Efficient and rapid specification of spinal cord neuronal subtypes derived from human pluripotent stem cells

Abstract

The present invention relates to the targeted generation of specific cell populations. Motor neuron (MN) subtypes show differential vulnerability in disease and spinal cord injury. Generated MNs with specific rostral-caudal identity represent an important resource for cell therapy approaches. However, this strategy is still hampered by slow and inefficient targeted differentiation due to imprecise control in the in vitro specification of cell fate. We now use embryoid body-based differentiation of hPSCs and show that HOX clock expression can be controlled to generate subtypes of spinal cord MNs. Accordingly, the present invention relates to an in vitro or ex vivo method for generating spinal neuronal subtypes comprising exposing axial precursors to retinoic acid (RA), an agonist of the Hedgehog signaling pathway, and optionally an FGFR agonist and / or an activator of the TGF pathway, wherein progressively more caudal motor neuron identity is acquired by delayed exposure to retinoic acid and / or by exposure to retinoic acid in combination with an FGFR agonist and / or an activator of the TGF pathway.

Description

[Technical Field]

[0001] The present invention relates to methods for generating human cell types with defined rostro-caudal identities. [Background technology]

[0002] Patterning of the bilaterian body is regulated by the differential expression of HOX transcription factors along its rostral-caudal axis, and the initiation of the Hox gene expression pattern is closely linked to the rostral-caudal extension of the body axis.

[0003] As axial progenitors contribute to the progressive expansion of caudal mesodermal and neuroectodermal structures, the 3'→5' sequence of HOX gene activation translates into a collinear spatial pattern of expression for the anteriorly expressed 3' HOX genes, while the 5' HOX genes are later activated and expressed caudally (Deschamps and Duboule, 2017; Henrique et al., 2015). Extrinsic factors, such as retinoic acid (RA), Wnt, or fibroblast growth factors (FGF), play important roles in controlling HOX patterns, while cell-intrinsic changes in the chromatin state within the complex correlate with the temporal order of its transcriptional induction ( Bel-Vialar et al., 2002 ; Deschamps and Duboule, 2017 ; Liu et al., 2001 ; Mazzoni et al., 2013 ; Narendra et al., 2015 ; Neijts et al., 2017 ; Philippidou and Dasen, 2013 ). However, it remains unclear whether the progressive opening of chromatin along the complex acts as an internal timer that is started and stopped by exogenous cues, or whether the order of secreted factors that progressively activate more caudal HOX genes defines the tempo of induction ( Bel-Vialar et al., 2002 ; Del Corral and Storey, 2004 ; Deschamps and Duboule, 2017 ; Ebisuya and Briscoe, 2018 ; Lippmann et al., 2015 ; Mazzoni et al., 2013 ; Wymeersch et al., 2019 ).This may result in the currently limited control over the differentiation of pluripotent stem cells (PSCs) into caudal cell types with several well-defined rostrocaudal identities (Diaz-Cuadros et al., 2020; Du et al., 2015; Duval et al., 2019; Faustino Martins et al., 2020; Frith et al., 2018; Gouti et al., 2014; Li et al., 2005; Lippmann et al., 2015; Matsuda et al., 2020; Maury et al., 2015; Ogura et al., 2018; Peljto et al., 2010; Verrier et al., 2018). Indeed, the two models suggest different strategies for controlling PSC differentiation in vitro. The "intrinsic model" predicts that efficient specification of posterior identity relies on precise synchronization between the timing of differentiation and internal HOX timers. Alternatively, a second mechanism predicts that exposure of axial precursors to relevant exogenous cues synchronizes the HOX clock and generates progeny with a defined rostral-caudal identity.

[0004] Spinal motor neurons (sMNs), located in the spinal cord, are one of two fundamental classes of motor neurons; the other being hindbrain-derived cranial motor neurons. sMNs rely on a precise HOX code to acquire their corresponding rostral-caudal subtype identity. Indeed, differential expression of HOX transcription factors along the vertebrate spinal cord is a major product of HOX gene regulation. In spinal motor neurons, this Hox code governs the specification of subtype-specific properties that control the formation of the locomotor circuit (Dasen, 2017; Philippidou and Dasen, 2013). In particular, Hox expression profiles within motor neurons control their subtype specification, column and pool segmentation, and muscle group innervation targets. However, whether the spinal HOX code and associated MN subtypes are conserved in humans remains unclear, thus hindering a faithful evaluation of HOX regulation and its relationship to cellular "rostral-caudal" identity during hPSC differentiation.

[0005] The targeted generation of specific cell populations is a major tool for developmental studies, disease modeling, and regenerative medicine. However, this strategy remains hampered by slow and inefficient targeted differentiation, due in large part to imprecise control over cell fate specification in vitro.

[0006] In particular, MN subtypes exhibit differential vulnerability to disease and spinal cord injury. Therefore, de novo generation of motor neurons with specific rostrocaudal identities provides a much-needed resource for modeling these incurable diseases and a more controlled cell source for putative cell therapy approaches.

[0007] Our previous application (see International Publication No. WO 2016012570) presented a method for obtaining enriched populations of sMNs and cMNs from hPSCs. It was specifically demonstrated that culturing neuralized hPSCs in the presence of high concentrations of an activator of the Wnt signaling pathway, combined with retinoic acid (RA) and SAG, enabled the obtaining of a population containing over 80% spinal motor neuron precursors within 10 days of culture, which could efficiently differentiate into spinal motor neurons (60–70% of cells) in the presence of a γ-secretase inhibitor. However, despite the fact that MN subtypes are differentially impaired in disease and the lack of an efficient method for generating MN subtypes from hPSCs, this application did not provide any clues regarding the specific rostral-caudal and subtype identities of sMNs or any suggestions or methods for obtaining such specific sMNs.

[0008] Retinoic acid (RA), wingless-type MMTV integration site protein family (Wnt), fibroblast growth factor (GDG), and growth differentiation factor (GDF) signaling have also been shown to intricately regulate HOX expression during posterior CNS development (Liu et al., 2001; Nordstrom et al., 2006). Currently, the most efficient procedure for determining sMNs (Maury et al. 2015, Du et al. 2015 doi: 10.1038 / ncomms7626) generates anterior cervical MNs. However, due primarily to the lack of knowledge regarding the combination of cues and the time required to generate MN subtypes, no efficient method has been reported to date for generating MN subtypes with defined rostral-caudal identities. In particular, the generation of upper limb, thoracic, and lumbar MNs, especially limb-innervating MNs, remains elusive. The mRNA profiles of HOX proteins are distinct and broader than their protein domains. Therefore, mRNA profiles (especially in motor neuron precursors) do not reflect the expression of Hox proteins in spinal motor neurons. As a result, assessment of MN subtypes based on mRNA expression, for example, by PCR analysis, therefore lacks the necessary accuracy for identifying MN subtypes (Dasen et al., Nature 2003, Lippmann et al., 2015). The inadequate characterization of antibodies in humans also hinders proper characterization of MN subtypes. Furthermore, despite several attempts, the resulting differentiation (based on 2D adherent differentiation of hPSCs) has resulted in a low percentage of MNs, but with mixed identities and often contaminated by non-neuronal cells, demonstrating poor control over the differentiation process (Lippmann et al. 2015, Patani et al. 2011). These limitations preclude systematic studies of MN subtypes in healthy conditions and even in disease states where selective MN populations are impaired.

[0009] Thus, there remains a significant need for improved and efficient procedures that allow obtaining enriched populations of sMNs with specific rostrocaudal phenotypes. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2016012570 [Patent Document 2] International Application No. PCT / EP 2015 / 066944 [Non-patent literature]

[0011] [Non-Patent Document 1] Chung et at. (2008) Cell Stem Cell 2:113-117 [Non-patent document 2] Maury Y, Come J, Piskorowski RA, Salah-Mohellibi N, Chevaleyre V, Peschanski M, Martinat C, Nedelec S. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat Biotechnol. 2015 Jan;33(1):89-96 [Non-patent document 3] Bienz (2005) Curr. Biol. 15:R64-67 [Non-patent document 4] Bennett et al. (2002) J. Biol. Chem. 277:30998-3104 [Non-Patent Document 5] Miyabayashi et al. (2007) Proc. Natl. Acad. Sci. USA 104:5668-5673

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[0012] We now use embryoid body-based differentiation of hPSCs and provide evidence that the duration of the time window between Wnt and RA defines the final positional identity of the progeny, and that varying concentrations, duration, and combinations of caudalizing factors (fibroblast growth factors (FGFs) and growth differentiation factors (GDFs)) control the speed at which temporally colinear activation of HOX genes occurs. The pace of the HOX clock is therefore dynamically encoded by parameters of exposure to exogenous cues.

[0013] From a biotechnological perspective, exogenous control of this HOX clock provides a simple means to generate cell types with defined "rostral-caudal" identities from PSCs, shortening the time requirements for generating cells generated at different times in development during axial elongation.

[0014] Thus, the methods of the present invention allow for the simultaneous generation of human MN subtypes with defined rostrocaudal identities with unprecedented efficiency and precision. In particular, the methods allow for fine tuning of the rostrocaudal identity of sMNs. [Means for solving the problem]

[0015] Accordingly, the present invention relates to an in vitro or an ex vivo method for generating human cell / neuron subpopulations with a defined rostro-caudal identity, comprising culturing axial precursors in a culture medium comprising retinoic acid (RA), an agonist of the Hedgehog signaling pathway, and optionally an FGFR agonist and / or an activator of the TGF / Activin / Nodal signaling pathway (including GDF11 or GDF8), wherein progressively more caudal motor neuron identity is obtained by delayed addition of retinoic acid to the culture medium and / or by addition of retinoic acid in combination with an FGFR agonist and / or an activator of the TGF / Activin / Nodal signaling pathway.

[0016] More specifically, the present invention provides an in vitro or ex vivo method comprising: - exposing the pluripotent stem cells to a culture medium comprising an activator of the Wnt signaling pathway and advantageously an inhibitor of the BMP and Activin / TGB pathways to obtain axial precursors; - exposing the axial precursors to retinoic acid (RA) and an agonist of the Hedgehog signaling pathway, optionally in combination with an FGFR agonist and / or an activator of the TGF / Activin / Nodal signaling pathway, in a culture medium to obtain spinal motor neuron precursors; - optionally exposing the spinal motor neuron precursors to a Notch pathway inhibitor in culture medium; wherein progressively more caudal motor neuron identity is generated by delaying exposure to retinoic acid relative to exposure to a Wnt signaling pathway activator, or by exposing axial precursors to retinoic acid in combination with increased exposure to an FGFR agonist and / or an activator of the TGF / Activin / Nodal signaling pathway, wherein increased exposure generally relates to increased concentration in the culture medium and / or increased duration in the culture medium.

[0017] Typically, human pluripotent stem cells (hPSCs) are exposed to inhibitors of the bone morphogenetic protein (BMP) signaling pathway and inhibitors of the transforming growth factor (TGF) / activin / nodal signaling pathway, prior to or in parallel with activators of the Wnt signaling pathway.

[0018] Generally, caudal thoracic and lumbar motoneurons are also obtained by exposing axial precursors to retinoic acid, FGFR agonists, and activators of the TGF / activin / nodal signaling pathway.

[0019] Optionally, an FGFR agonist is added to the culture medium at a concentration of at least 15 ng / ml, particularly at least 50 ng / ml, or at least 100 ng / ml for at least 24 hours, and an activator of the TGF / activin / nodal signaling pathway (e.g., GDF11 or GDF8) is added to the culture medium at a concentration of at least 20 ng / ml for at least 24 hours.

[0020] In some embodiments of the methods of the present invention: - an inhibitor of the bone morphogenetic protein (BMP) signaling pathway, an inhibitor of the transforming growth factor (TGF) / activin / nodal signaling pathway, and an activator of the Wnt signaling pathway are added to the culture medium starting from D0 to about D3 or D4; - an agonist of the sonic hedgehog signaling pathway can be added to the culture medium from about D3-D7 to about D9, typically between D3-D9, D4-D9, D5-D9, D6-D9, or D7-D9; - A Notch pathway inhibitor is added from about D9 to D14 or longer.

[0021] Generally, RA is added to the culture medium for a period of about 2 to 11 days, optionally between about D3 and D9, optionally at a concentration of at least 10 nM.

[0022] In some embodiments of the method: - upper limb motoneurons are obtained by adding RA alone between D4 and D9 or between D3 and D9 to the culture medium, or in combination with an FGFR agonist at a concentration of at least 100 ng / ml or an activator of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml between D3 and D4; - anterior thoracic motor neurons are obtained by adding RA to the culture medium starting from D5 or later until D9 (or later), or between D3 and D9 (or later), alone or in combination with an FGFR agonist at a concentration of at least 60 ng or an activator of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml, for 48 hours or more (preferably 48 hours) starting from D3; - caudal thoracic motoneurons are obtained by adding RA to the culture medium between D3 and D9 and by combining an FGFR agonist at a concentration of at least 100 ng / ml with GDF11 at a concentration of at least 20 ng / ml between D3 and D4; - Lumbar motor neurons are obtained by adding RA to the culture medium between D5 and D9 (or later) and by combining an FGFR agonist at a concentration of at least 100 ng / ml with GDF11 at a concentration of at least 20 ng / ml, which are added to the culture medium starting from D3 or D4 until D5.

[0023] In some embodiments, the FGFR agonist is FGF2 or 8.

[0024] In some embodiments, the agonist of the sonic hedgehog signaling pathway is a smoothened agonist (SAG), and optionally, the SAG is added at a concentration of at least 300 nM.

[0025] In some embodiments, the Notch pathway inhibitor is DAPT, optionally at a concentration of at least 5 μM. The Notch pathway inhibitor is typically added from about D9 to about D14 or later.

[0026] In some embodiments, the activator of the Wnt signaling pathway is selected from the compound Chir-99021 or the Wnt3a protein. DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] Unless otherwise stated, the present invention encompasses any combination of the various embodiments as described herein.

[0029] In describing the embodiments and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0030] As used herein, "about" or "around" means within 10% of a stated concentration range or within 10% of a stated time frame.

[0031] As used herein, "effective amount" means an amount of an agent sufficient to elicit a defined cellular effect according to the present invention.

[0032] Previous work by Maury et al. identified spinal cord precursors with putative characteristics of axial precursors capable of generating motor neurons; however, neither the competence of these precursors nor the rostrocaudal identity of their progeny was documented. Indeed, the expression patterns of HOX transcription factors had not previously been documented, and markers for MN pools and columns in the human spinal cord remained unknown. We have now characterized the expression patterns of HOX transcription factors in human embryos. Thus, we have defined combinations of transcription factors that can be used as markers for distinct rostrocaudal locations within the spinal cord and identified the molecular mechanisms governing that specification.

[0033] The present invention thus provides a method for generating spinal motor neurons with a defined rostral-caudal identity, which involves the inventors' discovery that differentiation of hPSCs into axial precursors under defined conditions by exposure to an activator of a Wnt signaling inhibitor, typically in combination with prior or concurrent exposure to inhibitors of the BMP and TGF pathways, followed by immediate application of a retinoic acid receptor agonist, optionally in combination with an FGFR agonist and an activator of the TGF / Activin / Nodal signaling pathway, enables precise positional patterning of the resulting posterior neuroectoderm and neuroepithelium along the hindbrain-spinal cord axis, as reflected by HOX family gene expression.

[0034] The present invention provides a method for generating human motor neuron subpopulations with defined rostral-caudal identity, comprising: a) exposing human pluripotent stem cells (PSCs) to an activator of the Wnt signaling pathway and an inhibitor of the BMP and Activin / TGB pathways, advantageously added to obtain axial precursors; b) exposing the resulting axial precursors to retinoic acid (RA) and an agonist of the sonic hedgehog signaling pathway, optionally in combination with an FGFR agonist and / or an activator of the TGF / activin / nodal signaling pathway, to obtain motor neuron precursors; c) optionally exposing the motor neuron precursors to a Notch pathway inhibitor to obtain post-mitotic human motor neurons; Including, The present invention relates to methods, particularly in vitro or ex vivo methods, in which progressively more caudal motor neuron identities are generated by delaying exposure to retinoic acid relative to exposure to a Wnt signaling pathway activator, or by exposing axial precursors to retinoic acid in combination with increased exposure to an FGFR agonist and / or an activator of the TGF / Activin / Nodal signaling pathway, where increased exposure generally relates to increased concentration in the culture medium and / or increased duration in the culture medium.

[0035] Various embodiments of such methods, which can be implemented alone or in combination, are illustrated in the detailed description that follows.

[0036] Identification of molecular markers based on the present invention Generally, the methods of the present invention allow for obtaining enriched cell populations that express a combination of molecular markers as disclosed herein, particularly as defined in Table 2. An enriched cell population refers to an ex vivo cell population that contains a higher proportion of a particular cell type or cells with particular characteristics found in vivo (e.g., in a tissue).

[0037] Techniques for identifying the expression of molecular markers are well known to those skilled in the art and include immunostaining, Western blotting, and flow cytometry using antibodies specific for the markers.

[0038] The mRNA expression levels within a population can be detected using any of several routine methods in the art, including, but not limited to, qRT-PCR, RNA-blot, RNA sequencing, and RNAse protection.

[0039] Suitable quantitative methods for assessing molecular markers of interest as described herein also include well-known techniques, such as qRT-PCR, RNA-sequencing, RNA blot, RNAse protection, etc., for assessing gene expression at the RNA level. Quantitative methods for assessing marker expression at the protein level in a cell population are also known in the art. For example, flow cytometry is commonly used to determine the fraction of cells in a given cell population that express (or do not express) one or two protein markers of interest (e.g., Brachyury and Sox2).

[0040] pluripotent stem cells As used herein, the term "pluripotent cells" refers to undifferentiated cells that can give rise to a variety of different cell lineages.

[0041] Preferably, the pluripotent cells are human pluripotent cells.

[0042] Preferably, the pluripotent cells are stem cells.

[0043] In the context of the present invention, stem cells include embryonic stem cells (ESCs); embryonic and extraembryonic tissue stem cells, such as fetal stem cells including amniotic fluid stem cells, and induced pluripotent stem cells. In particular, stem cells according to the present invention include fetal stem cells and induced pluripotent stem cells.

[0044] The term "embryonic stem cells" refers to pluripotent stem cells derived from the epiblast tissue of the inner cell mass of a blastocyst or early morula stage embryo. Embryonic stem cells can also be defined by the expression of several transcription factors, such as Oct-4, Nanog, and Sox2, and cell surface proteins, such as glycolipids SSEA3 and SSEA4, and keratin sulfate antigens Tra-1-60 and Tra-1-81.

[0045] In a particular embodiment, the term "stem cells" according to the present invention does not include stem cells derived from human embryos. More specifically, stem cells according to the present invention are preferably not derived directly from a human embryo or did not require the destruction of a human embryo. In particular, embryonic stem cells 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 derived from publicly available and previously established stem cell lines that do not require the destruction of a human embryo, as described, for example, in Chung et al. (2008) Cell Stem Cell 2:113-117.

[0046] In particular, human embryonic stem cells derived from one of the cell lines exemplified in Table 1 fall within the meaning of the term "stem cells" as used in the present invention.

[0047] [Table 1]

[0048] The term "fetal stem cells" refers to stem cells derived from the fetus itself or, preferably, from extraembryonic tissues that emerge during pregnancy, including umbilical cord blood, amniotic fluid, Wharton's gel, amniotic membrane, and placenta.

[0049] As used herein, the term "induced pluripotent stem cells" or "iPS cells" refers to a type of pluripotent stem cell that is artificially derived (e.g., induced by complete or partial reversion) from a differentiated cell (e.g., a non-pluripotent cell), typically an adult somatic cell, such as an adult fibroblast.

[0050] In some embodiments, the stem cells used in the context of the present invention are induced pluripotent stem cells (iPSCs). Suitable induced pluripotent stem cells (iPSCs), such as line WTSIi002 (male, RRID: CVCL_AH3, also known as HPSI0913i-eika_2), are generally available from the European Bank for Pluripotent Stem Cells (EBISC). WTC-mEGF-safe harbor locus (AAVS1)-cl6, produced by the Allen Cell Institute, was obtained from the Coriell Laboratory (catalog number AICS-0036-006, male, RRID: CVCL_JM19).

[0051] In some embodiments, the pluripotent cells are obtained from an individual suffering from a neurodegenerative genetic disease, such as hereditary amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA). In other embodiments, the pluripotent cells contain a genetic mutation, e.g., involved in a neurodegenerative genetic disease, such as hereditary amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA). Advantageously, in these embodiments, the population of motor neuron precursors and / or the population of motor neurons also contain the mutation, and can therefore provide a good cellular model of the disease.

[0052] Advantageously, the human pluripotent stem cells used in the present invention are dissociated into single cells on day 0 and then reassembled into free-floating aggregates of PSC stem cells (in the form of embryoid bodies, also called PSC aggregates) in non-adherent culture conditions, such as microwell plates or ultra-low attachment plates. Such procedures are described in the "Materials and Methods" section of the Results. The protocol is also detailed in Maury et al. (Maury Y, Com J, Piskorowski RA, Salah-Mohellibi N, Chevaleyre V, Peschanski M, Martinat C, Nedelec S. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat Biotechnol. 2015 Jan;33(1):89-96).

[0053] Axis precursor As used herein, the term "axial precursors" refers to progenitor cells derived from hPSCs that generate neural precursor cells and neurons of the spinal cord.

[0054] Axial precursors are typically induced by adding Wnt activators to the hPSC culture medium, allowing their conversion into axial precursors capable of generating progeny expressing different HOX combinations located at different rostrocaudal locations within the spinal cord.

[0055] Axial precursors may be specifically characterized by co-expression of SOX2 and CDX2. In some embodiments, typically 2-3 days after exposure to a Wnt agonist, axial precursors also typically co-express TBXT (BRACHYURY or BRA). The SOX2+ / CDX2+ / BRA+ profile characterizes the population of axial precursors that generate the spinal cord and somites in the mouse embryo.

[0056] Generally, at D2, D3, and D4 (i.e., approximately 2 to at least 4 days after the onset of exposure to Wnt pathway activators), axial precursors exhibit elevated expression of CDX1 and 2, TBXT (BRACHYURY), FGF17, RXRG, SP5 / 8, WNT5A / B, WNT8A, FGF8, GRSF1, CYSTM1, and HES3, along with SOX2 (correlated with the loss of most pluripotency markers, such as Nanog, Klf2, Klf4, Prdm14, Fgf4, Oct4 (also in early NMPs), and Sox2 (also in the caudal epiblast and neuroectoderm)).

[0057] Axis precursors also generally do not express representative markers of node cells, mesoderm, and allantois, such as Ccno, Nodal, Nog, Noto, and Shh, respectively; Aldh1a2, Cited1, Meox1, Msgn1, Tbx3 and 6, Osr1, Pax2, Mesp1 / 2, Foxf1, and Hand1-2; and Mixl1, Evx1, Gata6, Tbx2, Tbx4, Mixl1, and Evx1 (notably Figures 2B–2C and Table S1 in the Results section, and Cambray and Wilson, 2007; Edri et al., 2019; Gouti et al., 2014; Gouti et al., 2017; Henrique et al., 2015; Koch et al., 2017; Wymeersch et al., 2016). see Wymeersch et al., 2019 ).

[0058] Motor neurons and precursors As used herein, the term "motor neuron" or "motoneuron" refers to an efferent neuron that innervates a muscle cell or an autonomic ganglion. Motor neurons include two basic classes: spinal motor neurons and cranial motor neurons.

[0059] As used herein, "spinal motor neurons" refer to motor neurons located within the spinal cord, while "cranial motor neurons" refer to motor neurons located in the brainstem. Unless otherwise specified, the term "motor neurons" as used herein also serves as an extension of "spinal motor neurons." As known to those skilled in the art, spinal motor neurons can be characterized by specific patterns of marker expression. Generally, spinal motor neurons can be characterized by, among other things, ISL1 / 2+ (ISL LIM homeobox 1 or 2 positive) and / or HB9+ (homeobox protein 9 positive). Additional markers that may be expressed by spinal motor neurons include Lhx3 (LIM homeobox 3), FOXP1 (forkhead box P1), Lhx1 (LIM homeobox 1), CHAT (choline acetyltransferase), and VACHT (vesicular acetylcholine transporter). Therefore, identification of spinal motor neurons can be performed by any technique known to those skilled in the art that involves detection of the above markers, such as immunostaining using specific anti-ISL1 / 2 and / or anti-HB9 antibodies.

[0060] Spinal motor neurons, and more specifically, functional spinal motor neurons, can also be characterized using electrophysiological techniques, such as whole-cell patch clamp recording. Generally, whole-cell patch clamp assays can be performed as follows: patch pipettes (3-4 MΩ) containing 135 mM methyl potassium sulfate, 5 mM KCl, 0.1 mM EGTA-Na, 10 mM HEPES, 2 mM NaCl, 5 mM ATP, 0.4 mM GTP, 10 mM phosphocreatine (pH 7.2; 280-290 mOsm), and an extracellular solution (expressed in mM) containing 125 mM NaCl, 2.5 mM KCl, 10 mM glucose, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 2 mM Na-pyruvate, 2 mM CaCl₂, and 1 mM MgCl₂, saturated with 95% O₂ and 5% CO₂, at pH 7.3, are used. The membrane potential is corrected for the liquid junction potential, which can generally be measured as approximately 3 mV. Series resistance (typically less than 10 MΩ) is monitored and compensated for throughout each experiment using an amplifier circuit. 0.5 μM tetrodotoxin (TTX) is applied to the bath after dilution from a concentrated stock solution into the external solution. To maintain constant osmolarity and pH, 30 mM TEACl (tetraethylammonium chloride) can be added to the modified external solution (95 mM NaCl instead of 125 mM). Glutamate can be added along with an equal concentration of NaOH to maintain a neutral pH. Leak currents are subtracted from the current family using a positive / negative (P / N) protocol, and each set of currents is averaged, e.g., four times. All data are acquired using Axograph X software and can be analyzed, e.g., using Igor Pro. Functional immature spinal motor neurons can therefore be characterized by the small number of immature spikes they emit in response to a 600 pA current injection, typically with a shape and magnitude of 3-10 mV.Functional mature spinal motor neurons can be characterized by projecting trains of action potentials with a shape and magnitude typically between 60-80 mV (see International Application No. PCT / EP2015 / 066944). Functional mature spinal motor neurons can also be characterized by detecting choline acetyltransferase (CHAT) expression using conventional immunostaining and Western blot techniques.

[0061] The term "motor neuron progenitor" or "motoneuron progenitor" refers to a cell population that can give rise to one of two major groups of motor neurons (i.e., spinal motor neurons and cranial motor neurons). The term "spinal motor neuron precursor" generally refers to Olig2-positive cells. As known to those skilled in the art, spinal motor neuron precursors can be characterized by specific patterns of marker expression. Generally, the generated spinal motor neuron precursors can be characterized by OLIG2+ (oligodendrocyte lineage transcription factor 2 positive), NKX6.1+ (NK6 homeobox 1 positive), SOX1+, and SOX2+.

[0062] Identification of spinal motor neuron precursors can therefore be performed by any technique known to those skilled in the art, involving the detection of the above-mentioned markers, such as immunostaining, typically using specific anti-OLIG2 and / or anti-NKX6.1 antibodies, SOX1 and 2. Subtypes of MN precursors along the rostro-caudal axis can be characterized by their expression of HOX mRNA by RT-PCR (and immunostaining for HOXC9) (see below and especially in the Results section).

[0063] Human spinal cord motoneuron (hsMN) rostral-caudal subpopulation In the results contained herein, we demonstrate that HOX transcription factors are regionally expressed along the rostrocaudal axis of the human spinal cord. Within these distinct HOX domains, distinct MN subtypes, identifiable by transcription factor combinations, are generated in typical locations.

[0064] It has been shown that (postmitotic) human MNs express ISL1 and / or HB9, both along the spinal cord. Within MNs, specific HOX expression exhibits a rostral-caudal pattern: cervical MNs express HOXA / C5, whereas upper limb MNs express HOXC6, thoracic MNs express HOXC9, and lumbar MNs express HOXC10. Caudal upper limb MNs co-expressed HOXC6 and HOXC8, and anterior thoracic MNs co-expressed HOXC8 and HOXC9. HOXD9, along with HOXC10, was associated with caudal thoracic MNs and anterior lumbar MNs.

[0065] We found that high levels of FOXP1 (FOXP1 high We also found that MNs expressing FOXP1 were observed at the upper limb, anterior thoracic (HOXC6 and HOXC8 / HOXC9), and lumbar (HOXC10) levels. These MNs formed the lateral motor column (LMC), where limb-innervating MNs are located. high Within the LMC, SCIP / HOXC8 MNs were observed in the caudal upper limb spinal cord. In contrast, SCIP / HOXC8 / HOXC9 MNs were located in the anterior thoracic region. Their location and transcriptional coding identify them as putative hand-controlling MNs.

[0066] Thus, in some embodiments of the methods of the present invention, subpopulations of spinal motor neurons (including spinal motor neuron precursors) identified in Table 2 below are obtainable.

[0067] [Table 2]

[0068] In some embodiments, at least 50% of the cells in an isolated population of human spinal motor neurons (obtained according to the teachings disclosed herein) express one of the above Hox transcription factor profiles, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, etc. percent of the cells in the isolated cell population exhibit the desired Hox transcription factor profile.

[0069] Activators of the Wnt signaling pathway and acquisition of axial precursors In the context of the present invention, the term "activator of the Wnt signaling pathway" refers to any compound (natural or synthetic) that causes increased activation of the Wnt signaling pathway, which is a series of molecular signals generated as a result of binding of any member of the Wnt family to a cell surface receptor. Generally, activators of the Wnt signaling pathway induce the 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 for determining whether a given compound is an activator of the Wnt signaling pathway are well known to those skilled in the art. Generally, a compound is considered to be an activator of the Wnt signaling pathway if the level of nuclear β-catenin increases after culturing cells in the presence of the compound compared to cells cultured in the absence of the compound. The level of nuclear β-catenin can be measured by Western blot using an antibody specific for β-catenin.

[0070] The activator of the Wnt signaling pathway can be a Wnt agonist or a molecule that activates any of the downstream steps of the Wnt signaling pathway. The activator of Wnt signaling can be a natural or synthetic compound. When the activator of the Wnt signaling pathway is a protein, it can be a purified protein, a recombinant protein, or a synthetic protein.

[0071] Examples of activators of the Wnt signaling pathway include the compound CHIR-99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, as described in Bennett et al. (2002) J. Biol. Chem. 277:30998-3104), 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 (Coghlan et al. (2000) Chem. Biol. 7:793-803), and compound BIO (6-bromoindirubin-3'-oxime, as described in Sato et al. (2004) Nat. Med. 10:55-63).

[0072] 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.

[0073] Generally, the compound CHIR-99021 is added to the culture medium at a concentration in the range of 0.5 to 5 μM, preferably in the range of 1 to 4 μM, even more preferably about 3 μM or about 1 μM.

[0074] Generally, a Wnt activator is added to a culture of hPSCs, in particular a culture of human embryoid bodies (as identified above, but see also Maury et al., incorporated herein by reference, and International Application No. PCT / EP2015 / 066944), for a period sufficient to detect expression of markers of axial precursors as described above, in particular expression of CDX2, and in particular to obtain embryoid bodies with a SOX2+ / CDX2+ / BRA+ profile.

[0075] Embryoid bodies can be obtained using techniques well known to those skilled in the art. Generally, pluripotent cells can be provided to aggregate and form embryoid bodies, typically by centrifugation at 1200 g for 5 minutes in a V SHAPE 384.

[0076] Thus, in some embodiments, the Wnt activator is typically applied in the culture medium for 2-4 days (optionally together with inhibitors of the BMP and TGFβ pathways, as detailed below), particularly in the range of 3-4 days, and typically 4 days. According to the methods of the present invention, the day on which the Wnt activator is added to the cultured embryoid bodies may be considered day 0 (D0).

[0077] Exposure of embryoid bodies to inhibitors of the bone morphogenetic protein (BMP) signaling pathway and the transforming growth factor (TGF) / activin / nodal signaling pathway In some embodiments, hPSCs, particularly human embryoid bodies, as defined in the "Pluripotent Cells" section, are exposed to both (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 (also referred to herein for short as the TGF pathway).

[0078] As used herein, the term "inhibitor of the BMP signaling pathway" refers to any compound (natural or synthetic) that reduces the activation of the BMP signaling pathway, which is a series of molecular signals generated as a result of any member of the BMP (bone morphogenetic protein) family binding to a cell surface receptor. Generally, inhibitors of the BMP signaling pathway induce a decrease in the phosphorylation level of the proteins Smad 1, 5, and 8, as described in Gazzero and Minetti (2007) Curr. Opin. Pharmacol. 7:325-333. Techniques. Techniques for determining whether a given compound is an inhibitor of the BMP signaling pathway are well known to those skilled in the art. Generally, a compound is considered to be an inhibitor of the BMP signaling pathway if the level of phosphorylated Smad 1, 5, or 8 decreases after culturing cells in the presence of the compound compared to cells cultured in the absence of the compound. The level of phosphorylated Smad proteins can be measured by Western blot using an antibody specific for the phosphorylated form of the Smad protein.

[0079] The inhibitor of the BMP signaling pathway can be a BMP antagonist or a molecule that inhibits any of the downstream steps of the BMP signaling pathway. The inhibitor of BMP signaling can be a natural or synthetic compound. When the inhibitor of the BMP signaling pathway is a protein, it can be a purified protein, a recombinant protein, or a synthetic protein.

[0080] Inhibitors of the BMP signaling pathway include noggin, chordin, follistatin, inhibitory Smad 6 (l-Smad 6), inhibitory Smad 7 (l-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), 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), and compound DMH2 (Hao et al. (2010) ACS Chem Biol. 5:245-253), 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), 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), compound LDN193189 (or DM-3189, Cuny et al. al. (2008) Bioorg. Med. Chem. Lett. 18:4388-4392), and compound K02288 (3-[6-amino-5-(3,4,5-trimethoxyphenyl)-pyridin-3-yl]-phenol described in Sanvitale et al. (2013) PLoS ONE 8:62721), but preferably the inhibitor of the BMP signaling pathway is compound LDN193189.

[0081] Generally, the compound LDN193189 is added to the culture medium containing hPSCs / AT at a concentration in the range of 0.05-1 μM, preferably 0.1-0.5 μM, 0.15-0.25 μM, even more preferably about 0.2 μM.

[0082] As used herein, the term "inhibitor of the TGF / activin / nodal signaling pathway" refers to any compound (natural or synthetic) that causes a decrease in activation of the TGF / activin / nodal signaling pathway, which is the series of molecular signals generated as a result of any member of the TGF / activin / nodal family binding to a cell surface receptor.

[0083] Generally, inhibitors of the TGF / activin / nodal signaling pathway induce a decrease in the phosphorylation level of the protein Smad2, as described in Shi and Massague (2003) Cells 113:685-700. Techniques for determining whether a given compound is an inhibitor of the TGF / activin / nodal signaling pathway are well known to those skilled in the art. Generally, a compound is considered to be an inhibitor of the TGF / activin / nodal signaling pathway if the level of phosphorylated Smad2 decreases after culturing cells in the presence of the compound compared to cells cultured in the absence of the compound. The level of phosphorylated Smad protein can be measured by Western blot using an antibody specific for the phosphorylated form of the Smad protein.

[0084] The inhibitor of the TGF / activin / nodal signaling pathway can be a TGF / activin / nodal antagonist or a molecule that inhibits any of the downstream steps of the TGF / activin / nodal signaling pathway. The inhibitor of TGF / activin / nodal signaling can be a natural or synthetic compound. When the inhibitor of the TGF / activin / nodal signaling pathway is a protein, it can be a purified protein, a recombinant protein, or a synthetic protein.

[0085] The inhibitor of the TGF / activin / nodal signaling pathway may be selected from the group consisting of the compound SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyrlidn-2-yl-1H-imidazol-2-yl)-benzamide hydrate), Lefty-A protein, and Cerberus.

[0086] Preferably, the inhibitor of the TGF / Activin / Nodal signaling pathway is the compound SB431542.

[0087] Generally, the compound SB431542 is added to a culture medium containing hPSCs at a concentration in the range of 10-75 μM, preferably in the range of 20-50 μM, and even more preferably at about 40 μM.

[0088] 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 at a concentration in the range of 0.15 to 0.25 μM, e.g., about 0.2 μM, and the compound SB431542 is present in the culture medium at a concentration in the range of 20 to 50 μM, e.g., about 40 μM.

[0089] The inhibitor of the BMP signaling pathway and the inhibitor of the TGF / activin / nodal signaling pathway are applied to the culture medium for about 2 to 4 days, particularly 3 to 4 days, and generally for about 4 days.

[0090] Also advantageously, the PSCs and axial precursors (provided for neuralization after retinoic acid exposure) are in the form of embryoid bodies, as described above. Embryoid bodies can be obtained from hPSCs using techniques well known to those skilled in the art. Generally, pluripotent cells can be centrifuged, e.g., at 1200 g for 5 minutes, particularly in a V SHAPE 384, to aggregate the cells and form embryoid bodies.

[0091] Advantageously, the inhibitor of the BMP signaling pathway and the inhibitor of the TGF / Activin / Nodal signaling pathway are applied to a culture medium containing PSCs (typically in the form of embryoid bodies together with a Wnt activator). Thus, the culture medium typically comprises an inhibitor of the BMP signaling pathway, an inhibitor of the TGF / Activin / Nodal signaling pathway, and an activator of the Wnt signaling pathway, as defined above. Thus, in some embodiments, hPSCs (particularly in the form of human embryoid bodies or free-floating aggregates) are cultured in a culture medium, in which the inhibitor of the BMP signaling pathway and the inhibitor of the TGF / Activin / Nodal signaling pathway are added together with or subsequent to the addition of the Wnt activator.

[0092] In a preferred embodiment, PSCs (see above), typically in the form of embryoid bodies, are cultured in a culture medium in which a Wnt activator is added together with an inhibitor of the BMP signaling pathway and an inhibitor of the TGF / activin / nodal signaling pathway. In such an embodiment, the inhibitor of the BMP signaling pathway and the inhibitor of the TGF / activin / nodal signaling pathway (as previously described) can be added to the culture medium for a period of 3 to 4 days (i.e., from D0 to D3 or from D0 to D4), and in particular, the inhibitor of the TGF / activin / nodal signaling pathway can be added from D0 to D3, while the inhibitor of the BMP signaling pathway can be added (maintained) in the culture medium until D3 or D4, and the Wnt activator can be added to the culture medium for a period of 2 to 4 days, particularly 3 to 4 days (i.e., from D0 to D2, from D0 to D3, or from D0 to D4, preferably from D0 to D3 or D4). Generally, when RA, and / or FGFR agonists, and / or activators of the TGF / activin / nodal signaling pathway are present in the culture medium, inhibitors of the TGF / activin / nodal signaling pathway are no longer present in the culture medium after D3.

[0093] Agonists of the Retinoic Acid and Hedgehog Signaling Pathways: As used herein, the term "retinoic acid" (RA) refers to an active form (synthetic or natural) of vitamin A known to have the ability to induce neuronal differentiation. Examples of forms of retinoic acid that can be used in accordance with the present invention include, but are not limited to, retinoic acid, retinol, retinal, 11-cis-retinal, all-trans retinoic acid (ATRA), 13-cis-retinoic acid, and 9-cis-retinoic acid.

[0094] Generally, retinoic acid is added to the culture medium at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM, or in the range of 100 nM to 500 nM. Generally, RA is added to the culture medium at a concentration of at least 100 nM.

[0095] Several retinoic acid receptor agonists can also be used in the methods of the present invention; an exemplary class of suitable retinoic acid receptor agonists is retinoids and retinoid analogs, including, but not limited to, all-trans retinoic acid (ATRA), retinol acetate, EC23 (4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl)-benzoic acid; CAS No: 104561-41-3), BMS453 (4-[(1E)-2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]-benzoic acid; CAS No: 166977-43-10), fenretinide (N-(4-hydroxyphenyl)retinamide; CAS No: 65646-68-6), AM580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid; CAS No: 102121-60-8), tazarotene (6-[2-(3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)ethynyl]-3-pyridinecarboxylic acid ethyl ester; CAS No: 118292-40-3), and TTNPB (4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid; CAS No: 71441-28-6).Other exemplary retinoic acid receptor agonists that may be used include AC261066 (4-[4-(2-butoxyethoxy)-5-methyl-2-thiazolyl]-2-fluorobenzoic acid; CAS No: 870773-76-5), AC55649 (4'-octyl-[1,1'-biphenyl]-4-carboxylic acid; CAS No: 59662-49-6), adapalene (6-(4-methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic acid; CAS No: 106685-40-9), AM80 (4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)amino]carbonyl]benzoic acid; CAS No: 94497-51-5), BMS753 (4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl)carbonyl]amino]benzoic acid; CAS No: 215307-86-1), BMS961 (3-fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)acetyl]amino]-benzoic acid; CAS No: 185629-22-5), CD1530 (4-(6-hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic acid; CAS No: 107430-66-0), CD2314 (5-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)-3-thiophenecarboxylic acid; CAS No: 170355-37-0), CD437 (6-(4-hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic acid; CAS No: 125316-60-1), and Ch55 (4-[(1E)-3-[3,5-bis(1,1-dimethylethyl)phenyl]-3-oxo-1-propenyl]benzoic acid; CAS No: 110368-33-7).

[0096] In the context of the present invention, the term "hedgehog signaling pathway agonist" refers to any compound (natural or synthetic) that causes an increase in activation of the hedgehog signaling pathway (which is the series of molecular signals generated as a result of any member of the hedgehog family binding to a cell surface receptor). Generally, a hedgehog signaling pathway agonist induces activation of the hedgehog signaling pathway.

[0097] Agonists of the Hedgehog signaling pathway can be Hedgehog agonists or molecules that activate any of the downstream steps in the Hedgehog signaling pathway. Agonists of Hedgehog signaling can be natural or synthetic compounds. When the agonist of the Hedgehog signaling pathway is a protein, it can be a purified protein, a recombinant protein, or a synthetic protein.

[0098] Examples of agonists of the hedgehog signaling pathway include purmorphamine (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 (Chen et al. (2002) Proc. Natl. Acad. Sci. USA 99:14071), and the agonist β-glucanase inhibitor ... and 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).

[0099] Preferably, the agonist of the hedgehog signaling pathway is selected from SHH (sonic hedgehog) or SAG (smoothened agonist). Most preferably, the agonist of the hedgehog signaling pathway is SAG.

[0100] In some advantageous embodiments, the agonist of the hedgehog signaling pathway, in particular SAG, is added to the culture medium at a concentration of at least 300 nM, in particular between 100 nM and 1 μM or 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM.

[0101] Typically, an agonist of the Hedgehog signaling pathway is added to the culture medium for a period of time sufficient to induce the expression of motor neuron precursor markers, such as Olig2n and NKX6.1. Techniques for detecting the expression of motor neuron precursor markers are well known to those skilled in the art and include immunostaining, Western blotting, and RT-PCR using antibodies specific for the markers, such as anti-Olig2 and / or anti-NKX6.1 antibodies, or primers for detecting NKX6.1. Typically, this period can extend for at least 2 days, more preferably about 2-6 days, particularly 2-5 days, and typically about 5 days.

[0102] In some embodiments of the methods of the present invention, the agonist of the Hedgehog signaling pathway is generally added to the culture medium at least 2 to 7 days after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway. Advantageously, the agonist of the Hedgehog signaling pathway is added to the culture medium between days D3 and D9, between days D4 and D9, between D5 and D9, between D6 and D9, or between D7 and D9, preferably between D3 and D9.

[0103] Markers of neural plate identity are well known to those skilled in the art and generally include SOX1, PAX6, etc. Such markers are efficiently induced after retinoic acid exposure.

[0104] Exposure of axial precursors to a combination of RA and an agonist of the hedgehog signaling pathway allows for the generation of spinal motor neuron precursors as previously defined.

[0105] In some embodiments, post-mitotic spinal motor neurons can be rapidly obtained by exposing spinal motor neuron precursors to a differentiation culture medium containing an inhibitor of the Notch signaling pathway, in particular a gamma secretase inhibitor.

[0106] As used herein, the term "inhibitor of the Notch signaling pathway" refers to any compound (natural or synthetic) that directly or indirectly reduces the activation of the Notch signaling pathway (a series of molecular signals generated as a result of a ligand binding to any member of the Notch family). Preferably, the inhibitor of the Notch signaling pathway is an inhibitor of gamma-secretase. As is well known to those skilled in the art, gamma-secretase inhibitors indirectly inhibit the Notch pathway. Techniques for determining whether a given compound is an inhibitor of gamma-secretase are well known to those skilled in the art and are described, for example, in Yang et al. (2008) Molecules Brain 1:15 or Wang et al. (2009) Molecules 14:3589-2599.

[0107] Inhibitors of Notch signalling, particularly gamma secretase inhibitors, can be natural or synthetic compounds. When the inhibitor of the Notch signalling pathway is a protein, it can be a purified protein, or a recombinant protein, or a synthetic protein.

[0108] Inhibitors of the Notch signaling pathway include γ-secretase inhibitors, particularly 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), compound W (3,5-bis(4-nitrophenoxy)benzoic acid, as described in Okochi et al. (2006) J. Biol. Chem. 281:7890-7898), compound L-685,458 ((5S)-(ylethoxycarbonylamino)-6-phenyl-(4 / 4-diaminophenyl)-2-methyl-2-propanol, as described in Shearman et al. (2000) Biochemistry 39:8698), and the like. : ?)-hydroxy-(2 / : ?)-benzylhexanoyl)-L-leucyl-L-phenylalaninamide.

[0109] Preferably, the inhibitor of the Notch signalling pathway is the compound DAPT.

[0110] Generally, DAPT is present in the differentiation culture medium at a concentration in the range of 1 to 25 μM, more preferably in the range of 5 to 20 μM, especially about 10 μM.

[0111] The differentiation culture medium may also contain other compounds known to contribute to the induction of differentiation of motor neuron precursors into motor neurons. Such compounds are well known to those skilled in the art. Preferably, culture medium C3 further contains BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor). Preferably, BDNF and GDNF are present in the differentiation culture medium at concentrations of 5 to 100 ng / ml each, preferably 10 to 50 ng / ml, and more preferably about 10 ng / ml each.

[0112] In the context of the present invention, the period of exposure of spinal motor neuron precursors to differentiation culture medium is sufficient to induce the expression of motor neuron markers, particularly HB9, ISL1 / 2, HOXA5, HOXA4, and optionally Lhx3 or FOXP1. Techniques for detecting the expression of motor neuron markers are well known to those skilled in the art and include immunostaining, flow cytometry, RT-PCR, or in situ hybridization. Generally, the period may extend for at least 5 days, preferably 5-7 days, and more preferably about 5 days. Generally, an inhibitor of the Notch signaling pathway is added 9 days after initiating the culture of hPSCs in culture medium containing an activator of the Wnt signaling pathway, and the cells are maintained in culture medium for at least 5-7 days, particularly 5 days.

[0113] In some embodiments, the culture period in the presence of retinoic acid and an agonist of the Hedgehog signaling pathway and the culture period in the presence of an inhibitor of the Notch signaling pathway may partially overlap, such that during the overlapping period, the culture differentiation medium also contains retinoic acid and an agonist of the Hedgehog signaling pathway.

[0114] Acquisition of a defined rostrocaudal phenotype of MNs We demonstrate that motor neurons with progressively more caudal identity are generated by exposing axial precursors to agonists of the Hedgehog signaling pathway and retinoic acid. 1. By delaying RA exposure, or 2. By further exposing the axial precursors to an activator of the FGF pathway (FGFR agonist), and / or an activator of the TGF / activin / nodal signaling pathway (also referred to herein for short as the TGF pathway), It was demonstrated that it is possible to generate it.

[0115] Several different fibroblast growth factor FGF isoforms can be used in accordance with the present invention as FGFR agonists. Non-limiting examples of suitable FGFR agonists include FGF1, FGF2, FGF3n, FGF8a, FGF8b, FGF10, FGF22, FGF8f, FGF17, and FGF18; typically, the FGFR agonist is FGF2 or FGF8 (including FGF8a and FGF8b), particularly FGF2.

[0116] The TGF-activin / nodal signaling pathway is particularly associated with activation of Smad2 and Smad3 in response to the interaction of activin, TGF-β, and nodal ligand with the type I receptors ALK4, ALK5, and ALK7. Accordingly, the present invention includes type I receptor ALK4, ALK5, and ALK7 agonists, such as TGFβ family members (including TGFβ, activin, and GDF), and nodal ligands. Non-limiting examples of such activators of the TGF pathway include GDF11 or GDF8.

[0117] Therefore, retinoic acid is generally added to the culture medium for at least 3 to 11 days, and advantageously until at least day 9 (D9), after initiating the culture of hPSCs using a culture medium containing an activator of the Wnt signaling pathway.

[0118] According to the present invention: Upper limb motor neurons, including FOXP1 highly innervated motor neurons, are: 1. By adding RA to the culture medium from about D3 to about D9 after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway, or 2. From about D3 to about D9 after initiation of culturing hPSCs in a culture medium containing an activator of the Wnt signaling pathway, by adding RA to the culture medium in combination with adding an FGFR agonist or an activator of the TGF / activin / nodal signaling pathway (typically GDF11 or GDF8) between D3 and D4 (optionally, the FGFR agonist is added to the culture medium at a concentration of at least 100 ng / ml, particularly between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, particularly about 120 ng / ml, or the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including 20 to 70 ng / ml), It is possible to obtain it.

[0119] Anterior thoracic motor neurons: 1. By adding RA to the culture medium from about at least D5 and up to about D9, particularly from D5 to D9, after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway; or 2. From about D3 or D4 to about D9, particularly from D3 to D9, after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway, adding RA to the culture medium in combination with adding an FGFR agonist or an activator of the TGF / activin / nodal signaling pathway (e.g., GDF11 or GDF8) about 24 to 48 hours before adding RA to the culture medium (optionally, the FGFR agonist is added to the culture medium at a concentration of at least 60 ng, particularly between 60 and 250 ng / ml, between 60 and 200 ng / ml, preferably between 60 and 150 ng / ml, particularly about 120 ng / ml, or the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including 20 to 70 ng / ml); It is possible to obtain it.

[0120] Caudal thoracic motor neurons by adding RA to the culture medium from about D3 to about D9 after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway, in combination with adding a combination of an FGFR agonist and an activator of the TGF / activin / nodal signaling pathway to the culture medium from about D3 to about D4, whereby optionally the FGFR agonist is added at a concentration of at least 100 ng / ml, particularly between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, particularly about 120 ng / ml; and the activator of the TGF / activin / nodal signaling pathway (such as GDF11 or GDF8) is added to the culture medium at a concentration of at least 20 ng / ml, typically including 20 to 70 ng / ml; It can be obtained by

[0121] FOXP1-expressing lumbar motor neurons, including highly arm-innervating motor neurons, adding RA to the culture medium from about D5 to about D9 after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway in combination with adding a combination of an FGFR agonist and an activator of the TGF / activin / nodal signaling pathway (e.g., GDF11 or GDF8) to the culture medium from about D3 or D4 to D5, particularly from D3 to D5, whereby optionally the FGFR agonist is added at a concentration of at least 100 ng / ml, particularly between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, particularly about 120 ng / ml; and the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including 20 to 70 ng / ml; It is possible to obtain it.

[0122] In a particularly preferred embodiment of the present invention, a method for generating a population of spinal motor neurons with a defined rostral-caudal identity comprises the steps of:

[0123] 1. Generation of axial precursors: From day 0 (D0), culturing hPSCs, particularly in the form of human embryoid bodies, in a culture medium comprising at least a Wnt agonist, particularly chosen from CHIR-9921 or Wnt3a protein, and optionally an inhibitor of the BMP signaling pathway and an inhibitor of the TGF / Activin / Nodal signaling pathway (e.g., LDN-193189 and SB431542, respectively), optionally comprising: - the compound CHIR-99021 is added to the culture medium at a concentration ranging from 0.5 to 5 μM, preferably ranging from 1 to 4 μM, and even more preferably at a concentration of about 3 μM; - the compound LDN193189 is added to a culture medium containing hPSCs at a concentration in the range of 0.05 to 1 μM, preferably 0.1 to 0.5 μM, 0.15 to 0.25 μM, even more preferably about 0.2 μM; - compound SB431542 is added to a culture medium containing hPSCs at a concentration in the range of 10 to 75 μM, preferably in the range of 20 to 50 μM, and even more preferably at a concentration of about 40 μM; The inhibitor of the BMP signaling pathway and the inhibitor of the TGF / activin / nodal signaling pathway are added to the culture medium for a period ranging from 3 to 4 days (i.e., D0 to D3 or D0 to D4), and in particular, the inhibitor of the TGF / activin / nodal signaling pathway can be added from D0 to D3; and The Wnt activator is added to the culture medium for a period ranging from 2 to 4 days, preferably 3 to 4 days (i.e., D0 to D2, D0 to D3, or D0 to D4). Process.

[0124] 2. Acquisition of spinal motor neurons with defined rostral-caudal identity: a) adding RA to the culture medium from about D3 to about D9 after initiation of culturing hPSCs in a culture medium containing an activator of the Wnt signaling pathway, and adding an agonist of the Hedgehog signaling pathway at a concentration of at least 300 nM, particularly 100 nM to 1 μM or 100 to 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, between days D3 and D9, between day 4 (D4) and day 9 (D9), between D5 and D9, between D6 and D9, or between D7 and D9, particularly between D3 and D9; or b) From about D3 to about D9 after initiation of culturing hPSCs in a culture medium containing an activator of the Wnt signaling pathway, RA is added to the culture medium, and from D3 to D9 or D10 or more, from day 4 (D4) to day 9 (D9) or D10 or more, from D5 to D9 or D10 or more, from D6 to D9 or D10 or more, or from D7 to D9 or D10 or more, particularly from D3 to D9 or D10 or more, at a concentration of at least 300 nM, particularly between 100 nM and 1 μM or between 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, in combination with the addition of an FGFR agonist or an activator of the TGF / activin / nodal signaling pathway between D3 and D4. Optionally, however, the FGFR agonist is added to the culture medium at a concentration of at least 100 ng / ml, in particular between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, in particular about 120 ng / ml, and the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including between 20 and 70 ng / ml; Optionally, retinoic acid is added to the culture medium at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM, or in the range of 100 nM to 500 nM. Generally, RA is added to the culture medium at a concentration of at least 100 nM.

[0125] Obtaining upper limb motoneurons containing FOXP1 highly innervated motoneurons a') adding RA to the culture medium from about at least D5 and up to about D9, particularly from D5 to D9, after initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway, and adding an agonist of the Hedgehog signaling pathway at a concentration of at least 300 nM, particularly between 100 nM and 1 μM, or between 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably 500 nM, between D3 and D9, between day 4 (D4) and day 9 (D9), between D5 and D9, between D6 and D9, or between D7 and D9, particularly between D3 and D9, or b') After initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway, RA is added to the culture medium at least from about D3 or D4 and up to about D9, particularly from D3 to D9, and a Hedgehog signaling pathway agonist at a concentration of at least 300 nM, particularly from 100 nM to 1 μM or from 100 to 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, between days D3 and D9, between day 4 (D4) and day 9 (D9), between D5 and D9, between D6 and D9, or between D7 and D9, particularly between D3 and D9, in combination with the addition of an FGFR agonist or an activator of the TGF / Activin / Nodal signaling pathway, 24 to 48 hours before adding RA to the culture medium. Optionally, however, the FGFR agonist is added to the culture medium at a concentration of at least 60 ng, in particular between 60 and 250 ng / ml, between 60 and 200 ng / ml, preferably between 60 and 150 ng / ml, in particular about 120 ng / ml, and the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including between 20 and 70 ng / ml; Optionally, retinoic acid is added to the culture medium at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM, or in the range of 100 nM to 500 nM. Generally, RA is added to the culture medium at a concentration of at least 100 nM.

[0126] Obtaining anterior thoracic motoneurons, including FOXP1 highly innervated motoneurons and FOXP1 / SCIP motoneurons a''') After initiation of culturing hPSCs in culture medium containing an activator of the Wnt signaling pathway, RA is added to the culture medium from about D3 to about D9, and between days D3 and D9, between day 4 (D4) and day 9 (D9), between D5 and D9, between D6 and D9, or between D7 and D9, especially between D3 and D9, at a concentration of at least 300 nM, especially between 100 nM and 1 μM or between 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, in combination with the addition of a combination of an FGFR agonist and an activator of the TGF / Activin / Nodal signaling pathway to the culture medium from about D3 to about D4. wherein optionally the FGFR agonist is added at a concentration of at least 100 ng / ml, particularly between 100 and 250 ng / ml, between 100 and 250 ng / ml, typically between 100 and 200 ng / ml or between 100 and 150 ng / ml, particularly about 120 ng / ml; and the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including between 20 and 70 ng / ml; Optionally, retinoic acid is added to the culture medium at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM, or in the range of 100 nM to 500 nM. Generally, RA is added to the culture medium at a concentration of at least 100 nM.

[0127] Obtaining caudal thoracic motoneurons a'''') After initiating the culture of hPSCs in a culture medium containing an activator of the Wnt signaling pathway, RA is added to the culture medium from about D5 to about D9, and a concentration of at least 300 nM, particularly 100 nM to 1 μM or 100 to 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, of an agonist of the Hedgehog signaling pathway is added to the culture medium from D3 to D9, from day 4 (D4) to day 9 (D9), between D5 and D9, between D6 and D9, or between D7 and D9, particularly between D3 and D9, in combination with the addition of a combination of an FGFR agonist and an activator of the TGF / Activin / Nodal signaling pathway to the culture medium by about D3 or D4 and D5, particularly between D3 and D5. However, optionally, the FGFR agonist is added at a concentration of at least 100 ng / ml, particularly between 100 and 250 ng / ml, between 100 and 200 ng / ml, or between 100 and 150 ng / ml, particularly about 120 ng / ml; and the activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, typically including 20 to 70 ng / ml; Optionally, retinoic acid is added to the culture medium at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM, or in the range of 100 nM to 500 nM. Generally, RA is added to the culture medium at a concentration of at least 100 nM.

[0128] Obtaining lumbar motoneurons containing FOXP1-rich motoneurons highly innervating the arm

[0129] 3. Optionally, differentiation of spinal motor neuron precursors into post-mitotic spinal motor neurons: Approximately 9 days after initiating the culture of hPSCs in culture medium containing an activator of the Wnt signaling pathway, an inhibitor of the Notch signaling pathway, typically DAPT, is added for a period of at least 5-7 days or more, typically 5 days, optionally with the proviso that DAPT is present in the culture medium at a concentration in the range of 1-25 μM, more preferably in the range of 5-20 μM, particularly about 10 μM.

[0130] A representative scheme of an advantageous method for obtaining spinal motor neurons with a defined rostral-caudal identity is presented in particular in Figure 4A.

[0131] Culture medium In the context of the present invention, the term "culture medium" refers to a liquid medium suitable for the in vitro culture of mammalian cells. The culture medium used in the methods of the present invention may be based on a commercially available medium, such as DMEM / F12 sold by Invitrogen, or a mixture of DMEM / F12 and Neurobasal in a 1:1 ratio, also sold by Invitrogen.

[0132] The culture medium used in the methods of the present invention may also contain various supplements, such as B27 supplement (Invitrogen) and N2 supplement (Invitrogen).

[0133] B27 supplements contain, among other components, SOD, catalase, and other antioxidants (GSH), as well as unique fatty acids such as linoleic acid, linolenic acid, and lipoic acid.

[0134] The culture medium used in the method of the invention can in particular be based on N2B27 medium.

[0135] The term "N2B27" refers to the medium described in Liu et al. (2006) Bioc em. Biop ys. Res. Commun. 346:131-139, which contains DMEM / F12 medium and Neurobasal medium (1:1 ratio), N2 supplement (1 / 100), B27 supplement (1 / 50), and β-mercaptoethanol (1 / 1000).

[0136] Preferably, the culture medium used in the methods of the present invention further comprises the ROCK inhibitor Y-27632, generally at a concentration of about 5 μM.

[0137] In the method of the present invention, the culture medium can be partially or completely renewed at regular intervals, if necessary. Generally, the culture medium can be replaced with fresh culture medium every other day for the periods described below.

[0138] In some embodiments, the present invention also encompasses the following culture media:

[0139] A culture medium suitable for obtaining upper limb motoneurons, including FOXP1 highly innervated motoneurons, comprises at least the following: - an agonist of the hedgehog signaling pathway at a concentration of at least 300 nM, in particular between 100 nM and 1 μM or between 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, an FGFR agonist (optionally, the FGFR agonist is added to the culture medium at a concentration of at least 100 ng / ml, in particular between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, in particular about 120 ng / ml), or an activator of the TGF / activin / nodal signaling pathway (for example GDF11 or GDF8) at a concentration of at least 20 ng / ml, typically including 20 to 70 ng / ml, and - retinoic acid at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM, or in the range of 100 nM to 500 nM. Generally, RA is added to the culture medium at a concentration of at least 100 nM.

[0140] A suitable culture medium for obtaining anterior thoracic motoneurons contains at least the following: - an agonist of the hedgehog signalling pathway at a concentration of at least 300 nM, in particular between 100 nM and 1 μM or 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM. - FGFR at a concentration of at least 60 ng, in particular between 60 and 250 ng / ml, between 60 and 200 ng / ml, preferably between 60 and 150 ng / ml, in particular about 120 ng / ml, or an activator of the TGF / activin / nodal signalling pathway (e.g. GDF11 or GDF8) at a concentration of at least 20 ng / ml, generally including 20 to 70 ng / ml. retinoic acid at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM or in the range of 100 nM to 500 nM, generally at a concentration of at least 100 nM.

[0141] A suitable culture medium for obtaining caudal thoracic motoneurons contains at least the following: - an agonist of the hedgehog signalling pathway at a concentration of at least 300 nM, in particular between 100 nM and 1 μM or 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM. - an FGFR agonist is added at a concentration of at least 100 ng / ml, in particular between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, in particular about 120 ng / ml; and an activator of the TGF / activin / nodal signaling pathway is added to the culture medium at a concentration of at least 20 ng / ml, generally including between 20 and 70 ng / ml. retinoic acid at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM or in the range of 100 nM to 500 nM, generally at a concentration of at least 100 nM.

[0142] A culture medium suitable for obtaining lumbar motor neurons, including FOXP1 highly arm-innervating motor neurons, comprises at least the following: - an agonist of the hedgehog signaling pathway at a concentration of at least 300 nM, in particular between 100 nM and 1 μM or between 100 and 1 μM, preferably in the range of 300 to 750 nM, even more preferably about 500 nM, - FGFR agonists and activators of the TGF / activin / nodal signaling pathway, where the FGFR agonists are at a concentration of at least 100 ng / ml, in particular between 100 and 250 ng / ml, between 100 and 200 ng / ml, preferably between 100 and 150 ng / ml, in particular about 120 ng / ml; and activators of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml, typically including between 20 and 70 ng / ml, retinoic acid at a concentration in the range of 5 to 1000 nM, preferably in the range of 10 to 500 nM or in the range of 100 nM to 500 nM, generally at a concentration of at least 100 nM.

[0143] The invention is further illustrated by the following figures and examples. [Brief explanation of the drawings]

[0144] [Figure 1]This figure shows that axial precursors derived from aged hPSCs generate progressively more caudal motor neuron subtypes. (A) Schematic summary of the data in Figure S1. MNs (shown here in gray) defined by ISL1 or HB9 expression are organized into motor columns within the ventral horn of the spinal cord. The HOX expression profile within MNs and the localization of MNs expressing high levels of FOXP1 and SCIP are shown in the human embryonic spinal cord at 6.3 and 7.5 weeks of gestation. Changes in shape suggest an increase or decrease in the number of MNs expressing a given marker. FOXP1-high MNs are selectively observed in the lateral motor column (LMC) of the upper limb thoracic and lumbar spinal cord. SCIP-high MNs are a subset of LMC MNs within the caudal upper limb spinal cord. (B) Differentiation conditions used in C–F (with controlled exposure times to RA / SAG). (C) Immunostaining for ISL1, HB9 (MNs), NEFL (neurons), HOX transcription factors, FOXP1, and SCIP was performed on cryostat sections of EBs at day 14 of differentiation. Delaying RA application resulted in more caudal MNs. FOXP1 and SCIP MNs were primarily generated at RA days 4 and 5 (further defining the rostrocaudal identity of MNs in the HOXC8+ condition). Scale bar: 100 μM (D–F). Percentage of MNs (ISL1+ cells) expressing the indicated markers. Data are presented as mean ± SD. Each circle represents an independent experiment. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. See also Figures S1 and S2. [Figure 2]Temporal transcriptome analysis of hPSC-derived axial precursors. (A) Experimental design. (B) Comparison of the 20 most enriched genes in D2 precursors with hESCs. The precursors acquired a transcriptome signature similar to one of the mouse axial precursors. (C) Fold enrichment of the 20 most enriched genes in neuromesodermal precursor (NMP) genes defined by (Goui et al., 2017) compared with D3 precursors and hESCs. (D) Immunostaining for axial precursor markers on hiPSC-derived D2 and D3 precursors (WTS2 lineage). Cells express three markers. Scale bar: 40 μM. (E) Temporal transcriptional changes in HOX genes encoding HOXs locally expressed in human MNs in vivo. (F) Reactome pathway analysis of 232 genes upregulated 2-fold (p-value < 0.05) from D3 to D2. Y-axis: FDR = false positive rate. X-axis = enrichment score calculated for a given Reactome pathway. (G) Temporal evolution of upregulated MAPK target genes and FGF ligands in aging axial progenitors. ETV5 is a transcription factor activated by ERK1 / 2 MAPK in many other systems. Other genes encode feedback negative regulators of the MAPK pathway. All data are shown as mean ± SD. (H) Schematic of transcriptional and immunostaining analysis of day 2 and 3 progenitors. [Figure 3]This figure shows that FGF pathway inhibition impairs the temporal induction of HOX and caudal MN specification. (A) Differentiation conditions. PD173074 (FGFR1-3 inhibitor) or PD032590 (MEK1 / 2 inhibitor) was added from D3 to D7. (B) Percentage of MNs (ISL1+ cells) in different conditions (mean ± SD). (C) Immunostaining for ISL1 (MNs) and HOX transcription factors was performed on cryostat sections of embryoid bodies at day 14 of differentiation. MEK and FGFR inhibitors prevent the specification of HOXC8 and HOXC9 MNs. Instead, HOXA5 and HOXC6 MNs are generated. Scale bar: 100 μM. (C) Quantification of HOXC6 and HOXC9 MNs at day 14 of differentiation. (D) Real-time PCR analysis of HOX mRNA in precursors at days 3, 4, and 5 of differentiation. MEK and FGFR inhibitors prevent the temporal increase in caudal HOX expression. Data are shown as mean ± SD; each circle represents an independent experiment. *P≦0.05, **P≦0.01, and ***P≦0.001. [Figure 4]Dynamic pacing of HOX induction and specification of individual MN subtypes upon varying FGF2 and GDF11 levels. (A) Differentiation conditions. Exogenous cues, FGF2, GDF11, or FGF2 + GDF11, were added at various concentrations or for different durations on day 3 of differentiation. (B) Immunostaining for HOX was performed on cryostat sections of embryoid bodies on day 14 of differentiation. FGF2, GDF11, and FGF2 / GDF11 induce more caudal MN subtypes. Scale bar: 100 μM. (C, D, E) Percentage of MNs (ISL1+ cells) expressing the indicated markers. The effects of duration of FGF2 treatment (C), FGF2 concentration (D), and duration of GDF11 or FGF2 + GDF11 (E) were monitored. (F) Real-time quantitative PCR analysis of the expression of locally expressed HOX genes in human MNs in vivo. On D4 (24 hours after treatment) and D5 (48 hours after treatment), HOX mRNA was monitored upon addition of FGF2, GDF11, or a combination of GDF11 (25 ng / ml) and FGF2 (120 ng / ml). Data are normalized to the control condition treated with RA alone on day 3 at each time point. Data are shown as mean ± SD. Each circle represents an independent experiment. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. See also Figure 5. [Example]

[0145] result Materials and Methods Human Embryonic Spinal Cord Histology Human fetal embryos at 6.3 weeks gestation were obtained from pregnant women referred to the Gynecology and Obstetrics Department of the Antoine Beclere Hospital (Clamart, France) for legally induced abortions in the first trimester, as previously described (Lambrot et al., 2006). All women provided written consent for the scientific use of fetal tissue. None of the abortions were due to fetal abnormalities. Fetal age was determined by measuring limb and foot lengths (Evtouchenko et al., 1996). This project was approved by the local medical ethics committee and the French Agency for Biomedical Sciences (reference number PFS 12-002). Alternatively, human embryonic spinal cords (n = 2) at 7.5 weeks gestation were collected in accordance with US (National Institutes of Health, US Food and Drug Administration) and New York State guidelines, as well as the institutionally approved ethical guidelines of Columbia University for anonymous tissue. Material was obtained after induced abortion and classified based on external morphology according to the Carnegie developmental stages. Gestational age was determined by the patient's last menstrual period or by ultrasound if the ultrasound estimate differed by one week from the obstetrician's. In all cases, spinal cords were removed as intact as possible, fixed in freshly cooled 4% PFA on ice for 1.5 hours, washed extensively with PBS, and then cryoprotected overnight in 30% sucrose. After fixation, spinal cords were measured and cut into anatomical sections to facilitate embedding in OCT Compound (Leica) and stored at -80°C before cutting on a cryostat. Sections (16 μM) were cut along the entire length of the spinal cord.

[0146] Human pluripotent stem cell lines The human SA001 embryonic stem cell (ESC) line (male, RRID: CVCL_B347) was obtained from Cellectis and used in accordance with current French regulations (Agency of Biomedicine, license number AFSB1530532S). The induced pluripotent stem cell (iPSC) line WTSIi002 (male, RRID: CVCL_AH30, also known as HPSI0913i-eika_2) was obtained from the European Bank for Pluripotent Stem Cells (EBISC). WTC-mEGFP-Safe harbor locus (AAVS1)-cl6, generated by the Allen Cell Institute, was obtained from Coriell (catalog number AICS-0036-006, male, RRID: CVCL_JM19). ~ were obtained from the European Bank for Pluripotent Stem Cells (EBISC). Approval for experiments using iPSCs was obtained from the relevant ethical committee (declaration DC-2015-2559). All lines were cultured on Matrigel (Corning) in mTSER1 medium (Stem Cell Technologies) at 37°C and amplified using an EDTA (Life Technologies) clamp-based passaging method. The lines were tested for possible mycoplasma contamination every other week (MycoAlert™ Mycoplasma Detection Kit, Lonza, LT07-118). No contamination was detected during the study period. PSCs were thawed in the presence of Y-27632 (10 μM, Stemgent or Stem Cell Technologies), and the culture medium was changed daily.

[0147] Differentiation of human pluripotent stem cells Human PSC embryoid body-based differentiation was performed as previously described (Maury et al., 2015). hPSCs were dissociated using chilled Accutase (Life Technologies) at 37°C for 3–5 min and resuspended in differentiation medium N2B27 (Advanced DMEM F12, Neurobasal vol:vol (Life Technologies)) supplemented with N2 (Life Technologies), vitamin A-free B27 (Life Technologies), 1% penicillin / streptomycin, 0.1% β-mercaptoethanol (Life Technologies), Y-27632 (10 μM, Stemgent or Stem Cell Technologies), CHIR-99021 (3 μM or 4 μM, Selleckchem), SB431542 (20 μM, Selleckchem), and LDN 193189 (0.1 μM, Selleckchem). cells 2×10 5 Embryoid bodies (EBs) were formed by seeding cells at 1000 / ml into ultra-low attachment 6-well plates (Corning). All differentiation conditions were fed with the same medium on days 2 and 3, except for removing SB431542 on day 3. Differentiation then proceeded according to the schema presented in the figure. SAG (smoothened agonist, Merck Millipore), FGF2 (recombinant human FGF basic, Peprotech), RA (Sigma-Aldrich), GDF11 (recombinant human / mouse / rat GDF11, Peprotech), PD0325901 (Selleckchem), PD173074, SCH772984 (Selleckchem), and DAPT (Stemgent) were added at the indicated time points. See Table S2 for concentrations. Medium was changed every other day (unless otherwise specified).

[0148] Embryoid body processing for immunostaining EBs were harvested, rinsed with PBS, and then fixed with 4% PFA for 5 minutes at 4°C, followed by three 5-minute rinses with PBS. EBs were cryoprotected with 30% sucrose and embedded in OCT (Leica) before sectioning using a cryostat. Alternatively, day 2, 3, and 4 progenitors were seeded onto Matrigel (Corning, diluted according to the manufacturer's recommendations)-coated coverslips and allowed to adhere for 30 minutes before being fixed with 4% PFA for 5 minutes at 4°C.

[0149] immunostaining All immunostaining was performed as follows: cells or slices were incubated with a saturation solution (PBS / 10% FBS / 0.2% Triton) for 10 minutes. Primary antibodies (Table S3) were diluted in staining solution (PBS / 2% FBS / 0.2% Triton) and incubated overnight at 4°C in a humidified chamber. After four PBS washes (10 minutes each), secondary antibodies (Alexa488, Alexa568, and Alexa647, Life Technologies, 1:1,000) were added for 1 hour at room temperature. After three PBS washes, DAPI (Invitrogen, 1:3,000) was added to the cells for 5 minutes. Cells or slices were then embedded in Fluoromount (Sigma-Aldrich or Cliniscience).

[0150] Image acquisition Samples were visualized and imaged using a ZEISS LSM 880 confocal laser scanning microscope (Carl Zeiss Microscopy) controlled by Zen black software, a confocal microscope TCS SP5 II (Leica), or a DM6000 microscope (Leica) equipped with a CoolSNAP EZ CDD camera controlled by MetaMorph software (MetaMorph Inc.) Alternatively, images were acquired using an automated microscope Cell Discoverer 7 (Zeiss) equipped with an Axiocam 506m camera with Zen black software (Zeiss).

[0151] Quantitative RT-PCR analysis Total RNA was extracted (RNAeasy Plus Mini Kit, Qiagen), and cDNA was synthesized using SuperScript III (Invitrogen). Quantitative real-time PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems) with Sybr Green PCR Master Mix (Applied Biosystems). Alternatively, QuantStudio 5 Real-Time PCR System (Thermofisher Scientific) and qPCR Brilliant II SYBR MM with low ROX (Agilent) were used. Primers are listed in Table 3 in the Appendix. All expression data were normalized to Cyclophilin A mRNA. All analyses were performed with three technical replicates per plate. Relative expression levels were determined by calculating 2-ΔΔCt.

[0152] Transcriptome analysis hESCs were collected after dissociation and before exposure to differentiation medium. Progenitors were collected on days 2, 3, and 4 of differentiation. For each of the eight samples, total RNA was extracted and then reverse-transcribed using the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermofisher Scientific). cDNA libraries were amplified and barcoded using the AmpliSeq Transcriptome Human Gene Expression core panel and the Ion Xpress Barcode Adapter (Thermofisher Scientific). After amplicon quantification using the Agilent High Sensitivity DNA kit, samples were pooled into sets of eight. Emulsion PCR and enrichment were performed on the Ion OT2 system instrument using the Ion PI Hi-Q OT2 200 kit (Thermofisher Scientific). Samples were loaded onto an Ion PI v3 Chip and sequenced on an Ion Proton System using the Ion PI Hi-Q sequencing 200 kit chemistry (200 bp read length; Thermofisher Scientific). Ion Proton reads (FASTQ files) were imported into the RNA-seq pipeline of Partek Flow software (v6, Partek Inc.) using hg19 as the reference genome. The number of reads per sample ranged from 7.5 million to 12 million reads. To determine differentially expressed genes between groups, mapped reads were quantified using the Partek E / M algorithm normalized by total counts / sample (resulting counts represent gene expression levels (reads / million) across 20,800 distinct genes present in the AmpliSeq Human Gene Expression panel). Evaluation of differential expression between two conditions was performed using the EdgeR package under R.Pathway enrichment analysis was performed for up-regulated genes (FC≥2.0, p-value<0.05) between the two time points by obtaining information from the Reactome database. Significant enrichment was calculated using the hypergeometric test and Benjamini-Hochberg correction for multiple comparisons. Enrichment scores were calculated as described (Wang et al., 2017).

[0153] Normalized transcriptome data are presented in Table S1. Raw data are available upon request.

[0154] Quantitative and statistical analysis All statistics were computed using GraphPad Prism software. One-way analysis of variance (ANOVA) with Kruskall-Wallis post-hoc analysis was performed after performing the normality test provided by Prism. The number of n, variance index, and p-value are shown in the figure legends. In all figures, n represents independent differentiations initiated from independent, freshly thawed hPSC vials. For each condition, at least four independent EBs were imaged, and all cells were quantified using automated image analysis. Images were exported and, if necessary, saved as TIFFs using Fiji (Schindelin et al., 2012). Quantitative analysis of images was performed using CellProfiler software (Carpenter et al., 2006) (open source software from the Broad Institute, available at www.cellprofiler.org). DAPI-stained nuclei were segmented into primary objects using the CellProfiler segmentation pipeline, and nuclear masks were used to define the objects on the target channels. A threshold for defining nuclei positive for a given target was obtained using EB sections negative for the target of interest. All images across conditions were then automatically analyzed as a batch to ensure unbiased analysis. Analysis of FOXP1 and SCIP immunostaining intensity was performed using CellProfiler in conjunction with the software FCS express 7 (DeNovo Software, Glendale, CA, USA). Nuclei were segmented into primary objects as described above, and FOXP1 and SCIP fluorescence intensities were calculated for each primary object. Next, using FCS express 7 software, fluorescence intensity plots for FOXP1 and SCIP were generated to visualize the intensity levels of the different markers for each individual cell and to determine the percentage of cells above a given threshold. A CellProfiler pipeline for quantification is available upon request.

[0155] result HOX expression profile and motor neuron subtypes in the human embryonic spinal cord Differential expression of HOX transcription factors along the vertebrate spinal cord is a major product of HOX gene regulation. In spinal motor neurons, this Hox code directs the specification of subtype-specific properties that control the formation of locomotion circuits (Dasen, 2017; Philippidou and Dasen, 2013). Whether the spinal HOX code and associated MN subtypes are conserved in humans remains unclear, hindering a reliable assessment of HOX regulation and its relationship to cellular "rostrocaudal" identity during hPSC differentiation. Therefore, we mapped seven HOX transcription factors that show colinear expression patterns in human embryos at 6.3 and 7.5 weeks of development and direct MN subtype specification in mice and chicks (Philippidou and Dasen, 2013) (Figure 1A). Similar to animal models, human MNs expressed either ISL1 or HB9 along the spinal cord (Figure 1A) (Amoroso et al., 2013). Within MNs, HOX expression showed a rostrocaudal pattern similar to that in mice and chicks (Dasen et al., 2003; Liu et al., 2001): cervical MNs expressed HOXA / C5, whereas upper limb MNs expressed HOXC6, thoracic MNs HOXC9, and lumbar MNs HOXC10. Caudal upper limb MNs coexpressed HOXC6 and HOXC8, and anterior thoracic MNs coexpressed HOXC8 and HOXC9. HOXD9, together with HOXC10, was associated with caudal thoracic MNs and anterior lumbar MNs (Figure 1A). In mesenchymal neurites, this Hox code directs the formation of distinct motor columns that innervate common muscle groups, and motor pools that innervate single muscles (Philippidou and Dasen, 2013). To be able to assess in vitro whether changes in HOX expression lead to the specification of the corresponding neuronal subtypes, we mapped MN subtype markers relative to HOX expression domains (Figure 1A). Similar to mice and chicks, MNs express high levels of FOXP1 (FOXP1 highMNs expressing FOXP1 were observed at the upper limb-thoracic (HOXC6 and HOXC8 / HOXC9) and lumbar (HOXC10) levels (Fig. 1A). These MNs formed the lateral motor column (LMC), where limb-innervating MNs are located (Amoroso et al., 2013; Routal and Pal, 1999). This FOXP1 high Within the LMC, SCIP / HOXC8 MNs were found in the caudal upper limb spinal cord. In contrast, SCIP / HOXC8 / HOXC9 MNs were located in the anterior thoracic region. Their location and transcriptional coding identify them as putative hand-controlling MNs (Bell et al., 2017; Mendelsohn et al., 2017).

[0156] Overall, HOX transcription factors are expressed regionally along the rostrocaudal axis of the human spinal cord. Within these distinct HOX domains, distinct MN subtypes (identifiable by combinations of transcription factors) are generated in typological locations. This data provided a readout for assessing the mechanisms controlling HOX expression in axial precursors and its impact on cell type specification during hPSC differentiation.

[0157] Aging axial precursors generate more caudal neuronal subtypes We first sought to test whether axial precursors (i.e., precursors with the capacity to generate caudal cell types with distinct rostrocaudal identities) could be generated using 3D differentiation of human embryonic stem cells (hESCs) by monitoring MN subtype specification. We previously reported a sequence of extrinsic cues that triggers the targeted generation of spinal cord MNs from human PSCs through a putative axial precursor stage ( Maury et al., 2015 ). Exposure of embryoid bodies (EBs) to a Wnt pathway agonist (CHIR99021) and inhibitors of the BMP and TGFβ pathways (SB431542 and LDN193189) generated precursors expressing CDX2, a marker of axial precursors and a regulator of caudal HOX gene induction ( Bel-Vialar et al., 2002 ; Bialecka et al., 2010 ; Gouti et al., 2014 ; Maury et al., 2015 ; Mazzoni et al., 2013 ; Neijts et al., 2017 ). Spinal cord MNs (70% of cells, on average) were generated on days 2 or 4 following exposure to retinoic acid (RA) and sonic hedgehog pathway (SAG) agonists, which promote neurogenesis and induce axial precursors to ultimately become ventral MNs, respectively (Briscoe and Novitch, 2008; Maury et al., 2015; Ribes et al., 2009). Here, we assessed the rostrocaudal identity of MNs generated under these conditions (Figure 1B). Efficient generation of spinal cord MNs was confirmed, as previously demonstrated, by staining for HB9, ISL1, and the pan-neuronal marker neurofilament light chain (NEFL) along with quantification of ISL1+ cells (Maury et al., 2015) (Figure 1C). Analysis of HOX expression revealed that exposure to RA / SAG from day 2 (D2) to D9 resulted in HOXC6 MNs, which corresponded to anterior upper limb MNs; an identity acquired by most MNs after addition of RA / SAG at D3–9 (74.6% of HOXC6 MNs and 17.3% of HOXC8 MNs) ( Fig. 1D , Fig. 1E ).Addition of RA / SAG on D4–9 generated MNs with a caudal upper limb identity (41.8% of HOXC6 / C8), 37.1% of which expressed high levels of FOXP1 (corresponding to limb-innervating MNs in the spinal cord) (Figure 1D–F). Overall, these results suggested that MN subtype identity depends either on i) the duration of exposure to RA (shorter RA exposure promotes caudalization) or ii) the timing of RA exposure, as previously suggested (Bel-Vialar et al., 2002; Del Corral and Storey, 2004; Lippmann et al., 2015). To distinguish between these possibilities, D3 precursors were exposed to RA for shorter periods (D3–5, D3–8, and D3–9). None of these shorter RA treatments promoted the birth of more caudal MNs. These results revealed that the day on which precursors are exposed to RA / SAG is the primary trigger for caudalization. Further delays in RA / SAG induced even more caudal MN subtypes. At D5, MNs with an anterior thoracic identity were generated (59.8% of HOXC8 / C9) (Fig. 1B-E), of which 27.6% acquired a FOXP1+ limb-innervating identity (Fig. 1F). HOXC9 / FOXP1 / SCIP (located in the human anterior thoracic spinal cord and likely corresponding to hand-innervating MNs) were almost exclusively observed under this condition (Fig. 1F, S2F-G). Thus, RA / SAG (D6 or D7) significantly increased the expression of HOXC9 and FOXP1. high As evidenced by the loss of MNs, MNs were defined to acquire a mid-thoracic identity (Figures 1B-F, S2F-G). Using an induced PSC (iPSC) line, we confirmed that the gradual progression of caudalization of MN identity occurred when the addition of RA / SAG was gradually delayed. Importantly, increasing concentrations of CHIR (3-4 μM) were required to generate a homogenous population of CDX2-expressing precursors at D3 and observe the subsequent specification of caudal MN subtypes (Figures S2H-J). This suggests lineage differences, an important finding for future research.

[0158] Overall, Wnt activation in combination with TGF-BMP pathway inhibition converts hPSCs into precursors capable of expressing distinct HOX combinations and generating progeny found in distinct rostrocaudal locations within the human embryo. These precursors are therefore qualified as axial precursors. The duration of the time window between Wnt and RA defines the final positional identity of the progeny.

[0159] Concurrent induction of caudal HOX genes and FGF target genes in aging axial precursors Next, we sought to define the molecular changes that occur over time in aging axial precursors before RA exposure. We performed comparative transcriptome analysis of hESCs and hESC-derived axial precursors at days 2, 3, and 4 of differentiation (Fig. 2A and Table S1). Pathway analysis of genes that were enriched more than two-fold (p < 0.05) in precursors compared with hESCs suggested activation of the Wnt pathway in parallel with transcriptional activation of HOX genes (not shown). Consistent with their axial potential, D2, D3, and D4 progenitors are highly enriched in transcripts that characterize cells of the mouse caudal lateral epiblast (in which axial progenitors reside), including SOX2, CDX1 and 2, TBXT (BRACHYURY), FGF17, RXRG, SP5 / 8, WNT5A / B, WNT8A, FGF8, GRSF1, CYCTM1, and HES3; the loss of most pluripotency markers, and the absence of representative markers of the nodal cell, mesoderm, and allantois (Figures 2B-C and Table S1) (Cambray and Wilson, 2007; Edri et al., 2019; Gouti et al., 2014; Gouti et al., 2017; Henrique et al., 2015; Koch et al., 2017; Wymeersch et al., 2017). We demonstrated that D2, D3, or D4 precursors express 122 of the 142 genes that define early and late NMPs (Gouti et al., 2017; Henrique et al., 2015; Tzouanacou et al., 2009; Wymeersch et al., 2019). Furthermore, their transcriptomes are highly similar to those of one of the mouse bipotential neuromesodermal precursors (NMPs), which represent a population of axial precursors (Gouti et al., 2014; Gouti et al., 2017; Henrique et al., 2015; Tzouanacou et al., 2009; Wymeersch et al., 2016). Of the 142 genes defining early and late NMPs (Gouti et al., 2017), D2, D3, or D4 precursors expressed 122 of these genes, including the most enriched genes when comparing precursors to hESCs (Figure 2C).Accordingly, D2 and D3 axial precursors co-expressed SOX2, CDX2, and low levels of TBXT at the protein level, a signature associated with NMPs (Attardi et al., 2018; Denham et al., 2015; Diaz-Cuadros et al., 2020; Edri et al., 2019; Faustino Martins et al., 2020; Gouti et al., 2014; Gouti et al., 2017; Lippmann et al., 2015; Metzis et al., 2018; Olivera-Martinez et al., 2012; Wymeersch et al., 2019) (Figure 2D). Overall, these results demonstrated that Wnt-induced hPSC-derived axial precursors resemble those in the mouse anterior caudal lateral epiblast, which feeds the spinal cord outgrowth ( Edri et al., 2019 ; Gouti et al., 2014 ; Gouti et al., 2017 ; Henrique et al., 2015 ; Koch et al., 2017 ; Liu et al., 2001 ; Wymeersch et al., 2016 ).

[0160] Next, comparative analysis of the transcriptome of aging progenitors suggested temporally colinear activation of HOX genes, which exhibited a localized expression pattern within the spinal cord. At D2, progenitors expressed HOXA5 and low levels of HOXC6 (which belongs to the 3' half of the HOX complex with an anterior boundary in the anterior spinal cord) (Fig. 1A, S1). At D3, HOXC8 was well expressed, and HOXC9 expression had begun, both of which belong to the central part of the HOXC complex expressed in the central part of the spinal cord (Fig. 1A, S1). Their expression further increased in progenitors (3.8-fold and 3.4-fold, respectively) at day 4, when exposed to RA / SAG, inducing a portion of HOXC8 and HOXC9 MNs (Fig. 1C-D). The non-expressing HOX genes represented a group of 10–13 paralogs (the latest and most caudally expressed HOXs) (Gaunt, 1991; Izpisua-Belmonte et al., 1991; Philippidou and Dasen, 2013) and Figure 1A). The temporality of colinear activation in axial precursors is therefore consistent with the generation of more caudal MNs when RA / SAG is delayed (Figure 1). Thus, Wnt induced temporally colinear activation of HOX genes, which paralleled changes in the rostrocaudal potential of precursors.

[0161] To define pathways activated in parallel with the sequential induction of HOX genes, we performed pathway analysis on genes that increased more than twofold (p<0.05) between D2 and D3. We detected enrichment for 232 genes corresponding to annotations associated with mitogen-activated protein kinase (MAPK) pathway activation, due to the gradual increase in expression of representative MAPK target genes (ETV5, DUSP4, DUSP6, IL17DR, or SPRY2) (Figure 2F-H). MAPKs are classical mediators of FGF signaling (Lunn et al., 2007). Consistent with the increase in MAPK target genes, we observed increased expression of FGF8 and FGF17—two secreted FGF ligands previously described to increase over time in the caudal epiblast of chick embryos (Figure 2G-H) (Liu et al., 2001; Wymeersch et al., 2019).

[0162] Thus, the sequential colinear expression of HOX genes is paralleled by increased expression of FGF ligands and MAPK target genes within axial precursors. Because FGF promotes caudal Hox genes in other systems ( Bel-Vialar et al., 2002 ; Dasen et al., 2003 ; Liu et al., 2001 ), we hypothesized that paracrine or autocrine FGF signaling could trigger the sequential induction of HOX genes.

[0163] FGF signaling is required for sequential activation of HOX and caudal MN specification We focused on testing whether endogenous FGF signaling is required for the temporal shift in the rostrocaudal potential of axial precursors and HOX gene induction. To this end, we blocked FGF signaling in aging precursors before RA exposure and tested the effects on caudal HOX gene induction and MN subtype specification. From D3 to D7, precursors were exposed to i) PD173074 (a selective FGFR1 / 3 antagonist) or ii) PD0325901 (which inhibits the MAPK kinase MEK1 / 2) (Figure 3A). Although fewer EBs were collected in the PD173074 condition, the efficiency of MN generation was not affected by the two inhibitors (Figure 3B). In the control condition, RA / SAG induced MNs expressing HOXC8 (97.5%) and C9 (87.6%) at D7. In contrast, adding the inhibitors at D3 and RA / SAG at D7 generated MNs with an anterior upper limb identity (HOXC6). This more anterior identity is normally acquired when RA is added to early D3 precursors, indicating that the inhibitor blocks the temporal change in the rostrocaudal potential (Figure 3A-C). Next, we tested whether the dissociation between precursor age and offspring rostrocaudal identity is associated with impaired continuity of HOX gene induction in axial precursors. After inhibiting MEK1 / 2 at D3, we monitored the expression of HOXC6, C8, and C9 mRNA. In control conditions, the three genes increased over time, whereas MEK inhibition blocked this increase at D4 or D5 (Figure 3D). Thus, inhibition of FGF signaling in aging axial precursors blocks the temporal induction of HOX and the specification of caudal cell types. Our transcriptome analysis showed a temporal increase in FGF8 and FGF17 expression, suggesting that increasing FGF concentrations and / or duration of exposure may determine the pace of the HOX clock.

[0164] FGF levels determine the pace of the HOX clock and MN subtype specification To test whether environmental FGF levels determine the pace of the HOX clock, early D3 precursors were exposed to RA / SAG along with FGF2 at different concentrations and for different durations. In all FGF2-exposed conditions, a more caudal MN identity was induced, even without the need for a delay in RA addition (Figures 4A–4D). Thus, the extent of caudalization varied with FGF parameters. Exposing precursors to increasing concentrations of FGF2 from D3 to D9 induced progressively more caudal MN subtypes. Caudal upper limb HOXC8 MNs were induced at 15 ng / ml (a 68.2%, 8.6-fold increase), and HOXC9+ / HOXC6- thoracic MNs were induced at 60 ng / ml (a 58.9%, 82.5-fold increase), whereas at 120 ng / ml, HOXC6 MNs were slightly reduced (Figures 4A–4D, S6A–H). Similar results were obtained with FGF8 (Figure S3B). Addition of 120 ng / ml FGF2 for 24 or 48 hours induced MNs with caudal upper limb or mid-thoracic identity (49.7% HOXC6, 79.4% HOXC8, 14.7% HOXC9, 47.9% FOXP1 at 24 hours). high / SCIP) (at 48 hours, 7.5% HOXC6, 76.4% HOXC9, 13.2% FOXP1 high / SCIP) (Figures 4A-4C, S3C-H), FGF acted directly and rapidly on the axial precursors.

[0165] To determine whether this caudalization effect was manifested by accelerated induction of limb and thoracic HOX genes, real-time PCR analysis was performed 24 and 48 hours after FGF2 treatment. Early increases in HOXC8, HOXC9, HOXD9, and HOXC10 expression were observed compared with RA / SAG controls (Fig. 4F).

[0166] Thus, a precocious increase in FGF signaling promoted the induction of caudal HOX genes in early axial precursors, resulting in the specification of more caudal cell types within the same differentiation timeline (14 days). These results demonstrate that the level or duration of FGF signaling dynamically determines the tempo of colinear HOX activation during human axial precursor differentiation.

[0167] FGF and GDF11 act cooperatively to further accelerate the HOX clock FGF2 accelerated HOX induction but generated MNs only up to the midthoracic level, suggesting that early axial precursors may not have the capacity to generate more caudal segments. Alternatively, other exogenous factors are required to further accelerate HOX gene induction and promote a more caudal identity. GDF11 is a member of the TGF family involved in controlling axial outgrowth and MN subtype specification. It is also required for the expression of the 5'-most HOX genes, starting with 10 paralogs, in late NMP-like cells in vivo and in vitro (Aires et al., 2019; Gaunt et al., 2013; Lippmann et al., 2015; Liu, 2006; Liu et al., 2001; McPherron et al., 1999; Peljto et al., 2010). D3 early precursors were exposed to a combination of RA and GDF11 (25 ng / ml) for 24 or 48 hours. 24-hour GDF11 exposure induced caudal upper limb and anterior thoracic MNs (58.7% HOXC8, 41.0% HOXC9); the later category increased after 48 hours (Figures 4A, 4E, and S4A). However, as with FGF2, no more caudal identity was observed. In chick spinal cord explants, exposure to a combination of FGF2 and GDF11 promoted more caudal MNs than either factor individually (Liu et al., 2001). Therefore, we tested whether this combination accelerated the clock and promoted caudal thoracic or lumbar identity. At 24 h after FGF2 / GDF11 treatment, HOXC9, HOXD9, and HOXC10 mRNA were strongly induced (65.4-, 2774.77-, and 3329.44-fold, respectively, compared with the control) and further increased after 48 h (Figure 4F). Similarly, 24 h (D3–D4) treatment induced caudal thoracic MNs (88.2% HOXC9 and 46.7% HOXD9) at day 14 and lumbar HOXC10 MNs (11.1%) 48 h after exposure (Figure 4B, Figure 4E, S4B–E).

[0168] Thus, axial precursors have the capacity to generate distinct spinal rostrocaudal identities from the upper limb to the lumbar region. Varying the parameters of exposure to FGF and GDF11 (concentration, duration, and combination) dynamically determines the pace at which the HOX clock progresses, leading to the specification of MN subtypes that are generated early or late within the same timeline of differentiation.

[0169] Table S1 - List of transcriptome data and markers defining axial precursors and other developmentally relevant cell types The first panel shows the normalized number of reads for each gene for hESC, D2, D3, and D4 replicates. The next panel shows a list of genes enriched (FC≧2.0, p-value<0.05) in the indicated comparison. The final panel shows a list of genes specifying specific cell types (allantoic, mesoderm precursors) and the references used to construct these lists.

[0170] [Table 3A]

[0171] [Table 3B]

[0172] [Table 4A]

[0173] [Table 4B]

[0174] [Table 5]

[0175] Consideration Overall, we report the selective generation of axial precursors from hPSCs, demonstrated by a combination of specific markers, detailed transcriptome analysis, and their ability to generate cell types found at distinct rostro-caudal levels. Importantly, even though the concentration of CHIR required to potently induce an axial precursor state that subsequently reliably induces caudal cell types was optimal, lineage differences were observed. By effectively accessing axial precursors, we demonstrate that varying the concentration, duration, and combination of the caudalizing factors FGF and GDF11 controls the speed at which temporally colinear activation of HOX genes occurs. Thus, the pace of the HOX clock is dynamically encoded by parameters of exposure to exogenous cues. The sequential changes in chromatin structure that occur along HOX complexes during their activation do not appear to act as an intrinsic timer that can be turned on and off by exogenous factors ( Bel-Vialar et al., 2002 ; Del Corral and Storey, 2004 ; Kimelman and Martin, 2012 ; Lippmann et al., 2015 ; Mazzoni et al., 2013 ; Narendra et al., 2015 ; Noordermeer et al., 2011 ; Noordermeer et al., 2014 ; Soshnikova and Duboule, 2009 ; Tschopp et al., 2009 ). Our results may provide a molecular basis for the observation that heterochronic grafting of "aged" axial precursors to "younger" caudal stem zones reverts their HOX profile to a "young" one (McGrew et al., 2008; Wymeersch et al., 2019). Secreted factors determine the pace of the HOX clock, ensuring a community effect to synchronize HOX expression among neighboring precursors, which may enable the emergence of expression domains at the tissue level (Durston, 2019).Furthermore, FGFs and GDFs also control the maintenance of stem cell pools and axial outgrowth (Aires et al., 2019; Boulet and Capecchi, 2012; Jurberg et al., 2013; Mallo et al., 2009; McPherron et al., 1999). A general mechanism controlling rostral-caudal outgrowth of the axial axis, along with Hox gene induction, is a parsimonious way to link morphogenesis and patterning (Denans et al., 2015; Young et al., 2009). From a biotechnological perspective, exogenous control of the HOX clock provides a simple means to generate cell types with defined "rostral-caudal" identities from PSCs. Such a method shortens the time requirements for generating cells generated at different developmental times during axial outgrowth. This allows for the simultaneous generation of human MN subtypes with defined rostral-caudal identities with unprecedented efficiency and precision. In particular, we provide the first evidence for the generation of putative hand-controlling MNs (Mendelsohn et al., 2017). MN subtypes exhibit differential vulnerability to disease and spinal cord injury. Our results provide a much-needed resource for modeling these incurable diseases and access to a more controlled cell source for putative cell therapy approaches (Abati et al., 2019; An et al., 2019; Baloh et al., 2018; Nijssen et al., 2017; Ragagnin et al., 2019; Sances et al., 2016; Steinbeck and Studer, 2015; Tung et al., 2019). Because HOX plays a central role in directing cellular diversification in the three lineages, controlled manipulation of the HOX clock holds great potential for MN subtypes as well as cell engineering.Our strategy may be extendable to other axial stem cell derivatives, such as the paraaxial mesoderm (which generate different muscles of the body), and has obvious applications for modeling neuromuscular diseases (Bakooshli et al., 2019; Diaz-Cuadros et al., 2020; Faustino Martins et al., 2020; Frith et al., 2018; Machado et al., 2019; Matsuda et al., 2020; Osaki et al., 2020; Pourquie et al., 2018; Steinbeck et al., 2015; Verrier et al., 2018). More broadly, the temporal generation of different neuronal or glial types from the same progenitor domain is a widely used strategy to increase cellular diversity in the nervous system (Dias et al., 2014; Kohwi and Doe, 2013; Oberst et al., 2019a; Rossi et al., 2017). Exogenous cues play a critical role in unravelling these temporal sequences (Kawaguchi, 2019; Oberst et al., 2019a; Oberst et al., 2019b; Syed et al., 2017; Tiberi et al., 2012). Exogenously manipulating the timing of these lineages should improve the generation of early- and late-born cells for both disease modeling and basic cell therapy research.

[0176] [References] TIFF0007765472000008.tif231170TIFF0007765472000009.tif234170TIFF0007765472000010.tif234170TIFF0007765472000011.tif234170TIFF0007765472000012.tif234170TIFF0007765472000013.tif234170TIFF0007765472000014.tif234170TIFF0007765472000015.tif234170TIFF0007765472000016.tif234170TIFF0007765472000017.tif234170TIFF0007765472000018.tif234170

Claims

1. 1. An in vitro or ex vivo method for obtaining a subpopulation of human motor neurons of a specific rostral-caudal phenotype, the method comprising culturing axial precursors in a culture medium and adding retinoic acid (RA) and a Hedgehog signaling pathway agonist to the culture medium to obtain spinal motor neuron precursors of a specific rostral-caudal phenotype, wherein the longer the addition of retinoic acid to the culture medium during the culturing of the axial precursors is delayed, the more caudal-type motor neurons are obtained.

2. 2. The method of claim 1, wherein RA and the Hedgehog signaling pathway agonist are added in combination with an FGFR agonist and / or a TGF / Activin / Nodal signaling pathway activator, and the concentration or duration of exposure to the FGFR agonist or TGF / Activin / Nodal signaling pathway activator correlates with the number of caudal-type motor neurons obtained.

3. - exposing human pluripotent stem cells (hPSCs) to a culture medium containing a Wnt signaling pathway activator to obtain axial precursors; - exposing the axial precursors to retinoic acid (RA) and a hedgehog signaling pathway agonist in the culture medium to obtain spinal motor neuron precursors; Including, the longer the exposure of the axial precursors to retinoic acid is delayed relative to exposure to the Wnt signaling pathway activator, the more caudal-type motor neurons are obtained. The method according to claim 1 or 2.

4. 4. The method of claim 3, wherein human pluripotent stem cells (hPSCs) are exposed to a Wnt signaling pathway activator, an inhibitor of the BMP pathway, and an inhibitor of the TGFβ pathway in a culture medium to obtain axial precursors.

5. 5. The method of any one of claims 1 to 4, wherein the spinal motor neuron precursors are further exposed to a Notch pathway inhibitor in the culture medium.

6. 6. The method of claim 5, wherein the Notch pathway inhibitor is added between days 9 and 14.

7. 7. The method of any one of claims 3 to 6, wherein human pluripotent stem cells (hPSCs) are exposed to a Wnt signaling pathway activator in combination with a bone morphogenetic protein (BMP) signaling pathway inhibitor and a transforming growth factor (TGF) / activin / nodal signaling pathway inhibitor.

8. - the bone morphogenetic protein (BMP) signaling pathway inhibitor, the transforming growth factor (TGF) / activin / nodal signaling pathway inhibitor, and the Wnt signaling pathway activator are added to the culture medium from day 0 to day 3 or day 4; - a hedgehog signaling pathway agonist is added to the culture medium on days 3 and 7 and up to day 9 or 10; The method of claim 7.

9. 9. The method of any one of claims 1 to 8, wherein the caudal thoracic and lumbar motoneurons are obtained by exposing axial precursors to retinoic acid, an FGFR agonist, and a TGF / activin / nodal signaling pathway activator.

10. A method described in any one of claims 2 to 9, wherein an FGFR agonist is added to the culture medium at a concentration of at least 15 ng / ml for at least 24 hours, and a TGF / activin / nodal signaling pathway activator is added to the culture medium at a concentration of at least 20 ng / ml for at least 24 hours.

11. A method according to any one of claims 2 to 10, wherein the FGFR agonist is added to the culture medium at a concentration of at least 60 ng / ml.

12. The method of claim 2, wherein the FGFR agonist is added to the culture medium at a concentration of at least 100 ng / ml.

13. 13. The method of any one of claims 1 to 12, wherein RA is added to the culture medium for a period of 2 to 11 days.

14. 14. The method of claim 1, wherein RA is added to the culture medium between days 3 and 9.

15. 15. The method of any one of claims 1 to 14, wherein RA is added at a concentration of at least 10 nM.

16. - upper limb motoneurons are obtained by adding RA alone to the culture medium between days 4 and 9, or by adding RA between days 3 and 9 and either an FGFR agonist at a concentration of at least 100 ng / ml or an activator of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml to the culture medium between days 3 and 4; - anterior thoracic motor neurons are obtained by adding RA alone to the culture medium any time between days 5 and 9, or by adding RA between days 3 and 9 and an FGFR agonist at a concentration of at least 60 ng or an activator of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml to the culture medium 24 to 48 hours before adding RA to the culture medium; - caudal thoracic motoneurons are obtained by adding to the culture medium between days 3 and 9 RA, and between days 3 and 4 an FGFR agonist at a concentration of at least 100 ng / ml and an activator of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml; - lumbar motoneurons are obtained by adding RA between days 5 and 9, and an FGFR agonist at a concentration of at least 100 ng / ml and an activator of the TGF / activin / nodal signaling pathway at a concentration of at least 20 ng / ml between days 3 or 4 and 5, 16. The method according to any one of claims 1 to 15.

17. The method of any one of claims 2 to 16, wherein an FGFR agonist is added to the culture medium, and the FGFR agonist is FGFR2.

18. 18. The method of any one of claims 1 to 17, wherein the hedgehog signaling pathway agonist is a smoothened agonist (SAG).

19. 19. The method of claim 18, wherein the hedgehog signaling pathway agonist is SAG at a concentration of at least 300 nM.

20. A method described in any one of claims 6 to 19, wherein a Notch pathway inhibitor is added to the culture medium, and the Notch pathway inhibitor is DAPT.

21. 21. The method of claim 20, wherein the Notch pathway inhibitor is DAPT at a concentration of at least 5 μM.

22. A method described in any one of claims 3 to 21, wherein a Wnt signaling pathway activator is added to the culture medium, and the Wnt signaling pathway activator is Chir-99021 compound or Wnt3a protein.

23. A method described in any one of claims 2 to 22, wherein a TGF / activin / nodal signaling pathway activator is added to the culture medium, and the TGF / activin / nodal signaling pathway activator is GDF11 or GDF8.

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