WO2023104792A1 - Amplificateurs de maturation neuronale - Google Patents

Amplificateurs de maturation neuronale Download PDF

Info

Publication number
WO2023104792A1
WO2023104792A1 PCT/EP2022/084590 EP2022084590W WO2023104792A1 WO 2023104792 A1 WO2023104792 A1 WO 2023104792A1 EP 2022084590 W EP2022084590 W EP 2022084590W WO 2023104792 A1 WO2023104792 A1 WO 2023104792A1
Authority
WO
WIPO (PCT)
Prior art keywords
neurons
stimulator
cells
cortical
inhibitor
Prior art date
Application number
PCT/EP2022/084590
Other languages
English (en)
Inventor
Pierre Vanderhaeghen
Pierre CASIMIR
Ryohei IWATA
Original Assignee
Vib Vzw
Katholieke Universiteit Leuven
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vib Vzw, Katholieke Universiteit Leuven filed Critical Vib Vzw
Publication of WO2023104792A1 publication Critical patent/WO2023104792A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/71—Oxidoreductases (EC 1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to the field of in vitro methods of inducing maturation of immature human pluripotent stem cell derived neurons into fully mature, electrically excitable neurons.
  • the presently disclosed subject matter also provides for cells generated by such methods and uses of such cells for drug discovery purposes and/or treating neurodegenerative disorders.
  • hESCs Human embryonic stem cells
  • hESCs Human embryonic stem cells
  • blastocyst embryos blastocyst embryos
  • hESCs have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues.
  • embryonic germ layers ectoderm, mesoderm, endoderm
  • a number of studies have reported the differentiation of ESCs into a range of embryonic tissues. These results were obtained either by stimulating the cells with particular molecules or by simulating the environmental cues of the early embryo.
  • in vitro differentiation of embryonic stem cells (ESCs) or pluripotent stem cells (PSCs) has several clinically relevant applications. Having the ability to culture large amounts of specific cell types provides a platform for the screening of chemical libraries. Disease causing mutations can be engineered in the cells or cells can be obtained from patients and be used in a drug discovery process to find candidate medicaments that can restore or compensate for morphological, genetic or physiological defects.
  • in vitro derived differentiated cells offer a source of transplantable cells for example for regenerative approaches or treatment of neurological diseases. Indeed, following transplantation of ESC-derived neuronal precursors into the central nervous system (CNS) of mammals, said precursors have been shown to integrate into the host tissue and yield functional improvement (Sun 2016 Neural Regen Res 11).
  • NSC neuronal stem cell
  • a neuron also called neurogenesis
  • NSCs can be in vitro converted to cortical neurons in 1-2 weeks.
  • the cortical neurons are not electrically excitable and do not show the typical architecture of a mature cortical neuron that is intertwined in complex neuronal networks.
  • converting immature into mature cortical neurons takes several months. This makes it impractical to use neuronal cell cultures for drug screenings, neurodegenerative disease modelling etc.
  • the inventors of current application have studied the differences in cortical neuron maturation between mice and human. They could attribute the faster maturation of mice cortical neurons to a significantly higher mitochondrial metabolism. Surprisingly by contacting immature human cortical neurons with activators of mitochondrial oxidative phosphorylation, the maturation of said human neurons could be drastically accelerated.
  • TIGAR an endogenous inhibitor of glycolysis
  • TIGAR accelerates OXPHOS by increasing the level of pyruvate among others.
  • boosting mitochondrial metabolism has not been shown to be required for neuron maturation.
  • Zheng et al 2017 eLife
  • Zheng et al performed RNAseq experiments on immature and mature neurons and found no difference in transcript level of the genes encoding the glycolysis and Krebs cycle enzymes.
  • the stimulator statistically significantly increases mitochondrial oxidative phosphorylation.
  • the stimulator is selected from the list consisting of a hexokinase 2 inhibitor, lactate dehydrogenase A inhibitor, phosphofructokinase inhibitor, pyruvate dehydrogenase kinase inhibitor, lactate dehydrogenase B or a lactate dehydrogenase B agonist, PPAR agonist, mPTP blocker, mTOR activator, Sirtl antagonist, AMPK inhibitor, dexpramipexole, galactose, glutamine, pyruvate, free fatty acid, bezafibrate and mdivi-1.
  • the cortical neurons are derived from human pluripotent stem cells.
  • a stimulator of mitochondrial oxidative phosphorylation for use in treating a neurological disorder, more particularly a neurological disorder associated with neuronal loss.
  • the neurological disorder is a brain injury, malformation cortical development, intellectual disability, epilepsy, autism, fragile X syndrome, schizophrenia, stroke or a neurodegenerative disease.
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease and Niemann-Pick's disease.
  • an in vitro method for generating mature cortical neurons from immature cortical neurons derived from human pluripotent stem cells comprising contacting the immature cortical neurons with a stimulator of mitochondrial oxidative phosphorylation to obtain a population of mature neurons wherein at least about 10% of the mature neurons express NPAS4.
  • the contact of the immature neurons with the stimulator of oxidative phosphorylation is at least 10 days.
  • a population of mature cortical neurons generated by the above method In one embodiment said population is provided for use as a medicament, more particularly for use in treating a neurological disorder, more particularly a neurological disorder associated with neuronal loss.
  • the neurological disorder is a brain injury, malformation cortical development, intellectual disability, epilepsy, autism, fragile X syndrome, schizophrenia, stroke or a neurodegenerative disease.
  • said neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease and Niemann-Pick's disease.
  • a method of transplanting cortical neurons in a subject in need thereof comprising the steps of contacting a culture of cortical neurons with a stimulator of mitochondrial metabolism, and injecting the obtained cells in the brain of the subject.
  • the cortical neurons are derived from in vitro differentiation of pluripotent stem cells.
  • the subject has a neurological disorder, more particularly a neurological disorder associated with neuronal loss, more particularly the subject has a brain injury, malformation cortical development, intellectual disability, epilepsy, autism, fragile X syndrome, schizophrenia, stroke or a neurodegenerative disease.
  • said neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease and Niemann-Pick's disease.
  • Figure 1 shows that mitochondria growth and dynamics follow a species-specific timeline during cortical neuronal maturation.
  • NNN NeuroDl-dependent Newborn Neuron
  • D,E Representative images of mitochondrial morphology in NNN-labelled neurons in (D) mouse and (E) human.
  • FIG. 2 shows that mitochondrial OXPHOS activity increases during neuronal maturation and displays human-mouse interspecies differences.
  • A Timeline of the oxygraph experiments in mouse and human neurons.
  • B-D Changes of oxygen consumption rate (OCR) during neuronal development. Quantified OCR from at least 2 biological replicate experiments. Data are shown as mean ⁇ SEM.
  • B Resting status.
  • C OCR following a treatment with ADP fuelling oxidative phosphorylation (OXPHOS). Electron transport chain complex I and Il-linked (CI+CI l-linked).
  • D Electron transport system (ETS) capacity (maximum respiration) under uncoupled condition (uncoupler, CCCP treated). CI+CI l-linked.
  • ETS Electron transport system capacity (maximum respiration) under uncoupled condition (uncoupler, CCCP treated). CI+CI l-linked.
  • Mouse Dunn's multiple comparisons test.
  • Figure 3 illustrates species-specific metabolism features of cortical neurons.
  • A Schematic representation of the experimental setup to measure glycolysis and TCA cycle intermediate metabolites labelling by U-13C-labeled glucose at 30h post tracer addition. 13C enrichment patters of the metabolites in (Orange) Mouse and (Blue) Human.
  • FIG. 4 shows that increasing mitochondria metabolism accelerates human neuronal maturation.
  • A Schematic of metabolic pathways targeted by indicated chemical compounds.
  • GSK GSK-2837808A.
  • LDHA/B lactate dehydrogenase A/B.
  • PDH pyruvate dehydrogenase.
  • PDK/P pyruvate dehydrogenase kinase/phosphatase.
  • B-C Quantified OCR of human neurons following treatments with DMSO, GSK, AlbuMAX or AlbuMAX+GSK from more than three biological replicate experiments (C) according to the experimental scheme shown in B. Data are shown as mean ⁇ SEM.
  • VDAC1 outer mitochondria membrane protein
  • NDUFB8 Complex I subunit
  • SDHB Complex II subunit
  • UQCRC2 Complex III subunit
  • COXII Complex IV subunit
  • ATP5A ATP synthase subunit.
  • J-K Impact of LDHA inhibition and/or AlbuMAX treatment on viability of human neurons represented by quantified cell density (J) and quantification of act-Casp3+ cells among DAPI+ cells (K). Data in J is represented by box plots displaying minimum to maximum values.
  • the box extends from the 25th to the 75th percentile. There is no significant difference of cell density among conditions.
  • Figure 5 further shows that increasing mitochondria metabolism accelerates human neuronal maturation.
  • A-B Representative images of KCI-induced NPAS4 expression in NNN-labelled human neurons over time (B) according to the experimental set up shown in A. Arrow: NPAS4+ neurons.
  • C-D Quantification of the proportion of NPAS4-positive neurons among all neurons (human-specific nuclear antigen) induced by KCI following treatment with indicated chemical compounds from three independent biological replicates (D) according to the experimental design shown in C.
  • G-H Quantification of the KCI-induced proportion of NPAS4+ cells among DAPI+ cells following AlbuMAX+GSK or mock (DMSO) treatment or at 5 days after withdrawal of AlbuMAX+GSK from three biological replicate experiments (H) according to the scheme shown in G. At day 40, MACS-sorted cells were plated on coverslip without mouse astrocytes and at day 59, media were changed to fresh media without chemical compounds.
  • M-N Quantification of number of synapsin I puncta on dendrites from three independent biological replicates following AlbuMAX+GSK or DMSO treatment (N) according to the experimental scheme shown in M. Data are shown as mean ⁇ SEM.
  • Unpaired t test for all statistical testing: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, **** or ttttp ⁇ 0.0001.
  • Figure 6 shows that increasing mitochondrial metabolism accelerates neuronal growth and complexification.
  • A Experimental scheme and (B) representative images of NNN-labelled human neurons following treatment with indicated chemical compounds.
  • C Total dendritic length. Data are shown as mean ⁇ SEM. Dunn's multiple comparisons test.
  • D Number of intersection as determined by the Sholl analysis of dendritic branching.
  • E Cell body area. Tukey's multiple comparisons test.
  • L-M Quantification of total dendritic length (L) and sholl analysis of dendritic branching (M) of NNN-labelled mouse neurons treated with DMSO (mock) or Rotenone from three biological replicates.
  • Figure 7 shows that in vivo maturation of human cortical neurons is enhanced by increased mitochondria function.
  • D Representative images of LDHB overexpressing (right) and GFP control (left) transplanted human neurons in mouse cortex at 28-29 days post transplantation.
  • E-F Quantification of total dendritic length (E) and sholl analysis of dendritic branching (F) of LDHB overexpressing and GFP control transplanted human neurons from three biological replicate experiments.
  • Embryonic stem cells are pluripotent stem cells (PSCs) derived from the inner cell mass of blastocyst stage embryos (Compagnucci et al 2014 Cell Mol Life Sci 71). ESCs are characterized by the ability to self-renew, the lack of contact inhibition, atypical cell cycle regulation and the potential to differentiate into ectoderm, mesoderm and endoderm in vivo, but also in vitro (Evans and Kaufman 1981 Nature 292; Martin 1981 PNAS 78; Doetschman 1985 J Embryol Exp Morphol 87). ESCs not only have the ability to be propagated in culture for years, they also retain their capacity to be pluripotent.
  • ESCs By responding to specific in vitro signals, ESCs differentiate spontaneously into a large variety of cell types (Robertson 1987 Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, ed. EJ Robertson, IRL Press, Washington DC; Smith et al 1988 Nature 336).
  • hESCs human ESCs
  • a particularly efficient strategy is the use of small molecules inhibiting SMAD signalling (e.g. dual SMAD inhibition) to trigger differentiation of hESCs into PAX6+ central nervous system (CNS) neural precursors (Chambers et al 2009 Nature Biotechnol 27).
  • SMAD signalling e.g. dual SMAD inhibition
  • CNS central nervous system
  • Neural subtype specification can be further modulated using additional small molecules targeting pathways such as Wnt signalling. Timed exposure to compounds activating Wnt signalling under dual-SMAD inhibition conditions induces SQX10+ neural crest lineages.
  • MAPK/ERK kinase accelerates the differentiation of cortical neurons from pluripotent stem cells contacted with (i) one or more inhibitors of transforming growth factor beta (TGFP)/Activin-Nodal signalling; (ii) one or more inhibitors of bone morphogenetic protein (BMP) signalling; (iii) one or more inhibitors of Wnt signalling; (iv) one or more inhibitors of FGF signalling; and (v) one or more inhibitors of Notch signalling (WO2017132596A1).
  • TGFP transforming growth factor beta
  • BMP bone morphogenetic protein
  • the process of differentiation of an ESC or PSC into a neuron is called neurogenesis or the birth of a new neuron.
  • the obtained neurons express neuron-specific markers including beta-3 tubulin, double-cortin (DCX), NeuroD-family transcription factors (NeuroDl/NeuroD2), and are post-mitotic (i.e. they lack the ability to undergo mitosis).
  • DCX double-cortin
  • NeuroD-family transcription factors NeuroD-family transcription factors
  • the obtained neurons are not electrically excitable, do not grow dendrites and axons and do not make synapses as mature neurons do in the human brain.
  • a further neuronal maturation step is needed to obtain fully mature neurons that are physiologically identical to mature in vivo neurons.
  • mice neurons showed a significantly increased mitochondrial activity compared to that in human neurons.
  • adding compounds that stimulate mitochondrial metabolism or activity, more particularly oxidative phosphorylation, to a culture of immature human cortical neurons significantly shortened the maturation process.
  • the present invention provides methods for inducing maturation of neurons, more particularly cortical neurons.
  • methods are provided of shortening the maturation process of an immature to a mature neuron or of reducing the time needed for an immature neuron to mature into an electrically excitable mature neuron.
  • the methods comprise the step of stimulating or enhancing mitochondrial metabolism or activity in the immature neurons. Said stimulation can be achieved by transfecting the neurons as such that mitochondrial metabolism or activity is stimulated. Alternatively, said stimulation is achieved by the administration of at least one stimulator of mitochondrial metabolism or activity.
  • a stimulator of mitochondrial metabolism or activity more particularly a compound or molecule that statistically significantly increases mitochondrial oxidative phosphorylation, for the maturation of cortical neurons or alternatively phrased for generating mature cortical neurons from immature cortical neurons or for stimulating, increasing or accelerating the maturation of immature cortical neurons into mature cortical neurons.
  • the statistically significant increase is determined in comparison to a control situation in the absence of the stimulator.
  • the methods of the first aspect comprise the step of contacting neurons with a stimulator of mitochondrial metabolism or activity, more particularly with an enhancer of oxidative phosphorylation.
  • neuron or neurons as used herein refer to cortical neuron(s). In another embodiment of the invention, neuron or neurons as used herein refer to human neuron(s). In yet another embodiment of the invention, neuron or neurons as used herein refer to human cortical neuron(s).
  • Stimulating mitochondrial metabolism or activity refers to the induction of a statistically significant higher mitochondrial metabolism or activity compared to a control situation.
  • a control situation can be a neuron not transfected by or not treated with a stimulator of mitochondrial metabolism.
  • Said mitochondrial metabolism or activity can be evaluated on the level of the single mitochondria or can be evaluated on the level of the cell. In the latter case, an increased mitochondrial metabolism or activity can also be obtained by increasing the number of mitochondria (and thus the total mitochondrial volume in the cell) or the size of the mitochondria.
  • a stimulator of mitochondrial metabolism or activity as used herein thus also refers to a stimulator of mitochondrial size (e.g. by stimulating mitochondrial fusion or inhibiting mitochondrial fission) or mitochondrial number per cell (e.g. by stimulating biogenesis of mitochondria).
  • said statistically significant higher mitochondrial metabolism or activity refers to a statistically significantly higher oxidative phosphorylation compared to a control situation.
  • said stimulation of mitochondrial metabolism or activity is obtained by enhancing or increasing mitochondrial oxidative phosphorylation.
  • said stimulation of mitochondrial metabolism or activity is stimulation or activation of mitochondrial oxidative phosphorylation.
  • Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained.
  • the null hypothesis is the default assumption that nothing happened or changed.
  • an observed result has to be statistically significant, i.e. the observed p-value is less than the pre-specified significance level a.
  • the p- value of a result, p is the probability of obtaining a result at least as extreme, given that the null hypothesis were true.
  • a is 0.05.
  • a is 0.01.
  • a is 0.001.
  • a is 0.0001.
  • a “neuronal cell” or “neuron” is a nerve cell of the nervous system that typically consists of a cell body that contains the nucleus and surrounding cytoplasm; several short, radiating dendrites; and one long axon, which terminates in twig-like branches (telodendrons), and which may have branches (collaterals) projecting along its course. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.
  • neurons include, without limitation, neurons of the dorsal root ganglia (DRG), motor neurons, cortical neurons, peripheral neurons, sensory neurons, neurons of the spinal cord, and ventral interneurons, all of which may be cholinergic, dopaminergic, or serotonergic.
  • DRG dorsal root ganglia
  • motor neurons cortical neurons
  • peripheral neurons peripheral neurons
  • sensory neurons neurons of the spinal cord
  • ventral interneurons all of which may be cholinergic, dopaminergic, or serotonergic.
  • neurons form local and long-distance networks, which is a key component for proper brain function.
  • immature neurons Prior to the establishment of neuronal connections, immature neurons are generated from neuronal progenitor cells (NPCs). These immature neurons that are not electrically excitable and that do not have the appearance of a fully functional neuron that is connected within complex neuronal networks, first have to undergo a long maturation process. This process comprises a series of sequential steps comprising neuro
  • Electronically excitable refers to the ability of the neuron to induce an action potential, i.e. a rapid and reversible reversal of the electrical potential difference across the plasma membrane of the neuron.
  • an action potential i.e. a rapid and reversible reversal of the electrical potential difference across the plasma membrane of the neuron.
  • the membrane potential rapidly changes from its resting level of approximately -70 mV to around +50 mV and, subsequently, rapidly returns to the resting level again.
  • the neuronal action potential forms an important basis for information processing, propagation and transmission.
  • Cortical neuron refers to a neuron from the cerebral cortex, the outer layer of neural tissue of the cerebrum of the brain in humans and other mammals.
  • the cerebral cortex is a highly ordered brain structure with neurons organized into distinct layers each displaying unique afferent (registering signals) and efferent (transmitting signals) connections.
  • the cerebral cortex plays a vital role in memory, attention, perception, awareness and consciousness.
  • Cortical neurons can be broadly divided into two classes. Pyramidal neurons constitute >85% of cortical neurons, they are glutamatergic, and send long-range projections to other cortical or subcortical targets. The remaining 15% of cortical neurons are GABA-ergic interneurons that display only local connectivity.
  • Cortical projection neuron cell identity can be identified by the expression of one or more cortical neuron fate markers selected from the group consisting of Emxl (general marker of all cortical projection neurons), TBR1, TLE4, DCX, CTIP2, SATB2, FOXP2, RORB, CUX1, CUX2, BRN2 and BRN3 (layer specific markers) and combinations thereof.
  • the term "marker” or "cell marker” refers to a gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker. Markers may refer to a "pattern" of markers such that a designated group of markers may identity a cell or cell type from another cell or cell type.
  • Immature neuron refers to a post-mitotic neuron expressing one or more of the fate markers selected from the list consisting of beta-3 tubulin, double-cortin (DCX) and NeuroD-family transcription factors (NeuroDl/NeuroD2). Moreover, immature neurons differ from mature neurons by not being electrically excitable, not displaying long or branched dendrites, not displaying long or branched axons, not displaying synapses and not having the ability to make synapses. Immature neurons also have a small cell body.
  • a "mature neuron” as used herein refers to a neuron that is electrically excitable, grows cell body, dendrites and axons and makes synapses. This cell state can be identified by the expression of the NPAS4 marker as used herein.
  • Mitochondria are complex organelles serving a plethora of cellular functions including, but not limited to calcium buffering, anaplerosis, cell death regulation and ROS balancing. Their core function, however, is considered to be their energy generating capacity. Mitochondria can consequently be seen as the "power houses" of the cell.
  • OXPHOS oxidative phosphorylation
  • Cl-V complexes
  • the endergonic (requiring energy) process of phosphorylation of ADP to ATP is thus coupled to the exergonic (releasing energy) process of electron transfer to oxygen. Coupling is achieved through the proton pumps generating and utilizing the protonmotive force in a proton circuit across the inner mitochondrial membrane.
  • Mitochondria can be in a resting state, an OXPHOS state and in an ETS state. The resting state represents the state of mitochondria in which the respiration level compensates for the proton leakage.
  • An increase in ADP content increases the rate of phosphorylation by CV, attenuating the proton gradient and stimulating electron transport and oxygen consumption.
  • OXPHOS This state is called the OXPHOS state.
  • OXPHOS mitochondria are in a partially coupled (or partially uncoupled) state.
  • ETS electron transfer system
  • stimulating mitochondrial metabolism or activity refers to stimulating or activating mitochondrial oxidative phosphorylation. This can be achieved by multiple independent ways.
  • stimulating mitochondrial metabolism or activity or stimulating mitochondrial oxidative phosphorylation is obtained by modulating the activity of specific enzymes involved in glycolysis and/or Krebs cycle. In one particular embodiment, the modulating action leads to an enhanced level of pyruvate or acetyl CoA.
  • the specific enzymes of which the action should be stimulated according to the present invention can be added as such.
  • Non-limiting examples of such enzymes are lactate dehydrogenase B (LDHB) and pyruvate dehydrogenase (PDH).
  • the specific enzymes can be modulated in a pharmacological way, i.e. by adding biological or chemical compounds.
  • Non-limiting examples of chemical compounds modulating said specific enzymes are hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors.
  • Lactate dehydrogenase A of LDHA catalysed the conversion of pyruvate into lactate.
  • Non-limiting examples of an LDHA inhibitor are GSK-2837808A, CHK-336, quinoline-3-sulfonamides, NCI-006, KAT- 001, KAT-002, KAT-003, KAT-004, KAT-005, KAT-006, KAT-007, KAT-008, KAT-009, KAT-010, KAT-011, KAT-012, 3-bromopyruvate and GNE-140.
  • the cDNA sequence of LDHA is depicted in SEQ ID No. 1, while the protein sequence is depicted in SEQ ID No. 2.
  • the lactate dehydrogenase B or LDHB gene encodes the B subunit of the lactate dehydrogenase enzyme, which catalyzes the conversion of lactate into pyruvate.
  • the cDNA sequence of LDHB is depicted in SEQ ID No. 3, while the protein sequence is depicted in SEQ ID No. 4.
  • Phosphofructokinases are regulatory glycolytic enzymes that convert fructose 6-phosphate and ATP into fructose 1,6-bisphosphate (through PFK-1), fructose 2,6-bisphosphate (through PFK-2) and ADP.
  • PFK inhibitors are ML251, suramin and polysin.
  • Pyruvate dehydrogenase kinase (also pyruvate dehydrogenase complex kinase, PDC kinase or PDK; EC 2.7.11.2) is a mitochondrial kinase which inactivates the enzyme pyruvate dehydrogenase by phosphorylating it using ATP and therefore inhibits the conversion of pyruvate to Acetyl CoA.
  • PDK inhibitors are PS10, JTT-25, VP-Y, CMI X-11S, AZD-7545, KULA-18 and ACER- 001.
  • the specific enzymes can be modulated in a genetic way, i.e.
  • genes encoding for said enzymes Downregulation of genes is well known in the art and can for example be obtained by RNA interference or by the CRISPR technology. Depending on the CRISPR approach, gene transcription can be inhibited (CRISPRi) or can be activated (CRISPRa). Production of gene products can also be increased by overexpressing the genes in cortical neurons. All genetic modulations can be obtained in a conditional or inducible manner. The skilled one is familiar with these well-established methods to down and/or up-regulate gene expression in neurons. The invention thus also envisages to downregulate the expression of LDHA or PDK encoding genes in order to stimulate or enhance mitochondrial metabolism or activity.
  • LDHB lactate dehydrogenase B
  • PPARGC1A lactate dehydrogenase B
  • PDH lactate dehydrogenase B
  • LDHB encoding the protein as depicted in SEQ ID No. 4 is expressed.
  • the LDHB gene as depicted in SEQ ID No. 3 is expressed.
  • Mitochondrial metabolism or activity can also be stimulated by the expression of TP53 inducible glycolysis and apoptosis regulator (TIGAR) or other inhibitors of glycolysis.
  • TIGAR is generally regarded as an anti-apoptotic gene expressed in response to p53-induced cell death.
  • As a bisphosphatase TIGAR reduces intracellular fructose-2,6-bisphosphate levels, resulting in an inhibition of glycolysis (Bensaad et al 2006 Cell 126).
  • stimulation of mitochondrial metabolism or activity more particularly stimulation or activation of mitochondrial oxidative phosphorylation is achieved by administering one or more compounds selected from the list consisting of PPAR agonists, mPTP blockers, mTOR activators, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine and pyruvate.
  • Peroxisome proliferator-activated receptors are ligand-activated transcription factors that are involved in regulating glucose and lipid homeostasis, inflammation, proliferation and differentiation.
  • PPAR agonists are lanifibranor, clofibrate, gemfibrozil, ciprofibrate, bezafibrate, fenofibrate, GW7647, aleglitazar, muraglitazar, saroglitazar and tesaglitazar.
  • the mitochondrial permeability transition pore (mPTP or MPTP; also referred to as PTP, mTP or MTP) is a protein that is formed in the inner membrane of the mitochondria. Opening allows increase in the permeability of the mitochondrial membranes to molecules of less than 1500 Daltons in molecular weight and release of Ca 2+ into the cytosol.
  • Non-limiting examples of blockers of mitochondrial permeability transition pore are Cyclosporin A (CsA), Sanglifehrin A (SfA), ADP, a nonimmunosuppressant derivative of CsA, N-methyl-Val-4-cyclosporin A (MeValCsA), a nonimmunosuppressive agent, 2-aminoethoxydiphenyl borate (2-APB), bongkrekic acid and NIM811.
  • Sirtl has been shown to be implicated in metabolic control and mitochondrial biogenesis.
  • Non-limiting examples of SIRT1 agonists are resveratrol, SRT1720, SRT2104 and piceatannol.
  • mTOR coordinates energy consumption by the mRNA translation machinery and mitochondrial energy production by stimulating synthesis of nucleus-encoded mitochondria-related proteins including TFAM, mitochondrial ribosomal proteins and components of complexes I and V.
  • mTOR activators are MHY1485, 3-benzyl-5-((2-nitrophenoxy) methyl)- dihydrofuran-2(3H)-one (3BDO) and NV-5138.
  • 5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate-activated protein kinase is an enzyme (EC 2.7.11.31) that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low.
  • Non-limiting examples of AMPK (AMP- activated protein kinase) inhibitors are glycogen synthase kinase 3, heat shock factor 1 and dorsomorphin.
  • stimulation of mitochondrial metabolism or activity more particularly stimulation or activation of mitochondrial oxidative phosphorylation is achieved by administering free fatty acids.
  • mitochondrial fatty acid -oxidation (FAO) and OXPHOS are tightly linked, with FAO providing reducing equivalents to the OXPHOS pathway.
  • a non-limiting example of a fatty acid preparation is Albumax, a commercially available lipid-rich BSA (bovine serum albumin) formulation.
  • octanoate which is a medium-chain fatty acid (MCFA) that is rich in milk and tropical dietary lipids
  • palmitoyl-carnitine which is an ester derivative of carnitine (long-chain acylcarnitine) involved in the metabolism of fatty acids.
  • MCFA medium-chain fatty acid
  • palmitoyl-carnitine is transported into the mitochondria to deliver palmitate for fatty acid oxidation and energy production.
  • stimulating mitochondrial metabolism or activity refers to stimulating or increasing mitochondrial size and/or number of mitochondria in a cortical neuron (i.e. total volume of mitochondria). This can for example be done by contacting cells with bezafibrate that activates PGClalpha and thereby increases mitochondria biogenesis (and thus increases total volume of mitochondria).
  • a cortical neuron i.e. total volume of mitochondria.
  • Another non-limiting example is the compound mdivi-1 that was identified as a mitochondrial fission inhibitor. Contacting cells with mdivi-1 results in an increased mitochondria size.
  • the stimulator of mitochondrial metabolism or activity or the activator or enhancer of oxidative phosphorylation is selected from the list consisting of hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, lactate dehydrogenase B (LDHB), LDHB agonists, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors, PPAR agonists, mPTP blockers, mTOR activators, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine, pyruvate, free fatty acids, bezafibrate and mdivi-1.
  • said free fatty acids comprise or consist of octanoate or palmitoyl-carnitine or Albumax.
  • said stimulator of mitochondrial metabolism or activity or the activator or enhancer of oxidative phosphorylation is GSK-2837808A and/or Albumax.
  • the stimulator of mitochondrial metabolism or activity or size or volume more particularly the activator of mitochondrial oxidative phosphorylation herein disclosed increases OXPHOS capacity with at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 250% or increases OXPHOS capacity in a range between 1.5 and 3 times or 1.5 and 2.5 times compared to a control situation where said stimulator was not present.
  • OXPHOS can be measured in cells by several techniques, for example but without the intention to limit, by enzymology and oxygraphy.
  • Enzymology refers to the technique of measuring enzyme activities by spectrophotometric methods. Substrates or products linked to the respiratory chain complex of interest are monitored by absorbance over time, thus allowing the determination of the rate of activity. The technique is well established in clinical practice globally and considered the gold standard for the biochemical diagnosis of mitochondrial diseases.
  • Oxygraphy is an alternative technique.
  • the technique measures the amount of oxygen being consumed in a sample in an airtight chamber, and is thus a proxy for OXPHOS activity.
  • the activity of multiple systems can be indirectly inferred, including that of the TCA cycle, OXPHOS and of the glycerophosphate dehydrogenase complex (Bird et al 2019 Metabolites 9).
  • the methods according to the first aspect for inducing maturation of a cortical neuron comprise the step of stimulating mitochondrial metabolism in a cortical neuron or contacting the cortical neuron with amounts of a stimulator of mitochondrial metabolism or activity to produce a mature cortical neuron.
  • said stimulator is an enhancer or activator of OXPHOS.
  • said enhancer or activator of OXPHOS is selected from the list consisting of hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, LDHB, LDHB agonists, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors, PPAR agonists, mPTP blockers, mTOR activators, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine, pyruvate, free fatty acids, bezafibrate and mdivi-1.
  • said enhancer or activator of OXPHOS is a combination of at least 2, at least 3, at least 4 or at least 5 enhancers or activators of OXPHOS selected from the list consisting of hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, LDHB, LDHB agonists, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors, PPAR agonists, mPTP blockers, mTOR activators, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine, pyruvate, free fatty acids, bezafibrate and mdivi-1.
  • said free fatty acids comprise or consist of octanoate or palmitoylcarnitine.
  • said enhancer or activator of OXPHOS is GSK-2837808A and/or Albumax.
  • a method is provided of creating or generating a population of mature cortical neurons wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the mature cortical neurons, more particularly mature cortical neurons express NPAS4, the method comprising the step of contacting cortical neurons derived from pluripotent stem cells with an activator of oxidative phosphorylation.
  • said activator is selected from the list consisting of hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, LDHB, LDHB agonists, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors, PPAR agonists, mPTP blockers, mTOR activators, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine, pyruvate, free fatty acids, bezafibrate and mdivi-1.
  • said free fatty acids comprise or consist of octanoate or palmitoylcarnitine.
  • said enhancer or activator of OXPHOS is GSK-2837808A and/or Albumax.
  • contacting cells with a compound refers to exposing cells to a compound, for example, placing the compound in a location that will allow it to touch the cell.
  • the contacting may be accomplished using any suitable methods. For example, contacting can be accomplished by adding the compound to a tube of cells. Contacting may also be accomplished by adding the compound to a culture medium comprising the cells.
  • the effective amounts of the stimulator of mitochondrial metabolism or activity or of the enhancer/activator of OXPHOS are contacted to the cells for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more days.
  • the differentiation from a pluripotent cell to a mature neuron can be divided in neurogenesis and neuron maturation.
  • ESCs give rise to neural stem cells (NSCs, or neural progenitors), found in the developing embryonic brain, but also in restricted areas of the adult brain, such as the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) in the hippocampus (Min and Song 2011 Neuron 70).
  • NSCs are multipotent, as opposed to the pluripotency of embryonic stem cells, as they can differentiate into neurons and glial cells in response to specific cues (Inokuchi 2011 Curr Op Neurobiol).
  • Neurogenesis or the birth of new neurons is thus defined as the formation of new neurons from neural stem and progenitor cells. However these neurons are immature. They are not electrically excitable, do not make synapses and do not express the NPAS4 marker.
  • Neuronal maturation is the process wherein immature neurons obtain the skills to interact and communicate with other neurons. It is the phase in the neuron differentiation process during which neurons gain their morphological, electrophysiological and molecular characteristics to fulfil their functions as the central components of the nervous system.
  • neurogenesis and neuron maturation are completely different processes and are regulated in completely different way.
  • Neurogenesis takes place during fetal life, while most neuronal maturation occurs during postnatal ages. The fact that it happens at completely different periods implies that the mechanisms are likely to be distinct.
  • many neurodevelopmental disorders including intellectual disability (ID), epilepsy and Autism Spectrum Disorder (ASD) occur postnatally and are the result of defects in neuronal development while neurogenesis is most often normal in these cases.
  • ID intellectual disability
  • ASD Autism Spectrum Disorder
  • neurogenesis involves Notch signalling and proneural factors such as Neurogenins, while neuronal maturation involves NeuroD factors and neuron specific transcription factors (Tbrl, Mytll, CTIP2, ). Additionally, neurogenesis involves a decrease in the size of mitochondria (increased fission) (Iwata et al Science 2020), while it is show herein that neuronal maturation implies an increase in mitochondria size.
  • a pluripotent stem cell or PSC is an embryonic stem cell or ESC.
  • Procedures for differentiating ESC or PSC preferably employ culture conditions that attempt to mimic the in vivo environment driving the development of a particular lineage, such as by the addition of specific growth factors or inhibition of certain signalling pathways.
  • a number of sources may contribute to the growth factor environment, including: 1) endogenous expression from the cells themselves, 2) the sera and/or media that the pluripotent stem cells are cultured and/or subsequently differentiated in, and 3) the addition of exogenous growth factors.
  • endogenous expression from the cells themselves include: 1) endogenous expression from the cells themselves, 2) the sera and/or media that the pluripotent stem cells are cultured and/or subsequently differentiated in, and 3) the addition of exogenous growth factors.
  • BMP bone morphogenetic protein
  • the methods of the second aspect of inducing differentiation of a pluripotent stem cell to a mature cortical neuron comprise the steps of: culturing at least one pluripotent stem cell; contacting the pluripotent stem cell with at least one antagonist of the bone morphogenetic protein (BMP) signalling pathway in an amount effective to produce cortical neuronal cells; contacting the obtained cortical neuronal cells with a stimulator of mitochondrial metabolism or activity, more particularly an activator of oxidative phosphorylation.
  • BMP bone morphogenetic protein
  • the at least one pluripotent stem cell is a population of PSCs.
  • Suitable antagonists of the BMP signalling pathway include, but are not limited to Noggin, chordin, follistatin, dorsomorphin and Xnr3.
  • Other non-limiting examples of inhibitors of BMP signalling are disclosed in W02010096496, WO2011149762, WO2013067362, WO2014176606, WO2015077648, Chambers et al 2009 Nature Biotechnol 27 , and Chambers et al 2012 Nature Biotechnol 30.
  • “Antagonist” and “inhibitor” are used interchangeably herein.
  • the cells are contacted with an antagonist of BMP signalling at a concentration of between 10 and 500 nM, between 25 and 475 nM, between 50 and 450 nM, between 100 and 400 nM, between 150 and 350 nM, between 200 and 300 nM, or between 1 and 20 nM, between 1.5 and 10 nM, between 2 and 8 nM or between 2 and 6 nM.
  • said inhibitor or antagonist of BMP signalling is Noggin and is administered at a concentration of between 2.5 and 4 nM.
  • the stimulator of mitochondrial metabolism or activity more particularly the activator of oxidative phosphorylation as used in the method of the second aspect refers to any stimulator of mitochondrial metabolism or activity disclosed herein.
  • the term "inducing differentiation of a pluripotent stem cell” means activating, initiating or stimulating a pluripotent stem cell to undergo differentiation.
  • differentiation refers to the cellular process by which cells become structurally and functionally specialized during development. Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signalling pathways involving proteins embedded in the cell surface.
  • the pluripotent stem cell of the present invention may be obtained from any animal, but is preferably obtained from a mammal (e.g. human, domestic animal or commercial animal). In one embodiment, the pluripotent stem cell is a murine pluripotent stem cell.
  • the pluripotent stem cell is obtained from a human.
  • a pluripotent stem cell or PSC is an embryonic stem cell or ESC.
  • a "differentiated neuronal cell” is a partially-differentiated or fully-differentiated neuronal cell or neuron.
  • the present invention also provides a population of cells produced by any of the methods of the first or second aspect.
  • Said population of cells comprise the mature cortical neurons produced by any method of the invention.
  • some or all of these cells express a fluorescent protein such as GFP.
  • said population is a population of in vitro differentiated cells expressing one or more cortical neuron markers, more particularly a marker for mature cortical neurons, even more particularly expressing the NPAS4 marker.
  • at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the population of cells are mature cortical neuron expressing NPAS4.
  • a population of cells refers to a group of at least two cells.
  • a cell population can include at least 10, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 cells, at least 5,000 cells or at least 10,000 cells or at least 100,000 cells or at least 1,000,000 cells.
  • the population may be a pure population comprising one cell type, such as a population of mature cortical neurons. Alternatively, the population may comprise more than one cell type, for example a mixed cell population. In a particular embodiment, the population consists of human cells.
  • Several diseases and disorders are associated with neural tissue loss. Loss of neurons can result for example from traumatic brain injuries, from strokes or from diseases associated with neurotoxicity such as Alzheimer's and Parkinson's disease.
  • Cell transplantation is a potential strategy to repair and promote the recovery of the injured brain. Indeed, when transplanted into a normal or damaged CNS, human ESCs or PSCs can differentiate, migrate and are capable of making innervations (Hentze et al 2007 Trends Biotechnol 25). Gao et al (2006 Exp Neurol 201) have reported that NSCs from human ESCs survive and differentiate to neurons after transplantation into the injured brain and that injured animals with cell transplantation had improved cognitive functional recovery.
  • the application also provides the methods disclosed herein comprising an additional step of transplanting the obtained neuronal cells in the brain of a subject in need thereof.
  • methods to treat a neurological disorder comprising the steps of: contacting immature cortical neurons with a stimulator of mitochondrial metabolism or activity; and transplanting the obtained cortical neurons in a subject in need thereof.
  • a population of cortical neurons is provided for use to treat neurological disorders, wherein said population of cortical neurons are immature cortical neurons that are treated with a stimulator of mitochondrial metabolism or activity.
  • a method of transplanting cortical neurons in a subject in need thereof comprising the steps of:
  • the immature cortical neurons are derived from PSCs, more particularly from human PSCs, even more particularly from in vitro differentiation of (human) pluripotent stem cells.
  • a pluripotent stem cell or PSC is an embryonic stem cell or ESC.
  • the treated cortical neurons are transplanted into a patient in need thereof immediately after the treatment step.
  • the treated cortical neurons are transplanted into a patient in need thereof at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more days after the treatment step, i.e.
  • said population of cortical neurons treated with a stimulator of mitochondrial metabolism or activity are mature cortical neurons obtained by any of the methods of the first or second aspect.
  • the subject has a neurological disorder associated with neuronal loss or with suboptimal neuron maturation.
  • the stimulator is any of the stimulators of mitochondrial metabolism or activity as disclosed herein, more particularly a hexokinase 2 inhibitor, lactate dehydrogenase A inhibitor, LDHB, LDHB agonist, phosphofructokinase inhibitor, pyruvate dehydrogenase kinase inhibitor, PPAR agonist, mPTP blocker, mTOR activator, Sirtl antagonist, AMPK inhibitor, dexpramipexole, galactose, glutamine, pyruvate, free fatty acid, bezafibrate and/or mdivi-1.
  • the present invention also provides a method for treating neurological disorders in a subject thereof, comprising inducing differentiation of PSCs into mature cortical neurons, in accordance with the methods described herein, and transplanting the mature cortical neurons into the subject, thereby treating the neurological disorder. More particularly, said method comprises the following step: obtaining or generating a culture of pluripotent stem cells; contacting the culture of pluripotent stem cells with a BMP signalling antagonist, in an amount effective to produce cortical neurons; contacting the cortical neurons with a stimulator of mitochondrial metabolism or activity; and transplanting the treated cortical neurons into the subject, in an amount effective to treat the neurological disorder.
  • cortical neurons treated with a stimulator of mitochondrial metabolism or activity of the present invention may be transplanted into a subject in need of treatment by standard procedures known in the art.
  • cortical neurons may be prepared for transplantation and then transplanted into a subject.
  • the cortical neurons are transplanted into the subject at at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more days after the initial contact with the stimulator of mitochondrial metabolism or activity.
  • the stimulator of mitochondrial metabolism or activity is an enhancer or activator of (mitochondrial) oxidative phosphorylation.
  • said enhancer or activator is selected from the list consisting of hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors, PPAR agonists, lactate dehydrogenase B (LDHB) or LDHB agonists, mPTP blockers, mTOR activators, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine, pyruvate, free fatty acids, bezafibrate and mdivi-1.
  • said free fatty acids comprise or consist of octanoate or palmitoylcarnitine.
  • the subject is a human.
  • the neurological disorder is a brain injury, nervous tissue degeneration, intellectual disorder, epilepsy, autisms, schizophrenia, fragile X syndrome or malformation of cortical development (MCD).
  • Nervous tissue degeneration in the central nervous system may be caused by or associated with, a variety of disorders, conditions and factors, including, without limitation, primary neurologic conditions (e.g. neurodegenerative diseases), CNS traumas and injuries, and acquired secondary effects of non-neural dysfunction (e.g. neural loss secondary to degenerative, pathologic or traumatic events).
  • CNS traumas include, without limitation, blunt trauma, hypoxia and invasive trauma.
  • acquired secondary effects of non-neural dysfunction include, without limitation, cerebral palsy, congenital hydrocephalus, muscular dystrophy, stroke, and vascular dementia, as well as neural degeneration resulting from any of the following: an injury associated with cerebral haemorrhage, developmental disorders (e.g.
  • a defect of the brain such as congenital hydrocephalus), diabetic encephalopathy, hypertensive encephalopathy, intracranial aneurysms, ischemia, kidney dysfunction, subarachnoid haemorrhage, trauma to the brain and spinal cord, treatment by such therapeutic agents as chemotherapy agents and antiviral agents, vascular lesions of the brain and spinal cord, and other diseases or conditions prone to result in nervous tissue degeneration.
  • MCD cortical development
  • MCD Malformations of cortical development
  • MCD are a complex family of rare disorders that result from alterations of one or combined developmental steps, including proliferation of neural progenitors, migration of neuroblasts to the cortical plate, layer organization and neuronal maturation (Barkovich et al 2012 Brain 135).
  • Malformations of cortical development (MCD) are increasingly recognized as an important cause of epilepsy, cognitive deficits and developmental delay. It is estimated that up to 40% of children with refractory epilepsy have a cortical malformation (Pang et al 2008 Neurologist 14).
  • the majority of patients with MCD develop the first clinical manifestations of epilepsy during the first year of life, a period of brain development characterized by an intense neuronal maturation and synaptogenesis. (Represa 2019 Front Neurosci 13).
  • Non-limiting examples of neurodegenerative disease are Alzheimer's disease, Parkinson's disease, Huntington's disease, progressive supranuclear palsy (PSP), progressive supranuclear palsyparkinsonism (PSP-P), Richardson's syndrome, argyrophilic grain disease, corticobasal degeneration Pick's disease, frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), post-encephalitic parkinsonism, Parkinson's disease complex of Guam, Guadeloupean parkinsonism, Down's syndrome, dementia pugilistica, familial British dementia, familial Danish dementia, myotonic dystrophy, Hallevorden-Spatz disease, Niemann Pick type C, chronic traumatic encephalopathy, tangle- only dementia, white matter tauopathy with globular glial inclusions, subacute sclerosing panencephalitis, SLC9A6-related mental retardation, non-Guamanian motor neuron disease with neurofibrillary
  • Said stimulator of mitochondrial metabolism or activity is also provided for use in maturing immature neurons, more particularly immature cortical neurons, even more particularly immature human cortical neurons, into electrically excitable neurons.
  • “Maturing” or “to mature” as used herein includes the process of accelerating, stimulating or inducing the maturation process.
  • the maturation process as used herein means the generation of mature neurons, more particularly cortical neurons, even more particularly human cortical neurons from immature neurons, more particularly cortical neurons, even more particularly human cortical neurons.
  • said stimulator is an enhancer or activator of (mitochondrial) oxidative phosphorylation.
  • said enhancer or activator is selected from the list consisting of hexokinase 2 inhibitors, lactate dehydrogenase A inhibitors, phosphofructokinase inhibitors, pyruvate dehydrogenase kinase inhibitors, PPAR agonists, mPTP blockers, mTOR activators, LDHB or LDHB agonists, Sirtl antagonists, AMPK inhibitors, dexpramipexole, galactose, glutamine, pyruvate, free fatty acids, bezafibrate and mdivi-1.
  • said free fatty acids comprise or consist of octanoate or palmitoyl-carnitine.
  • the stimulator of mitochondrial metabolism is also provided for use to treat a neurological disorder.
  • said neurological disorder is one of the disorders described above or is a neurological disease associated with a defective cortical neuron maturation.
  • Defective as used herein refers to a lack, retardation, suboptimal or prolonged cortical neuron maturation compared to a control cortical neuron maturation process in a healthy subject.
  • In vitro neurogenesis and neuron maturation enable the investigation of the mechanisms underlying neuron differentiation during the development of the human brain and the study of species-specific features of corticogenesis. However, it additionally offers many novel opportunities to model brain diseases.
  • One example relates to Timothy syndrome, a monogenic disorder caused by a mutation in an L-type voltage-gated calcium channel, which is strongly associated with developmental delay and autism.
  • Examination of cortical cells from induced PSC derived from patients with Timothy syndrome revealed several interesting phenotypes, including defects in calcium signalling and neuronal activity, defects in the generation of specific types of neurons (i.e. callosal projection neurons), as well as defects in dendrite remodelling (van den Ameele et al 2014Trends Neurosc 7).
  • a screening method for identifying a therapeutic candidate for treating a neurological disease, comprising the steps of: (a) obtaining or generating at least one culture of immature cortical neurons comprising a mutation associated with said neurological disease and at least one control culture of immature cortical neurons not comprising the disease-associated mutation;
  • step (b) contacting the immature cortical neurons from step (a) with a stimulator of mitochondrial metabolism or activity to obtain mature cortical neurons;
  • step (e) identifying the test compound as a therapeutic candidate for treating the neurological disease if the difference in the parameter or read-out of step (c) is statistically significantly different, reduced or increased in the presence of the test compound.
  • a screening method for identifying a therapeutic candidate for treating a neurological disorder, comprising the step of
  • pluripotent stem cells comprise a mutation in a gene associated with the neurological disorder, and at least one control culture of pluripotent stem cells not comprising the disorder-associated mutation;
  • step (f) identifying the test compound as a therapeutic candidate for treating the neurological disorder if the difference in read-out of step (d) is statistically significantly different, reduced or increased in the presence of the test compound.
  • Non-limiting examples of said read-out or parameter is calcium signalling, neuronal activity, dendrite remodelling, neurotransmitter release, amyloid precursor protein (APP) production or processing, Tau phosphorylation or Tau neurofibrillary tangle formation.
  • Compounds tested in the disclosed screening methods are not limited to a specific type of the compound.
  • compound libraries (comprising at least two different compounds) are screened.
  • Compound libraries are a large collection of stored compounds utilized for high throughput screening and comprise biological and/or chemical compounds.
  • Compounds in a compound library can have no relation to one another, or alternatively have a common characteristic.
  • the screening methods are not limited to the types of compound libraries screened.
  • compound libraries may be used. Examples include, but are not limited to libraries comprising small molecules, compounds with known functions, FDA approved drugs, compounds pre-screened on bioactivity, natural compounds, allosteric compounds, peptides, antibody fragments, synthetic compounds, or can be combinatorial chemical libraries or drug repurposing libraries.
  • high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such libraries are then screened in one or more assays, to identify those library members that display the desired characteristic activity.
  • the therapeutic candidate is selected using a "high content screening" (HCS) method that uses a series of experiments as the basis for high throughput compound discovery.
  • HCS high content screening
  • HCS is an automated system to enhance the throughput of the screening process.
  • the present invention is not limited to the speed or automation of the screening process.
  • the method is neither limited to large or high-throughput or any scale, and can be refined based on the availability of test compounds or other variable features of the screening assay.
  • the compounds thus identified can serve as conventional "hit compounds" or can themselves be used as potential or actual therapeutics.
  • the term “and/or” as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • an indefinite or definite article is used when referring to a singular noun e.g. "a” or “an”, “the”, this includes a plural of that noun unless something else is specifically stated.
  • the terms first, second, third and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order.
  • stem cell any cell type that can self-renew and, if it is an embryonic stem (ES) cell, can give rise to all cells in an individual, or, if it is a multipotent or neural stem cell, can give rise to all cell types in the nervous system, including neurons, astrocytes and oligodendrocytes.
  • ES embryonic stem
  • neural progenitor cell is meant a daughter or descendant of a neural stem cell, with a more differentiated phenotype and/or a more reduced differentiation potential compared to the stem cell.
  • precursor cell it is meant any other cell being or not in a direct lineage relation with neurons during development but that under defined environmental conditions can be induced to transdifferentiate or redifferentiate or acquire a neuronal phenotype.
  • cell culture refers to a growth of cells in vitro in an artificial medium for research or medical treatment.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures. 1 As used herein, the term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.
  • disease refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • Treatment refers to any rate of reduction or retardation, more particularly statistically significant reduction or retardation, of the progress of the disease or disorder compared to the progress or expected progress of the disease or disorder when left untreated. More desirable, the treatment results in no or zero progress of the disease or disorder (i.e. "inhibition” or “inhibition of progression”) or even in any rate of regression of the already developed disease or disorder.
  • Reduction refers to a statistically significant reduction of effects in the presence of the stimulator of mitochondrial metabolism of the present invention compared to the absence of the stimulator. More particularly, a statistically significant reduction upon administering the stimulator of the invention compared to a control situation wherein the stimulator is not administered. In a particular embodiment, said statistically significant reduction is an at least 25%, 30%, 35%, 40%, 45% or 50% reduction compared to the control situation.
  • Increasing or “increase” or “enhancing” or “promoting” or “stimulating” are used herein interchangeable and refer to a statistically significant increase, more particularly said statistically significant increase is an at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% increase compared to the control situation.
  • An effective amount as used herein is an amount that produces a desired effect.
  • the term "derived from” or “originated from” or “established from” or “differentiated from” when made in reference to any cell disclosed herein refers to a cell that was obtained from (e.g., isolated, purified, etc.) a parent cell in a cell line, tissue (such as a dissociated embryo, or fluids using any manipulation, such as, without limitation, single cell isolation, cultured in vitro, treatment and/or mutagenesis using for example proteins, chemicals, radiation, infection with virus, transfection with DNA sequences, such as with a morphogen, etc., selection (such as by serial culture) of any cell that is contained in cultured parent cells.
  • a derived cell can be selected from a mixed population by virtue of response to a growth factor, cytokine, selected progression of cytokine treatments, adhesiveness, lack of adhesiveness, sorting procedure, and the like.
  • Example 1 Mitochondria growth and dynamics during cortical neuronal maturation follow a speciesspecific timeline.
  • This system combines the expression of inducible CreER under the control of NeuroDl, with Cre-dependent (floxed) reporters eGFP and/or truncated CD8 (tCD8), enabling identification or MACS purification of the labelled cells (Figure 1A).
  • Cre-dependent (floxed) reporters eGFP and/or truncated CD8 (tCD8) enabling identification or MACS purification of the labelled cells
  • tCD8 truncated CD8
  • Example 3 Increasing mitochondria activity in human neurons leads to accelerated neuronal maturation, growth and complexification.
  • LDH lactate dehydrogenase
  • the neurons were pre-treated with the LDHA inhibitor GSK-2837808A (referred to as GSK) (Billiard et al 2013 Cancer Metab 1) for 19 days. Applying the chemical LDHA inhibitor resulted in increased mitochondria activity in human neurons, as measured by oxygen consumption ( Figure 4B- C).
  • Example 4 Increased mitochondrial activity accelerates human neuronal maturation in vivo
  • KnockOut-DMEM (Thermo Fisher Scientific, Cat#10829018) with KnockOut Serum Replacement (20%, Thermo Fisher Scientific, Cat#10828028), Non-Essential Amino Acids Solution (lx, Thermo Fisher Scientific, Cat#11140050), L-Glutamine (2mM, Thermo Fisher Scientific, Cat#25030081), 2- Mercaptoethanol (lOOpM, Merck, Cat#M3148) and Human FGF-basic (lOng/ml, PeproTech, Cat#AF-100- 18B).
  • DMEM/F12 + GlutaMAX (Thermo Fisher Scientific, Cat#10565042) with N2-supplement (lx, Thermo Fisher Scientific, Cat#A1370701), B27 supplement minus Vitamin A (lx, Thermo Fisher Scientific, Cat#12587010), Bovine Albumin Fraction V (0.05%, Thermo Fisher Scientific, Cat#15260037), 2- Mercaptoethanol (lOOpM, Merck, Cat#M3148), MEM Non-Essential Amino Acids Solution (lx, Thermo Fisher Scientific, Cat#11140050) and Sodium Pyruvate (ImM, Thermo Fisher Scientific, Cat#11360070).
  • Nb/B27 medium N2-supplement (lx, Thermo Fisher Scientific, Cat#A1370701), B27 supplement minus Vitamin A (lx, Thermo Fisher Scientific, Cat#12587010), Bovine Albumin Fraction V (0.05%, Thermo Fisher Scientific, Cat#15260037), 2-
  • Neurobasal-A (Thermo Fisher Scientific, Cat#10888022) with B27 supplement (lx, Thermo Fisher Scientific, Cat#17504044), Sodium Pyruvate (0.227mM, Thermo Fisher Scientific, Cat#11360070), Glucose (2.5mM, Thermo Fisher Scientific, Cat#A2494001) and GlutaMAX supplement (lx, Thermo Fisher Scientific, Cat# 35050061).
  • Neurobasal Thermo Fisher Scientific, Cat#21103049) with B27 supplement (lx, Thermo Fisher Scientific, Cat#17504044) and GlutaMAX supplement (lx, Thermo Fisher Scientific, Cat# 35050061).
  • Neurobasal-A (Thermo Fisher Scientific, Cat#10888022) with B27 supplement (lx, Thermo Fisher Scientific, Cat#17504044), Sodium Pyruvate (0.227mM, Thermo Fisher Scientific, Cat#11360070), Glucose (25mM, Thermo Fisher Scientific, Cat#A2494001) and GlutaMAX supplement (lx, Thermo Fisher Scientific, Cat# 35050061).
  • mice All mouse experiments were performed with the approval of the KU Leuven Committee for animal welfare. Mice were housed under standard conditions (12h I ight: 12h dark cycles) with food and water ad libitum. Embryos (aged E13.5 - E14.5) of the mouse strain ICR (CD1, Charles River Laboratory) or Swiss (Janvier Labs) were used for in utero experiments and primary culture. The plug date was defined as embryonic day (E)0.5, and the day of birth was defined as P0. The data obtained from all embryos were pooled without discrimination of sexes for the analysis of in utero electroporation, given the difficulty to determine sex identity at embryonic stages.
  • Human ESC H9; WiCell Cat # NIHhESC-10-0062; female donor
  • irradiated mouse embryonic fibroblasts in the ES cell medium until the start of cortical cell differentiation.
  • Cortical cell differentiation was performed as described previously (Linaro et al 2019 Neuron 104).
  • days before start of neuronal cell differentiation day -2
  • hESCs were dissociated with Accutase (Thermo Fisher Scientific, Cat#00-4555-56) and plate on Matrigel-coated (BD, Cat#354277) plates at low confluency (5,000 cells/cm2) in hES medium with lOpM ROCK inhibitor (Merck, Cat#688000).
  • the medium On day 0 of the differentiation, the medium was changed to DDM/B27 medium with recombinant mouse Noggin (lOOng/ml, R&D systems, Cat#1967-NG). The medium was changed every other day until day 6. From day 6, the medium was changed every day until day 16. At day 16, medium was changed to DDM/B27 medium and changed every day until day 25. At day 25, the differentiated cortical cells were dissociated using Accutase and cryopreserved in mFreSR (STEMCELL technologies, Cat#05855).
  • cortical cells were validated for neuronal and cortical markers by immunostaining using antibodies for TUBB3 (1:2,000; BioLegend, Cat#MMS-435P), TBR1 (1:1,000; Abeam, Cat#abl83032), CTIP2 (1:1,000; Abeam, Cat#abl8465), FOXG1 (1:1,000; Takara, Cat#M227), SOX2 (1:2,000; Santa Cruz, Cat#sc-17320), FOXP2 (1:500; Abeam, Cat#abl6046), SATB2 (1:2,000; Abeam, Cat#ab34735), and CUX1 (1:1,000; Santa Cruz, Cat#sc-13024).
  • mESC Mouse ESC (mESC) (E14Tg2A; ATCC Cat # CRL) were cultured and differentiated as described previously (Gaspard et al 2008 Nature 455) with some modifications.
  • mESCs were dissociated with Accutase and plated on Gelatin or GFR-Matrigel (Corning, Cat#354230) coated 5 cm dish.
  • the medium was changed to DDM/B27 medium with recombinant mouse Noggin (100 ng/ml).
  • the medium was changed to DDM/B27+Noggin with Cyclopamine (lpM, Calbiochem, Cat#239803). The medium was changed every day until day 6.
  • cortical cells were validated for neuronal and cortical markers by immunostaining using antibodies for TUBB3 (1:2,000; BioLegend, Cat#MMS-435P), TBR1 (1:1,000; Abeam, Cat#abl83032), CTIP2 (1:1,000; Abeam, Cat#abl8465), FOXG1 (1:1,000; Takara, Cat#M227) and SATB2 (1:2,000; Abeam, Cat#ab34735).
  • IUE was performed as described previously (Iwata et al 2020 Science 862). On the same day, electroporated embryos were used for primary mouse cortical cell culture as described above. 4-OHT (0.25pM) was added to the medium from day in vitro (DIV) 0 to DIV2. Half of the medium was changed every 2-3 days. Doxycycline hyclate (lpg/ml, Sigma-Aldrich, Cat#D9891) was added to the medium 24h before fixation. At indicated time points, cells were fixed in 4% PFA in PBS for lh, at 4°C.
  • the coverslips were washed in PBS, incubated in PBS with 0.3% Triton X-100 with DAPI for 30 min at room temperature, then washed in PBS and mounted on a Superfrost slide with Glycergel mounting medium.
  • Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37°C with 5% CO2.
  • the cortical cells were dissociated with Accutase and plated on Matrigel coated 6-well plates at 260,000-300,000 cells/cm2.
  • One fourth of the cells were infected with the following lentiviral vectors: LV-NeuroDl promoter-CreERT2-WPRE and LV-CAG-DIO-EGFP-WPRE.
  • 4-OHT (0.25pM) was added to the medium for 48h.
  • AraC Cytosine p-D-arabinofuranoside hydrochloride, Sigma-Aldrich, Cat#C6645-25MG
  • the cells were dissociated using Accutase and plated on mouse-astrocyte coated coverslips at 100,000 cells/cm2 with a ratio of non-infected to infected cells of 4:1.
  • the cells were fixed in 4% PFA in PBS for lh at 4°C and further processed for immunostaining using anti-GFP and anti-Mitochondria antibodies, as described above.
  • Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37°C with 5% CO2. At seven days after thawing (day 32), cells were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm2) with LV-NeuroDl promoter-CreERT2-WPRE and LV-CAG-DIO-tCD8-WPRE.
  • CD8+ MACS dissociated cells were incubated with magnetic beads conjugated anti-human CD8 (Miltenyi Biotec, Cat#130-045-201) in MACS buffer, mixture of autoMACS Rinsing Solution (Miltenyi Biotec, Cat#130-091-222) and MACS BSA Stock Solution (Miltenyi Biotec, Cat#130-091-376), at 4°C for 10 min.
  • CD8 positive selection were carried out with LS columns (Miltenyi Biotec, Cat#130-042-401) according to the manufacturer's instructions.
  • the sorted cells were plated on mouse astrocyte-coated coverslips at 110,000 cells/cm2.
  • DDM/B27+Nb/B27 medium was changed to following: (1: DMSO) Nb/B27 low glucose medium with DMSO (1:4,000, Merck, Cat#D2650); (2: GSK) Nb/B27 low glucose medium with GSK-2837808A (5pM, MedChemExpress, Cat#HY-100681); (3: AlbuMAX+GSK) Nb/B27 low glucose medium with GSK-2837808A (5pM) and AlbuMAX (0.5%, Thermo Fisher Scientific, Cat#11020021). These media were changed every other day until further analysis.
  • Human neuron xenotransplantation in neonatal brain was performed as described previously (Linaro et al 2019 Neuron 104) with some modifications.
  • Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37°C with 5% CO2.
  • days after thawing day 32
  • cells were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm2) with LV-hSynl-EmGFP-WPRE.
  • the medium was changed to DDM/B27+Nb/B27 medium with lOpM DAPT and cultured for three additional days.
  • the cells were treated with 5mM AraC for 24h.
  • the cortical cells were dissociated using NeuroCult Enzymatic Dissociation Kit following manufacturer's instructions and suspended in the injection solution containing 20mM EGTA (Merck, Cat#03777) and 0.1% Fast Green (Merck, Cat#210-M) in PBS at 100,000-200,000 cells/pl.
  • mice Approximately l-2pl of cell suspension was injected into the lateral ventricles of each hemisphere of neonatal (postnatal day 0 or 1) immunodeficient mice (Rag2-/-) using glass capillaries pulled on a horizontal puller (Sutter P-97). At indicated time points (5 weeks, 6 months and 15 months post xenotransplantation, the mice were perfused with 4% PFA and 0.5% GA in 0.1M PM and the brains were collected.
  • human cortical cells (day 36-38) were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm2) with LV- hSynl-tTA-WPRE and LV-TRE-EGFP-WPRE or LV-TRE-LDHB-IRESEGFP-WPRE.
  • the medium was changed to DDM/B27+Nb/B27 medium with lOpM DAPT and cultured for three additional days.
  • the cortical cells were dissociated using NeuroCult Enzymatic Dissociation Kit following manufacturer's instructions and suspended in the injection solution containing 20mM EGTA in PBS at 100,000-200,000 cells/pl. Approximately l-2pl of cell suspension was injected into the lateral ventricles of each hemisphere of neonatal (postnatal day 0 or 1) Rag2-/- using glass capillaries. 28-29 days post xenotransplantation, the mice were perfused with 4% PFA in PBS and brains were collected, the sections were used as described in the previous section.
  • Human cortical cells (frozen at day 25) were thawed and plated on Matrigel-coated plates using DDM/B27+Nb/B27 medium at 37°C with 5% CO2. Seven days after thawing (day 32), the cells were dissociated using Accutase and plated on Matrigel-coated plate at high confluency (450,000-700,000 cells/cm 2 ) with LV-NeuroDl promoter-CreERT2-WPRE and LV-CAG-DIO-tCD8-WPRE. Four days later (day 36), the medium was changed to DDM/B27+Nb/B27 medium with 4-OHT (lpM) and DAPT ( 10 .M ).
  • the DDM/B27+Nb/B27 medium was changed to the following medium: (1: DMSO) Nb/B27 low glucose medium with DMSO (1:4,000); (2: GSK) Nb/B27 low glucose medium with GSK-2837808A (5pM, MedChemExpress, Cat#HY-100681); (3: AlbuMAX+GSK) Nb/B27 low glucose medium with GSK-2837808A (5pM) and AlbuMAX (0.5%, Thermo Fisher Scientific, Cat#11020021). These media were changed every other day. At indicated time points, oxygraphy was performed as described previously (Bird et al 2019 Metabolites 9). Cells were dissociated using NeuroCult Enzymatic Dissociation Kit.
  • the cells were washed with Collection solution and incubated in Dissociation solution at 37°C for 7 min.
  • the Inhibition solution was added to the plate and the cells transferred to a 10ml tube (SARSTEDT, Cat#62.9924.284).
  • the cell pellet was resuspended in 200pl of Miro5 buffer before counting.
  • Miro5 buffer were added to adjust to 2,000,000-40,000,000 cells/ml.
  • Oxygen saturation was maintained > 40pM throughout the experiment, and between 170 and 200pM before the measurement of isolated complex IV (CIV) activity.
  • CIV complex IV
  • the reported values of maximum OXPHOS (phosphorylating) and ETC (non-phosphorylating) were calculated as succinate minus antimycin A (non- mitochondrial respiration) and CCCP minus antimycin A respectively. Detection of KCI-induced NPAS4 expression
  • the sorted cells were plated on mouse astrocyte-coated coverslips or Poly- L-ornithine-, Laminin- and horse serum-coated coverslips at 110,000 cells/cm2.
  • the DDM/B27+Nb/B27 medium was changed twice a week.
  • 50mM KCI depolarization solution was prepared by adding 0.41 volume of pre-mixture solution (170mM KCI, ImM MgCI2, 2mM CaCI2, lOmM HEPES (pH7.2)) to DDM/B27+Nb/B27 medium with FPL64176 (5pM, Tocris, Cat#1403) and D-AP5 (5pM, Tocris, Cat#0106) to prevent excitotoxicity (Qiu et al 2016 Elife 5). The DDM/B27+Nb/B27 medium was removed and add pre-warmed 50mM KCI depolarization solution.
  • pre-mixture solution 170mM KCI, ImM MgCI2, 2mM CaCI2, lOmM HEPES (pH7.2)
  • FPL64176 5pM, Tocris, Cat#1403
  • D-AP5 5pM, Tocris, Cat#0106
  • DDM/B27+Nb/B27 medium was changed to the following: (1: DMSO) Nb/B27 low glucose medium with DMSO (1:4,000); (2: GSK) Nb/B27 low glucose medium with GSK-2837808A (5pM); (3: AlbuMAX+GSK) Nb/B27 low glucose medium with GSK- 2837808A (5pM) and AlbuMAX (0.5%). These media were changed every other day.
  • mice cells were obtained from primary culture of timed pregnant mouse embryos at E13.5 as described before and were plated on coverslips in 12-well plates that had previously been coated with laminin- and poly-D-lysin overnight at 37°C and horse serum lh at 37°C, at 200,000 cells/cm2 using NbB27x medium with Penicillin-Streptomycin (50U/ml) and with LV-NeuroDl promoter-CreERT2 and LV-CAG- DIO-GFP-WPRE.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Neurology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Neurosurgery (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Developmental Biology & Embryology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Ophthalmology & Optometry (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne le domaine des procédés in vitro d'induction de la maturation de neurones issus de cellules souches pluripotentes humaines immatures en neurones électriquement actifs entièrement matures. La présente invention concerne également des cellules générées par de tels procédés et des utilisations de ces cellules pour la découverte de médicaments et/ou le traitement de troubles neurodégénératifs.
PCT/EP2022/084590 2021-12-07 2022-12-06 Amplificateurs de maturation neuronale WO2023104792A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP21212778 2021-12-07
EP21212778.1 2021-12-07

Publications (1)

Publication Number Publication Date
WO2023104792A1 true WO2023104792A1 (fr) 2023-06-15

Family

ID=78822364

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2022/084590 WO2023104792A1 (fr) 2021-12-07 2022-12-06 Amplificateurs de maturation neuronale

Country Status (1)

Country Link
WO (1) WO2023104792A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096496A2 (fr) 2009-02-17 2010-08-26 Memorial Sloan-Kettering Cancer Center Procédés de conversion neurale de cellules souches embryonnaires humaines
WO2011149762A2 (fr) 2010-05-25 2011-12-01 Memorial Sloan-Kettering Cancer Center Procédé de différenciation en nocicepteur de cellules souches embryonnaires humaines et ses utilisations
WO2013067362A1 (fr) 2011-11-04 2013-05-10 Memorial Sloan-Kettering Cancer Center Neurones dopaminergiques (da) du mésencéphale pour greffe
WO2014176606A1 (fr) 2013-04-26 2014-10-30 Memorial Sloan-Kettering Center Center Interneurones corticaux et autres cellules neuronales produits par la différentiation dirigée de cellules pluripotentes et multipotentes
WO2015077648A1 (fr) 2013-11-21 2015-05-28 Memorial Sloan-Kettering Cancer Center Spécification de dérivés de placode crânienne fonctionnelle à partir de cellules souches pluripotentes humaines
WO2017117571A1 (fr) * 2015-12-31 2017-07-06 President And Fellows Of Harvard College Neurones et compositions et méthodes de production associées
WO2017132596A1 (fr) 2016-01-27 2017-08-03 Memorial Sloan-Kettering Cancer Center Différenciation de neurones corticaux à partir de cellules souches pluripotentes humaines

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096496A2 (fr) 2009-02-17 2010-08-26 Memorial Sloan-Kettering Cancer Center Procédés de conversion neurale de cellules souches embryonnaires humaines
WO2011149762A2 (fr) 2010-05-25 2011-12-01 Memorial Sloan-Kettering Cancer Center Procédé de différenciation en nocicepteur de cellules souches embryonnaires humaines et ses utilisations
WO2013067362A1 (fr) 2011-11-04 2013-05-10 Memorial Sloan-Kettering Cancer Center Neurones dopaminergiques (da) du mésencéphale pour greffe
WO2014176606A1 (fr) 2013-04-26 2014-10-30 Memorial Sloan-Kettering Center Center Interneurones corticaux et autres cellules neuronales produits par la différentiation dirigée de cellules pluripotentes et multipotentes
WO2015077648A1 (fr) 2013-11-21 2015-05-28 Memorial Sloan-Kettering Cancer Center Spécification de dérivés de placode crânienne fonctionnelle à partir de cellules souches pluripotentes humaines
WO2017117571A1 (fr) * 2015-12-31 2017-07-06 President And Fellows Of Harvard College Neurones et compositions et méthodes de production associées
WO2017132596A1 (fr) 2016-01-27 2017-08-03 Memorial Sloan-Kettering Cancer Center Différenciation de neurones corticaux à partir de cellules souches pluripotentes humaines

Non-Patent Citations (40)

* Cited by examiner, † Cited by third party
Title
BARDY CEDRIC ET AL: "Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 112, no. 20, 13 April 2015 (2015-04-13), XP093022650, ISSN: 0027-8424, DOI: 10.1073/pnas.1504393112 *
BARDY CEDRIC: "Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro: Methods (Online Supplement)", PNAS, 13 April 2015 (2015-04-13), XP093022653, Retrieved from the Internet <URL:pnas.1504393112.sapp.pdf> [retrieved on 20230209] *
BARKOVICH ET AL., BRAIN, vol. 135, 2012
BENSAAD ET AL., CELL, vol. 126, 2006
BILLIARD ET AL., CANCER METAB, vol. 1, 2013
BIRD ET AL., METABOLITES, vol. 9, 2019
CHAMBERS ET AL., NATURE BIOTECHNOL, vol. 27, 2009
CHAMBERS ET AL., NATURE BIOTECHNOL, vol. 30
CHARRIER ET AL., CELL, vol. 149, 2012
COMPAGNUCCI ET AL., CELL MOL LIFE SCI, vol. 71, 2014
CRINO PETER B.: "The mTOR signalling cascade: paving new roads to cure neurological disease", NATURE REVIEWS NEUROLOGY, vol. 12, no. 7, 24 June 2016 (2016-06-24), London, pages 379 - 392, XP093022969, ISSN: 1759-4758, Retrieved from the Internet <URL:http://www.nature.com/articles/nrneurol.2016.81> DOI: 10.1038/nrneurol.2016.81 *
DOETSCHMAN, J EMBRYOL EXP MORPHOL, vol. 87, 1985
EBERTGREENBERG, NATURE, vol. 493, 2013
ESPUNYD-CAMACHO ET AL., NEURON, vol. 77, 2013
EVANSKAUFMAN, NATURE, vol. 292, 1981
GAO ET AL., EXP NEUROL, vol. 201, 2006
GASPARD ET AL., NATURE, vol. 455, 2008
GORDON AARON ET AL: "Long-term maturation of human cortical organoids matches key early postnatal transitions", NATURE NEUROSCIENCE, vol. 24, no. 3, 28 March 2021 (2021-03-28), pages 331 - 342, XP037389353, ISSN: 1097-6256, DOI: 10.1038/S41593-021-00802-Y *
HENTZE ET AL., TRENDS BIOTECHNOL, vol. 25, 2007
IWATA ET AL., SCIENCE, vol. 862, 2020
IWATA RYOHEI ET AL: "Species-specific mitochondria dynamics and metabolism regulate the timing of neuronal development", BIORXIV, 27 December 2021 (2021-12-27), XP093022600, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.12.27.474246v1> [retrieved on 20230209], DOI: 10.1101/2021.12.27.474246 *
LINARO ET AL., NEURON, vol. 104, 2019
MARCHETTO ET AL., ELIFE, vol. 8, 2019
MAROOF ET AL., CELL STEM CELL, vol. 12, 2013
MARTIN, PNAS, vol. 78, 1981
MENENDEZ ET AL., PNAS, vol. 108, 2011
MICA ET AL., CELL REP, vol. 3, 2013
PANG ET AL., NEUROLOGIST, vol. 14, 2008
QIU ET AL., ELIFE, vol. 5, 2016
ROBERTSON: "Teratocarcinomas and Embryonic Stem Cells: A Practical Approach", 1987, IRL PRESS
SAMBROOK ET AL.: "current Protocols in Molecular Biology", 2012, COLD SPRING HARBOR PRESS
SATIR TUGCE MUNISE ET AL: "Accelerated neuronal and synaptic maturation by BrainPhys medium increases A[beta] secretion and alters A[beta] peptide ratios from iPSC-derived cortical neurons", SCIENTIFIC REPORTS, vol. 10, no. 1, 17 January 2020 (2020-01-17), XP093022647, Retrieved from the Internet <URL:https://www.nature.com/articles/s41598-020-57516-7> DOI: 10.1038/s41598-020-57516-7 *
SHEAR ET AL., BRAIN RESEARCH, vol. 1026, 2004
SMITH ET AL., NATURE, vol. 336, 1988
THOMSON ET AL., SCIENCE, vol. 282, 1998, pages 1145 - 1147
TSO ET AL., J BIOL CHEM, vol. 289, 2014
VAN DEN AMEELE ET AL., TRENDS NEUROSC, vol. 7, 2014
YICHEN SHI ET AL: "Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks", NATURE PROTOCOLS, vol. 7, no. 10, 13 September 2012 (2012-09-13), GB, pages 1836 - 1846, XP055328305, ISSN: 1754-2189, DOI: 10.1038/nprot.2012.116 *
ZHENG ET AL., ELIFE, 2017
ZHOU ET AL., CELL DEATH & DISEASE, 2019

Similar Documents

Publication Publication Date Title
US20230365928A1 (en) Cortical interneurons and other neuronal cells produced by the directed differentiation of pluripotent and multipotent cells
JP6588500B2 (ja) 移植用中脳ドーパミン（ｄａ）ニューロン
Tsai et al. Cell contact regulates fate choice by cortical stem cells
EP2772534B1 (fr) Culture de cellule souche épithéliale colorectale, et transplantation d&#39;épithélium colorectal
AU2020244551B2 (en) Directed differentiation of astrocytes from human pluripotent stem cells for use in drug screening and the treatment of Amyotrophic Lateral Sclerosis (ALS)
CA2815223A1 (fr) Procedes de differenciation pour la production de populations de cellules gliales
US20230233617A1 (en) Methods for differentiating stem cells into dopaminergic progenitor cells
Brunet et al. Early acquisition of typical metabolic features upon differentiation of mouse neural stem cells into astrocytes
AU2014330807A1 (en) Directed differentiation of astrocytes from human pluripotent stem cells for use in drug screening and the treatment of Amyotrophic Lateral Sclerosis (ALS)
US20070065941A1 (en) Neuronal cells obtained by electric pulse treatment of es cells
KR20130011023A (ko) 전능성 줄기세포로부터 희소돌기아교전구세포의 제조 방법
US10696945B2 (en) Methods for treating a brain tissue damage by cultured adult pluripotent olfactory stem cells
WO2023104792A1 (fr) Amplificateurs de maturation neuronale
KR20210077634A (ko) 인간 만능 줄기세포로부터 3d 오가노이드를 이용하여 미세교세포를 다량 확보하는 미세교세포의 분화방법
Luo et al. Developmental deficits and early signs of neurodegeneration revealed by PD patient derived dopamine neurons
Jovanovic et al. Directed differentiation of human pluripotent stem cells into radial glia and astrocytes bypasses neurogenesis
Petralla AGC-1 deficiency, a rare genetic demyelinating and neurodegenerative disease: a study on oligodendrocyte precursor cells in cell lines, a mouse model and human iPS-derived brain cells
Kumari Role of SMAD2 and SMAD3 on Adipose Tissue Development and Function
Leeder Primitive Neural Stem Cells in the Mouse Brain
Dadwal Metformin promotes tissue repair and functional recovery in a model of postnatal brain injury

Legal Events

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

Ref document number: 22830536

Country of ref document: EP

Kind code of ref document: A1