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WO2025224322A1 - Protogénine en tant que nouveau marqueur de surface pour des cellules souches neurales corticales précoces - Google Patents

Protogénine en tant que nouveau marqueur de surface pour des cellules souches neurales corticales précoces

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Publication number
WO2025224322A1
WO2025224322A1 PCT/EP2025/061382 EP2025061382W WO2025224322A1 WO 2025224322 A1 WO2025224322 A1 WO 2025224322A1 EP 2025061382 W EP2025061382 W EP 2025061382W WO 2025224322 A1 WO2025224322 A1 WO 2025224322A1
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cells
prtg
neural
cortical
cell
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Yechiel ELKABETZ
Amèlia ARAGONÉS HERNÁNDEZ
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells

Definitions

  • the present invention relates to a method for generating an enriched population of early cortical neural stem cells (NSCs) or a subpopulation thereof, the method comprising: isolating cells that are positive for the cell surface marker protogenin (PRTG) from an initial population of neural progenitor cells, wherein said isolating is conducted at a time point between about day 4 and about day 12, preferably on day 5, after initiation of neural induction, thereby obtaining an enriched population of early cortical neural stem cells (NSCs); and optionally re-culturing said enriched population of early cortical neural stem cells (NSCs) in a neural induction medium or other culture medium, wherein said re-culturing preferably produces progeny of said early cortical neural stem cells (NSCs
  • PRTG cell surface marker protogenin
  • NSCs Initially, there is the expansion of the NSC pool through proliferative symmetric divisions, whereas later, through differentiative asymmetric divisions, NSCs give rise to the diverse cell populations that reside within the cortical layers. Throughout this process, NSCs undergo extensive modifications in their transcriptomic profile and chromatin landscape contributing to the formation of heterogeneous progenitor populations. These NSC subtypes are more restricted in their differentiation capacity, and thus more limited in the types of neurons they can generate. Although in recent years much progress has been made towards understanding temporal cell-fate specification during human corticogenesis, the mechanisms responsible for the temporal lineage specification of NSCs remain largely unknown.
  • the present invention relates in a first aspect to a method for generating an enriched population of early cortical neural stem cells (NSCs) and/or a subpopulation thereof, the method comprising: isolating cells that are positive for the cell surface marker protogenin (PRTG) from an initial population of neural progenitor cells, wherein said isolating is conducted at a time point between about day 4 and about day 12, preferably on day 5, after initiation of neural induction, thereby obtaining an enriched population of early cortical neural stem cells (NSCs); and optionally re-culturing said enriched population of early cortical neural stem cells (NSCs) in a neural induction medium or other culture medium, wherein said re-culturing preferably produces progeny of said early cortical neural stem cells (NSCs).
  • PRTG cell surface marker protogenin
  • neural stem cells refers to are self-renewing, multipotent cells that firstly generate the radial glial progenitor cells that generate the neurons and glia of the central nervous system of all animals. NSCs are capable of self-maintenance (self-renewal), meaning that with each cell division, one daughter cell will also be a stem cell. The non-stem cell progeny of NSCs are termed “neural progenitor cells”. Neural progenitor cells generated from a single multipotent NSC are capable of differentiating into neurons, astrocytes, and oligodendrocytes.
  • NSCs are “multipotent” because their progeny have multiple neural cell fates.
  • NSCs can be functionally defined as a cell with the ability to: 1) proliferate, 2) self-renew, and 3) produce functional progeny that can differentiate into the three main cell types found in the central nervous system: neurons, astrocytes, and oligodendrocytes.
  • cortical neural stem cells refers to neural stem cells (NSCs) that are commonly found in, or isolated from, the cerebral cortex (i.e., the outermost layer of the brain (the cerebrum)).
  • cortical neural stem cells refers to a early differentiation state of “cortical neural stem cells”, preferably a state where the “cortical neural stem cells” are capable of symmetric division.
  • subpopulation refers to a (more specific) subset of the referred (more generic) cell population.
  • Protogenin also known as “PRTG”, “protein Shen-Dan”, “protogenin homolog” or “IGDCC5” (immunoglobulin superfamily, DCC subclass, member 5), is a single-pass transmembrane protein that belongs to the immunoglobulin superfamily (IgSF).
  • DCC colorectal cancer
  • Neogenin which are receptors for Netrin1 and RGMa, respectively, as well as to cell adhesion molecules such as L1 and neural cell adhesion molecule (NCAM).
  • PRTG also refers to any known isoforms thereof.
  • nucleotide sequences of the PRTG-encoding genes and mRNAs from different species and the amino acid sequences of the respectively encoded proteins are known in the art and can be retrieved from publicly available databases such as the NCBI database (https://www.ncbi.nlm.nih.gov/) or UniProt (https://www.uniprot.org/).
  • NCBI database https://www.ncbi.nlm.nih.gov/
  • UniProt https://www.uniprot.org/
  • the mRNA (more specifically cDNA) and corresponding amino acid sequence of the human PRTG-encoding gene is defined by NCBI Reference Sequence: NM_173814.6; see also UniProt accession code Q2VWP7.
  • the “isolating” cells that are positive for a cell surface marker may be established by any method known in the art that can be suitably employed for that purpose, such as in particular cell sorting (e.g., fluorescence-activated cell sorting (FACS)).
  • FACS fluorescence-activated cell sorting
  • the term “isolating” may be an “enriching”, meaning that the isolated population may not be entirely pure with respect to the targeted population but also comprise certain amounts of the unwanted species as impurity.
  • the isolation yields a target cell population (i.e., PRTG-positive cells) which make up at least, with increasing preference, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the total amounts of cells comprised in the isolated population.
  • the term “initiation of neural induction” refers to the provision of culture conditions by which the differentiation of the stem cells towards neural cell linages is effected.
  • said neural induction is initiated as described in the herein disclosed examples.
  • alternative approaches may also be suitable.
  • the term “about” means, with increasing preference, ⁇ 18h, ⁇ 12h, ⁇ 10h, ⁇ 8h, ⁇ 6h, ⁇ 4h, ⁇ 2h, ⁇ 1h of the referred times.
  • said initial population of neural progenitor cells has been obtained from, or is obtained by: (i) culturing primate stem cells in a primate stem cell medium until the formation of embryonic bodies (EBs); and (ii) culturing the EBs as obtained in step (i) in a neural induction medium comprising an inhibitor of WNT, an inhibitor of TGF- ⁇ , and an inhibitor of BMP, thereby initiating neural induction, whereby the EBs differentiate into neural progenitor cells.
  • a neural induction medium comprising an inhibitor of WNT, an inhibitor of TGF- ⁇ , and an inhibitor of BMP, thereby initiating neural induction, whereby the EBs differentiate into neural progenitor cells.
  • the nature of the primate stem cells to be used herein is not particularly limited as long as they display the capability of differentiating into embryonic bodies (EBs) when subjected to the method of the present invention.
  • the primate stem cells are human primate stem cells.
  • the composition of the primate stem cell medium is not particularly limited as long as the culturing of primate stem cells in the medium under suitable conditions results in the formation of embryonic bodies (typically with 1, 2, 3, 4, 5 or 6 days, i.e., less than one week).
  • Embryonic bodies are three-dimensional aggregates of pluripotent stem cells. They compromise the three embryonic germ layers. EBs are formed by the homophilic binding of the Ca2 + dependent adhesion molecule E-cadherin, which is highly expressed on undifferentiated primate stem cells. When cultured as single cells in the absence of anti-differentiation factors, primate stem cells spontaneously aggregate to form EBs.
  • the composition of the “neural induction medium” is not particularly limited as long as the culturing of EBs in the medium under suitable conditions results in the differentiation of EBs into neural progenitor cells.
  • WNT signalling is an important pathway for neural induction as well as for the axis patterning process. It has roles in both cell proliferation and fate specification. Inhibition of WNT pathway establishes anterior fates. Studies in mice mutants of the WNT co-receptors Lrp5 and Lrp6 show the expansion of the anterior neuroectoderm. Loss of the WNT inhibitor Dickkopf 1 (Dkk1) in mouse prevents the formation of forebrain.
  • Dkk1 Dickkopf 1
  • TGF- ⁇ Transforming growth factor beta
  • TGF- ⁇ Transforming growth factor beta
  • HGNC symbols TGFB1, TGFB2, TGFB3 HGNC symbols TGFB1, TGFB2, TGFB3
  • BMPs Bone morphogenetic proteins
  • the inhibitors of TGF ⁇ , BMP and/or WNT signalling may either inhibit the nucleic acid encoding TGF ⁇ , BMP and/or a member of the WNT signalling and/or inhibit the protein TGF ⁇ , BMP or a protein being a member of the WNT signalling pathway.
  • the inhibitor of the nucleic acid molecule is preferably selected from a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease.
  • TAL transcription activator-like effector
  • the inhibitor of the protein is preferably selected from a small molecule, an antibody or antibody mimetic, and an aptamer, wherein the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.
  • the "small molecule” as used herein is preferably an organic molecule.
  • Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds.
  • Organic compounds can be natural or synthetic.
  • the organic molecule is preferably an aromatic molecule and more preferably a heteroaromatic molecule.
  • aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms.
  • Aromatic molecules are very stable, and do not break apart easily to react with other substances.
  • a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g.
  • the molecular weight is preferably in the range of 200 Da to 1500 Da and more preferably in the range of 300 Da to 1000 Da.
  • the "small molecule" in accordance with the present invention may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da such as less than about 500 Da, and even more preferably less than about Da amu.
  • the size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry.
  • the small molecules may be designed, for example, based on the crystal structure of the target molecule, where sites presumably responsible for the biological activity can be identified and verified in in vivo assays such as in vivo high- throughput screening (HTS) assays.
  • antibody as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity to the target, e.g. TGF ⁇ , are comprised in the term "antibody".
  • Antibody fragments or derivatives comprise, inter alia, Fab or Fab’ fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains, such as VhH or V-NAR-domains, as well as multimeric formats such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated Fab’-multimers (see, for example, Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 198; Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol.
  • the multimeric formats in particular comprise bispecific antibodies that can simultaneously bind to two different types of antigen.
  • the first antigen can be found on the protein of the invention.
  • the second antigen may, for example, be a tumor marker that is specifically expressed on cancer cells or a certain type of cancer cells.
  • Non- limting examples of bispecific antibodies formats are Biclonics (bispecific, full length human IgG antibodies), DART (Dual-affinity Re-targeting Antibody) and BiTE (consisting of two single-chain variable fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).
  • the term "antibody” also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanised (human antibody with the exception of non-human CDRs) antibodies.
  • Various techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010, loc. cit.
  • polyclonal antibodies can be obtained from the blood of an animal following immunisation with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g. in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999 and include the hybridoma technique originally described by Köhler and Milstein, 1975, the trioma technique, the human B-cell hybridoma technique (see e.g.
  • recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display.
  • a suitable system for the expression of the recombinant (humanised) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for a desired epitope, e.g. of TGF ⁇ . Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies.
  • antibody mimetics refers to compounds which, like antibodies, can specifically bind antigens, such as for example, TGF ⁇ in the present case, but which are not structurally related to antibodies.
  • Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
  • an antibody mimetic may be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and prododies. These polypeptides are well known in the art and are described in further detail herein below.
  • affibody refers to a family of antibody mimetics which is derived from the Z- domain of staphylococcal protein A. Structurally, affibody molecules are based on a three-helix bundle domain which can also be incorporated into fusion proteins. In itself, an affibody has a molecular mass of around 6kDa and is stable at high temperatures and under acidic or alkaline conditions. Target specificity is obtained by randomisation of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).
  • adnectin (also referred to as “monobody”), as used herein, relates to a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like ⁇ -sandwich fold of 94 residues with 2 to 3 exposed loops, but lacks the central disulphide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255).
  • Adnectins with the desired target specificity i.e., for example, against TGF ⁇ , can be genetically engineered by introducing modifications in specific loops of the protein.
  • anticalin refers to an engineered protein derived from a lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci U S A.96(5):1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255).
  • Anticalins possess an eight-stranded ⁇ -barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means of four structurally variable loops at the open end.
  • Anticalins although not homologous to the IgG superfamily, show features that so far have been considered typical for the binding sites of antibodies: (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with differing shape.
  • DARPin refers to a designed ankyrin repeat domain (166 residues), which provides a rigid interface arising from typically three repeated ⁇ -turns. DARPins usually carry three repeats corresponding to an artificial consensus sequence, wherein six positions per repeat are randomised. Consequently, DARPins lack structural flexibility (Gebauer and Skerra, 2009).
  • avimer refers to a class of antibody mimetics which consist of two or more peptide sequences of 30 to 35 amino acids each, which are derived from A-domains of various membrane receptors and which are connected by linker peptides. Binding of target molecules occurs via the A-domain and domains with the desired binding specificity, e.g. for TGF ⁇ , can be selected, for example, by phage display techniques. The binding specificity of the different A-domains contained in an avimer may but does not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).
  • Nanofitin is an antibody mimetic protein that is derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius. Nanofitins usually have a molecular weight of around 7kDa and are designed to specifically bind a target molecule, such as e.g. TGF ⁇ , by randomising the amino acids on the binding surface (Mouratou B, Béhar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods Mol Biol.; 805:315-31).
  • a target molecule such as e.g. TGF ⁇
  • affilin refers to antibody mimetics that are developed by using either gamma- B crystalline or ubiquitin as a scaffold and modifying amino-acids on the surface of these proteins by random mutagenesis. Selection of affilins with the desired target specificity, i.e., for example, against TGF ⁇ , is effected, for example, by phage display or ribosome display techniques. Depending on the scaffold, affilins have a molecular weight of approximately 10 or 20kDa.
  • affilin also refers to di- or multimerised forms of affilins (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).
  • a “Kunitz domain peptide” is derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI).
  • BPTI bovine pancreatic trypsin inhibitor
  • APP amyloid precursor protein
  • TFPI tissue factor pathway inhibitor
  • Kunitz domains have a molecular weight of approximately 6kDA and domains with the required target specificity, i.e., for example, against TGF ⁇ , can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).
  • the term "Fynomer®” refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain.
  • Fyn SH3-derived polypeptides are well-known in the art and have been described e.g. in Grabulovski et al. (2007) JBC, 282, p.
  • trispecific binding molecule refers to a polypeptide molecule that possesses three binding domains and is thus capable of binding, preferably specifically binding to three different epitopes. At least one of these three epitopes is an epitope of the protein of the fourth aspect of the invention.
  • the two other epitopes may also be epitopes of the protein of the fourth aspect of the invention or may be epitopes of one or two different antigens.
  • the trispecific binding molecule is preferably a TriTac.
  • a TriTac is a T-cell engager for solid tumors which comprised of three binding domains being designed to have an extended serum half-life and be about one-third the size of a monoclonal antibody.
  • the term "probody” refers to a protease-activatable antibody prodrug.
  • a probody consists of an authentic IgG heavy chain and a modified light chain.
  • a masking peptide is fused to the light chain through a peptide linker that is cleavable by tumor-specific proteases. The masking peptide prevents the probody binding to healthy tissues, thereby minimizing toxic side effects.
  • Aptamers are nucleic acid molecules or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications (Osborne et. al. (1997), Current Opinion in Chemical Biology, 1:5-9; Stull & Szoka (1995), Pharmaceutical Research, 12, 4:465-483).
  • Nucleic acid aptamers are nucleic acid species that normally consist of (usually short) strands of oligonucleotides. Typically, they have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Peptide aptamers are usually peptides or proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range).
  • variable peptide loop typically comprises 10 to 20 amino acids, and the scaffold may be any protein having good solubility properties.
  • the bacterial protein Thioredoxin-A is the most commonly used scaffold protein, the variable peptide loop being inserted within the redox-active site, which is a -Cys-Gly-Pro-Cys-loop (SEQ ID NO: 1) in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge.
  • Peptide aptamer selection can be made using different systems, but the most widely used is currently the yeast two- hybrid system. Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies.
  • small interfering RNA also known as short interfering RNA or silencing RNA, refers to a class of 18 to 30, preferably 19 to 25, most preferred 21 to 23 or even more preferably 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology.
  • siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene.
  • RNAi RNA interference
  • siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.
  • siRNAs naturally found in nature have a well-defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.
  • siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest.
  • any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • the double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, it is preferred that at least one RNA strand has a 5'- and/or 3'-overhang.
  • one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides.
  • the other end may be blunt-ended or has up to 6 nucleotides 3'- overhang.
  • any RNA molecule suitable to act as siRNA is envisioned in the present invention. The most efficient silencing was so far obtained with siRNA duplexes composed of 21-nt sense and 21- nt antisense strands, paired in a manner to have a 2-nt 3'- overhang.
  • the sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair.
  • 2'-deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides but are often cheaper to synthesize and probably more nuclease resistant.
  • siRNA Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP) (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol.437: Drug Delivery Systems – Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics).
  • IV intravenously
  • IP intraperitoneally
  • si/shRNAs to be used in the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • siRNAs or shRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs.
  • RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi.
  • Further molecules effecting RNAi include, for example, microRNAs (miRNA).
  • Said RNA species are single-stranded RNA molecules.
  • Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference.
  • exogenous miRNA may be employed as an inhibitor of, for example, TGF ⁇ after introduction into the respective cells.
  • a ribozyme from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA is an RNA molecule that catalyses a chemical reaction.
  • ribozymes catalyse either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyse the aminotransferase activity of the ribosome.
  • Non-limiting examples of well-characterised small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes, whereas the group I intron is an example for larger ribozymes.
  • the principle of catalytic self-cleavage has become well established in recent years.
  • the hammerhead ribozymes are characterised best among the RNA molecules with ribozyme activity.
  • the conformational change induced in the aptamer upon binding the target molecule can regulate the catalytic function of the ribozyme.
  • An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked.
  • CRISPR/Cas9 as well as CRISPR-Cpf1, technologies are applicable in nearly all cells/model organisms and can be used for knock out mutations, chromosomal deletions, editing of DNA sequences and regulation of gene expression.
  • the regulation of the gene expression can be manipulated by the use of a catalytically dead Cas9 enzyme (dCas9) that is conjugated with a transcriptional repressor to repress transcription a specific gene, here, for example, the TGF ⁇ gene.
  • dCas9 catalytically dead Cas9 enzyme
  • Inhibitors provided as inhibiting nucleic acid molecules that target the gene of interest, e.g., the TGF ⁇ gene or a regulatory molecule involved in target gene expression are also envisaged herein.
  • Such molecules, which reduce or abolish the expression of the target gene or a regulatory molecule include, without being limiting, meganucleases, zinc finger nucleases and transcription activator-like (TAL) effector (TALE) nucleases.
  • TAL transcription activator-like effector
  • the present invention relates to a method of determining whether a test agent is an inhibitor or activator of the generation of early cortical neural stem cells (NSCs), the method comprising: (i) culturing primate stem cells in a primate stem cell medium until the formation of embryonic bodies (EBs); (ii) culturing the EBs as obtained in step (i) in a neural induction medium comprising an inhibitor of WNT, an inhibitor of TGF- ⁇ , and an inhibitor of BMP in the presence of the test agent; (iii) quantifying the cells that are positive for the cell surface marker protogenin (PRTG); and (iv) determining the test agent as an inhibitor if the number of PRTG-positive cells is reduced as compared to the absence of the test compound, or identifying the test agent as an activator if the number of PRTG-positive cells is increased as compared to the absence of the test compound.
  • EBs embryonic bodies
  • PRTG cell surface marker protogenin
  • test agent or “test compound”, as interchangeably referred to herein, is an “inhibitor” of the generation of early cortical neural stem cells (NSCs) if the number of cells that are positive for the cell surface marker protogenin (PRTG) is reduced by, with increasing preference, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% as compared to the absence of the test agent.
  • NSCs cell surface marker protogenin
  • test agent or “test compound”, as interchangeably referred to herein, is an activator of the generation of early cortical neural stem cells (NSCs) if the number of cells that are positive for the cell surface marker protogenin (PRTG) is increased by, with increasing preference, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% as compared to the absence of the test agent.
  • the method of the second aspect may also be conducted as a high-throughput assays, wherein several test agents are tested in parallel. This is generally performed in wells of microtiter plates.
  • Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact the test agents with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices.
  • mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well.
  • said mixture of test compounds may be de-convoluted to identify the one or more test agents in said mixture giving rise to said activity.
  • the nature of the test agent/compound is not particularly limited.
  • the test agent is preferably a small molecule as defined in connection with the first aspect.
  • activator as used in the context of the test agent also encompasses, and preferably refers to an “accellerator”, i.e., an agent which enhances the cell divison rate per time by, with increasing preference, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% as compared to the absence of the test agent.
  • said primate stem cells are: (i) primate embryonic stem cells (ESCs); or (ii) primate induced pluripotent stem cells (iPSCs).
  • the primate embryonic stem cells are human embryonic stem cells
  • the primate induced pluripotent stem cells are human induced pluripotent stem cells (iPSCs).
  • said isolating is conducted by cell sorting, preferably by fluorescence-activated cell sorting (FACS) or magnetic cell separation (e.g., Magnetic-activated cell sorting (MACS)), using an PRTG-binding agent, preferably a PRTG-binding antibody.
  • FACS fluorescence-activated cell sorting
  • MCS Magnetic-activated cell sorting
  • the term “cell sorting” refers to a method by which cells are mixed with a detectable binding partner (e.g., a fluorescently detectable anti-PRTG antibody or other binding agent specifically binding to PRTG) in solution.
  • a detectable binding partner e.g., a fluorescently detectable anti-PRTG antibody or other binding agent specifically binding to PRTG
  • the cells may be first contacted with a primary antibody specifically binding to PRTG (i.e., an anti-PRTG antibody), followed by a fluorescently detectable secondary antibody which specifically binds the primary antibody.
  • a primary antibody specifically binding to PRTG i.e., an anti-PRTG antibody
  • a fluorescently detectable secondary antibody which specifically binds the primary antibody.
  • Any conventional cell sorting method may be used.
  • Fluorescence-activated cell sorting (FACS) is an example of a cell sorting method.
  • Fluorescence activated cell sorting refers to a method by which the individual cells of a sample are analyzed and sorted according to their optical properties (e.g., light absorbance, light scattering and fluorescence properties, etc.) as they pass in a narrow stream in single file through a laser beam.
  • Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.
  • said isolating is conducted at a time point between about day 4 and about day 12, preferably between about day 4 and about day 6, more preferably on day 5, after initiation of neural induction, and whereby the cells of the enriched population of early cortical neural stem cells (NSCs) are characterized by: (i) having a higher proportion, preferably an at least, with increasing preference, 10%, 20%, 30%, 40%, 50%, or at least 60% higher proportion of cells which are positive for one or more telencephalic-specific markers as compared to that in: (i-a) the initial population of neural progenitor cells prior to said isolating; and/or (i-b) the PRTG negative cells, as determinable, for example, by immunostaining; wherein preferably said telencephalic- specific marker(s) is/are selected from OTX2, NES, GLI3, CDH2 (N-cadherin), VIM, PAX6, FEZF1, LRP1, and
  • said optional re- culturing said enriched population of early cortical neural stem cells (NSCs) in a neural induction medium is conducted for a duration of between about 8 days to about 12 days, preferably for about 10 days, thereby obtaining cells that are characterized by: (i) having a higher, preferably an at least 10% higher proportion of PAX6-positive and/or FOXG1-positive cells as compared to that in: (i-a) the initial population of neural progenitor cells prior to said isolating; and/or (i-b) correspondingly re-cultured PRTG negative cells; as determinable, for example, by immunostaining; (ii) having an increased ability for neural rosette formation as compared to: (ii-a) the initial population of neural progenitor cells prior to said isolating; and/or (ii-b) correspondingly re-cultured PRTG negative; (iii) having a detectable expression
  • the present invention relates to an early cortical neural stem cell (NSC) or an enriched population of early cortical neural stem cells (NSCs) or a subpopulation or progeny thereof obtained or obtainable by the method according to the first aspect of the invention.
  • NSC early cortical neural stem cell
  • NSCs enriched population of early cortical neural stem cells
  • the present invention relates to composition comprising the early cortical neural stem cell (NSC) or enriched population of early cortical neural stem cells (NSCs) according to the third aspect of the invention.
  • the present invention relates to a composition according to the fourth aspect for use as a medicament.
  • the present invention relates to a composition according to the fourth or fifth aspect for use: (i) in treating, preventing, ameliorating at least one symptom, or slowing the progression of a disease that would benefit from an administration of said composition; and/or (ii) in cell replacement therapy, preferably in the treatment of a primate CNS disorder.
  • the disease that would benefit from an administration of said composition is selected from: - a disease of the CNS, preferably a neurodegenerative disease of the CNS, more preferably selected from Alzheimer's disease, Parkinson's disease (PD) and multiple sclerosis (MS); - a neuroinflammatory disease; - a neurodevelopmental disease, preferably a zikka- or mutated gene-induced microcephaly; - an acute brain disease (e.g., stroke) or brain injury, preferably a stroke occurring during a perinatal stage (i.e., in a prenatal embryo, preferably at a time point on or after week 20 of gestation) or an early post-birth stage (preferably until week 4 after birth); - a brain disease characterized by a shortage of cortical cells, malignant cortical cells (e.g., a brain disease characterized by a malignant conversion of cortical cells to a stem-cell like state, such as cancer stem cells) and/or a defective
  • the present invention relates to an in-vitro or ex-vivo use of an PRTG-binding agent for detection or imaging of early cortical NSCs.
  • the present invention relates to a use of PRTG as a cell surface marker for (i) in vitro, in vivo, in situ, or ex vivo detection and/or localization of early cortical NSCs; (ii) isolation of early cortical NSCs; (iii) enriching for early cortical NSCs; and/or (iv) maintaining, and optionally expanding, symmetrically dividing early cortical NSCs.
  • the present invention relates to a use of PRTG as a cell surface marker for enriching outer radial glial (oRG) cells.
  • Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing primate (including human) cortex that contribute to its cellular diversity and evolutionary expansion (Andrews et al. (2020), eLife; 9:e58737). Evolutionary expansion of the human neocortex is partially attributed to a relative abundance of oRG cells.
  • oRG cells display a characteristic division mode, mitotic somal translocation (MST), in which the soma rapidly translocates toward the cortical plate immediately prior to cytokinesis.
  • MST mitotic somal translocation
  • oRG cells are derived from ventricular radial glia (vRG), the primary neural stem cells present in all mammals. oRG cells reside primarily within the outer subventricular zone (oSVZ), closer to the cortical plate than vRG cells, and lack the apical ventricular contact characteristic of vRG cell (Ostrem et al (2014), 8(3):656-664). Without wishing to be bound by any theory, it is expected by the inventors that the cell generated by the herein disclosed method according to the first aspect of the invention can be further differentiated (via further intermediate forms/differentiation stages) into oRG cells.
  • vRG ventricular radial glia
  • the invention relates to a PRTG-binding agent for use in an in vivo method of diagnosing a disease in a subject, wherein said disease is a disease that is causatively linked with an aberrant expression of early cortical neural stem cells (NSCs), the method comprising: (i) administering to said subject a PRTG-binding agent, (ii) detecting and/or quantifying PRTG-positive cells by means of detecting the bound PRTG-binding agent; (iii) wherein the subject is diagnosed as being positive for the disease if the level of detected PRTG is, with increasing preference, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 90% higher or lower as compared to the level detected in a healthy reference subject or mean level detected
  • NSCs cortical neural stem cells
  • the present invention relates to the use of a compound (or a gene expression of a certain group of genes) capable of (i) maintaining early cortical neural stem cells (NSCs) in a symmetrically dividing state; and/or (ii) inhibiting asymmetric division of early cortical neural stem cells (NSCs), to expand early cortical neural stem cells (NSCs).
  • NSCs early cortical neural stem cells
  • NSCs early cortical neural stem cells
  • NSCs early cortical neural stem cells
  • Figure 5 scRNA-seq merged data depicting main clusters and cell compositions of the different time point.
  • UMAP Uniform Manifold Approximation and Projection
  • Color-coded scale represents relative expression levels of each gene (row) across clusters and it is based on the Z scores, with upregulation in red, downregulation in blue, and undetermined directionality in white.
  • Figure 7 Selected potential candidates for early cortical NSCs.
  • Figure 8 Expression of candidate surface markers through 2D differentiation.
  • FIG. 10 PRTG and MCAM expression during human corticogenesis.
  • Cross reference with in vivo data shows enrichment of both marker genes in the early stages of human cortical development (Bhaduri et al., 2020).
  • Both PRTG and MCAM are highly enriched in early RG cells being co-expressed with other known early markers such as LIN28A and DLK1. Note that MCAM has a wider expression than PRTG being expressed also in late RG cells.
  • Figure 11 Surface markers expression profiles of neural cell types. Representative FACS analysis of PRTG and MCAM expression in hiPSCs-derived neural stem cells at various stages of differentiation. Samples incubated with the secondary antibody were used as negative controls to set the appropriate negative gates ( ⁇ 1%) (top). Scatter plots showing coimmunostaining of both cell surface markers and the frequency of each cell population (bottom).
  • Figure 13 Gating strategy followed to sort and collect samples for RNA-seq.
  • RNA-seq analysis identifies specific gene expression profiles based on PRTG sorting. Heatmap representing expression of marker genes for pluripotent, neural stem cells, and differentiated cells, as well as different brain regions (Subpallium, Neocortex, Medial pallium, Diencephalon and Mid/Hindbrain). Color-coded scale represents relative expression levels of each gene (row) across clusters. Note the increased cortical cell identity in day 12 PRTG sorted cells. Surprisingly, at day 35 PRTG detection demarcates non-cortical stem cells, including diencephalic and mid/hindbrain NSCs, instead of cortical lineages.
  • Figure 17 PRTG sorting strategy and phenotype of replated cells at day 12 of neural induction.
  • B) Bright-field images of the replated cultures a few hours after sorting. Note the inability of most PRTG negative cells to attach to the culture plate, indicating a high cell death rate of this subpopulation. Scale bar 200 ⁇ m.
  • Figure 22 PRTG expression analysis in early days of neural induction. Representative FACS analysis of PRTG expression in hiPSCs-derived neural stem cells at early stages of differentiation. Samples incubated with the secondary antibody were used as negative controls to set the appropriate negative gates ( ⁇ 1%) (top).
  • FIG. 24 Sorting for PRTG at day 5 enriches for anterior neural identity.
  • A) Immunostaining images of sorted populations at day 5 for OTX2 and GBX2. Scale bar 50 ⁇ m.
  • Figure 25 Sorting for PRTG at day 5 enriches for anterior forebrain.
  • A) Immunostaining images of sorted populations at day 5 for PAX6 and TFAP2A. Scale bar 50 ⁇ m.
  • FIG. 28 Sorting strategy at day 5 for scRNA-seq analysis.
  • Upregulated genes of interest are shown in red and the downregulated ones in blue.
  • D UMAP plots depicting expression of selected differentially expressed genes of interest.
  • Figure 31 PRTG prospectively isolates distinct telencephalic subpopulations.
  • C UMAP plots depicting expression of selected marker genes which were used to annotate the scRNA-seq clusters.
  • Example 1 The formation of the vertebrate nervous system Neurulation -the process by which the early nervous system is established- starts directly after gastrulation once the three germ layers have been formed: the ectoderm, the endoderm, and the mesoderm.
  • the ectodermal germ layer specializes into a thickened pseudostratified epithelium – the neural plate – instructed by the anterior endoderm and the notochord.
  • the neural plate starts bending and neural folds form bilaterally creating the neural groove.
  • epithelial fusion of the tips of the neural folds culminates in the closure and formation of the neural tube.
  • the neural tube undergoes a series of expansions and constrictions to form the primary brain vesicles: the Prosencephalon (Forebrain), the Mesencephalon (Midbrain), and the Rhombencephalon (Hindbrain).
  • Mangold and Spemann postulated a first model based on their intra-species transplantation experiments involving developing amphibian embryos. These were based on dissecting and transplanting the dorsal portion of the developing embryo that was thought to serve as an ‘organizing center’ into the ventral portion of another embryo. This resulted in the formation of a second set of dorsal axial structures on the ventral side of the host embryo, including a well-organized secondary neural plate.
  • Such seminal finding introduced the concept of an organizer center as a specific population of cells capable of inducing and assembling the various tissues in the embryo along the three body axes: 1) the anterior (rostro)–posterior (caudal) (AP) axis; 2) the dorso–ventral (DV) axis; and 3) the left–right (LR) axis.
  • the neural inducing factors generated by the Spemann-Mangold organizer responsible for inducing neural tissues were unknown.
  • Noggin was identified as the protein which was secreted by the organizer and had the dorsalizing effect (Harland and Smith, 1992). Nevertheless, it was not until 1995 that the final piece of the puzzle was discovered.
  • Wilson and Hemmati-Brivanlou showed that the bone morphogenetic protein 4 (BMP4) is secreted from the ventral side of the embryo, opposite to the organizer, and diffuses throughout the embryo inducing ectodermal tissues. BMP4 activity is inhibited by Noggin thus inducing ectodermal cells in the area of the organizer to develop into neural fate instead of becoming skin cells (Wilson and Hemmati-Brivanlou, 1995).
  • BMP4 activity is inhibited by Noggin thus inducing ectodermal cells in the area of the organizer to develop into neural fate instead of becoming skin cells (Wilson and Hemmati-Brivanlou, 1995).
  • Peter Nieuwkoop He observed that ectoderm can differentiate into anterior neural structures (forebrain) in the absence of any external factors, while posterior fates need to be actively induced by caudalization factors.
  • FGF signaling was also identified as a main player inhibiting the TGFB/BMP signaling pathway in the early embryo (Streit et al, 2000), thus potentially having a synergistic effect with the organizer-secreted BMP antagonists.
  • FGF signaling reduces directly the activity of BMP signaling by promoting phosphorylation of the linker domain and degradation of SMAD1 (Pera EM et al, 2003), and indirectly by inducing the transcription factor ZEB2, which in turn binds to and represses the transcriptional activity of SMADS (Sheng G et al, 2003).
  • the canonical WNT pathway involvement in neural induction remains controversial.
  • WNT signaling seems to induce epidermis at expenses of neural fate by regulating the response of ectodermal cells to FGF signaling (Wilson et al, 2001).
  • intricate spatiotemporal modulation and interplay of WNT, TGFB/BMP and FGF signaling pathways is required for early neural induction and patterning of the AP identity.
  • SHH plays a key role in stablishing the DV axis.
  • SHH is secreted from the ventral regions of the neural tube- the notochord and floor plate- generating a signaling gradient that is essential for proper DV patterning of the CNS, specifically the development of the ventral forebrain, midbrain and hindbrain (Ruiz i Altaba et al, 2002).
  • Vertebrate telencephalic development Once the neural plate is established, early in its development the most anterior primordial sheet of cells give rise to the prosencephalon, which is subsequently subdivided into the telencephalon and the diencephalon, as above mentioned.
  • the telencephalon arises from the most anterior rostral end of the prosencephalon and it is further subdivided into two distinct regions across the DV axis: the pallium, the dorsal region that will primarily become the cerebral cortex; and the subpallium, the ventral region that, in turn, will primarily become the basal ganglia.
  • the pallium is further comprised by four major domains that form cortical structures: dorsal, medial, lateral and ventral pallium.
  • the dorsal pallium will give rise to the neocortex (isocortex), while the medial pallium develops into the cortical hem that will give rise to the hippocampus (archicortex).
  • telencephalic patterning progressive molecular and cellular specification Early patterning of the developing telencephalon is orchestrated by an intricate interplay of morphogenetic gradients of growth factors working in concert with multiple components, including cell– cell interactions, to regulate regional identities. Mainly, SHH is produced ventrally, FGF8 is produced rostrally (most prominently in the cortex) and several BMP and WNT proteins are mainly produced caudo-medially (Charron et al., 2005).
  • telencephalic cell types activate a spatially specific signaling code that activates a pattern of transcription factors (TFs) that control many aspects of the subsequent development.
  • TFs transcription factors
  • these TFs can also modulate the secretion of morphogens creating regulatory loops to instruct lineage commitment, thus establishing specific telencephalic cell types from a common primordium (Lee et al., 2014).
  • FOXG1 acts in concert with FGF signaling and that it is required for FGF8 expression. Conversely, studies also suggest that FGF8 induces and maintains FOXG1 expression in the anterior neural plate (Shimamura and Rubenstein, 1997). Moreover, FOXG1 then restricts expression of BMP4 to the telencephalic midline (Ohkuboa et al., 2002). FOXG1 has also been shown to exert control over telencephalic progenitors to induce proliferation by cell autonomous mechanisms that include the regulation of PAX6 (Manuel et al., 2011).
  • SIX3 shows a defined spatiotemporal expression pattern in the developing telencephalon
  • PAX6 has a role in mediating the early regional subdivision of the prospective prosencephalon into the telencephalon and diencephalon
  • OTX2 together with GBX2 are among the earliest genes expressed in the neuroectoderm, and have been suggested to determine the midbrain-hindbrain boundary in vertebrates, dividing the anterior versus posterior domains (Crossley et al., 2001).
  • OTX2 plays essential roles in rostral brain development being required for the development of the forebrain and midbrain, and is counteracted by the effects of GBX2 which is necessary for anterior hindbrain development.
  • Otx2-null mice lack forebrain and midbrain regions due to a defective anterior neuroectoderm specification during gastrulation (Acampora et al., 1995).
  • mice lacking Gbx2 show developmental failure of the hindbrain development and display a caudal expansion of the midbrain (Inoue et al., 2012). Patterning along the DV axis DV patterning of the telencephalon is established early in forebrain development.
  • GLI3 is initially expressed broadly throughout the telencephalic primordium and then is progressively downregulated in the ventral telencephalon (Gunhaga et al., 2003). In Gli3-deficient mice, they found that the development of the dorsal telencephalon is completely disrupted resulting in a compromised neocortex and the failed formation of the choroid plexus, the cortical hem and the hippocampus (Grove et al., 1998).
  • SHH promotes ventral cell fates in the forebrain by antagonizing the dorsalizing effects of GLI3 (Hebert and Fishell, 2008).
  • Another crucial gene for ventral specification is the TF NKX2-1, which defines and delineates MGE from LGE progenitors (Butt et al., 2008). Mice lacking NKX2-1 display a ventral to dorsal change of fate within the basal telencephalon, with precursor cells generating LGE instead of MGE.
  • GSX2 (Gsh2 in mouse) accompanies the emergence LGE with a lower expression level in MGE.
  • GSX2 is a downstream target of SHH and that its function is required to repress pallial fates. Lack of GSX2 in mice results in profound defects in telencephalic development (Corbin et al., 2000). In the absence of GSX2, it has been observed a loss of DV regionalization in mice, shown by the expansion of MGE (Sussel et al., 1999). In the dorsal telencephalon, PAX6 also plays an essential role in creating the sharp border between the pallium (dorsal) and subpallium (ventral), mainly being expressed in the prospective neocortex.
  • the cerebral cortex has been divided into: the isocortex and the allocortex.
  • the isocortex or neocortex corresponds to the six stratified layers, while the allocortex, represented by the archicortex (hippocampus) and paleocortex (olfactory cortex), exhibit a laminar structure composed of 3 layers (Triarhou, L.C., 2021). It was at the beginning of the 20 th century that many studies were undertaken to exclusively analyze the cytoarchitecture of the cortical areas, being Ramón y Cajal the one who described the intrinsic organization of the cerebral cortex in human and vertebrates.
  • the six layers of neocortex are: layer I or plexiform layer -also known as molecular layer or marginal zone (MZ)- containing mainly nerve axons and a few scattered Cajal-Retzius cells; layer II or external granular layer composed of a varying density stellate (granular) cells and pyramidal cells; layer III or external pyramidal layer containing predominantly pyramidal cells of varying sizes; layer IV or internal granular layer consisting mostly of the stellate cells and a smaller portion of the pyramidal cells; layer V or internal pyramidal layer containing mainly medium-sized to large pyramidal cells; and layer VI or fusiform/multiform layer composed by different types of neuron types, mostly fusiform cells with less dominant pyramidal cells and interneurons.
  • MZ molecular layer or marginal zone
  • the deep layers are the first ones to be generated and to achieve their stratification and functional maturation of their neurons.
  • Neuronal maturation proceeds successively from the deeper layers to the upper layers (layers II/III/IV), from the oldest generated neurons to most superficial and recently born neurons.
  • Neurons mature into two main subtypes: pyramidal or non-pyramidal neurons. Neurons that retain their original contact with layer I become pyramidal neurons (which represent 70% the of cortical neurons), while neurons that lose that contact become stellate cells, non-pyramidal neurons or interneurons (Cadwell et al, 2019). How can we study cortical development?
  • these neural rosette structures corresponding to early anterior NSCs can be induced by using the BMP antagonist Noggin. More importantly, they can be propagated and expanded in culture under specific conditions thus allowing functional characterization of the NSCs and their progeny (Elkabetz et al., 2008). Additional to EB formation capacity, it was shown that PSCs in adherent monocultures are able to commit efficiently to a neural fate in the absence of serum or growth factors due to autocrine signaling (Ying et al., 2003).
  • the NE stage corresponds to the first NSC population which begin diving symmetrically to expand the pool of progenitors and, consequently, become the E-RG population.
  • E-RG rosettes contain highly proliferative NSCs exhibiting broad differentiation potential, and are able to divide asymmetrically to give rise to IP and deep layer neurons.
  • M-RG rosettes emerge becoming restricted from generating earlier fates, and will give rise to upper layer neurons.
  • stemness and an increase tendency to differentiate into neurons are Generally, there is a decrease in stemness and an increase tendency to differentiate into neurons.
  • induced pluripotent stem cells In 2006, Yamanaka’s group reported for the first time that the introduction of four transcription factors- Oct4, Sox2, c-Myc, and Klf4- were sufficient to reprogram mouse fibroblasts into pluripotent stem cells, termed induced pluripotent stem cells (iPSCs).
  • hiPSCs human iPSCs
  • Yamanaka's group successfully differentiated human fibroblasts into iPSCs through the transduction of the same 4 transcription factors previously used in mice cells (Oct4, Sox2, Klf4 and c-Myc) by means of a retroviral system (Takahashi et al., 2007).
  • Thomson’s group used a different set of transcription factors- Oct4, Sox2, Nanog and Lin28- that were transduced by means of a lentiviral system (Yu et al., 2007).
  • the experimental pipeline is focused on identifying a cell surface marker to enable a cell-sorting approach for the isolation of these cells in vitro.
  • Main objectives - Identification of a candidate surface marker for the isolation of early cortical NSCs - Validation of the candidate surface marker - Molecular and cellular characterization of the sorted populations 2.
  • MATERIALS AND METHODS 2.1 Reagents ⁇ 2-Mercaptoethanol (Gibco TM , cat. no.: 31350010) ⁇ Accutase® solution (Sigma-Aldrich®, cat. no.: A6964) ⁇ Apo-transferin human (Sigma-Aldrich®, cat.
  • VT0270, VT0250, VT0220 and VT0200 ⁇ 500 mL Vacuum Filter/Storage Bottle System, 0.22 ⁇ m Pore 33.2cm2 PES Membrane, Sterile ( Corning®, cat. no.: 431097) ⁇ 250 mL Vacuum Filter/Storage Bottle System, 0.22 ⁇ m Pore 19.6cm2 CN Membrane, Sterile (Corning®, cat. no.: 430756) ⁇ Screw cap tube, 50 ml, (Lx ⁇ ): 114 x 28 mm, PP, with print (Sarstedt, cat.
  • HEK273T cells were grown in uncoated plates and cultured in self-made HEK medium containing KnockOut DMEM (10829018, Gibco) supplemented with 10% Fetal Bovine Serum (16140071, Gibco), 2mM Glutamine (35050038, Thermo Scientific), 50 ⁇ M 2-Mercaptoethanol (31350010, Gibco) and Penicillin/Streptomycin (15140122, Gibco).
  • KnockOut DMEM 10829018, Gibco
  • Fetal Bovine Serum 16140071, Gibco
  • 2mM Glutamine 35050038, Thermo Scientific
  • 50 ⁇ M 2-Mercaptoethanol 31350010, Gibco
  • Penicillin/Streptomycin 15140122, Gibco.
  • Detached cells were washed with DPBS and centrifuged at 14,000 rpm for 3 min. Then, cells were resuspended freezing mix containing 10% DMSO (D2650, Sigma-Aldrich) in KSR media (description below). The desired number of cells in a total volume of 1 ml were transferred into a Cryotube (Greiner). Cryotubes were then placed in a slow freezing box, where one 1 °C drops per 1 minute which was stored at the -80°C. The next day, all Cryotubes were placed in the liquid nitrogen tank for long-term storage.
  • KSR medium used for monolayer neural differentiation was made by adding 75 ml of KSR supplement, 5 ml of GlutaMAX, 5 ml of MEM-NEAA, 5 ml of Penicillin-Streptomycin, and 0.5 ml of beta- mercaptoethanol to 409.5 ml of Knockout DMEM (for a total volume of 500 ml). The medium was then filtered by using a vacuum-driven 0.2- ⁇ m filter unit and store at 4°C for up to one month.
  • N2 medium used for neural differentiation was made by adding the following components to 490 ml of double distilled water: 6.5 g of DMEM/F-12 powder, 0.775 g of D-Glucose, 1 g of Sodium bicarbonate, 5 mg of Apo-transferrin, 12.5 mg of insulin, 30 ⁇ l of 500 ⁇ M Sodium selenite, 100 ⁇ l of 830 nM Putrescine, 100 ⁇ l of 100 ⁇ M Progesterone and 5 ml of Penicillin-Streptomycin (for a total volume of 500 ml).
  • the medium was stirred at room temperature until all components were dissolved, then filtered by using a vacuum-driven 0.2- ⁇ m filter unit and store at 4°C for up to one month.
  • 2.4.3 Neurobasal (NB) medium The NB medium used for neural differentiation was made by adding, 5 ml of GlutaMAX, 5 ml of MEM- NEAA, 5 ml of Penicillin-Streptomycin, and 0.5 ml of beta-mercaptoethanol to 484.5 ml of Neurobasal medium (for a total volume of 500 ml). The medium was then filtered by using a vacuum-driven 0.22- ⁇ m filter unit and store at 4°C for up to one month.
  • Neural induction was initiated on day 2 by changing the medium to 1 ⁇ 4 KSR and 3 ⁇ 4 N2+NB containing 1% B27 without retinoic acid, 10 ⁇ M of ROCK inhibitor, and adding the inhibitor molecules SB-431542 (10 ⁇ M), Noggin (250 ng/ml) and XAV939 (3.3 ⁇ M).
  • EBs are transferred from the low-attachment plates to the 6 cm dishes that had been previously coated with polyornithine (PO) (15 ⁇ g/ml), laminin (Lam) (1 ⁇ g/ml) and fibronectin (FN) (1 ⁇ g/ml).
  • PO polyornithine
  • Lam laminin
  • FN fibronectin
  • the pre-coated dishes were dried around the edges in order to limit and restrict the surface area to the center of the dish where EBs will be able to attach and grow. Then, EBs were scraped, collected and distributed into the PO/Lam/FN coated- dishes (1:2) to allow their flattening, growth and rosette-formation.
  • medium was change with fresh 1 ⁇ 2 N2 + 1 ⁇ 2 NB media containing 1% B27 without retinoic acid and SB-431542 (5 ⁇ M), Noggin (125 ng/ml) and XAV939 (3.3 ⁇ M).
  • medium was changed replacing all inhibitors for FGF8 (100 ng/ml) and BDNF (20 ng/ml).
  • organoids were transferred to a 24-well low-attachment plates in 500 ⁇ l of N2 medium supplemented with the inhibitor molecules SB-431542 (10 ⁇ M), Noggin (250 ng/ml) and XAV939 (3.3 ⁇ M). Every 2 days, 300 ⁇ l of media was replaced for fresh one. On day 11, organoids were embedded in matrigel drops (30 ⁇ l) and transferred into a 6-well low-attachment plates.
  • Organoid samples were transferred with 2.5 ml of N2 medium containing 1% B27 without retinoic acid. Every 2 days media was changed. On day 15, organoids were transferred to an orbital shaker (at 86 rpm) to allow for better oxygenation, and N2 medium was supplemented with 1% B27 with retinoic acid. From day 50 onwards, for long term organoid differentiation, 1% matrigel was added into the medium every time the media was changed- every 2-3 days. 2.5.3 Processing organoid samples for immunostainings Organoid samples were taken at the desired date were fixed with 4% paraformaldehyde solution (E15713-S, Science Service) for 30 minutes to 1 hour depending on the size.
  • E15713-S Science Service
  • organoids were submerged in OCT (4583, Tissue-Tek®) for embedding and stored at -80oC until processing. Prior to cryosectioning, the prepared blocks containing the organoids were placed at -20oC to allow the tissue to acclimate, and then were sectioned into 10- ⁇ m-thick slices. Samples were then stored at -80oC until used for immunofluorescence analysis.
  • PB DPBS containing 1% BSA and 10% FBS
  • cells were incubated overnight at 4oC with the desired primary antibody combination diluted in PB (listed in table 3.1).
  • PB DPBS containing 1% BSA and 10% FBS
  • DAPI fluorescent conjugated secondary antibodies
  • S econdary antibodies Distributor Cat. No. Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Invitrogen A-21123 Antibody, Alexa Fluor 546 Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Invitrogen A-21121 Antibody, Alexa Fluor 488 Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Invitrogen A-21143 Antibody, Alexa Fluor 546 Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Invitrogen A-21242 Antibody, Alexa Fluor 647 Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Invitrogen A-21141 Antibody, Alexa Fluor 488 Goat anti-Mouse IgM Heavy Chain Secondary Antibody, Invitrogen A-21238 Alexa Fluor 647 Goat anti-Rabbit IgG (H+L)
  • Cell viability was determined by trypan blue dye exclusion for a viability above 80% before use for analysis and sorting experiments.
  • Surface antigens were labeled by incubating with MCAM and PRTG primary antibodies diluted in HBSS with 5% FBS and ROCKi (10 ⁇ M) (table 2.3) for 15 minutes on ice, followed by two washes in HBSS and incubation for 10 minutes with the appropriate fluorescent secondary antibodies diluted in HBSS with 5% FBS and ROCKi (10 ⁇ M) (table 2.4). After incubation, cells were washed twice and resuspended in HBSS with ROCKi (10 ⁇ M) for sorting.
  • MCAM the anti-Mouse IgG1 on the 488-emission wavelength was used, and for PRTG anti-Mouse IgG2a on the 546-emission wavelength.
  • data were additionally analysed by using FlowJo software (Tree Star, Ashland, OR, http://www.treestar.com), and collected cells were either replated in conditioned medium or pelleted down and snap frozen for downstream analysis.
  • sorted cells were replated at very high density (400,000 cells per cm2) and differentiated for 14 days with NB medium supplemented with BDNF (20 ng/ml), ascorbic acid (0.2 mM), GDNF (20 ng/ml) and DAPT (10 ⁇ M).
  • cDNA reaction (20 ⁇ l) Cycle no T [°C] Time [h:min:sec] 2 ⁇ l RT buffer x10 1 25 10:00 2 ⁇ l Random Primers x10 2 37 2:00:00 0.8 ⁇ l dNTPs mix x25 (100mM) 3 85 0:5 0.25 ⁇ l RNase inhibitor 4 4 hold 0.25 ⁇ l RT enzyme 5 ⁇ l RNA (250 ng) Up to 20 ⁇ l Nuclease free water Table 2.5. Brief protocol for cDNA synthesis. Master mix reaction and thermocycler conditions for conducting the cDNA preparation.
  • RNA-seq libraries were generated by using the TruSeq RNA Library Preparation Kits (Illumina), as described by the manufacturer. Briefly, the main steps are described below. 2.11.1 Purification and fragmentation of mRNA 100 ng per sample of total RNA was diluted with nuclease-free ultra-pure water to a final volume of 50 ⁇ l with the RBP barcode label.50 ⁇ l of RNA Purification Beads was added to each sample to bind the poly-A RNA to the oligo dT magnetic beads.
  • Samples were added to the thermocycler for mRNA denaturation (65°C for 5 minutes, 4°C hold) to denature the RNA and facilitate binding of the poly-A RNA to the beads was done. Next, samples were incubated at room temperature for 5 minutes to allow the RNA to bind to the beads. Without disturbing the beads, the supernatant was discarded. Beads were washed by adding 200 ⁇ l of Bead Washing Buffer per sample and incubated at room temperature for 5 minutes. Without disturbing the beads, the supernatant was discarded, and 50 ⁇ l of Elution Buffer was added to each sample.
  • Samples were placed on the pre-programmed thermal cycler (80°C for 2 minutes, 25°C hold) to elute the mRNA from the beads.50 ⁇ l of Bead Binding Buffer was added to each sample and were incubated at room temperature for 5 minutes. Supernatant was then removed and beads were washed by adding 200 ⁇ l of Bead Washing Buffer. After 5 minutes of incubation at room temperature, the supernatant was discarded and 19.5 ⁇ l of Elute, Prime, Fragment Mix was added per sample which were added to the thermocycler (94°C for 8 minutes, 4°C hold) to elute, fragment, and prime the RNA.
  • RNA-Seq processing and analysis Raw RNA-Seq reads were processed by trimming using Trimmomatic v0.36 (Bolger et al., 2014). The following parameters were used: leading:3; trailing:3; sliding window:4:15; minlen:36. Trimmed reads were then mapped to the human reference genome hg38 with gencode v29 as a reference transcriptome (https://www.gencodegenes.org/human/release_29.html) using STAR v2.6.1d (Dobin et al., 2013).
  • FPKM values for each gene and corresponding isoforms were estimated with RSEM v1.3.1 (Li and Dewey, 2011) and aligned to the reference transcriptome by using the STAR aligned bam. Principal component analysis was run on the logged FPKM expression values (base 10 with a pseudocount of 1) using the top 10,000 genes with the highest variance. 2.13 Single cell RNA sequencing 2.13.1 Sample preparation For generating the 2D monolayer differentiation dataset, neural rosettes were picked at the three different timepoints (day 12, day 35, and day 50). Cells were collected in 15 ml falcon tubes containing Accutase and were incubated in a water bath for 5 minutes at 37oC.
  • a cell suspension of 16500 cells per sample (1000/ ⁇ l) was taken aiming at a recovery of 10000 cells per sample, as recommended in the protocol.
  • Cells were then used to generate the Gel Bead-In-Emulsions and then followed by library preparation as suggested by v3.1 single cell kit protocol as mentioned by manufactured. Briefly, the main steps are described below. 2.13.3 GEM generation and barcoding 70 ⁇ l Master Mix was added to the cell suspension and was gently dispensed into the bottom center of each well in row labeled 1 without introducing bubbles.
  • samples were placed on a 10x Magnetic Separator until the solution was cleared and 150 ⁇ l of the supernatant was transferred to new tubes. Then, 20 ⁇ l of SPRIselect reagent (0.8X) was added to each sample and incubated for 5 minutes at room temperature. Then, samples were placed on a 10x Magnetic Separator until the solution was cleared and the supernatant was removed. Beads were washed twice by adding 200 ⁇ l of 80% ethanol to the pellet while on the magnet for 30 seconds and removed. Samples were then air-dried for 1 minute and immediately 50.5 ⁇ l of Elution Solution was added to each sample.
  • SPRIselect reagent 0.8X
  • scRNA-Seq data were processed using the Cellranger v3.1.0 software (Zheng, G., et al 2017) was used to cluster and determine valid cell barcodes, identify unique molecular identifier (UMI) corresponding to identify and quantify unique RNA molecules for each individual cell, and map reads to the reference genome hg38 and nd ensembl reference transcriptome version 93 (http://ftp.ensembl.org/pub/release-93/gtf/homo_sapiens/Homo_sapiens.GRCh38.93.gtf.gz).
  • UMI unique molecular identifier
  • Neural induction of ZIP13K2 (hiPSC line) towards cortical lineages is conducted in a 2D setting using the Triple-inhibitor (Triple-i) protocol which was established in the lab (Rosebrock et al, 2022).
  • the triple-i protocol combines WNT inhibition using XAV 939, and TGF- ⁇ and BMP inhibition using SB-431542 and Noggin, respectively.
  • neural rosette formation a hallmark of early cortical differentiation- is visible, representing the early NSC stage.
  • Such rosette structures are manually picked and replated weekly in order to propagate the NSCs.
  • each cluster based on the expression of a panel of well- known marker genes for cell state and brain regions, including dorsal pallium (neocortex), medial pallium, subpallium, and diencephalic, as well as more posterior regions such as mid-hindbrain and non- neural lineages (epithelial and mesenchymal) ( Figure 2, 3, 4).
  • dorsal pallium neocortex
  • medial pallium medial pallium
  • subpallium subpallium
  • diencephalic as well as more posterior regions such as mid-hindbrain and non- neural lineages (epithelial and mesenchymal)
  • Figure 2, 3, 4 the expression of dividing and non-dividing NSCs.
  • stem cell marker must be higher expressed in the stem cell populations compared to the intermediate progenitor or neuronal cell populations
  • cortical identity must be regionally restricted and highly expressed in those cells with cortical identity (forebrain specific)
  • early marker must have a clear peak expression in early days followed by a decrease in its expression at later stages
  • representative must be expressed in at least 50% of the cells in the target population
  • surface marker to be able to conduct fluorescence-activated single cell sorting (FACS) restricts the gene to be expressed in the membrane.
  • FACS fluorescence-activated single cell sorting
  • markers like LGR5, as well as CEMPI2 are expressed in higher levels at day 50 than at day 12 which, in turn, rendering the markers unfavorable for tracking possible reprogramming events when conducting such experiments.
  • the staining pattern of the marker is the punctate type instead of being expressed in the cell membrane which hinders the possibility of conducting FACS and sorting for such marker.
  • day 35 PRTG negative is an exception, sharing a higher correlation with day 12 samples. This could suggest the ability of PRTG to segregate a more advanced and committed subpopulation versus a an ‘‘earlier phenotype’’ population on day 35.
  • the PRTG sorted negative subpopulation shares a higher correlation than expected with the undifferentiated hiPSCs (day 0 unsorted), whereas the PRTG positive, the MCAM positive and the MCAM negative subpopulations of day 12 are highly correlated and segregate from the day 0 undifferentiated cells, reflecting expected differences in general transcriptional identity.
  • the PRTG positive sorted subpopulation- compared to the PRTG negative subpopulation- showed a higher expression level of cortical markers including FOXG1, SP8, LHX2 and SIX3, together with a lower expression of posterior markers such as BARHL1 and GBX2. Also presenting lower expression levels of pluripotency - POU5F1 (OCT4) and NANOG- and neuronal markers- DCX, STMN2 and TUBB3. Meaning that by sorting for PRTG expression we are able to purify our cortical culture by sorting out pluripotent cells and neurons, as well as posterior NSCs which remain in the PRTG negative subpopulation.
  • MCAM positive and MCAM negative populations seem to share to a large extent their transcriptional signature meaning that sorting for MCAM at day 12 is insufficient to segregate distinct subpopulations.
  • MCAM positive and MCAM negative populations seem to share to a large extent their transcriptional signature meaning that sorting for MCAM at day 12 is insufficient to segregate distinct subpopulations.
  • the double sorted populations to the PRTG single sorted they share their general transcriptional identity.
  • cortical markers indicating a shift in PRTG expression as a readout from cortical identity to non-cortical identity.
  • a clear dichotomy is drawn on day 35, while the PRTG positive subpopulation exclusively expresses posterior markers such as GBX2, IRX3, PAX3 and EN2, the PRTG negative population exclusively expresses telencephalic markers.
  • PRTG alone efficiently labels NSC populations enriching for cortical NSCs
  • MCAM does not show a clear segregation of subpopulations based on regional identity.
  • MCAM does not provide a clear additional enrichment when sorting for both markers concomitantly.
  • PRTG expression demarcates different transcriptional identities whereas MCAM positive and negative sorted cells seem to have a very similar transcriptomic signature.
  • PRTG expression begins after four days of cortical neural induction Given that PRTG expression peaks at day 12, distinguishing a more cortical NSC population, we were interested to know at what time point PRTG starts being expressed to better understand if its appearance correlates with early cell type specification of cortical lineages. In order to address this question, we conducted FACS analysis at various early time points after neural induction. We began by examining the PRTG expression pattern from day 2 to day 10 (here shown 4-6, Figure 22). We see that expression of PRTG starts coming up at day 4, accounting for 16.9% of the total population.
  • WNT5B has been reported to be specifically expressed in the neocortex together with other WNTs
  • SEMA3A is also expressed in the developing neocortex, specifically in the upper layers.
  • both molecules are highly differentially expressed in a gradual manner, from positive high to middle low and absent in the negative population.
  • NSCs undergo extensive modifications in their transcriptomic profile and chromatin landscape contributing to the formation of heterogeneous progenitor populations. These NSC subtypes are more restricted in their differentiation capacity, and thus more limited in the types of neurons they can generate.
  • NSC populations were derived and studied to dissect the differentiation process from neuroepithelial cells towards the diverse cortical cell types, recapitulating in vivo development (Ziller et al., 2015; Edri et al., 2015).
  • Such studies provided the first glimpse into cell-fate decisions and specification during the ontogeny of in vitro derived cortical neural stem cells.
  • the regulatory mechanisms that orchestrate the stage-specific differentiation process remain poorly understood.
  • PRTG is a cell adhesion molecule, part of the immunoglobulin superfamily, that was first identified during embryonic development in chick (Toyoda et al., 2005) and soon after in mouse (Vesque et al., 2006).
  • One of the more recent studies reports that expression of PRTG emerges during mouse development in the neural tube by day E7.75, being strong until day E9.5 and starting to decrease after E10.5.
  • Co-expression of PRTG together with Sox2 during E7.5–E10.5 in mouse embryos confirms expression of PRTG in early neural progenitors (Wong et al., 2010).
  • MCAM mesenchymal stem cells
  • PRTG expression at day 18 is already reduced. While low expression remains in rosette cells, a higher expression level of PRTG is found in non-cortical cells that lack EMX1 expression. Again, supporting the idea that the remaining expression of PRTG on later days will eventually be marking non-cortical cells in culture.
  • telencephalic markers such as OTX2, PAX6, FOXG1 and SIX3 and complete absence of posterior marker GBX2 in the re-sorted populations.
  • sorting for PRTG high positive cells at day 12 allows for the prospective isolation of early cortical NSCs while excluding unwanted lineages such as posterior NSCs.
  • PRTG in culture correlates with the specification of telencephalic lineages during early neural induction.
  • FACS-purified PRTG positive cells at day 5 display higher levels of the anterior marker OTX2 and lower levels of the posterior marker GBX2, as well as lower levels of neural crest/placodal marker TFAP2A (Dincer et al., 2013) compared to the negative subpopulation.
  • This initial immunostaining analysis of the sorted populations show clear differences indicating PRTG’s ability in segregating anterior neural ectodermal cells from other lineages such as placodal ectodermal cells.
  • scRNA-seq we further provide evidence that the establishment of cortical NSC identity in culture can be detected by the emergence of high PRTG expression as early as day 5.
  • PRTG might act as a cellular receptor by interacting with DNAJB11 (also known as ERdj3).
  • DNAJB11/PRTG signaling might play a role in maintaining the stemness potential of neural progenitors and suppressing premature neuronal differentiation during development (Wong et al., 2010).
  • PRTG exercise its role is not clear, it is not surprising that it can act as a receptor since it has been previously demonstrated that cell adhesion molecules can function as signaling receptors, as is the case of NCAM (Paratcha et al., 2003).
  • PRTG can be used as a readout in a FACS-based screening platform to identify and isolate early cortical NSCs upon perturbation.
  • the differentiation paradigm may be perturbed at different stages with a TF lentiviral library.
  • PRTG would then be used in order to detect and isolate those cells that maintain or achieve an early cortical NSC identity upon overexpression of a specific TF. Sorted cells would then be analyzed by means of DNA-sequencing to identify the integrated TF responsible for the effect, and by means of RNA-sequencing to corroborate the early cortical NSC identity.
  • Semaphorin-3A guides radial migration of cortical neurons during development. Nat Neurosci, 11(1), 36-44. https: / /doi.org/10.1038/nn2018 Chen, S., Xiong, J., Chen, B., Zhang, C., Deng, X., He, F., Yang, L., Chen, C., Peng, J., & Yin, F. (2022).
  • Autism spectrum disorder and comorbid neurodevelopmental disorders (ASD-NDDs): Clinical and genetic profile of a pediatric cohort. Clin Chim Acta, 524, 179-186. https://doi.Org/10.1016/i.cca.2021.ll.014 Cheung, A. F., Pollen, A.
  • CD146/MCAM defines functionality of human bone marrow stromal stem cell populations. Stem Cell Res Tber, 7, 4. https://doi.org/10.1186/sl3287-015-0266-z Harrison-Uy, S. J., k Pleasure, S. J. (2012). Wnt signaling and forebrain development. Cold Spring Harb Perspect Biol, 4(7), a008094. https: / / doi.org/10.1101 /cshperspect.a008094 Haubst, N., Berger, J., Radjendirane, V., Graw, J., Favor, J., Saunders, G. F., Stoykova, A., k Gotz, M. (2004).
  • FOXG1 dose tunes cell proliferation dynamics in human forebrain progenitor cells. Stem Cell Reports, 17(3), 475-488.
  • CD146/MCAM defines functionality of human bone marrow stromal stem cell populations. Stem Cell Res Tber, 7, 4. https://doi.org/10.1186/sl3287-015-0266-z Harrison-Uy, S. J., k Pleasure, S. J. (2012). Wnt signaling and forebrain development. Cold Spring Harb Perspect Biol, 4(7), a008094. https: / / doi.org/10.1101 /cshperspect.a008094 Haubst, N., Berger, J., Radjendirane, V., Graw, J., Favor, J., Saunders, G. F., Stoykova, A., k Gotz, M. (2004).
  • FOXG1 dose tunes cell proliferation dynamics in human forebrain progenitor cells. Stem Cell Reports, 17(3), 475-488.
  • RSEM accurate transcript quantification from RNA-Seq data with or without a reference genome.
  • BMC Bioinformatics 12, 323. https://doi.org/10.1186/1471-2105-12-323 Lindvall, O., k Kokaia, Z. (2010). Stem cells in human neurodegenerative disorders—time for clinical translation? J Clin Invest, 120(1), 29-40. https://doi.org/10.1172/JCI40543 Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 26(4), 402-408.
  • Foxgl is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis.
  • NCAM neural cell adhesion molecule
  • Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature, 404(6778), 567-573. https: / /doi.org/10.1038/35007001 Qi, Y., Zhang, X. J., Renier, N., Wu, Z., Atkin, T., Sun, Z., Ozair, M. Z., Tchieu, J., Zimmer, B., Fattahi, F., Ganat, Y., Azevedo, R., Zeltner, N., Brivanlou, A.
  • BF-1 Telencephalon-restricted expression of BF-1, a new member of the HNF- 3/fork head gene family, in the developing rat brain. Neuron, 6(5), 957-966. https://doi.org/10.1016/0896-6273l92i9021Q-5 Tormin, A., Li, O., Brune, J. C., Walsh, S., Schutz, B., Ehinger, M., Ditzel, N., Kassem, M., & Scheding, S. (2011). CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization. Blood, 777(19), 5067-5077.
  • Gli3 repressor controls cell fates and cell adhesion for proper establishment of neurogenic niche.
  • Cell Rep 5(4), 1093-1104. https://doi.Org/10.1016/i.celrep.2014.07.006 Watanabe, Y., & Nakamura, H. (2012). Nuclear translocation of intracellular domain of Protogenin by proteolytic cleavage. Dev Growth Differ, 54(2), 167-176. https://doi.Org/10.llll/i.1440- 169X.2011.01315.x Wigg, K. G., Feng, Y., Crosbie, J., Tannock, R., Kennedy, J.

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Abstract

La présente invention concerne un procédé de génération d'une population enrichie de cellules souches neurales (CSN) corticales précoces ou d'une sous-population de celles-ci, le procédé comprenant : l'isolement de cellules qui sont positives à la protogénine (PRTG) de marqueur de surface cellulaire à partir d'une population initiale de cellules progénitrices neurales, ladite isolation étant effectuée à un instant compris entre environ le jour 4 et environ le jour 12, de préférence le jour 5, après l'initiation de l'induction neuronale, ce qui permet d'obtenir une population enrichie de cellules souches neurales (CSN) corticales précoces ; et, éventuellement, la reculture de ladite population enrichie de cellules souches neurales (CSN) corticales précoces dans un milieu d'induction neuronal ou un autre milieu de culture, ladite reculture produisant de préférence une descendance desdites cellules souches neurales (CSN) corticales précoces.
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