Directed differentiation of astrocytes from human pluripotent stem cells for treatment and drug screening of amyotrophic lateral sclerosis (ALS)

Abstract

The invention relates to directed differentiation of astrocytes from human pluripotent stem cells for treatment and drug screening of amyotrophic lateral sclerosis (ALS). Disclosed are methods for identifying agents that affect human astrocyte functionality using ex vivo differentiated pluripotent stem cells (PSCs). In addition, the use of human progenitor astrocytes or human astrocytes for the treatment of amyotrophic lateral sclerosis (ALS) in a subject is also disclosed.

Description

This application is a divisional application of an invention patent with application date of September 23, 2014, application number 201480054641.3, and invention name “Directed differentiation of astrocytes from human pluripotent stem cells for the treatment and drug screening of amyotrophic lateral sclerosis (ALS)”. Technical Field The present invention, in some embodiments thereof, relates to methods for identifying agents that affect the functionality of human astrocytes using differentiated pluripotent stem cells (PSCs) in vitro. In addition, the use of human astrocytes for treating amyotrophic lateral sclerosis (ALS) is also provided. Background Art ALS is a motor neuron (MN) disease characterized by the loss of upper and lower MNs (Moloney et al., Front Neurosci 2014, 8:252). It affects approximately 30,000 individuals in the United States, approximately 150,000 patients worldwide, and approximately 400 individuals in Israel. 5-10% of ALS cases are familial ALS, but most ALS cases are sporadic and the cause is unknown. Transgenic mice and rats carrying ALS-related mutant human SOD1 genes reproduce many of the characteristics of the human disease. It is important to note that to date, there is no effective treatment available except for an FDA-approved compound, Riluzole, which only extends lifespan by up to three months.

[0003] ALS is diagnosed when significant cell loss has already occurred. MN axons grow up to one meter in length. The MN axons connect the cell body to its target (muscle). The ability to properly rewire and ensure functional connections after MN replacement therapy remains a daunting task with uncertain results, and it has not been demonstrated to date that this will be possible in humans. Therefore, attempts to avoid further cell loss after diagnosis will have a significant impact on MN survival and the patient's quality of life and lifespan.

[0004] However, cellular abnormalities in ALS are not limited to MNs. There are many observations of astrocyte dysfunction in ALS patients, both sporadic ALS (sALS) and familial (fALS). In vitro co-culture of astrocytes from ALS patients with normal motor neurons appears to accelerate the death of the motor neurons.

[0005] Therefore, there is a widely recognized need to generate human astrocytes (propagated and expanded from human PSC cells) that address these defects. Summary of the invention According to one aspect of the present invention, there is provided a method for screening an agent for preventing or treating a motor neuron (MND) disease (e.g., amyotrophic lateral sclerosis (ALS)), the method comprising: (a) contacting a population of astrocytes with the agent, wherein the astrocytes are differentiated in vitro from pluripotent stem cells (PSCs); (b) co-culturing the astrocyte population or conditioned culture medium thereof of step (a) with a neuronal population; and (c) quantifying the effect of the agent on enhancing the survival or neurological function of the neuronal population.

[0007] According to some embodiments of the invention, the neuronal population is hypoxic, under oxidative stress, under glutamate toxicity, or under glutamate / NMDA / AMPA / kainate toxicity.

[0008] According to some embodiments of the invention, the population ratio of astrocytes to neurons is greater than 1:1, 10:1, 100:1, 1000:1, or 10,000:1.

[0009] According to some embodiments of the invention, the astrocytes express each of GFAP, GLAST, AQP4 or a combination thereof.

[0010] According to some embodiments of the invention, the astrocytes exhibit secretion of neurotrophic factors selected from the group consisting of BDNF, GDNF and VEGF.

[0011] According to some embodiments of the present invention, the oxidative stress is selected from reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ) and any derivatives thereof.

[0012] According to some embodiments of the invention, the quantification is performed by counting the number of neurons under apoptosis.

[0013] According to some embodiments of the invention, apoptosis of the neurons is detected by caspase-3a labeling, Annexin V, Tubulin-B3, HB9 or DAPI.

[0014] According to some embodiments of the invention, the agent is a small molecule.

[0015] According to some embodiments of the invention, normal human progenitor astrocytes or astrocytes should provide an environment that prevents motor neuron death.

[0016] According to another aspect of the present invention, a method for treating or preventing the progression of ALS in a subject in need thereof is provided; the method comprises administering to a subject in need thereof human progenitor astrocytes (hAPCs) or astrocytes differentiated in vitro from pluripotent stem cells (PSCs).

[0017] According to some embodiments of the invention, the administering involves the cerebrospinal fluid, brain or spinal cord parenchyma of a subject in need thereof.

[0018] According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising the cell population of the present invention as an active agent, and a pharmaceutically acceptable carrier.

[0019] According to some embodiments of the invention, the cell population is not genetically manipulated.

[0020] According to some embodiments of the invention, the human progenitor astrocyte or astrocyte is non-autologous to the subject.

[0021] According to some embodiments of the invention, the human progenitor astrocytes or astrocytes are allogeneic to the subject.

[0022] According to some embodiments of the present invention, there is provided a use of human progenitor astrocytes or astrocytes differentiated ex vivo from pluripotent stem cells (PSCs) for the preparation of a medicament identified for the treatment of ALS.

[0023] Unless otherwise specified, all technical and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used to implement or test embodiments of the present invention, exemplary methods and / or materials are still described below. In the event of a conflict, this patent specification (including definitions) shall prevail. In addition, the materials, methods and examples are illustrative only and are not intended to be necessary limitations. BRIEF DESCRIPTION OF THE DRAWINGS This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] Some embodiments of the present invention are described herein (by way of example only) with reference to the accompanying drawings and images. With specific reference now to the drawings in detail, it is emphasized that the details shown are illustrative and for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description with the drawings makes it apparent to those skilled in the art how embodiments of the present invention may be implemented.

[0026] In the attached picture: Figure 1The kinetics of astrocyte gene expression by hESC-derived astrocytes are described. The expression of each gene at days 0, 7, 14, 21, 28, and 35 is shown. Adult brain and human fetal brain are used as controls. The following colors - orange, blue, green, yellow, red, and light blue represent the gene expression kinetics of GFAP, BDNF, PDGFRα, GLAST, GDNF, and ALDH1L1, respectively. For each gene, fetal brain expression is used as an internal reference gene (relative quantification, RQ=1).

[0027] Figure 2 AC describes the expression of astrocyte markers by hESC-derived astrocytes. A.CD44, B.CXCR4 and C.GLAST were measured by FACS at 0, 7, 14, 21, 28, 35 and 43 days. PDGF and CNTF are known factors that promote APC differentiation and differentiation. The effects of untreated (NT), PDGF, CNTF and PDGF+CNTF treatment on hESC-APC differentiation (marker expression %) were tested. Blue line: NT, red line: CNTF, green line: CNTF+PDGF, light blue: PDGF. The population obtained by the hPSC-derived astrocyte protocol produces an astrocyte-enriched population.

[0028] Figure 3A -E illustrates that hESC-derived astrocytes express markers of mature astrocytes. A. Grayscale of GFAP-positive cells. B. Grayscale of GLAST-positive cells. C. Overlay montage image of A+B, red for GFAP-positive cells, green for GLAST-positive cells and blue for Dapi-positive cells. Yellow staining indicates co-staining of GFAP and GLAST. Magnification of DC insert (same observation on yellow staining). E. Green for aquaporin-4-positive cells (AQP4) and red for GLAST-positive cells. Scale bars in AC and E: 100 μm and scale bar in D: 30 μm.

[0029] Figure 4 AC illustrates the production and secretion of neurotrophic factors by hPSC-derived astrocytes, as measured in the cell contents and culture medium after astrocyte activation, respectively. A. GDNF cell contents and secretion after IFN-γ activation (red). B. BDNF cell contents and secretion after IFN-γ activation (orange). C. VEGF cell contents and VEGF secretion after LPS activation (purple).

[0030] Figure 5AB illustrate the kinetics of 500 μM and 2 mM glutamate uptake by hESC-derived astrocytes. A. From left to right (green bars): 0', 10', 30', 60', 90' and 120' after addition of 500 μM glutamate to hESC-derived astrocytes at day 100 of differentiation. Light red bars: 1 st Negative control, medium containing 500 μM glutamate 60' without astrocytes. Dark red bar -2 nd Negative control, glutamate levels in medium grown with human fibroblasts after 120'. Right bar (blue): positive control, glutamate levels in medium grown with human spinal cord derived astrocytes. B. From left to right (light green bars): 0', 10', 30', 60', 90' and 120' after addition of 2 mM glutamate to hESC derived astrocytes at day 100 of differentiation. Blue bar: negative control, medium containing 2 mM glutamate after 60' in the absence of astrocytes.

[0031] Figure 6 AD describes the neuroprotective effect of human PSC-derived astrocytes on motor neurons under oxidative stress.

[0032] A. Use H 2 O 2 MNs were supplemented with 50 μM and 150 μM for 6 h or were left untreated. During this period, MNs were either left untreated or supplemented with astrocyte supernatant (24 h conditioned medium) or astrocytes. 2 O 2 The percentage of caspase-3a in MNs is indicated by the grey bar (right). 2 O 2 The percentage of caspase-3a in the incubated MNs is indicated by the red bar (50 μM H 2 O 2 ) and olive green bars (150 μΜ H 2 O 2 ), or after addition of astrocyte-conditioned medium by orange and light green bars, and in the presence of astrocytes by pink and light green bars. Caspase-3 activation (red) in B and C. Motor neurons were stained with beta3 tubulin (green) and nuclei were stained with DAPI. D. Image of the high-content analysis device for image quantification and analysis.

[0033] Figure 7AC confirms that injection of human progenitor astrocytes (hAPC) increases the survival of hSOD1 mice. A. Compare the survival of hSOD1 mice at two time points (day 130 and day 140). As shown at day 140, the survival of mice transplanted with "day 7" cells (grown for 7 days without growth factors) increases. B. Compare the Kaplan-Meir survival curves of all experimental groups. C. Compare the Kaplan-Meir curves generated for disease onset measured by rotation <106 for all experimental groups.

[0034] Figure 8 AC demonstrates that early human-derived astrocytes increase motor performance in hSOD1 mice. A. Comparison of the kinetics of disease progression described by BBB scores for all groups. 0 = normal mice, 5 = paralyzed mice. B. Comparison of the kinetics of disease progression described by rotarod scores for all experimental groups, 180 seconds = normal mice, 0 = mice unable to hold the rotarod. C. Mice on the rotarod instrument.

[0035] Fig. 9 Early human-derived astrocytes were confirmed to maintain body weight in transplanted hSOD1 cells. The kinetics of body weight changes throughout the course of disease progression were compared in all groups.

[0036] Fig.10 Kapaln Meier Log rank statistical analysis was described. Log Rank survival analysis was performed on 50% of the population. Fig.11 A general protocol for a neuroprotection assay using the addition of astrocyte condition medium or astrocytes is shown. DETAILED DESCRIPTION The present invention, in some embodiments thereof, relates to methods for identifying agents that affect the functionality of human astrocytes using differentiated pluripotent stem cells (PSCs) in vitro. In addition, the use of human astrocytes or human progenitor astrocytes for treating amyotrophic lateral sclerosis (ALS) in a human subject is also disclosed.

[0038] In order to obtain a large number of human astrocytes, the inventors of the present invention proposed to differentiate them from human pluripotent stem cells (PSCs), which can be propagated indefinitely before differentiation.

[0039] The present invention further discloses a unique and powerful protocol for producing a highly homogeneous astrocyte population (>90% GFAP, S100b) from human pluripotent stem cells (hPSCs). The differentiation protocol, together with expandable culture techniques, allows the production of large numbers of astrocytes in vitro. These hPSC-derived astrocytes exhibit gene expression patterns and functional properties similar to primary human astrocytes, including: I) secretion of neurotrophic factors (BDNF, GDNF, and VEGF) that protect motor neurons. II) The ability to take up glutamate and III) protect neurons from oxidative stress in vitro. These hPSC-derived astrocytes can be cryopreserved and used as needed.

[0040] The therapeutic properties of the human astrocytes were evaluated in vivo, and for this purpose, the cells were transplanted intrathecally into G93A high copy number hSOD1 mice (a mouse model for ALS disease). Human astrocyte transplantation resulted in significant improvements in motor performance in all functional tests (P<0.05). In addition, positive effects on survival and disease duration were observed in transplanted mice.

[0041] ALS is characterized by the death of motor neurons in the cortex, brainstem and spinal cord. In about 5-10% of the cases, ALS is familial, and the rest are sporadic. The course of the disease is difficult to distinguish between familial and sporadic ALS. The disease reveals itself in late life at an average age of 56 years, and once diagnosed, it leads to complete paralysis and death within 2-5 years. In a subgroup of the familial patients, mutations were found in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). SOD1 is a cytoplasmic enzyme involved in free radical detoxification. In addition, the overproduction of alleles encoding pathogenic human SOD1 proteins in motor neurons leads to late-onset progressive neurodegenerative diseases. Research has led to the identification of intrinsic pathogenic features of ALS motor neurons, including the formation of protein aggregates, cytoskeletal abnormalities, proteomic dysfunction, and increased sensitivity to cell death signals. In addition, the overproduction of alleles encoding pathogenic human SOD1 proteins in other cells can lead to non-cell-autonomous motor neuron pathology, and the cells are often found in vivo associated with motor neurons, such as but not limited to glial cells (e.g., astrocytes, Schwann cells, etc.). Examples of alleles encoding pathogenic SOD1 proteins include, but are not limited to, SOD1G93A, SOD1G85R, and SOD1G37R.

[0042] Neurodegenerative diseases such as ALS, Alzheimer's disease and Parkinson's disease are untreatable conditions that are commonly manifested as a serious health care burden. An enhanced understanding of the biology of these diseases is needed to develop neuroprotective treatments and final restorative treatments. Until recently, neurodegenerative diseases have been regarded as specialized neuronal disorders to a large extent. However, recent research results have raised objections to this concept, suggesting that the non-cellular spontaneous mechanisms of neurodegeneration mediated by astrocytes in acute and chronic conditions. Human stem cell biology allows the interaction of human astrocytes-neurons under simulated physiological and injury paradigms. Although there is a robust platform for producing neural stem cells and functional neurons, the generation of enriched and functional human astrocytes is relatively being studied. It is assumed that human astrocytes may protect diseased neurons and increase their survival mechanisms by them, including: preventing glutamate neurotoxicity, secreting neurotrophic factors (NTFs) such as BDNF and GDNF, and by the neurovascular modification of VEGF secretion.

[0043] The present invention is based in part on the generation of enriched and functional human pluripotent stem cell-derived progenitor astrocytes and astrocyte populations in vitro, and the establishment of an agent screening platform for ALS, in which the neuroprotective effects of human astrocytes on motor neurons under oxidative stress were tested.

[0044] In some embodiments of the present invention, methods and compositions are provided for identifying, characterizing, and optimizing lead compounds that exhibit neuroprotection and enhance human astrocyte functionality.

[0045] Agents that can be tested using the assays of the invention include small molecule agents, chemicals, peptides, proteins, polynucleotide agents (eg, siRNA agents).

[0046] The cell is contacted with the agent, which can be carried out by any in vitro method known in the art, including, for example, adding the agent to the cell so that the agent is directly contacted with the cell. According to some embodiments of the present invention, the cell is incubated with the agent. For the ratio between time period / cell concentration / agent concentration / cell and agent, etc., the conditions for incubating the cell are selected, which enable the agent to induce cell changes, such as changes in the transcription and / or translation rate of a specific gene analyzed. According to a specific embodiment, the cell is contacted with the agent for at least 1 day, 3 days, 5 days, 7 days, 10 days, 14 days or at least 21 days.

[0047] Candidate neuroprotective lead compounds can be added to the culture medium of the cells of the invention and assayed for desired properties. Compounds that exhibit desired properties, such as compounds that increase the number of live motor neurons in the culture medium relative to the number of live motor neurons in the absence of the candidate neuroprotective lead compound, can be selected for further characterization and optimization. Compounds can be selected for their ability to increase motor neuron viability in co-cultures with human astrocytes or in culture medium conditioned thereto.

[0048] The high throughput screen can also be used to identify compounds that can correct any disease-associated phenotype, such as survival, apoptosis, necrosis, axon degeneration, axon guidance, axon morphology, dendritic morphology, receptor density, synaptogenesis, neurogenesis, synaptic density, synaptic transmission, synaptic signaling, receptor trafficking, protein trafficking, protein aggregation, proteasome activity, receptor expression, oxidative stress (ROS), FGFR-signaling, and FGF signaling.

[0049] Characterization and optimization of candidate lead compounds selected for their ability to modulate cellular phenotypes (e.g., neuroprotection), including but not limited to further screening, generation of derivatives and analogs, analysis of absorption distribution, metabolism and excretion (ADME) characteristics, safety testing and efficacy testing. Specifically, optimization efforts may include the development of compounds designed to pass the blood-brain barrier.

[0050] Drug candidate compounds (or "test agents") can be selected independent small molecules (e.g., lead compounds from previous drug screening) or, in some cases, drug candidate compounds to be screened come from combinatorial libraries, i.e., a collection of multiple chemical compounds generated by chemical synthesis or biosynthesis by combining many chemical "building blocks". For example, linear combinatorial chemical libraries such as polypeptide libraries are formed by combining a collection of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized by such combinatorial mixing of chemical building blocks. Even, in theory, the systematic combinatorial mixing of 100 interchangeable chemical building blocks results in the synthesis of 100 million tetrameric compounds or 10 billion pentamer compounds.

[0051] As used herein, the term "survival" means any process by which cells avoid death. As used herein, the term survival also refers to preventing cell loss as demonstrated by necrosis, apoptosis, or preventing cell loss by other mechanisms. As used herein, increased survival indicates a decrease of at least 10%, 25%, 50%, 75%, 100% or more in cell mortality relative to an untreated control. The survival rate can be measured by counting cells in the culture medium that can be stained with a dye specific for dead cells, such as propidium iodide.

[0052] Cells used in the assays of the present invention include astrocytes differentiated ex vivo from pluripotent stem cells.

[0053] For example, human embryonic stem cells (such as described in WO2006 / 040763) can be separated from human blastocysts or delayed blastocyst stages. Human blastocysts are usually obtained from preimplantation embryos in the human body or from in vitro fertilization (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. For separating human ES cells, the zona pellucida is taken out from the blastocyst and separated by immunosurgery from the inner cell mass (ICM), wherein the trophectoderm cells are cracked and removed from the complete ICM by gently blowing and aspirating. The ICM is then inoculated into a tissue culture bottle containing an appropriate culture medium, and the culture medium enables it to grow outward. After 9-15 days, the growth from the ICM is dissociated into clumps by mechanical dissociation or by enzymatic degradation, and then the cells are re-inoculated on fresh tissue culture medium. The colonies showing undifferentiated morphology are selected separately by micropipette, mechanical dissociation into clumps and re-inoculated. The resulting ES cells are then routinely divided every 1-2 weeks. For further details on methods for preparing human ES cells, see Thomson et al., [U.S. Patent No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4:706, 1989]; Gardner et al., [Fertil. Steril. 69:84, 1998].

[0054] It will be appreciated that commercially available stem cells may also be used in this aspect of the invention. Human ES cells may be purchased from the NIH Human Embryonic Stem Cell Registry (<http: / / escr.nih.gov> ). Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, SAO1, TE03 (I3), TE04, TE06 (I6), HES-1, HES-2, HES-3, UC01, UC06, WA01, WA07 and WA09.

[0055] The stem cells used in the present invention may also be derived from induced pluripotent stem cells (IPSc).

[0056] Regardless of their origin, the stem cells used according to the present invention are at least 50% purified, 75% purified or at least 90% purified. When using human embryonic stem cell lines, the human ES cell colonies are separated from their trophoblasts (fibroblast-like cells irradiated by x-rays) by mechanical and / or enzymatic means to provide a substantially pure stem cell population.

[0057] Once human stem cells are obtained, they can be processed to differentiate into astrocytes. One exemplary method is described in the Materials and Methods section below. However, it will be understood that the present methods contemplate other methods for generating astrocytes, which are known in the art.

[0058] The culture medium used is selected according to the stem cell used. Therefore, for example, the culture medium suitable for ES cell growth can be, for example, DMEM / F12 (Sigma-Aldrich, St.Lewis, MO) or alpha MEM culture medium (LifeTechnologies Inc., Rockville, MD, USA), which is supplemented with supporting enzymes and hormones. These enzymes can be, for example, insulin (ActRapid; Novo Nordisk, Bagsvaerd, DENMARK), progesterone and / or Apo transferrin (BiologicalIndustries, Beit Haemek, Israel). Other components are listed in the embodiment section.

[0059] As used herein, the phrase "retinoic acid" refers to the active form of vitamin A (synthetic or natural) that is capable of inducing neural cell differentiation. Examples of forms of retinoic acid that can be used in accordance with the present invention include, but are not limited to, retinoic acid, retinol, retinal, 11-cis-retinal, all-trans-retinoic acid, 13-cis retinoic acid, and 9-cis retinoic acid (all available from Sigma-Aldrich, St.Lewis, MO).

[0060] In some embodiments of the invention, retinoic acid is used in a concentration range of 1-50 μΜ.

[0061] The culture medium may be further supplemented with growth factors, which may be present at least in part of the culture period to promote cell proliferation and facilitate differentiation into neuronal glial cell lines. According to one embodiment of this aspect of the invention, the growth factors include, for example, EGF (5-50 ng / ml) and bFGF (5-50 ng / ml) (R&D Systems, Minneapolis, MN, Biotest, Dreieich, Germany).

[0062] As used herein, the phrase "neurosphere" refers to spheroidal clusters or balls containing primarily neural stem cells and early multipotent progenitor cells that can differentiate into neurons, oligodendrocytes and astrocytes, as well as other glial cells.

[0063] The cells were cultured until mature neurospheres were formed.

[0064] The phrase "mature neurosphere" as used herein refers to a neurosphere in which some of the neural stem cells have differentiated into specialized oligodendrocyte progenitor cells with acquired oligodendrocyte lineage markers (e.g., Sox10, Nkx2.2., NG2, A2B5), while others have differentiated into neural progenitor cells or astrocyte progenitor cells.

[0065] According to one embodiment of the invention, the cells are cultured for, e.g., 10-30 (e.g., 20-30) days, at the end of which isolated neurospheres are formed. The neurospheres or cells dissociated therefrom are then adhered to an adhesion substrate (e.g., matrigel or extracellular matrix components (e.g., collagen, laminin, and fibronectin)) and further expanded with growth factors and finally differentiated on a cationic adhesion substrate (e.g., poly-D-lysine or poly-ornithine containing fibronectin (FN)) or an adhesion substrate (e.g., matrigel or extracellular matrix components (e.g., collagen, laminin, and fibronectin)) after removal of the growth factors.

[0066] The term "human astrocyte progenitor cell (hAPC)" or "human progenitor astrocyte" as used herein means a cell capable of generating progeny as mature astrocytes. Typically, the cell expresses some of the phenotypic markers that are characteristic of the astrocyte lineage.

[0067] The terms "administered" and "administering" refer to the process by which a therapeutically effective amount of astrocytes or agents contemplated herein are delivered to a subject for prophylactic and / or therapeutic purposes. The astrocytes and agents are administered in accordance with good medical practice, taking into account the clinical condition of the subject, the site and method of administration, the dose, the patient's age, sex, weight, and other factors known to the physician.

[0068] The term "treatment" refers to reversing, alleviating or inhibiting the progression of the disease to which the term is applied or one or more symptoms of the disease. Treatment includes management and care of the subject at diagnosis or after. Treatment can be performed in an acute or chronic manner. Depending on the condition of the subject, the term may refer to preventing the disease, and includes preventing the onset of the disease or preventing symptoms associated with the disease. The term also refers to reducing the severity of the disease or symptoms associated with the disease before being affected by the disease. The prevention or reduction of the severity of the disease before being affected refers to administering astrocytes or an agent of the same composition to a subject who has not been affected by the disease at the time of administration. "Prevention" also refers to preventing the recurrence of the disease or one or more symptoms associated with the disease. The goal of treatment is to combat the disease and includes administering the astrocytes to prevent or delay the onset of the symptoms or complications, or to alleviate the symptoms or complications, or to eliminate or partially eliminate the disease. The terms "therapy" and "therapeutically" refer to the act of treating, as "treatment" is defined above.

[0069] The present invention relates to such cells and populations thereof, which can be transplanted into patients to treat a wide range of neurodegenerative diseases.

[0070] According to some embodiments of the invention, the cells and populations thereof have not been genetically manipulated (ie, transformed with an expression construct) to produce the cells and cell populations described herein.

[0071] The terms or phrases "transplantation," "cell replacement," or "grafting" are used interchangeably herein and refer to the introduction of cells of the present invention into a target tissue.

[0072] The cells may be transplanted into the central nervous system or into a ventricular cavity or subdurally onto the surface of the host brain or onto the host cerebrospinal fluid. Conditions for successful transplantation may include: (i) viability of the implant; (ii) retention of the implant at the site of transplantation; and (iii) minimal pathological response at the site of transplantation. Methods for transplanting different neural tissues, such as fetal brain tissue, into the host brain have been described in: "Neural grafting in the mammalian CNS", Bjorklund and Stenevi, eds. (1985); Freed et al., 2001; Olanow et al., 2003). These procedures include intraparenchymal transplantation, i.e., in the host brain (as opposed to extra-brain or extraparenchymal transplantation), which is achieved by injecting or depositing tissue in the host brain so as to oppose the brain parenchyma during transplantation. Intraparenchymal transplantation can be achieved using two approaches: (i) injecting cells into the host brain parenchyma or (ii) surgically preparing a cavity to expose the host brain parenchyma and then placing the graft into the cavity. Both methods provide substantial placement between the transplant and the host brain tissue when transplanting, and both are beneficial to the anatomical integration between the transplant and the host brain tissue. If the transplant is required to become a component of the host brain and survive in the life of the host, this is very important. Alternatively, the transplant can be placed in a chamber (e.g., cerebral ventricle) or placed on cerebrospinal fluid under the dura mater, that is, placed on the surface of the host brain separated from the host brain substance by intervening pia mater or arachnoid and pia mater. Transplantation into the chamber can be completed by injecting the cells. For subdural transplantation, the cells can be injected around the brain surface after an incision is prepared in the dura mater. Injection into the selected host brain region can be carried out by drilling and piercing the dura mater to allow the needle of the microinjector to be inserted. Preferably, the microinjector is mounted on a stereotaxic frame and three-dimensional stereotaxic coordinates are selected for placing the needle in the desired part of the brain or spinal cord. The cells can also be introduced into the shell, basal nucleus, hippocampal cortex, striatum, substantia nigra or caudate area and spinal cord of the brain.

[0073] The cells may also be transplanted into healthy areas of tissue. In some cases, the exact location of the damaged tissue area may be unknown, and the cells may be inadvertently transplanted into a healthy area. In other cases, it may be preferred to administer the cells to a healthy area to avoid any further damage to the area. In either case, after transplantation, the cells may migrate to the damaged area.

[0074] For transplantation, the cell suspension is drawn into a syringe and administered to an anesthetized transplant recipient. Multiple injections can be performed using this procedure.

[0075] The cell suspension procedure thus allows the cells to be transplanted into any desired site in the brain and spinal cord, is relatively non-invasive, and allows multiple transplantations at several different sites or at the same site simultaneously using the same cell suspension.

[0076] For transplantation into a cavity, which may be preferred for spinal cord transplantation, tissue is removed from an area near the outer surface of the central nervous system (CNS) to form a transplant cavity, such as by removing the bone covering the brain and using materials such as gelatin sponge to stop bleeding as described by Stenevi et al. (Brain Res. 114: 1-20., 1976). Suction can be used to create the cavity. The graft is then placed in the cavity.

[0077] In view of the fact that non-autologous cells may induce an immune response when given to the body, several approaches have been developed to reduce the possibility of rejection of non-autologous cells. These approaches include suppressing the recipient's immune system. Examples of immunosuppressants include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazine (sulphasalazopyrine)), sodium chloroaurate (gold salts), D-penicillamine, leflunomide, azathioprine, anakinra, infliximab, etanercept, TNF, alpha blockers (biological agents targeting inflammatory cytokines), and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tacrolimus, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, and tramadol.

[0078] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to animals, including warm-blooded animals, such as mammals.

[0079] As used herein, the term "healthy subject" refers to a subject, particularly a mammal, who does not have diagnosed ALS or symptoms of ALS.

[0080] As used herein, "pharmaceutical composition" refers to a preparation of one or more active ingredients or cells described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of cells or compounds to an organism.

[0081] The term "active ingredient" herein refers to an agent that enhances the survival or neurological function of the motor neuron population.

[0082] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably and refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are included within these phrases.

[0083] The term "excipient" herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0084] A "therapeutically effective amount" relates to an amount or dose of astrocytes or an agent thereof that will result in one or more desired effects, specifically, one or more beneficial effects. The therapeutically effective amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the substance to elicit a desired response in the subject. The dosing regimen may be adjusted to provide the optimal therapeutic response (e.g., a beneficial effect, more specifically a sustained beneficial effect). For example, several divided doses may be administered daily or the dose may be proportionately reduced, as indicated by the exigencies of the therapeutic situation.

[0085] Induced pluripotent and multipotent stem cell lines are referred to herein as "induced stem cell lines" (iSC lines). Induced pluripotent stem cells are referred to as iPS cells or iPSCs.

[0086] As used herein, "correcting" a phenotype means changing the phenotype so that it more closely approximates a normal phenotype.

[0087] Techniques for formulating and administering drugs may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0088] As used herein, the term "about" refers to ± 10%.

[0089] The terms "comprises," "comprising," "includes," "including," "having" and conjugates thereof, mean "including but not limited to."

[0090] The term "consisting of" means "including and limited to."

[0091] The term "consisting essentially of" means that the composition, method, or structure may include additional ingredients, steps, and / or parts, but only if the additional ingredients, steps, and / or parts do not materially change the basic and novel characteristics of the claimed composition, method, or structure.

[0092] Throughout this application, various embodiments of the present invention can be expressed in range form. It should be understood that the description in range form is only for convenience and brevity, and should not be construed as a hard limit to the scope of the invention. Therefore, the description of the range should be considered to specifically disclose all possible sub-ranges and each numerical value within the range. For example, a range description such as 1-6 should be considered to specifically disclose sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and each number within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this applies.

[0093] Whenever a numerical range is shown herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrases "range is / ranges between a first indicated number and a second indicated number" and "range is / ranges from a first indicated number to a second indicated number" are used interchangeably herein and are intended to include the first and second indicated numbers and all fractions and integers therebetween.

[0094] The term "method" as used herein refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to those manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or those which are readily developable from known manners, means, techniques and procedures.

[0095] It is to be understood that certain features of the invention, which for clarity are described in the text of separate embodiments, may also be provided in combination in a single embodiment. Conversely, multiple features of the invention, which for brevity are described in the text of a single embodiment, may also be provided independently or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features described in the text of various embodiments are not to be considered essential features of those embodiments, unless the embodiments are ineffective without those elements.

[0096] Various embodiments and aspects of the present invention as hereinafter delineated and as claimed in the claims section below find experimental support in the following examples. Example

[0097] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0098] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, e.g., "Molecular Cloning: A laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III, Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series," Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodology set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Vols. I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology", Vols. I-III Coligan JE, eds. (1994); Stites et al. (eds.), Basic and Clinical Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), Selected Methods in Cellular Immunology, WH Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD and Higgins SJ, ed. (1985); "Transcription and Translation" Hames, BD and Higgins SJ, ed. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference. .

[0099] Materials and methods Generation of astrocytes for assays Human ES cells were cultured on a feeder layer of human neonatal foreskin fibroblasts (HEF) at 37°C and 5% CO. 2 The growth medium (ES1) consisted of DMEM / F12 (Sigma), 14% knockout serum replacement (KSR), non-essential amino acids (1 / 100), 0.1 mM beta-mercaptoethanol (all three from Invitrogen / Gibco), 1 mM sodium pyruvate, 2 μg / ml heparin (Sigma), and 8 ng / ml human recombinant basic FGF (FGF-2; Preprotech). Five days after seeding, hES cell colonies were detached at 37°C for 45-90 minutes using 1.2 mg / ml collagenase IV (Worthington). The aggregates were further cultured as above or subjected to differentiation as described (Izrael et al. Mol Cell Neurosci 34, 310-323 (2007)).

[0100] Step 1 - Transformation: For this transformation step, hES cell colonies were transferred to tissue culture plates in medium T (Transformation), which consisted of 50% (v / v) ES1 medium and 50% (v / v) ITTSPP / B27 medium. ITTSPP / B27 itself is a mixture of one volume of DMEM / F12 containing 2% B27 supplement (Invitrogen) and one volume of DMEM / F12 containing 25 μg / ml human insulin (ActRapid, Novo Nordisk), 50 μg / ml human Apo-transferrin (Biological Industries, Israel), 6.3 ng / ml progesterone, 10 μg / ml putrescine, 50 ng / ml sodium selenite and 40 ng / ml triiodothyronine (T3) (all from Sigma), with 100 U / ml penicillin and 100 μg / ml streptomycin. Medium T was supplemented with 20 ng / ml r-human EGF (R&D Systems) and 4 ng / ml r-human bFGF. After 1 day, large non-adherent hES cell colonies and aggregates were transferred to 6 cm bacterial (non-adherent) plates and grown in medium T supplemented with 20 ng / ml r-human EGF (R&D Systems) and 2 ng / ml r-human bFGF.

[0101] Step 2: All-trans retinoic acid (ATRA) treatment: The culture medium was changed to ITTSPP / B27, supplemented with 20 ng / ml EGF and 10 μΜ ATRA. The culture medium was changed every day for 7 days.

[0102] Step 3: Suspension culture: In this step, it matures neurospheres (NS), and the culture is continued for 18 days in ITTSPP / B27 medium containing 20 ng / ml EGF. The medium is changed every other day.

[0103] Neural stem cell proliferation and differentiation Step 4: Matrigel Adhesion 1: Transfer the balls / clusters to 6-well tissue culture plates that have been coated with BD Matrigel (Matrigel with reduced growth factors, BD Biosciences) diluted 1:30 in DMEM / F12 for 1.5 h at room temperature (RT). The culture medium was ITTSPP / B27 with 20 ng / ml EGF, and any non-adherent aggregates were removed after one day. The culture medium was changed every other day for 7-9 days.

[0104] Step 5: Matrigel Adhesion 2: Neurospheres / clusters that do not contain fibroblasts and epithelial cells are selected and picked onto new Matrigel plates. Neurospheres (no more than 10NS) are dissociated and partially dissociated using PBS (without Ca+ and Mg2+) containing 0.025% trypsin (Invitrogen) at 37°C for 2-3 min (passage 1). Trypsin is neutralized by one volume of trypsin inhibitor (Invitrogen) and four volumes of ITTSPP / B27 containing 2mg / ml BSA. The cells are spun at 1200 rpm for five minutes in an Eppendorf centrifuge. The dissociated small cell clusters are re-seeded onto Matrigel-coated plates (at a ratio of 1:1-1:3) on ITTSPP / B27 containing 20ng / ml EGF (step 4). After 2-3 weeks, the cells were dissociated by trypsin treatment as above (passage 2) and the resulting hES-NS cells were seeded on plates coated with poly-D-lysine (PDL, 100 μg / ml) or matrigel-coated plates, and ITTSPP / B27 containing mouse laminin (20 μg / ml) or PDL (20 μg / ml) and fibronectin 5 μg / ml with 20 ng / ml EGF for one day. At each new seeding, 1 μg / ml mouse laminin (Sigma) was added to the culture medium.

[0105] Steps 6 and 7: Expansion on poly-D-lysine or matrigel: After one day, the medium was changed to N2 / B27 medium, which consists of DMEM / F12 containing 0.5% (v / v) N2 supplement (Invitrogen), 1% (v / v) B27 supplement. The growth factors EGF and bFGF were added at 10 ng / ml each. After 7-14 days, the cells were split by trypsinization (1:2 or 1:3, passage 2 (P2)) to obtain a monolayer. After one week, the cells were collected with trypsin and cultured at 0.5×10 6 Cells were cryopreserved in ampoules (P3) and further passaged weekly when indicated.

[0106] To enrich for astrocytes and promote their commitment, the cells can be grown on Matrigel, Cultrex, or Geltrex.

[0107] Step 8: GF Depletion: At different passages, terminal differentiation was initiated by depletion of growth factors. Whenever growth factors were depleted, 50 μg / ml vitamin C was added to the N2 / B27 medium. Cells were thawed in N2 / B27 medium supplemented with growth factors bFGF and EGF. Growth factors were depleted one day prior to splitting and plating into 96-well plates.

[0108] Neuroprotection assay using astrocyte conditioned medium or astrocytes The general scheme is shown in Fig.11 The effect of the compound can be tested in the following steps (yellow circles): 1. Compounds may be added during the differentiation of hAPCs into mature astrocytes, and thereafter the astrocytes or their conditioned medium exposed to the compound may be added to neurons.

[0109] 2. At the last medium change 24-72 hours before induction of oxidative stress.

[0110] 3. In co-cultures during oxidative stress.

[0111] 4. Combinations are possible, i.e. 1+2, 1+3, 2+3, etc.

[0112] Step 1: Generating motor neurons from the rodent spinal cord Spinal cords from rat embryos (E15) were used for each experiment. Meninges were removed from all spinal cords. The spinal cord tissue was cut into small pieces and divided into 15 ml tubes (5 embryos per tube). Trypsin (2 ml, 0.25%) (Gibco # 250050014) was added to each tube and incubated at 37 ° C for 15 min. Cells were resuspended with NB culture medium (7 ml) and FCS (1 ml), centrifuged at 300g for 5 minutes and the supernatant was removed. The precipitation was resuspended with NB culture medium (2 ml). Single cells were counted and inoculated (120K cells / well) onto 96-well plates (Costar # cc-3596) coated with matrigel (BD # FAL354230). Cells were cultured in NB culture medium supplemented with 50 ng / ml NGF (Alomone # n-100), and the culture medium was changed every 2 days. The characterization of motor neurons was carried out after three days of culture. The neurons were stained with tubulin-b3, neurofilament and HB9 (motoneuron specific transcription factor). At least 70% of the neurons were positive for these antibodies.

[0113] Step 2: Astrocyte seeding The astrocytes were seeded on day 8-10 (after spinal cord surgery) and the cells were cultured using NB medium supplemented with 50 ng / ml NGF (Alomone #n-100).

[0114] Step 3: Astrocyte conditioned medium generation The culture medium from hPSC-derived astrocytes (30-150 day old astrocytes) was collected; diluted 1:1 with fresh N2B27 supplemented with vitamin C. The culture medium was called ACM (Astrocyte Conditioned Medium).

[0115] Step 4: Oxidative stress induction (performed on days 9-10) 50 μM or 150 μM H2O2 (Sigma #216763) was diluted with N2B27 (150 μl) supplemented with vitamin C or ACM and added to each well of the plate seeded with astrocytes. The plate was incubated at 37°C for 6 hours.

[0116] The plates were fixed with 4% PFA (100 μl / well) for 10 min, then washed twice and stored at 4°C until staining.

[0117] Step 5: Analysis of Motor Neuron Cell Death: dyeing: The cells were incubated with blocking buffer supplemented with Tx-100 0.3% (sigma) for 1 hour at room temperature (RT) and then washed with PBS (- / -).

[0118] The cells were incubated with the following antibodies for 1 hour at RT: 1 st Antibody-Rb-anti-caspase-3a (Promega #G7481) (1:250) M-anti-β-Tub3 (Convence#MMS-435P)(1:500) Rb-anti-HB9 (Abcam #AB-ab92606) (1:100) The cells were washed twice with PBS(- / -) and incubated with the following antibodies for 1 hour at RT: 2 nd Antibody-goat-anti-Rb-568 (Invitrogen #A11036) (1:1000) Goat-anti-mouse-488 (Invitrogen #A11029) (1:1000) The cells were washed twice with PBS (- / -), labeled with DAPI (1:1000), incubated for 5 min, and washed twice with PBS (- / -). HCS analysis was performed using ScanArray-Cellomics (Thermo-scientific inc).

[0119] The effect of ACM or the addition of hPSC-derived astrocytes can be tested with other stress inducers, such as: - Motor neuron excitotoxicity. Specifically, glutamate toxicity (glutamate uptake capacity tested in addition to motor neuron cell viability) - Additional reactive oxygen species toxicity -AMPA / Kainate Toxicity Culture medium composition: N2B27 Medium: - DMEM / F12 (Sigma #01-170) 96.5%.

[0120] -N2 (Invitrogen #17502-048) 0.5%.

[0121] -B27 (Invitrogen #17504044) 1%.

[0122] -Pen / Srep / Ampho B (Biological ind. #03-033-1B) 1%.

[0123] -GlutaMAX (Invitrogen #35050038) 1%.

[0124] NB medium: - Neurobasal medium (Gibco #21103049) 97%.

[0125] -B27 (Invitrogen #17504044) 1%.

[0126] -Pen / Srep / Ampho B (Biological ind. #03-033-1B) 1%.

[0127] -GlutaMAX (Invitrogen #35050038) 1%.

[0128] Characterization of human PSC-derived astrocytes using FACS analysis: Samples for FACS: Samples of hPSC-derived astrocytes from different developmental stages were stained; Day 0 = hPSC-derived astrocytes grown in the presence of EGF and bFGF Day 7-14 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 7-14 days Day 14-28 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 14-28 days Day 28-42 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 28-42 days Day 42-56 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 42-56 days FACS staining: Reagents: FACS Buffer: PBS (Gibco #14190-094) 99.5% BSA (sigma#A9418) 0.5% Antibody: -GLAST (Miltenyi) -A2B5(Miltenyi) -CD44 (Miltenyi) -CXCR4 (Miltenyi) -CD140(BD) -CD9 (Miltenyi) -CD133 (Miltenyi) -TRA1-60(Miltenyi) -EpCAM (Miltenyi) -ALDHILI (Miltenyi) -GFAP (after fixation) (Sigma) -Aqua4 (after fixation +Tx-100) Cell preparation for adherent cells: The cells were trypsinized for 3 minutes using trypsin (Gibco) and inactivated with DMEM / F12 (Sigma) containing DTI (Gibco) supplemented with 2 mg / ml BSA (Sigma, Cat#: A9418). The cells were collected into a conical tube along with the culture medium, centrifuged at 300 g for 5 minutes at RT and the supernatant was discarded. The cells were resuspended using cold PBS buffer (Gibco, Cat#: 14190-094), centrifuged at 300 g for 5 minutes at RT and the supernatant was discarded.

[0129] fixed: Resuspend the cells with PFA 0.5% (diluted, EMC) and incubate at 4°C for 30 minutes. Centrifuge the cells at 300 g for 5 minutes and discard the supernatant. Wash the cells with 1 ml PBS, centrifuge at 300 g for 5 minutes and discard the supernatant.

[0130] Permeabilization for intracellular antigen staining: The cells were incubated with Tx-1000.5% at 4°C for 30 minutes to permeabilize the cell membrane. The cells were centrifuged at 300 g for 5 minutes and the supernatant was discarded. The cells were resuspended with PBS, centrifuged at 300 g for 5 minutes and the supernatant was discarded.

[0131] Immunostaining: FACS buffer (FB) (1 × 10 5 cells / tube, resuspend the cells in a volume of 100 ul).

[0132] For unconjugated primary antibodies: Dilute the unconjugated first antibody and the unconjugated isotype control antibody with FB, respectively. Dilute the first antibody at the optimal dilution according to the manufacturer's instructions. Resuspend the cells with the diluted first antibody or the diluted isotype control antibody and incubate at 4°C for 30min. Centrifuge the cells at 300g for 5 minutes and discard the supernatant. Wash the cells with FB and centrifuge at 300g for 5 minutes, discard the supernatant. Dilute the corresponding fluorescent dye-conjugated second antibody with FB. Resuspend the cells with the diluted second antibody and incubate at 4°C for 30 minutes. Dilute the second antibody at the optimal dilution according to the manufacturer's instructions, and this incubation must be completed in the dark. Resuspend the cells with FB and centrifuge at 300g for 5 minutes, discard the supernatant. Resuspend the cells with 0.5ml FB and analyze on a flow cytometer.

[0133] For fluorescent dye-conjugated primary antibodies: Dilute the fluorescent dye-conjugated primary antibody with FB according to the manufacturer's instructions. Resuspend the cells with the conjugated primary antibody and incubate for 15 min at 4°C in the dark. Centrifuge the cells at 300g for 5 minutes and discard the supernatant. Resuspend the cells with FB and centrifuge at 300g for 5 minutes, discard the supernatant. Resuspend the cells with 0.5 ml FB and analyze on a flow cytometer.

[0134] Gene expression in human PSC-derived astrocytes: Samples for RNA extraction: RNA samples were extracted from hPSC-derived astrocytes at different developmental stages; Day 0 = hPSC-derived astrocytes grown in the presence of EGF and bFGF Day 7-14 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 7-14 days Day 14-28 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 14-28 days Day 28-42 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 28-42 days Day 42-56 = hPSC-derived astrocytes grown in the absence of EGF and bFGF for 42-56 days Extraction of RNA from hPSC-derived astrocytes: To the cell sample (1*10 6 -10*10 6 0.05% trypsin (Invitrogen #25200-056) was added to 100 μL of 4% lysine (10 μg / mL). The cells were incubated at 37°C for 5 minutes. Trypsin was inactivated by adding 2 volumes of 2 mg / ml BSA (Sigma). The cells were centrifuged at 300 rpm and the supernatant was removed. The pellet was resuspended with 1 ml of RLT (Qiangen) and 10 μL of beta-mercaptoethanol and stored at -80°C.

[0135] Real-time RT-PCR Reagents and instruments Gene expression assay probes: -A2B5 (ABI, Life technologies) -GFAP Hs00909236(life technologies) -BDNF (ABI, Life technologies) -PDGFR Hs00998018(life technologies) -GLAST (ABI, Life technologies) -GDNF (ABI, Life technologies) -ALDH1L1 Hs00201836(life technologies) -TRA-1-60 (ABI, Life technologies) -Nanog Hs04260366(life technologies) -Oct4(ABI, Life technologies) -Glul Hs00365928(life technologies) -IGF-1 (ABI, Life technologies) -NGF(ABI, Life technologies) -Aqua4 (ABI, Life technologies) -Connexin 30 (ABI, Life technologies) -CD44(ABI, Life technologies) Human total RNA (Ambion).

[0136] Human fetal RNA (Clontech).

[0137] TaqMan PCR master mix, 50 ml (Applied Biosystems).

[0138] Fast Optical 96-well reaction plates (Applied Biosystems).

[0139] • T-Professional Basic Gradient for RT-PCR (Biometra).

[0140] • High Capacity cDNA Reverse Transcription Kit (200 reactions) (Applied Biosystems).

[0141] Step One Plus Real-Time PCR System (Applied Biosystems).

[0142] Reverse transcription Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and a T-Professional Basic Gradient Apparatus. The following procedure is based on the Invitrogen protocol.

[0143] Prepare the following RNA / primer mixes in separate tubes: RT buffer 2μ1 Total RNA 1 μg RT random primers 1 μl RNase inhibitor 1 μl Reverse transcriptase 1 μl 100mM dNTP mix 0.8μl DEPC H 2 0 to 10 μl Program the automated thermal cycler to: 1.25℃10min 2.37℃120min 3.85℃5min 4.4℃ Pause The cDNA was stored at -20°C until used in real-time PCR.

[0144] Real-time PCR The following mixtures were prepared in each well of an optical plate (for 10 μl reaction mixture).

[0145] 5μl TaqMan Mix 0.5μl probe + primers 0.5 μl cDNA 4 μl HO 2 O Data obtained by qPCR for individual genes were analyzed using adult fetal brain RNA as an internal control.

[0146] hPSC-derived astrocyte neurotrophic factor secretion assay Elisa solution: Commercially available kit used: -BDNF (promega #G7610) -GDNF (promega#G7620) -IGF-1 (R&D Systems #DG100) -VEGF (R&D Systems #DVE00) -NGF (promega#G7630) hPSC-derived astrocytes were grown in the absence of growth factors (EGF+bFGF) for at least 20 days. The hPSC-derived astrocyte supernatant (conditioned medium) and / or cell contents were collected after the last medium replenishment (24h-72h).

[0147] Elisa for each factor was performed according to the manufacturer's protocol.

[0148] Glutamate intake regimen: Commercially available kits: - EnzyChrom Glutamate Assay Kit (Bioassay Systems #EGLT-100) was used.

[0149] Cell culture samples used: hPSC-derived astrocytes deprived of growth factors (EGF+bFGF) for at least 20 days, positive control - human astrocytes (Gibco), negative control - human fibroblasts. Glutamate (0.5-3 mM) was added to each experimental well.

[0150] At least 0.5 ml of medium samples were taken from the test cells at the following time points: T = 0', 10', 30', 60', 90', 120' and stored at 4°C until further processing. Glutamate uptake was performed according to the manufacturer's protocol (Bioassay Systems #EGLT-100).

[0151] Human astrocytes for transplantation: Steps 1-8 were performed as described above ("Generation of Astrocytes for Assay") to generate human astrocytes. For transplantation, human astrocyte precursor cells were differentiated by removal of growth factors. Two cell populations were used for the experiments: one was a cell population grown for 7 days without growth factors ("Day 7") and the other was a cell population grown for 42 days ("Day 42"). 5 Resuspend the "Day 7" and "Day 42" human astrocytes in DMEM / F12 at a concentration of 10 cells / μL.

[0152] Cell transplantation Experimental design Five transplantation experimental groups were tested. In group #1, 67 ± 2 day old SOD1 G93A Mice were transplanted (n=10 SOD1 G93A Group #2, using "Day 7" differentiated human astrocytes to 67 ± 2 day old SOD1 G93A Mice were transplanted (n=12 SOD1 G93A Group #3, at 67 ± 2 days of age SOD1 G93A Mice and SOD1 at day 97 ± 2 G93A Two "Day 7" cell injections were performed on mice (n=13). Group #4 was treated with vehicle only (DMEM / F12 sham injection group) for 67±2 days old SOD1 G93A Mice were injected (n=10) and group #5 did not receive any treatment (untreated group, n=4). Mice were immunosuppressed via daily IP injections of 10 mg / Kg cyclosporine (Sandimmun, Novartis) starting 3 days before transplantation until the end of the experiment, and in addition, 15 mg / kg CellCept (Roche) was given twice a day (per OS) starting 3 days before transplantation until day 7 after transplantation.

[0153] Human astrocyte transplantation Immunosuppressed animals were transplanted on days 67±2 (all groups) and 97±2 (group #3). 7 μl of culture medium or 2.0×10 6Cells were injected intrathecally through the cisterna magna. Cells were delivered using a Hamilton Gastight syringe (10 μL) (Hamilton; Reno, NV) with a 30-gauge 45° bevel needle. After the transplantation phase was completed, cell viability was assessed using a Nucleocounter and found to be greater than 85%.

[0154] SOD1 G93A Mouse Transgenic mice carrying the human SOD1 gene with the G93A mutation (B6SJL-Tg(SOD1*G93A)1Gur / J) were used. Male and female mice were evenly distributed between experimental groups. Mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained as an in-house colony.

[0155] Animal Care and Handling All procedures were performed in strict accordance with Israeli guidelines; measures were taken to minimize any possible pain or discomfort to the animals. Mice were housed at standard temperature (21°C) and in a controlled light environment with food and water available ad libitum and maintained in racks in ventilated cages in the same room. To avoid dehydration, access to a cage water basin was provided when animals began to show symptoms of illness.

[0156] Behavioral and motor performance analysis Forelimb grip Animal weighing and all behavioral data collection began one week before transplantation and were performed twice a week until the end. The forelimb muscle grip was measured separately using a "grip dynamometer". The grip test was performed by having the animal grasp a thin bar connected to the dynamometer. After this, the animal was pulled away from the dynamometer until the hind limbs or forelimbs released the bar. This provides the force value of the maximum grip. The force measurements were recorded in three independent experiments and the average value was used for analysis.

[0157] Rotarod performance test Motor function was performed twice a week. Motor function was tested using an accelerating rotarod apparatus (180 sec, rotarod 7650; Ugo Basile, Comerio, Italy). The time when the mice failed from the rotarod was recorded. The animals were trained for one week before recording. Recording began one week before implantation.

[0158] BBB-Clinical Score According to the table below, scores are given on a scale of 0 to 5. Mice may be given an "intermediate" score (ie, 0.5, 1.5, 2.5, 3.5) when the clinical signs are between the two specified scores.

[0159] Rating on a scale of 0-5: When the clinical signs were between two defined scores, mice were given an "intermediate" score (ie, 0.5, 1.5, 2.5, 3.5). In most cages, mice reached a clinical score of 3.5-4, and their clinical signs worsened from that point onwards.

[0160] weight Body weight was measured twice a week.

[0161] Survival / terminal and seizure analyses To determine the end stage of the disease in a reliable and ethical manner, the end stage was determined by the inability of the mouse to right itself within 30 seconds when placed in lateral recumbency.

[0162] Onset of illness was defined by a decrease in rotarod performance of less than 160 seconds.

[0163] Statistical analysis The SOD1 G93A Kaplan-Meier analysis of mice to analyze survival, disease onset and duration data. WeightRotarod, BBB and grip results were analyzed by repeated measures ANOVA. In some cases, Student t test was performed to compare data between animal groups. All data are shown as mean ± SEM, and the significance level was set at p ≤ 0.05.

[0164] Histological and biochemical analysis Tissue processing Animals were sacrificed at the terminal stage by transcardial perfusion of 0.3% saline followed by ice-cold 4% paraformaldehyde (Fisher Scientific; Pittsburgh, PA). The spinal cord was removed from the animals and then cryoprotected with 30% sucrose (Fisher) / 1M phosphate buffer at 4°C for 3 days. The tissue was embedded in OCT (Fisher), snap frozen with dry ice and stored at -80°C until processing. Spinal cord tissue blocks were cut in sagittal or transverse sections at 30 μm thickness. Sections were collected on slides and stored at -20°C until analysis. A subset of spinal cord sections were collected in PBS for free floating histochemistry.

[0165] Quantification of graft survival and migration HuNA (human nuclear antigen; Millipore; Temecula, CA; monoclonal; 1:400) was used to selectively identify graft-derived human cells (both hGRP and hF).

[0166] Example 1 In vitro generation and characterization of human pluripotent stem cell (PSC)-derived astrocytes Differentiation of human astrocyte precursor cells (APCs) into mature astrocytes was tested in vitro for each astrocyte cell line by: a. Real-time PCR (qPCR). The following probes (mRNA) were tested: BDNF, GDNF, PDGFR, GFAP, GLAST, and ALDH1L1. Figure 1 The kinetics of astrocyte lineage gene expression in hESC-derived astrocytes are shown. In order to promote the differentiation of astrocyte precursor cells to a mature phenotype, growth factors were removed from the culture medium. Samples were collected and tested at day 0 (with growth factors) and every two weeks after the growth factors were removed. Human fetal brain was used as an internal reference sample for all tested genes (relative quantification (RQ) = 1) and was used as a positive control together with the adult brain. It is important to note that the adult brain consists of more than 50% astrocytes. In this study, significant increases were found in all tested astrocyte genes during cell differentiation, and in many cases, the expression of astrocyte genes (i.e., GFAP, BDNF, PDGFRα, and GLAST) was significantly higher than that of human fetal brain ( Figure 1 ). These results demonstrate that the protocol results in an enriched population of human astrocytes.

[0167] b. Fluorescence activated cell sorting (FACS, flow cytometry) analysis using early and late glial restriction markers (e.g., CD44, CXCR4, and GLAST). In the FACS analysis shown ( Figure 2 ), tested the dynamics of early astrocyte markers such as CD44 and CXCR4, as well as GLAST as a late astrocyte marker. CXCR4, also known as fusin or CD184, is a chemokine receptor with potential chemotactic activity in the CNS. It was found that more than 95% of the cells expressed CXCR4 and CD44 from day 0 to day 21, and these levels decreased as astrocytes matured. At the same time, the level of GLAST increased as astrocytes matured ( Figure 2 ). CXCR4 expression indicates a high migration potential of the hESC-derived astrocyte precursor cells, which is a required ability for the cells to reach their destination.

[0168] c. Immunohistochemistry (IHC) using astrocyte-specific antibodies: GFAP, GLAST, S100b and aquaporin 4. Quantitative staining by our ScanArray high-capacity screening device (HCS). In Figure 3, hPSC-derived astrocytes were stained at day 50 of in vitro differentiation. As can be seen in the picture, most of the cells are positive for astrocyte-specific markers (GFAP, GLAST and AQP4).

[0169] Example 2 Testing the biological functionality of the hPSC-derived astrocytes Several experiments were performed to demonstrate that hPSC-derived human astrocytes exhibit the functional properties of mature, healthy astrocytes. These in vitro results illustrate possible in vivo mechanisms of action. For the purposes described, the following functional mechanisms were tested: a. Neurotrophic factor (NTF) secretion: Elisa for BDNF, GDNF, and VEGF was performed on astrocyte upper culture medium and detergent extracts. Different astrocyte activators such as IFN-γ and LPS were used. Figure 4 It was confirmed that upon activation of hPSC-derived astrocytes, GDNF, BDNF, and VEGF were secreted into the culture medium ( Figure 4 , the middle column respectively).

[0170] b. Glutamate uptake: Astrocyte glutamate uptake capacity was tested by measuring glutamate levels in the culture medium of astrocytes. A colorimetric assay (Enzychrom) was used to measure glutamate levels in the culture medium. Fibroblasts were used as negative controls, while astrocytes from adult tissue were used as positive controls. Data show that glutamate uptake by human PSC-derived astrocytes ( Figure 5 ). In this study, the kinetics of glutamate uptake at two glutamate concentrations (0.5mM and 2mM) were tested at the following time points: 0', 10', 30', 60', 90' and 120' after the addition of glutamate. It was found that the hPSC-derived astrocytes took up glutamate from the two concentrations of culture medium in a time-dependent manner. This indicates the ability of the specific sample of astrocytes to take up glutamate.

[0171] c. MN neuroprotection assay: In this assay, hydrogen peroxide (H 2 O 2) stimulated MNs of mouse or human origin. The effect of adding hPSC-derived astrocyte conditioned medium and / or astrocytes on MN survival was tested after induction of oxidative stress. The number of live / dead MNs was evaluated by using a high-capacity screening device (Cellomics-Scan Array). This assay can be used as a valuable tool for finding new drugs that affect MN survival in ALS disease. The data show that the conditioned medium from hPSC-derived astrocyte tissue culture has an effect on MNs under oxidative stress (using H 2 O 2 ) under the neuroprotective effect of rodent spinal MN. Figure 6 As shown in Figure , the protective effects of conditioned medium from hPSC-derived astrocytes on mouse MN cultures and the effects of adding hPSC-derived astrocytes directly to mouse MN cultures were tested. 2 O 2 concentration (50 μM and 150 μM). 2 O 2 After adding hPSC-derived astrocyte-conditioned medium at 100 μg / mL, a significant reduction in MN death was found. Surprisingly, the neuroprotective effect of adding hPSC-derived astrocytes to MN cultures under oxidative stress was even greater than that of adding their conditioned medium ( Figure 6 ), indicating that its presence is more significant in reducing oxidative stress than its secretions alone. These results further confirm the in vitro neuroprotective ability of our hPSC-derived astrocytes. The hPSC-derived astrocyte protocol obtains highly purified human astrocytes. The human astrocytes display functional astrocyte properties in vitro, such as secretion of neurotrophic factors and glutamate uptake, properties known to increase motor neuron survival. The potential of hPSC-derived astrocytes in protecting motor neurons under oxidative stress was directly verified using an in vitro model. This assay is a valuable tool for finding drugs that increase the neuroprotective potential of human astrocytes in vivo.

[0172] Example 3 Finding the optimal conditions for transplanting hPSC-derived astrocytes Before conducting experiments on SOD1 animals, optimization experiments on different transplantation aspects were performed.

[0173] a. Optimizing cell injection methods i. Different implantation sites were tested (ie, direct injection into the ventral horn of the spinal cord, intrathecal injection, or intracerebroventricular injection).

[0174] ii. The number of hPSC-derived astrocytes (per injection / per site) was tested.

[0175] b. Perform evaluation histological methods for measuring neuronal damage and detecting implanted cells for optimization of large-scale efficacy studies.

[0176] c. Evaluate the efficacy of behavioral and electrophysiological tests used to follow up the disease.

[0177] d. hPSC-derived astrocyte survival, migration and kinetics of cell differentiation after transplantation were tested using immunohistochemistry.

[0178] Example 4 Transplantation of hPSC-derived astrocytes into an ALS animal model (SOD1 mice) The neuroprotective effect of hPSC-derived astrocytes on MNs was tested in vivo, i.e., on improved locomotion and survival of animals. For these purposes, the hSOD1 G93A C57BL6 / SJL background (high copy number) mouse model was used and treated as described in Materials and Methods.

[0179] As in Figure 7 As shown in , injection of early human-derived astrocytes increased the survival of hSOD1 mice. Figure 8 Significant improvement in BBB rotarod performance was demonstrated at "Day 7 (pool)" compared to sham injection (P<0.05).

[0180] As in Fig. 9 As shown in FIG, the "d7" group significantly maintained its body weight compared to the sham-injected group (P<0.05).

[0181] As in Fig.10 As shown in , T1 / 2 statistical analysis revealed a significant increase in survival as observed in both injected groups compared to the sham injected group at "Day 7" (P=0.046).

[0182] Although the invention is described in conjunction with its specific embodiments, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0183] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference. In addition, the citation or identification of any reference in this application should not be construed as an admission that the reference can be used as prior art for the present invention. To the extent that section headings are used, they should not be construed as necessary limitations.

Claims

1. Use of a cell population comprising human progenitor astrocytes or human astrocytes in the preparation of a medicament for treating or preventing the progression of ALS in a human subject in need thereof, wherein the human progenitor astrocytes and human astrocytes have been differentiated in vitro from human pluripotent stem cells (PSCs), the human pluripotent stem cells (PSCs) being selected from embryonic stem cells and induced pluripotent stem cells, the human pluripotent stem cells (PSCs) having been treated with retinoic acid and growth factors including bFGF and EGF, and wherein the human progenitor astrocytes or human astrocytes show secretion of neurotrophic factors selected from BDNF, GDNF and VEGF, wherein the human progenitor astrocytes or human astrocytes are cells that have not been genetically modified, and wherein the human progenitor astrocytes or human astrocytes are formulated for intrathecal administration.

2. The use according to claim 1, wherein the human progenitor astrocytes or human astrocytes express each of GFAP, GLAST, AQP4 or a combination thereof.

3. The use according to claim 1, wherein the human progenitor astrocytes or human astrocytes show glutamate uptake ability. 4 . The use according to claim 1 , wherein the human progenitor astrocytes or human astrocytes are non-autologous to the subject.

5. The use according to claim 1, wherein the human progenitor astrocytes or human astrocytes are allogeneic to the subject.

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