WO2020091688A1

WO2020091688A1 - A spinal organoid and method of obtaining thereof

Info

Publication number: WO2020091688A1
Application number: PCT/SG2019/050472
Authority: WO
Inventors: Shi Yan NG; Jin Hui HOR
Original assignee: Agency For Science, Technology And Research
Priority date: 2018-11-01
Filing date: 2019-09-19
Publication date: 2020-05-07

Abstract

There is provided an organoid comprising motor neurons derived from stem cells, said motor neurons characterized by the expression of insulin gene enhancer protein (ISL1). In particular, the organoid is a spinal organoid. Also provided is a method of obtaining an organoid, comprising culturing stem cells in a culture medium comprising one or more reagents capable of inducing neuralization of the stem cells e.g. CHIR99021 and LDN-193189; contacting the stem cells to a reagent capable of caudalizing the stem cells e.g. retinoic acid; and contacting the stem cells to a reagent capable of ventralizing the stem cells e.g. purmorphamine. In addition, the present invention relates to a method of screening agents capable of prolonging motor neuron survival using said organoids, as well as a method of treating spinal muscular atrophy with a CDK4/6 inhibitor e.g. PD 0332991.

Description

A SPINAL ORGANOID AND METHOD OF OBTAINING THEREOF

TECHNICAL FIELD

The present disclosure relates broadly to an organoid and a method of obtaining an organoid. BACKGROUND

Motor neuron diseases are a group of neurodegenerative disorders that selectively affect motor neurons in both children and adults. General symptoms of motor neuron diseases include muscle weakness, muscle spasms, pain, difficulty swallowing, breathing difficulties, total body paralysis etc. There are no known curative treatments for the majority of motor neuron diseases.

One of the most common form of motor neuron disease affecting children is Spinal Muscular Atrophy (SMA). It is a genetic disease caused by homozygous mutations or deletions in the SMN1 gene, resulting in drastically reduced amounts of the SMN (survival of motor neuron) protein. SMA manifests clinically as a childhood motor neuron disease, with the death of spinal motor neurons and subsequent denervation of skeletal muscles resulting in arrested childhood developmental milestones, paralysis and eventually death in severe SMA. The SMN2 gene in humans primarily gives rise to truncated and partially functional protein lacking exon 7, known as SMNA7. As such, copy number variation in the SMN2 gene is known to affect clinical severity of SMA patients. SMA is classified into four categories (SMA Type I to Type IV), with Type I as the most severe and Type IV being adult-onset. While most Type I patients have between 1 and 2 copies of SMN2, Type IV patients can have between 4 and 6 copies of SMN2.

Although SMN is ubiquitously expressed, it is still not completely understood why motor neurons are one of the most severely affected cell types. The roles of SMN have not been exhaustively characterized, but it is best known as a component of the spliceosome, and widespread splicing defects have been reported in SMA and SMN-deficient cultures. Due to its importance as a splicing regulator and the observation that SMN-null mice are embryonic lethal, it has been suggested that SMA is also a neurodevelopmental disorder, where motor neurons in the spinal cord do not properly form, and those that eventually survive would rapidly degenerate postnatally.

While animal models have provided some understanding into the mechanisms of neurodevelopment of motor neuron diseases, there is limited value in the use of animal models due to issues of translatability arising from differences between animals and human and limitations in the recapitulation of the microenvironment (e.g. cellular features, phenotype, genotype, neurodevelopment, neuropathology etc.). Thus, there is a need for other models that are capable of mimicking the microenvironment of a motor neuron disease or providing a simple model of a motor neuron disease and its biological processes.

SUMMARY In one aspect, there is provided an organoid comprising, motor neurons derived from stem cells, said motor neurons characterized by the expression of ISL1 (insulin gene enhancer protein).

In one embodiment of the organoid disclosed herein, the motor neurons are further characterized by the expression of one or more markers selected from the group consisting of FOXP1 (Forkhead box protein P1 ) and SMI32 (neurofilament FI).

In one embodiment of the organoid disclosed herein, the motor neurons comprise one or more types of motor neurons selected from the group consisting of cervical motor neurons characterized by the expression of FIOXB4, brachial motor neurons characterized by the expression of FIOXC8, and lumbar motor neurons characterized by the expression of FIOXC10. In one embodiment of the organoid disclosed herein, the motor neurons consist of cervical motor neurons characterized by the expression of HOXB4, and brachial motor neurons characterized by the expression of HOXC8.

In one embodiment of the organoid disclosed herein, the motor neurons are limb-innervating motor neurons characterized by co-expression of FOXP1 and ISL1.

In one embodiment of the organoid disclosed herein, the motor neurons are cholinergic motor neurons characterized by co-expression of ISL1 (Insulin gene enhancer protein) and choline acetyltransferase (ChAT).

In one embodiment of the organoid disclosed herein, the motor neurons are capable of neurite outgrowth when the organoid is co-cultured with myoblasts.

In one embodiment of the organoid disclosed herein, the motor neurons are capable of forming neuromuscular junctions (NMJs) when the organoid is co cultured with myoblasts.

In one embodiment of the organoid disclosed herein, the organoid further comprises cells characterized by the expression of SOX1 (SRY-Box 1 ).

In one embodiment of the organoid disclosed herein, the organoid further comprises motor neurons characterized by the expression of TUJ1 (neuron- specific Class III b-tubulin).

In one embodiment of the organoid disclosed herein, the organoid comprises one or more rosette structures.

In one embodiment of the organoid disclosed herein, the organoid further comprises interneurons characterized by the expression of at least one interneuron marker selected from the group consisting of CHX10 (Ceh-10 Homeodomain-Containing Homolog), Calbindin, PAX2 (paired box gene 2) and LHX1 (Homeobox Protein Lim-1 ); and/or astrocytes characterized by the expression of at least one astrocytic marker selected from the group consisting of S100p (S100 calcium-binding protein B), AQP4 (aquaporin-4) and GFAP (glial fibrillary acidic protein).

In one embodiment of the organoid disclosed herein, the organoid further comprises V 2a interneurons characterized by the expression of CHX10 marker. In one embodiment of the organoid disclosed herein, the organoid further comprises Renshaw cells characterized by the expression of Calbindin, PAX2 and LHX1 markers.

In one embodiment of the organoid disclosed herein, the organoid further comprises astrocytes characterized by the expression of S100b.

In one embodiment of the organoid disclosed herein, the number of motor neurons comprises at least 40% of the total number of cells in the organoid.

In one embodiment of the organoid disclosed herein, the organoid is a spinal organoid.

In one embodiment of the organoid disclosed herein, the spinal organoid is a ventral spinal organoid comprising neurons that are found in the ventral horn of a spinal cord.

In one embodiment of the organoid disclosed herein, the stem cells are pluripotent stem cells.

In one embodiment of the organoid disclosed herein, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

In one embodiment of the organoid disclosed herein, the stem cells are obtained from a healthy (non-diseased) subject.

In one embodiment of the organoid disclosed herein, the stem cells are obtained from a subject having a neurodegenerative disease.

In one embodiment of the organoid disclosed herein, the neurodegenerative disease is a motor neuron disease.

In one embodiment of the organoid disclosed herein, the motor neuron disease is selected from the group consisting of Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS).

In one embodiment of the organoid disclosed herein, the motor neuron disease is SMA.

In one embodiment of the organoid disclosed herein, the motor neurons derived from stem cells of a subject having SMA are characterized by a higher expression of Cyclin Dependent Kinase (CDK) and/ or cyclin (CCN) as compared to motor neurons derived from stem cells of a healthy (non-diseased) subject. In one embodiment of the organoid disclosed herein, the CDK is one or more selected from the group consisting of CDK1 , CDK2, CDK4, and CDK6.

In one embodiment of the organoid disclosed herein, the CCN is one or more selected from the group consisting of CCNA2, CCNB1 , CCNB2, CCND1 , CCND2, CCNE1 , and CCNE2.

In one embodiment of the organoid disclosed herein, the motor neurons derived from stem cells of a subject having SMA are characterized by a higher expression of CCNA2, CCNB1 , CCNB2, CCNE2 and CDK6 as compared to motor neurons derived from stem cells of a healthy (non-diseased) subject.

In one embodiment of the organoid disclosed herein, the motor neurons derived from stem cells of a subject having SMA are characterized by a higher expression of proliferative marker Ki67 as compared to motor neurons derived from stem cells of a healthy (non-diseased) subject.

In one embodiment of the organoid disclosed herein, the motor neurons derived from stem cells of a subject having SMA are further characterized by the expression of apoptotic marker cCASP3.

In one embodiment of the organoid disclosed herein, the organoid is obtained in-vitro.

In one embodiment of the organoid disclosed herein, the expression comprises gene expression and/ or protein expression.

In one embodiment of the organoid disclosed herein, the organoid is used as a platform for screening agents capable of prolonging motor neuron survival, wherein the organoid is to be contacted to one or more agents in a culture.

In one aspect, there is provided a method of obtaining an organoid, the method comprising, culturing stem cells in a culture medium comprising one or more reagents capable of inducing neuralization of the stem cells; contacting the stem cells to a reagent capable of caudalizing the stem cells; contacting the stem cells to a reagent capable of ventralizing the stem cells; and forming the organoid.

In one embodiment of the method disclosed herein, the one or more reagents capable of inducing neuralization of the stem cells is selected from the group consisting of an agent for blocking Bone Morphogenetic Protein (BMP) signaling and an agent for activating Wnt pathways. In one embodiment of the method disclosed herein, the agent for blocking Bone Morphogenetic Protein (BMP) signaling comprises one or more selected from the group consisting of LDN-193189, dorsomorphin and Noggin.

In one embodiment of the method disclosed herein, the agent for activating Wnt pathways comprises CHIR99021 and/or BIO.

In one embodiment of the method disclosed herein, the step of contacting the stem cells to a reagent capable of inducing neuralization of the stem cells comprises simultaneously contacting the stem cells with the agent for blocking Bone Morphogenetic Protein (BMP) signaling and the agent for activating Wnt pathways

In one embodiment of the method disclosed herein, the reagent capable of caudalizing the stem cells comprises retinoic acid.

In one embodiment of the method disclosed herein, the reagent capable of ventralizing the stem cells is a Sonic Hedgehog (SHH) pathway agonist.

In one embodiment of the method disclosed herein, the SHH pathway agonist is purmorphamine.

In one embodiment of the method disclosed herein, the reagent capable of caudalizing the stem cells is contacted to the stem cells between day 3 to day 15 of the culture, wherein day 0 is the day of seeding the stem cells.

In one embodiment of the method disclosed herein, the reagent capable of caudalizing the stem cells is contacted to the stem cells on or after the expression of one or more neuroepithelial stem cell markers in the stem cells.

In one embodiment of the method disclosed herein, the one or more neuroepithelial stem cell markers is selected from the group consisting of SOX1 and Nestin.

In one embodiment of the method disclosed herein, the reagent capable of ventralizing the stem cells is contacted to the stem cells between day 10 to day 17 of the culture, wherein day 0 is the day of seeding the stem cells.

In one embodiment of the method disclosed herein, the method further comprises contacting the stem cells to one or more neurotrophic factors capable of promoting neuronal maturation of the stem cells. In one embodiment of the method disclosed herein, the one or more neurotrophic factors capable of promoting neuronal maturation is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and glial cell- derived neurotrophic factor (GDNF).

In one embodiment of the method disclosed herein, the one or more neurotrophic factors capable of promoting neuronal maturation of the stem cells is contacted to the stem cells from day 17 onwards of the culture, wherein day 0 is the day of seeding the stem cells.

In one embodiment of the method disclosed herein, the method further comprises dissociating the stem cells into single cells prior to culturing the stem cells in the culture medium.

In one embodiment of the method disclosed herein, the method further comprises seeding the stem cells at a density of at least 150,000 cells/ml in a suspension culture.

In one embodiment of the method disclosed herein, the method further comprises encapsulating the stem cells in a support matrix.

In one embodiment of the method disclosed herein, the stem cells are encapsulated in the support matrix for one or more day, or two or more days, or three or more days, or four or more days, or at least one day, or at least two days, or at least three days, or at least four days, or at most one day, or at most two days, or at most three days, or at most four days.

In one embodiment of the method disclosed herein, the support matrix is a growth matrix capable of providing support matrices to the stem cells and/ or acting as an extracellular matrix for the stem cell and/ or acting as a basement membrane matrix for the stem cells.

In one embodiment of the method disclosed herein, the support matrix is a gelatinous protein mixture derived from mouse tumour cells.

In one embodiment of the method disclosed herein, the method further comprises transferring the cells in the support matrix into a dynamic cell culture device.

In one embodiment of the method disclosed herein, the dynamic cell culture device is a spinner flask. In one embodiment of the method disclosed herein, the stem cells are pluripotent stem cells.

In one embodiment of the method disclosed herein, the stem cells are induced pluripotent stem cells (iPSCs).

In one embodiment of the method disclosed herein, the stem cells are obtained from a healthy (non-diseased) subject.

In one embodiment of the method disclosed herein, the stem cells are obtained from a subject having a neurodegenerative disease.

In one embodiment of the method disclosed herein, the neurodegenerative disease is a motor neuron disease.

In one embodiment of the method disclosed herein, the motor neuron disease is selected from the group consisting of SMA and ALS.

In one embodiment of the method disclosed herein, the organoid is a spinal organoid.

In one embodiment of the method disclosed herein, the spinal organoid is a ventral spinal organoid.

In one embodiment of the method disclosed herein, the spinal organoid comprises motor neurons characterized by the expression of ISL1.

In one embodiment of the method disclosed herein, the spinal organoid comprises one or more cells selected from the group consisting of motor neurons, interneurons, and astrocytes.

In one embodiment of the method disclosed herein, the spinal organoid comprises neurons that are found in the ventral horn of a spinal cord.

In one embodiment of the method disclosed herein, the spinal organoid comprises cells which are organized into one or more rosette structures.

In one embodiment of the method disclosed herein, the method is an in- vitro method.

In one aspect, there is provided a method of screening agents capable of prolonging motor neuron survival comprising, contacting one or more agents to the organoid disclosed herein or the organoid produced by the method disclosed herein, In one aspect, there is provided an organoid obtained using the methods disclosed herein.

In one aspect, there is provided a spinal organoid comprising one or more cells selected from the group consisting of cholinergic motor neurons characterized by the expression of ISL1 , FOXP1 , and SMI32 markers, V 2a interneurons characterized by the expression of CHX10 marker, Renshaw cells characterized by the expression of Calbindin marker and astrocytes characterized by the expression of S100p.

In one aspect, there is provided a method of treating spinal muscular atrophy comprising administering an effective amount of a CDK4/6 inhibitor to a subject in need thereof.

In one aspect, there is provided a use of a CDK inhibitor in the manufacture of a medicament for the treatment of spinal muscular atrophy, wherein the CDK inhibitor is a CDK4/6 inhibitor.

In one embodiment of the use disclosed herein, the CDK4/6 inhibitor is PD

0332991.

DEFINITIONS

The term“organoid” as used herein is to be interpreted broadly to include a three-dimensional (3D) multicellular in vitro tissue construct that substantially mimics its corresponding in vivo tissue or organ. Therefore, an organoid may contain multiple cell types and mimics physiological conditions/ structures observed in vivo in a tissue or an organ of a subject. This is in contrast to two- dimensional (2D) cultures that typically contain fewer cell types and do not mimic physiological conditions/ structures observed in vivo in a tissue or an organ. The organoid may be used to study aspects of that tissue or organ in a tissue culture dish.

The term“in vitro " refers to an artificial environment and to processes or reactions that occur within an artificial environment, or elsewhere outside a living organism. In vitro environments may include, but are not limited to, test tubes, cell cultures, bioreactors etc.

The term“in vivo” as used herein refers to the natural environment within the body of an organism (e.g., an animal or a human) and to processes or reaction that occur within a natural environment.

The term“cell culture” as used herein refers to any in vitro culture of cells.

The term“organoid culture” as used herein refers to any in vitro culture of organoids.

The terms“culture media” as used herein refer to media that are suitable to support the growth of cells or organoids of interest in vitro.

The term“stem cell” as used herein refers to a cell that is totipotent or pluripotent or multipotent and are capable of differentiating into one or more different cell types.

The term“embryonic stem cell” as used herein refers to a cell of a stem cell line, or a cell isolated from an embryo or placenta or umbilical cord.

The term“induced pluripotent stem cell” (“iPSC”) as used herein refers to a stem cell induced from a somatic cell, e.g., a differentiated somatic cell, and that has a higher potency than said somatic cell. iPSCs are capable of self- renewal and differentiation into mature cells.

The term“pluripotent” as used herein refers to a cell that is capable of differentiating into any differentiated cell type.

The term“neural cell” or“neuronal cell” as used herein refers to a cell that in vivo would become part of the nervous system and in culture is obtained by, for example, the methods as disclosed herein.

The term“progenitor cell” as used herein refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue. The term“neural progenitor cell” as used herein refers to a non-fully differentiated cell capable of forming a part of the nervous system.

The term“motor neuron” as used herein refers to a neuron whose cell body is located in the motor cortex, brainstem or the spinal cord, and whose axon (fiber) projects to the spinal cord or outside of the spinal cord to directly or indirectly control effector organs, mainly muscles and glands. The term“rosette structure” or“rosette” as used herein refers to a halo or spoke-wheel arrangement of cells.

The term “neurite outgrowth” as used herein refers to observation of elongated, membrane-enclosed protrusions of cytoplasm from neural cells.

The term“neural induction” as used herein refers to a process by which embryonic cells in the ectoderm make a decision to acquire a neural fate (to form the neural plate) rather than give rise to other structures such as epidermis or mesoderm.

The term“inducing differentiation” as used herein in reference to a cell refers to changing the default cell type (genotype and/or phenotype) to a non default cell type (genotype and/or phenotype). Thus“inducing differentiation in a stem cell” refers to inducing the cell to divide into progeny cells with characteristics that are different from the stem cell, such as genotype (i.e. change in gene expression) and/or phenotype (i.e. change in expression of a protein).

The term“cell differentiation” as used herein in reference to a pathway refers to a process by which a less specialized cell (i.e. stem cell) develops or matures or differentiates to possess a more distinct form and/or function into a more specialized cell or differentiated cell, (e.g. motor neuron, interneuron, astrocytes etc.).

The term“caudalize”,“caudalizing” or“caudalization” as used herein refers to the initiation of posterior pathways of neural development in the dorsalized ectoderm during embronic development, for example, dorsalized ectorderm develops various levels of posterior neural tissues, depending on the extent of caudalization. The term“caudalizing agent/ factor” refers to a compound that induces, or contributes to, development in the direction of the bottom, tail, or inferior position of an object or subject.

The term“ventralize” or“ventralizing” or“ventralization” as used herein refers to the development in the direction of the front or anterior position of an object or subject. The term“ventralizing agent/ factor” refers to a compound that induces, or contributes to, development in the direction of the front or anterior position of an object or subject. The term“neural maturation” as used herein refers to a developmental process, independent of morphogenetic (shape) change, that is required for a neuron to attain its fully functional state.

The term“neurotrophic factor” as used herein refers to a factor involved in the nutrition or maintenance of neural tissue. Neurotrophic factors may further the development and differentiation of committed neural progenitor cells, or they may induce or enhance the growth and survival of differentiated neural cells. Examples of neurotrophic factor include but are not limited to GDNF (glial cell line-derived neurotrophic factor), BDNF (brain-derived neurotrophic factor), NGF (nerve growth factor), NT3 (neurotrophin-3), and CNTF (ciliary neurotrophic factor).

The term“marker” or“cell marker” as used herein refers to gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker only. Markers may refer to a“pattern” or“group” of markers such that a designated group of markers may identify a cell or cell type from another cell or cell type.

The term“expressing” or“expression” as used herein in relation to a gene or protein refers to making an mRNA or protein which can be observed using assays known in the art, such as RTPCR (reverse transcription polymerase chain reaction), microarray assays, antibody staining assays, and the like.

The term “gene expression” as used herein refers to the process of converting genetic information encoded in a gene into RNA (ribonucleic acid) (e.g., mRNA, rRNA, tRNA, or snRNA) through“transcription” of the gene (i.e. , via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through“translation” of mRNA. Gene expression can be regulated at many stages in the process.“Up-regulation” or“activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up- regulation or down-regulation are often called “activators” and “repressors,” respectively. The term“inhibitor” as used herein in relation to inhibiting a signaling target or a signaling target pathway refers to a compound that interferes with (i.e. reduces or eliminates or suppresses) a resulting target molecule or target compound or target process when compared to an untreated cell or a cell treated with a compound that does not inhibit a treated cell or tissue.

The term“healthy subject” as used herein refers to a subject that can be confirmed not to have a specific disease (e.g. neurodegenerative disease) based on a set of signs, symptoms, tests and criteria used by a person skilled in the art (e.g. medical/ veterinary professional) to diagnose the disease. In some examples, the subject may be a mammal which includes but is not limited to human, non-human primates, rodents (such as rats, mice etc.), and the like.

The term“subject having a disease” as used herein refers to a subject e.g. patient that can be confirmed to have the disease (e.g. neurodegenerative disease) based on a set of signs, symptoms, tests and criteria used by a person skilled in the art (e.g. medical/ veterinary professional) to diagnose the disease.

The term “neurodegeneration” as used herein refers to a condition of deterioration of neurons, wherein the neurons change to a lower or less functionally-active form. Examples of conditions associated with neuron degeneration include peripheral neuropathies, demyelinating conditions, and the primary neurologic conditions (e.g., neurodegenerative diseases), CNS (central nervous system) and PNS (peripheral nervous system) traumas and injuries, and acquired secondary effects of non-neural dysfunction (e.g., neural loss secondary to degenerative, pathologic, or traumatic events) described herein.

The term“treating” as used herein is to be interpreted broadly to mean attempting to inhibit the progression of a disease (e.g. spinal muscular atrophy) temporarily or attempting to stop the progression of the disease permanently. The disease may not need to be effectively treated eventually.

The term“effective amount” as used herein is to be interpreted broadly as an amount that is sufficient to carry out its intended effect. For example, when an “effective amount” is used to refer to the administration of a compound, it can refer to the situation where the compound is administered at a dosage and/or for a period of time necessary to achieve the desired result. The term“micro” as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns.

The term“nano” as used herein is to be interpreted broadly to include dimensions less than about 1000 nm.

The term“derived from” as used herein shall be taken to indicate that a specified product, in particular a molecule such as, for example, a polypeptide, protein, gene or nucleic acid molecule, antibody molecule, Ig fraction, or other molecule, or a biological sample comprising said molecule, may be obtained directly/ indirectly from a particular source, organism, tissue, organ or cell.

The term“contacting” as used herein in the context of contacting a cell or organoid with an agent or reagent e.g. a compound refers to placing the compound in a location that will allow it to touch the cell or organoid. Contacting may be accomplished using any suitable method. For example, contacting may be done by adding the compound to a tube containing the cell or organoid. Contacting may also be accomplished by adding the compound to a culture of the cell or organoid.

The terms “coupled” or “connected” as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

The term“associated with”, used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.

The term“adjacent” used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween.

The term "and/or", e.g.,“X and/or Y” is understood to mean either“X and

Y” or“X or Y” and should be taken to provide explicit support for both meanings or for either meaning. Further, in the description herein, the word“substantially” whenever used is understood to include, but not restricted to,“entirely” or“completely” and the like. In addition, terms such as“comprising”,“comprise”, and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a“one” feature is also intended to be a reference to“at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as“comprising”, “comprise”, and the like. Therefore, in embodiments disclosed herein using the terms such as“comprising”,“comprise”, and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as“consisting”,“consist”, and the like. Further, terms such as“about”, “approximately” and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1 % to 5% is intended to have specifically disclosed sub-ranges 1 % to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1 %, 2%, 3%, 4% and 5%. The intention of the above specific disclosure is applicable to any depth/breadth of a range.

Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. Flowever, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

DETAILED DESCRIPTION

Non-limiting embodiments of an organoid and a method of obtaining an organoid are disclosed hereinafter.

In various embodiments, the organoid comprises motor neurons derived from stem cells, said motor neurons characterized by the expression of ISL1 (insulin gene enhancer protein). Expression of ISL1 may refer to gene expression of the ISL1 gene (or mRNA) which encodes for the insulin gene enhancer protein and/or protein expression of the insulin gene enhancer protein. In various embodiments, the organoid is obtained/ prepared in-vitro.

In various embodiment, the motor neurons are further characterized by the expression of one or more markers such as TUJ1 (neuron-specific Class III b- tubulin), FOXP1 (forkhead box protein P1 ) and SMI32 (neurofilament H).

In various embodiments, the organoid further comprises one or more other types of cells which are found together with motor neurons in a native tissue microenvironment e.g. spinal cord.

Advantageously, the organoid provides a 3D structure which is capable of recapitulating/ reproducing/ mimicking the tissue microenvironment with its cell- cell, cell-extracellular matrix, and cell-niche interactions. For example, the diversity of cells found in spinal cord neurogenesis may provide a more“in-vivo” like culture system as compared to conventional 2D culture

In various embodiments, the number of motor neurons e.g. ISL expressing (ISL⁺) motor neurons comprises at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the total number of cells in the organoid. In various embodiments, the organoid further comprises neural progenitor cells characterized by the expression of SOX1 (SRY-Box 1 ) and/or Nestin markers. In various embodiments, the organoid further comprises neurons characterized by the expression of TUJ1 marker. TUJ1 marker may be present in newly generated immature postmitotic neurons and differentiated neurons and in some mitotically active neuronal precursors.

In various embodiments, the organoid comprises one or more rosette structures formed on the organoid body. The one or more rosette structures are formed by SOX1 expressing cells organized into a halo or spoke-wheel arrangement. Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. The formation of one or more rosette structure indicates that the cells in the organoid are able to recapitulate spinal cord neurogenesis.

In various embodiments, the organoid comprises one or more types of cells found along the rostro-caudal axis and/ or dorso-ventral axis of a spinal cord. In various embodiments, the organoid comprises one or more types of motor neurons selected from the group consisting of cervical motor neurons, brachial motor neurons, thoracic motor neurons and lumbar motor neurons. Cervical/ brachial motor neurons may be characterized by the expression of homeobox (HOX) markers such as HOX4 to HOX8. Thoracic motor neurons may be characterized by the expression of markers such as HOX9. Lumbar motor neurons may be characterized by the expression of markers such as HOX10 to HOX13. In various embodiments, cervical motor neurons are characterized by the expression of HOXB4 marker, brachial motor neurons are characterized by the expression of HOXC8 marker, and lumbar motor neurons are characterized by the expression of HOXC10 marker. In various embodiments, the motor neurons consist of cervical motor neurons characterized by the expression of HOXB4, and brachial motor neurons characterized by the expression of HOXC8.

In various embodiments, the motor neurons are limb-innervating motor neurons. Nerve-innervating neurons may be characterized by expression of FOXP1 marker, or co-expression of FOXP1 and ISL1 markers. In various embodiments, the motor neurons are cholinergic motor neurons characterized by co-expression of ISL1 and choline acetyltransferase (ChAT). The motor neurons are capable of neurite outgrowth when the organoid is co-cultured with myotubes derived from myoblasts. In addition, the motor neurons are capable of forming neuromuscular junctions (NMJs) when the organoid is co-cultured with myotubes derived from myoblasts.

In various embodiments, the organoid further comprises one or more types of interneurons. The interneurons may be excitatory or inhibitory interneurons. In various embodiments, the organoid further comprises interneurons such as V2a interneurons and/or Renshaw cells. V2A interneuron is a type of excitatory interneuron and Renshaw cell is a type of inhibitory interneuron. In various embodiments, the interneurons are characterized by the expression of at least one interneuron marker selected from the group consisting of CHX10 (Ceh-10 Homeodomain-Containing Homolog), Calbindin, PAX2 (paired box gene 2) and LHX1 (Homeobox Protein Lim-1 ). In various embodiments, V 2a interneurons are characterized by the expression of CHX10 marker. In various embodiments, Renshaw cells are characterized by the expression of Calbindin, PAX2 and LHX1 markers.

In various embodiments, the organoid further comprises astrocytes. In various embodiments, astrocytes are characterized by the expression of at least one astrocytic marker selected from the group consisting of S100p (S100 calcium-binding protein B), AQP4 (aquaporin-4) and GFAP (glial fibrillary acidic protein). In various embodiments, astrocytes are characterized by the expression of S100p marker.

Advantageously, the organoid derived from stem cells comprises motor neurons, astrocytes as well as interneurons that form part of the motor circuit, thus mimicking the microenvironment in-vivo. Without wishing to be bound by theory, it is believed that the organoids are derived from the cells’ innate ability to self-organize. Due to its diversity of cell types, the organoid may be used for modeling neurodevelopmental disorders, studying neurodegenerative disorders. For example, the organoid may be useful for studying hyperexcitability of motor neurons in diseases e.g. SMA and ALS and may also be used for studying neural development in human having such diseases.

In various embodiments, the cells in the organoid are derived from stem cells. The stem cells may be pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) e.g.“true” embryonic stem cell (ES cells) derived from embryos, embryonic stem cells made by somatic cell nuclear transfer (ntES cells) and embryonic stem cells from unfertilized eggs (parthenogenesis embryonic stem cells, or pES cells). The stem cells may be derived from sources which include but are not limited to embryonic or fetal tissue, post-fetal tissue, adult tissue and differentiated tissue. The stem cell may be obtained from a subject e.g. human. The stem cell may be obtained from the same species as the subject or different species from the subject. The stem cell may be from a healthy (i.e. non-diseased) or diseased subject.

In various embodiments, the cells in the organoid are derived from iPSCs. iPSCs may be induced from somatic cells from different species, such as mice, humans, rats, marmosets, rhesus monkeys, pigs, and rabbits. In various embodiments, the iPSCs are derived from BJ fibroblast e.g. human foreskin tissue.

In various embodiments, the stem cells are obtained from a subject having a neurodegenerative disease. In one embodiment, the neurodegenerative disease is a motor neuron disease selected from the group comprising or consisting of SMA, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, hereditary spastic paraplegia, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy and post- polio syndrome. In one embodiment, the motor neuron disease is selected from the group consisting of SMA and ALS. In one embodiment, the motor neuron disease is SMA.

In various embodiments, the organoid is derived from stem cells e.g. iPSCs which are obtained from a subject having SMA, said stem cells differentiating into neuronal cell types e.g. motor neurons, interneurons, astrocytes etc. to form the organoid. In various embodiments, the organoid derived from stem cells of a subject having SMA comprises motor neurons characterized by a higher expression (or increased expression) of Cyclin Dependent Kinase (CDK) and/ or cyclin (CCN) as compared to an organoid derived from stem cells of a healthy subject. In various embodiments, the level of expression of Cyclin Dependent Kinase (CDK) and/ or cyclin (CCN) in motor neurons of an organoid derived from stem cells of a subject having SMA is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than the level of expression of Cyclin Dependent Kinase (CDK) and/ or cyclin (CCN) in motor neurons of an organoid derived from stem cells of a healthy subject. In various embodiments, the level of expression of Cyclin Dependent Kinase (CDK) and/ or cyclin (CCN) in motor neurons of an organoid derived from stem cells of a subject having SMA is at least 1 -fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold higher than the level of expression of Cyclin Dependent Kinase (CDK) and/ or cyclin (CCN) in motor neurons of an organoid derived from stem cells of a healthy subject. The CDK may be one or more selected from the group consisting of CDK1 , CDK2, CDK4, and CDK6. The CCN may be one or more selected from the group consisting of CCNA2, CCNB1 , CCNB2, CCND1 , CCND2, CCNE1 , and CCNE2. In various embodiments, the organoid derived from stem cells of a subject having SMA comprises motor neurons characterized by a higher expression of CCNA2, CCNB1 , CCNB2, CCNE2 and CDK6 as compared to an organoid derived from stem cells of a healthy subject.

In various embodiments, the organoid derived from stem cells of a subject having SMA comprises a higher percentage of motor neurons characterized by the expression of proliferative marker Ki67 as compared to an organoid derived from stem cells of a healthy subject. In various embodiments, the motor neurons in the organoid derived from stem cells of a subject having SMA are further characterized by the expression of apoptotic marker cCASP3. In various embodiments, the level of expression of proliferative marker Ki67 in motor neurons of an organoid derived from stem cells of a subject having SMA is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than the level of expression of proliferative marker Ki67 in motor neurons of an organoid derived from stem cells of a healthy subject. In various embodiments, the level of expression of proliferative marker Ki67 in motor neurons of an organoid derived from stem cells of a subject having SMA is at least 1 -fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the level of expression of proliferative marker Ki67 in motor neurons of an organoid derived from stem cells of a healthy subject.

In various embodiments, the organoid is maintained in culture for at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, or at least 90 days. In some example embodiments, the organoid may be maintained in culture for at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at least 43 days, or at least 44 days.

In various embodiments, the organoid is a spinal organoid. In one embodiment, the organoid is a ventral spinal organoid comprising or consisting of neurons that are found in the ventral horn of a spinal cord. For example, the spinal organoid is a ventral spinal organoid comprising or consisting of cholinergic motor neurons, V2a interneurons, Renshaw cells and astrocytes. Advantageously, the organoid may recapitulate spinal cord-like tissues consisting of multiple ventral spinal cord cell types. These various cell types represent the complexity of the ventral spinal cord and may provide a platform for modelling neurodegenerative diseases e.g. motor neuron neurodegeneration diseases and understanding spinal cord development. In some example embodiments, the organoid does not comprise/ is not a dorsal spinal organoid.

In various embodiments, the organoid is used as a platform for screening agents that affect the cells within the organoid e.g. drug screening platform. The organoid may provide a platform for testing small molecules that modulate e.g. promote or inhibit motor neuron survival. The organoid may also provide a platform for testing compounds/ agents other than small molecules e.g. large molecules. Small molecules refer to molecules with small molecular weights. The term“molecular weight” refers to the sum of the atomic weights of all atoms constituting a molecule and can be numerically expressed in Dalton (Da). Low molecular weight molecules may have a molecular weight of less than 900 Da and large molecular weight molecules may have a molecular weight greater than 900 Da. Using the organoid as a platform for screening agents may be advantageous over traditional 2D cultures because the organoid comprises multiple cell types e.g. spinal cord cell types and may provide a more physiologically relevant model as compared to 2D cultures which typically contain fewer cell types.

In various embodiments, the organoid is used as a platform for screening agents capable of prolonging motor neuron survival by exposing the organoid to one or more agents in an organoid culture. In various embodiments, the one or more agent is introduced to the organoid culture and the organoid is exposed to the one or more agents in the organoid culture over a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days or more (as would be appropriately determined by the person skilled in the art). In various embodiments, the agent is a CDK inhibitor. A CDK inhibitor is any chemical that inhibits the function of CDKs. CDK inhibitors may be categorized based on their target specificity and may include broad CDK inhibitors (i.e. compounds which target a broad spectrum of CDKs), specific CDK inhibitors (i.e. compounds which target a specific type of CDK), and multiple target inhibitors (i.e. compounds which target CDKs as well as additional kinases such as VEGFR or PDGFR). In various embodiments, a CDK inhibitor is one or more selected from the group consisting of pan-CDK inhibitor (CDKi), CDK1 inhibitor (CDK1 i), CDK2 inhibitor (CDK2i), CDK4 inhibitor (CDK4i) and CDK4/6 inhibitor. In one embodiment, the CDK4/6 inhibitor may be PD 0332991 (Palbociclib, or analog or derivative thereof). Advantageously, the organoid may be used as a platform for therapeutic discovery and could be used as an additional screening step before moving into in-vivo models.

In various embodiments, a method of obtaining an organoid is provided.

The method may comprise culturing stem cells in a culture medium comprising one or more reagents capable of inducing neuralization of the stem cells; contacting the stem cells to a reagent capable of caudalizing the stem cells; contacting the stem cells to a reagent capable of ventralizing the stem cells; and forming the organoid. In various embodiments, the method is an in-vitro method.

In various embodiments, the method further comprises a step of obtaining the stem cells. In various embodiments, the stem cells are pluripotent stem cells. In various embodiments, the stem cells are induced pluripotent stem cells (iPSCs). The stem cells may be obtained from a healthy (non-diseased) subject or a subject having a neurodegenerative disease (such as motor neuron disease). In various embodiments, the motor neuron disease is selected from the group consisting of SMA and ALS.

In various embodiments, the stem cells are dissociated into single cells prior to seeding and culturing the stem cells in the culture medium. Dissociation of cells may be accomplished using mechanical means e.g. mechanical tools such as cell scraper or using enzymatic means. Enzymatic means of dissociation include but are not limited to using trypsin/EDTA, TrypLE selection agent, IV type collagenase and neutral protease.

In various embodiments, the stem cells are seeded at a density of at least about 150,000 cells/ml, at least about 200,000 cells/ml, at least about 250,000 cells/ml, at least about 300,000 cells/ml, at least about 350,000 cells/ml, or at least about 400,000 cells/ml in a suspension culture. For example, using a seeding density of about 150,000 cells/ml, about 30,000 cells can be seeded in one well of a 96-well plate based on a working volume of 200 mI. A seeding density of at least about 150,000 cells/ml may advantageously result in better derivation of mature cell types e.g. spinal cell types as compared to a lower seeding density. In some example embodiments, a seeding density of at least about 150,000 cells/ml may result in higher expression of spinal cell markers such as motor neuron and interneuron markers as compared to a lower seeding density. The interneurons may include V2a interneurons characterized by the expression of CHX10 marker and V3 interneurons characterized by the expression of Sim1 (Single-Minded Family BHLH Transcription Factor 1 ) marker. The motor neurons may be characterized by the expression of ISL1 , FOXP1 and/or ChAT markers. In various embodiments, the one or more reagents capable of inducing neuralization of the stem cells comprises an agent for blocking Bone Morphogenetic Protein (BMP) signaling and an agent for activating Wnt pathways. In one embodiment, the one or more reagents capable of inducing neuralization of the stem cells is selected from the group consisting of an agent for blocking BMP signaling and an agent for activating Wnt pathways. In one embodiment, inducing neuralization of the stem cells comprises contacting the stem cells with the agent for blocking BMP signaling and the agent for activating Wnt pathways simultaneously/ concurrently over a period.

In various embodiments, blocking agents of BMP signaling broadly refers to any molecule which may inhibit BMP signaling. For example, a blocking agent of BMP signaling may be directed against any component involved in BMP signaling (e.g. a BMP receptor) or against any element of the downstream signal transduction cascade associated with human BMPs signaling, thereby indirectly affecting BMP activity. For example, a blocking agent of BMP signaling may be directed against a BMP receptor e.g. the BMP type I receptors ALK2, ALK3 or ALK6. Blocking agents of BMP signaling include but are not limited to dorsomorphin, LDN-193189, a BMP receptor antagonist, the protein complex Inhibin, BMP-3, Noggin, Chordin and Chordin-like molecules, Follistatin and Follistatin-related gene (FLRG), Ventroptin, twisted gastrulation (Tsg), Dan, Cerberus, Gremlin, Dante, Caronte, Protein related to Dan and Cerberus (PRDC), Sclerostin and sclerostin-like, Coco, Cer1 , Uterine sensitization- associated gene 1 (USAG-1 ) or connective tissue growth factor (CTGF), or any combination thereof. In some embodiments, the agent for blocking BMP signaling is one or more selected from the group consisting of Noggin, LDN-193189 and dorsomorphin. In one embodiment, the agent for blocking BMP signaling comprises LDN-193189 (i.e. 4-(6-(4-(Piperazin-1 -yl)phenyl)pyrazolo[1 ,5- a]pyrimidin-3-yl)quinoline).

In various embodiments, an agent for activating Wnt pathways broadly refers to any factor that effects, increases, induces, initiates, or stimulates release of a Wnt protein; a factor that effects or produces biochemical signaling within a Wnt signaling pathway; and a factor that increases, induces, initiates, or stimulates signaling within a Wnt signaling pathway. Examples of Wnt pathway activator include but are not limited to an exogenous, soluble, biologically active Wnt protein that binds to a Wnt receptor and activates the Wnt pathway, and a small molecule GSK-3 antagonist that activates the Wnt pathway selected from the group consisting of: 6-Bromoindirubin-3'-oxime (BIO); N-(4-Methoxybenzyl)- N'-(5-nitro-1 ,3-thiazol-2-yl)urea (AR-AO 14418); 3-(2,4-Dichlorophenyl)-4-(1 - methyl-1 H-indol-3-yl)-1 H-pyrrole-2,5-dione (SB 216763); 4-Benzyl-2-methyl- 1 ,2,4-thiadiazolidine-3,5-dione (TDZD-8); CHIR-91 1 (also known as CHIR99021 ), and CHIR-837. In some embodiments, the agent for activating Wnt pathways is CHIR99021 and/or BIO. In one embodiment, the agent for activating Wnt pathways comprises CHIR99021 (i.e. 6-((2-((4-(2,4-dichlorophenyl)-5-(4- methyl-1 H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile, or C25H18CI2N8).

In various embodiments, the reagent capable of caudalizing the stem cells includes but is not limited to a retinoid (e.g., retinoic acid), a Wnt protein, or one of their receptors. In one embodiment, the reagent capable of caudalizing the stem cells comprises retinoic acid.

In various embodiments, the reagent capable of ventralizing the stem cells comprises an activator of a hedgehog signaling pathway, e.g. Sonic Hedgehog (SHH) pathway agonist. “Hedgehog” refers to any member of the hedgehog family of proteins. “Hedgehog signaling pathway” refers to the cascade of biochemical signaling that includes, is initiated by, or directly or indirectly results from release of a hedgehog protein, particularly signaling that relates to direction or position of cells in an embryo during development and differentiation. An “activator” of a hedgehog signaling pathway includes a factor that effects, increases, induces, initiates, or stimulates release of a hedgehog protein; a factor that effects or produces biochemical signaling within a hedgehog signaling pathway; and a factor that increases, induces, initiates, or stimulates signaling within a hedgehog signaling pathway. For example, the activator of hedgehog signaling may be a hedgehog protein (e.g., DHH (desert hedgehog), SHH (sonic hedgehog), or IHH (Indian hedgehog)), a hedgehog receptor, or an agonist of a hedgehog signaling pathway. An“agonist” of a hedgehog signaling pathway, as used herein, is a factor that has affinity for, and stimulates physiologic activity at, cell receptors normally stimulated by naturally-occurring substances, such that signaling in a hedgehog signaling pathway within the cell is increased, initiated, stimulated, or induced. In one embodiment, the reagent capable of ventralizing the stem cells comprises purmorphamine (C31 H32N6O2) which is a SHH pathway agonist.

In various embodiments, the method further comprises contacting the stem cells to one or more neurotrophic factors capable of promoting neuronal maturation of the stem cells. In various embodiments, the stem cells are contacted to one or more neurotrophic factors capable of promoting neuronal maturation of the stem cells upon the appearance of neurites e.g. short neurites in the stem cells. In various embodiments, one or more neurotrophic factors is supplemented to the culture medium of the stem cells. Examples of neurotrophic factors capable of promoting neuronal maturation include but are not limited to GDNF, NGF, NT3, CNTF, and BDNF. In one embodiment, the one or more neurotrophic factors is selected from the group consisting of BDNF and GDNF.

In various embodiments, the stem cells are seeded on day 0, wherein day 0 is defined as the day on which the stem cells are introduced into a culture dish (e.g. by pipetting a volume of cell suspension into a well of the culture dish) for culturing in a culture medium. In various embodiments, the one or more reagents capable of inducing neuralization of the stem cells is contacted to the stem cells from day 0, from day 1 , from day 2, from day 3, from day 4, or from day 5 onwards. In one embodiment, the stem cells are cultured in a culture medium comprising one or more reagents capable of inducing neuralization of the stem cells from day 0. In various embodiments, the one or more reagents capable of inducing neuralization of the stem cells is contacted to the stem cells for at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In one embodiment, the stem cells are cultured in a culture medium comprising one or more reagents capable of inducing neuralization of the stem cells from day 0 to day 10.

In various embodiments, the reagent capable of caudalizing the stem cells is contacted to the stem cells no later than day 3, no later than day 4, no later than day 5, no later than day 6 or no later than day 7 of the culture. In various embodiments, the reagent capable of caudalizing the stem cells is contacted to the stem cells for at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 1 6 days, at least 1 7 days, at least 1 8 days, at least 1 9 days, at least 20 days, or at least 21 days. In one embodiment, the reagent capable of caudalizing the stem cells is contacted to the stem cells from day 3 to day 15 of the culture. In various embodiments, the reagent capable of caudalizing the stem cells is contacted to the stem cells on or after the expression of one or more neuroepithelial stem cell markers such as SOX1 and nestin in the stem cells.

In various embodiments, the reagent capable of ventralizing the stem cells is contacted to the stem cells no later than day 7, no later than day 8, no later than day 9, no later than day 10, no later than day 1 1 , no later than day 12, no later than day 13, or no later than day 14 of the culture. In various embodiments, the reagent capable of ventralizing the stem cells is contacted to the stem cells for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, or at least 14 days.

In various embodiments, the steps of contacting stem cells with the reagent capable of caudalizing the stem cells and the reagent capable of ventralizing the stem cells overlap with each other. That is, the cells in the culture are concurrently contacted with the caudalizing agent and ventralizing agent over a period. In one embodiment, the reagent capable of caudalizing the stem cells is contacted to the stem cells from day 3 to day 15 of the culture and the reagent capable of ventralizing the stem cells is contacted to the stem cells from day 10 to day 17 of the culture.

In various embodiments, cells expressing neuroepithelial stem cell markers such as SOX1 and Nestin are formed in the cell culture no later than day 7, no later than day 8, no later than day 9, no later than day 10, no later than day 1 1 , no later than day 12, no later than day 13, or no later than day 14 of the culture. In various embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the cell culture express SOX1 marker. The presence of cells expressing neuroepithelial stem cell markers indicates that the stem cells have been neutralized. In one embodiment, cells expressing neuroepithelial stem cell markers such as SOX1 and Nestin are formed on or before day 10 of culture.

In various embodiments, the organoid is formed no later than day 14, no later than day 15, no later than day 16, no later than day 17, no later than day 18, no later than day 19, no later than day 20, or no later than day 21 of the culture. In various embodiments, the organoid exhibits an apical-basal polarity such that the apical region is substantially populated by cells expressing SOX1 marker and the basal region is substantially populated by motor neuron progenitors expressing ISL1 marker.

In various embodiments, cells expressing SOX1 organize into one or more rosette structures no later than day 18, no later than day 19, no later than day 20, no later than day 21 , no later than day 22, no later than day 23, or no later than day 24 of the culture. In various embodiments, the one or more rosette structures are present in the organoid after day 28, after day 35, or after day 42 of the culture.

In various embodiments, motor neurons expressing ISL1 are formed in the cell culture no later than day 18, no later than day 19, no later than day 20, no later than day 21 , no later than day 22, no later than day 23, or no later than day 24 of the cell culture. In various embodiments, as the stem cells undergo differentiation, the number of motor neurons expressing ISL1 increases with time while the number of cells expressing SOX1 decreases with time.

In various embodiments, neurons expressing TUJ1 marker are formed in the cell culture no later than day 10, no later than day 1 1 , no later than day 12, no later than day 13, no later than day 14, no later than day 15, or no later than day 16 of the cell culture. In various embodiments, neurons expressing TUJ1 marker persist in the organoid after day 14, after day 21 , after day 28, after day 35, or after day 42 of culture.

In various embodiments, the step of contacting the stem cells to one or more neurotrophic factors follows after the steps of caudalizing and ventralizing the stem cells. In various embodiments, the step of contacting the stem cells to one or more neurotrophic factors does not overlap with the steps of caudalizing and ventralizing the stem cells. In various embodiments, the stem cells are contacted to one or more neurotrophic factors upon the appearance of neurites e.g. short neurites in the stem cells. In various embodiments, the culture medium for maintaining the supplemented with the one or more neurotrophic factors for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks. In one embodiment, the stem cells are contacted to the one or more neurotrophic factors from day 17 of the culture onwards.

In various embodiments, the method further comprises encapsulating the stem cells in a support matrix. The support matrix is a growth matrix capable of providing support matrices to the stem cells and/ or acting as an extracellular matrix for the stem cell and/ or acting as a basement membrane matrix for the stem cells. For example, the support matrix may be a gelatinous protein mixture derived from mouse tumour cells. Examples of a support matrix include but are not limited to Matrigel™ or Geltrex™. In various embodiments, the volume of support matrix used for encapsulating the stem cells is at least about 10 mI, at least about 15 mI, at least about 20 mI, at least about 25 mI, at least about 30 mI, at least about 35 mI, at least about 40 mI, at least about 45 mI, or at least about 50 mI. In one embodiment, the volume of support matrix used for encapsulating the stem cells is about 15 mI. In various embodiments, prior to encapsulating the stem cells in the support matrix, the stem cells are cultured in a stationary suspension culture for at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, or at least 14 days. In various embodiments, the stem cells are encapsulated in the support matrix and maintained under stationary culture condition for one or more day, or two or more days, or three or more days, or four or more days, or at least one day, or at least two days, or at least three days, or at least four days, or at most one day, or at most two days, or at most three days, or at most four days.

In various embodiments, the stem cells are encapsulated and cultured in the support matrix for a period in static culture conditions before transferring and culturing the stem cells encapsulated in the support matrix in a dynamic cell culture device which is capable of enhancing nutrient absorption. Examples of dynamic cell culture devices include but are not limited to a spinner flask and a bioreactor. In one embodiment, the dynamic cell culture device is a spinner flask.

In various embodiments, the stem cells are culture in a static suspension culture during a first phase of the culture, followed by encapsulation and static culture in a support matrix during a second phase, followed by transferring to a cell culture device e.g. spinner flask for a third phase of the culture. In various embodiments, the first phase of the culture lasts for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In various embodiments, the second phase of the culture lasts for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In various embodiments, the third phase of the culture lasts for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks.

In various embodiments, the method further comprises maintaining the organoid in culture for at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, or at least 90 days.

In various embodiments, there is provided a co-culture system comprising myoblast cells e.g. myoblasts from C2C12 cell line co-cultured with an organoid as disclosed herein or an organoid obtained by the method as disclosed herein. In various embodiments, the organoid comprises limb-innervating motor neurons characterized by the expression of FOXP1 marker. In various embodiments, the organoid comprises cholinergic motor neurons characterized by the co expression of ISL1 and ChAT markers. In various embodiments, the myoblast cells are differentiated to form myotubes. In various embodiments, the organoid is added to the co-culture system after the formation of myotubes from myoblasts. In various embodiments, the organoid comprises motor neurons which are capable of forming neurite outgrowth when co-cultured with the myotubes. In various embodiments, the organoid comprises motor neurons characterized by the expression of SMI-32 marker and are capable of forming neuromuscular junctions. In various embodiments, the motor neurons are capable of causing myotube contraction of the myotubes, thus demonstrating innervation by the motor neurons.

In various embodiments, there is provided an organoid obtained using the methods as disclosed herein. In various embodiment, the organoid is a spinal organoid. In various embodiments, the spinal organoid comprises one or more cells selected from the group consisting of motor neurons, interneurons, and astrocytes. In various embodiments, the spinal organoid comprises cells which are organized into one or more rosette structures. In various embodiments, the spinal organoid comprises motor neurons characterized by the expression of ISL1 . In various embodiment, the spinal organoid is a ventral spinal organoid comprising neurons that are found in the ventral horn of a spinal cord.

In various embodiments, there is provided a method of screening agents capable of prolonging motor neuron survival comprising contacting one or more agents to the organoid as disclosed herein or the organoid produced by the method as disclosed herein. In various embodiments, the agent is a CDK inhibitor. In various embodiments, the CDK inhibitor is one or more selected from the group consisting of pan-CDK inhibitor (CDKi), CDK1 inhibitor (CDK1 i), CDK2 inhibitor (CDK2i), CDK4 inhibitor (CDK4i) and CDK4/6 inhibitor. In one embodiment, the CDK4/6 inhibitor is PD 0332991 . In various embodiments, the agent is contacted to the organoid over a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days.

In various embodiments, there is provided a method of treating SMA comprising administering an effective amount of a CDK4/6 inhibitor to a subject in need thereof. In various embodiments, there is provided use of a CDK inhibitor in the manufacture of a medicament for the treatment of SMA, wherein the CDK inhibitor is a CDK4/6 inhibitor. In various embodiments, the CDK4/6 inhibitor is PD 0332991 .

In various embodiments, there is provided an organoid e.g. spinal organoid comprising one or more cells selected from the group consisting of cholinergic motor neurons, V 2a interneurons, Renshaw cells and astrocytes. In various embodiments, the spinal organoid comprises motor neurons which express ISL1 , FOXP1 , and SMI32 markers, V2a interneurons which express CHX10 marker, Renshaw cells which express Calbindin marker, and astrocytes which express S100p. In various embodiments, the spinal organoid comprises functional motor neurons which is shown by co-culturing with the mouse myoblast cell line C2C12. Neuron outgrowth from the organoid were shown to be significant and evident. Co-culture of organoids and C2C12 myotubes resulted in myotube contraction, which is typically a measure of motor neuron functionality. Formation of neuromuscular junctions was also evident in various embodiments of the spinal organoid. In various embodiments, by treating iPSCs to caudalizing signal (retinoic acid) and ventralizing signal (Purmorphamine), iPSCs were induced towards spinal progenitors that differentiated into spinal organoids. In various embodiments, the organoid can be used for drug screening. Using the organoid as disclosed herein for drug screening may be advantageous over the traditional 2D culture, as the organoid contains multiple spinal cord cell types and is a more physiologically relevant model (for example, in a co-culture with myoblast, neuromuscular junction is observed). 2D cultures typically contain fewer cell types. In some cases, differentiation protocols of the method as disclosed herein may generate organoids containing up to 90% motor neurons.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 A is a qPCR (quantitative polymerase chain reaction) plot of pluripotency marker OCT4 showing similar reduction in OCT4 in organoids made with 10,000 and 30,000 cells.

FIG. 1 B is a qPCR plot of pluripotency marker NANOG showing similar reduction in NANOG in organoids made with 10,000 and 30,000 cells.

FIG. 1 C is a qPCR plot of V2a marker CFIX10 showing significantly higher expression of CFIX10 observed at days 21 and 28 in the 30,000-cell condition.

FIG. 1 D is a qPCR plot of V3 marker Sim1 showing significantly higher expression of Sim1 observed at days 21 and 28 in the 30,000-cell condition. FIG. 1 E is a qPCR plot of motor neuron marker ISL1 showing significantly higher expression of ISL1 observed at day 28 in the 30,000-cell conditions.

FIG. 1 F is a qPCR plot of motor neuron marker FOXP1 showing significantly higher expression of FOXP1 observed at day 28 in the 30,000-cell conditions.

FIG. 1 G is a qPCR plot of motor neuron marker ChAT showing significantly higher expression of ChAT observed at day 28 in the 30,000-cell conditions.

FIG. 2A are microscope images showing representative images of immunostaining of organoids for Nestin and SOX1 markers. Immunostaining of organoids at day 28 suggests normal neural progenitor populations in 10,000- and 30,000-cell conditions.

FIG. 2B are microscope images showing representative images of immunostaining of organoids for SMI-32 and ISL1 markers. Immunostaining of organoids at day 28 suggests normal motor neuron populations in 10,000- and 30,000-cell conditions.

FIG. 2C are microscope images showing representative images of immunostaining of organoids for TUJ1 and cCASP3 markers. Staining with cleaved Caspase-3 (cCASP3) demonstrated that the cores of the organoids were also not apoptotic.

FIG. 3 is a schematic chart illustrating differentiation of spinal organoids from iPSC in an example embodiment.

FIG. 4 are microscope images showing representative images of co staining of SOX1 and Nestin which demonstrates successful generation of neural progenitors in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole). Scale bars, 50pm.

FIG. 5 are microscope images showing representative images of BJ-iPS spinal organoids at respective time points stained with SOX1 and TUJ1. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 6 is a bar chart showing percentage of SOX1 ⁺ cells at day 14, 21 , 28 and 35 in BJ-iPS spinal organoids relative to total cell number. ^* indicates p < 0.05, ^** indicates p < 0.01 , ^*** indicates p < 0.001. FIG. 7 are microscope images showing representative images of BJ-iPS spinal organoids demonstrating SOX1 ⁺ and ISL1 ⁺ in an apical-to-basal patterning. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 8 are microscope images showing representative images of BJ-iPS spinal organoids at respective time points stained with ISL1 and SMI-32. Cellular nuclei were counterstained with DAPI. The bottom row images are enlarged views of the highlighted area (square outline) of the corresponding top row images. Scale bars, 100pm.

FIG. 9 is a bar chart showing percentage of ISL1 ⁺ cells at day 21 , 28 and 35 in BJ-iPS spinal organoids relative to total cell number. ^* indicates p < 0.05, ^** indicates p < 0.01 , ^*** indicates p < 0.001.

FIG. 10 are microscope images showing representative images illustrating the presence of FIOXB4⁺ cells in spinal organoids. Cellular nuclei were counterstained with DAPI. The bottom row images are enlarged views of the highlighted area (square outline) of the corresponding top row images. Scale bars, 100pm.

FIG. 1 1 are microscope images showing representative images illustrating the presence of FIOXC8⁺ cells in spinal organoids. Cellular nuclei were counterstained with DAPI. The bottom row images are enlarged views of the highlighted area (square outline) of the corresponding top row images. Scale bars, 100pm.

FIG. 12 are microscope images showing representative images of co staining of FOXP1 and ISL1 markers which demonstrates presence of limb- innervating neurons in spinal organoids. Scale bars, 100pm.

FIG. 13 are microscope images showing representative images of spinal organoids at day 42 stained with ISL1 and ChAT. Cellular nuclei were counterstained with DAPI. The bottom row images are enlarged views of the highlighted area (square outline) of the corresponding top row images. Scale bars, 100pm.

FIG. 14 is a microscope image showing a representative image of a spinal organoid stained with CFIX10⁺ cells. Scale bars, 100pm. FIG. 15 is a microscope image showing a representative image of a spinal organoid stained with CALB⁺ cells. Scale bars, 100pm.

FIG. 16 are microscope images showing representative images of co staining of 8100b and TUJ1 which shows presence of astrocytes in spinal organoids. Scale bars, 100pm.

FIG. 17 is a bar chart of quantitative-PCR analysis of SOX10, BRN3A and TLX3 markers demonstrating a lack of dorsal cell types in the spinal organoids generated.

FIG. 18 are microscope images showing representative images of immunostaining showing homogeneous generation of HOXB4⁺ cervical spinal cell types.

FIG. 19 is a time-coursed qPCR analysis chart showing increased GDF1 1 expression in organoid cultures, but not in conventional 2D cultures.

FIG. 20A is a time coursed qPCR analysis chart of SOX1 marker in a ventral spinal organoid.

FIG. 20B is a time coursed qPCR analysis chart of OLIG2 marker in a ventral spinal organoid.

FIG. 20C is a time coursed qPCR analysis chart of HOXA6 marker in a ventral spinal organoid.

FIG. 20D is a time coursed qPCR analysis chart of HOXC8 marker in a ventral spinal organoid.

FIG. 20E is a time coursed qPCR analysis chart of HOXC10 marker in a ventral spinal organoid. Time-coursed qPCR analysis of ventral spinal organoids revealed diversity of cell types within the organoids, including derivation of thoracic (HOXA6⁺), brachial (HOXC8⁺) and slight increase in lumbar (HOXC10) expression.

FIG. 20F is a time coursed qPCR analysis chart of LHX1 marker in a ventral spinal organoid.

FIG. 20G is a time coursed qPCR analysis chart of ISL1 marker in a ventral spinal organoid.

FIG. 20H is a time coursed qPCR analysis chart of PAX2 marker in a ventral spinal organoid. FIG. 21 are microscope images showing representative images of motor neurons (stained with SMI-32) co-cultured on mouse myotubes differentiated from C2C12 cell line. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 22 is a bar chart showing mean neurite lengths from the SMI-32 motor neurons in FIG. 21 , determined using Neurite Tracer in ImageJ.

FIG. 23 are microscope images showing representative images of visualization of NMJs by staining acetylcholine receptors with a-BTX (bungarotoxin) and co-staining adjacent motor axons with SMI32. The bottom row images are enlarged views of the highlighted area (square outline) of the corresponding top row images. Scale bars, 10pm.

FIG. 24 are microscope images showing representative images of co staining of SOX1 and TUJ1 in SMA Type I (1 -38G) and SMA Type II (1 -51 N) spinal organoids at respective time points. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 25 is a bar chart showing quantification of SOX1 ⁺ levels percentage of SMA Type I and Type II spinal organoids at respective time points relative to total cell number. The values were not significant.

FIG. 26 are microscope images showing representative images of SMA Type I and Type II spinal organoids at respective time points stained with ISL1 and SMI-32. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 27 is a bar chart showing percentage of ISL1 ⁺ at day 21 , 28 and 35 in SMA Type I and Type II spinal organoids relative to total cell number. ^** indicates p < 0.01 , n.s. non-significant.

FIG. 28 is a bar chart showing mRNA fold change comparing SMA HB9⁺ motor neurons to wild-type HB9⁺ motor neurons. The motor neurons were purified based on HB9 immunoreactivity. mRNA expression levels of CDKs and cyclins were measured by RNA-seq and qPCR respectively. The dotted line indicates relative expression of wild-type H B9⁺ motor neurons.

FIG. 29 is a bar chart showing mRNA fold change of SMA 1 -38G ISL1 ⁺ motor neurons relative to BJ ISL1 ⁺ motor neurons. qPCR analysis of ISL1 ⁺ motor neurons was derived from day 28 organoids. FIG. 30 is a bar chart showing imRNA fold change of si-SMN relative to si- NT (non-targeting siRNA control). Knockdown of SMN in wild-type motor neuron cultures revealed upregulation of cell cycle genes. ^* indicates p < 0.05; ^** indicates p < 0.01 and ^*** indicates p < 0.001.

FIG. 31 is a bar chart showing Ki67 and ISL1 immunostaining analysis of wild-type (BJ iPS and 18a), SMA Type I (1 -38G) and SMA Type II (1 -51 N) motor neuron cultures at day 28. The percentages of ISL1 ⁺Ki67⁺ cells amongst all ISL1 ⁺ motor neurons are shown.

FIG. 32 is a bar chart showing Ki67 and ISL1 immunostaining analysis of si-NT and si-SMN. Knockdown of SMN in wild-type cell line (BJ-iPS) increased the percentage of ISL1 ⁺ motor neurons co-expressing Ki67.

FIG. 33 are microscope images showing co-staining of ISL1 and Ki67 showing increased Ki67⁺ cells upon SMN knockdown in BJ-iPS motor neuron cultures. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 34 is a bar chart showing Ki67 and cCASP3 immunostaining analysis of wild-type motor neurons demonstrating higher cCASP3 expression in Ki67⁺ motor neurons than Ki67 motor neurons. ^*** indicates p < 0.001.

FIG. 35 is a chemical structure of a pan-CDK inhibitor, CDKi hydrochloride.

FIG. 36A is a bar chart showing ISL1 immunostaining analysis and demonstrating increased percentage of ISL1 ⁺ SMA type I motor neurons (1 -38G) upon pan-CDK inhibitor treatment for 3 days. ^* indicates p < 0.05.

FIG. 36B is a bar chart showing ISL1 immunostaining analysis and demonstrating increased percentage of ISL1 ⁺ SMA type II motor neurons (1 -51 N) upon pan-CDK inhibitor treatment for 3 days. ^* indicates p < 0.05.

FIG. 37A is a bar chart showing quantification of ISL1 ⁺ SMA type I motor neurons (1 -38G) treated with various CDKs inhibitors treatment. The dotted line indicates percentage of motor neurons relative to DMSO (dimethyl sulfoxide) treated motor neurons.

FIG. 37B is a bar chart showing quantification of ISL1 ⁺ SMA type II motor neurons (1 - 51 N) treated with various CDKs inhibitors treatment. The dotted line indicates percentage of motor neurons relative to DMSO treated motor neurons. FIG. 38 is a bar chart showing ISL1 immunostaining analysis of various CDKs knockdown in SMA type II motor neurons. The dotted line indicates percentage of motor neurons survival relative to non-targeting siRNA treated motor neurons.

FIG. 39 are microscope images showing representative images of SMA type II motor neurons treated with various CDKs siRNA and stained with ISL1 and SMI-32. Cellular nuclei were counterstained with DAPI. Scale bars, 50pm.

FIG. 40 is a Western blot of SMA type II motor neurons treated with various CDKs inhibitors, indicating that SMN levels remained the same.

FIG. 41 is a bar chart showing quantification of SMN levels of SMA type II motor neurons treated with various CDKs inhibitors relative to a-tubulin expression. The values were not significant. ^* indicates p < 0.05 and ^** indicates p < 0.01.

FIG. 42A is a bar chart showing qPCR analysis confirming efficient knockdown of CDK1 compared to a non-targeting siRNA control, in SMA motor neurons. ^* indicates p < 0.05; ^** indicates p < 0.01 and ^*** indicates p < 0.001.

FIG. 42B is a bar chart showing qPCR analysis confirming efficient knockdown of CDK2 compared to a non-targeting siRNA control, in SMA motor neurons. ^* indicates p < 0.05; ^** indicates p < 0.01 and ^*** indicates p < 0.001.

FIG. 42C is a bar chart showing qPCR analysis confirming efficient knockdown of CDK4 compared to a non-targeting siRNA control, in SMA motor neurons. ^* indicates p < 0.05; ^** indicates p < 0.01 and ^*** indicates p < 0.001.

FIG. 42D is a bar chart showing qPCR analysis confirming efficient knockdown of CDK6 compared to a non-targeting siRNA control, in SMA motor neurons. ^* indicates p < 0.05; ^** indicates p < 0.01 and ^*** indicates p < 0.001.

FIG. 43 are microscope images showing co-staining of ISL1 and SMI-32 in SMA type I spinal organoids treated with DMSO and PD0332991. Cellular nuclei were counterstained with DAPI. Scale bars, 100pm.

FIG. 44 is a bar chart showing SMA type I spinal organoids demonstrating increased MN survival. ^* indicates p < 0.05 and ^** indicates p < 0.01.

FIG. 45 is a bar chart showing SMA type II spinal organoids demonstrating increased MN survival. ^* indicates p < 0.05 and ^** indicates p < 0.01. EXAMPLES Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures.

[Dear Inventors, please note that the Examples section is populated based on the manuscript provided in the TDF.]

Experimental Procedures

Culture and differentiation of human pluripotent stem cells Wild-type BJ fibroblast-derived iPSCs (BJ-iPS) and SMA patient-derived iPSCs (Type II SMA 1 -51 N and Type I SMA 1 -38G) were cultured feeder-free on Matrigel-coated plates in MACS iPS-Brew media (Miltenyi Biotec). BJ cells are human diploid foreskin fibroblasts. Routine passaging using ReLeSR (Stem Cell Technologies) was performed once every 6-7 days. Pluripotent stem cells were differentiated towards the spinal motor neuron fate following established protocols described previously accordingly to“Ng, S.Y. et al. Genome-wide RNA- Seq of human motor neurons implicates selective ER stress activation in spinal muscular atrophy. Cell Stem Cell 17, 569-584 (2015)”.

Spinal organoids were made by dissociating iPS cells into single cells, and either 10,000 or 30,000 cells per well was seeded in a 96-well low attachment plate. Comparison of spinal organoids made with 10,000 versus 30,000 cells revealed that higher cell density promotes motor neuron maturation and cellular diversity. Eventually, 30,000 cells per well were used because that resulted in better derivation of mature spinal cell types (see FIGS. 1 and 2). The embryoid bodies were then encapsulated in 15 mI Matrigel droplets at day 10 before transferring to spinner flasks at day 14 for neuronal maturation in the presence of growth factors BDNF and GDNF. Organoids were harvested by day 42 for analysis even though they can be maintained for at least 90 days (see FIGS. 1 and 2).

RNA extraction and expression analysis

Cells were harvested in Trizol reagent for RNA extraction following manufacturer’s instructions. Purified RNA was converted to cDNA using the High- Capacity cDNA Reverse Transcription kit (Ambion), and quantitative PCR (qPCR) was performed on the QuantStudio 5 Real-Time PCR System using FAST SYBR Master mix (all from Applied Biosystems). Gene expressions were normalized to GAPDH and ACTB expression unless otherwise stated. A list of the qPCR primers used is provided in Table 1 below.

Table 1. List of human primers used in qPCR studies

RNA interference in motor neuron cultures

Motor neuron cultures were dissociated with Accutase and seeded at 75,000 cells per well in a 96-well plate. Non-targeting siRNA or siRNAs against genes of interest were individually complexed with Lipofectamine RNAiMAX (Invitrogen) following manufacturer’s instructions. For each well, 10 pmol of siRNAs and 0.5 mI of Lipofectamine RNAiMAX were used. Cells were either harvested for RNA and protein analyses or fixed for immunostaining 3 days after siRNA transfection.

Treatment of motor neuron cultures with small molecule inhibitors CDKi hydrochloride (Sigma), specific CDK1 inhibitor (Santa-Cruz), specific CDK2 inhibitor (Santa-Cruz), specific CDK4 inhibitor (Santa-Cruz), and PD 0332991 (Santa-Cruz), were reconstituted in DMSO and diluted in media at the desired concentrations: CDKi (10 mM), CDK1 i (0.1 mM), CDK2i (1 mM), CDK4i (0.1 mM) PD (0.1 mM). Motor neurons at day 23 were plated at 75,000 cells per well of a 96-well plate. Treatment with the respective small molecules began at day 25, for a total of 3 days. Biological triplicates were performed with a minimum of 5 technical replicates each. Treatment of SMA spinal organoids with small molecule inhibitors

SMA spinal organoids were treated with either DMSO or PD 0332991 (0.1 mM) on a low-attachment plate at day 28 for a total of 7 days. SMA spinal organoids were then harvest at Day 35 for cryosectioning.

SDS-PAGE and Western blot

Protein lysates were resolved in 12% SDS-PAGE gels in T ris-Glycine-SDS buffer. Proteins were then transferred to a PVDF membrane and blocked in buffer containing 5% milk. Primary antibodies were diluted in 5% milk and incubated with the membranes overnight at 4 °C. The following primary antibodies (and their respective dilutions) were used: mouse SMN (1 :1000) (BD Pharmingen, 610647), mouse a-tubulin (1 :500) (Santa Cruz Biotechnologies, sc-32293). Membranes were washed thrice in TBST buffer. The corresponding horseradish peroxidase secondary antibodies (Santa Cruz) were then diluted 1 :2000 in 5% milk and incubated at room temperature for 90 minutes. Blots were washed thrice before exposing to ECL (Enhanced ChemiLuminescence) for imaging.

Immunofluorescence, image acquisition and image analysis

Cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized in 0.1 % Triton X-100 for 15 minutes and blocked in buffer containing 5% FBS (fetal bovine serum) and 1% BSA (bovine serum albumin) for an hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1 :1000) (Abeam, ab87775), mouse Nestin (1 :1000) (Abeam, ab22035), rabbit ISL1 (1 :1500) (Abeam, ab109517), rabbit cleaved Caspase-3 (1 :1000) (Cell Signaling Technology, #9661 ), mouse Ki67 (1 :1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1 :1000) (Calbiochem, NE- 1023), mouse SMN (1 :400) (BD Pharmingen, 610647), rabbit Ki67 (1 :250) (Abeam, ab16667), mouse TUJ1 (1 :2000) (Biolegend, #801202), goat SOX10 (1 :100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1 :200) (Abeam, ab133521 ), rabbit HOXC8 (1 :200) (Abeam, ab86236), rabbit Calbindin (1 :1000) (Abeam, ab11426), mouse FoxP1 (1 :100) (R&D Systems, MAB45341 ), sheep Chx10 (1 :200) (Abeam, ab16141 ). Cells were washed thrice in PBS. The respective secondary antibodies (Molecular Probes, Invitrogen) were diluted 1 :1500 in blocking buffer and incubated at room temperature, in the dark, for 90 minutes. DAPI was used at 0.1 gg/ml to visualize cellular nuclei.

Images of cultured cells on 96-well plates were acquired using the high content microscope Operetta (Perkin Elmer) using the 20x objective. Image analyses including cell counts and intensity measurements were performed using Columbus (Perkin Elmer). Nuclei were detected based on DAPI staining, with dead cells filtered based on abnormally high DAPI intensity and small (<20 pm²) nuclei area. Intensity of ISL1 staining within nuclei was determined and a cut-off above background intensity was used to identify motor neurons.

Spinal organoids were fixed in 4% PFA (paraformaldehyde) overnight, and dehydrated in 15% sucrose and 30% sucrose for 24 hours each before cryosectioning at 10pm per section. These were then stained respectively with antibodies listed above and images were acquired with an upright fluorescence microscope (Nikon Eclipse Ni) using the 10x objective. ISL1 ⁺ and SOX1 ⁺ cells were quantified by automated counting performed by image analysis software (ImageJ, N IH). All quantifications were normalized to total DAPI counts.

Co-culture of spinal organoids with mouse mvotubes C2C12 cell line (ATCC) was maintained in myoblast media consisting of DMEM (high glucose) with 20% fetal bovine serum (FBS) on gelatin-coated dishes. To differentiate the C2C12 cell line towards myotubes, confluent C2C12 myoblasts were cultured in DMEM (high glucose) with 2% FBS on Matrigel- coated plates. Myotubes were visibly formed 7 days after differentiation and were conditioned in N2B27 media a day before seeding organoids. 5 organoids at day 21 were seeded into each well of a 6-well plate and extensive neurite elongation was observed 3 days after co-culture. A video was taken at day 3 of co-culture where myotube contraction was observed in multiple parts of the well adjacent to the organoids. Neurite outgrowth was measured by image analysis software (ImageJ, NIH).

Visualization of neuromuscular junctions

At day 28, co-cultures were washed twice with PBS and alpha- Bungarotoxin (BTX) labelling (Life Technologies, B35451 ) of AChRs was performed on the co-cultures with 5pg/ml in PBS for 15 mins at RT. After BTX staining, cells were washed twice with PBS prior to acetone/methanol (ratio 1 :1 ) fixation for 6 mins at -20°C. Permeabilization was done in 0.1 % Triton X-100 for 15 minutes and blocked in buffer containing 5% FBS and 1 % BSA for an hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. The following primary antibodies (and their respective dilutions) were used: mouse SMI-32 (1 :1000) (Calbiochem, NE-1023). Cells were washed thrice in PBS. The respective secondary antibodies (Molecular Probes, Invitrogen) were diluted 1 :1500 in blocking buffer and incubated at room temperature, in the dark, for 90 minutes. DAPI was used at 0.1 pg/ml to visualize cellular nuclei. Images of neuromuscular junctions from the cocultures were acquired with an inverted confocal microscope (Olympus FLUOVIEW FV1000) using the 20x objective.

Statistical Analyses At least three biological replicates were performed for each experiment. Statistical analysis comparing two groups were performed by means of a two- tailed unpaired Student’s t test. P values lower than 0.05 were considered significant. All results are presented as mean ± standard deviation unless otherwise specified.

Results Derivation of spinal organoids from pluripotent stem cells

To generate spinal organoids, iPSCs were first dissociated into single cells, 30,000 cells per well were seeded in a 96-well low attachment plate (see FIGS. 1 and 2), and neuralization of iPSCs was induced by blocking Bone Morphogenic Protein (BMP) signaling by LDN-193189 treatment while simultaneously activating Wnt pathways with CHIR99021 treatment. Retinoic acid (RA) treatment begun at day 3 to caudalize the cultures, while Purmorphamine, a Sonic Hedgehog pathway agonist, was used as a ventralizing signal from days 10-17 (see FIG. 3). To ensure that neutralization was successful, cells were also seeded on Matrigel-coated plates, and immunostaining was performed on day 10 cultures. It was observed that cultures were homogeneously expressing neuroepithelial stem cell markers SOX1 and Nestin (see FIG. 4). At day 10, cells in each well were encapsulated with Matrigel. These were allowed to grow as stationary cultures until day 14, where the cell-Matrigel droplets were transferred into spinner flasks. To promote neuronal maturation, organoids were cultured in media supplemented with neurotrophic factors and ascorbic acid (AA) from day 17 onwards (see FIG. 3).

To investigate the cellular composition and cytoarchitecture of the spinal organoids, cryosectioning and immunostaining of organoids were performed at days 14, 21 , 28 and 35. At day 14, 86% of the cells were expressing SOX1 , demonstrating homogeneity within the spinal organoid (see FIGS. 5 and 6). As the spinal organoids continued to mature, SOX1 ⁺ cells organized into rosette structures by day 21 and continued to be present in day 28 and 35 spinal organoids (see FIG. 5). A typical apical-to-basal patterning of the organoids was observed where the apical region is marked by a layer of proliferative SOX1 ⁺ cells while ISL1 ⁺ motor neurons are present at the basal region (see FIG. 7).

As differentiation progressed, reduced number of SOX1 ⁺ cells were observed with the simultaneous appearance of ISL1 ⁺ motor neurons at day 21 , showing maturation of the spinal organoids (see FIGS. 8 and 9). ISL1 ⁺ motor neurons continued to rise in day 28 and 35 spinal organoids. TUJ1 ⁺ can also be observed to be appearing at day 14 of the spinal organoids and continued to persist in day 21 , 28 and 35 spinal organoids (see FIG. 5). Together, the results demonstrate that spinal organoids are able to recapitulate spinal cord neurogenesis.

Diverse spinal cord cell types observed in spinal organoids

The spinal cord is organized both rostro-caudally, as well as along the dorso-ventral axis. Motor neurons along the rostro-caudal axis are classified as cervical, brachial, thoracic or lumbar depending on the muscle groups they innervate. In order to determine if the spinal organoids recapitulate the diversity of neural cells along the rostro-caudal axis, immunostaining was performed for FIOXB4 (cervical marker), FIOXC8 (brachial/thoracic marker) and FIOXC10 (lumbar marker) in the day 28 organoids.

It was found that while there were some clusters of FIOXB4⁺ cells, many cells in the spinal organoid are FIOXC8⁺ (see FIGS. 10 and 1 1 ). FIOXC10 staining was not detected, indicating that lumbar motor neurons are not present in the organoids (data not shown). It was also found that a conventional 2D protocol resulted in a homogeneous layer of FIOXB4-expressing cervical subtype cells (see FIG. 18), with no FIOXC8 or FIOXC10 immunoreactivity. The acquisition of caudal identity in the spinal cord is orchestrated by Growth and Differentiation Factor 1 1 (GDF1 1 ). In this respect, an increased expression of GDF1 1 was measured in the organoid cultures over time while the 2D cultures were consistently lacking GDF1 1 expression (see FIG. 19). Quantitative PCR analysis also confirmed the immunostaining data, showing increased expression of HOXC8 over time in the organoids (see FIGS. 20A to 20H, in particular, FIG. 20D).

Along the dorso-ventral axis of the spinal cord, motor neurons are found in the ventral horns while sensory neurons are located in the dorsal horns. It was confirmed that motor neurons in the spinal organoids were FOXP1 limb- innervating neurons (see FIG. 12) and cholinergic based on ISL1 and ChAT co expression (see FIG. 13) at day 42. These neurons are also functional because co-culture of these organoids with mouse myotubes resulted in neurite outgrowth of about 650 pm within 3 days of co-culture (see FIGS. 21 and 22) and myotube contractions could be observed. C2C12 myotubes that were not co-cultured with organoids did not show any contraction. To confirm that neuromuscular junctions (NMJs) were formed, acetylcholine receptors at NMJs were labeled with alpha bungarotoxin (a-BTX) and SMI-32⁺ axons with a-BTX stains were observed at close proximity by confocal microscopy (see FIG. 23).

Apart from motor neurons, the presence of other ventral spinal cord cells was also detected, such as CFIX10-expressing cells which indicate the formation of V2a interneurons (see FIGS. 1 C and 14). Presence of Calbindin⁺ cells, along with increased expression of PAX2 and LHX1 also suggests that V1 inhibitory interneurons known as Renshaw cells were present (see FIGS. 15, 20F and 20H). Astrocytes, marked by S100p expression, can also be detected by day 35 (see FIG. 16). Flowever, SOX10⁺ dorsal root ganglia progenitors or BRN3A⁺ sensory neurons were not detected in the organoids by immunostaining or qPCR, suggesting that the cultures are more representative of ventral spinal organoids (see FIG. 17). Additionally, the inventors did not detect any increased expression of TLX3, a transcription factor expressed in the dorsal spinal cord (see FIG. 17), confirming the lack of dorsal cell types.

SMA ventral spinal organoids do not show a defect in neurogenesis

Using iPSCs derived from SMA Type I (1 -38G) and Type II (1 -51 N) patients, ventral spinal organoids were derived using the method described above. It has been suggested that SMA is also a neurodevelopmental disorder because histopathological analyses of spinal cords from patients have shown loss of anterior horn motor neurons, as well as immature and mismigrated neurons. Therefore, to investigate plausible defects in neurogenesis in SMA organoids, organoids were harvested for cryosectioning and immunostaining every 7 days starting at day 14, until they reach day 35. Similarly, like the wild- type (WT) organoids (see FIG. 5), SOX1 ⁺ cells appeared at day 14 and organized into rosette structures by day 21 in SMA organoids (see FIGS. 24 and 25). Reduced number of SOX1 ⁺ progenitor cells with rising appearance of ISL1 ⁺ motor neurons were also observed in the SMA organoids at day 21 (see FIGS. 24 and 26). Correspondingly, TUJ1 ⁺ neurons appeared in day 14 and continued to be present in day 21 , 28 and 35 SMA organoids (see FIG. 24). By day 28, 45.3% of all cells in WT organoids were made up of ISL1 ⁺ motor neurons. Likewise, similar percentages of motor neurons were observed in SMA organoids, with 42.2% in 1 -38G organoids and 44.7% in 1 -51 N organoids (see FIG. 27).

A cellular hallmark of SMA is the rapid degeneration of motor neurons. When WT and SMA organoids were cultured to day 35, it was observed that while the WT motor neurons increased slightly to 54.5% in the organoid, motor neuron numbers declined to 13.5% in 1 -38G organoids (p = 0.0015) and 35.9% in 1 -51 N organoids (p = 0.0061 ) (see FIGS. 26 and 27). This indicates that SMA motor neurons were unviable shortly after formation, with the Type I organoids showing a more severe degenerative phenotype compared to the Type II organoids. This observation was similarly observed on motor neurons derived using a conventional differentiation protocol and confirms that ventral spinal organoids recapitulate the cellular features of SMA.

SMA motor neurons express high levels of cell cycle CDKs and cvclins

The inventors used a 2-dimensional culture to profile the expression of purified HB9⁺ motor neurons derived from WT and 1 -38G iPSCs and found specific transcriptional events in diseased motor neurons. The ability to isolate pure populations of motor neurons for RNA-seq (i.e. whole transcriptome shotgun sequencing) circumvented the problem of intrinsic heterogeneity of iPSC-derived cultures, which was detrimental to whole transcriptome analyses because specific yet small changes in the diseased motor neuron population are often masked by the other contaminating cell types in the culture. From the RNA-seq study, it was found that mRNAs corresponding to CDK1 , CDK2, CDK4, Cyclins A2, B1 , B2 and D1 were upregulated in purified 1 -38G motor neurons compared to controls.

To confirm this, SMA and wild-type iPSCs were differentiated towards the spinal motor neuron fate, and quantitative PCR was performed on cDNA samples of purified HB9⁺ motor neurons by FACS sorting. This sorting removed variability of differentiation efficiencies between different cell lines and revealed higher expression of the cell cycle genes CDK1, CDK2, CCNA2, CCNB1 and CCNB2 (see FIG. 28), confirming the RNA-seq results. RNA was also isolated from purified ISL1 ⁺ motor neurons in organoids made from 1 -38G and BJ iPSCs at day 28 for qPCR analysis and similar upregulation was found in several CDKs and cyclins (see FIG. 29). This confirms that the spinal organoids recapitulated the molecular phenomenon that was observed in the 2-dimensional cultures.

The inventors postulated that loss of SMN protein was responsible for the upregulation of cell cycle genes, and to investigate this, siRNA-mediated knockdown of SMN was performed in WT motor neuron cultures. Quantitative PCR analysis revealed that loss of SMN was indeed responsible for the upregulation of Cyclins A2, B1 and B2 as observed in the SMA iPSC-derived motor neurons. In addition, depletion of SMN also resulted in upregulation of CDK6 and Cyclin E2 expression in the motor neuron cultures (see FIG. 30).

Low levels of SMN trigger cell cycle re-entry in motor neurons

Depletion of SMN resulting in cell cycle gene activation suggests that motor neurons deficient in SMN aberrantly re-enter the cell cycle. To confirm this, the inventors investigated the cell cycle status of motor neurons in WT and SMA cultures by determining the percentage of motor neurons that are co-expressing the proliferative marker Ki67. While 7-8% of wild-type ISL1 ⁺ motor neurons co- label Ki67, it was found that 28.9% and 22.5% (p < 0.01 ) of SMA Type I and Type II motor neurons co-express Ki67 respectively (see FIG. 31 ). Since motor neurons are post-mitotic cells and Ki67 expression is a hallmark of dividing cells, this indicates that loss of SMN triggers cell cycle re-entry in motor neurons. Furthermore, depletion of SMN protein by siRNAs in wild-type BJ-iPS motor neuron cultures also led to a significant increase in the percentage of Ki67- expressing motor neurons (see FIGS. 32 and 33).

To demonstrate that reactivation of the cell cycle is deleterious to motor neurons, the inventors compared the rate of apoptosis of Ki67⁺ motor neurons versus Ki67 motor neurons by co-staining with cleaved Caspase-3 (cCASP3). While 30.7% of Ki67 motor neurons co-express the apoptotic marker cCASP3, it was found that 71 .8% of Ki67⁺ motor neurons co-express cCASP3, indicating that reactivation of the cell cycle in motor neurons resulted in apoptosis (see FIG. 34). Inhibition of CDKs rescue SMA motor neurons

Since SMN-deficient motor neurons re-activate the cell cycle, it was hypothesized if inhibition of cell cycle components such as CDKs would rescue SMA motor neurons from cell death. SMA motor neurons were first treated for 3 days with a pan-CDK inhibitor (CDKi) (see FIG. 35) known to selectively inhibit CDK1 /Cyclin B, CDK2/Cyclin E and CDK4/Cyclin D1 complexes. It was found that treatment with the pan-CDK inhibitor (CDKi) was able to promote SMA motor neuron survival by up to 30% compared to a DMSO control (see FIGS. 36A and 36B).

To expand the study of CDK inhibition and its effects on neuroprotection in SMA, a series of motor neuron survival experiments was carried out using specific inhibitors of CDKs: CDK1 Inhibitor (CDK1 i), CDK2 Inhibitor II (CDK2i), CDK4 Inhibitor (CDK4i), as well as PD 0332991 (PD), a CDK4/6 inhibitor. It was found that while CDK1 and CDK2 inhibition had no significant effect on SMA motor neuron survival compared to DMSO, inhibition of CDK4 and CDK6 by CDK4i and PD resulted in up to 50% increase in motor neuron survival (see FIGS. 37A and 37B) in both the SMA Type I and II cultures. In addition to the small molecule inhibitor study, the inventors performed siRNA-mediated knockdown of each specific CDK on the SMA motor neurons (see FIGS. 42A to 42D) and quantified motor neuron numbers following siRNA treatment. Confirming results from the CDK small molecule inhibitor experiment, it was found that specific knockdown of CDK4 and CDK6 led to 27.1 % and 18.4% increase in SMA motor neuron survival respectively while depletion of CDK1 and CDK2 yielded insignificant results (see FIGS. 38 and 39). The results show that this neuroprotection was not due to an elevated SMN protein expression that could have resulted from CDK inhibition or knockdown (see FIGS. 40 and 41 ).

Motor neuron death in SMA ventral spinal organoids reversed with CDK inhibitor

Since neural organoids represent a more“in v/Vo-like” culture system compared to conventional two-dimensional cultures, the inventors investigated if ventral spinal organoids could be a good model for testing of therapeutic compounds. As a proof of principle, the efficacy of PD 0332991 in reversing the SMA motor neuron degenerative phenotype was tested. To this end, SMA organoids were treated at day 28 with either DMSO (control) or 0.1 mM PD for 7 days, and histology was performed to assess the treated and control organoids at day 35. At least 5 organoids in each treatment condition were analyzed by cryosectioning and immunostaining and it was found that SMA ventral spinal organoids remained highly neuronal based on SMI-32 labeling. It was also found that ISL1 ⁺ motor neuron survival in PD-treated SMA organoids was increased by 27.6% in 1 -38G and 29.1 % in 1 -51 N compared to DMSO treated organoids (see FIGS. 43 to 45). This confirms that PD 0332991 was specific in rescuing SMA motor neurons, even in the context of a ventral spinal organoid culture.

Summary of Findings In this study, the inventors reported the formation of ventral spinal organoids from iPSCs derived from SMA and healthy individuals to study neurodevelopmental, as well as neurodegenerative aspects of the disease. Detailed characterization of these organoids revealed that motor neurons, astrocytes, as well as interneurons that form part of the motor circuit were derived, mimicking the microenvironment in vivo. This would be especially useful for studying hyperexcitability of motor neurons in diseases such as SMA and Amyotrophic Lateral Sclerosis (ALS). It has been demonstrated that this abnormal neuronal firing can be caused by changes in the premotor circuits, where interneurons such as Renshaw cells exert inhibitory feedback control on motor neuron firing. However, dorsal cell types such as sensory neurons and dorsal interneurons were absent, limiting the use of this ventral spinal organoids to the studying of motor neurons and premotor circuits rather than sensory-motor connectivity.

Organoids have immense potential for studying human neural development. Various neural organoids, including cerebral (forebrain) organoids, retinal organoids and midbrain organoids have been generated for that purpose Therefore, the inventors made use of the spinal organoids as disclosed herein to first address neurodevelopmental processes in SMA. It remains controversial whether SMA is a neurodevelopmental disease. In the most severe forms of SMA, the very early onset of disease before 6 months of age and the lack of developmental milestones achievement suggest a developmental defect of the motor unit. Presence of fetal forms of acetylcholine receptors in neuromuscular junctions of SMA Type I patients, as well as loss of spinal motor neurons and presence of misguided and immature motor neurons appear to support that. It has also been reported that SMA mice have fewer spinal motor neurons at birth compared to healthy littermates, further suggestive of a neurogenesis defect. Therefore, using spinal organoids, the inventors sought to investigate manifestations of neurodevelopmental phenotypes in SMA.

Time-coursed analysis of healthy and SMA ventral spinal organoids revealed similar neurogenesis dynamics, where similar percentages of healthy and SMA SOX1 ⁺ neural progenitor cells were observed at each time point, indicative of a normal neural progenitor population. Likewise, similar percentages of healthy and SMA ISL1 ⁺ motor neurons were measured at days 21 and 28, highlighting that motor neuron formation is not impaired. SMA motor neurons rapidly degenerate between days 28 to 35, and the severity of motor neuron loss correlated with their clinical classifications, demonstrating that these spinal organoids have the capacity to model the spectrum of motor neuron degenerative phenotype with cells derived from Type I and Type II SMA patients.

While neural organoids have been widely used to model neurodevelopmental disorders, their utility in studying neurodegenerative disorders is less common. As effective models of neurodegeneration, the inventors further tested the utility of spinal organoids as disease models by conducting two-dimensional (2D) and organoid experiments simultaneously. Using SMA patient-derived motor neurons and SMN knockdown approaches, it was found that SMN-deficient motor neurons aberrantly reactivate the cell cycle, in agreement with recently published reports that SMN deficiency in motor neurons induces p53 activation. It has been previously reported that postmitotic neurons re-enter cell cycle as a response to DNA damage and a simultaneous increase in p53 expression. It was also found that blocking cell cycle progression by means of a CDK4/6 inhibitor significantly prolonged SMA motor neuron survival both in 2D and ventral spinal organoid cultures. This confirmed that neural organoids are amenable for small molecule screening approaches and could be considered as an additional screening step before moving into in vivo models.

APPLICATIONS Various embodiments of the disclosure provided herein provide an organoid and a method of obtaining an organoid.

Advantageously, various embodiments of the organoid e.g. spinal organoid provide a platform for evaluating the neurodevelopmental defects in neurodegenerative diseases such as SMA. SMA is caused by genetic mutations in the SMN1 gene, resulting in drastically reduced levels of Survival of Motor Neuron (SMN) protein. Although SMN is ubiquitously expressed, spinal motor neurons are one of the most affected cell types. To investigate why motor neurons are more affected than other neural types, a spinal organoid model was developed. The inventors found that by deriving spinal organoids from stem cells e.g. patient induced pluripotent stem cells (iPSCs), neurodevelopment was not significantly altered.

Even more advantageously, various embodiments of the organoid e.g. spinal organoid provide a suitable platform for testing small molecules that promote motor neuron survival. The inventors demonstrated overt motor neuron degeneration in SMA spinal organoids, and this degeneration can be prevented using a small molecule inhibitor of CDK4/6, indicating that spinal organoids are an ideal platform for therapeutic discovery.

It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

1. An organoid comprising,

motor neurons derived from stem cells, said motor neurons characterized by the expression of ISL1 (insulin gene enhancer protein).

2. The organoid of claim 1 , wherein the motor neurons are further characterized by the expression of one or more markers selected from the group consisting of FOXP1 (Forkhead box protein P1 ) and SMI32 (neurofilament FI).

3. The organoid of claim 1 or 2,

wherein the motor neurons comprise one or more types of motor neurons selected from the group consisting of cervical motor neurons characterized by the expression of FIOXB4, brachial motor neurons characterized by the expression of FIOXC8, and lumbar motor neurons characterized by the expression of HOXC10.

4. The organoid of any one of claims 1 to 3,

wherein the motor neurons consist of cervical motor neurons characterized by the expression of FIOXB4, and brachial motor neurons characterized by the expression of FIOXC8.

5. The organoid of any one of claims 1 to 4, wherein the motor neurons are limb-innervating motor neurons characterized by co-expression of FOXP1 and

ISL1.

6. The organoid of any one of claims 1 to 5, wherein the motor neurons are cholinergic motor neurons characterized by co-expression of ISL1 (Insulin gene enhancer protein) and choline acetyltransferase (ChAT).

7. The organoid of any one of claims 1 to 6, wherein the motor neurons are capable of neurite outgrowth when the organoid is co-cultured with myoblasts.

8. The organoid of any one of claims 1 to 7,

wherein the motor neurons are capable of forming neuromuscular junctions (NMJs) when the organoid is co-cultured with myoblasts.

9. The organoid of any one of claims 1 to 8, further comprising cells characterized by the expression of SOX1 (SRY-Box 1 ).

10. The organoid of any one of claims 1 to 9, further comprising motor neurons characterized by the expression of TUJ1 (neuron-specific Class III b-tubulin).

1 1. The organoid of any one of claims 1 to 10, wherein the organoid comprises one or more rosette structures.

12. The organoid of any one of claims 1 to 11 , further comprising

interneurons characterized by the expression of at least one interneuron marker selected from the group consisting of CHX10, Calbindin, PAX2 and LHX1 ; and/or

astrocytes characterized by the expression of at least one astrocytic marker selected from the group consisting of S100p, AQP4 and GFAP.

13. The organoid of claim 12, further comprising V 2a interneurons characterized by the expression of CHX10 marker.

14. The organoid of claim 12 or 13, further comprising Renshaw cells characterized by the expression of Calbindin, PAX2 and LHX1 markers.

15. The organoid of any one of claims 12 to 14, further comprising astrocytes characterized by the expression of S100p.

16. The organoid of any one of claims 1 to 15, wherein the number of motor neurons comprises at least 40% of the total number of cells in the organoid.

17. The organoid of any one of claims 1 to 16, wherein the organoid is a spinal organoid.

18. The organoid of claim 17, wherein the spinal organoid is a ventral spinal organoid comprising neurons that are found in the ventral horn of a spinal cord.

19. The organoid of any one of claims 1 to 18, wherein the stem cells are pluripotent stem cells.

20. The organoid of claim 19, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

21. The organoid of any one of claims 1 to 20, wherein the stem cells are obtained from a healthy (non-diseased) subject.

22. The organoid of any one of claims 1 to 20, wherein the stem cells are obtained from a subject having a neurodegenerative disease.

23. The organoid of claim 22, wherein the neurodegenerative disease is a motor neuron disease.

24. The organoid of claim 23, wherein the motor neuron disease is selected from the group consisting of Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS).

25. The organoid of claim 24, wherein the motor neuron disease is SMA.

26. The organoid of claim 24 or 25, wherein the motor neurons derived from stem cells of a subject having SMA are characterized by a higher expression of Cyclin Dependent Kinase (CDK) and / or cyclin (CCN) as compared to motor neurons derived from stem cells of a healthy (non-diseased) subject.

27. The organoid of claim 26, wherein the CDK is one or more selected from the group consisting of CDK1 , CDK2, CDK4, and CDK6.

28. The organoid of claim 26 or 27, wherein the CCN is one or more selected from the group consisting of CCNA2, CCNB1 , CCNB2, CCND1 , CCND2, CCNE1 , and CCNE2.

29. The organoid of any one of claims 26 to 28, wherein the motor neurons derived from stem cells of a subject having SMA are characterized by a higher expression of CCNA2, CCNB1 , CCNB2, CCNE2 and CDK6 as compared to motor neurons derived from stem cells of a healthy (non-diseased) subject.

30. The organoid of any one of claims 24 to 29, wherein the motor neurons derived from stem cells of a subject having SMA are characterized by a higher expression of proliferative marker Ki67 as compared to motor neurons derived from stem cells of a healthy (non-diseased) subject.

31. The organoid of claim 30, wherein the motor neurons derived from stem cells of a subject having SMA are further characterized by the expression of apoptotic marker cCASP3.

32. The organoid of any one of claims 1 to 31 , wherein the organoid is obtained in-vitro.

33. The organoid of any one of claims 1 to 32, wherein the expression comprises gene expression and/ or protein expression.

34. The organoid of any one of claims 1 to 33, for use as a platform for screening agents capable of prolonging motor neuron survival, wherein the organoid is to be contacted to one or more agents in a culture.

35. A method of obtaining an organoid, the method comprising,

culturing stem cells in a culture medium comprising one or more reagents capable of inducing neuralization of the stem cells;

contacting the stem cells to a reagent capable of caudalizing the stem cells;

contacting the stem cells to a reagent capable of ventralizing the stem cells; and

forming the organoid.

36. The method of claim 35, wherein the one or more reagents capable of inducing neuralization of the stem cells is selected from the group consisting of an agent for blocking Bone Morphogenetic Protein (BMP) signaling and an agent for activating Wnt pathways.

37. The method of claim 36, wherein the agent for blocking Bone Morphogenetic Protein (BMP) signaling comprises one or more selected from the group consisting of LDN-193189, dorsomorphin and Noggin.

38. The method of claim 36 or 37, wherein the agent for activating Wnt pathways comprises CHIR99021 and/or BIO.

39. The method of any one of claims 36 to 38, wherein contacting the stem cells to a reagent capable of inducing neuralization of the stem cells comprises simultaneously contacting the stem cells with the agent for blocking Bone Morphogenetic Protein (BMP) signaling and the agent for activating Wnt pathways

40. The method of any one of claims 35 to 39, wherein the reagent capable of caudalizing the stem cells comprises retinoic acid.

41 . The method of any one of claims 35 to 40, wherein the reagent capable of ventralizing the stem cells is a Sonic Hedgehog (SHH) pathway agonist.

42. The method of claim 41 , wherein the SHH pathway agonist is purmorphamine.

43. The method of any one of claims 35 to 42, wherein the reagent capable of caudalizing the stem cells is contacted to the stem cells between day 3 to day 15 of the culture, wherein day 0 is the day of seeding the stem cells.

44. The method of any one of claims 35 to 43, wherein the reagent capable of caudalizing the stem cells is contacted to the stem cells on or after the expression of one or more neuroepithelial stem cell markers in the stem cells.

45. The method of claim 44, wherein the one or more neuroepithelial stem cell markers is selected from the group consisting of SOX1 and Nestin.

46. The method of any one of claims 35 to 45, wherein the reagent capable of ventralizing the stem cells is contacted to the stem cells between day 10 to day 17 of the culture, wherein day 0 is the day of seeding the stem cells.

47. The method of any one of claims 35 to 46, further comprising contacting the stem cells to one or more neurotrophic factors capable of promoting neuronal maturation of the stem cells.

48. The method of claim 47, wherein the one or more neurotrophic factors capable of promoting neuronal maturation is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF).

49. The method of claim 47 or 48, wherein the one or more neurotrophic factors capable of promoting neuronal maturation of the stem cells is contacted to the stem cells from day 17 onwards of the culture, wherein day 0 is the day of seeding the stem cells.

50. The method of any one of claims 35 to 49, further comprising dissociating the stem cells into single cells prior to culturing the stem cells in the culture medium.

51. The method of any one of claims 35 to 50, further comprising seeding the stem cells at a density of at least 150,000 cells/ml in a suspension culture.

52. The method of any one of claims 35 to 51 , further comprising encapsulating the stem cells in a support matrix.

53. The method of claim 52, wherein the stem cells are encapsulated in the support matrix for one or more day, or two or more days, or three or more days, or four or more days, or at least one day, or at least two days, or at least three days, or at least four days, or at most one day, or at most two days, or at most three days, or at most four days.

54. The method of claim 52 or 53, wherein the support matrix is a growth matrix capable of providing support matrices to the stem cells and/ or acting as an extracellular matrix for the stem cell and/ or acting as a basement membrane matrix for the stem cells.

55. The method of any one of claims 52 to 54, wherein the support matrix is a gelatinous protein mixture derived from mouse tumour cells.

56. The method of any one of claims 52 to 55, further comprising transferring the cells in the support matrix into a dynamic cell culture device.

57. The method of claim 56, wherein the dynamic cell culture device is a spinner flask.

58. The method of any one of claims 35 to 57, wherein the stem cells are pluripotent stem cells.

59. The method of any one of claims 35 to 58, wherein the stem cells are induced pluripotent stem cells (iPSCs).

60. The method of any one of claims 35 to 59, wherein the stem cells are obtained from a healthy (non-diseased) subject.

61. The method of any one of claims 35 to 59, wherein the stem cells are obtained from a subject having a neurodegenerative disease.

62. The method of claim 61 , wherein the neurodegenerative disease is a motor neuron disease.

63. The method of claim 62, wherein the motor neuron disease is selected from the group consisting of SMA and ALS.

64. The method of any one of claims 35 to 63, wherein the organoid is a spinal organoid.

65. The method of claim 64, wherein the spinal organoid is a ventral spinal organoid.

66. The method of claim 64 or 65, wherein the spinal organoid comprises motor neurons characterized by the expression of ISL1.

67. The method of any one of claims 64 to 66, wherein the spinal organoid comprises one or more cells selected from the group consisting of motor neurons, interneurons, and astrocytes.

68. The method of any one of claims 64 to 67, wherein the spinal organoid comprises neurons that are found in the ventral horn of a spinal cord.

69. The method of any one of claims 64 to 68, wherein the spinal organoid comprises cells which are organized into one or more rosette structures.

70. The method of any one of claims 35 to 69, wherein the method is an in- vitro method.

71. A method of screening agents capable of prolonging motor neuron survival comprising,

contacting one or more agents to the organoid of any one of claims 1 to 34 or the organoid produced by the method of any one of claims 35 to 70,

72. An organoid obtained using the methods of any one of claims 35 to 70.

73. A spinal organoid comprising one or more cells selected from the group consisting of cholinergic motor neurons characterized by the expression of ISL1 , FOXP1 , and SMI32 markers, V2a interneurons characterized by the expression of CHX10 marker, Renshaw cells characterized by the expression of Calbindin marker and astrocytes characterized by the expression of S100b.

74. A method of treating spinal muscular atrophy comprising administering an effective amount of a CDK4/6 inhibitor to a subject in need thereof.

75. Use of a CDK inhibitor in the manufacture of a medicament for the treatment of spinal muscular atrophy, wherein the CDK inhibitor is a CDK4/6 inhibitor.

76. The use of claim 75, wherein the CDK4/6 inhibitor is PD 0332991.