC, Dong X, Stephanopoulos G, Hiller K. Fragment formula calculator (FFC): determination of chemical formulas for fragment ions in mass spectrométrie data. Anal Chem. 2014 Feb 18;86(4):2221-8

Wemig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R. 2008. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA 105:5856-61

Wise DR, Thompson CB. 2010. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem Sci 35:427-33

Wu X, Walker J, Zhang J, Ding S, Schultz PG. 2004. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem Biol 11:1229-38.

Yan Q, Gong L, Deng M, Zhang L, Sun S, Liu J, Ma H, Yuan D, Chen PC, Hu X and others. 2010. Sumoylation activates the transcriptional activity of Pax-6, an important transcription factor for eye and brain development. Proc Natl Acad Sci U S A 107:21034-9

Yuan SH, Martin J, Elia J, Flippin J, Paramban Rl, Hefferan MR, Vidal JG, Mu Y, Killian RL, Israel MA and others. 2011. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6:e17540

Claims (47)

1, Method for obtaining a neural stem cell (NSC), the method comprising b) cultivating induced pluripotent stem cells (iPSCs) in a medium comprising (i) an activin/transforming growth factor-ß (TGF-ß) signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a SHH-pathway activator; and c) cultivating the cells obtained in b) in a medium comprising (i) an activin/TGF-ß signaling inhibitor; (ii) a canonical WNT-signaling activator; (iii) a BMP signaling inhibitor; and (iv) a SHH-pathway activator; and d) further cultivating the cells obtained in c) in a medium comprising (i) a canonical WNT-signaling activator; (ii) a SHH-pathway activator; and (iii) an antioxidant; and" e) maintaining the cells obtained in d) in a medium comprising (i) a FGF signaling activator; (ii) an EGF signaling activator; and (iii) a LIF signaling activator, and thereby obtaining a NSC. :2:., The method of claim 1, wherein the NSC is a human NSC (hNSC).

3·. The method of claim 1 or 2, wherein the iPSC is a human iPSC (hiPSC).

4. The method of any one of claims 1-3, wherein the iPSC has been obtained from a fibroblast.

5. The method of claim 4, wherein the fibroblast has been obtained from a human.

6. The method of any one of claims 1-5, wherein activin/TGF-ß inhibitor is an inhibitor of the TGF-ß type I receptor activin receptor-like kinase(s).

7. The method of claim 6, wherein the activin/TGF-ß inhibitor inhibits ALK5, ALK4 and/or ALK7.

8. The method of any one of claims 1-7, wherein the activin/TGF-ß inhibitor is selected from the group consisting of A-83-01, D4476, GW788388, LY364947, R268712, SB-431542, SB-505124, SD208, SB-525334 and ALK5 Inhibitor II (CAS: 446859-33-2).

9. The method of any one of claims 1-8, wherein WNT-signaling is activated by the addition of a GSK-3 inhibitor.

10. The method of claim 9, wherein the GSK-3 inhibitor is selected from the group consisting of CHIR 99021, SB-216763, 6-bromoindirubin-3'-oxime, Tideglusib, GSK-3 inhibitor 1, AZD1080, TDZD-8, TWS119, CHIR-99021, CHIR-98014, SB 415286, SB 216763, LY2090314, AR-A014418 and IM-12, preferably CHIR 99021.

11. The method of any one of claims 1-10, wherein the BMP signaling inhibitor is selected from the group consisting of chordin, noggin, DMH1 (CAS 1206711-16-1), K 02288, dorsomorphin and LDN 193189.

12. The method of any one of claims 1-11, wherein the SHH-pathway activator is selected from the group consisting of purmorphamine, SHH, SAG Analog and Gli-2.

13. The method of any one of claims 1-12, wherein step d) can further comprise a step d.2) comprising culturing the cells obtained in step d) in a medium comprising a FGF signaling activator.

14. The method of any one of claims 1-13, wherein the FGF signaling activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4 and FGF8.

15. The method of any one of claims 1-14, wherein the EG F signaling activator is selected from the group consisting of epidermal growth factor (EGF), transforming growth factor a (TGFa), amphiregulin (AR), heparin-binding EGF-like growth factor (ΗΒ-EGF), betacellulin (BTC), epigen (ERG) and epiregulin (EPR).

16. The method of any one of claims 1-15, wherein the LIF signaling activator in step e) is LIF, preferably human LIF.

17. The method of any one of claims 1-16, wherein the method further comprises differentiation of the cells obtained in step e) into neurons or glial cells, preferably astrocytes.

18. The method of any one of claims 1-17, wherein the method further comprises differentiation of the cell obtained in step e) into a (i) Map2; (ii) TH; (iii) GABA; (iv) vGlut; (v) dcx; (vi) synaptophysin; (vii) postsynaptic density protein 95 (PSD 95); (viii) Tuj1; and/or (ix) NCAM expressing cell.

19. The method of any one of claims 1-17, wherein the method further comprises differentiation of the cell obtained in step e) into a (i) GFAP; (ii) S100b; (iii) vimentin; (iv) aquaporin 4; and/or (v) EAAT2 expressing cell.

20. The method of claim 17 or 18, wherein the differentiation comprises culturing cells obtained in step e) in a medium comprising (i) at least two different neurotrophins; and (ii) an antioxidant.

21. The method of claim 20, wherein the at least two different neurotrophins are selected from the group consisting of BDNF, NGF, GDNF, NT-3, NT-4, or CNTF, preferably GDNF and BDNF.

22. The method of any one of claims 17, 18, 20-21, wherein the process of differentiation takes at least 4 weeks.

23. The method of any one of claims 17, 18, 20-22, wherein differentiation results in the generation of GABAergic cells, with a percentage of 10 %, 15 %, 20 %, 25 %, 30 %, 35 % or higher of the total amount of cells.

24. The method of any one of claims 17, 18, 20-23, wherein differentiation results in the generation of glutamatergic cells, with a percentage of 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 % or higher of the total amount of cells.

25. The method of any one of claims 17, 18, 20-24, wherein differentiation results in the generation of dopaminergic cells, with a percentage of 3 %, 7 %, 10 %, 12 % or higher of the total amount of cells.

26. The method of any one of claims 17, 18, 20-25, wherein differentiation does virtually not result in the generation of astrocytes, wherein preferably the culture comprises less than 20 %, 15 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 % astrocytes of the total amount of cells.

27. The method of claim 17 or 19, wherein the differentiation comprises culturing the cells obtained in step e) in a medium comprising (i) serum.

28. The method of claim 27, wherein the serum is selected from the group consisting of fetal calf serum (FCS) and fetal bovine serum (FBS), preferably FCS.

29. The method of any one of claims 17, 19, 27, 28, wherein the process of differentiation takes 45 days.

30. The method of any one of claims 17, 19, 27-29, wherein differentiation results in the generation of S100ß positive cells, with a percentage of 50 %, 60 % 70%, 75 %, 80 %, 85 %, 90 %, 95 % or higher of the total amount of cells.

31. The method of any one of claims 17, 19, 27-30, wherein differentiation results in the generation of vimentin positive cells, with a percentage of 50 %, 60 % 70%, 75 %, 80 %, 85 %, 90 %, 95 % or higher of the total amount of cells.

32. NSC obtainable by a method as defined in any one of claims 1-16.

33. NSC of claim 32, wherein the NSC expresses PAX6 with a fold change of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 or more relative to the iPSC.

34. NSC of claim 32 or 33, wherein the NSC expresses SOX1 with a fold change of at least 2, 3, 4, 5, 6, 7 or more relative to the iPSC.

35. NSC of any one of claims 32-34, wherein the NSC lacks expression of at least one of Nanog, Klf4, LIN28A, Oct4 or Myc.

36. NSC of any one of claims 32-35, wherein the NSC expresses K167 with a fold change of at least 1.1 or more relative to the iPSC.

37. NSC of any one of claims 32-36, wherein the NSC expresses SOX2 with a fold change of at least 1.1 or more relative to the iPSC.

38. NSC of any one of claims 32-37, which can be passaged for more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22 or 23 or more times.

39. Neuron obtainable by a method as defined in any one of claims 17,18, 20-26.

40. Astrocyte obtainable by a method of any one of claims 17, 19, 27-31.

41. Pharmaceutical composition comprising a NSC of any one of claims 32-38, neuron of claim 39 or astrocyte of daim 40.

42. Pharmaceutical composition of claim 41, NSC of any one of claims 32-38, neuron of claim 39 or astrocyte of claim 40 for use in therapy.

43. A preparation obtainable by a method of any of claims 1 -31.

44. A preparation comprising a NSC of any one of claims 32-38, neuron of claim 39 or astrocyte of claim 40.

45. In vitro method or test system, wherein the method or test system comprises (i) NSCs as defined in any one of claims 32-38, (ii) neurons as defined in claim 39 or (iii) astrocytes as defined in claim 40.

46. In vitro method or test system of claim 45, for use in testing efficiency or toxicity of a molecule of interest in therapy, wherein the method or test system further comprises (iv) contacting the NSCs, neurons and or astrocytes with the molecule of interest.

47. In vitro method or test system of claim 46, wherein the method or test system further comprises (iv) correlating the result obtained to the efficiency or toxicity of the molecule of interest in therapy.

48. In vitro method or test system of any one of claims 45-47, for use in screening, expression profiling or disease modeling.