WO2018115302A1 - Moyens et procédés de génération d'astrocytes - Google Patents

Moyens et procédés de génération d'astrocytes Download PDF

Info

Publication number
WO2018115302A1
WO2018115302A1 PCT/EP2017/084133 EP2017084133W WO2018115302A1 WO 2018115302 A1 WO2018115302 A1 WO 2018115302A1 EP 2017084133 W EP2017084133 W EP 2017084133W WO 2018115302 A1 WO2018115302 A1 WO 2018115302A1
Authority
WO
WIPO (PCT)
Prior art keywords
astrocytes
astrocyte
ipscs
cells
cell
Prior art date
Application number
PCT/EP2017/084133
Other languages
English (en)
Inventor
Marc EHRLICH
Tanja KUHLMANN
Original Assignee
Westfälische Wilhelms-Universität Münster
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Westfälische Wilhelms-Universität Münster filed Critical Westfälische Wilhelms-Universität Münster
Publication of WO2018115302A1 publication Critical patent/WO2018115302A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for producing astrocytes. Furthermore, the invention relates to an astrocyte obtainable by a method of the present invention. In addition, the present invention relates to a pharmaceutical composition. Also encompassed are astrocytes of the present invention for use in therapy and for use in a method of treating a disease. The present invention also relates to a method for identifying agents, which modulate astrocyte viability or function as well as a kit.
  • Genome editing has been widely used to introduce sequence-specific alterations in the genome of cells to derive reporter or gene-knockout cell lines for various purposes.
  • Most current approaches entail application of the CRISPR/Cas9 system, which was originally discovered as a bacterial immune response to foreign DNA and was subsequently remodeled to allow double strand breaks in specific regions of mammalian genomes (Cong et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823).
  • Genome editiing technology may be a promising tool to cure neurodegenerative diseases.
  • FTD is a group of neurodegenerative diseases characterized by profound degeneration of the frontal and temporal lobes leading to early-onset dementia with impairment of language, behavior and cognition (Boxer and Miller (2005) "Clinical features of frontotemporal dementia” Alzheimer Dis Assoc Disord 19 SuppI 1 , S3-6).
  • FTD is the second most common dementia after Alzheimer's disease (AD) and can be caused by mutations in MAPT on chromosome 17 encoding the microtubule-associated protein TAU, which stabilizes microtubules, promotes neural outgrowth and protects DNA from heat damage and oxidative stress (Goedert and Spillantini (201 1 ). Pathogenesis of the tauopathies. J Mol Neurosci 45, 425-431 ; Noble et al. (2013) "The importance of tau phosphorylation for neurodegenerative diseases” Front Neurol 4, 83).
  • MAPT microtubule-associated protein tau gene
  • the present invention relates to a method for producing astrocytes, the method comprising
  • NSCs neural stem cells
  • iPSCs induced pluripotent stem cells
  • SOX10 transcription factor
  • the present invention additionally relates to an astrocyte obtainable by a method of the present invention.
  • the present invention relates to a pharmaceutical composition comprising an astrocyte of the present invention.
  • the present invention relates to an astrocyte of the present invention for use in therapy.
  • the present invention also relates to an astrocyte for use in a method of treating a disease of a subject or for use in method of therapy of a disease of a subject, the method comprising
  • the present invention further relates to a use of transcription factor SOX10 for the generation of astrocytes from NSCs or iPSCs.
  • the present invention also relates to a method for identifying agents, which modulate astrocyte viability or function comprising
  • test agent alters the viability or function of the astrocytes compared to the viability or function of the astrocytes before contacting
  • test agent is suitable to modulate astrocyte viability or function.
  • the present invention relates to a kit comprising
  • c) optionally means for culturing NSCs and/or iPSCs.
  • the present invention also relates to a method for identifying molecules promoting or inhibiting astrocyte differentiation and/or death of astrocytes, the method comprising
  • Figure 1 Genetic correction of the MAPT locus in neural progenitor cells using CRISPR/Cas9 genome editing and characterization of disease phenotypes in differentiated FTD and Ctrl neurons and astrocytes.
  • FIG. 1 Schematic drawing depicting the genetic correction of the FTD-causing N279K mutation in exon 10 of the MAPT gene in iPS cell-derived neural progenitor cells (NPCs) using CRISPR/Cas9 technology.
  • Figure 2 Characterization of disease phenotypes in FTD NPC-derived astrocytes and in healthy Ctrl neurons after co-culture with FTD NPC-derived astrocytes.
  • D qRT-PCR expression analysis of differentially regulated genes (DRGs) in FTD and Ctrl astrocytes.
  • Figure 3 Screening for and characterization of CRISPR/Cas9 gene- corrected neural progenitor cells and calcium imaging of FTD and Ctrl astrocytes.
  • Genomic DNA was isolated from puromycin-resistant CRISPR/Cas9/ssODN transfected FTD N279K MAPT NPCs.
  • a 228 bp fragment spanning the mutation- containing region in exon 10 of MAPT was amplified via PCR and was digested with Mboll. Mboll does not cut the rescued wildtype N279 MAPT sequence in indicated clones (red arrows).
  • Figure 4 Protein and gene expression in FTD and Ctrl astrocytes and in Ctrl neurons after coculture with FTD or Ctrl astrocytes.
  • A-B Western blot expression analysis of (A) TAU (clone TAU-5) and (B) GFAP protein in FTD and Ctrl astrocytes. GAPDH was used as loading control. Independent replicates are shown for each line.
  • C Quantification of TALI and GFAP protein expression in FTD and Ctrl astrocytes.
  • astrocytes when contacting neural stem cells (NSCs) or induced pluripotent stem cells (iPSCs) with transcription factor SOX10 (SOX10) astrocytes can be obtained.
  • This effect could not have been forseen because SOX10 is usually considered to be restricted to the oligodendrocyte lineage and has not been described to have an important role in the develo ⁇ ment of astrocytes.
  • the astrocyte cultures and the method to obtain astrocytes as described herein have/has different advantages.
  • the astrocytes cultures obtained by the methods as described herein are very pure, since up to about 75 % to about 90 % of the total number of cells in the obtained cultures are astrocytes.
  • a further advantage of the method of the present invention and the so obtained astrocytes cultures is that the astrocyte cultures can be easily expanded. Thus, once generated the need for repetition of the obtaining procedures of the astrocytes is low. In this way costs, time and variance of astrocytes obtained in different experiments are also reduced.
  • a further advantage is that these methods can be performed to provide for patient-specific therapies, since these astrocytes can be obtained from NSC or iPSC.
  • cells such as fibroblasts can be obtained from a patient. From these fibroblasts then NSCs or iPSCs can be generated.
  • Techniques for such generation of NSCs and iPSCs from fibroblasts are known o the skilled person and for example described in Ring et al. (2012) "Direct Reprogramming of Mouse and Human Fibroblasts into Multipotent Neural Stem Cells with a Single Factor" Cell Stem Cell, vol. 1 1 , issue 1 , pages 100-109 or Lowry et al.
  • fibroblasts also other primary cells than fibroblasts can be used, which is also known to the skilled person. After obtaining astrocytes with the method as described herein these astrocytes may be introduced to the very same patient from whom the fibroblasts have been obtained, thereby expandinging a patient specific therapy.
  • the present present invention relates to a method for producing astrocytes, the method comprising
  • NSCs neural stem cells
  • iPSCs induced pluripotent stem cells
  • SOX10 transcription factor
  • ..astrocytes refers any glial cell that is an astrocyte.
  • astrocytes are involved in brain development (Reemst et al. (2016) "The Indispensable Roles of Microglia and Astrocytes during Brain Development” Front Hum Neurosci.10:566) are part of the tripartite synapse, can influence extrasynaptic neuronal currents (Pal et al. (2015) "Astrocytic Actions on Extrasynaptic Neuronal Currents” Front Cell Neurosci.
  • ALS amyotrophic lateral sclerosis
  • multiple sclerosis Lee et al. (2016) "Astrocytes and Microglia as Non-cell Autonomous Players in the Pathogenesis of ALS” and Correale and Farez (2015) “The Role of Astrocytes in Multiple Sclerosis Progression” Exp Neurobiol. 25(5):233-240).
  • Astrocytes can be characterized in any way that may be suitable. Suitable ways of characterizing astrocytes are also known to the skilled artesian. For example, astrocytes may be characterized by their morphology as e.g. described by Matyash and Kettenmann (2010) "Heterogeneity in astrocyte morphology and physiology" Brain Res Rev. 63(1 -2):2-10. Additionally or alternatively astrocytes can also be characterized by the expression of certain markers. Such markers, which can be expressed by astrocytes, can include e.g.
  • GFAP glial fibrillary acidic protein
  • S100b S100 calcium-binding protein B
  • vimentin aquaporin 4
  • ALDH1 L1 Aldehyde Dehydrogenase 1 Family Member L1
  • Glutamine synthetase EAAT1
  • EAAT2 Excitatory amino-acid transporter 2 also termed Glutamate transporter 1
  • the person skilled in the art also knows how to detect the expression of such astrocytic markers or other markers as described herein e.g. via utilizing commercially available antibodies recognizing such a marker.
  • the method of the present invention comprises using neural stem cells (NSC) as a starting cell population.
  • a neural stem cell "NSC” as described herein can be any NSC.
  • NSCs can, for example, be obtained from pluri- or multipotent stem cells in several different stages of neural development. They may have also been directly isolated from a subject. NSCs are multipotent. Therefore, neural stem cells have the capacity to differentiate further into multiple types of cells, such as neurons, astrocytes and oligodendrocytes. Multipotency can be tested by differentiating said cells into different lineages such as astrocytes, oligodendroctyes and neurons. Such methods are known to the skilled artesian.
  • 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 are also 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.
  • NSCs can also be identified by the expression of certain/several markers, which are also known in the art. Such markers can, for example, include Nestin and SOX2, Pax6, CD133, Prominin 2, PDGF receptor a just to name a few.
  • pluripotent stem cells can be obtained by methods which do not destroy the embryo.
  • pluripotent stem cells as described herein refer to cells, which have been obtained by a method not destroying an embryo. Such methods are known to the skilled artesian and for example described in Dittrich et al. (2015) ,, ⁇ -embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine" Raven Press
  • NSCs can, for example, be obtained from induced plu potent stem cells (iPSCs) as described herein and as also known to the skilled artesian. Therefore, the present invention also encompasses that the PSCs are induced PSCs (iPSCs).
  • iPSCs induced plupotent stem cells
  • How NSCs can be obtained from induced pluhpotent stem cells is, for example, described in Choi et al. (2014) "Neural stem cells differentiated from iPS cells spontaneously regain pluripotency" Stem Cells. 32(10):2596-604 or Lee-Kubli and Lu (2015) "Induced pluhpotent stem cell-derived neural stem cell therapies for spinal cord injury" Neural Regen Res. 10(1 ): 10-16.
  • iPSCs as such can be utilized as a starting population. Without being bound to theory it is thought that these iPSCs can be directly differentiated into astrocytes without the need of passing through an explicit NSC stage. Yet, as outlined above these iPSCs can also first be differentiated into NSCs and then further be differentiated into astrocytes.
  • IPCs Induced pluhpotent stem cells
  • Induced pluhpotent stem cells are an important advancement in stem cell research, as they allow obtaining pluhpotent stem cells without the use or destruction of embryos. How such iPSCs can be generated is known to the skilled artesian and is, for example, described in Tanabe et al. (2014) “Induction of pluripotency by defined factors” Proc. Jpn. Acad., 2014, Ser. B 90, Takahashi et al. (2007) "Induction of pluhpotent stem cells from adult human fibroblasts by defined factors” Cell; 131 (5):861 -72, Lowry et al. (2007) "Generation of human induced pluhpotent stem cells from dermal fibroblasts" PNAS, vol.
  • the NSCs and/or iPSCs are human NSCs and/or human iPSCs.
  • the NSCs and/or iPSCs can be in principle from any animal or subject as described herein. Exemplary further animals include canine, feline, equine, ape, macaque, mouse or rat.
  • iPSCs can be derived/generated from any suitable non- pluripotent cell.
  • iPSCs can be obtained from a fibroblast, keratinocyte, T-cell, G-CSF mobilized CD34+ cell, myeloid cell, or a renal epithelial cell from the urine.
  • iPSCs can for example be obtained from primary amniotic fluid cells, umbilical cord blood mononuclear cells. Particularly, the iPSCs can be obtained from a fibroblast.
  • the present invention further contemplates that the NSCs or iPSCs are contacted with transcription factor SOX10 (SOX10).
  • SOX10 transcription factor
  • the term "SOX10" as used herein is known to the skilled person.
  • the SOX genes form a gene family related by homology to the high-mobility group (HMG) box region of the testis- determining gene SRY (Pusch et al. (1998) "The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor” Hum Genet. 103(2):1 15-23).
  • HMG high-mobility group
  • Sox10 is an essential component of the myelin-specific regulatory network (Hornig et al. (2013) "The transcription factors Sox10 and Myrf define an essential regulatory network module in differentiating oligodendrocytes" PLoS Genet. 9(10):e1003907).
  • SOX10 or "Sox10” or “sox10” which can be used interchangebly herein includes SOX10 from any species such as SOX10 (Sox10, sox10) from human, canine, feline, equine, ape, macaque, mouse or rat.
  • SOX10 is human SOX10.
  • SOX10 further embraces any SOX10 polypeptide or any SOX10 nucleic acid molecule.
  • polypeptide when used herein means a peptide, a protein, or polypeptides, which are used interchangeable and which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds.
  • peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine.
  • Peptides, oligopeptides and proteins may be termed polypeptides.
  • polypeptide and protein are often used interchangeably herein.
  • polypeptide also refers to, and does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
  • 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 include DNA, cDNA, genomic DNA, RNA, synthetic forms, for example, PNA, and mixed polymers, both sense and antisense strands, or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • the polynucleotide of the present invention is preferably composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • the polynucleotide can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, the term “nucleic acid molecules" embraces chemically, enzymatically, or metabolically modified forms.
  • a SOX10 polypeptide can comprise a sequence of SEQ ID NO. 1 or 2. It is further contemplated that the SOX10 polypeptide can comprise a sequence having at least 85 %, 86, %, 87 %, 88%, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the sequence of SEQ ID NO. 1 and/or 2.
  • SOX10 nucleic acid molecule can be any suitable SOX10 nucleic acid molecule.
  • the SOX10 nucleic acid molecule can be mRNA or DNA.
  • a nucleic acid molecule can comprise a nucleic acid molecule encoding a polypeptide comprising a sequence of SEQ ID NO. 1 or 2.
  • the SOX10 nucleic acid molecule can encode a polypeptide comprising a polypeptide having at least 85 %, 86, %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of SEQ ID NO. 1 and/or 2.
  • the nucleic acid molecule can also comprise a nucleic acid molecule comprising a sequence of SEQ ID NO. 3.
  • the SOX10 nucleic acid molecule can comprise a nucleic acid molecule having at least 85 %, 86, %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of SEQ ID NO. 3.
  • identity or “sequence identity” as used herein is meant a property of sequences that measures their similarity or relationship.
  • sequence identity or “identity” as used in the present invention means the percentage of pair-wise identical residues - following (homology) alignment of a sequence of a polypeptide or nucleic acid sequence of the invention with a sequence in question - with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • BLAST which stands for Basic Local Alignment Search Tool (Altschul (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res. 25(17):3389-402, Altschul (1993) "A protein alignment scoring system sensitive at all evolutionary distances” J Mol Evol. 36(3):290-300, Altschul (1990) "Basic local alignment search tool” J Mol Biol. 1990 Oct 5;215(3):403-10), can be used to search for local sequence alignments.
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity.
  • HSP High-scoring Segment Pair
  • An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
  • the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
  • the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
  • the present invention encompasses that the NSCs or iPSCs are contacted with transcription factor SOX10 (SOX10).
  • SOX10 transcription factor
  • the term "contacting" as used herein can mean any contacting that is suitable for the purposes of the present invention.
  • contacting may comprise introducing of SOX10 as described herein into NSCs or into iPSCs.
  • the skilled person knows means and methods to introduce a transcription factor such as SOX10 into a NSC or iPSC.
  • any form of introduction of SOX10 into a cell may be applied.
  • a vector is utilized.
  • Exemplary vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Often such vectors comprise an origin of replication, a multicloning site, and a selectable marker.
  • the vector may integrate into the DNA of the NSC or iPSC or may remain extrachromosomally within the NSC or iPSC.
  • SOX10 can be encoded by a recombinant expression vector. It is further encompassed that the vector comprises an expression control sequence functional in the NSCs or the iPSCs operatively linked to SOX10.
  • exemplary recombinant expression vectors include a naked plasmid, a herpesvirus, a retrovirus, a lentivirus, a sendai virus, an adenovirus and an adeno-associated virus.
  • the NPCs or the iPSCs can be transduced with a lentivirus.
  • naked DNA histone free DNA
  • recombinant DNA DNA molecules formed by laboratory methods
  • RNA or protein can be used to introduce SOX10 as described herein into a NSC or iPSC.
  • the introduction of SOX10 can include the induction of SOX10 expression in the NSCs or in the iPSCs.
  • SOX10 expression can, for example, be analyzed by measuring SOX10 protein expression by utilizing commercially available anti-SOX10 antibodies.
  • the NSCs or the iPSCs are transduced or transfected with SOX10.
  • transfection means the delivery of DNA or RNA into eukaryotic cells. Transfection can be transient or stable. When cells are transiently transfected with plasmids, the DNA is introduced into the nucleus of the cell, but does not integrate into the chromosome. This means that many copies of the gene of interest can be present, which can lead to high levels of expressed protein. With stable or permanent transfection, the transfected DNA is either integrated into the chromosomal DNA or maintained as an episome. Transfection can be e.g. a plasmid DNA transfection.
  • RNA such as mRNA, in vitro transcribed RNA, viral RNA, RNA oligos, siRNA, and ribozymes
  • Transfection can be mediated by e.g. chemical transfection methods, physical transfection methods, micro-injections, electroporation, or phototransfection using a multi-photon laser.
  • suitable techniques for transfection which are e.g. described in Kim and Eberwine (2010) geoMammalian cell transfection: the present and the future" Anal Bioanal Chem; 397(8): 3173-3178.
  • transduction means a transfection process by which foreign DNA is introduced into a cell by a virus or viral vector.
  • viral vectors include a herpesvirus, a retrovirus, a lentivirus, a sendai virus, an adenovirus and an adeno- associated virus.
  • the NPCs or the iPSCs can be transduced with a lentivirus.
  • the NSCs or iPSCs can be contacted with SOX10 by protein transduction, microinjection, electroporation, protein transduction using protein transduction domain (PTD)-mediated transduction, a non-covalent carrier or a lipid carrier.
  • PTD protein transduction domain
  • the transduction can be performed for about 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or about 1 day.
  • the method of the present invention may further comprise
  • BMP signaling inhibitor as used herein is defined as a compound/molecule reducing or blocking the activity of the BMP signaling pathway.
  • the BMP signaling pathway is known to the skilled artesian and, for example, described in Rahman et al. (2015) "TGF-b/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation” Bone Research 3, Art.no. 15005).
  • BMPs can signal through both canonical and non-canonical pathways. In the canonical signaling pathway they initiate the signal transduction cascade by binding to cell surface receptors and forming a heterotetrameric complex comprised of two dimers of type I and type II serine/threonine kinase receptors.
  • ALK1 -7 type I receptors for the TGF- ⁇ family of ligands, three of which bind BMPs: type 1A BMP receptor (BMPR-1A or ALK3), type 1 B BMP receptor (BMPR-1 B or ALK6), and type 1A activin receptor (ActR-1A or ALK2).
  • BMPs type 1A BMP receptor
  • BMPR-1B type 1 B BMP receptor
  • ActR-1A type 1A activin receptor
  • ActR-2B type 2B activin receptor
  • BMPR-1A, BMPR- 1 B, and BMPR-2 are specific to BMPs, ActR-1A, ActR-2A, and ActR-2B can function as receptors for activins, which are also members of the TGF- ⁇ superfamily.
  • BMP4 was found to activate TAK-1 , a serine-threonine kinase of the MAPKKK family.
  • BMP signaling has been found to affect PI3K/Akt, P/kc, Rho-GTPases, and others.
  • the inhibitors of BMP signaling can only block/reduce the canonical signaling BMP pathway.
  • the BMP signaling inhibitor can be a canonical BMP signaling inhibitor.
  • the BMP signaling inhibitor is an inhibitor for the BMP type 1 receptor isotypes Alk2 and/or Alk3.
  • the inhibitor may achieve this effect by reducing or blocking the transcription of the gene encoding BMP (compared to transcription before addition of the inhibitor) and/or reducing/blocking the translation of the imRNA encoding BMP (compared to translation before addition of the inhibitor). It can also be that BMP performs its biochemical function with decreased efficiency in the presence of the inhibitor (compared to biochemical function before the addition of the inhibitor) or that BMP performs its cellular function with reduced efficiency in the presence of the inhibitor (compared to cellular function before addition of the inhibitor).
  • the term "inhibitor” encompasses both molecules/compounds that have a directly reducing/blocking effect on the specific BMP signaling pathway but also molecules that are indirectly inhibiting, e.g. by interacting, for example, with molecules that positively regulate (e.g. activate) this 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 expression/amount of e.g. pathway effector proteins (e.g. compared to the expression/amount measured before addition of the inhibitor).
  • the compound/molecule that can be used as an inhibitor can be any compound/molecule, which can reduce or block the BMP signaling pathway or which inhibits an activator of the BMP signaling (pathway).
  • Exemplary inhibitors can include suitable binding molecules as described herein, which are directed e.g. against activators of the BMP signaling pathway.
  • the binding molecule can be an antibody.
  • Such antibody can, for example, be a monovalent or divalent antibody fragment.
  • the divalent antibody fragment can comprise two binding sites with different specificities.
  • the binding molecule 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 molecule 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 aptamer can also be a molecule only comprising nucleotides.
  • 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 (ALK2, ALK3 and ALK6).
  • BMP bone morphogenetic protein
  • the inhibitor can also be an antagonist of the pathway/signaling pathway 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 inhibitor.
  • 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.
  • BMP signaling inhibitors include DMH1 (CAS 120671 1 -16-1 ), K 02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol; CAS No.: 1431985-92-0), LDN 193189 (4-[6-[4-(1 -Piperazinyl)phenyl]pyrazolo[1 ,5- a]pyrimidin-3-yl]-quinoline hydrochloride, CAS No.: 1062368-24-4), ML347 (5-[6-(4- Methoxyphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl]quinolone, CAS No.
  • dorsomorphin dihydrochloride (6-[4-[2-(1 -Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)- pyrazolo[1 ,5-a]pyrimidine dihydrochloride; CAS No.: 1219168-18-9).
  • the BMP signaling inhibitor can also be LDN-193189.
  • the BMP signaling inhibitor such as LDN 193189 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 ⁇ .
  • LDN 193189 can for example be obtained from Axon Medchem.
  • the medium comprising the BMP signaling inhibitor as described herein may further comprise a SHH signaling activator. It is also contemplated herein that cells are first cultivated in a medium comprising a BMP signaling inhibitor and subsequently in a medium comprising a SHH signaling activator. It is further encompassed that the cells are first cultivated in a medium comprising a SHH signaling activator and subsequently in a medium comprising a BMP signaling inhibitor.
  • activator is defined as a compound/molecule enhancing or achieving the activity of the SHH signaling pathway.
  • the "Hedgehog signaling pathway” or “SHH pathway” is well known in the art and has been described in Rimkus (2016) "Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors” Cancers (Basel); 8(2). pii: E22.
  • the sonic hedgehog (Shh) signaling pathway is a major regulator of cell differentiation, cell proliferation, and tissue polarity. Downstream effectors of the Shh pathway include smoothened (SMO) and glioma-associated oncogene homolog (GLI) family of zinc finger transcription factors.
  • the term "activator of the Hedgehog signaling pathway” also refers to an activator of a molecules that form part of the SHH signaling pathway.
  • the SHH signaling activator may achieve this effect by enhancing or inducing the transcription of the gene encoding SHH (compared to transcription before addition of the activator) and/or enhancing the translation of the mRNA encoding SHH (compared to translation before addition of the activator). It can also be that SHH performs its biochemical function with enhanced efficiency in the presence of the activator (compared to biochemical function before addition of the activator) or that SHH performs its cellular function with enhanced efficiency in the presence of the activator (compared to cellular function before addition of the activator).
  • the term "activator” encompasses both molecules/compounds that have a directly activating effect on the SHH 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 SHH signaling pathway.
  • Methods for testing if a compound/molecule is capable to induce or enhance the activity of a target molecule and/or pathway is known to the skilled artesian.
  • an activator of a SHH can be tested by performing Western Blot analysis of the amount of e.g. pathway effector proteins such as Gli proteins.
  • 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 or SHH C24II.
  • the SHH- pathway activator can also be selected from the group consisting of purmorphamine, SHH, SAG and Gli-2.
  • the medium comprising the BMP signaling inhibitor as described herein may further comprise at least 1 , 2, 3, 4, 5, 6 or more growth factors. It is also contemplated herein that cells are first cultivated in a medium comprising a BMP signaling inhibitor and/or a SHH signaling inhibitor and subsequently in a medium comprising at least 1 , 2, 3, 4, 5, 6 or more growth factors. It is further encompassed that the cells are first cultivated in a medium comprising at least 1 , 2, 3, 4, 5, 6 or more growth factors and subsequently in a medium comprising a BMP signaling inhibitor and/or a SHH signaling inhibitor.
  • a "growth factor” as used herein encompasses any suitable growth factor. Growth factors are also known to the skilled artesian. Typically, a growth factor is capable of stimulating cellular growth proliferation, healing, and/or cellular differentiation. Usually it is a protein or a steroid hormone.
  • Exemplary growth factors include Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Insulin-like growth factor (IGF), Epidermal growth factor (EGF), Leukemia inhibitory factor (LIF), Vascular endothelial growth factor (VEGF), Transforming growth factor (TGF), Platelet-derived growth factor (PDGF), Brain-derived neurotrophic factor (BDNF), Nerve growth factor (NGF), Neurotrophin-3 (NT3), and/or Neurotrophin-4 (NT4). It is further encompassed that the medium can comprise NT3, PDGF, FGF and IGF.
  • FGF Fibroblast growth factor
  • GDNF Glial cell line-derived neurotrophic factor
  • IGF Insulin-like growth factor
  • EGF Epidermal growth factor
  • LIF Leukemia inhibitory factor
  • VEGF Vascular endothelial growth factor
  • TGF Transforming growth factor
  • PDGF Platelet-derived growth factor
  • BDNF Brain-derived neurotrophic factor
  • the medium comprising the BMP signaling inhibitor as described herein may further comprise one or more of
  • the cells are kept in the medium as described herein for at least 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days or more days. It is also encompassed that the cells are kept in the medium for at most 60 days, 55 days, 50 days, 45 days, 40 days, 35 days, 30 days, 25 days, 25 days or less days. For example, the cells can be kept in the medium for 45 days.
  • the cells that are cultivated in a medium as described herein are splitted at least every three, four, five, six, seven, eight or nine days, for example after each 7 days.
  • the medium comprising the BMP signaling inhibitor as described herein may not comprise an inhibitor of SMAD signaling under the proviso that NPCs are contacted with transcription factor SOX10 (SOX10).
  • the method as described herein can further comprise
  • differentiation medium means a medium that promotes cells to change from one type to another. Often a less specialized type such as NPC or iPSC is then becoming more specialized in form and function such as e.g. astrocyte.
  • the astrocyte differentiation medium can comprise serum.
  • serum as used herein is known to the skilled artesian and different sera are commercially available.
  • 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. Biochrom. Serum can, for example, be employed at a concentration of about 0.5 %, 1 %, 1.5 %, 2 %, 2.5 %, 3 % 3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 % to about 9 %.
  • Serum e.g. FCS can also be employed in a concentration of 4 %.
  • such astrocyte differentiation medium may for example comprise one or more of
  • one or more growth factors for example CNTF and IGF;
  • an "antioxidant” as used herein means a molecule that inhibits the oxidation of other molecules. Accordingly, an antioxidant refers to an inhibitor of a molecule involved in cellular oxidative processes.
  • the terms “oxidation” and “antioxidant” are well known in the art.
  • Exemplary antioxidants include 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.
  • the antioxidant can be ascorbic acid (AA).
  • 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.
  • 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)
  • the cells can be kept in the astrocyte differentiation medium for at least 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days or more days. It is further envisioned that the cells can be kept in the astrocyte differentiation medium for at most 60 days, 55 days, 50 days, 45 days, 40 days, 35 days, 30 days, 25 days, 25 days or less days. So, for example, the cells can be kept in the astrocyte differentiation medium for 40-45 days.
  • the method as described herein may additionally or alternatively further comprise
  • "Maintaining" or “expanding” of cells can, for example, include passaging of cells.
  • the growth of cells in culture proceeds from the lag phase following seeding to the log phase, where the cells proliferate exponentially.
  • the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is greatly reduced or ceases entirely. To keep them at an optimal density for continued growth and to stimulate further proliferation, the culture has to divide and fresh medium supplied (passaging of cells).
  • astrocytes In the cell culture as obtained by a method as described herein more than 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 97 %, 99 % or 100 % of the total number of cells are astrocytes.
  • astrocytes In the obtained cell culture about 75 % to about 90 % of the total number of cells can be astrocytes.
  • the present invention also relates to an astrocyte obtainable by a method as described herein.
  • the present invention also relates to an astrocyte obtained by a method as described herein.
  • the astrocytes of the culture obtained by the method as described herein or the astrocyte of the present invention can express one or more of
  • the astrocytes of the culture obtained by the method as described herein or the astrocyte of the present invention can additionally or alternatively be capable of uptaking L-glutamate and/or propagating calcium waves.
  • the present invention also relates to a preparation obtainable by a method as described herein.
  • a preparation can relate to a purification/isolation/recovery of an astrocyte of the present invention from the medium and/or from a cell extract.
  • the term preparation can also comprise a cell present in any of the method steps as described herein.
  • a preparation can also comprise a cell present in step a), b), c), d), e) as described herein.
  • the preparation can also comprise culture medium as described herein.
  • the present invention also relates to a pharmaceutical composition comprising an astrocyte as described herein.
  • the present invention also relates to an astrocyte as described herein for use in therapy.
  • the therapy can for example be a therapy of a neurodegenerative disease.
  • exemplary neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, hepatic encephalopathy (HE), hyperammonemia (HA), ischemia, amyotrophic lateral sclerosis, Huntington's disease and Alexander disease or frontotemporal dementia (FTD).
  • the present invention also relates to astrocytes for use in a method of treating a disease of a subject or for use in method of therapy of a disease of a subject, the method comprising
  • the present invention also relates to a method of treating a subject in need thereof, the method comprising
  • 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. In principle any neurodegenerative disease can be encompassed by the present invention.
  • Preferred neurodegenerative diseases are such diseases, which comprise a mutation in a certain gene.
  • the cell can be an autologous cell.
  • the autologous cell may be any suitable cell.
  • Exemplary, autologous cells or cells in general are fibroblasts, keratinocytes, T-cells, G-CSF mobilized CD34+ cells, myeloid cells, or renal epithelial cells from the urine.
  • the autologous cell can be a fibroblast.
  • the method can further comprise
  • the method can additionally or alternatively further comprise
  • the method can additionally or alternatively further comprise
  • the autologous cell may be gene edited (step a.1 ).
  • Gene editing is known to the skilled artesian and for example described in Ratner et al. (2016) Overview of CRISPR-Cas9 Biology" Cold Spring Harb Protoc. 1 ;2016(12):pdb.top08884 and Gaj et al. (2016) “Genome-Editing Technologies: Principles and Applications” Cold Spring Harb Perspect Biol. 8(12). pii: a023754.
  • Gene editing can thus mean that DNA is inserted, deleted or replaced in the genome of a cell.
  • engineered nucleases can be used. Such nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
  • induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations.
  • exemplary engineered nucleases include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the CRISPR-Cas system.
  • gene editing can comprise exchanging a nucleic acid sequence present in the NSCs or iPSCs with a nucleic acid sequence formally not present in the NSCs or iPSCs.
  • gene editing can comprise exchanging a nucleic acid sequence in the NSCs or iPSCs with a nucleic acid sequence, which was not present in the NSCs or iPSCs before exchanging.
  • the present invention encompasses that e.g. a autologous cell, astrocyte, NPC or iPSC carrying a mutation in a gene is gene edited to express a non-mutated gene.
  • any gene and especially mutated genes may be targeted by the methods as described herein.
  • Exemplary (possibly mutated) genes that can be a target of gene editing can include MAPT (microtubule associated protein tau), APP (amyloid precursor protein), PSEN1 (presenilin-1 ), SNCA (a-synuclein), LRRK2 (leucine-rich repeat kinase 2), UCHL-1 (ubiquitin carboxyl-terminal hydrolase isozyme L1 ), PARK2 (E3 ubiquitin-protein ligase parkin), PINK1 (PTEN-induced putative kinase 1 ), or DJ-1 (protein deglycase DJ-1 ).
  • MAPT microtubule associated protein tau
  • APP amloid precursor protein
  • PSEN1 presenilin-1
  • SNCA a-synuclein
  • LRRK2 leucine-rich repeat kinase 2
  • UCHL-1 ubiquitin carboxyl
  • the introducing of astrocytes may be performed by any method that is suitable for that purpose.
  • One exemplary method is transplantation of the astrocytes e.g. intoa brain area affected by a disease of the subject.
  • the present invention also relates to a use of transcription factor SOX10 for the generation of astrocytes from NSCs or iPSCs.
  • the use can further comprise a use of a BMP signaling inhibitor.
  • the embodiments as described herein for the methods of the present invention also apply mutatis mutandis to the uses as described herein.
  • the present invention also relates to a method for identifying agents, which modulate astrocyte viability or function comprising
  • test agent alters the viability or function of the astrocytes compared to the viability or function of the astrocytes before contacting
  • the alteration of viability or function of astrocytes can for example be measured by a cellular parameter suitable to indicate astrocyte viability or function. So for example, astrocyte viability can be measured by the occurrence of astrocyte death as described herein. The less astrocyte death is detected the more viable are the astrocytes. Astrocyte function can, for example, be detected by the determination of the uptake of glutamate or the ability to propagate calcium waives as e.g. described in the Examples. The higher the uptake of glutamate or the ability of propagating calcium waives more functional is an astrocyte. Usually for comparision a control as described herein is utilized.
  • the present invention also relates to a method for identifying molecules promoting or inhibiting astrocyte differentiation and/or death of astrocytes, the method comprising
  • control as referred to herein can be the same culture without the addition of the molecule of interest.
  • molecule of interest and “test agent” can be used interchangeably herein.
  • the control may also be a culture of NSCs or iPSCs obtained from the very same subject without the addition of the molecule of interest.
  • the control may also be a culture obtained from NSCs or iPSCs obtained from a healthy subject.
  • Astrocyte differentiation and/or death of astrocytes can be measured by different means.
  • the differentiation astrocytes and their death can be measured by analyzing expression levels of astrocytic markers as described herein.
  • the differentiation into astrocyes and/or astrocyte death can be measured by comparing expression of astrocyte specific markers as described herein. The comparison can be performed with regard to a control e.g. between similar cultures e.g. from the very same subjects with and without the addition of the molecule of interest as described herein.
  • a control e.g. between similar cultures e.g. from the very same subjects with and without the addition of the molecule of interest as described herein.
  • Higher levels of expression of astrocyte marker(s) compared to a control indicate the presence of more astrocytes and greater survival/lesser death of astrocytes.
  • lower levels of expression of astrocyte specific markers compared to a control indicate the presence of less astrocytes and lesser survival/greater death of astrocytes. Again comparison can be performed using the same culture with or without the molecule of interest or between different cultures e.g. of diseased and healthy subjects.
  • astrocytes can also be measured by methods well-known to the skilled artesian. For example, cell cultures may be stained for caspase 3 or TUNEL eventually in addition to staining for astrocyte specific markers.
  • the present invention further relates to a method for identifying agents, which modulate viability or function of neurons, the method comprising
  • test agent alters the viability or function of the neurons compared to the viability or function of the neurons before contacting.
  • the autologous cell as described herein can for example comprise a mutation in a gene.
  • the neuron as described herein can for example be a respiratory stressed neuron.
  • the present invention also relates to a kit comprising
  • c) optionally means for culturing NSCs and/or iPSCs.
  • the present invention also relates to a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of astrocytes as described herein to a subject in need thereof.
  • the present invention is further characterized by the following items:
  • a method for producing astrocytes comprising
  • NSCs neural stem cells
  • iPSCs induced pluripotent stem cells
  • SOX10 transcription factor
  • the recombinant expression vector is selected from the group consisting of a naked plasmid, a herpesvirus, a retrovirus, a lentivirus, a sendai virus, an adenovirus and an adeno- associated virus.
  • the BMP signaling inhibitor is selected from the group consisting of K02288, LDN-193189, DMH1 , LDN-212854, ML347, and dorsomorphin dihydrochloride, preferably the BMP signaling inhibitor is LDN-193189.
  • SHH- pathway activator is selected from the group consisting of purmorphamine, SHH, SAG and Gli-2.
  • the growth factor is selected from the group consisting of Fibroblast growth factor (FGF), Glial cell line- derived neurotrophic factor (GDNF), Insulin-like growth factor (IGF), Epidermal growth factor (EGF), Leukemia inhibitory factor (LIF), Vascular endothelial growth factor (VEGF), Transforming growth factor (TGF), Platelet-derived growth factor (PDGF), Brain-derived neurotrophic factor (BDNF), Nerve growth factor (NGF), Neurotrophin-3 (NT3), and/or Neurotrophin-4 (NT4).
  • FGF Fibroblast growth factor
  • GDNF Glial cell line- derived neurotrophic factor
  • IGF Insulin-like growth factor
  • EGF Epidermal growth factor
  • LIF Leukemia inhibitory factor
  • VEGF Vascular endothelial growth factor
  • TGF Transforming growth factor
  • PDGF Platelet-derived growth factor
  • BDNF Brain-derived neurotrophic factor
  • NGF Nerve growth factor
  • NT3 Neurotrophin-3
  • [151] 28. The method any one of the preceding items, wherein the cells are kept in the medium for at most 60 days, 55 days, 50 days, 45 days, 40 days, 35 days, 30 days, 25 days, 25 days or less days.
  • one or more growth factors preferably CNTF and IGF;
  • antioxidant is selected from the group consisting of ascorbic acid, glutathione, lipoic acid, superoxide dismutase 1 , superoxide dismutase 2, superoxide dismutase 3, epigallocatechin gallate, curcumine, melatonin, hydroxytyrosol, ubiquinone, catalase, vitamin E and uric acid.
  • 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-cA P), 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-methyladen
  • [159] 36 The method of any one of the preceding items, wherein the cells are kept in the astrocyte differentiation medium for at least 20 days, 25 days, 30 days, 35 days, 40 days,45 days, 50 days, 55 days, 60 days or more days.
  • [168] 45 The astrocyte of item 43 or 44, wherein the astrocyte is capable of uptaking L-glutamate and/or propagating calcium waves.
  • [170] 47 A pharmaceutical composition comprising an astrocyte of any one of items 43-45.
  • [172] 49 The astrocyte for use of item 48, wherein the therapy is a therapy of a neurodegenerative disease.
  • astrocyte for use of item 49, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, hepatic encephalopathy (HE), hyperammonemia (HA), ischemia, amyotrophic lateral sclerosis, Huntington's disease and Alexander disease or frontotemporal dementia.
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, hepatic encephalopathy (HE), hyperammonemia (HA), ischemia, amyotrophic lateral sclerosis, Huntington's disease and Alexander disease or frontotemporal dementia.
  • MAPT microtubule associated protein tau
  • APP amynuclein
  • SNCA D-synuclein
  • LRRK2 leucine-rich repeat kinase 2
  • UCHL-1 ubiquitin carboxyl-terminal hydrolase isozyme L1
  • PARK2 E3 ubiquitin-protein liga
  • astrocytes prepared by the method of any one of items 1 -42, b) contacting said astrocytes with a test agent, and
  • test agent alters the viability or function of the astrocytes compared to the viability or function of the astrocytes before contacting
  • test agent is suitable to modulate astrocyte viability or function.
  • [185] 62 A method for identifying agents, which modulate viability or function of neurons, the method comprising
  • test agent alters the viability or function of the neurons compared to the viability or function of the neurons before contacting.
  • c) optionally means for culturing NSCs and/or iPSCs.
  • [189] 66 A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of astrocytes as defined in items 43- 45 to a subject in need thereof.
  • Fibroblasts from an individual carrying the N279K mutation in MAPT here referred to as FTD
  • FTD human neural progenitor cells
  • NPCs Neural progenitor cells
  • NPCs were cultured in expansion medium consisting in equal parts of DMEM-F12 (Invitrogen) and neurobasal medium (Invitrogen) with 1 :200 N2 supplement (Invitrogen), 1 :100 B27 supplement lacking vitamin A (Invitrogen), 1 % penicillin/streptomycin/glutamine, 3 ⁇ CHIR99021 (Axon Medchem), 0.5 ⁇ SAG (Cayman Chemical) and 150 ⁇ Ascorbic acid (AA; Sigma).
  • Cells were grown on matrigel-coated (Matrigel, growth factor reduced, high concentration; BD Bioscience) 12-well plates (Nunc) and split once a week at ratios of 1 :15 to 1 :20 by treatment with accutase.
  • sgRNA single guide RNA harboring vector pX260 (Addgene plasmid #42229) was used to edit the N279K MAPT mutation on chromosome 17 (17q21 .1 ).
  • sgRNAs were designed to target exon 10 of MAPT using the Target Finder Software (Feng Zhang Laboratory, MIT, Cambridge, USA, http://crispr.mit.edu.; MAPT10_1 : 5'- GTACTCACACTGCCGCCTCC-3' (SEQ ID NO. 4); MAPT10_2: 5'- AGGCGTCCTTGCGAGCAAGC-3') (SEQ ID NO. 5). Annealed oligos were ligated into pX260.
  • a 156-nt single-stranded DNA oligonucleotide(ssODN) containing the wild-type nucleotide sequence (TTGCGAGCAA GCAGGCGGGTCCAGGGTGGC GTGTCACTCA TCCTTTTTTC TGGCTACCAA AGGTGCAGAT AATTAATAAG AAGCTGGATC TTAGCAACGT CCAGTCCAAG TGTGGCTCAA AGGATAATAT CAAACACGTC CCGGGAGGCG GCAGTG; (SEQ ID NO. 6) was used as template for homology directed repair (HDR).
  • the vector (1.5 pg of pX260- MAPT10_1 and 1.5 pg or 2 pg of pX260- MAPT10_2) and the ssODN (1 .5 pg or 2 pg, respectively) were co-transfected into NPCs using Fugene 6 (Promega) as transfection reagent according to the manufacturer ' s manual. After selection for transfected cells with addition of 5 pg/ml puromycin in the medium for 24 hours and expansion, emerging single cell colonies were transferred to 96-well plates for further expansion.
  • Genomic DNA was prepared by isopropanol precipitation and successful HDR-mediated mutation correction was assessed by amplification of an exon 10 sequence of MAPT using the PCR primers (5'-CGAGCAAGCAGCGGGTCC-3' (SEQ ID NO. 7)) and (5'- GTACGACTCACACCACTTCC-3' (SEQ ID NO. 8)) and consecutive restriction digests with Mboll, distinguishing wild-type from mutant MAPT, followed by genomic sequencing. To ensure that the gene-corrected cell lines originated from N279K MAPT cells, short tandem repeat (STR) loci were compared using the PowerPlex Fusion System (Promega).
  • STR short tandem repeat
  • NPCs were tranduced with different dilutions of lentivirus containing the pLEX307 vector, which was a gift from David Root (Addgene plasmid # 41392), expressing SOX10.
  • Virus was produced by co-transfection of 293T cells with pLEX307-SOX10 and packaging plasmids and viral particles were subsequently concentrated by ultracentrifugation and stored at -80°C. Cells were infected with lentivirus for 24 hr and in the presence of 5 ⁇ g/ml protamine sulfate (Sigma).
  • DMEM-F12 Two days after lentiviral transduction, medium was changed to DMEM-F12 with 1 :100 N2 supplement (Invitrogen), 1 :100 B27 supplement lacking vitamin A (Invitrogen), 1 % penicillin/streptomycin/glutamine, 1 ⁇ SAG, 10 ng/ml PDGF (Peprotech), 2.5 ng/ml bFGF (Peprotech), 10 ng/ml NT3 (Peprotech), 10 ⁇ g/ml IGF (Sigma), 200 ⁇ AA, 1 :1000 Trace Element B (Corning), 0.5 MM LDN 193189 (Axon Medchem) and additionally with 5 ⁇ g/ml puromycin for one week to remove non-transduced cells.
  • 1 :100 N2 supplement Invitrogen
  • 1 :100 B27 supplement lacking vitamin A Invitrogen
  • 1 % penicillin/streptomycin/glutamine 1 ⁇ SAG
  • astrocyte differentiation medium consisting of DMEM-F12 with 1 :100 N2 supplement (Invitrogen), 1 :100 B27 supplement lacking vitamin A (Invitrogen), 1 % penicillin/streptomycin/glutamine, 4% FCS (Biochrom) 10 ⁇ g/ml IGF, 10 ng/ml CNTF (Peprotech), 200 ⁇ M AA and 50 ⁇ dbcAMP (Sigma).
  • Astrocyte cultures were split once a week using accutase and replated on fresh Matrigel-coated plates. After 40 to 45 days in astrocyte differentiation medium cells were either fixed in 4% PFA or were lyzed in RLT buffer (Qiagen) or in ice-cold RIPA buffer.
  • GFAP Primary Antibodies
  • Dako Dako, Z0334, rabbit, 1 :4000
  • Synaptophysin Dako, M0776, mouse, 1 :500
  • GFP Abeam, ab6556, rabbit, 1 :2000
  • MAP-2 Santa Cruz, SC-20172, rabbit, 1 :1000
  • ⁇ - Tubulin Covance, MMS-435P, mouse, 1 :750
  • AT8 Thermo Scientific, MN1020, mouse, 1 :150
  • the glutamate clearance capacity of astrocyte cultures was assessed using the Gluatamte Colorimetric Assay Kit (BioVision, USA) according to the manufacturer ' s instructions. Briefly, cells were plated at a density of 105 cells in matrigel-coated 12- well plates one day prior to performing the assay. 24 hr after plating, cells were washed once with HBSS (Gibco) and subsequently incubated with HBSS containing 500 ⁇ glutamate. After incubation for 2hr at 37°C, 20 ⁇ of cell supernatant or medium control were transferred into a 96-well plate, diluted with assay buffer to a volume of 50 ⁇ and incubated with 100 ⁇ of the reaction mix for 30 min at 37°C. Absorbance of the product was measured at 450 nm using a microplate reader. For quantification of glutamate concentrations, a standard curve was created in each assay based on measurement of cell-free HBSS containing known glutamate concentrations.
  • astrocytes were plated on matrigel-coated glass coverslips at a density of 3 x 104 cells per well of a 12-well plate. Three days after plating, astrocytes were loaded with the fluorescent calcium indicator Oregon Green 488 BAPTA-1 (Thermo Fisher Scientific, Waltham, MA) for at least 45 min at 37°C.
  • RNA extraction was performed according to the manufacturer ' s protocol, including an on-column DNA digestion (RNase free DNase Set; Qiagen).
  • cDNA was generated by reverse transcription of isolated RNAs using the High Capacity cDNA reverse Transcription Kit (Applied Biosystems). 0.375- 4 ng cDNA were subsequently used for RT-PCR reactions performed on a StepOne Plus real time cycler (Applied Biosystems) with the Power SYBR Green PCR master mix (Applied Biosystems). Specificity of the primers used for RT-PCR reactions was determined beforehand, by agarose gelelektrophoresis.
  • qRT-PCRs were run as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 sec at 95°C and 1 min at 55-60°C. Expression levels were calculated applying the 2-AAct method and normalizing to GAPDH and the biological reference sample. Sequences of primers used are listed in Table 1 .
  • the beadchips were washed as recommended and subsequently stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (lllumina) and the associated software. All samples were hybridized as biological replicates. Data were processed by mapping bead intensities to the corresponding gene information using BeadStudia 3.2 (lllumina) and background correction was achieved by applying the Affymetrix Robust Multiarray Analysis (RMA) background correction model (Irizarry et al. (2003) ..Summaries of Affymetrix GeneChip probe level data" Nucleic Acids Res 31 , e15). Variance stabilization was performed using log2 scaling.
  • RMA Affymetrix Robust Multiarray Analysis
  • pPERK Santa Cruz, sc-32577, rabbit, 1 :1000; TAU5: Invitrogen, AHB0042, mouse, 1 :1000; ANXA2: Cell Signaling, #8235, rabbit, 1 :1000; Ubiquitin: Dako, Z0458, rabbit, 1 : 2000; GADPH: Sigma, G9545, rabbit, 1 :200000; ⁇ -ACTIN: Sigma, A5441 , mouse, 1 :200000) was carried out overnight at 4°C. After washing the membrane three times with TBST, HRP-conjugated secondary antibodies were applied for 1 hr at RT. After washing the membrane again three times with TBST, chemiluminescent HRP substrate solution was applied to the membrane.
  • Proteins were detected using the ChemiDocTM XRS+ System (BioRad) and expression levels were quantified by densitometric analysis with the Image LamTM software (BioRad). For detection of TAU and pPERK, protein samples were precipitated prior to immunoblotting. In short, volumes of protein samples corresponding to 80 ⁇ g of protein were mixed with nine times the sample volume of cold (-20°C) Acetone (Roth) and incubated overnight at - 20°C. The next day, samples were centrifuged (20 min/ 21000 rcf/ 4°C), the supernatant was decanted and pellets were left to dry for approximately 1 hr at RT. The dried pellet was subsequently resuspended in water and 6x Laemmli buffer was added.
  • astrocytes were plated a density of 8 x 103 cells per well into 96-well plates. After 12 days in maturation medium, 0.5 ⁇ and 1 ⁇ rotenone in N2 medium consisting of DMEM-F12 with 1 :100 N2 supplement and 1 % penicillin/streptomycin/glutamine were added to the cells for 48 hours. Viability was assessed by measuring LDH leakage in the cell culture supernatant using the Cytotoxicity Detection Kit Plus (Roche). Absorbance was recorded at 490 nm and was expressed as the percentage of absorbance in Ctrl cells after subtraction of background absorbance. [205] Neuronal differentiation
  • NPCs were differentiated into neurons by incubation with N2B27 medium supplemented with 1 ⁇ SAG (Cayman Chemical), 2 ng/ml BDNF (Peprotech), 2 ng/ml GDNF (Peprotech) and 100 ⁇ AA (Sigma) for 6 days and afterwards with N2B27 medium supplemented with 2 ng/ml BDNF, 2 ng/ml GDNF, 0.5 ng/ml TGF-&3 (Peprotech), 100 ⁇ dbcAMP (Sigma) and 100 ⁇ AA.
  • N2B27 medium supplemented with 1 ⁇ SAG (Cayman Chemical), 2 ng/ml BDNF (Peprotech), 2 ng/ml GDNF (Peprotech) and 100 ⁇ AA (Sigma) for 6 days and afterwards with N2B27 medium supplemented with 2 ng/ml BDNF, 2 ng/ml GDNF, 0.5 ng/ml TGF-
  • Activin A 5 ng/ml Activin A were added to the medium at days 7 through 9.
  • cells were detached, singularized by treatment with acctuase and replated at densities of 2.2 x 105 neurons in 12-well plates and 1 .5 x 105 neurons in 24-well plates containing glass coverslips.
  • cells were fixed in 4% PFA for immunocytochemical analyses or lyzed in RLT buffer (Qiagen) for RNA preparation.
  • Ctrl-1 NPCs were labeled with GFP by lentiviral transduction with the LVTHM vector (Wiznerowicz and Trono, 2003) as previously described (Hargus et al. (2014) "Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain" Cell Rep 8, 1697-1703). Labeled Ctrl-1 NPCs were subsequently differentiated into neurons applying the protocol described above. After 10 days of neuronal ifferentiation, cells were detached and singularized by treatment with acctuase.
  • Neurons were added to FTD- 1 , FTD-2 or Ctrl-2 astrocyte cultures at densities of 3.5 x 106 neurons in a 10 cm dish and 1 x 105 neurons per well in 24-well plates containing glass coverslips.
  • Astrocytes had been plated three days before at day 31 of astrocyte differentiation at densities of 5 x 105 cells in matrigel-coated 10 cm dishes and 1.5 x 104 cells per well in matrigel-coated 24-well plates.
  • Neurons were co-cultured with astrocytes in neuronal differentiation medium for 21 days. After that, cells were either fixed in 4% PFA or cells were detached and singularized by treatment with trypsin.
  • astrocytes To assess the influence of astrocytes on the vulnerability of neurons to rotenone- induced stress, co-cultured cells were incubated with N2B27 medium containing 400 nm rotenone (Sigma) for 48 hr. Cells were subsequently fixed and stained for ⁇ - Tubulin. The number of neurons per visual field was determined using Image J and the cell counter plugin. Neurite density of neurons after co-culture with astrocytes was determined by staining of fixed cultures with an anti-GFP antibody. Images were taken with a Zeiss LSM 700 confocal microscope and image analysis was performed using the Image J software and the color pixel plugin. The percentage of GFP positive signals over all signals was determined for 10 images per co-culture setup and per biological replicate.
  • NPCs were differentiated from human iPS cells carrying the N279K MAPT mutation (FTD NPCs) and genome editing was performed in different NPC clones addressing exon 10 of the MAPT gene (Fig. 1A).
  • FTD NPCs FTD NPCs
  • Fig. 1A We used dual expression of sgRNAs and Cas9 from a single vector, transfection of a single-stranded DNA oligonucleotide (ssODN) providing the wildtype sequence as well as short-term puromycin selection to identify NPC clones carrying the gene-corrected MAPT locus (MAPT N279).
  • ssODN single-stranded DNA oligonucleotide
  • MAT N279 gene-corrected MAPT locus
  • Hierarchical clustering analysis demonstrated that gene- corrected lines clustered closest with their parental lines and separated from an unrelated, non-isogenic healthy control (Ctrl) NPC line (C1 ), which was also included in the analysis (Fig. 1 D).
  • the gene-edited and parental NPCs could be efficiently expanded and showed characteristic, strong expression of the NPC markers SOX1 and NESTIN (Fig. 1 E).
  • Two parental lines (FTD-1 and FTD-2) and two Ctrl lines (C1 and FTD-1 GC-1 henceforth termed Ctrl-1 and Ctrl-2) were used in this study for differentiation and co-culture experiments.
  • FTD NPCs demonstrate efficient differentiation into neurons and astrocytes in vitro
  • astrocyte marker genes such as GFAP, ALDH1 L1 , SLC1A2, SLC1A3 and S1003 ⁇ 4 in FTD and Ctrl astrocytes via qRT-PCR revealing similar expression profiles, except for ⁇ ⁇ , which was more highly expressed in FTD astrocytes (Fig. 1 L).
  • FTD astrocytes show disease-associated changes in TAU expression, proteasome activation, oxidative stress and alterations in whole genome expression profiles
  • GO Gene Ontology
  • GPRC5C integral to plasma membrane
  • HLA-DRB5 multicellular organism development
  • upregulated genes were associated with neurological system processes (CHRNA1 , CRYZ, EYA1 , NPY, PCDHB5), synapse organization and biogenesis (CHRNA1 , PCDHB5) and synaptic transmission (CHRNA1 , NPY, PCDHB5).
  • Additional upregulated genes included genes that have been linked to astrocyte migration (MMP14), neural survival (EN1 , NPY), microtubule maintenance and TAU-associated pathology in AD (GAS7), protein accumulation in AD (Dysferlin, DYSF) and regulation of NF-kappB, which is involved in various cellular processes including activation of cultured astrocytes (TCEAL7, CXCL12; Fig. 2D; Angelucci et al. (2014) involveThe effect of neuropeptide Y on cell survival and neurotrophin expression in in vitro models of Alzheimer's disease" Can J Physiol Pharmacol 92, 621 -630, Fuchs et al.
  • Annexin A2 is a calcium-dependent multifunctional phospholipid-binding protein associated to the plasma membrane and endosomal compartments (Luo and Hajjar (2013) derAnnexin A2 system in human biology: cell surface and beyond" Semin Thromb Hemost 39, 338-346).
  • N279K MAPT astrocytes also modified neural programs and increased stress responses in previously healthy, co-cultured neurons.
  • CRISPR/Cas9 genome editing can successfully be applied in NPCs to analyse disease phenotypes in neurodegenerative diseases such as FTD.
  • NPCs may provide certain advantages over genetic correction in PSCs, as NPCs comprise robust, easily expandable somatic progenitor populations, which are already committed to neural differentiation (Ehrlich et al. (2015). Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein. Stem Cell Reports 5, 83-96; Hargus et al. (2014) "Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain" Cell Rep 8, 1697-1703; Koch et al.
  • 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.
  • Gauthier-Kemper et al. (201 1 ) spreadThe frontotemporal dementia mutation R406W blocks tau's interaction with the membrane in an annexin A2-dependent manner" J Cell Biol 192, 647-661

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Neurosurgery (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de production d'astrocytes ; un astrocyte pouvant être obtenu par le procédé selon la présente invention ; et une composition pharmaceutique les contenant. Les astrocytes selon la présente invention se prêtent à une utilisation en thérapie et à une utilisation dans une méthode destinée à traiter une maladie. Un procédé d'identification d'agents, qui modulent la viabilité ou la fonction astrocytaire, ainsi qu'un kit sont en outre décrits.
PCT/EP2017/084133 2016-12-22 2017-12-21 Moyens et procédés de génération d'astrocytes WO2018115302A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
LU93394 2016-12-22
LU93394 2016-12-22

Publications (1)

Publication Number Publication Date
WO2018115302A1 true WO2018115302A1 (fr) 2018-06-28

Family

ID=58046721

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/084133 WO2018115302A1 (fr) 2016-12-22 2017-12-21 Moyens et procédés de génération d'astrocytes

Country Status (1)

Country Link
WO (1) WO2018115302A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021097552A1 (fr) * 2019-11-22 2021-05-27 Critical Outcome Technologies Inc. Peptides, composés, compositions et procédés pour inhiber la dimérisation de sox9
CN116478923A (zh) * 2022-04-26 2023-07-25 浙江霍德生物工程有限公司 一种星形胶质细胞的制备方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009115295A1 (fr) 2008-03-17 2009-09-24 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Vecteurs et procédés de génération de cellules souches pluripotentes induites (ips) sans vecteur à l'aide d'une recombinaison dirigée
WO2009144008A1 (fr) 2008-05-27 2009-12-03 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Génération de cellules souches pluripotentes induites (ips)
EP2218778A1 (fr) 2009-02-16 2010-08-18 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Conversion de cellules souches germinales unipotentes en cellules souches pluripotentes sans facteur exogène
WO2011091048A1 (fr) * 2010-01-19 2011-07-28 The Board Of Trustees Of The Leland Stanford Junior University Conversion directe de cellules en cellules d'autres lignées
WO2012058243A2 (fr) * 2010-10-26 2012-05-03 Case Western Reserve University Conversion de reprogrammation cellulaire de cellules somatiques différenciées en cellules gliales
WO2014104409A1 (fr) * 2012-12-28 2014-07-03 Kyoto University Méthode d'induction des astrocytes
WO2015133879A1 (fr) * 2014-03-07 2015-09-11 국립대학법인 울산과학기술대학교 산학협력단 Composition d'induction d'une transdifférenciation directe en cellules progénitrices d'oligodendrocytes à partir de cellules somatiques et son utilisation
EP3118306A1 (fr) * 2014-03-07 2017-01-18 UNIST (Ulsan National Institute of Science and Technology) Composition d'induction d'une transdifférenciation directe en cellules progénitrices d'oligodendrocytes à partir de cellules somatiques et son utilisation

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009115295A1 (fr) 2008-03-17 2009-09-24 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Vecteurs et procédés de génération de cellules souches pluripotentes induites (ips) sans vecteur à l'aide d'une recombinaison dirigée
WO2009144008A1 (fr) 2008-05-27 2009-12-03 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Génération de cellules souches pluripotentes induites (ips)
EP2218778A1 (fr) 2009-02-16 2010-08-18 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Conversion de cellules souches germinales unipotentes en cellules souches pluripotentes sans facteur exogène
WO2011091048A1 (fr) * 2010-01-19 2011-07-28 The Board Of Trustees Of The Leland Stanford Junior University Conversion directe de cellules en cellules d'autres lignées
WO2012058243A2 (fr) * 2010-10-26 2012-05-03 Case Western Reserve University Conversion de reprogrammation cellulaire de cellules somatiques différenciées en cellules gliales
WO2014104409A1 (fr) * 2012-12-28 2014-07-03 Kyoto University Méthode d'induction des astrocytes
WO2015133879A1 (fr) * 2014-03-07 2015-09-11 국립대학법인 울산과학기술대학교 산학협력단 Composition d'induction d'une transdifférenciation directe en cellules progénitrices d'oligodendrocytes à partir de cellules somatiques et son utilisation
EP3118306A1 (fr) * 2014-03-07 2017-01-18 UNIST (Ulsan National Institute of Science and Technology) Composition d'induction d'une transdifférenciation directe en cellules progénitrices d'oligodendrocytes à partir de cellules somatiques et son utilisation

Non-Patent Citations (73)

* Cited by examiner, † Cited by third party
Title
"POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS", 1983, ACADEMIC PRESS, pages: 1 - 12
ALTSCHUL: "A protein alignment scoring system sensitive at all evolutionary distances", J MOL EVOL., vol. 36, no. 3, 1993, pages 290 - 300, XP008057491, DOI: doi:10.1007/BF00160485
ALTSCHUL: "Basic local alignment search tool", J MOL BIOL., vol. 215, no. 3, 5 October 1990 (1990-10-05), pages 403 - 10, XP002949123, DOI: doi:10.1006/jmbi.1990.9999
ALTSCHUL: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, no. 17, 1997, pages 3389 - 402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
ANGELUCCI ET AL.: "The effect of neuropeptide Y on cell survival and neurotrophin expression in in vitro models of Alzheimer's disease", CAN J PHYSIOL PHARMACOL, vol. 92, 2014, pages 621 - 630
AUGUSTYNIAK: "Reprogramming of somatic cells: possible methods to derive safe, clinical-grade human induced pluripotent stem cells", ACTA NEUROBIOL EXP (WARS, vol. 74, no. 4, 2014, pages 373 - 82
BOXER; MILLER: "Clinical features of frontotemporal dementia", ALZHEIMER DIS ASSOC DISORD, vol. 19, no. 1, 2005, pages 3 - 6
C. D. POZNIAK ET AL: "Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression", PROCEEDINGS NATIONAL ACADEMY OF SCIENCES PNAS, vol. 107, no. 50, 22 November 2010 (2010-11-22), US, pages 21795 - 21800, XP055313664, ISSN: 0027-8424, DOI: 10.1073/pnas.1016485107 *
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 1062368-24-4
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 1062368-49-3
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 1206711-16-1
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 1219168-18-9
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 1431985-92-0
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 483367-10-8
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 612542-14-0
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 912545-86-9
CHOI ET AL.: "Neural stem cells differentiated from iPS cells spontaneously regain pluripotency", STEM CELLS, vol. 32, no. 10, 2014, pages 2596 - 604
CHURKO ET AL.: "Generation of human iPSCs from human peripheral blood mononuclear cells using non-integrative Sendai virus in chemically defined conditions", METHODS MOL BIOI., vol. 1036, 2013, pages 81 - 8
CHURKO ET AL.: "Generation of human iPSCs from human peripheral blood mononuclear cells using non-integrative Sendai virus in chemically defined conditions", METHODS MOL BIOL., vol. 1036, 2013, pages 81 - 8
CONG ET AL.: "Multiplex genome engineering using CRISPR/Cas systems", SCIENCE, vol. 339, 2013, pages 819 - 823, XP055400719, DOI: doi:10.1126/science.1231143
CORREALE; FAREZ: "The Role of Astrocytes in Multiple Sclerosis Progression", EXP NEUROBIOL., vol. 25, no. 5, 2015, pages 233 - 240
DI GIORGIO ET AL.: "Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation", CELL STEM CELL, vol. 3, 2008, pages 637 - 648
DITTRICH ET AL.: "Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine", GEBURTSHILFE FRAUENHEILKD, vol. 75, no. 12, 2015, pages 1239 - 1242
DREWS: "Generation of iPSC lines from primary human amniotic fluid cells", STEM CELL RESEARCH, vol. 15, no. 3, 2015, pages 712 - 714
EHRLICH ET AL.: "Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein", STEM CELL REPORTS, vol. 5, 2015, pages 83 - 96, XP055467602, DOI: doi:10.1016/j.stemcr.2015.06.001
FONG ET AL.: "Genetic correction of tauopathy phenotypes in neurons derived from human induced pluripotent stem cells", STEM CELL REPORTS, vol. 1, 2013, pages 226 - 234, XP055441675, DOI: doi:10.1016/j.stemcr.2013.08.001
FUCHS ET AL.: "Engrailed signaling in axon guidance and neuron survival", EUR J NEUROSCI, vol. 35, 2012, pages 1837 - 1845, XP055043542, DOI: doi:10.1111/j.1460-9568.2012.08139.x
GAJ ET AL.: "Genome-Editing Technologies: Principles and Applications", COLD SPRING HARB PERSPECT BIOL., vol. 8, no. 12, 2016, pages a023754
GALVIN ET AL.: "The muscle protein dysferlin accumulates in the Alzheimer brain", ACTA NEUROPATHOL, vol. 112, 2006, pages 665 - 671, XP019442888, DOI: doi:10.1007/s00401-006-0147-8
GAUTHIER-KEMPER ET AL.: "The frontotemporal dementia mutation R406W blocks tau's interaction with the membrane in an annexin A2-dependent manner", J CELL BIOL, vol. 192, 2011, pages 647 - 661
GHETTI ET AL.: "Invited review: Frontotemporal dementia caused by microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging", NEUROPATHOL APPL NEUROBIOL, vol. 41, 2015, pages 24 - 46
GOEDERT; SPILLANTINI: "Pathogenesis of the tauopathies", J MOL NEUROSCI, vol. 45, 2011, pages 425 - 431, XP019971785, DOI: doi:10.1007/s12031-011-9593-4
HARGUS ET AL.: "Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain", CELL REP, vol. 8, 2014, pages 1697 - 1703
HORNIG ET AL.: "The transcription factors Sox10 and Myrf define an essential regulatory network module in differentiating oligodendrocytes", PLOS GENET., vol. 9, no. 10, 2013, pages e1003907
HUANGFU ET AL.: "Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2", NATURE BIOTECHNOLOGY, vol. 26, 2008, pages 1269 - 1275, XP002534948, DOI: doi:10.1038/nbt.1502
IRIZARRY ET AL.: "Summaries of Affymetrix GeneChip probe level data", NUCLEIC ACIDS RES, vol. 31, 2003, pages e15, XP002460628, DOI: doi:10.1093/nar/gng015
JANSEN ET AL.: "The ubiquitin proteasome system in glia and its role in neurodegenerative diseases", FRONT MOL NEUROSCI, vol. 7, 2014, pages 73
KALMAN; SZABO: "Appearance of annexin II immunopositivity in reactive astrocytes but not in microglia", NEUROBIOLOGY (BP, vol. 9, 2001, pages 139 - 145
KIM ET AL.: "Direct reprogramming of human neural stem cells by OCT4", NATURE, vol. 461, 2009, pages 649 - 643
KIM; EBERWINE: "Mammalian cell transfection: the present and the future", ANAL BIOANAL CHEM, vol. 397, no. 8, 2010, pages 3173 - 3178, XP019839356
KOCH ET AL.: "A rosette-type, selfrenewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration", PROC NATL ACAD SCI U S A, vol. 106, 2009, pages 3225 - 3230, XP002563719, DOI: doi:10.1073/pnas.0808387106
KOCH ET AL.: "A rosette-type, selfrenewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration", PROC NATL ACAD SCI, vol. 106, 2009, pages 3225 - 3230, XP002563719, DOI: doi:10.1073/pnas.0808387106
LAM ET AL.: "Mechanism of glial activation by S100B: involvement of the transcription factor NfkappaB", NEUROBIOL AGING, vol. 22, 2001, pages 765 - 772
LEE ET AL., ASTROCYTES AND MICROGLIA AS NON-CELL AUTONOMOUS PLAYERS IN THE PATHOGENESIS OF ALS, 2016
LEE ET AL.: "Astrocytes and Microglia as Non-cell Autonomous Players in the Pathogenesis of ALS", EXP NEUROBIOL., vol. 25, no. 5, 2016, pages 233 - 240
LEE-KUBLI; LU: "Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury", NEURAL REGEN RES., vol. 10, no. 1, 2015, pages 10 - 16
LOWRY ET AL.: "Generation of human induced pluripotent stem cells from dermal fibroblasts", PNAS, vol. 105, no. 8, 2007, pages 2883 - 2888, XP002555951, DOI: doi:10.1073/pnas.0711983105
LOWRY ET AL.: "Generation of human induced pluripotent stem cells from dermal fibroblasts", PNAS, vol. 105, no. 8, 2008, pages 2883 - 2888, XP002555951, DOI: doi:10.1073/pnas.0711983105
LUO; HAJJAR: "Annexin A2 system in human biology: cell surface and beyond", SEMIN THROMB HEMOST, vol. 39, 2013, pages 338 - 346
MANDAL ET AL.: "Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9", CELL STEM CELL, vol. 15, 2014, pages 643 - 652, XP055348680, DOI: doi:10.1016/j.stem.2014.10.004
MATYASH; KETTENMANN: "Heterogeneity in astrocyte morphology and physiology", BRAIN RES REV., vol. 63, no. 1-2, 2010, pages 2 - 10, XP027032595
NOBLE ET AL.: "The importance of tau phosphorylation for neurodegenerative diseases", FRONT NEUROL, vol. 4, 2013, pages 83
PAL ET AL.: "Astrocytic Actions on Extrasynaptic Neuronal Currents", FRONT CELL NEUROSCI., vol. 9, 2015, pages 474
PAL ET AL.: "Astrocytic Actions on Extrasynaptic Neuronal Currents", FRONT CELL NEUROSCI., vol. 9, 9 December 2015 (2015-12-09), pages 474
PEKNY: "Astrocytes: a central element in neurological diseases", ACTA NEUROPATHOL, vol. 131, 2016, pages 323 - 345, XP035878631, DOI: doi:10.1007/s00401-015-1513-1
PHATNANI; MANIATIS: "Astrocytes in neurodegenerative disease", COLD SPRING HARB PERSPECT BIOL, vol. 7, 2015
PUSCH ET AL.: "The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor", HUM GENET., vol. 103, no. 2, 1998, pages 115 - 23
RAHMAN ET AL.: "TGF-b/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation", BONE RESEARCH, vol. 3, 2015
RATNER ET AL.: "Overview of CRISPR-Cas9 Biology", COLD SPRING HARB PROTOC., vol. 1, no. 12, 2016
RATTAN: "Protein synthesis, posttranslational modifications, and aging", ANN. NY ACAD. SCI., vol. 663, 1992, pages 48 - 62, XP009082490, DOI: doi:10.1111/j.1749-6632.1992.tb38648.x
REEMST ET AL.: "The Indispensable Roles of Microglia and Astrocytes during Brain Development", FRONT HUM NEUROSCI., vol. 10, 2016, pages 566
REINHARDT ET AL.: "Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling", PLOS ONE, vol. 8, 2013, pages e59252, XP055234383, DOI: doi:10.1371/journal.pone.0059252
RIMKUS: "Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors", CANCERS (BASEL, vol. 8, no. 2, 2016, pages E22
RING ET AL.: "Direct Reprogramming of Mouse and Human Fibroblasts into Multipotent Neural Stem Cells with a Single Factor", CELL STEM CELL, vol. 11, no. 1, 2012, pages 100 - 109, XP028400409, DOI: doi:10.1016/j.stem.2012.05.018
ROBERT KRENCIK ET AL: "Specification of transplantable astroglial subtypes from human pluripotent stem cells", NATURE BIOTECHNOLOGY, vol. 29, no. 6, 1 June 2011 (2011-06-01), US, pages 528 - 534, XP055259166, ISSN: 1087-0156, DOI: 10.1038/nbt.1877 *
SCHRODER; KAUFMAN: "The mammalian unfolded protein response", ANNU REV BIOCHEM, vol. 74, 2005, pages 739 - 789
SEIFTER: "Analysis for protein modifications and nonprotein cofactors", METH. ENZYMOL., vol. 182, 1990, pages 626 - 646, XP009082492, DOI: doi:10.1016/0076-6879(90)82049-8
SLOWINSKI ET AL.: "Frontotemporal dementia and Parkinsonism linked to chromosome 17 with the N279K tau mutation", NEUROPATHOLOGY, vol. 27, 2007, pages 73 - 80
T. E. CREIGHTON: "PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES", 1993, W. H. FREEMAN AND COMPANY
TAKAHASHI ET AL.: "Induction of pluripotent stem cells from adult human fibroblasts by defined factors", CELL, vol. 131, no. 5, 2007, pages 861 - 72, XP002555952, DOI: doi:10.1016/j.cell.2007.11.019
TANABE ET AL.: "Induction of pluripotency by defined factors", PROC. JPN. ACAD., 2014
WANG ET AL.: "Generation of Induced Pluripotent Stem Cells with High Efficiency from Human Umbilical Cord Blood Mononuclear Cells", GENOMICS, PROTEOMICS & BIOINFORMATICS, vol. 11, no. 5, 2013, pages 304 - 311
ZWAKA; THOMSON: "Homologous recombination in human embryonic stem cells", NAT BIOTECHNOL, vol. 21, 2003, pages 319 - 321, XP002427413, DOI: doi:10.1038/nbt788

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021097552A1 (fr) * 2019-11-22 2021-05-27 Critical Outcome Technologies Inc. Peptides, composés, compositions et procédés pour inhiber la dimérisation de sox9
CN116478923A (zh) * 2022-04-26 2023-07-25 浙江霍德生物工程有限公司 一种星形胶质细胞的制备方法
CN116478923B (zh) * 2022-04-26 2024-01-02 浙江霍德生物工程有限公司 一种星形胶质细胞的制备方法

Similar Documents

Publication Publication Date Title
Ehrlich et al. Distinct neurodegenerative changes in an induced pluripotent stem cell model of frontotemporal dementia linked to mutant TAU protein
Papadimitriou et al. 3D culture method for Alzheimer's disease modeling reveals interleukin-4 rescues Aβ42-induced loss of human neural stem cell plasticity
Qi et al. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells
Ren et al. Maintenance of nucleolar homeostasis by CBX4 alleviates senescence and osteoarthritis
Zhang et al. Pharmacological reprogramming of fibroblasts into neural stem cells by signaling-directed transcriptional activation
JP6774333B2 (ja) 年齢改変細胞および年齢改変細胞を作製するための方法
US20230323294A1 (en) Differentiation of cortical neurons from human pluripotent stem cells
JP7055638B2 (ja) 幹細胞からの筋肉系列細胞の生成
Rubio et al. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming
JP2019502407A (ja) 神経オルガノイド組成物および使用方法
US20150250824A1 (en) Methods and compositions for expansion of stem cells and other cells
Park et al. Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons
JP2023156413A (ja) 背側化シグナル伝達物質又は腹側化シグナル伝達物質による錐体視細胞又は桿体視細胞の増加方法
US20190322981A1 (en) Means and methods for the generation of oligodendrocytes
Zhou et al. Conversion of fibroblasts to neural cells by p53 depletion
JP2023169391A (ja) 細胞凝集体、細胞凝集体の混合物及びそれらの製造方法
JP2018076385A (ja) 神経前駆細胞を用いた細胞治療における移植補助剤
Canals et al. Transcription factor-based strategies to generate neural cell types from human pluripotent stem cells
WO2018115302A1 (fr) Moyens et procédés de génération d'astrocytes
Rojas et al. Mature iPSC-derived astrocytes of an ALS/FTD patient carrying the TDP43 A90V mutation display a mild reactive state and release polyP toxic to motoneurons
WO2017223241A1 (fr) Production d'astrocytes à l'aide de petites molécules
Mutukula Modeling cortical development and microcephaly in human pluripotent stem cells using combinatorial pathway inhibition
Finelli et al. Mature iPSC-derived astrocytes of an ALS/FTD patient carrying the TDP43 A90V mutation display a mild reactive state and release polyP toxic to motoneurons
Bhattarai et al. 3D Culture Method for Alzheimer’s Disease Modeling Reveals Interleukin-4 Rescues Ab42-Induced Loss of Human Neural Stem Cell Plasticity
Vu Studying Human Outer Radial Glia Development in Pluripotent Stem Cells-Derived Neural Cultures

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17826232

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17826232

Country of ref document: EP

Kind code of ref document: A1