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WO2017009766A1 - Cellules souches neuronales à auto-renouvellement à long terme - Google Patents

Cellules souches neuronales à auto-renouvellement à long terme Download PDF

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WO2017009766A1
WO2017009766A1 PCT/IB2016/054131 IB2016054131W WO2017009766A1 WO 2017009766 A1 WO2017009766 A1 WO 2017009766A1 IB 2016054131 W IB2016054131 W IB 2016054131W WO 2017009766 A1 WO2017009766 A1 WO 2017009766A1
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cells
medium
nsc
differentiation
signaling
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Jens Schwamborn
Silvia BOLOGNIN
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Universite du Luxembourg
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Definitions

  • 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.
  • the present invention relates to NSCs, neurons and astrocytes of the present invention for use in therapy.
  • 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 iPSC731 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.
  • iPSC iPSC replacement therapies
  • hESCs human embryonic stem cells
  • 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
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • the present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising
  • iPSCs induced pluripotent stem cells
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • the present invention relates to a NSC obtainable by a method of the present invention.
  • the present invention relates to a neuron obtainable by a method as of the present invention.
  • the present invention also relates to an astrocyte obtainable by a method of the present invention.
  • 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.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a NSC of the present invention, neuron of the present invention or astrocyte of the present invention.
  • the present invention relates to a pharmaceutical composition of the present invention for use in therapy.
  • the present invention further relates to a preparation obtainable by methods of the present invention.
  • 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 present invention relates to an in vitro method or test system, wherein the method or test system comprises
  • 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.
  • 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.
  • 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.
  • 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
  • MIDs Mass isotopomer distributions
  • 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.
  • Glutamine has no contribution to the serine pool (data not shown)
  • Mean ⁇ SEM, n 3 independent experiments in triplicate; p-value ⁇ 0.05.
  • 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).
  • 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).
  • FIG. 1 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).
  • Figure 12 Transplantation of hNSC derived neurons in NOD/SCID mice.
  • 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 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 (Kim et al. 2012; Koch et al. 2009; Li et al. 201 1 ; 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.
  • hNSCs were homogeneously maintained in a two-dimensional adherent cell system. Spontaneous differentiation of hNSC into other cell types was negligible. hLIF 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 cells in the hNSC under maintenance condition were not detected.
  • BMP bone morphogenetic protein
  • NSCs Neuronal cells
  • hNSCs differentiated into glial cells 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.
  • the present invention also provides for the preparation of neurons and glial cells from iPSCs via NSCs.
  • 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. (201 1 ) 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).
  • 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).
  • the present invention now provides for an improved 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.
  • 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. (201 1 ) 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.
  • 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 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 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).
  • astrocytes as well as MLDCs (multilineage 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.
  • 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 1 1 : 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.
  • astrocytes were solely described as supporting elements for neurons.
  • astrocytes are essential players involved in brain information processing (Araque et al. (1999) Tripartite synapses: glia, the unacknowledged partner.
  • 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).
  • ALS amyptrophic lateral sclerosis
  • astrocytes 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.
  • 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.
  • astrocytes and especially the generation of autologous astrocytes from iPSCs are of important clinical relevance.
  • 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).
  • 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).
  • the present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising
  • iPSCs induced pluripotent stem cells
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • 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
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • 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.
  • NSC neural stem cell
  • 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.
  • 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, 1 1 , 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, oligodendroctyes and neurons.
  • 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.
  • 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.
  • iPSCs induced pluripotent stem cells
  • 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.
  • stem cell markers can include Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) as well as stem cell-specific antigen 3 and 4 (SSEA3/4).
  • ALP alkaline phosphatase
  • SSEA3/4 stem cell-specific antigen 3 and 4
  • the chromatin methylation 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).
  • 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 germ line chimaeric animals through blastocyst injection.
  • 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.
  • PBMCs peripheral blood Mononuclear Cells
  • fibroblasts such as for example fibroblasts obtained from skin tissue biopsies.
  • the iPSC can be produced or derived or obtained from any somatic cell of a subject.
  • the iPSCs can be produced from somatic cells such as fibroblasts.
  • the iPSC can be a human iPSC (hiPSC).
  • the somatic cells such as fibroblasts have been obtained from a subject.
  • the fibroblast can be obtained from a human.
  • 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.
  • the subject is a vertebrate.
  • the subject can also be a human subject.
  • the subject can also be a subject suffering from a neurodegenerative disease.
  • any neurodegenerative disease can be encompassed by the present invention.
  • the term "neurodegenerative disease” can e.g. concern a group of hereditary and sporadic conditions characterised by progressive dysfunction, degeneration and death of specific populations of neurons, which are often synaptically interconnected.
  • 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, dentatorubropallidoluysial 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.
  • the neurodegenerative disease can thus include Parkinson's disease, Down syndrome, or Alzheimer's disease.
  • 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.
  • the methods of the present invention further require that cells such as iPSCs and NSCs are cultivated.
  • 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 (O2, CO2) 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 cell 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).
  • 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.
  • 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.
  • 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.
  • biologically derived matrices e.g. fibrin, collagen, feeder layers as further described herein
  • synthetic hydrogels e.g. PAA, PEG and as further described herein
  • 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).
  • matrices or scaffolds are grown or differentiated in matrices or scaffolds.
  • 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.
  • 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.
  • 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® 30-2002TM), DMEM-F12 medium (e.g. ATCC® 30-2006TM), RPMI medium (e.g. RPMI-1640 Medium (ATCC® 30-2001TM), Iscove's modified Dulbecco's medium (IMDM) (e.g.
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM-F12 e.g. ATCC® 30-2006TM
  • RPMI medium e.g. RPMI-1640 Medium (ATCC® 30-2001TM
  • media as used in the methods of the present invention can comprise further additives/supplements. Suitable additives/supplements are known to the person skilled in the art and also publicly availale.
  • 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.
  • the media used in the methods of the present invention can comprise a signaling activator or a signaling inhibitor.
  • activator 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.
  • 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.
  • 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 or TCFI protein, respectively.
  • 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.
  • the binding protein can be an antibody or a divalent antibody fragment comprising two binding sites with different specificities.
  • 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.
  • 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.
  • monovalent binding proteins include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties.
  • 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.
  • 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.
  • the activator can also be a nucleic acid molecule, such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer.
  • a nucleic acid molecule such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer.
  • a nucleic acid molecule such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer.
  • a nucleic acid molecule such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer.
  • a nucleic acid molecule such as a RNA, siRNA, miRNA or a non-proteinaceous aptamer.
  • aptamer is an oligonucleic acid that binds to a specific target molecule.
  • aptamers can be classified as: DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides.
  • the activator is a small molecule or protein/polypeptide.
  • 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.
  • 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 (or before the addition) of the activator.
  • 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.
  • 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 with 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/molecule is capable to reduce or block the activity of a target molecule and/or signaling pathway are known to the skilled artesian.
  • 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.
  • the compound/molecule that can be used as an inhibitor can be any compound/molecule, 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.
  • 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.
  • the inhibitor is a small molecule or protein/polypeptide.
  • 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.
  • an inhibitor of the BMP pathway can be dorsomorphine, which is a small- molecule antagonist for bone morphogenetic protein (BMP) type I receptors (AL 2, ALK3 and ALK6).
  • BMP bone morphogenetic protein
  • the inhibitor can also be an antagonist of the pathway/signaling patthway to be inhibited.
  • 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 (or before the addition) of the inhibitor.
  • the media as used in the methods of the present invention can for example comprise an activin/TGF- ⁇ inhibitor.
  • the medium used in step b) and step c) of the methods of the present invention comprises an activin/TGF- ⁇ inhibitor.
  • the activin/TGF- ⁇ 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 .
  • Receptor ligands including, for example, TGFB1 , TGFB2, TGFB3, ACTIVIN A, ACTIVIN 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), ACVR1 B (ALK4) and/or ACVR1 C (ALK7).
  • activator of the activin/TGF- ⁇ signaling pathway refers to an activator of any one of the above recited molecules that form part of this signaling pathway
  • inhibitor of the activin/TGF- ⁇ signaling pathway refers to inhibitors of any one of the above recited molecules that form part of this signaling pathway.
  • an activator can be an agonist of the ACVR2A and/or ACVR1 B (ALK4) receptor or an agonist of the TGF RII receptor and/or ALK5 receptor.
  • Such an inhibitor can be an antagonist of the ACVR2A and/or ACVR1 B (ALK4) receptor or an antagonist of the TGF RII receptor and/or ALK5 receptor.
  • inhibitors/activators of the activin/TGF- ⁇ signaling pathway are known to the skilled artesian and are commercially available.
  • the activin/TGF- ⁇ inhibitor is an inhibitor of the TGF- ⁇ type I receptor activin receptor-like kinase(s). Further envisioned by the present invention is that the activin/TGF- ⁇ inhibitor inhibits ALK5, ALK4 and/or ALK7.
  • an activin/TGF- ⁇ inhibitor is 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)-1 H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide; CAS No.: 452342-67-5), LY364947 ( 4-[3-(2-pyridinyl)-1 H-pyrazol-4-y
  • the activin/TGF- ⁇ inhibitor such as SB-431542 can be employed in a concentration of between about 0,01 ⁇ and about 1 M, more preferably between about 5 ⁇ and about 15 ⁇ , and most preferably the amount is about 10 ⁇ .
  • SB-431542 can be obtained from Ascent Scientific.
  • the media as used in the methods of the present invention can additionally or alternatively comprise a canonical WNT-signaling activator.
  • the medium used in step b), step c), step d) of the methods of the present invention comprises a canonical WNT-signaling activator.
  • media used in step d.1 ) and/or d.2) as described herein can comprise a WNT-signaling activator.
  • 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).
  • a Wnt ligand binds to Frizzled receptors, which triggers displacement of the multifunctional kinase GSK-3P from a regulatory APC/Axin/GSK-3p-complex.
  • ⁇ -catenin is targeted by coordinated phosphorylation by CK1 and the APC/Axin/GSK-3p-complex leading to its ubiquitination and proteasomal degradation through the ⁇ -TrCP/SKP pathway.
  • Wnt ligand 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-3P from APC/Axin.
  • Dvl Dishevelled
  • the transcriptional effects of Wnt ligand is mediated via Rac1 -dependent nuclear translocation of ⁇ -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 Wnt1 1 .
  • canonical WNT-signaling activator refers to an activator of any one of the above recited molecules that form part of this signaling pathway.
  • Exemplary canonical WNT-signaling activators include Norrin, R-spondin 2 or WNT protein.
  • 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.
  • GSK-3 inhibitors include CHIR 99021 (6-[[2-[[4-(2,4- Dichlorophenyl)-5-(5-methyl-1 H-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)-1 H-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.: 6124
  • the canonical WNT-signaling activator such as CHIR 99021 can be employed in a concentration of between about 0,01 ⁇ and about 1 M, more preferably between about 0, 1 ⁇ and about 5 ⁇ , and most preferably the amount is about 3 ⁇ .
  • CHIR 99021 can for example be obtained from Axon Medchem.
  • the media as used in the methods of the present invention can additionally or alternatively comprise a BMP signaling inhibitor.
  • the medium in step b), step c) used in the methods of the present invention compises a BMP signaling inhibitor.
  • 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 -1 1.
  • 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 (Bmprl b).
  • 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).
  • R-Smad receptor I mediated phosphorylation of Smadl , Smad5, or Smad8
  • 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 TGFpi activated tyrosine kinase 1 (TAK1 , a MAPKKK) and through mitogen activated protein kinase (MAPK), which also involves cross-talk between the BMP and Wnt pathways.
  • TGFpi activated tyrosine kinase 1 TGFpi activated tyrosine kinase 1
  • MAPKKK mitogen activated protein
  • the inhibitors of BMP signaling can only block/reduce the canonical BMP pathway.
  • the BMP signaling inhibitor can be a cancoical BMP signaling inhibitor.
  • One such inhibitor selective for cannocial BMP signaling pathway is dorsomorphin.
  • BMP signaling inhibitors include chordin, noggin, DMH1 (CAS 120671 1 -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 -ylethoxy)phenyl]-3-pyridin-4- ylpyrazolo[1 ,5-a]pyrimidine; CAS No.: 866405-64-3) and LDN 193189 (4-[6-[4-(1 - Piperazinyl)phenyl]pyrazolo[1 ,5-a]pyrimidin-3-yl]-quinoline hydrochloride, CAS No.: 1062368-24-4).
  • the BMP signaling inhibitor can also be dorsomorphin.
  • the BMP signaling inhibitor such as dorsomorphin can be employed in a concentration of between about 0,01 ⁇ and about 1 M, more preferably between about 0, 1 ⁇ and about 5 ⁇ , and most preferably the amount is about 0,1 ⁇ .
  • Dorsomorphin can for example be obtained from Tocris.
  • the media as used in the methods of the present invention can additionally or alternatively comprise a SHH-pathway activator.
  • the medium in step b), step c) and step d) of the methods of the present invention compise a SHH-pathway activator.
  • 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 GLI1 , 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.
  • Exemplary activators of the Hedgehog signaling include purmorphamine (PMA; 2-(1 -Naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine
  • SHH smoothened agonist
  • SAG 3-chloro-N-[trans-4- (methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]-benzo[b]thiophene-2- carboxamide; CAS No.: 912545-86-9
  • 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.
  • the SHH signaling pathway activator such as purmorphamine can be employed in a concentration of between about 0,25 ⁇ and about 1 M, more preferably between about 0,4 ⁇ and about 0,5 ⁇ , and most preferably the amount is about 0,5 ⁇ .
  • 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.
  • Embryoid bodies refers to aggregates of cells derived from pluripotent stem cells (iPSCs).
  • Embryoid bodies 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.
  • the EB medium comprises a "basic/basal” medium as described herein, which can compise supplements/additives as described herein.
  • the EB medium or the medium used in step b) of the methods of the present invention can for example be a DMEM medium.
  • 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 KnockoutTM DMEM medium from Gibco.
  • 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 replacement” is usually used to replace serum in a medium.
  • the serum replacement can be a knockout serum replacement.
  • the "knockout serum replacement” 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 KnockoutTM SR from Gibco.
  • 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 1 1 of the non-essential amino acids described herein.
  • the non-essential amino acids can comprise at least one of arginine, asparagine, glutamine, glycine, alanine, cysteine, aspartic acid, glutamic acid, proline, tyrosine and serine.
  • the non-essential amino acids can also comprise at least one of alanine, aspartic acid, cysteine or glutamamic acid.
  • 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.
  • 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.
  • the medium used in step b) of the methods of the present invention can additionally or alternatively comprise 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 ⁇ -mercaptoethanol, DL-Dithiothreitol (DTT), 2-Mercaptoethylamine-HCI, TCEP, TCEP-HCI or Cysteine-HCI.
  • the reducing agent can be ⁇ -mercaptoethanol.
  • 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).
  • 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.
  • the antibiotic such as a mix of penicillin and streptomycin can be present in a total concentration of 1 % (including both penicillin and streptomycin).
  • 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/basal” medium as described herein which can compise supplements/additives as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the medium can further comprise L-glutamine.
  • L-glutamine can be added at a concentration of 0.5 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM 3 mM 3.5 mM, 4 mM or more.
  • L-glutamine can be added at a concentration of 2 mM.
  • 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, Arner ES. (2001 ) "Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.” Free Radic Biol Med. 31 (1 1 ): 1287-312.
  • 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.
  • an antioxidant refers to an inhibitor of a molecule involved in cellular oxidative processes.
  • 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 an antioxidant, which is selected from the group consisting of ascorbic acid, glutathione, lipoic acid and uric acid.
  • the antioxidant can be ascorbic acid.
  • the antioxidant such as ascorbic acid can be utilized in an amount of about 50 ⁇ to about 1 mM, or between about 100 ⁇ and about 500 ⁇ , or the amount can about 150 ⁇ .
  • 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/ ⁇ .
  • Lipoic acid can be employed between about 200 and about 1000 ⁇ .
  • 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 ⁇ .
  • the antioxidant such as melatonin can be employed between about 10 and about 200 ⁇ .
  • the antioxidant such as hydroxytyrosol can be employed between about 10 and about 100 ⁇ .
  • the antioxidant such as ubiquinone can be employed between about 10 and about 50 ⁇ .
  • 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 ⁇ .
  • Cells can be cultivated in the media used in the methods of the present invention for any period of time.
  • 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.
  • the cells can be cultivated for 48 hours in the medium used in step b) of the method of the present invention.
  • 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.
  • 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.
  • cells can be cultivated for 48 hours in the medium used in step c).
  • 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.
  • cells obtained after/in the cultivating step d) of the method of the present invention can be plated on Matrigel coated plates.
  • 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.
  • Matrigel coated plates can for example be 12-well plates.
  • step d) of the method of the present invention can comprise d) further cultivating the cells obtained in c) cells in a medium comprising
  • 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.
  • 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.
  • 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
  • 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 (201 1 ) Roles of FGF signaling in stem cell self-renewal, senescence and aging. Aging (Albany NY); 3(10):920-33.
  • 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.
  • FGF fibroblast growth factor
  • 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.
  • Jak/Stat the Janus kinase/signal transducer and activator of transcription
  • PLCg phosphoinositide phospholipase C
  • PI3K phosphatidylinositol 3-kinase
  • Erk Erk pathways.
  • 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.
  • 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, FGF1 1 , 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).
  • 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/ml, 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.
  • step e) of the methods of the present invention may be performed in matrigel coated dishes such as 10 cm dishes.
  • cells 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.
  • 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/ ⁇ , 100 ng/ ⁇ , 150 ng/ ⁇ , 200 ng/ ⁇ , 250 ng/ ⁇ , 300 ng/ ⁇ , 350 ng/ ⁇ 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/ ⁇ .
  • the FGF receptor agonist can be for example obtained from Peprotech.
  • 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.
  • the medium used in step e) of the method of the present invention comprises an EGF 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.
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • 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.
  • Tyrosine 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.
  • SH2 Src homology domain 2
  • PTB phospho- tyrosine binding domains
  • EGF signaling activator(s) include epidermal growth factor (EGF), transforming growth factor a (TGFa), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), epigen (EPG) and epiregulin (EPR).
  • EGF, BTC and EPR bind to erbB4 while Neuregulins (NR) bind to either erbB3 or erbB4.
  • NR Neuregulins
  • any molecule that induces/increases EGF signaling can serve as an activator of EGF signaling.
  • the EGF signaling activator can also be selected from the group consisting of EGF, TGF-a, Epigen, heparin-binding EGF-like growth factor (HB-EGF), betacellulin and amphiregulin (AR).
  • 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/ ⁇ , 100 ng/ ⁇ , 150 ng/ ⁇ , 200 ng/ ⁇ , 250 ng/ ⁇ , 300 ng/ ⁇ , 350 ng/ ⁇ 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/ ⁇ .
  • the EGF signaling activator can, for example, be obtained from Peprotech.
  • the medium used in step e) of the method of the present invention comprises a (Leukemia Inhibitory Factor) LIF receptor-a agonist or LIF receptor-a signaling activator.
  • the LIF receptor-a signaling pathway is well known to the skilled artesian and for example described in Mathieu et al. (2012) LIF-Dependent Signaling: New Pieces in the Lego Stem Cell Rev.; 8(1 ): 1-15.
  • a LIF receptor agonist such as LIF binds to the specific LIF receptor (LIFR-a) which forms a heterodimer with a specific subunit common to all members of that family of receptors, the GP130 signal transducing subunit.
  • LIF LIF receptor
  • LIF receptor binds to the specific LIF receptor (LIFR-a) which forms a heterodimer with a specific subunit common to all members of that family of receptors, the GP130 signal transducing subunit.
  • JAK/STAT Japanese kinase/signal transducer and activator of transcription
  • MAPK mitogen activated protein kinase cascades.
  • removal of LIF can push stem cells toward differentiation, but upon presence of LIF they may retain their proliferative potential or pluripotency.
  • LIF is typically added to stem cell culture medium to reduce spontaneous differentiation.
  • 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.
  • the present invention also relates to a method for obtaining a neural stem cell (NSC), the method comprising
  • iPSCs induced pluripotent stem cells
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • the present invention relates to a method for obtaining a neural stem cell (NSC), the method comprising
  • iPSCs induced pluripotent stem cells
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • 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.
  • the present invention can further comprise f) further cultivating the cells obtained in e), d), d.1 ) or d.2)
  • 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.
  • NSCs the cells obtained in step e
  • d the cells obtained in step e
  • d the cells obtained in step e
  • d the cells obtained in step e
  • d the cells obtained in step e
  • d the cells obtained in step e
  • d d.1
  • d.2 d.2
  • neurons and glia cells For example, cells may be differentiated into neurons and/or astrocytes.
  • the method of the present invention can comprise differentiation of the cell obtained in step e), d), d.1 ) or d.2) into a
  • Tuj1 Tuj1
  • ix NCAM expressing cell.
  • Such a cell can be characterized as an immature or mature neuron depending on the observed marker expression.
  • 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 NM_001039538 (SEQ ID NO: 1 ) and the protein by Uniprot No. P1 1 137 (SEQ ID NO: 2).
  • Map2 embraces any Map2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • 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 NM_000360 (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.
  • GABA refers to ⁇ -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.
  • GABA embraces any polypeptide and can also comprise fragments or variants thereof.
  • 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.
  • 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 NM_000555 (SEQ ID NO: 7) and the protein by Uniprot No. O43602 (SEQ ID NO: 8).
  • the term dcx embraces any dcx nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • 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.
  • PSD95 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_001 128827 (SEQ ID NO: 1 1 ) 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.
  • Tuj1 also known as " ⁇ Tubulin” is a protein that in humans is encoded by the TUBB3 gene.
  • the protein ⁇ 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.
  • NCAM also known as “Neural cell adhesion molecule”
  • 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.
  • 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.
  • 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.
  • the neuron differentiation medium can additionally or alternatively comprise
  • neurotrophins 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).
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4 neurotrophin-4
  • CNTF ciliary neurotrophic factor
  • the term "at least two different neurotrophins” refers to two or more of the above recited molecules.
  • the at least two different neurotrophins are BDNF and GDNF (Gene Symbols: BDNF and GDNF, respectively; (Jiang et al. (Chin Med J (Engl) (201 1 ) 124: 1540-1544); Glavaski-Joksimovic et al. (J Neurosci res (2010) 88:2669-2681 ).
  • 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.
  • the neuron differentiation medium can additionally or alternatively comprise a (iii) activin/transforming growth factor- ⁇ (TGF- ⁇ ) signaling activator.
  • TGF- ⁇ signaling activators are elsewhere described herein.
  • Exemplary activators of the activin/TGF- ⁇ signaling pathway include TGF i , TGF 2, TGF 3, activin A, activin B, activin AB or nodal.
  • the activator of activin/TGF- ⁇ signaling pathway can be TGF 3.
  • the activator of the activin/TGF- ⁇ signaling pathway such as TGF 3 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.
  • TGF 3 can be obtained from Peprotec, for example.
  • the neuron differentiation medium can additionally or alternatively comprise a cAMP analogue.
  • 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; 1 14(Pt 1 1 ): 1971 -2.
  • 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.
  • the cAMP analogue can be dbcAMP.
  • the cAMP analogue such as dbcAMP can be utilized in an amount of 50 ⁇ to 1000 ⁇ such as e.g. in an amount of 500 ⁇ .
  • dbcAMP can be obtained from Sigma Aldrich, for example.
  • 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.
  • 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.
  • about 36 % of the total of cells/the total of cells in the culture can differentiate into GABA ergic cells (cells positive for GABA).
  • 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.
  • about 40 % of the total of cells/the total of cells in the culture can differentiate into glutamatergic cells (cells positive for vGlut+).
  • 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 cells (e.g. TH+ cells) among the total of cells/the total of cells in the culture (in the neuron differentiation medium) can be about 0.5 %, 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).
  • the present invention further contemplates that when cells (NSCs) are differentiated into neurons, differentiation does virtually not result in the generation of astrocytes.
  • the culture (differentiated to neurons) can comprise less than 20 %, 15 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 % astrocytes.
  • differentiation into neurons in the neuron differentiation medium as described herein can result in the generation of 10-60 % GABA+ cells and/or 10-70 % vGlut+ cells and/or 0.5-30 % TH+ cells and/or less than 20 % astrocytes of the total of cells/the total of cells in the culture.
  • the differentiation into neurons in the neuron differentiation medium as described herein can also result in the generation of 20-40 % GABA+ cells and/or 30-55 % vGlut+ cells and/or 5-15 % TH+ cells and/or less than 10 % astrocytes of the total of cells/the total of cells in the culture.
  • 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
  • astrocytes after differentiation of 45-50 days astrocytes can express S100 but can lack expression of GFAP. After a differentiation of 60 days astrocytes may express both S100 and GFAP.
  • astrocyte cultures obtained by the methods described herein comprise both, S100 + and GFAP+ astrocytes.
  • 60 days of differentiation astrocytes may additionally or alternatively express AQP4 and or EAAT2.
  • 80-90 days of differentiation astrocytes may be able to transport glutamate intracellular ⁇ . The person skilled in the art knows how to measure such a glutamate transport, whih is also described in the Examples herein.
  • GFAP also known as Glial fibrillary acidic protein is a protein that in humans is encoded by the GFAP gene.
  • Glial fibrillary acidic protein is an intermediate filament (IF) protein that is expressed by numerous cell types of the central nervous system (CNS) including astrocytes.
  • IF intermediate filament
  • Human GFAP mRNA is represented by the NCBI reference NM_001 131019 (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.
  • S100b or “S100” 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).
  • S100b embraces any S100b nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • 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.
  • IF intermediate filament
  • 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_001650 (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.
  • EAAT2 Excitatory amino-acid transporter 2
  • SLC1 A2 solute carrier family 1 member 2
  • SLC1A2/EAAT2 is a member of a family of the solute carrier family of proteins.
  • Human EEAT2 mRNA is represented by the NCBI reference NM_001 195728 (SEQ ID NO: 23) and the protein by Uniprot No. P43004 (SEQ ID NO: 24).
  • EAAT2 embraces any EAAT2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • 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).
  • MLDC multilineage cells
  • 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.
  • 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.
  • the ground medium can comprise serum.
  • 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).
  • FCS fetal calf serum
  • FBS fetal bovine serum
  • the serum can be FCS. Serum such as for example FCS may be obtained from e.g. Gibco.
  • 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.
  • serum can be employed at a concentration of about 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 1 1 %, 12 %, 13 %, 14 %, 15 %, 20 % or more.
  • serum can also be employed in a concentration of 10 % for example in the ground medium as described herein.
  • the differentiation of cells (NSCs) in the ground medium to generate astrocytes or MLDCs can be performed for any period of time.
  • cells may be differentiated for 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15 or more days.
  • 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, 1 10, 1 15 or more days.
  • Cells may also be differentiated in ground medium for less than 300, 250, 200, 185, 150, 130, 1 10 or less days.
  • cells can be differentiated in ground medium for 45-50 days, 60 days or 80-90 days.
  • the process of differentiation can take 45-50 days.
  • the process of differentiation can also take 60 days.
  • the process of differentiation can also take 80-90 days.
  • NSCs cells
  • differentiation can result in the generation of S100 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 s100b- positive.
  • NSCs cells
  • differentiation can result in the generation of GFAP-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 GFAP-positive.
  • NSCs cells
  • 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.
  • 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, 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19 or more relative to the iPSC (cell or culture from which it has been generated). It is further contemplated by the present invention that the so obtained NSC can express PAX6 with a fold change which is in the range of 8-40, or 8-30, or 9-20 relative to the iPSC (cell or culture from which it has been generated).
  • the so obtained NSC can express PAX6 with a fold change, which is less than 50, less than 40, less than 35, less than 30, less than 25 or less than 20 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).
  • 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.
  • the fold- change can be obtained with respect to mRNA levels of the markers as described herein. Such fold-changes may be measured using RT-qPCR.
  • 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). It is further contemplated by the present invention that the NSC expresses SOX1 with a fold change of at most 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5, 4, 3 or less 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).
  • 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). It is also envisioned by the present invention that the so obtained NSC can express SOX2 with a fold change of at most 2, 1 .9, 1 .8, 1 .7, 1 .6, 1 .5, 1 .4, 1 .3, 1 .2, 1 .1 , 1 .0 or less 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). 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 Ki-67 with a fold change of at most 10, 9, 8, 7, 6, 5, 4, 3.5, 3, 2.5, 2, 1.9, 1 .8, 1 .7, 1 .6, 1 .5, 1 .4, 1 .3, 1 .2, 1 .1 1 .0 or less 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).
  • the so obtained NSC can express nestin 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, 2, 2.2, 2.5, 2.7, 3, 3.3, 3.5, 3.7, 4, 5, 6 or more relative to the iPSC (cell or culture from which it has been generated). It is also envisioned by the present invention that the so obtained NSC can express nestin with a fold change of at most 6, 5, 4, 3, 2, 1 .5 or less relative to the iPSC (cell or culture from which it has been generated). The so obtained NSC can express nestin with a fold change of about 2 relative to the iPSC (cell or culture from which it has been generated).
  • 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).
  • the NSC lacks expression of at least one of Nanog, Klf4, LIN28A, Oct4 and/or Myc.
  • the NSC can lack expression of of Nanog and/or Oct4.
  • 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.
  • 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. O00570 (SEQ ID NO: 28).
  • SOX1 embraces any SOX1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • SOX2 also known as sex determining region Y-box 2
  • SOX2 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 cells. 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.
  • 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.
  • Klf4 also known as 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).
  • Klf4 embraces any Klf4 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • 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.
  • Oct4 also known as octamer- binding transcription factor 4
  • POU5F1 POU domain, class 5, transcription factor 1
  • 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_001 173531 (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.
  • 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. P01 106 (SEQ ID NO: 40).
  • the term Myc embraces any Myc nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
  • 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 NM_006617 (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.
  • the NSCs obtained by methods of the present invention can be passaged for more than 10, 1 1 , 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%.
  • 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.
  • the NSCs obtained by a method of the present invention can undergo about one population doubling in one passage.
  • cells can undergo about 20 population doublings. This is also shown in Figure 9a of the present application.
  • the present invention also relates to a neuron obtainable by methods of the present invention.
  • the present invention relates to an astrocyte obtainable by a method of the present invention.
  • 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.
  • therapy may also comprise administering a cell culture medium as described herein, in which cells have been grown/maintained/differentiated. This media are also referred to as conditioned media.
  • cells/media may be transplated/administered into the brain or areas of the peripheral ervous system of the subject.
  • cells/culture media/preparations/pharmaceutical compositions as disclosed herein are transplanted into a subject
  • cells are pre-differentiated for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days in a differentiation medium as described herein (neuronal/gound mdium) before transplantation.
  • a differentiation medium as described herein (neuronal/gound mdium)
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a NSC of the present invention, neuron of the present invention or astrocyte of the present invention.
  • 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.
  • the present invention further relates to a preparation obtainable by a method of the present invention.
  • 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.
  • preparation relates to a purification/isolation/ 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.
  • preparation can also comprise a cell present in any of the method steps of the present invention.
  • a preparation can also comprise a cell 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.
  • the present invention relates to an in vitro method or test system, wherein the method or test system comprises
  • 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
  • the compound of interest may for example be a binding protein such as an antibody or antibody molecule, a small molecule, a chemical compound, siRNA, mRNA, miRNA or any other compound.
  • a binding protein such as an antibody or antibody molecule, a small molecule, a chemical compound, siRNA, mRNA, miRNA or any other compound.
  • the in vitro method or test system of the present invention can be used in screening, expression profiling or disease modeling.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA and protein analysis can be detected by e.g. RNA and protein analysis.
  • RNA and protein analysis the skilled artesian knows how to determine if a cell expresses a certain marker.
  • expression of a certain molecule/marker can be determined by RNA and/or protein analysis.
  • 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 antibodies as described for example in Dainiak et al. (Adv Biochem Eng Biotechnol. 2007; 106: 1 -18).
  • MCS magnetic activated cell sorting
  • FACS flow cytometry activated cell sorting
  • 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 RT-qPCR) 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.
  • the measured expression of a marker level/amount as described herein may be normalized to a housekeeping gene.
  • the measured expression for a marker expressed by iPSCs as described herein is set at 1 and the expression of the same marker in the NSCs is related to thereto.
  • 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.
  • nucleic acid molecules can embrace chemically, enzymatically, or metabolically modified forms.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • 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.
  • polypeptide also refers to, and does not exclude, modifications of the polypeptide. Such modifications are known to the skilled artesian.
  • a "variant" of a polypeptide when used herein encompasses a polypeptide wherein one or more amino acid residues are substituted.
  • 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,
  • 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, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 ,
  • 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, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 67, 69.
  • 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,
  • 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, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 67, 69.
  • 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.
  • 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.
  • 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.
  • 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.
  • the BLASTP program uses as defaults a word size (W) of 3, and an expectation (E) of 10.
  • the BLOSUM62 scoring matrix Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) can be used.
  • 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.
  • oligonucleotides also called primers
  • 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.
  • 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).
  • PCR Polymerase Chain Reaction
  • a oligonucleotide can have a sequence of any of SEQ ID NO: 41 -66.
  • Exemplary means to detect a marker/protein as described herein can include suitable binding proteins as described herein.
  • the present invention is further characterized by the following items:
  • NSC neural stem cell
  • iPSCs induced pluripotent stem cells
  • TGF- ⁇ activin/transforming growth factor- ⁇
  • BMP bone morphogenetic protein
  • NSC human NSC
  • activin/TGF- ⁇ inhibitor is an inhibitor of the TGF- ⁇ type I receptor activin receptor-like kinase(s).
  • activin/TGF- ⁇ 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).
  • 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, TWS1 19, CHIR-99021 , CHIR-98014, SB 415286, SB 216763, LY2090314, AR-A014418 and IM-12, preferably CHIR 99021 .
  • non-essential amino acids comprise at least one of arginine, asparagine, glutamine, glycine, alanine, cysteine, aspartic acid, glutamamic acid, proline, tyrosine and serine.
  • antioxidant 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.
  • 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.
  • FGF signaling activator is FGF2 such as basic FGF2.
  • step d further comprises
  • 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 (HB-EGF), betacellulin (BTC), epigen (EPG) and epiregulin (EPR).
  • EGF epidermal growth factor
  • TGFa transforming growth factor a
  • AR amphiregulin
  • HB-EGF heparin-binding EGF-like growth factor
  • BTC betacellulin
  • EPG epigen
  • EPR epiregulin
  • [292] 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.
  • TGF- ⁇ transforming growth factor- ⁇
  • cAMP analogue is selected from the group consisting of 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, preferably the cAMP analogue is dbcAMP.
  • forskolin 8-(4-chloro-phenylthio)-2'-0-methyladeno
  • [298] 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.
  • [300] 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.
  • [301] 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.
  • FCS fetal calf serum
  • FBS fetal bovine serum
  • [304] 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 %.
  • [311] 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.
  • NSC of item 78 wherein the NSC expresses PAX6 with a fold change of at most 50 or less relative to the iPSC.
  • 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.
  • Astrocyte obtainable by a method of any one of items 53, 55, 68-77.
  • composition comprising a NSC of any one of items 78- 84, neuron of item 85 or astrocyte of item 86.
  • a preparation comprising a NSC of any one of items 78-84, neuron of item 85 or astrocyte of item 86.
  • 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.
  • iPSC were detached from plates using dispase (1 mg/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 ⁇ Medium" (KnockoutTM DMEM (Gibco), KnockoutTM SR (Gibco), NEAA (Gibco), penicillin/streptomycin (Invitrogen) and ⁇ -mercaptoethanol (Gibco)) supplemented with 10 MSB-431542 (Ascent Scientific), 1 ⁇ dorsomorphin (Tocris), 3 ⁇ CHIR 99021 (Axon Medchem) and 0.5 ⁇ (Alexis).
  • 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 ⁇ SB- 431542, 1 ⁇ dorsomorphin, 3 ⁇ CHIR 99021 and 0.5 ⁇ PMA.
  • 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 ⁇ SB- 431542, 1 ⁇ dorsomorphin, 3 ⁇ CHIR 99021 and 0.5 ⁇ PMA.
  • the medium was exchanged by N2B27 medium supplemented with 3 ⁇ CHIR 99021 , 0.5 ⁇ PMA, 150 ⁇ ascorbic acid and 20 ng/ml basic (b) FGF2 (Peprotech).
  • Human NSC Maintenance Medium was composed by DMEM HAM's F12 medium (Gibco) supplemented with 200ng/Ml EGF (Peprotech), 200ng ⁇ l bFGF2, N2 supplement, B27 supplement (with vitamin A), glutamine, Penicillin/Streptomycin and hLIF (150U/ml).
  • Neuronal differentiation medium composed by N2B27 Medium supplemented with 10 ng/mL BDNF (Peprotech), 10 ng/mL GDNF (Peprotech), 1 ng/mL TGF- 3 (Peprotech), 200 ⁇ ascorbic acid and 500 ⁇ dbcAMP (Sigma Aldrich). Cultures were tested/harvested after 4 weeks of neuronal differentiation.
  • RNAeasy kit Quality of service (Life Technologies). 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).
  • 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.
  • GO-biological process analyzer implemented in the DAVID analysis platform (http://david.abcc.ncifcrf.gQv/) (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 were then introduced into the REVIGO webserver (Supek et al. (201 1 ).
  • RNAs and miRNAs total RNA 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 CCTCACTTCACTGCACTGTA (SEQ ID NO: 43), CAGGTTTTCTTTCCCTAGCT (SEQ ID NO: 44)
  • NANOG TGAACCTCAGCTACAAACAG (SEQ ID NO: 45), TGGTGGTAGGAAGAGTAAAG (SEQ ID NO: 46)
  • SOX2 CCCAGCAGACTTCACATGT (SEQ ID NO: 47), CCTCCCATTTCCCTCGTTTT (SEQ ID NO: 48)
  • Ki67 ATACGTGAACAGGAGCCAG (SEQ ID NO: 49), C CTTG G AATCTTG AG CTTTCTC (SEQ ID NO: 50) SOX1 : AATTTTATTTTC G G C GTTG C (SEQ ID NO: 51 ), TGGGCTCTGTCTCTTAAATTTGT (SEQ ID NO: 52)
  • PAX6 ATGTGTGAGTAAAATTCTGGGCA (SEQ ID NO: 55), GCTTACAACTTCTGGAGTCGCTA (SEQ ID NO: 56)
  • GFAP CTGCTCAATGTCAAGCTGG (SEQ ID NO: 57), AATGGTGATCCGGTTCTCC (SEQ ID NO: 58)
  • MAP2 G G AG AC AG AG AG ATG AG AATTC CT (SEQ ID NO: 59), GAATTGGCTCTGACCTGGT (SEQ ID NO: 60)
  • GTGCTAAACCTGCTCTTCTC (SEQ ID NO: 61 ), TTCAAACGTCTCAAACACCT (SEQ ID NO: 62)
  • SLC6A1 1 (GABA): CAACAACTGCTACAGGGAC (SEQ ID NO: 63), GAG AAG ATG G C AAAC C C AG (SEQ ID NO: 64)
  • SLC17A7 Glutamate transporter: C C ATG ACTAAG C AC AAGACTC (SEQ ID NO: 65), AGATGACACCTCCATAGTGC (SEQ ID NO: 66).
  • 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 cell scraper and transferred in tubes containing 0.2 ml -20 °C chloroform. The extracts were shaken at 1400 rpm for 20 min at 4 °C (Thermomixer Eppendorf) and centrifuged at 16,000*g 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 MPS. Dried polar metabolites were dissolved in 15 ⁇ 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 ⁇ 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.
  • 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): NaCI 130, KCI 3, NaHC0 3 10, CaCI 2 1 .5, MgCI 2 1 , Glucose 1 1 , HEPES 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, MgCI 2 4, Na 2 GTP 0.3, HEPES 10, pH 7.4 with KOH. Measurements were done with an EPC10 amplifier and Patchmaster software (HEKA, Lambrecht, Germany).
  • 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).
  • Astrocytes DIV 80-90 and HEK 293 cells were incubated for 10 minutes at 37°C in basic media containing 50 ⁇ 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.
  • mice were under deep anesthesia (intraperitoneal injection of 17 ⁇ 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 .10 5 -2.10 5 cells) were injected into the lateral ventricle over 5 minutes using a Hamilton 7005KH 5 ⁇ syringe. Following stereotactic coordinates in relation to bregma were used:
  • 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 ⁇ sagittal brain sections were cut using a Vibratom (Leica VT 1200 S). Free-floating sections were permeabilized in Tris-buffered saline solution with 0.1 M 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.
  • Tris-buffered saline solution with 0.1 M Tris, 150mM NaCI, pH 7.4 / 0.5% Triton-X 100 / 0.1 % Na-Azide / 0.1 % Na-Citrate / 5% normal goat serum (TBS+/+/+
  • 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.
  • secondary Alexa-fluorophore conjugated antibodies Invitrogen
  • Hoechst 33258 (1 : 10000, Invitrogen
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • TCA tricarboxylic acid cycle
  • M3 citrate isotopologue results from both, pyruvate dehydrogenase and PC activity ( Figure 5a).
  • MLDCs multi-lineage differentiation cultures
  • PC activity in the pure astrocyte culture was observed (17 % enrichment vs. 4 % enrichment of M3 aspartate) ( Figure 5b).
  • M3 and M5 isotopologue abundance of citrate originating from M3 oxaloacetate as the precursor was observed (Figure 5c).
  • Lactate 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 1 mM concentration) resulting in higher MO abundance and second, pyruvate and thus lactate might be produced from serine via serine dehydratase.
  • 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.
  • NSCs neuronal differentiation
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cell line-derived neurotrophic factor
  • TGFB-3 transforming growth factor beta-3
  • dbcAMP dibutyryl- cAMP
  • ascorbic acid was used.
  • 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 %), glutamatergic (vGlutl , 40.34%) and dopaminergic neurons (TH, 12.68%) (Figure 10d-f), whereas differentiation into GFAP-positive cells was low (Figure 10g).
  • 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 Nestin- positive 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.
  • mice In total, 1 * 10 6 to 2 * 10 6 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.
  • hNSC 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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

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Abstract

La présente invention concerne des procédés pour l'obtention d'une cellule souche neuronale (NSC). Ces NSC peuvent être différenciées additionnellement en neurones et en cellules gliales. L'invention concerne également des NSC, des neurones et des astrocytes pouvant être obtenus par les procédés de la présente invention, ainsi que des préparations et des compositions pharmaceutiques comprenant ces cellules. De plus, la présente invention concerne des NSC, des neurones et des astrocytes selon la présente invention, destinées à être utilisés dans des applications thérapeutiques.
PCT/IB2016/054131 2015-07-10 2016-07-11 Cellules souches neuronales à auto-renouvellement à long terme Ceased WO2017009766A1 (fr)

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