WO2024209036A1 - Generating highly pure glutamatergic neuronal populations using the pro-neural factor ascl1 - Google Patents

Abstract

Glutamatergic neurons produce, accumulate and release in synapses the neurotransmitter glutamate, which is the main excitatory neurotransmitter in the mammalian central nervous system. Said neurons are involved in most of the brain's fundamental processes such as cognition, learning, memory, and sensory perception. There is an interest to identify transcription factor that would allow the differentiation towards glutamatergic neurons. The inventors surprisingly show that overexpression of ASCL1 induces the generation of a highly pure population of glutamatergic neurons. Said observation goes totally in the opposite direction of what has been previously taught, since ASLC1 was mainly described as inducing GABAergic neurons in the forebrain. Therefore, the present invention relates to methods for generating highly pure glutamatergic neuronal populations using the pro-neural factor ASCL1.

Description

GENERATING HIGHLY PURE GLUTAMATERGIC NEURONAL POPULATIONS

USING THE PRO-NEURAL FACTOR ASCL1

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular neurology.

BACKGROUND OF THE INVENTION:

Directed neuronal differentiation of human induced pluripotent stem cells (hiPSCs), neural progenitors, or fibroblasts using transcription factors has allowed for the rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor has been indeed widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modelling, drug screening, and neuronal replacement therapies. In particular, there is an interest to identify transcription factor that would allow the differentiation towards glutamatergic neurons. Glutamatergic neurons produce, accumulate and release in synapses the neurotransmitter glutamate, which is the main excitatory neurotransmitter in the mammalian central nervous system. It is involved in most of the brain's fundamental processes such as cognition, learning, memory, and sensory perception. Asci 1 (Mashl) and Neurogenin2 (Neurog2), which are the mammalian homologs of Drosophila achaete-scute complex and atonal, respectively, are the two main proneural factors that initiate and regulate neurogenesis in vertebrate nervous systems (Bertrand, Nicolas, Diogo S. Castro, and Franqois Guillemot. "Proneural genes and the specification of neural cell types. " Nature Reviews Neuroscience 3.7 (2002): 517-530). In mice, Ascii and Neurog2 are respectively required to specify GAB Aergic and glutamatergic neurons in the forebrain and sympathetic and sensory neurons of the peripheral nervous system (Parras, Carlos M., et al. "Divergent functions of the proneural genes Mashl and Ngn2 in the specification of neuronal subtype identity. " Genes & development 16.3 (2002): 324-338). In said context, it has been shown that Neurog2 expression in hiPSCs is sufficient to promote lineage-conversion into glutamatergic neurons (Fernandopulle, Michael S., et al. "Transcription factor-mediated differentiation of human iPSCs into neurons." Current protocols in cell biology 79.1 (2018): e51.; Ho, Seok- Man, et al. "Rapid Ngn2 -induction of excitatory neurons from hiP SC -derived neural progenitor cells." Methods 101 (2016): 113-124.; Nehme, Raida, et al. "Combining NGN2 programming with developmental patterning generates human excitatory neurons with NMDAR-mediated synaptic transmission." Cell reports 23.8 (2018): 2509-2523.; Zhang, Yingsha, et al. "Rapid single-step induction of functional neurons from human pluripotent stem cells. " Neuron 78.5 (2013): 785-798.).

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to methods for generating highly pure glutamatergic neuronal populations using the pro-neural factor ASCL1.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors surprisingly show that overexpression of ASCL1 induces the generation of a highly pure population of glutamatergic neurons. Said observation goes totally in the opposite direction of what has been previously taught, since ASLC1 was mainly described as inducing GABAergic neurons in the forebrain (Parras, Carlos M., et al. "Divergent functions of the proneural genes Mashl and Ngn2 in the specification of neuronal subtype identity. " Genes & development 16.3 (2002): 324-338).

The first object of the present invention relates to a method of generating a highly pure population of glutamatergic neurons comprising the steps consisting of i) expressing a polynucleotide encoding for the transcription factor ASCL1 in a population of human induced neural progenitor cells (hiNPCs) and ii) differentiation said population of cells into a highly pure population of glutamatergic neurons.

As used herein, the term “glutamatergic neuron” has its general meaning in the art and refers to neurons that produce, accumulate in vesicles and release in synapses the neurotransmitter glutamate, which is the main excitatory neurotransmitter in the mammalian central nervous system. The glutamatergic neuron is involved in most of the brain’s fundamental processes such as cognition, learning, memory, and sensory perception. Dysregulation of glutamatergic neurotransmission is associated with many neurological disorders including epilepsy, schizophrenia, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and stroke. The glutamatergic neuron is characterized by the expression of the Vesicular Glutamate Transporter 1 (VGLUT1) and 2 (VGLUT2).

As used herein, the term "population" refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest (e.g. a population of glutamatergic neurons comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the markers of glutamatergic neurons).

As used herein, the expression “highly pure population of cells” indicates that the population of cells contains at least 90% of the desired cells. Accordingly, a “highly pure population of glutamatergic neurons” means that the population contain 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of glutamatergic neurons.

As used herein, the term “human induced neural progenitor cell” or “hiNPC” has its general meaning in the art and refers to a neural progenitor cell that is differentiated from human iPSC. As used herein, the term “induced pluripotent stem cell” or “iPSC” has its general meaning in the art and refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell — typically an adult somatic cell — by inducing a “forced” expression of specific genes. Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. Different protocols can be used to obtain hiNPC. In particular, most useful approaches rely solely on small molecule to produce highly proliferative hiNPC populations (Chambers, Stuart M., et al. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. " Nature biotechnology 27.3 (2009): 275-280 f. A key characteristic of these hiNPCs is their capability of robust and homogenous expansion. At the same time, hiNPCs should be able to efficiently differentiate into neuronal and glial cell types (Reinhardt, Peter, et al. "Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. " PloS one 8.3 (2013): e59252.).

As used herein, the term “ASCL1” has its general meaning in the art and refers to the Achaete- scute homolog 1 protein encoded by ASCL1 gene. The term is also known as ASH-1, Class A basic helix-loop-helix protein 46 or bHLHa46. ASCL1 is a transcription factor that plays a key role in neuronal differentiation. An exemplary amino acid sequence for ASCL1 is shown as SEQ ID NO: !. SEQ ID NO : 1 >sp | P50553 | ASCL1_HUMAN Achaete- scute homolog 1 OS=Homo sapiens OX=9606 GN=ASCL1 PE=1 SV=2

ME S S AKME S GGAGQQ PQ PQ PQQ P FL PPAAC F FATAAAAAAAAAAAAAQS AQQQQQQQQQQ QQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSS PELMRCKRRLNFSGFGYSLPQQQPAAV ARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAV SAAFQAGVLS PTI S PNYSNDLNSMAGS PVSSYSSDEGSYDPLS PEEQELLDFTNWF

As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogues thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and singlestranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the doublestranded form.

In some embodiments, the population of hiNPCs is engineered to express a polynucleotide that encodes for a polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

As used herein, the term “engineered” refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered cell” refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the cell prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the cell prior to the manipulation; and/or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the cell prior to the manipulation. In some embodiments, an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a polynucleotide, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell. Those of ordinary skill in the art will appreciate that reference to an “engineered cell” herein may, in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such cell.

As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), considering the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. According to the invention, a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

It is contemplated that a polynucleotide can be introduced into the hiNPCs as naked DNA or in a suitable vector.

Naked DNA generally refers to the DNA contained in a plasmid expression vector in proper orientation for expression. Physical methods for introducing a polynucleotide construct into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, nucleofection, and the like. Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

In some embodiments, the polynucleotide is introduced into the population of hiNPCs by a viral vector that is an adeno-associated virus (AAV), a retrovirus, lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is a retroviral. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, the polynucleotide of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based and are configured to carry the essential sequences for incorporating foreign polynucleotide, for selection and for transfer of the polynucleotide into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a polynucleotide encoding a viral gag and a pol gene and another vector that can provide a polynucleotide encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.

Typically, the vector of the present invention includes "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

Another polynucleotide sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. To increase the expression, polynucleotides of the present invention may be operably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), P-actin, ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter, elongation factor (EF)-la and the spleen focus-forming virus (SFFV) promoter.

In some embodiments, the polynucleotide sequence that encodes for the ASCL1 polypeptide is placed under the control of an inducible promoter. Exemplary inducible promoters include, for example, promoters that respond to heavy metals, to thermal shocks, to hormones, promoters that respond to chemical agents, such as glucose, lactose, galactose or antibiotic (e.g., tetracycline or doxycycline). A tetracycline-inducible promoter is an example of an inducible promoter that responds to an antibiotic. The tetracycline-inducible promoter comprises a minimal promoter linked operably to one or more tetracycline operator(s) (i.e. TetO). The presence of tetracycline or one of its analogues leads to the binding of a transcription activator to the tetracycline operator sequences, which activates the minimal promoter and hence the transcription of the associated cDNA. Tetracycline analogue includes any compound that displays structural homologies with tetracycline and can activate a tetracycline-inducible promoter. Exemplary tetracycline analogues include, for example, doxycycline, chlorotetracycline and anhydrotetracycline.

In some embodiments, the sequence of the polynucleotides is codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

Typically, hiNPCs are cultured in an appropriate culture medium for allowing the differentiation into glutamatergic neurons.

As used herein, the terms “culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,” “expand,” “expanding,” etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. The term does not imply that all cells in the culture survive or grow or divide, as some may naturally senesce, etc. Cells are typically cultured in media, which can be changed during the culture.

As used herein, the term "culture medium", refers to a chemical composition that supports the growth and/or differentiation of a cell, suitably of a mammalian cell. Typical culture media include suitable nutrients (e.g. sugars, amino acids, proteins, and the like) to support the growth and/or differentiation of a cell. Typically, the culture medium is a neuronal medium. As used herein, the term “neuronal medium” has its general meaning in the art and refers to a medium that supports the culture of neurons. In some examples, the neuronal medium includes one or more ingredients selected from: a cell culture medium containing growth-promoting factors and/or a nutrient mixture (e.g., DMEM/F12, MEM/D-valine, neurobasal medium etc., including mixtures thereof); media supplements containing hormones, proteins, vitamins and/or amino acids (e.g., N2 supplement, B27 supplement, non-essential amino acids (NEAA), L-glutamine, Glutamax, BSA, insulin, all trans retinoic acid, etc. including mixtures thereof); and optionally small molecule inhibitors (e.g., SB431542 (BMP inhibitor), LDN193189 (TGF-pi inhibitor), CHIR99021(GSK3p inhibitor), etc., including mixtures thereof). Ingredients may also include one or more of beta- mercaptoethanol, transferrin; sodium selenite; and cAMP. Suitable concentrations of each of these ingredients are known to those of skill in the art and/or may be empirically determined. In some embodiments, the cells are cultured in the BrainPhys™ Neuronal Medium that is based on the formulation published by Cedric Bardy and Fred H. Gage (C Bardy et al. Proc Natl Acad Sci USA, 2015).

In some embodiments, when the polynucleotide that encodes for the ASCL1 polypeptide is placed under the control of an inducible promoter that is responder to a chemical agent, the culture medium is thus supplemented with an amount of said chemical agent. For instance, when the polynucleotide is placed under the control of a tetracycline-inducible promoter, then the culture medium is supplemented with a tetracycline analogue (for example, doxycycline, chlorotetracycline and anhydrotetracycline).

Typically, the cells are cultured in appropriate culture system such as plates or dishes. Culture conditions may vary, but standard tissue culture conditions are preferably used. Typically, cells are incubated in 5% CO2 incubators at 37°C in the appropriate culture medium.

In some embodiments, the hiNPCs are cultured with feeder cells. In some embodiments, the neural differentiation may be performed in the absence or presence of co-cultured astrocytes. In some embodiments, the differentiating includes culturing the hiNPC on a surface coated with feeder cells or polyomithine/ laminin.

Typically, the duration of the culturing step is in the range of about 5 to 25 days, more preferably about 14 to 35 days (2-5 weeks). In some embodiments, the hiNPCs differentiate effectively into mature and synaptically active glutamatergic neurons within 3 to 5 weeks, although this may vary depending on the cell line and the reprogramming protocol.

The method of the present invention is particularly suitable for the preparation of large amounts of glutamatergic neurons that can be subsequently used e.g. for research or therapeutics applications. In particular, the glutamatergic neurons can be used in neurodevelopment studies, disease modelling, drug screening, and/or neuronal replacement therapies.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Comparison of ours and previously described models to generate hiNs. A) Schemes illustrating the main steps for the generation of hiNs i) via ASCL1 expression in human-induced neural progenitor cells (hiNPCs; Method 1); ii) via NEUR0G2 expression in human-induced pluripotent stem cells (hiPSCs; Method 2); and iii) via chemically-induced methods where hiPSCs are first driven into a neural progenitor state (hiNPCs) that spontaneously differentiate into neurons and astrocytes in 2D or 3D (cerebral organoids) cultures. B) Quantification of the two mature neuronal subtypes (glutamatergic and GABAergic) identified in these different models using single-cell RNA-sequencing.

EXAMPLE:

Methods

Generation of ASCLl-induced human neurons (ASCLl-hiNs)

HiNPCs are first virally transduced with the FUW-M2rtTA (Addgene #20342) lentiviral construct and a passage later, the Tet-O-FUW-Ascl 1 (Addgene #27150) lentiviral construct was transduced. These cells are maintained in neural progenitor medium (NPM; StemCell Technologies, Inc) and expanded prior to differentiation. For differentiation in human induced neurons (hiNs), hiNPCs are plated onto PLO/laminin-coated imaging plates at density 50,000 cells/cm² in NPM. After 24h, complete BrainPhys medium (BP) is added 1 : 1 together with 2 pg/mL doxycycline (Sigma-Aldrich) to induce TetO-regulated Ascii gene expression. The following day, 1 pg/mL puromycin (Sigma-Aldrich) was added to start cell selection. After 2- 3 days (depending on the efficiency of antibiotic selection), 50,000 human cortical astrocytes were added in each well with BrainPhys (StemCell Technologies) containing doxycycline. After 24 hours, 2 pM of Ara-C (Cytosine P-D-arabinofuranoside) (Sigma-Aldrich) was added to arrest the proliferation of astrocytes. Half of the medium in each well was changed biweekly with fresh BrainPhys medium containing doxycycline until the 14th day. After that, the biweekly medium change was performed only with BrainPhys. Differentiation was allowed to continue for another 2-4 weeks prior to subjecting the cells to various experimental manipulations.

Human cortical astrocytes (HA; ScienCell # 1800) were sourced from ScienCell Research Laboratories, CA, USA and grown up to 10 passages on poly-l-lysine coated 75 cm2 flasks using astrocyte medium (ScienCell # 1801) supplemented with astrocyte growth supplement (AGS, ScienCell #1852) and 10 ml of fetal bovine serum (ScienCell # 1800).

This culture system was characterized at 4weeks using snRNA-seq showing that 70% of cells expressed the pan-neuronal markers SOX11, SNAP25, DCX and RBFOX3. Of those, more than 90% of cells express genes common to human forebrain glutamatergic neurons, and less than 5% of GABAergic neurons. Therefore, this method allows the generation of highly pure glutamatergic populations in a relatively short period of time from hNPCs.

Using multi-electrode array (MEA) electrophysiological recordings, we observe a high number of neuronal spikes and spike bursts that are highly synchronized, indicating the formation of a complex neuronal network in vitro. This is not observed for the other methods commonly used in the literature to generate hiNs (NEUROG2 expression in hiPSCs or chemically-induced differentiation from hiNPCs to neurons and astrocytes).

Results

We generate pure glutamatergic neuronal cultures by direct lineage-reprogramming of human NPCs (hNPCs) using doxycycline-inducible expression of ASCL1. After validation that ASCL1 expression efficiently reprogrammed hNPCs into highly pure neurons (hereafter ASCLl-hiNs), we added exogenous human cerebral cortex astrocytes to support functional neuronal maturation and synaptic connectivity (Christopherson, Karen S., et al. "Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. " Cell 120.3 (2005): 421-433). After 4 weeks of differentiation and snRNA-seq analyses (n=3114 from 2 independent culture batches), we observed that ASCLl-hiNs were composed of glutamatergic neurons (-92%) with a small proportion of GABAergic neurons (-2%) or of cells co-expressing low levels of markers of both neuronal subtypes (-6%) (Figure 1).

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of generating a highly pure population of glutamatergic neurons comprising the steps consisting of i) expressing a polynucleotide encoding for the transcription factor ASCL1 in a population of human induced neural progenitor cells (hiNPCs) and ii) differentiation said population of cells into a highly pure population of glutamatergic neurons.

2. The method of claim 1 wherein the population of hiNPCs is engineered to express a polynucleotide that encodes for a polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

3. The method according to claim 1 or 2 wherein the polynucleotide is introduced into the population of hiNPCs by a viral vector.

4. The method according to any one of claims 1 to 3 wherein the polynucleotide sequence that encodes for the ASCL1 polypeptide is placed under the control of an inducible promoter.

5. The method of claim 4 wherein when the polynucleotide that encodes for the ASCL1 polypeptide is placed under the control of an inducible promoter that is responder to a chemical agent, the culture medium is thus supplemented with an amount of said chemical agent to induce the expression of the ASCL1 polypeptide.

6. The method of claim 5 wherein when the polynucleotide is placed under the control of a tetracycline-inducible promoter, then the culture medium is supplemented with a tetracycline analogue (for example, doxycycline, chlorotetracycline and anhydrotetracycline) to induce the expression of the ASCL1 polypeptide.

7. The method according to any one of claims 1 to 6 wherein the cells are cultured in the BrainPhys™ Neuronal Medium.

8. The method according to any one claims 1 to 7 wherein the hiNPCs are cultured with feeder cells.

9. The method according to any one of claims 1 to 8 wherein the neural differentiation is performed in presence of co-cultured astrocytes.

10. The method according to any one of claims 1 to 9 wherein the differentiating step includes culturing the hiNPCs on a surface coated with feeder cells or poly ornithine/ laminin.