WO2016012570A1

WO2016012570A1 - Method for producing motor neurons from pluripotent cells

Info

Publication number: WO2016012570A1
Application number: PCT/EP2015/066944
Authority: WO
Inventors: Cécile MARTINAT; Marc Peschanski; Yves MAURY; Stéphane NEDELEC
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite D'evry Val D'essonne
Priority date: 2014-07-23
Filing date: 2015-07-23
Publication date: 2016-01-28

Abstract

The present invention concerns a method for producing a population of motor neuron progenitors comprising the following steps: a) culturing neuralized pluripotent cells in a culture medium C₁ comprising an activator of the Wnt signaling pathway during a period of time T₁ and b) culturing said cells in a culture medium C₂ comprising retinoic acid and an agonist of the Hedgehog signaling pathway during a period of time T₂.

Description

Method for producing motor neurons from pluripotent cells

The present invention concerns the production of motor neurons from pluripotent cells.

The targeted differentiation of human pluripotent stem cells (hPSC) into specific cell populations is a major avenue for developmental studies, disease modeling and regenerative medicine. However, a more systematic use of these cells for biological and clinical approaches is often impeded by slow and inefficient differentiations due in large part to the imprecise control over cell fate specification in vitro. This is typically the case of the generation of motor neurons from hPSCs.

Spinal (sMN) and cranial (cMN) motor neurons, respectively located in the spinal cord and the hindbrain, are the two fundamental classes of motor neurons and the selective target of several incurable diseases such as Amyotrophic Lateral Sclerosis (ALS) or Spinal Muscular Atrophy (SMA). Controlling the differentiation of hPSCs into both cell types is therefore essential to study the formation of specific motor circuits and their impairment in diseases. However, human cMNs are not yet accessible. Furthermore, human sMN generation is still inefficient (10 to 50% of total cells) and long (21 to 50 days) despite a long standing interest in obtaining them and numerous available protocols. Currently, the most efficient procedures to specify sMNs combine retinoic acid (RA) and Hedgehog (HH) agonists, after a neuralization with TGF3/BMP inhibitors (Amoroso et al. (2013) J. Neurosci. 33:574-586; Wada et al. (2009) PLoS ONE 4:e6722), but these procedures are long and only sMNs are obtained.

Accordingly, there is still an important need for improved procedures enabling obtaining enriched populations of sMNs from hPSCs and for procedures enabling obtaining populations of cMNs from hPSCs.

Using stage 4 caudal explants and stage 4 forebrain explants, Nordstrom et al. demonstrated that, during mouse and chick development, the combinatorial action of Wnt, Fibroblast growth factor (FGF) and RA differentially specifies hindbrain and spinal progenitors that, upon HH exposure, differentiate respectively into cranial or spinal motor neurons (Nordstrom et al. (2006) PLoS Biology 4:e252). However, due to the complexity to systematically assess the combinatorial action of these cues during hPSC differentiation, the potential implication of these pathways for the generation of human motor neuron subtypes from hPSCs remains unclear.

The present invention arises from the unexpected finding by the inventors, that the culture of neuralized hPSCs in the presence of an activator of the Wnt signaling pathway enabled obtaining motor neurons as soon as 14 days after the beginning of the culture. The inventors also demonstrated that according to the concentration of the activator of the Wnt signaling pathway used, either spinal motor neurons or cranial motor neurons could be obtained.

In particular, the inventors demonstrated that, by culturing neuralized hPSCs in the presence of low concentrations of activators of the Wnt signaling pathway, typically in the presence of 1 μΜ of the compound Chir-99021 , in combination with 1 0 nM RA and 500 nM of the Smoothened Agonist SAG, it was possible to obtain a population of cranial motor neurons progenitors that could efficiently differentiate into cranial motor neurons in the presence of gamma-secretase inhibitors. To the inventors' knowledge, it is the first time that cranial motor neurons are obtained from hPSCs.

The inventors also demonstrated that, by culturing neuralized hPSCs in the presence of high concentrations of activators of the Wnt signaling pathway, typically in the presence of 3 μΜ of the compound Chir-99021 , in combination with 1 00 nM RA and 500 nM SAG, it was possible to obtain a population comprising more than 80% of spinal motor neurons progenitors 1 0 days after the beginning of the culture, that could efficiently differentiate into spinal motor neurons in the presence of gamma-secretase inhibitors. To the inventor's knowledge, it is the first time that such an enriched population of spinal motor neurons is obtained in such a short time.

The present invention thus concerns a method, preferably an ex vivo method, for producing a population of motor neuron progenitors comprising the following steps:

a) culturing neuralized pluripotent cells in a culture medium Ci comprising an activator of the Wnt signaling pathway during a period of time T_{1 ;} and

b) culturing said cells in a culture medium C₂ comprising retinoic acid and an agonist of the Hedgehog signaling pathway during a period of time T₂.

In particular, the present invention concerns a method, preferably an ex vivo method, for producing a population of spinal motor neuron progenitors comprising the following steps:

a) culturing neuralized pluripotent cells in a culture medium d comprising a high concentration of an activator of the Wnt signaling pathway during a period of time T_{1 ;} and b) culturing the cells obtained in step a) in a culture medium C₂ comprising retinoic acid and an agonist of the Hedgehog signaling pathway during a period of time T₂.

The present invention also concerns a method, preferably an ex vivo method, for obtaining a population of spinal motor neurons comprising the steps of:

A) producing a population of spinal motor neuron progenitors by the method defined above, and B) differentiating said population of spinal motor neuron progenitors into spinal motor neurons.

The present invention also concerns a method, preferably an ex vivo method, for producing a population of cranial motor neuron progenitors comprising the following steps: a) culturing neuralized pluripotent cells in a culture medium Ci comprising a low concentration of an activator of the Wnt signaling pathway during a period of time T_{1 ;} pre-b) culturing the cells obtained in step a) in a culture medium C_pre-2 comprising an agonist of the Hedgehog signaling pathway during a period of time Τ_ρΓβ-₂, and

b) culturing the cells obtained in step pre-b) in a culture medium C₂ comprising retinoic acid and an agonist of the Hedgehog signaling pathway during a period of time T₂.

The present invention also concerns a method, preferably an ex vivo method, for obtaining a population of cranial motor neurons comprising the steps of:

A) producing a population of cranial motor neuron progenitors by the method defined above, and

B) differentiating said population of cranial motor neuron progenitors into cranial motor neurons.

Detailed description of the invention

Motor neurons

As used herein, the term "motor neuron" or "motoneuron" refers to an efferent neuron which innervated a muscle cell or autonomic ganglia. Motor neurons include two fundamental classes: spinal motor neurons and cranial motor neurons.

As used herein, the term "spinal motor neuron" refers to a motor neuron located in the spinal cord. As well-known by the skilled person, spinal motor neurons can be characterized by the expression of a specific pattern of markers. Typically, spinal motor neurons can be characterized by being ISL1 /2+ (ISL LIM Homeobox 1 or 2 positive), HB9+ (Homeobox protein 9 positive). Additional markers that can be expressed by spinal motor neurons include Lhx3 (LIM homeobox 3), FOXP1 (forkhead box P1 ), CHAT (Choline acetyltransferase) and VACHT (vesicular acetylcholine transporter). Identification of spinal motor neurons can thus be carried out by any technique well-known from the skilled person involving the detection of the above markers, such as immunostaining using specific anti-ISL1 /2 and/or anti-HB9 antibodies.

As used herein, the term "cranial motor neurons" refers to motor neurons located in the brainstem. As well-known from the skilled person, cranial motor neurons can be characterized by the expression of a specific pattern of markers. Typically, cranial motor neurons can be characterized by being ISL1/2+, HB9 negative and PHOX2B+ (paired-like homeobox 2b positive). Additional markers that can be expressed by cranial motor neurons include TBX20. Identification of cranial motor neurons can thus be carried out by any technique well-known from the skilled person involving the detection of the above markers, such as immunostaining using specific anti-ISL1/2 and/or anti-PHOX2B antibodies, RT-PCR or in situ hybridization for all the above mentioned markers.

Spinal and/or cranial motor neurons, more particularly, functional spinal and/or cranial motor neurons can also be characterized using electrophysiology techniques, such as whole-cell patch clamp recording. Typically, whole-cell patch clamp assays can be performed as follows: patch pipettes (3-4 ΜΩ) containing 135 mM KMethylSulfate, 5 mM KCI, 0.1 mM EGTA-Na, 10 mM HEPES, 2 mM NaCI, 5 mM ATP, 0.4 mM GTP, 10 mM phosphocreatine (pH 7.2; 280-290 mOsm) and an extracellular solution containing (in mM) 125 mM NaCI, 2.5 mM KCI, 10 mM glucose, 26 mM NaHC0₃, 1 .25 mM NaH₂P0₄, 2 mM Na Pyruvate, 2 mM CaCI₂ and 1 mM MgCI₂ and saturated with 95% 0₂ and 5% C0₂ are used, at a pH of 7.3. Membrane potentials are corrected for liquid junction potential, which can be typically measured to be ~3 mV. Series resistance (typically less than 10 ΜΩ) are monitored and compensated throughout each experiment with the amplifier circuitry. 0.5 μΜ tetrodotoxin (TTX) is bath-applied following dilution into the external solution from concentrated stock solutions. To maintain constant osmolarity and pH, 30 mM TEACI (tetraethylammonium chloride) can be added to a modified external solution (95 mM NaCI instead of 125 mM). Glutamate can be added with equal concentration NaOH to maintain neurtral pH. Leak current is subtracted from current families using a positive/negative (P/N) protocol and each set of currents is for example averaged four times. All data can be acquired with Axograph X software and analyzed for example with Igor pro. Functional immature spinal motor neurons can thus be characterized by firing small immature spikes in response to, for example a 600 pA current injection with shape and magnitude typically from 3 to 10 mV, as shown in Figure 1 . Functional mature spinal motor neurons can be characterized by firing trains of action potentials with shape and magnitude typically from 60 to 80 mV, as shown in Figure 2. Functional mature spinal motor neurons may also be characterized by dectecting choline acetyl transferase (CHAT) expression, using conventional immunostaining and Western Blot techniques.

In the context of the invention, the term "motor neuron progenitor" or "motoneuron progenitor" refers to a cell population that can give rise to one of the two main groups of motoneurons. As used herein, the term "spinal motor neuron progenitor" refers to Olig2 positive cells. As well-known from the skilled person, spinal motor neurons progenitors can be characterized by the expression of a specific pattern of markers. Typically, generated spinal motor neuron progenitors can be characterized by being OLIG2+ (oligodendrocyte lineage transcription factor 2 positive), NKX6.1 + (NK6 homeobox 1 positive), cells expressing as well HOXA2 (homeobox A2), HOXA4 (homeobox A4) and HOXA5 (homeobox A5) mRNAs. Identification of spinal motor neuron progenitors can thus be carried out by any technique well-known from the skilled person involving the detection of the above markers, such as immunostaining using specific anti-OLIG2 and/or anti-NKX6.1 antibodies, and RT-PCR for HOXA2, HOXA4 and/or HOXA5 mRNAs.

As used herein, the term "cranial motor neuron progenitor" refers to Olig2 negative, NKX6.1 +, NKX2.2+ (NK2 homeobox 2 positive) cells. As well-known from the skilled person, cranial motor neuron progenitors can be characterized by the expression of NKX2.2, NKX6.1 and HOXA2 mRNAs. Typically, cranial motor neuron progenitors can be characterized by being HOXA2+, HOXA4- and HOXA5- cells. Identification of cranial motor neuron progenitors can thus be carried out by any technique well-known from the skilled person involving the detection of the above markers, such as immunostaining using specific anti-NKX2.2 and/or anti-NKX6.1 antibodies, and RT-PCR for HOXA2 mRNAs.

Advantageously, the populations of motor neuron progenitors, in particular of spinal motor neuron progenitors, obtained with the methods of the invention are highly enriched in motor neuron progenitors, in particular in spinal motor neuron progenitors, since they comprise more than 70% of spinal motor progenitors, which is in sharp contrast with the populations obtained with protocols of the prior art.

Similarly, advantageously, the populations of motor neurons, in particular of spinal motor neurons or cranial motor neurons, obtained with the methods of the invention are highly enriched respectively in spinal motor neurons and cranial motor neurons, since they respectively comprise more than 70% (even up to 82% or up to 94%) of spinal motor neurons and more than 50% of cranial motor neurons, which is in sharp contrast with the populations obtained with protocols of the prior art.

Pluripotent cells

As used herein, the term "pluripotent cells" refers to undifferentiated cells which can give rise to a variety of different cell lineages.

Preferably, the pluripotent cells are human pluripotent cells.

Preferably, the pluripotent cells are stem cells. In the context of the present invention, stem cells encompass embryonic stem cells (ESCs); fetal stem cells including stem cells of the embryo proper and of extra-embryonic tissues such as amniotic fluid stem cells, and induced pluripotent stem cells. In particular, stem cells according to the invention encompass fetal stem cells and induced pluripotent stem cells.

The term "embryonic stem cells" refers to pluripotent stem cells derived from the epiblast tissue of the inner cell mass of a blastocyst or earlier morula stage embryos. Embryonic stem cells may also be defined by the expression of several transcription factors, such as Oct-4, Nanog and Sox2, and cell surface proteins such as the glycolipids SSEA3 and SSEA4 and the keratin sulphate antigens Tra-1 -60 and Tra-1 -81 .

In a particular embodiment, the term "stem cells" in accordance with the invention does not comprise stem cells from human embryos. More particularly, the stem cells according to the invention are preferably not directly derived from a human embryo or did not necessitate the destruction of a human embryo. In particular, embryonic stem cells which have been derived from publicly available and previously established stem cell lines fall within the meaning of the term "stem cells" as used in the present invention, in particular embryonic stem cells which have been derived from publicly available and previously established stem cell lines which did not necessitate the destruction of a human embryo, as for example described in Chung et at. (2008) Cell Stem Cell 2:1 13- 1 17).

In particular, human embryonic stem cells derived from one of the cell lines described in Table 1 fall within the meaning of the term "stem cells" as used in the present invention.

Table 1 : Human embryonic stem cell lines

The term "fetal stem cells" refers to stem cells derived either from the fetus proper or preferably from the extra-embryonic tissues emerging during gestation including umbilical cord blood, amniotic fluid, Wharton's jelly, the amniotic membrane and the placenta.

As used herein, the term "induced pluripotent stem cell" or "iPS cell" refers to a type of pluripotent stem cell artificially derived (e.g., induced by complete or partial reversal) from a differentiated cell (e.g. a non-pluripotent cell), typically an adult somatic cell such as an adult fibroblast. In a particular embodiment, the stem cells used in the context of the invention are induced pluripotent stem cells.

In a particular embodiment, the pluripotent cells are obtained from an individual suffering from a motor neurons neurodegenerative genetic disease, such as hereditary amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA). In another particular embodiment, the pluripotent cells contain a genetic mutation responsible for a motor neurons neurodegenerative genetic disease, such as hereditary amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA). Advantageously, in this embodiment, the population of motor neurons progenitors and/or the population of motor neurons also contain said mutation and can therefore provide a good cellular model of the disease.

Culture medium

In the context of the invention the term "culture medium" refers to a liquid medium suitable for the in vitro culture of mammalian cells. The culture media used in the methods of the invention may be based on a commercially available medium such as DMEM/F12 from Invitrogen or a mixture of DMEM/F12 and Neurobasal in a 1 :1 ratio, from Invitrogen.

The culture media used in the methods of the invention may also comprise various supplements such as B27 supplement (Invitrogen) and N2 supplement (from Invitrogen).

The B27 supplement contains, amongst other constituents, SOD, catalase and other anti-oxidants (GSH), and unique fatty acids, such as linoleic acid, linolenic acid, lipoic acids.

The culture media used in the methods of the invention can in particular be based on a N2B27 medium.

The term "N2B27" refers to the medium described in Liu et al. (2006) Bioc em. Biop ys. Res. Commun. 346:131 -139, which comprises DMEM/F12 and Neurobasal media in a 1 :1 ratio, N2 supplement (1/100), B27 supplement (1 /50) and β- mercaptoethanol (1/1000).

Preferably, the culture media used in the methods of the invention further comprise the ROCK inhibitor Y-27632, typically at a concentration of 5 μΜ.

Preferably, the culture media used in the methods of the invention do not comprise

FGF2.

In the methods of the invention, if necessary, the culture media can be renewed, partly or totally, at regular intervals. Typically, the culture media can be replaced with fresh culture media every other day, for the periods of time mentioned below. Neuralization

In the context of the invention, the term "neuralized pluripotent cells" refers to cells that after neural induction display a neural plate identity.

Markers of neural plate identity are well-known from the skilled person and include typically SOX2, PAX6 and NESTIN. Techniques to identify neuralized pluripotent cells are well-known from the skilled person and include immunostaining with anti-SOX2, anti- PAX6 and/or anti-nestin antibodies. Typically, after a neural induction treatment, cells are fixed with PFA 4% and rinsed with PBS, then incubated with primary anti-PAX6 and/or anti-SOX2 and /or anti-nestin antibodies, diluted in PBS / 2% FBS / 0.2% Triton overnight at 4°C and with secondary antibodies for 1 h at room temperature. The presence of PAX6 and/or Nestin can then be detected by microscopy and image acquisition.

Neuralized pluripotent cells are preferably obtained from pluripotent cells as defined in the section "Pluripotent cells" above.

Neuralized pluripotent cells can be obtained by any technique well-known from the skilled person. Preferably, said neuralized pluripotent cells are obtained by culturing, during a period of time T₀, pluripotent cells as defined in the section "Pluripotent cells" above in a culture medium C₀ comprising (i) an inhibitor of the Bone Morphogenetic Protein (BMP) signaling pathway and (ii) an inhibitor of the Transforming Growth Factor (TGF)/activin/nodal signaling pathway. In other words, in a particular embodiment, the method for producing motor neuron progenitors according to the invention further comprises a step pre-a) of culturing, during a period of time T₀, pluripotent cells as defined in the section "Pluripotent cells" above in a culture medium C₀ comprising (i) an inhibitor of the Bone Morphogenetic Protein (BMP) signaling pathway and (ii) an inhibitor of the Transforming Growth Factor (TGF)/activin/nodal signaling pathway.

As used herein, the term "inhibitor of the BMP signaling pathway" refers to any compound, natural or synthetic, which results in a decreased activation of the BMP signaling pathway, which is the series of molecular signals generated as a consequence of any member of the BMP (bone morphogenetic protein) family binding to a cell surface receptor. Typically, an inhibitor of the BMP signaling pathway provokes a decrease in the levels of phosphorylation of the proteins Smad 1 , 5 and 8, as described in Gazzero and Minetti (2007) Curr. Opin. Pharmacol. 7:325-333. Techniques to determine whether a given compound is an inhibitor of the BMP signaling pathway are well-known from the skilled person. Typically, a compound is deemed to be an inhibitor of the BMP signaling pathway if, after culturing cells in the presence of said compound, the level of phosphorylated Smad 1 , 5 or 8 is decreased compared to cells cultured in the absence of said compound. Levels of phosphorylated Smad proteins can be measured by Western blot using antibodies specific for the phosphorylated form of said Smad proteins.

The inhibitor of the BMP signaling pathway may be a BMP antagonist or a molecule which inhibits any downstream step of the BMP signaling pathway. The inhibitor of the BMP signaling may be a natural or a synthetic compound. When the inhibitor of the BMP signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein.

The inhibitor of the BMP signaling pathway may be selected from the group consisting of noggin, chordin, follistatin, inhibitory Smad 6 (l-Smad 6), inhibitory Smad 7 (I- Smad 7), dorsomorphin (6-[4-(2-Piperidin-1 -ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1 ,5- a]pyrimidine; described in Yu et al. (2008) Nat Chem Biol. 4:33-41 ), the compound DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl)quinoline; described in Hao et al. (2010) ACS Chem Biol. 5:245-253), the compound DMH2 (4-(2-(4-(3-(quinolin-4- yl)pyrazolo[1 ,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; described in Hao et al. (2010) ACS Chem Biol. 5:245-253), the compound DMH3 (A/,/V-dimethyl-3-(4-(3-(quinolin-4- yl)pyrazolo[1 ,5-a]pyrimidin-6-yl)phenoxy)propan-1 -amine; described in Hao et al. (2010) ACS Chem Biol. 5:245-253), the compound DMH4 (4-(2-(4-(3-phenylpyrazolo[1 ,5- a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; described in Hao et al. (2010) ACS Chem Biol. 5:245-253), the compound LDN193189 (or DM-3189, 4-[6-(4-Piperazin-1 - ylphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl]quinoline; described in Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18:4388-4392), and the compound K02288 (3-[6-amino-5-(3,4,5- trimethoxy-phenyl)-pyridin-3-yl]-phenol; described in Sanvitale et al. (2013) PLoS ONE 8:62721 ),

Preferably, the inhibitor of the BMP signaling pathway is the compound LDN193189.

Typically, the compound LDN193189 is present in the culture medium C₀ in a concentration ranging from 0.05 to 1 μΜ, preferably from 0.1 to 0.5 μΜ, from 0.15 to 0.25 μΜ, even more preferably at about 0.2 μΜ.

As used herein, the term "inhibitor of the TGF/activin/nodal signaling pathway" refers to any compound, natural or synthetic, which results in a decreased activation of the TGF/activin/nodal signaling pathway, which is the series of molecular signals generated as a consequence of any member of the TGF/activin/nodal family binding to a cell surface receptor. Typically, an inhibitor of the TGF/activin/nodal signaling pathway provokes a decrease in the levels of of phosphorylation of the protein Smad 2, as described in Shi and Massague (2003) Cell 113:685-700. Techniques to determine whether a given compound is an inhibitor of the TGF/activin/nodal signaling pathway are well-known from the skilled person. Typically, a compound is deemed to be an inhibitor of the TGF/activin/nodal signalling pathway if, after culturing cells in the presence of said compound, the level of phosphorylated Smad 2 is decreased compared to cells cultured in the absence of said compound. Levels of phosphorylated Smad proteins can be measured by Western blot using antibodies specific for the phosphorylated form of said Smad proteins.

The inhibitor of the TGF/activin/nodal signaling pathway may be a TGF/activin/nodal antagonist or a molecule which inhibits any downstream step of the TGF/activin/nodal signaling pathway. The inhibitor of the TGF/activin/nodal signaling may be a natural or a synthetic compound. When the inhibitor of the TGF/activin/nodal signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein.

The inhibitor of the TGF/activin/nodal signaling pathway may be selected from the group consisting of the compound SB431542 (4-(5-Benzol[1 ,3]dioxol-5-yl-4-pyrlidn-2-yl- 1 H-imidazol-2-yl)-benzamide hydrate), the Lefty-A protein and Cerberus.

Preferably, the inhibitor of the TGF/activin/nodal signaling pathway is the compound SB431542.

Typically, the compound SB431542 is present in the culture medium C₀ in a concentration ranging from 10 to 75 μΜ, preferably ranging from 20 to 50 μΜ, even more preferably at about 40 μΜ.

In a particularly preferred embodiment, the inhibitor of the BMP signaling pathway is the compound LDN193189 and the inhibitor of the TGF/activin/nodal signaling pathway is the compound SB431542. Typically, the compound LDN193189 is present in the culture medium C₀ at a concentration of about 0.2 μΜ and the compound SB431542 is present in the culture medium C₀ at a concentration of about 40 μΜ.

In the context of the invention, the period of time T₀ is a period of time sufficient to induce the expression of neural plate markers, in particular of PAX6 and/or nestin. Techniques to detect the expression of markers of motor neuron progenitors are well- known from the skilled person and include immunostaining, western blotting, and flow cytometry, using antibodies specific for said markers, such as anti-PAX6 and/or anti- nestin antibodies.

In a preferred embodiment, the period of time T₀ is of 4 days.

In a particular embodiment of the invention, the pluripotent cells which are submitted to neuralization are in the form of embryoid bodies. Embryoid bodies can be obtained using techniques well-known from the skilled person. Typically, pluripotent cells can be submitted to centrifugation in V shape 384, for example at 1200 g for 5 min, to force cell aggregation and form embryoid bodies.

Activator of the Wnt signaling pathway

In the context of the invention, the term "activator of the Wnt signaling pathway" refers to any compound, natural or synthetic, which results in an increased activation of the Wnt signaling pathway, which is the series of molecular signals generated as a consequence of any member of the Wnt family binding to a cell surface receptor. Typically, an activator of the Wnt signaling pathway provokes an accumulation of β- catenin in the cytoplasm and its eventual translocation into the nucleus, as described in Bienz (2005) Curr. Biol. 15:R64-67. Techniques to determine whether a given compound is an activator of the Wnt signaling pathway are well-known from the skilled person. Typically, a compound is deemed to be an activator of the Wnt signaling pathway if, after culturing cells in the presence of said compound, the level of nuclear β-catenin is increased compared to cells cultured in the absence of said compound. Levels of nuclear β-catenin can be measured by Western blot using antibodies specific for β-catenin.

The activator of the Wnt signaling pathway may be a Wnt agonist or a molecule which activates any downstream step of the Wnt signaling pathway. The activator of the Wnt signaling may be a natural or a synthetic compound. When the activator of the Wnt signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein.

Examples of activators of the Wnt signaling pathway include but are not limited to the group consisting of the compound CHIR-99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5- methyl-1 H-imidazol-2-yl)-2 pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, as described in Bennett et al. (2002) J. Biol. Chem. 277:30998-3104), the WNT3A protein, the compound IQ-1 (2-(4-Acetylphenylazo)-2-(3,3-dimethyl-3,4-dihydro-2H-isoquinolin-1 - ylidene)-acetamide, as described in Miyabayashi et al. (2007) Proc. Natl. Acad. Sci. USA 104:5668-5673), the compound SB-216763 (3-(2,4-Dichlorophenyl)-4-(1 -methyl-1 H-indol- 3-yl)-1 H-pyrrole-2,5-dione as described in Coghlan et al. (2000) Chem. Biol. 7:793-803) and the compound BIO (6-bromoindirubin-3'-oxime, as described in Sato et al. (2004) Nat. Med. 10:55-63).

Preferably, the activator of the Wnt signaling pathway is selected from the group consisting of the compound CHIR-99021 and the WNT3A protein. Most preferably, the activator of the Wnt signaling pathway is the compound CHIR-99021 . Typically, the compound CHIR-99021 is present in the culture medium Ci in a concentration ranging from 0.5 to 5 μΜ, preferably ranging from 1 to 4 μΜ, even more preferably at about 3 μΜ or at about 1 μΜ.

In the context of the invention, the period of time T-_\ is a period of time suitable to induce the expression of markers of motor neuron progenitors, such as OLIG2, NKX6.1 , NKX2.2, HOXA2, HOXA4 and/or HOXA5. Techniques to detect the expression of markers of motor neuron progenitors are well-known from the skilled person and include immunostaining, by using antibodies specific for said markers, such as anti-OLIG2 and/or anti-NKX6.1 , or RT-PCT to detect NKX2.2, NKX6. 1, HOXA2, HOXA4 or HOXA5 mRNAs.

The inventors demonstrated that Wnt activation not only shortened the time required for motor neuron progenitors specification (in only 8 days) but also increased their final rate in the population. Indeed, the population obtained with the method of the invention included 82% of motor neuron progenitors 10 days after the beginning of the cultures, whereas only 55% of motor neuron progenitors could be obtained in 16 days in a method wherein activators of the Wnt signaling pathway was not used.

The inventors further demonstrated that the time required for motor neuron progenitors specification could be shortened when the Wnt signaling pathway was immediately activated at the pluripotent cell stage. Indeed, when the Wnt signaling pathway was activated at day 0, in other words when the activator of the Wnt signaling pathway was added in the culture medium on the same day as the compounds added for neuralization, motor neuron progenitors appeared as early as 8 days after the beginning of the culture and peaked 10 days after the beginning of the culture, four days ealier than when the activator of the Wnt signaling pathway was added 2 days after the beginning of the culture.

The inventors also demonstrated that better results could be obtained when the neuralized pluripotent cells were cultured in the culture medium d comprising an activator of the Wnt signaling pathway during 2 to 4 days. Accordingly, in a particular embodiment the period of time is of 2 to 4 days. In a particularly preferred embodiment, the period of time Ti is of 4 days.

In a particular embodiment, the neuralized pluripotent cells are cultured in the culture medium d comprising an activator of the Wnt signaling pathway as defined above during a period of time starting the same day as the neuralization induction. In other words, in a particular embodiment, the period of time starts at day 0 of the method of the invention.

In another particular embodiment, the neuralized pluripotent cells are cultured in the culture medium Ci comprising an activator of the Wnt signaling pathway as defined above during a period of time T-_\ starting 2 days after the neuralization induction. In other words, in a particular embodiment, the period of time T-_\ starts at day 2 of the method of the invention.

In a particular embodiment, the period of time T-_\ lasts from day 2 to day 4 of the method of the invention.

In a particularly preferred embodiment of the invention, the period of time T-_\ lasts from day 0 to day 4 of the method of the invention.

In some embodiments, the period of time T₀ of neuralization and the period of time Ti of culture in the presence of an activator of the Wnt signaling pathway can overlap partially or completely. Accordingly, when said periods of time T₀ and ΤΊ overlap, the culture medium d also comprises the neuralization-inducing compounds defined in the section "Neuralization" above, or the culture medium C₀ also comprises an activator of the Wnt signaling pathway as defined above.

Retinoic acid and agonist of the Hedgehog signaling pathway As used herein, the term "retinoic acid" refers to an active form (synthetic or natural) of vitamin A, capable of inducing neural cell differentiation. Examples of retinoic acid forms which can be used in accordance with the invention include, but are not limited to, retinoic acid, retinol, retinal, 1 1 -cis-retinal, all-trans retinoic acid (ATRA), 13-cis retinoic acid and 9-cis-retinoic acid.

Typically, retinoic acid is present in the culture medium C₂ in a concentration ranging from 5 to 1000 nM, preferably ranging from 10 to 500 nM, even more preferably at about 10 nM or at about 100 nM.

In a particular embodiment, preferably when spinal motor neurons progenitors are produced, retinoic acid is present in the culture medium C₂ at a concentration superior to 10 nM, more preferably at a concentration of about 100 nM.

In another particular embodiment, preferably when cranial motor neurons progenitors are produced, retinoic acid is present in the culture medium C₂ at a concentration of 10 nM or less.

In the context of the invention, the term "agonist of the Hedgehog signaling pathway" refers to any compound, natural or synthetic, which results in an increased activation of the Hedgehog signaling pathway, which is the series of molecular signals generated as a consequence of any member of the Hedgehog family binding to a cell surface receptor. Typically, an agonist of the Hedgehog signaling pathway provokes activation of Hedgehog signaling pathway. The agonist of the Hedgehog signaling pathway may be a Hedgehog agonist or a molecule which activates any downstream step of the Hedgehog signaling pathway. The agonist of the Hedgehog signaling may be a natural or a synthetic compound. When the agonist of the Hedgehog signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein.

Examples of agonists of the Hedgehog signaling pathway include but are not limited to the group consisting of purmophamine (9-cyclohexyl-N-[4-(morpholinyl)phenyl]- 2- (1 -naphthalenyloxy)-9H-purin-6-amine; as described in Sinha and Chen (2006) Nat. Chem. Biol. 2:29-30), SHH (sonic hedgehog), SHH C241 1 (as described in Taylor et al. (2001 ) Biochemistry 40:4359-4371 ), the smoothened agonist SAG (N- Methyl- N'- (3- pyridinylbenzyl)- N'- (3- chlorobenzo[b] thiophene-2-carbonyl)-1 ,4-diaminocyclohexane; as described in Chen et al. (2002) Proc. Natl. Acad. Sci. USA 99:14071 -14076), and the compound Hh-Ag1 .5 (3- chloro- 4,7- difluoro-N- (4- methoxy-3- (pyridin-4-yl) benzyl) -N -( 4- (methylamino) cyclohexyl) benzo[b] thiophene- 2- carboxamide; as described in Frank- Kamenetsky et al. (2002) J. Biol. 1 :10.2-10.19).

Preferably, the agonist of the Hedgehog signaling pathway is selected from the group consisting of SHH and SAG. Most preferably, the agonist of the Hedgehog signaling pathway is SAG.

Typically, SAG is present in the culture medium C₂ in a concentration ranging from 100 to 1000 nM, preferably ranging from 250 to 750 nM, even more preferably at about 500 nM.

In a particular embodiment, the method of the invention may comprise, between steps a) and b), a step pre-b) of culturing the cells obtained in step a) in a culture medium Cpre-2 comprising an agonist of the Hedgehog signaling pathway as defined above during a period of time Τ_ρΓβ-₂.

Preferably, when the method of the invention comprises the step pre-b), SAG is present in the culture medium C_pre-2 in a concentration ranging from 100 to 1000 nM, preferably ranging from 200 to 1000 nM, more preferably ranging from 250 to 750 nM, even more preferably at about 500 nM.

In the context of the invention, the period of time T₂ is a period of time suitable to induce the expression of markers of motor neuron progenitors, such as OLIG2, NKX6.1 , NKX2.2, HOXA2, HOXA4 and/or HOXA5. Techniques to detect the expression of markers of motor neuron progenitors are well-known from the skilled person and include immunostaining, western blotting, and RT-PCR, using antibodies specific for said markers, such as anti-0LIG2 and/or anti-NKX6.1 antibodies, or primers to detect NKX2.2, NKX6. 1, HOXA2, HOXA4 and/or HOXA5 mRNAs.

Typically, the period of time T₂ may last for at least 5 days, more preferably for about 10 or 12 days.

In a particular embodiment, preferably when spinal motor neurons progenitors are produced, the period of time T₂ may last for at least 5 days, preferably for 6 to 12 days, more preferably for about 10 or 12 days, and the method of the invention preferably does not comprise the step pre-b).

In another particular embodiment, preferably when cranial motor neurons progenitors are produced, the period of time T₂ may last for 5 to 10 days, more preferably for about 8 or 10 days, and the method of the invention preferably comprises the step pre- b).

In the context of the invention, when the method of the invention comprises the step pre-b), the period of time T_pre-₂ is a period of time suitable to start the induction of the expression of markers of motor neuron progenitors, in particular of cranial motor neuron progenitors, such as NKX6.1 , NKX2.2 and/or HOXA2. Techniques to detect the expression of markers of motor neuron progenitors, in particular of cranial motor neuron progenitors are well-known from the skilled person and include immunostaining, western blotting, and RT-PCR, using antibodies specific for said markers, such as anti-NKX6.1 antibodies, or primers to detect NKX2.2 and/or HOXA2 mRNAs.

Typically, when the method of the invention comprises the step pre-b), the period of time T_pre-₂ may last for about 2 or 1 days, in particular for about 1 day.

In a particular embodiment, preferably when cranial motor neurons progenitors are produced, the method of the invention may comprise the step pre-b) and the period of time T_pre-₂ may last for 2 or 1 days, in particular for 1 day. More preferably, in that embodiment, the period of time T_pre-₂ may last for about 2 or 1 days, in particular for 1 day and the period of time T₂ may last for at least 5 days, preferably for 5 to 10 days, more preferably for about 8 or 10 days.

The inventors demonstrated that an optimal motor neuron progenitors specification could be obtained when the cells were contacted with retinoic acid and the agonist of the Hedgehog signaling pathway 2 to 4 days after the beginning of the culture with the culture medium comprising the activator of the Wnt signaling pathway.

In other words, in a particular embodiment of the invention, the period of time T₂ starts 2 to 4 days after the beginning of the period of time or T₀.

In another particular embodiment of the invention, when the method of the invention comprises the step pre-b), the period of time T_pre-₂ starts 2 days or 3 days, preferably 3 days after the beginning of the period of time T-_\ or T₀. Preferably, in that embodiment, the period of time T₂ starts 4 days after the beginning of the period of time T-_\ or T₀.

Accordingly, in some embodiments, the period of time ΤΊ of culture in the presence of an activator of the Wnt signaling pathway and the period of time T₂ of culture in the presence of retinoic acid and an agonist of the Hedgehog signaling pathway can overlap partially. Accordingly, in these embodiments, during the periods when said periods of time Ti and T₂ overlap, the culture medium d also comprises retinoic acid and an agonist of the Hedgehog signaling pathway. Similarly, in some embodiments, when the method of the invention comprises the step pre-b), the period of time ΤΊ of culture in the presence of an activator of the Wnt signaling pathway and the period of time T_pre-₂ of culture in the presence of an agonist of the Hedgehog signaling pathway, can overlap partially. Accordingly, in these embodiments, during the periods when said periods of time ΤΊ and T_pre-₂ overlap, the culture medium d also comprises an agonist of the Hedgehog signaling pathway.

Similarly, in some embodiments, the period of time T₀ of neuralization, the period of time Ti of culture in the presence of an activator of the Wnt signaling pathway and the period of time T₂ of culture in the presence of retinoic acid and an agonist of the Hedgehog signaling pathway can overlap. Accordingly, in these embodiments, during the periods when said periods of time T₀, ΤΊ and T₂ overlap, the culture medium Ci also comprises the neuralization-inducing compounds defined in the section "Neuralization" above and retinoic acid and an agonist of the Hedgehog signaling pathway. Similarly, in some embodiments, when the method of the invention comprises the step pre-b), the period of time T₀ of neuralization, the period of time T-_\ of culture in the presence of an activator of the Wnt signaling pathway and the period of time T_pre-₂ of culture in the presence of an agonist of the Hedgehog signaling pathway can overlap. Accordingly, in these embodiments, during the periods when said periods of time T₀, and T_pre-₂ overlap, the culture medium d also comprises the neuralization-inducing compounds defined in the section "Neuralization" above and an agonist of the Hedgehog signaling pathway.

In a particularly preferred embodiment of the invention, the method for producing a population of motor neuron progenitors of the invention comprises the following steps: pre-a) contacting, from day 0 of the method, pluripotent cells with an inhibitor of the BMP signaling pathway and an inhibitor of the TGF/activin/nodal signaling pathway, said pluripotent cells being cultured with said inhibitor of the BMP signaling pathway and said inhibitor of the TGF/activin/nodal signaling pathway during a period of time T₀ of 4 days, a) contacting, from day 0 of the method, the cells obtained in step pre-a) with an activator of the Wnt signaling pathway, said cells being cultured with said activator of the Wnt signaling pathway during a period of time ΤΊ of 4 days, and

b) contacting from day 2 of the method, the cells obtained in step a) with retinoic acid and an agonist of the Hedgehog signaling pathway, said cells being cultured with said retinoic acid and said agonist of the Hedgehog signaling pathway during a period of time T₂ of 12 days.

Typically, in the above preferred embodiment:

the cells are cultured from day 0 of the method and during a period of time of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, an inhibitor of the TGF/activin/nodal signaling pathway, and an activator of the Wnt signaling pathway;

- the cells are cultured from day 2 of the method and during a period of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, an inhibitor of the TGF/activin/nodal signaling pathway, an activator of the Wnt signaling pathway, retinoic acid and an agonist of the Hedgehog signaling pathway, and

- the cells are cultured from day 4 of the method and during a period of 10 days with a culture medium comprising retinoic acid and an agonist of the Hedgehog signaling pathway.

In another particularly preferred embodiment of the invention, the method for producing a population of motor neuron progenitors of the invention comprises the following steps:

pre-a) contacting, from day 0 of the method, pluripotent cells with an inhibitor of the BMP signaling pathway and an inhibitor of the TGF/activin/nodal signaling pathway, said pluripotent cells being cultured with said inhibitor of the BMP signaling pathway and said inhibitor of the TGF/activin/nodal signaling pathway during a period of time T₀ of 4 days, a) contacting, from day 0 of the method, the cells obtained in step pre-a) with an activator of the Wnt signaling pathway, said cells being cultured with said activator of the Wnt signaling pathway during a period of time of 4 days,

pre-b) contacting from day 2 or 3, preferably from day 3 of the method, the cells obtained in step a) with an agonist of the Hedgehog signaling pathway, said cells being cultured with said agonist of the Hedgehog signaling pathway during a period of time Τ_ρΓβ-₂ of 2 or 1 day, preferably of 1 day and

b) contacting from day 4 of the method, the cells obtained in step pre-b) with an agonist of the Hedgehog signaling pathway and retinoic acid, said cells being cultured with said agonist of the Hedgehog signaling pathway and said retinoic acid during a period of time T₂ of 10 days.

Typically, in the above preferred embodiment:

- the cells are cultured from day 2 of the method and during a period of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, an inhibitor of the TGF/activin/nodal signaling pathway, an activator of the Wnt signaling pathway and an agonist of the Hedgehog signaling pathway, and

Production of spinal or cranial motor neurons progenitors

The inventors unexpectedly demonstrated that according to the concentration of the activator of the Wnt signaling pathway and of retinoic acid used, either spinal or cranial motor neuron progenitors could be obtained.

Accordingly, in a particular embodiment of the method for preparing a population of motor neuron progenitors of the invention, the motor neuron progenitors obtained are spinal motor neuron progenitors.

Preferably, in that embodiment, the activator of the Wnt signaling pathway, as defined in the section "Activator of the Wnt signaling pathway" is present at a high concentration in the culture medium Ci . In a particularly preferred mode of that embodiment, the activator of the Wnt signaling pathway is the compound CHIR-99021 and the compound CHIR-99021 is present in the culture medium d at a concentration of at least 2 μΜ, more preferably at a concentration of at least 3 μΜ.

Still preferably, in that embodiment, retinoic acid is present in the culture medium C₂ at a concentration of at least 10 nM, more preferably at a concentration of at least 100 nM, still preferably at a concentration of 100 nM.

Advantageously, the above method enables obtaining a population of spinal motor neuron progenitors comprising more than 70% of spinal motor neuron progenitors. In particular, when activator of the Wnt signaling pathway is the compound CHIR-99021 and the compound CHIR-99021 is present in the culture medium Ci at a concentration of 3 μΜ, when the agonist of the Hedgehog signaling pathway is SAG and retinoic acid and SAG are present in the culture medium C₂ respectively at a concentration of 100 nM and 500 nM, the population of spinal motor neuron progenitors obtained comprises more than 80% of spinal motor neuron progenitors, in particular after 8 days of culture.

In a particularly preferred embodiment of the invention, the method for producing a population of spinal motor neuron progenitors of the invention thus comprises the following steps:

pre-a) contacting, from day 0 of the method, pluripotent cells with an inhibitor of the BMP signaling pathway, in particular LDN-193189, preferably at a concentration of 0.2 μΜ, and an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, said pluripotent cells being cultured with said inhibitor of the BMP signaling pathway and said inhibitor of the TGF/activin/nodal signaling pathway during a period of time T₀ of 4 days,

a) contacting, from day 0 of the method, the cells obtained in step pre-a) with the compound CHIR-99021 at a concentration of 3 μΜ, said cells being cultured with compound CHIR-99021 at a concentration of 3 μΜ during a period of time ΤΊ of 4 days, and

b) contacting from day 2 of the method, the cells obtained in step a) with retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM, said cells being cultured with retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM during a period of time T₂ of 12 days.

Typically, in the above preferred embodiment:

the cells are cultured from day 0 of the method and during a period of time of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 3 μΜ;

- the cells are cultured from day 2 of the method and during a period of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, CHIR-99021 at a concentration of 3 μΜ, retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM, and

- the cells are cultured from day 4 of the method and during a period of 10 days with a culture medium retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM. In another particular embodiment of the method for preparing a population of motor neuron progenitors of the invention, the motor neuron progenitors obtained are cranial motor neuron progenitors.

Preferably, in that embodiment, the activator of the Wnt signaling pathway, as defined in the section "Activator of the Wnt signaling pathway" is present at a low concentration in the culture medium Ci . In a particularly preferred mode of that embodiment, the activator of the Wnt signaling pathway is the compound CHIR-99021 and the compound CHIR-99021 is present in the culture medium d at a concentration of 1 μΜ or less, more preferably at a concentration of about 1 μΜ.

Preferably, in that embodiment, the method of the invention may comprise a step pre-b) of culturing the cells obtained in step a) in a culture medium C_pre-2 comprising an agonist of the Hedgehog signaling pathway during a period of time T_pre-_2.

Still preferably, in that embodiment, retinoic acid is present in the culture medium C₂ at a concentration of 10 nM or less, more preferably at a concentration of about 10 nM. In that embodiment, the culture medium C_pre-2 does not comprise retinoic acid.

In a particularly preferred embodiment of the invention, the method for producing a population of cranial motor neuron progenitors of the invention comprises the following steps:

a) contacting, from day 0 of the method, the cells obtained in step pre-a) with the compound CHIR-99021 at a concentration of 1 μΜ, said cells being cultured with compound CHIR-99021 at a concentration of 1 μΜ during a period of time ΤΊ of 4 days, and

pre-b) contacting from day 2 or 3, preferably from day 3 of the method, the cells obtained in step a) with SAG at a concentration of 500 nM, said cells being cultured with SAG at a concentration of 500 nM during a period of time T_pre-₂ of 2 or 1 days, preferably of 1 day and

b) contacting from day 4 of the method, the cells obtained in step pre-b) with retinoic acid at a concentration of 10 nM, said cells being cultured with retinoic acid at a concentration of 10 nM and SAG at a concentration of 500 nM during a period of time T₂ of 10 days.

Typically, in the above preferred embodiment:

the cells are cultured from day 0 of the method and during a period of time of 2 or 3 days, preferably of 3 days, with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 1 μΜ;

- the cells are cultured from day 2 or 3, preferably from day 3 of the method and during a period of 2 or 1 days, preferably of 1 day, with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN- 193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, CHIR-99021 at a concentration of 1 μΜ, and SAG at a concentration of 500 nM, and

- the cells are cultured from day 4 of the method and during a period of 10 days with a culture medium comprising retinoic acid at a concentration of 10 nM and SAG at a concentration of 500 nM.

In particular, in the above preferred embodiment:

- the cells are cultured from day 0 of the method and during a period of time of 3 days, with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 1 μΜ;

- the cells are cultured from day 3 of the method and during a period of 1 day, with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, CHIR-99021 at a concentration of 1 μΜ, and SAG at a concentration of 500 nM, and

- the cells are cultured from day 4 of the method and during a period of 10 days with a culture medium comprising retinoic acid at a concentration of 10 nM and SAG at a concentration of 500 nM. Differentiation

The inventors further demonstrated that the above methods for preparing a population of motor neuron progenitors enabled obtaining a population of motor neuron progenitors that could be very efficiently and very rapidly further differentiated into motor neurons.

Accordingly, the present invention also concerns a method, in particular an ex vivo method for obtaining a population of motor neurons comprising the steps of:

A) producing a population of motor neuron progenitors by the method for preparing a population of motor neuron progenitors according to the invention, and

B) differentiating said population of motor neuron progenitors into motor neurons.

In a particular embodiment, the method for obtaining a population of motor neurons is a method for obtaining a population of spinal motor neurons and the step A) of producing a population of motor neuron progenitors is a step of producing a population of spinal motor neuron progenitors by the method for preparing a population of spinal motor neuron progenitors according to the invention.

In another particular embodiment, the method for obtaining a population of motor neurons is a method for obtaining a population of cranial motor neurons and the step A) of producing a population of motor neuron progenitors is a step of producing a population of cranial motor neuron progenitors by the method for preparing a population of cranial motor neuron progenitors according to the invention.

Step B) of differentiating said population of motor neuron progenitors into motor neurons may be carried out by any method well-known from the skilled person.

However, the inventors demonstrated that a very good efficiency and rapidity could be obtained when the population of spinal motor neuron progenitors or cranial motor neuron progenitors respectively was differentiated into spinal motor neurons or cranial motor neurons respectively by culture in a differentiation culture medium comprising inhibitors of the Notch signaling pathway, in particular gamma secretase inhibitors.

Accordingly, in a preferred embodiment, the population of spinal motor neuron progenitors or cranial motor neuron progenitors respectively is differentiated into spinal motor neurons or cranial motor neurons respectively by culture, during a period of time T₃ in a culture medium C₃ comprising an inhibitor of the Notch signaling pathway, preferably a gamma-secretase inhibitor.

As used herein, the term "inhibitor of the Notch signaling pathway" refers to any compound, natural or synthetic, which results, directly or indirectly, in a decreased activation of the Notch signaling pathway, which is the series of molecular signals generated as a consequence of the binding of a ligand to any member of the Notch family. Preferably, the inhibitor of the Notch signaling pathway is an inhibitor of gamma secretase. As well-known from the skilled person, gamma secretase inhibitors undirectly inhibit Notch pathway. Techniques to determine whether a given compound is an inhibitor of gamma secretase are well-known from the skilled person and are for example described in Yang et al. (2008) Molecular Brain 1 :15 or Wang et al. (2009) Molecules 14:3589-2599.

The inhibitor of the Notch signaling, in particular the gamma-secretase inhibitor, may be a natural or a synthetic compound. When the inhibitor of the Notch signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein.

The inhibitor of the Notch signaling pathway may be selected from the group consisting of gamma-secretase inhibitors, in particular DAPT (N-[N-(3,5- Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; as described in Borghese et al. (2010) Stem Cells 28:955-964), the compound W (3,5-Bis(4-nitrophenoxy)benzoic acid; as described in Okochi et al. (2006) J. Biol. Chem. 281 :7890-7898), the compound L- 685,458 ((5S)-(ieri-Butoxycarbonylamino)-6-phenyl-(4/^:?)-hydroxy-(2/^:?)-benzylhexanoyl)-L- leucy-L-phenylalaninamide; as described in Shearman et al. (2000) Biochemistry 39 8698).

Preferably, the inhibitor of the Notch signaling pathway is the compound DAPT.

Typically, DAPT is present in the culture medium C₃ in a concentration ranging from 1 to 25 μΜ, more preferably at about 10 μΜ.

The culture medium C₃ may also comprise other compounds known to contribute to the induction of the differentiation of motor neuron progenitors into motor neurons. Such compounds are well-known from the skilled person. Preferably, the culture medium C₃ further comprises BDNF (brain-derived neurotrophic factor) and GDNF (glial cell derived neurotrophic factor). Preferably, BDNF and GDNF are present in the culture medium C₃ each at a concentration of 5 to 100 ng/ml, preferably 10 to 50 ng/ml, more preferably each at about 10 ng/ml.

In the context of the invention, the period of time T₃ is a period of time sufficient to induce the expression of makers of motor neurons, in particular of HB9, ISL1 /2, HOXA5, HOXA4, and optionally Lhx3 or FOXP1 in the case of spinal motor neurons, or PHOX2B, ISL1 /2 and TBX20 in the case of cranial motor neurons. Techniques to detect the expression of markers of motor neuron are well-known from the skilled person and include immunostaining, flow cytometry, RT-PCR or in situ hybridization. Typically, the period of time T₃ may last for at least 5 days, preferably for 5 to 7 days, more preferably for about 7 days.

The inventors demonstrated that efficient and rapid conversion of motor neuron progenitors into motor neurons could be obtained when the progenitors were contacted with the inhibitor of the Notch signaling pathway, in particular with the gamma-secretase inhibitor, about 9 days after the beginning of the culture with the culture medium comprising the activator of the Wnt signaling pathway.

In other words, in a particular embodiment of the invention, the period of time T₃ starts 9 days after the beginning of the period of time T

Accordingly, in some embodiments, the period of time T₂ of culture in the presence of retinoic acid and an agonist of the Hedgehog signaling pathway and the period of time T₃ of culture in the presence of an inhibitor of the Notch signaling pathway can overlap partially. Accordingly, during the periods when said periods of time T₂ and T₃ overlap, the culture medium C₃ also comprises retinoic acid and an agonist of the Hedgehog signaling pathway.

In a particularly preferred embodiment of the invention, the method for producing a population of motor neurons of the invention comprises the following steps:

pre-Aa) contacting, from day 0 of the method, pluripotent cells with an inhibitor of the BMP signaling pathway and an inhibitor of the TGF/activin/nodal signaling pathway, said pluripotent cells being cultured with said inhibitor of the BMP signaling pathway and said inhibitor of the TGF/activin/nodal signaling pathway during a period of time T₀ of 4 days,

Aa) contacting, from day 0 of the method, the cells obtained in step pre-Aa) with an activator of the Wnt signaling pathway, said cells being cultured with said activator of the Wnt signaling pathway during a period of time T-_\ of 4 days,

Ab) contacting from day 2 of the method, the cells obtained in step Aa) with retinoic acid and an agonist of the Hedgehog signaling pathway, said cells being cultured with said retinoic acid and said agonist of the Hedgehog signaling pathway during a period of time T₂ of 12 days, and

B) contacting from day 9 of the method, the cells obtained in step Ab) with an inhibitor of the Notch signaling pathway, and BDNF and GDNF, said cells being cultured with said inhibitor of the Notch signaling pathway during a period T₃ of 5 to 7 days.

Typically, in the above preferred embodiment:

- the cells are cultured from day 2 of the method and during a period of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, an inhibitor of the TGF/activin/nodal signaling pathway, an activator of the Wnt signaling pathway, retinoic acid and an agonist of the Hedgehog signaling pathway,

- the cells are cultured from day 4 of the method and during a period of 5 days with a culture medium comprising retinoic acid and an agonist of the Hedgehog signaling pathway;

- the cells are cultured from day 9 of the method and during a period of 5 to 7 days with a culture medium comprising retinoic acid, an agonist of the Hedgehog signaling pathway, an inhibitor of the Notch signaling pathway, and GNDF and BDNF, and

- the cells are optionally cultured from day 14 or day 16 of the method and during a period of 2 to 16 days with a culture medium comprising GDNF and BDNF and preferably without an inhibitor of the Notch signaling pathway.

Advantageously, the above method enables obtaining a population of spinal motor neurons comprising more than 70% of spinal motor neurons. In particular, when the activator of the Wnt signaling pathway is the compound CHIR-99021 and the compound CHIR-99021 is present in the culture medium Ci at a concentration of 3 μΜ, when the agonist of the Hedgehog signaling pathway is SAG and retinoic acid and SAG are present in the culture medium C₂ respectively at a concentration of 100 nM and 500 nM, and when the inhibitor of the Notch signaling pathway is a gamma-secretase inhibitor, in particular DAPT, and DAPT is present in the culture medium C₃ at a concentration of 10 μΜ, the population of spinal motor neurons obtained comprises more than 70% of spinal motor neurons, in particular after 14 days of culture.

In a particularly preferred embodiment of the invention, the method for producing a population of spinal motor neurons of the invention thus comprises the following steps: pre-Aa) contacting, from day 0 of the method, pluripotent cells with an inhibitor of the BMP signaling pathway, in particular LDN-193189, preferably at a concentration of 0.2 μΜ, and an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, said pluripotent cells being cultured with said inhibitor of the BMP signaling pathway and said inhibitor of the TGF/activin/nodal signaling pathway during a period of time T₀ of 4 days, Aa) contacting, from day 0 of the method, the cells obtained in step pre-Aa) with the compound CHIR-99021 at a concentration of 3 μΜ, said cells being cultured with compound CHIR-99021 at a concentration of 3 μΜ during a period of time ΤΊ of 4 days,

Ab) contacting from day 2 of the method, the cells obtained in step Aa) with retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM, said cells being cultured with retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM during a period of time T₂ of 12 days, and

B) contacting from day 9 of the method, the cells obtained in step Ab) with DAPT at a concentration of 10 μΜ, and BDNF and GDNF, each at a concentration of 10 ng/ml, said cells being cultured with DAPT at a concentration of 10 μΜ, and with BDNF and GDNF each at a concentration of 10 ng/ml during a period T₃ of 5 to 6 days.

Typically, in the above preferred embodiment:

- the cells are cultured from day 2 of the method and during a period of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, CHIR-99021 at a concentration of 3 μΜ, retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM,

- the cells are cultured from day 4 of the method and during a period of 5 days with a culture medium comprising retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM,

- the cells are cultured from day 9 of the method and during a period of 5 days with a culture medium comprising retinoic acid at a concentration of 100 nM, SAG at a concentration of 500 nM, DAPT at a concentration of 10 μΜ, and BNDF and GDNF each at a concentration of 10 ng/ml,

- the cells are cultured from day 14 of the method and during a period of 2 days with a culture medium comprising DAPT at a concentration of 10 μΜ, and

- the cells are optionally cultured from day 16 of the method with a culture medium comprising GDNF and BDNF at 10 ng/ml, and preferably without an inhibitor of the Notch signaling pathway. Advantageously, the method of the invention enables obtaining a population of cranial motor neurons comprising more than 50% of cranial motor neurons. In particular, when the activator of the Wnt signaling pathway is the compound CH IR-99021 and the compound CH IR-99021 is present in the culture medium Ci at a concentration of 1 μΜ, when the agonist of the Hedgehog signaling pathway is SAG and SAG is present in the culture medium C_pre-2 at a concentration of 500 nM, and retinoic acid and SAG are present in the culture medium C₂ respectively at a concentration of 1 0 nM and 500 nM, and when the inhibitor of the Notch signaling pathway is a gamma-secretase inhibitor, in particular DAPT, and DAPT is present in the culture medium C₃ at a concentration of 10 μΜ, the population of cranial motor neurons obtained comprises more than 50 % of cranial motor neurons, in particular after 14 days of culture.

In a particularly preferred embodiment of the invention, the method for producing a population of cranial motor neurons of the invention thus comprises the following steps: pre-Aa) contacting, from day 0 of the method, pluripotent cells with an inhibitor of the BMP signaling pathway, in particular LDN-193189, preferably at a concentration of 0.2 μΜ, and an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, said pluripotent cells being cultured with said inhibitor of the BMP signaling pathway and said inhibitor of the TGF/activin/nodal signaling pathway during a period of time T₀ of 4 days,

Aa) contacting, from day 0 of the method, the cells obtained in step pre-Aa) with the compound CH IR-99021 at a concentration of 1 μΜ, said cells being cultured with compound CHIR-99021 at a concentration of 1 μΜ during a period of time ΤΊ of 4 days, pre-Ab) contacting from day 2 of the method, the cells obtained in step Aa) with SAG at a concentration of 500 nM, said cells being cultured with SAG at a concentration of 500 nM during a period of time Τ_ρΓβ-₂ of 2 or 1 days, preferably of 1 day,

Ab) contacting from day 4 of the method, the cells obtained in step pre-Ab) with retinoic acid at a concentration of 10 nM, said cells being cultured with retinoic acid at a concentration of 1 0 nM and SAG at a concentration of 500 nM during a period of time T₂ of 10 days, and

B) contacting from day 9 of the method, the cells obtained in step Ab) with DAPT at a concentration of 1 0 μΜ, and BDNF and GDNF each at a concentration of 10 ng/ml, said cells being cultured with DAPT at a concentration of 1 0 μΜ and BDNF and GDNF each at a concentration of 1 0 ng/ml, during a period T₃ of 5 to 7 days.

Typically, in the above preferred embodiment: the cells are cultured from day 0 of the method and during a period of time of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 1 μΜ;

- the cells may be cultured from day 2 or 3, preferably from day 3 of the method and during a period of 2 or 1 days, preferably of 1 day, with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN- 193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, CHIR-99021 at a concentration of 1 μΜ and SAG at a concentration of 500 nM,

- the cells are cultured from day 4 of the method and during a period of 5 days with a culture medium comprising retinoic acid at a concentration of 10 nM and SAG at a concentration of 500 nM,

- the cells are cultured from day 9 of the method and during a period of 5 days with a culture medium comprising retinoic acid at a concentration of 10 nM, SAG at a concentration of 500 nM, DAPT at a concentration of 10 μΜ, and BDNF and GDNF each at a concentration of 10 ng/ml,

- the cells are cultured from day 14 of the method and during a period of 2 days with a culture medium comprising DAPT at a concentration of 10 μΜ, and BDNF and GDNF each at a concentration of 10 ng/ml, and

- the cells are optionally cultured from day 16 of the method with a culture medium comprising BDNF and GDNF each at a concentration of 10 ng/ml, and preferably without an inhibitor of the Notch signaling pathway.

In particular, in the above preferred embodiment:

the cells are cultured from day 0 of the method and during a period of time of 2 days with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 1 μΜ;

- the cells may be cultured from day 3 of the method and during a period of 1 day, with a culture medium comprising an inhibitor of the BMP signaling pathway, in particular LDN-193189 preferably at a concentration of 0.2 μΜ, an inhibitor of the TGF/activin/nodal signaling pathway, in particular SB-431542, preferably at a concentration of 40 μΜ, CHIR-99021 at a concentration of 1 μΜ and SAG at a concentration of 500 nM,

The present invention will be further illustrated by the following figures and examples.

Brief description of the figures

Figure 1 : Representative traces of whole cell current clamp recordings of an immature spinal motor neuron. The neuron was sitting at -50 mV and the spiking activity was examined following an injection of 600 pA.

Figure 2: Representative traces of whole cell current clamp recordings of a mature spinal motor neuron. The neuron was sitting at -50 mV and the spiking activity was examined following an injection of 600 pA.

Figure 3: Optimized prior art sMN differentiation protocol adapted in 384 well plate. NP: neural plate, sMN prog.: sMN progenitors, sMN: Spinal motor neurons. Values indicate the percentage of cells adopting the indicated cell fate (mean ± SD, n=3 independent differentiations). Figure 4: Screen protocol for the combinatorial action of RA, CHIR-99021 , FGF2 and SAG. Neuralized EBs (Smadi) in 384 well plates were exposed to various concentrations of RA, CHIR-99021 , FGF2 and SAG 500 nM for 2 days. On day 14, dissociated cells were stained for OLIG2.

Figure 5: Addition of CHIR-99021 increases sMN progenitors. Percentages of OLIG2+ cells per condition (4 well per condition).

Figure 6: Percentages of OLIG2+ cells at RA at 1 ,000 nM according to CHIR-99021 concentration irrespective of FGF (mean ± SD, 4 independent wells). Kruskal-Wallis oneway analysis of variance, ^*: p-value < 0.05. ^**: p-value < 0.01 . Scale bar = 10 μΜ. ns: non-significant.

Figure 7: Percentages of OLIG2+ depending on RA concentrations upon CHIR-99021 at 3 μΜ treatment irrespective of FGF (mean ± SD, 4 independent wells). Kruskal-Wallis one-way analysis of variance, ^*: p-value < 0.05. ^**: p-value < 0.01 . Scale bar = 10 μΜ.

Figure 8: Neuralized EBs (Smadi) were exposed to RA (100 nM), CHIR-99021 (3 μΜ) and SAG (500 nM) at different time points of differentiation. OLIG2 expression was analyzed at 4 time points from day 8 to 16.

Figure 9: Neuralized EBs (upon Smadi) were exposed to RA, CH IR-99021 and SAG at different time points of differentiation. The percentage of OLIG2+ cells in each condition was analyzed by high content image acquisition, n=4 well for each condition.

Figure 10: Quantitative analysis of OLIG2 expression over time depending on the day CHIR-99021 addition : DO : day 0, D2 : day 2, No Chir : no addition, (mean ± SD of 4 independent wells).

Figure 11 : Quantification of OLIG2+ cells on day 10 following the differentiation of different hPSC lines upon CHIR-99021 at 3 μΜ and RA at 100 nM. H9, n=6 independent differentiations, SA01 hES n=5, hiPS line 1 and 2 n=3.

Figure 12: Quantitative analysis of OLIG2+ and ISL1 /2+ cells over time (mean ± SD, 4 independent wells). Figure 13: Realtime RT-PCR analysis for genes expressed in sMN progenitors (OLIG2, PAX6, NGN2, LHX3) and sMNs (LHX3, ISL 1, HB9) 20h post DAPT addition on Day9 - fold changes compare to no DAPT treatment (mean ± SD - n=2 independent differentiations).

Figure 14: Quantitative analysis of OLIG2 (sMN progenitor marker) and Ki67 (marker of proliferating cells) shows that sMN progenitors and proliferating cells are very seldom present in the cultures while the culture are essentially neuronal.

Figure 15: Quantitative analysis of the differentiation efficiency on day 14 for different hPSC lines (mean ± SD of independent differentiations) ESC : H9 (n=4) and SA01 (n=6), hiPS lines (n=2).

Figure 16: Voltage clamp recording of a cell held at -60mV and then stepped to a series of voltages (-100 to +40 mV) in 10 mV increments. The peak inward current likely due to voltage-activated Na channels is plotted in below. Middle traces show the current family from the same cell following the application of 0.5 μΜ tetrototoxin (TTX). The steady state current is plotted as a function of voltage (dark squares, N=27) below. Right traces show currents from the same cell with TTX and 30 mM TEA showing the block of voltage- activated potassium channels. The steady state current is plotted below (light squares, N=15). Error bars denote S.E.M.

Figure 17: The addition of glutamate to the bath caused a transient depolarization of the membrane potential. Individual excitatory post-synaptic currents could be detected at the onset of the application (inset), indicating the presence of functional glutamate receptors. Scale bar: 20 μΜ.

Figure 18: Percentage of neurons (NF+ cells) and PHOX2B/ISL1 + neurons on day 16 in SA01 hES line treated with CHIR-99021 1 μΜ and RA 10 nM (mean ± SD, n=3 independent differentiations).

Figure 19: Increased expression of cMN markers PHOX2B and TBX20 in CHIR-99021 1 μΜ RA 10 nM condition. Realtime PCR analysis on day 14. Fold changes relative to CHIR-99021 3 μΜ RA 100 nM condition (sMN). Figure 20: Real time RT-PCR analysis of CDX1 and CDX2 gene induction on day 4. Fold change compare to control condition (Smadi, SAG, no CHIR-99021 , no RA - mean ± SD, n=3 independent differentiations).

Figure 21 : Real time RT-PCR analysis of HOXA2, HOXA4, HOXA5 gene on day 10 of differentiation compare to control condition (smadi, SAG, no CHIR-99021 , no RA- mean ± SD, n=3).

Example

Material and methods

Generation and culture of hPSC lines

Human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS) were cultured as described in Marteyn et al. (201 1 ) Cell Stem Cell 8:434-444. For the generation of the Hb9::GFP stable line, H9 hES (3x10⁶) cells were transfected with 5 μg of pHb9::GFP plasmid provided by Dr. Wichterle (Columbia university) using Amaxa (Lonza). Cells were replated on neomycin resistant MEF with 10 μΜ Y-27632 (rock inhibitor, Miltenyi, 130-095-563). Geneticin selection (InvitroGen, 10131027) started 4 days later at 25 μg/ml. Concentration was increased by two fold every 4 days to reach 100 μg/ml. After 15 days of selection, resistant ESC clones were passaged manually. Full- length transgene integration was verified by PCR. To generate iPS cells, Coriell fibroblasts (GM04603, GM03813, Candem, USA) were reprogrammed using retroviral vectors expressing OCT4, KLF4, SOX2 and c-Myc. The iPSC clones (iPS1 = GM03813, iPS2= GM04603) were then maintained on MEFs and passaged 15 times before differentiation. Selected clones were validated by G-banding analysis as described in Lefort et al. (2008) Nat. Biotechnol. 26:1364-1366.

Large scale differentiation in 384 well

On day 0, hPSCs were dissociated with accutase (InvitroGen) and resuspended in differentiation medium N2B27 (DMEM F12, Neurobasal vohvol, supplemented with N2 (Life technologies), B27 (Life technologies), Pen-strep 1 %, β-mercaptoethanol 0.1 % (Life technologies), ascorbic acid (0.5 μΜ, Sigma Aldrich) with Y-27632 (5 μΜ, STemGent), SB431542 (40 μΜ, Tocris) and LDN 193189 (0.2 μΜ, Miltenyi). 200 to 400 cells were seeded in V shape 384 (Brooks, Life Science System) with a Bravo Automated Liquid Handling Platform (Agilent). Plates were centrifuged (400 g, 5 min) to force cell aggregation and form a single embryoid body per well. SB431542 and LDN 193189 were kept for four days. Smoothened Agonist (SAG, 500 nM, Calbiochem), FGF2 (Peprotec), Chir-99021 (Tocris or Selleck), Retinoic Acid (Sigma Aldrich) Y-27632 (Miltenyi), compound W (Tocris), XAV-939 (Sigma Aldrich), L-685,458 (Tocris), IBMX (Enzolife), Forskolin (Tocris), Recombinant Human BMP4 (R&D System), SU5402 (VWR), U0126 (R&D system), PD0325901 (Miltenyi), FGF8, LY294002 (Ozyme), Wnt 3a (R&D system), were added at indicated time points and concentrations by automated medium addition (Bravo Automated Liquid Handling Platform, Agilent). BDNF (20 ng/ml, Peprotech) and GDNF (10 ng/ml, Peprotech) were added to differentiation medium at day 1 1 . Medium was changed automatically every other day (Bravo, Agilent). Quadruplicates were designed for each differentiation condition to analyze up to 96 independent conditions in parallel.

Automated EBs dissociation for single cell analysis

At appropriate timepoints for analysis, four EBs corresponding to one condition of differentiation were pooled in one well of a V shape 96 well plate (Bravo, head 96 LT) and incubated with trypsin for 10 min at 37°C. After addition of FBS (final 10%), medium was replaced with N2B27 with 25 μg/mL DNase I (ROCHE). EBs were dissociated by automated pipetting (BRAVO). Dissociated cells were either analyzed by flow cytometry or replated in 96 (1 well/ condition) or 384 (4 wells/ condition) well plates (both from Corning) coated with Poly-Ornithin (20 μg/ml, Sigma Aldrich) and 5 μg/ml laminin (InvitroGen).

Differentiation in larger format

Optimal conditions of differentiation were systematically replicated in larger format of differentiation (6 cm or 10 cm culture dishes) for subsequent gene expression profiling, histology on sectioned embryoid bodies, quantitative analysis of specific cell fate marker on dissociated cells and electrophysiology. Briefly, between 1 and 1 .5x10⁵ cells/ml were seeded in non-treated cell culture dishes to form embryoid bodies and differentiation procedures were identical to the one discovered by large-scale analysis in 384 well plates.

Immunostaining

In 384-well plates, adherent cells were fixed with PFA 4% (10 min at room temperature) and rinsed with PBS with the Cell Washer ELX405 automat (Biotech). For immunostaining on cryostat sections, embryoid bodies were fixed in PFA 4%, rinsed with PBS, cryoprotected with 30% sucrose and embedded in OCT prior sectioning. Primary antibodies were diluted in PBS/2%FBS/0, 1 % Triton and incubated overnight at 4 °C. Primary antibodies: mouse anti-Hb9 (81 .5C10, The Developmental Studies Hybridoma Bank, University of Iowa (DSHB), 1 :50), -Lhx3 (67.4E12, DSHB, 1 :50), -Ns1/2 (39.4D5, DSHB, 1 :100), -Nkx6.1 (F55A12, DSHB, 1 :100), -Evx1 (99.1 -3A2, DSHB, 1 :100), -Lhx1 /2 (4F2, DSHB, 1 :100), -PAX3 (1 :100, DSHB, 1 :100), mouse anti-Tujl (MMS435P, Covance, 1 :500), rabbit anti-Olig2 (AB9610.1 :1 ,000), rabbit anti-TrkA (06-574), mouse anti-Nestin (MAB5326, Millipore, 1 :1 ,000), mouse anti-Ki67 (MAB4190, Millipore, 1 :1 ,000), goat-anti CHAT (ab144P, Millipore, 1 :300), goat anti-Brn3a (Sc-31984, Santa Cruz, 1 :400), rabbit anti-Sox-9 (sc-20095, Santa Cruz, 1 :200), goat anti-FOXA2 (Sc-6554, Santa Cruz, 1 :300), goat-anti IsM (GT15051 , Neuromics, 1 :2,000) and chicken anti-neurofilament (CH22105, Neuromics, 1 :500), rabbit anti-Phox2b (provided by J.F. Brunet, 1 :1500), goat anti- Brachyury (AF2085, R&D Systems, 1 :1 :500), goat anti-Desmin (AF3844, R&D Systems, 1 :20), goat anti-Sox17 (AF373, R&D Systems, 1 :300), mouse anti-aSMA (1 A4, DAKO, 1 :100), mouse anti-SMI32 (NE1023, Calbiochem, 1 :300), rabbit anti-FoxP1 (ab16645, Abeam, 1 :5,000), mouse anti-Pax6 (AB78545, Abeam, 1 :200), rabbit anti-SOX2 (481400, Invitrogen, 1 :500), HuC/HuD (A21271 , Invitrogen, 1 :500), rabbit anti-GFP (A1 1 122, Invitrogen, 1 :1 ,000), and a-BTX-AlexaF594 conjugate (B13423, Invitrogen, 1 :500). Secondary antibodies were added for 1 h min at RT in presence of Hoechst or DAPI (Invitrogen, 1 :3,000): Alexa488, Alexa568 and Alexa647; (Life Technologies, 1 :1 ,000) and donkey anti-chicken cy5 (Jackson ImmunoResearch, 1 :1 ,000).

High content image acquisition

Multiwell plates were automatically imaged with an Array Scan VTI HCS technology (Thermofisher-Cellomics). Quantifications of signals were performed with the software VHS Scan, Thermofisher-Cellomics. Hoechst staining was used to identify individual nuclei and perform single-cell analysis.

Electrophysiology

EBs were dissociated into single cells and replated on coverslips coated with Poly- Ornithin (20 μg/ml, Sigma Aldrich) and 5 μg/ml laminin (InvitroGen). Patch clamp recording was performed with axopatch 770B amplifier after either 17 or 31 days in culture. MSs were identified by GFP fluorescence. The patch pipettes (3-4 ΜΩ) contained (in mM) 135 KMethylSulfate, 5 KCI, 0.1 EGTA-Na, 10 HEPES, 2 NaCI, 5 ATP, 0.4 GTP, 10 phosphocreatine (pH 7.2; 280-290 mOsm). The extracellular solution contained (in mM) 125 NaCI, 2.5 KCI, 10 glucose, 26 NaHC0₃, 1 .25 NaH₂P0₄, 2 Na Pyruvate, 2 CaCI₂ and 1 MgCI₂ and was saturated with 95% 0₂ and 5% C0₂. pH was 7.3. Membrane potentials were corrected for liquid junction potential, which was measured to be ~3 mV. Series resistance (typically less than 10 ΜΩ) was monitored and compensated throughout each experiment with the amplifier circuitry. 0.5 μΜ TTX was bath-applied following dilution into the external solution from concentrated stock solutions. To maintain constant osmolarity and pH, 30 mM TEACI was added to a modified external solution (95 mM NaCI instead of 125 mM). Glutamate was added with equal concentration NaOH to maintain neurtral pH. Leak current was subtracted from current families using a P/N protocol and each set of currents was averaged four times. All data was acquired with Axograph X software and analyzed with Igor pro.

Flow cytometry

After dissociation, Hb9::GFP positive cells were quantified using a cell MACSquant analyzer (Milteny Biotech).

Quantitative RT-PCR analysis

Total RNA were extracted (RNAeasy, Quiagen) and cDNA synthesized using Superscript III (InvitroGen). Quantitative real-time RT-PCR were performed using a Chromo4 Real-Time system (Bio-Rad) with Syber Green PCR Master Mix (Applied Biosystems).

Collapse assays

Collapse assays were performed as described in Nedelec et al. (2012) J. Neurosci. 32:1496-14506. Briefly, EBs differentiated into specific neuronal subtypes were plated on Poly-Ornithin (20 μg/ml, Sigma Aldrich) and 5 μg/ml laminin (InvitroGen) coated coverslips. 24h post plating, Ephrin-A5 (50 ng/ml, R&D system) or Netrin-1 (400 ng/ml, Adipogen) were added in the medium for 15 min. Cells were fixed with a 37°C 4% PFA/10% sucrose solution and percentages of growth cone collapse were evaluated.

Statistical analysis

Statistics were computed using Kruskall wallis or one-way analysis of variance (ANOVA) as indicated. All statistics were computed in Prism using P values as indicated. Independent differentiations are differentiations performed on different weeks and started with newly thawed or different passages of hPSCs.

Results

Miniaturized hPSC differentiations for the large scale analysis of differentiation conditions To conveniently monitor sMN generation, the inventors first generated a human embryonic stem cell (hESC) line containing an Hb9::GFP transgene that drives expression of GFP into sMNs (Wichterle et a/.(2002) Cell 110:385-397). The preexisting protocol to differentiate sMNs from hPSC in 25 days described in Amoroso et at. (2013) J. Neurosci. 33:574-586 was then adapted to 384 well plate format (Figure 3). hESCs were seeded and forced to form a single embryoid body (EB) per well. Then, exchange of media and addition of small molecules in individual wells were fully automated. At timepoints of interest along the differentiation process, EBs were dissociated into single cells and the impact of individual differentiation conditions was either analyzed by flow cytometry or by immunostaining for specific makers followed by automated image acquisition and analyses. Multiple parameters including hPSC dissociation methods, plates, volumes, or densities of hESC seeding were optimized yielding to a maximum of 32% sMNs in 25 days as expected with this protocol.

Then, to determine the roadblocks impeding efficient sMN generation, the inventors analyzed the successive steps of differentiation from neural plate (NESTIN+, PAX6+ cells), to sMN progenitors (OLIG2+ NKX6.1 +) and finally post mitotic sMNs. As shown in Chambers et at. (2009) Nat. Biotechnol. 27:275-280, the dual Smad inhibitors LDN-193189 and SB-431542 (Smadi) potently induced the specification of cells with a neural plate identity (>95% in 5 days). OLIG2+ sMN progenitors were first observed on day 1 1 and peaked on day 14 (52%). By day 25, sMNs reached their highest proportion (32%). Therefore, in the adapted prior art methods, from an optimal conversion of hESCs into neural plate cells, only half were converted to MN progenitors that differentiated only partially into post-mitotic sMNs.

The inventors thus tried to obtain an efficient production of sMNs both by improving progenitor generation and by increasing the transition from progenitors to sMNs.

Wnt and RA pathways cooperate to generate sMN progenitors

The inventors wondered whether the coordinated action of the Wnt, FGF, RA and HH pathways might be necessary to efficiently generate human sMN progenitors from hPSCs. Neuralized hESCs (upon Smadi) were exposed to increasing concentrations of the Wnt agonist, Chir-99021 (described in Bennett et al. (2002) J. Biol. Chem. 277:30998- 31004), RA and FGF2 in presence of the HH agonist, SAG (Figures 4 and 5). sMN progenitors were quantified by automated image acquisition and analysis in the 96 tested conditions. In the control condition (RA≥1 ,000 nM, SAG 500 nM) 42% of the cells expressed OLIG2 (Figure 6). While FGF2 had little effects, addition of Chir-99021 strikingly increased sMN progenitors (74% of the cells in Chir-99021 3 μΜ / RA 1 ,000 nM (Chir³RA¹⁰⁰⁰) conditions - Figure 7). The critical influence of Chir-99021 was further supported by the potent induction of OLIG2+ cells at RA concentrations under which sMN progenitors are normally not induced (RA = 10 nM - Figure 7). To determine whether Chir-99021 indeed mimics Wnt signaling, Chir-99021 was replaced with Wnt3a (300 ng/ml) which also potentiated the induction of OLIG2+ cells over control RA/SAG conditions. These results highlight the cooperative action of Wnt and RA pathways to specify human sMN progenitors.

Timing of Wnt, RA and HH signaling finely controls progenitor fate

Cell fate is controlled by the concentration but may also be controlled by the time at which cells are exposed to signaling molecules. The inventors tested whether tuning the relative activation of the Wnt, RA and HH pathways controls progenitor fate. Chir³, RA¹⁰⁰ and SAG⁵⁰⁰ were added at various days of differentiation and OLIG2 expression was monitored on day 8, 10, 14 and 16 (Figures 8 and 9). Following immediate activation of the Wnt pathway at ESC stage (day 0 of differentiation), sMN progenitors appeared as early as day 8 and peaked (82% of the cells) on day 10 (Figure 10), four days earlier than in the previous experiments when Chir-99021 was added on day 2. Wnt activation not only shortened the time required for progenitor specification but also increased their final rate (82% on day 10 versus 55% on day 16 in the absence of Chir-99021 - Figure 10).

The critical influence of the timing of extrinsic cue addition could be also illustrated by the effect of activating the HH pathway within 4 days of RA exposure in the specification of sMN progenitors. Addition of SAG on day 7 instead of day 2 or 4 decreased sMN progenitor rate (33% on day 10, 50% on day 16 - Figure 9). Altogether, this analysis revealed the critical influence of early Wnt signaling activation at the ESC stage (day 0) to rapidly specify sMN progenitors in cooperation with RA 100 nM and SAG (day 2). The robustness of this procedure was then further confirmed on independent hES and hiPS lines (84% OLIG2+/NKX6.1 + cells generated in average - Figure 1 1 ).

Rapid conversion of MN progenitors into sMN upon γ-secretase inhibition

To evaluate whether sMN generation would be increased in condition promoting sMN progenitors, the inventors monitored from day 8 to day 16 the expression of ISL1/2, a sMN marker (Figure 12). Surprisingly, generation of sMNs remained relatively poor as ISL1 /2+ cells represented only 36% of all cells on day 16 in conditions generating up to 83% sMN progenitors 6 days earlier. Therefore, increasing progenitors was not sufficient to consequently generate the corresponding post-mitotic neurons and extrinsic signals might be required to promote progenitor differentiation. In consequence, the inventors searched for molecules converting sMN progenitors into sMNs. The HB9::GFP hESC line was differentiated into sMN progenitors that were exposed to 30 different conditions modifying key developmental pathways. GFP induction was analyzed 4 days later by flow cytometry. The vast majority of conditions did not modify sMN generation. However, three γ-secretase inhibitors dramatically increased the proportion of sMNs. DAPT at 10 μΜ was the most potent increasing sMN rate by 4.2 fold (55%). γ-secretase inhibitors promote neurogenesis in different systems including hES differentiation by inhibiting Notch signaling and inducing proneural genes. On human sMN progenitors, DAPT indeed rapidly induced a genetic program that specify post mitotic sMN (NGN2, ISM , LHX3 and HB9) at mRNA and protein level (Figure 13).

A more systematic analysis of critical time points at which γ-secretase inhibition was optimal revealed again the importance of properly timing extrinsic cues. Addition of DAPT on day 1 1 or 14 instead of day 9 of differentiation was not as efficient in generating sMNs suggesting that OLIG2+ progenitors might progressively lose their ability to differentiate into sMNs. These results demonstrate that sMN progenitors poorly differentiate spontaneously into post mitotic MNs and that addition of extrinsic cues (i.e γ- secretase inhibitors) at appropriate time point, is necessary to rapidly convert them into sMN.

Efficient rapid generation of functional MNs

The inventors then determined the efficiency of the procedure and whether these rapidly generated sMNs correctly acquire functional properties. On day 14, 92% of the cells were neurons (Neurofilament+ (NF) or HuC/D+) and virtually no proliferating cells (Ki67+) or sMN progenitors (OLIG2+) remained (Figure 14). The vast majority of the cells (74% in average; up to 82%) coexpressed the generic sMN markers HB9 and ISL1 and acquired typical sMN properties such as the expression of FOXP1 (25%) or LHX3 (1 1 %), two markers of sMN subtypes. The robustness and the timeline of differentiation was verified with a second hESC line (SA01 ) and two independent human induced pluripotent stem cell (hiPSC) lines (Figure 16) and was further confirmed on 15 additional hPSC lines.

To verify the functionality of the neurons, whole-cell patch clamp recording was performed on Hb9::GFP expressing sMNs. On day 17, 100% of sMNs (n=13) fired small immature spikes in response to a 600 pA current injection with shape and magnitude (3 to 10 mV) typical of immature neurons (Figure 1 ). The immaturity of sMNs is further supported by the lack of expression of choline acetyl transferase (CHAT), the rate limiting enzyme for acetylcholine. To test whether sMNs would mature in vitro, dissociated cultures were maintained for an additional 13 days. Little cell proliferation was observed resulting in an almost pure population of neurons for extended periods of time. In this context, after 2 weeks in culture, CHAT staining was observed and sMNs displayed electrophysiological properties of more mature neurons (Figure 2). In response to steps of depolarizing current injection, 27 out of 29 recorded neurons fired trains of action potentials with the threshold, shape and magnitude of mature neurons (Figure 2). Furthermore, the spike frequency adaptation and increased firing frequency with increasing injected current resembled the ones of mouse sMNs described in Miles et at. (2004) J. Neurosci. 24:7848-7858. Whole-cell voltage clamp recordings performed before and after application of 0.5 μΜ TTX and 30 mM TEA revealed the presence of functional voltage activated sodium and potassium channels (Figure 16). In addition, bath application of the neurotransmitter glutamate triggered excitatory post synaptic currents (EPSCs) and induced a transient depolarization (Figure 17). Transient currents were also observed following GABA bath application.

In conclusion, despite their rapid generation, sMNs obtained by the present method were initially immature at birth but already displayed membrane properties typical of neurons and gradually matured in vitro to acquire typical characteristics of functional sMNs, such as CHAT expression, response to neurotransmitters and firing of action potentials. Therefore, compressing the developmental timeline did not impede the acquisition of functional properties.

Tempering Wnt and RA signaling induces cranial motor neurons

During embryonic development, subtle changes in time and concentration of the same combination of molecules direct the specification of distinct cell types. The inventors sought to test whether fine tuning of the Wnt, FGF, RA and HH signaling pathways would generate cMNs versus sMNs from hPSCs. cMNs are defined by the co-expression of PHOX2B and ISL1 .

Neuralized hES cells (Smadi) were exposed, in presence of SAG, to various concentrations of Chir-99021 , FGF, and RA followed by DAPT on day 9. On day 16, ISL1 , PHOX2B and Tuj1 (a neural marker) expression were assessed. As expected, ISL1 +, PHOX2B- sMNs were observed in the Chir³RA^{100 or 1000} conditions. In contrast, numerous ISL1 +/PHOX2B+ neurons were generated upon lower Wnt and RA concentrations (Chir¹ RA¹⁰). Reiteration of the differentiations and quantitative analyses indicated that in this condition PHOX2B, ISL1 + neurons represent 52% of the cells in average (up to 60% - Figure 18). Gene expression analysis on day 14 showed a strong upregulation of PHOX2B as well as TBX20, a specific cMN marker in Chir¹ RA¹⁰ compared to Chir³RA¹⁰⁰ (Figure 19). Therefore, tempering Wnt and RA signaling led to a discrete change in cell fate and the specification of cranial motor neurons.

To investigate whether these changes in Wnt and RA concentration led to discrete changes in key developmental pathways controlling in vivo the specification of the two distinct classes of motor neurons, the inventors monitored the differential expression profile of CDX and HOX genes that distinguish hindbrain and spinal progenitors. CDX1 and CDX2, two genes required for spinal cord specification and hindbrain fate repression, were strongly induced on day 4 upon Chir³RA¹⁰⁰ (sMN) but not Chir¹ RA¹⁰ (Figure 20). This differential expression of CDXs was mirrored on day 10 by the induction of HOX genes typical of hindbrain or spinal progenitors. Indeed, Chir³RA¹⁰⁰ induced progenitors expressing HOXA2, A4 and A5, a Hox code typical of the rostral spinal cord (Figure 21 ). In contrast, Chir¹ RA¹⁰ specified progenitors expressing HOXA2 but not HOXA4 and HOXA5, a HOX code typical of hindbrain progenitors (Figure 21 ).

These results show that graded Wnt and RA signaling leads to discrete changes in key developmental pathways (CDX and HOX) to generate sMN or cMN progenitors that are converted efficiently into the two cardinal classes of motor neurons upon γ-secretase inhibition.

Discussion

Wnt based control of neuronal diversity

The most efficient procedures of the prior art to generate spinal motor neurons from hPSCs were relying solely on RA to impose on differentiating cells a spinal fate and HH signaling to induce sMN progenitors which led to relatively slow and/or inefficient differentiations. The inventors herein demonstrated the critical role of a third developmentally important signal, Wnt, to greatly increase the speed and efficiency of sMN progenitor specification (around 80% of the cells in 9 days).

Importantly, in contrast to RA based MN differentiations that mainly drive cells towards a spinal fate, manipulating the level of Wnt signaling provides a means to generate progenitors with an hindbrain identity that can then be differentiated into cranial motor neurons. Wnt based differentiations are thus not only more efficient but also more flexible to access motor neuron subtypes through slight modulations of the same procedure.

Accessing progenitors with different identities open avenues for more systematic investigation of pathways controlling neuronal diversity and provide access to human cells not yet accessible for basic or translational studies. Acceleration of hPSC differentiation by extrinsic control of the developmental timeline

The inventors also herein showed how subtle changes both in concentrations and time of exposure to extrinsic cues greatly affect the output of hPSC differentiation. A two day delay in SAG or DAPT addition impeded sMN optimal generation and subtle changes in the concentration of Wnt agonist Chir-99021 induced spinal or hindbrain progenitors.

Surprisingly while many prior art protocols to generate neurons are long and thought to follow the timeline of human development (Nicholas et al. (2013) Cell Stem Cell 12:573-586), the inventors demonstrated that changes in the time at which progenitors are exposed to extrinsic factors greatly impacts on the speed at which the cells of interest are generated. While motor neurons are born around 3 weeks past the pluripotent cell stage in human embryos, in the optimized procedures of the invention, the generation of cMNs or sMNs occurs in 14 days without impeding the acquisition of typical molecular markers (ISLI1 , HB9, PHOX2B, FOXP1 , LHX3, CHAT...) or functional properties (axon response to guidance cues, response to neurotransmitters and electrophysiological signature). This rapid differentiations can be broken in 3 phases: 4-5 days to convert hPSC into cells with a neural plate identity, 4-5 days to guide the cells towards a more committed progenitor fate (OLIG2+, NKX2.2+...) and finally 4-5 days to generate post mitotic neurons. Therefore acceleration compared to normal embryonic development is not occurring by skipping typical progenitor stages but is rather mediated by the optimized timing of extrinsic factors addition. Indeed, early Wnt activation at PSC stage accelerates the appearance of sMN progenitors and γ-secretase inhibition is necessary to fasten the conversion of progenitors into their cognate post mitotic neurons. These findings suggest that the generation of other specific cell types from hPSCs could be accelerated relative to human development. Providing cost effective rapid and efficient procedures to differentiate hPSCs should greatly facilitate the use of hPSC-derived cells for future studies.

Synchronous generation of neuronal subtypes for comparative studies

Cell-type specificity is a hallmark of most human diseases. In the nervous system, thousands of neuronal subtypes are specified during embryonic development and individual neurological disorders such as Parkinson disease, ALS or SMA affect the function of very specific neuronal populations while closely related neurons are spared. Combining patient-specific hiPS cells and precise targeted differentiations into closely related cell types distinguished by their sensitivity to a disease should refine in vitro disease modeling and drug screenings. For instance, motor neuron diseases represent a group of symptomatically heterogeneous diseases due to the differential sensitivity of specific motor neuron subtypes in different disorders such as ALS or SMA. A better understanding of molecular and cellular pathways leading to selective motor neuron death or selective degeneration of specific motor neuron subtypes require parallel access to various motor neuron groups as well as closely related cells spared by motor neuron diseases. Here, the inventors revealed conditions to produce cranial motor neurons, a population affected in several motor neuron diseases such as bulbar SMA, bulbar palsy or ALS. Access to cranial motor neurons for future investigation of MN diseases might help deciphering whether the pathological mechanisms leading to cMN and/or sMN impairments are identical or whether rescuing both cell types might require different therapeutics.

Claims

1. An ex vivo method for producing a population of motor neuron progenitors comprising the following steps:

a) culturing neuralized pluripotent cells in a culture medium Ci comprising an activator of the Wnt signaling pathway during a period of time and

2. The method according to claim 1 , wherein said method further comprises the step pre-a) of culturing, during a period of time T₀, pluripotent cells in a culture medium C₀ comprising (i) an inhibitor of the Bone Morphogenetic Protein (BMP) signaling pathway and (ii) an inhibitor of the Transforming Growth Factor (TGF)/activin/nodal signaling pathway, in order to obtain neuralized pluripotent cells.

3. The method according to claim 1 or 2, wherein said activator of the Wnt signaling pathway is selected from the group consisting of the compound Chir-99021 and the Wnt3a protein, and is preferably the Wnt3a protein.

4. The method according to any one of claims 1 to 3, wherein the period of time is of 2 to 4 days.

5. The method according to any one of claims 1 to 4, wherein the agonist of the Hedgehog signaling pathway is the Smoothened Agonist SAG.

6. The method according to any one of claims 1 to 5, wherein the period of time T₂ starts 2 to 4 days after the beginning of the period of time T^

7. The method according to any one claims 1 to 6, wherein the motor neuron progenitors are spinal motor neuron progenitors.

8. The method according to claim 7, wherein the activator of the Wnt signaling pathway is present at a high concentration in the culture medium d and retinoic acid is present in the culture medium C₂ at a concentration superior to 10 nM.

9. The method according to claim 8, wherein: - the cells are cultured from day 0 of the method and during a period of time of 2 days with a culture medium comprising LDN-193189 at a concentration of 0.2 μΜ, SB-431542 at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 3 μΜ;

- the cells are cultured from day 2 of the method and during a period of 2 days with a culture medium comprising LDN-193189 at a concentration of 0.2 μΜ, SB-431542 at a concentration of 40 μΜ, CHIR-99021 at a concentration of 3 μΜ, retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM, and

- the cells are cultured from day 4 of the method and during a period of 10 days with a culture medium retinoic acid at a concentration of 100 nM and SAG at a concentration of 500 nM.

10. The method according to any one of claims 7 to 9, wherein the population of spinal motor neuron progenitors comprises more than 70% of spinal motor neuron progenitors.

11. The method according to any one claims 1 to 6, wherein the motor neuron progenitors are cranial motor neuron progenitors.

12. The method according to claim 1 1 , wherein the activator of the Wnt signaling pathway is present at a low concentration in the culture medium Ci and retinoic acid present in the culture medium C₂ at a concentration of 10 nM or less.

13. The method according to claim 12, wherein:

- the cells are cultured from day 0 of the method and during a period of time of 2 days with a culture medium comprising LDN-193189 at a concentration of 0.2 μΜ, SB-431542 at a concentration of 40 μΜ, and CHIR-99021 at a concentration of 1 μΜ;

- the cells are cultured from day 3 of the method and during a period of 1 day with a culture medium comprising LDN-193189 at a concentration of 0.2 μΜ, SB-431542 at a concentration of 40 μΜ, CHIR-99021 at a concentration of 1 μΜ, and SAG at a concentration of 500 nM, and

14. An ex vivo method for obtaining a population of motor neurons comprising the steps of:

A) producing a population of motor neuron progenitors by the method according to any one of claims 1 to 13, and

15. The method according to claim 14, wherein the population of motor neuron progenitors are differentiated into motor neurons by culture, during a period of time T_3, in a culture medium C₃ comprising an inhibitor of the Notch signaling pathway.