Compositions and methods for treating spinal cord injury

Abstract

SUMMARY OF THE DISCLOSURE Provided herein are methods, compositions, and devices for treating neurological disorders and diseases, including spinal cord injury. TIFF2024505051000028.tif58138

Description

[Background technology]

[0001] background Spinal cord injury (SCI) is a devastating and currently untreatable condition, except for symptomatic treatment of some of the resulting complications. Spinal cord injury results in complete or partial loss of motor, sensory, and autonomic function. As a result, patients often lose mobility and become wheelchair bound, in addition to suffering from numerous medical complications. It is estimated that over 12,000 Americans suffer from spinal cord injury (SCI) each year, with approximately 1.3 million people living with spinal cord injuries in the United States. Traumatic SCI most commonly affects individuals in their 20s and 30s, resulting in high levels of permanent disability in younger, previously healthy individuals. Individuals with SCI not only have impaired limb function, but also suffer from bowel and bladder dysfunction, decreased sensation, spasticity, autonomic dysreflexia, thrombosis, sexual dysfunction, increased infections, decubitus ulcers, and chronic pain, each of which can significantly affect quality of life and even become life threatening in some instances. The life expectancy of an individual who suffers from a cervical spinal cord injury at age 20 years is 20–25 years shorter than that of an individual of a similar age without SCI (NSCISC Spinal Cord Injury Facts and Figures 2013). To date, there are no treatments approved by the U.S. Food and Drug Administration (FDA) to induce neurological recovery after spinal cord injury (SCI). Several interventions, including glucocorticoids, modulation of voltage-gated channels, tetracycline antibiotics, and cell-based therapies, have been studied in clinical trials, but none have met the pivotal registration endpoints to date.

[0002] The clinical impact of spinal cord injury varies depending on the site and extent of the injury. Nervous systems that can be permanently destroyed below the level of injury are not only accompanied by loss of limb muscle regulation and the protective role of temperature and pain sensation, but also affect the cardiovascular system, breathing, sweating, bowel control, bladder control and sexual function (Anderson KD, Friden J, Lieber RL. Acceptable benefits and risks associated with surgically improving arm function in individuals living with cervical spinal cord injury. Spinal Cord.2009 Apr; 47(4):334-8). These losses result in a series of secondary problems that were acutely fatal until modern medicine, such as bedsores and urinary tract infections. Spinal cord injury often removes these unconscious regulatory mechanisms that maintain an appropriate level of excitability in the neural circuits of the spinal cord. As a result, spinal motor neurons can spontaneously become hyperactive, causing debilitating stiffness and unregulated muscle spasms or spasticity. This hyperactivity can cause chronic neurogenic pain and unpleasant sensations in the sensory system, including paresthesias, numbness, tingling, pain, and burning. In a recent survey of patients with spinal cord injury, recovery of ambulatory function was not the highest ranking function these patients wanted to regain, and in many cases, relief from the sequelae of spontaneous hyperactivity was most important (Anderson KD, Friden J, Lieber RL. Acceptable benefits and risks associated with surgically improving arm function in individuals living with cervical spinal cord injury. Spinal Cord.2009 Apr; 47(4):334-38).

[0003] There is a need for treatments for spinal cord injuries and associated pathologies. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Anderson KD, Friden J, Lieber RL. Acceptable benefits and risks associated with surgically improving arm function in individuals living with cervical spinal cord injury. Spinal Cord.2009 Apr; 47(4):334-8 [Non-Patent Document 2] Anderson KD, Friden J, Lieber RL. Acceptable benefits and risks associated with surgically improving arm function in individuals living with cervical spinal cord injury. Spinal Cord.2009 Apr; 47(4):334-38 Summary of the Invention

[0005] overview The examples and embodiments presented herein describe human embryonic stem cell (hESC) derived cells for the treatment of spinal cord injury (SCI), as described in more detail herein.

[0006] For example, the OPC compositions obtained according to the present disclosure can be used in cell therapy to improve one or more neurological functions in a subject in need of treatment. In one embodiment, the OPC cell population according to the present disclosure can be injected, implanted, or otherwise delivered to a subject in need thereof. In one embodiment, the cell population according to the present disclosure can be implanted or otherwise delivered to a subject in need thereof to treat spinal cord injury, stroke, or multiple sclerosis.

[0007] LCTOPC1 is a cell population containing a mixture of oligodendrocyte precursor cells and other characterized cell types obtained after directed differentiation of established and well-characterized lineages of hESCs. AST-OPC1 (previously known as GRNOPC1) is a cell population containing a mixture of oligodendrocyte precursor cells (OPCs) and other characterized cell types obtained after differentiation of undifferentiated human embryonic stem cells (uhESCs). Oligodendrocyte precursor cells (OPCs) are a subtype of glial cells in the central nervous system (CNS) that originate in the ventricular zone of the brain and spinal cord and mature into oligodendrocytes after migrating throughout the developing CNS. Mature oligodendrocytes produce the myelin sheath that insulates neuronal axons and remyelinate CNS lesions where the myelin sheath has been lost. Oligodendrocytes also contribute to neuroprotection through other mechanisms, including the production of neurotrophic factors that promote neuronal survival (Wilkins et al., 2001 Glia 36(1):48-57; Dai et al., 2003 J Neurosci.23(13):5846-53; Du and Dreyfus, 2002 J Neurosci Res.68(6):647-54). Unlike many progenitor cells, OPCs remain abundant in the adult CNS, where they retain the ability to generate new oligodendrocytes. Thus, OPCs and mature oligodendrocytes derived from OPCs are important therapeutic targets for demyelinating and hypomyelinating disorders (such as multiple sclerosis, adrenoleukodystrophy and adrenomeloneuropathy), other neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease) and acute neurological injuries (such as stroke and spinal cord injury (SCI)).

[0008] The OPC compositions obtained according to the present disclosure can be used in cell therapy to improve one or more neurological functions in a subject in need of treatment. In one embodiment, the OPC cell population according to the present disclosure can be injected or implanted into a subject in need thereof. In one embodiment, the cell population according to the present disclosure can be implanted into a subject in need thereof to treat spinal cord injury, stroke, or multiple sclerosis.

[0009] In certain embodiments, the OPC1 composition is administered after the subject suffers from a traumatic spinal cord injury. In some embodiments, the OPC1 composition is administered 14 to 90 days after the spinal cord injury, e.g., 14 to 75 days after the spinal cord injury, e.g., 14 to 60 days after the spinal cord injury, e.g., 14 to 30 days after injury, e.g., 20 to 75 days after injury, e.g., 20 to 60 days after injury, e.g., 20 to 40 days after injury. In some embodiments, the OPC1 composition is administered to treat about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about about 51, about 52, about 53, about 54, about 5, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89 or about 90 days after administration. In some embodiments, the OPC1 composition is administered between 14 days and the life of the subject.

[0010] Methods and compositions for obtaining cell populations comprising dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells can be found in WO 2020 / 154533, WO 2020 / 061371, U.S. Pat. No. 10,286,009, WO 2017 / 031092, WO 2017 / 173064, and WO 2018 / 053210, each of which is incorporated by reference in its entirety for all methods, compositions, cells, data, definitions, uses and all other information provided therein.

[0011] In one aspect, a method is provided for improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising administering to the subject a first dose of a composition comprising human pluripotent stem cell-derived oligodendrocyte precursor cells (OPCs), and optionally administering two or more doses of the composition.

[0012] In some embodiments, the method further comprises administering a second dose of the composition to the subject. In some embodiments, the method further comprises administering a third dose of the composition to the subject. In some embodiments, each administration comprises delivering the composition into the spinal cord of the subject, for example by injection. In some embodiments, each administration comprises delivering two or more fractions of a dose. In some embodiments, the SCI is a subacute cervical SCI. In some embodiments, the SCI is a chronic cervical SCI. In some embodiments, the SCI is a subacute thoracic SCI. In some embodiments, the SCI is a chronic thoracic SCI. In some embodiments, the first dose, the second dose and / or the third dose of the composition are administered in a dose of about 1×10 6 pieces~approx. 3×10 7 In some embodiments, the first dose of the composition comprises about 2×10 OPC cells. 6 In some embodiments, the first dose or the second dose of the composition comprises about 1×10 OPC cells. 7 In some embodiments, the second or third dose of the composition comprises about 2×10 OPC cells. 7The OPC cells are included. In some embodiments, each of the first, second and third doses of the composition is administered about 20 to about 45 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 14 to about 90 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 14 to about 75 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 14 to about 60 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 14 to about 30 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 20 to about 75 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 20 to about 60 days after the SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 20 to about 40 days after the SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 14 days after the SCI to the life span of the subject. In some embodiments, the injection is performed in the caudal half of the site of the SCI. In some embodiments, the injection is about 6 mm into the spinal cord of the subject. In some embodiments, the injection is about 5 mm into the spinal cord of the subject.

[0013] In another aspect, a method is provided for improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising administering to the subject a dose of a composition comprising oligodendrocyte precursor cells (OPCs) derived from human pluripotent stem cells.

[0014] In some embodiments, the dose of the composition is about 1×10 6 pieces~approx. 3×10 7 In some embodiments, the dose of the composition comprises about 2×10 OPC cells. 6The OPC cells. In some embodiments, administering the composition comprises injecting, implanting, or otherwise delivering the composition into the spinal cord of the subject. In some embodiments, the dose of the composition is administered about 7 days to about 14 days after the SCI. In some embodiments, the injection is performed in the caudal half of the site of the SCI. In some embodiments, the injection is about 6 mm into the spinal cord of the subject. In some embodiments, the injection is about 5 mm into the spinal cord of the subject. In some embodiments, the SCI is a subacute thoracic SCI. In some embodiments, the SCI is a chronic thoracic SCI. In some embodiments, the SCI is a subacute cervical SCI. In some embodiments, the SCI is a chronic cervical SCI. In some embodiments, improving one or more neurological functions comprises an improvement in ISNCSCI test upper extremity motor score (UEMS). In some embodiments, the improvement in UEMS occurs within about 6 months, within about 12 months, within about 18 months, within about 24 months or more after the injection. In some embodiments, the improvement is an increase in UEMS of at least 10% compared to baseline. In some embodiments, improving one or more neurological functions includes improving the Lower Extremity Motor Score (LEMS). In some embodiments, the improvement in LEMS occurs within about 6 months, within about 12 months, within about 18 months, within about 24 months or more after injection. In some embodiments, the improvement is an improvement in at least one motor level. In some embodiments, the improvement is an improvement in at least two motor levels. In some embodiments, the improvement is on one side of the subject's body. In some embodiments, the improvement is on both sides of the subject's body. In some embodiments, the dose of the composition is administered about 14 days to about 90 days after SCI. In some embodiments, the dose of the composition is administered about 14 days to about 75 days after SCI. In some embodiments, the dose of the composition is administered about 14 days to about 60 days after SCI. In some embodiments, the dose of the composition is administered about 14 days to about 30 days after SCI. In some embodiments, the dose of the composition is administered about 20 days to about 75 days after SCI. In some embodiments, the dose of the composition is administered about 20 days to about 60 days after the SCI.In some embodiments, the dose of the composition is administered from about 20 days to about 40 days after the SCI. In some embodiments, the dose of the composition is administered from about 14 days after the SCI to the lifetime of the subject.

[0015] In another aspect, there is provided a cell population comprising an increased percentage of cells positive for the oligodendrocyte precursor marker NG2 and reduced expression of non-OPC markers CD49f, CLDN6, and EpCAM, said cell population prepared according to the following method: culturing undifferentiated human embryonic stem cells (uhESCs) in a glial progenitor medium comprising a MAPK / ERK inhibitor, a BMP signaling inhibitor, and retinoic acid to obtain glial restricted cells; differentiating the glial restricted cells into oligodendrocyte precursor cells (OPCs) having an increased percentage of cells positive for the oligodendrocyte precursor marker NG2 and reduced expression of non-OPC markers CD49f, CLDN6, and EpCAM.

[0016] In some embodiments, the cell population is used to treat a thoracic spinal cord injury (SCI) in a subject. In some embodiments, the thoracic SCI is a subacute thoracic SCI. In some embodiments, the thoracic SCI is a chronic thoracic SCI. In some embodiments, the cell population is used to treat a cervical spinal cord injury (SCI) in a subject. In some embodiments, the cervical SCI is a subacute cervical SCI. In some embodiments, the cervical SCI is a chronic cervical SCI. In some embodiments, the composition is administered by implantation or other delivery methods. In some embodiments, the composition is administered to the subject by injection after SCI. In some embodiments, the injection is administered in the caudal half of the site of occurrence of the SCI. In some embodiments, the injection is about 6 mm into the spinal cord of the subject. In some embodiments, the injection is about 5 mm into the spinal cord of the subject. In some embodiments, the injection is administered about 14 days to about 90 days after the SCI. In some embodiments, the injection is administered about 14 days to about 75 days after the SCI. In some embodiments, the injection is administered about 14 days to about 60 days after the SCI. In some embodiments, the injection is administered from about 14 days to about 30 days after SCI. In some embodiments, the injection is administered from about 20 days to about 75 days after SCI. In some embodiments, the injection is administered from about 20 days to about 60 days after SCI. In some embodiments, the injection is administered from about 20 days to about 40 days after SCI. In some embodiments, the injection is administered from about 14 days after SCI to the lifetime of the subject.

[0017] In another aspect, there is provided a method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising administering to the subject a first dose of the cell population of claim 54, administering to the subject a second dose of the cell population, and optionally, administering to the subject a third dose of the cell population.

[0018] In some embodiments, the SCI is a subacute cervical SCI. In some embodiments, the SCI is a chronic cervical SCI. In some embodiments, the SCI is a subacute thoracic SCI. In some embodiments, the SCI is a chronic thoracic SCI. In some embodiments, the first dose, the second dose, and the third dose of the composition are each administered about 14 days to about 90 days after the SCI. In some embodiments, the first dose, the second dose, and the third dose of the composition are each administered about 14 days to about 75 days after the SCI. In some embodiments, the first dose, the second dose, and the third dose of the composition are each administered about 14 days to about 60 days after the SCI. In some embodiments, the first dose, the second dose, and the third dose of the composition are each administered about 14 days to about 30 days after the SCI. In some embodiments, the first dose, the second dose, and the third dose of the composition are each administered about 20 days to about 75 days after the SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 20 days to about 60 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 20 days to about 40 days after SCI. In some embodiments, each of the first, second and third doses of the composition is administered about 14 days to about the survival of the subject after SCI. [Brief description of the drawings]

[0019] Embodiments will now be described, by way of example only, with reference to the accompanying drawings.

[0020] [Figure 1] Figure 1 is a schematic timeline of the Phase 1 clinical trial. [Diagram 2] Figure 2 is a schematic diagram of patient screening, treatment, and follow-up during the Phase 1 clinical trial (CONSORT flow diagram). [Diagram 3]Figure 3 shows International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) screening and 5-year follow-up (*one ISNSCI performed at 4 years). In the figure, green represents normal movement and / or sensation, red represents absence of movement and / or sensation, and orange and light red represent present but abnormal sensation. [Figure 4] Figure 4 shows an example of a questionnaire administered for a long-term protocol, which required annual visits for years 2 to 5. After the annual visit in the fifth year, follow-up was by annual telephone questionnaire. [Diagram 5] FIG. 5 is a schematic diagram of the subject study. [Figure 6] Figure 6 is a schematic timeline of clinical trials. [Figure 7] FIG. 7 is a schematic diagram of patient screening and treatment during the clinical trial. [Figure 8] FIG. 8 is a schematic representation of the cohort structure and enrollment progress of a clinical trial consistent with the implementation of the present disclosure. [Figure 9] FIG. 9 is another Phase 1 clinical trial schematic timeline consistent with the implementation of the present disclosure. [Figure 10] FIG. 10 is an overview of two exemplary cell manufacturing processes consistent with implementations of the present disclosure. [Figure 11] FIG. 11 is a flow chart of a schematic diagram of the signaling sequence of a cell differentiation process consistent with the practice of the present disclosure. [Figure 12] FIG. 12 is a flowchart of a manufacturing process flow consistent with the practice of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description Before the compositions and methods are described, it should be understood that the disclosure is not limited to the specific processes, compositions, or methodologies described, as they may vary. It should also be understood that the terms used in this description are intended to describe only the specific versions or embodiments, and are not intended to limit the scope of the invention, which is limited only by the appended claims. For example, features shown with respect to one embodiment may be incorporated in other embodiments, and features shown with respect to a particular embodiment may be deleted from that embodiment. Thus, the disclosure contemplates that in some embodiments of the disclosure, any feature or combination of features described herein may be excluded or omitted. Furthermore, in light of the disclosure, numerous modifications and additions to the various embodiments proposed herein will be apparent to those skilled in the art, which do not depart from the disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the invention. It is not intended that any part of this specification be interpreted as a disclaimer of any portion of the full scope of the invention. Thus, the following description is intended to illustrate certain aspects of the disclosure and is not intended to exhaustively specify all permutations, combinations and variations thereof.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terms used in the description of the disclosure herein are intended to describe particular embodiments only and are not intended to be limiting of the disclosure.

[0023] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0024] It is specifically contemplated that the various features of the disclosure described herein may be used in any combination unless the context indicates to the contrary. Further, the present disclosure also contemplates that in some aspects of the disclosure, any feature or combination of features described herein may be excluded or omitted.

[0025] The methods disclosed herein may include one or more steps or actions to achieve the described method. The steps and / or actions of the method may be interchanged with one another without departing from the scope of the present invention. In other words, unless a specific order of steps or actions is required for the proper implementation of an embodiment, the order and / or use of specific steps and / or actions may be changed without departing from the scope of the present invention.

[0026] As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates not to include the plural forms.

[0027] As used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0028] The terms "about" and "approximately," when used herein when referring to measurable values ​​such as percentages, densities, volumes, and the like, are meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0029] As used herein, phrases such as "X to Y" and "about X to Y" should be interpreted to include X and Y. As used herein, phrases such as "about X to Y" mean "about X to about Y", and phrases such as "from about X to Y" mean "from about X to about Y".

[0030] The term "AST-OPC1" refers to a specific, characterized, in vitro differentiated cell population containing a mixture of oligodendrocyte precursor cells (OPCs) and other characterized cell types, obtained from undifferentiated human embryonic stem cells (uhESCs) according to the specific differentiation protocol disclosed herein.

[0031] Compositional analysis of AST-OPC1 by immunocytochemistry (ICC), flow cytometry and quantitative polymerase chain reaction (qPCR) demonstrates that the cell population is composed primarily of neural lineage cells of oligodendrocyte phenotype. Other neural lineage cells, i.e., astrocytes and neurons, are present at low frequencies. The only non-neuronal cells detected in the population are epithelial cells. Mesodermal, endodermal lineage cells and uhESCs are usually below the quantification or detection of the assay.

[0032] As used herein, the term "oligodendrocyte precursor cells" (OPCs) refers to cells of the neuroectodermal / glial lineage that have characteristics of cell types found in the central nervous system and can differentiate into oligodendrocytes. These cells typically express the characteristic markers nestin, NG2 and PDGF-Ra.

[0033] The terms "treatment", "treat", "treated" or "treating" as used herein can refer to both therapeutic treatment or preventative or prophylactic measures, the purpose being to prevent or slow down (alleviate) undesirable physiological symptoms, symptoms, disorders or diseases, or to obtain beneficial or desired clinical results. In some embodiments, the term can refer to both treating and preventing. In the present disclosure, beneficial or desired clinical results can include, but are not limited to, one or more of the following: alleviation of symptoms; reduction in the severity of the symptoms, disorders or diseases; stabilization (i.e., not worsening) of the condition of the symptoms, disorders or diseases; delay in onset or delay in progression of the symptoms, disorders or diseases; improvement of symptoms, disorders or disease symptoms; and remission (whether partial or complete), or enhancement or amelioration of the symptoms, disorders or diseases, whether detectable or undetectable. Treatment includes eliciting a clinically significant response. Treatment also includes prolonging survival compared to expected survival in the absence of treatment.

[0034] The term "subject" as used herein includes, but is not limited to, non-human vertebrates such as humans, non-human primates, and wild, domestic and farm animals, including any mammals such as cats, dogs, cows, sheep, pigs, horses, rabbits, rodents such as mice and rats. In some embodiments, the term "subject" refers to males. In some embodiments, the term "subject" refers to females.

[0035] As used herein, "implantation" or "transplantation" refers to the administration of a cell population into a target tissue using an appropriate delivery technique (e.g., using an injection device, an implantation device or other delivery device).

[0036] As used herein, "engraftment" and "engrafting" refer to the incorporation of implanted tissue or cells (i.e., "graft tissue" or "graft cells") into the subject's body. The presence of the graft tissue or graft cells at or near the implantation site 180 days or more after implantation indicates engraftment. In some embodiments, imaging techniques (e.g., MRI imaging, etc.) can be used to detect the presence of the graft tissue.

[0037] As used herein, "allogeneic" and "allogeneically derived" refer to a cell population that is derived from a source other than the subject and is therefore not genetically identical to the subject. In some embodiments, the allogeneic cell population is derived from cultured pluripotent stem cells. In some embodiments, the allogeneic cell population is derived from hESCs. In other embodiments, the allogeneic cell population is derived from induced pluripotent stem (iPS) cells. In yet other embodiments, the allogeneic cell population is derived from primate pluripotent (pPS) cells.

[0038] As used herein, "parenchymal cavitation" refers to the formation of a lesion or cavity within or near the site of CNS injury in an area normally occupied by parenchymal CNS tissue. The cavity or lesion may be filled with extracellular fluid and may contain small bands of macrophages, connective tissue, and blood vessels.

[0039] The terms "central nervous system" and "CNS", used interchangeably herein, refer to the complex of nervous tissue that regulates one or more activities of the body, and include, but are not limited to, the brain and spinal cord of a vertebrate animal.

[0040] The term "decorin" as used herein refers to a proteoglycan that in humans is encoded by the DCN gene. Decorin is a small cellular or pericellular matrix proteoglycan; this protein is a component of connective tissue, binds to type I collagen fibrils, and plays a role in matrix assembly.

[0041] The term "chronic" as used herein includes, but is not intended to be limited to, symptoms occurring in a subject for a period ranging from 90 days after injury to occurring during the subject's lifetime.

[0042] The term "subacute" as used herein includes, but is not intended to be limited to, symptoms occurring in a subject over the period of 14 to 90 days following injury.

[0043] Due to the injury itself and the subsequent secondary effects of edema, hemorrhage and inflammation, multiple pathologies are observed in the injured spinal cord (Kakulas BA. The applied neuropathology of human spinal cord injury. Spinal Cord. 1999 Feb;37(2):79-88). These pathologies include axonal severance, demyelination, parenchymal cavitation and the generation of heterotopic tissue such as fibrous scar tissue, gliosis and dystrophic calcification (Anderson DK, Hall ED. Pathophysiology of spinal cord trauma. Ann Emerg Med. 1993 Jun;22(6):987-92; Norenberg MD, Smith J, Marcillo A. The pathology of human spinal cord injury:defining the problems. J.Neurotrauma. 2004 Apr;21(4):429-40). Oligodendrocytes, which provide axons with both neurotrophic factors and myelination support, are prone to cell death after SCI and are therefore important therapeutic targets (Almad A, Sahinkaya FR, Mctigue DM. Oligodendrocyte fate after spinal cord injury. Neurotherapics 2011 8(2):262-73). Replacement of the oligodendrocyte population can support both surviving and damaged axons and also remyelinate axons to facilitate electrical conduction (Cao Q, He Q, Wang Yet al. Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury. J. Neurosci. 2010 30(8):2989-3001).

[0044] Oligodendrocyte precursor cells (OPCs) are a subtype of glial cells in the central nervous system (CNS) that arise in the ventricular zone of the brain and spinal cord and mature into oligodendrocytes after migrating throughout the developing CNS. Mature oligodendrocytes produce the myelin sheath that insulates neuronal axons and remyelinate CNS lesions that have lost their myelin sheath. Oligodendrocytes also contribute to neuroprotection through other mechanisms, including the production of neurotrophic factors that promote neuronal survival (Wilkins et al., 2001 Glia 36(1):48-57; Dai et al., 2003 J Neurosci.23(13):5846-53; Du and Dreyfus, 2002 J Neurosci Res.68(6):647-54). Additionally, OPCs are known to produce decorin, a secreted factor that has been shown to inhibit CNS scarring (Esmaeili, Berry et al. 2014, Gubbiotti, Vallet et al. 2016). Unlike many progenitor cells, OPCs remain abundant in the adult CNS, where they retain the ability to generate new oligodendrocytes. Thus, OPCs and mature oligodendrocytes derived from OPCs are important therapeutic targets for demyelinating and demyelinating disorders (such as multiple sclerosis, adrenoleukodystrophy and adrenal myeloneuropathy), other neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease) and acute neurological injuries (e.g., stroke and spinal cord injury (SCI)).

[0045] Proliferation and culture of undifferentiated pluripotent stem cells In certain embodiments, the present disclosure provides methods for producing large numbers of highly pure, characterized oligodendrocyte precursor cells from pluripotent stem cells. Derivation of oligodendrocyte precursor cells (OPCs) from pluripotent stem cells by the methods of the present invention provides a renewable and scalable source of OPCs for many important therapeutic, research, development and commercial purposes, including the treatment of acute spinal cord injury.

[0046] Methods for the proliferation and culture of undifferentiated pluripotent stem cells have been described previously. With regard to the tissue and cell culture of pluripotent stem cells, the reader is directed to any of the numerous publications available in the art, e.g., Teratocarcinomas and Embryonic Stem cells: A Practical Approach (E. J. Robertson, Ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al., Eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and R. I. Freshney, Culture of Animal Cells, Wiley-Liss, New York, 2000).

[0047] In some embodiments, the method may be performed on a pluripotent stem cell line. In other embodiments, the method may be performed on an embryonic stem cell line. In one embodiment, the method may be performed on a plurality of undifferentiated stem cells derived from H1, H7, H9, H13, or H14 cell lines. In another embodiment, the undifferentiated stem cells may be derived from an induced pluripotent stem cell (iPS) line. In another embodiment, the method may be performed on a primate pluripotent stem (pPS) cell line. In yet another embodiment, the undifferentiated stem cells may be derived from parthenotes, which are embryos stimulated to produce hESCs without fertilization.

[0048] In one embodiment, undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without added feeder cells (see, e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are typically supported by a nutrient medium containing factors that promote proliferation of cells without differentiation (see, e.g., U.S. Pat. No. 6,800,480). In one embodiment, a conditioned medium containing such factors can be used. Conditioned medium can be obtained by culturing the medium with cells that secrete such factors. Suitable cells include, but are not limited to, irradiated (4,000 Rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts or fibroblast-like cells derived from pPS cells (U.S. Pat. No. 6,642,048). The medium can be conditioned by seeding feeders in serum-free medium such as knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. Medium conditioned for 1-2 days can be supplemented with additional bFGF and used to support pPS cell cultures for 1-2 days (e.g., WO 01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0049] Alternatively, fresh or unconditioned media supplemented with added factors (e.g., fibroblast growth factor or forskolin) that promote the proliferation of cells in undifferentiated morphology can be used. Non-limiting examples include basal media such as X-VIVO™ 10 (Lonza, Walkersville, Md.) or QBSF™-60 (Quality Biological Inc. Gaithersburg, Md.) supplemented with bFGF at 40-80 ng / mL and optionally containing SCF (15 ng / mL) or Flt3 ligand (75 ng / mL) (see, e.g., Xu et al., (2005) Stem Cells 23(3):315). These media formulations have the advantage of supporting cell proliferation at 2-3 times the rate in other systems (see, e.g., WO 03 / 020920). In one embodiment, undifferentiated pluripotent cells such as hESCs can be cultured in media containing bFGF and TGFP. Non-limiting exemplary concentrations of bFGF include about 80 ng / ml. Non-limiting exemplary concentrations of TGFP include about 0.5 ng / ml.

[0050] In one embodiment, the undifferentiated pluripotent cells can be cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue (Thomson et al. (1998) Science 282:1145). The feeder cells can be derived from human or mouse sources, among others. Human feeder cells can be isolated from various human tissues or derived via differentiation of human embryonic stem cells into fibroblasts (see, e.g., WO 01 / 51616). In one embodiment, human feeder cells that may be used include, but are not limited to, placental fibroblasts (see, e.g., Genbacev et al. (2005) Fertil. Steril. 83(5):1517), fallopian tube epithelial cells (see, e.g., Richards et al. (2002) Nat. Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amit et al. (2003) Biol. Reprod. 68:2150), and endometrial cells (see, e.g., Lee et al. (2005) Biol. Reprod. 72(1):42).

[0051] A variety of solid surfaces can be used in the culture of undifferentiated pluripotent cells. These solid surfaces include, but are not limited to, standard commercially available cell culture plates, such as 6-well, 24-well, 96-well or 144-well plates. Other solid surfaces include, but are not limited to, microcarriers and disks. Solid surfaces suitable for growing undifferentiated pluripotent cells can be made of a variety of materials, including, but not limited to, glass or plastic, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, Melinex, Thermanox or combinations thereof. In one embodiment, a suitable surface can include one or more polymers, such as, for example, one or more acrylates. In one embodiment, the solid surface can be three-dimensional in shape. Non-limiting examples of three-dimensional solid surfaces are described, for example, in US Patent Application Publication No. 2005 / 0031598.

[0052] In one embodiment, the undifferentiated stem cells can be grown under feeder-free conditions on a growth substrate. In one embodiment, the growth substrate can be Matrigel® (e.g., Matrigel® or Matrigel® GFR), recombinant laminin, or vitronectin. In another embodiment, the undifferentiated stem cells can be passaged using various methods, such as using collagenase or by manual scraping. In another embodiment, the undifferentiated stem cells can be passaged using non-enzymatic means, such as using 0.5 mM EDTA in PBS, or by using ReLeSR™. In one embodiment, a plurality of undifferentiated stem cells are seeded or passaged at a seeding density that allows the cells to reach confluence in about 3 to about 10 days. In one embodiment, the seeding density is about 6.0×10 3 Cells / 1 cm of growth surface 2 ~Approx. 5.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 1.0 × 10 4 Cells / 1 cm of growth surface 2 , for example, about 5.0 × 10 4 Cells / 1 cm of growth surface 2 , for example, about 1.0 × 10 5 Cells / 1 cm of growth surface 2 , or for example about 3.0 x 10 5 Cells / 1 cm of growth surface 2 In another embodiment, the seeding density can range from about 6.0×10 3 Cells / 1 cm of growth surface 2 ~Approx. 1.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 6.0 × 10 3 Cells / 1 cm of growth surface 2 ~Approx. 9.0×10 3 Cells / 1 cm of growth surface 2 , for example, about 7.0 × 10 3 Cells / 1 cm of growth surface 2 ~Approx. 1.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 7.0 × 10 3 Cells / 1 cm of growth surface 2 ~Approx. 9.0×103 Cells / 1 cm of growth surface 2 , or for example about 7.0 x 10 3 Cells / 1 cm of growth surface 2 ~Approx. 8.0×10 3 Cells / 1 cm of growth surface 2 In yet another embodiment, the seeding density can range from about 1.0×10 4 Cells / 1 cm of growth surface 2 ~Approx. 1.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 2.0 × 10 4 Cells / 1 cm of growth surface 2 ~Approx. 9.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 3.0 × 10 4 Cells / 1 cm of growth surface 2 ~Approx. 8.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 4.0 × 10 4 Cells / 1 cm of growth surface 2 ~Approx. 7.0×10 4 Cells / 1 cm of growth surface 2 , or for example about 5.0 x 10 4 Cells / 1 cm of growth surface 2 ~Approx. 6.0×10 4 Cells / 1 cm of growth surface 2 In one embodiment, the seeding density can range from about 1.0×10 5 Cells / 1 cm of growth surface 2 ~Approx. 5.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 1.0 × 10 5 Cells / 1 cm of growth surface 2 ~Approx. 4.5×10 5 Cells / 1 cm of growth surface 2 , for example, about 1.5 × 10 5 Cells / 1 cm of growth surface 2 ~Approx. 4.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 2.0 × 10 5 Cells / 1 cm of growth surface 2 ~Approx. 3.5×10 5 Cells / 1 cm of growth surface2 , or for example about 2.5 x 10 5 Cells / 1 cm of growth surface 2 ~Approx. 3.0×10 5 Cells / 1 cm of growth surface 2 The range may be:

[0053] Any of a variety of suitable cell culture and subculture techniques can be used to culture cells according to the present disclosure. For example, in one embodiment, the culture medium can be changed at appropriate time intervals. In one embodiment, the culture medium can be changed completely every day, beginning about 2 days after subculture of the cells. In another embodiment, once the culture reaches about 90% colony coverage, a replacement flask can be sacrificed and counted using one or more suitable reagents, such as, for example, collagenase IV and 0.05% trypsin-EDTA, in sequence, to achieve a single cell suspension for quantification. In one embodiment, a plurality of undifferentiated stem cells can then be subcultured at a seeding density that allows the cells to reach confluence over an appropriate period of time, such as, for example, about 3 days to about 10 days, before seeding the cells on an appropriate growth substrate (e.g., Matrigel® GFR). In one embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and expanded on a recombinant laminin matrix. In one embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and expanded on a Matrigel® matrix. In one embodiment, ReLeSR™ can be used to subculture and expand undifferentiated stem cells on a Vitronectin matrix.

[0054] In one embodiment, the seeding density is about 6.0×10 3 Cells / 1 cm of growth surface 2 ~Approx. 5.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 1.0 × 10 4 Cells / 1 cm of growth surface 2 , for example, about 5.0 × 10 4 Cells / 1 cm of growth surface 2, for example, about 1.0 × 10 5 Cells / 1 cm of growth surface 2 , or for example about 3.0 x 10 5 Cells / 1 cm of growth surface 2 In another embodiment, the seeding density can range from about 6.0×10 3 Cells / 1 cm of growth surface 2 ~Approx. 1.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 6.0 × 10 3 Cells / 1 cm of growth surface 2 ~Approx. 9.0×10 3 Cells / 1 cm of growth surface 2 , for example, about 7.0 × 10 3 Cells / 1 cm of growth surface 2 ~Approx. 1.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 7.0 × 10 3 Cells / 1 cm of growth surface 2 ~Approx. 9.0×10 3 Cells / 1 cm of growth surface 2 , or for example about 7.0 x 10 3 Cells / 1 cm of growth surface 2 ~Approx. 8.0×10 3 Cells / 1 cm of growth surface 2 In yet another embodiment, the seeding density can range from about 1.0×10 4 Cells / 1 cm of growth surface 2 ~Approx. 1.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 2.0 × 10 4 Cells / 1 cm of growth surface 2 ~Approx. 9.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 3.0 × 10 4 Cells / 1 cm of growth surface 2 ~Approx. 8.0×10 4 Cells / 1 cm of growth surface 2 , for example, about 4.0 × 10 4 Cells / 1 cm of growth surface 2 ~Approx. 7.0×10 4 Cells / 1 cm of growth surface 2 , or for example about 5.0 x 104 Cells / 1 cm of growth surface 2 ~Approx. 6.0×10 4 Cells / 1 cm of growth surface 2 In one embodiment, the seeding density can range from about 1.0×10 5 Cells / 1 cm of growth surface 2 ~Approx. 5.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 1.0 × 10 5 Cells / 1 cm of growth surface 2 ~Approx. 4.5×10 5 Cells / 1 cm of growth surface 2 , for example, about 1.5 × 10 5 Cells / 1 cm of growth surface 2 ~Approx. 4.0×10 5 Cells / 1 cm of growth surface 2 , for example, about 2.0 × 10 5 Cells / 1 cm of growth surface 2 ~Approx. 3.5×10 5 Cells / 1 cm of growth surface 2 , or for example about 2.5 x 10 5 Cells / 1 cm of growth surface 2 ~Approx. 3.0×10 5 Cells / 1 cm of growth surface 2 The range may be:

[0055] Oligodendrocyte precursor cell composition As discussed above, the present disclosure provides compositions comprising a population of oligodendrocyte precursor cells (OPCs), and methods of making and using said compositions for use in treating acute spinal cord injury and other related CNS conditions. In some embodiments, the OPCs of the present disclosure can produce and secrete one or more biological factors that can enhance neural repair.

[0056] In one embodiment, the cell population can have a common genetic background. In one embodiment, the cell population can be derived from one host. In one embodiment, the cell population can be derived from a pluripotent stem cell line. In another embodiment, the cell population can be derived from an embryonic stem cell line. In one embodiment, the cell population can be derived from an hESC line. In one embodiment, the hESC line can be an H1, H7, H9, H13 or H14 cell line. In another embodiment, the cell population can be derived from an induced pluripotent stem cell (iPS) line. In one embodiment, the cell population can be derived from a subject in need thereof (e.g., the cell population can be derived from a subject in need of treatment). In yet another embodiment, the hESC line can be derived from a parthenogenetic cell, which is an embryo stimulated to produce hESC without fertilization.

[0057] In some embodiments, the OPCs of the disclosure express one or more markers selected from nestin, NG2, Olig1, and PDGF-Ra. In some embodiments, the OPCs of the disclosure express all of the markers nestin, NG2, Olig1, and PDGF-Ra.

[0058] In some embodiments, the OPCs of the present disclosure can secrete one or more biological factors. In some embodiments, the one or more biological factors secreted by the OPCs of the present disclosure can promote, but are not limited to, nerve repair, axonal outgrowth and / or glial differentiation, or any combination thereof. In some embodiments, the OPCs can secrete one or more factors that stimulate axonal outgrowth. In some embodiments, the OPCs can secrete one or more factors that promote glial differentiation by neural progenitor cells. In some embodiments, the OPCs can secrete one or more chemoattractants for neural progenitor cells. In some embodiments, the OPCs can secrete one or more inhibitors of matrix metalloproteinases. In some embodiments, the OPCs can secrete one or more factors that inhibit cell death after spinal cord injury. In some embodiments, the OPCs can secrete one or more factors that are upregulated after cell injury and aid in the clearance of misfolded proteins.

[0059] In certain embodiments, OPCs are capable of producing and secreting one or more biological factors selected from MCP-1, clusterin, ApoE, TIMP1 and TIMP2. In further embodiments, OPCs are capable of producing and secreting MCP-1 and one or more of the factors selected from clusterin, ApoE, TIMP1 and TIMP2. In yet further embodiments, OPCs are capable of producing and secreting all of the factors MCP-1, clusterin, ApoE, TIMP1 and TIMP2.

[0060] In one embodiment, the biological factor may be secreted by a composition comprising a population of OPCs at a concentration of more than about 50 pg / ml, such as more than about 100 pg / ml, such as more than about 200 pg / ml, such as more than about 300 pg / ml, such as more than about 400 pg / ml, such as more than about 500 pg / ml, such as more than about 1,000 pg / ml, such as more than about 2,000 pg / ml, such as more than about 3,000 pg / ml, such as more than about 4,000 pg / ml, such as more than about 5,000 pg / ml, such as more than about 6,000 pg / ml, or such as more than about 7,000 pg / ml.In one embodiment, the biological factor is about 50 pg / ml to about 100,000 pg / ml, for example, about 100 pg / ml, for example, about 150 pg / ml, for example, about 200 pg / ml, for example, about 250 pg / ml, for example, about 300 pg / ml, for example, about 350 pg / ml, for example, about 400 pg / ml, for example, about 450 pg / ml, for example, about 500 pg / ml, for example, about 550 pg / ml, for example, about 600 pg / ml, for example, about 650 pg / ml, for example, about 700 pg / ml, For example about 750pg / ml, such as about 800pg / ml, for example about 850pg / ml, such as about 900pg / ml, for example about 1,000pg / ml, such as about 1,500pg / ml, for example about 2,000pg / ml, such as about 2,500pg / ml, for example about 3,000pg / ml, such as about 3,500pg / ml, for example about 4,000pg / ml, such as about 4,500pg / ml, for example about 5,000pg / ml, such as about 5,500pg / ml, for example about 6,00 0pg / ml, for example about 6,500pg / ml, such as about 7,000pg / ml, for example about 7,500pg / ml, such as about 8,000pg / ml, for example about 8,500pg / ml, such as about 9,000pg / ml, for example about 10,000pg / ml, such as about 15,000pg / ml, for example about 20,000pg / ml, such as about 25,000pg / ml, for example about 30,000pg / ml, for example about 35,000pg / ml, such as about 40,000pg / ml, e.g. For example, the OPCs may be secreted by a composition comprising a population of cells comprising OPCs at a concentration in the range of about 45,000 pg / ml, for example about 50,000 pg / ml, such as about 55,000 pg / ml, for example about 60,000 pg / ml, for example about 65,000 pg / ml, such as about 70,000 pg / ml, for example about 75,000 pg / ml, such as about 80,000 pg / ml, for example about 85,000 pg / ml, such as about 90,000 pg / ml, for example about 95,000 pg / ml.

[0061] In one embodiment, the biological factor is about 1,000 pg / ml to about 10,000 pg / ml, for example, about 1,000 pg / ml to about 2,000 pg / ml, for example, about 2,000 pg / ml to about 3,000 pg / ml, for example, about 3,000 pg / ml to about 4,000 pg / ml, for example, about 4,000 pg / ml to about 5,000 pg / ml, for example, about 5,000 pg / ml In one embodiment, the OPCs may be secreted by the composition comprising the population of cells, at a concentration ranging from about 6,000 pg / ml to about 6,000 pg / ml, for example from about 6,000 pg / ml to about 7,000 pg / ml, for example from about 7,000 pg / ml to about 8,000 pg / ml, for example from about 8,000 pg / ml to about 9,000 pg / ml, or for example from about 9,000 pg / ml to about 10,000 pg / ml.

[0062] In one embodiment, the biological factor is about 10,000 pg / ml to about 100,000 pg / ml, for example, about 10,000 pg / ml to about 20,000 pg / ml, for example, about 20,000 pg / ml to about 30,000 pg / ml, for example, about 30,000 pg / ml to about 40,000 pg / ml, for example, about 40,000 pg / ml to about 50,000 pg / ml, for example, about 50,000 pg The OPCs may be secreted by the composition comprising the population of cells, at a concentration ranging from about 10,000 pg / ml to about 60,000 pg / ml, for example from about 60,000 pg / ml to about 70,000 pg / ml, for example from about 70,000 pg / ml to about 80,000 pg / ml, for example from about 80,000 pg / ml to about 90,000 pg / ml, or for example from about 90,000 pg / ml to about 100,000 pg / ml.

[0063] In some embodiments, clusterin may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 1,000 pg / ml to about 100,000 pg / ml. In some embodiments, clusterin may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 10,000 pg / ml to about 50,000 pg / ml. In some embodiments, MCP-1 may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 500 pg / ml to about 50,000 pg / ml. In some embodiments, MCP-1 may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 5,000 pg / ml to about 15,000 pg / ml. In some embodiments, ApoE may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In certain embodiments, ApoE may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 500 pg / ml to about 5,000 pg / ml. In some embodiments, TIMP1 may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In some embodiments, TIMP1 may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 500 pg / ml to about 5,000 pg / ml. In some embodiments, TIMP2 may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In some embodiments, TIMP2 may be secreted by a composition comprising a population of cells comprising OPCs at a concentration ranging from about 500 pg / ml to about 5,000 pg / ml.

[0064] Pharmaceutical Compositions The OPCs of the present disclosure can be administered to a subject in need of treatment, such as SCI treatment. Alternatively, the cells of the present disclosure can be administered to a subject in need of SCI treatment in a pharmaceutical composition with a suitable carrier and / or using a delivery system.

[0065] As used herein, the term "pharmaceutical composition" refers to a preparation containing one or more therapeutic agents in combination with other ingredients, such as physiologically suitable carriers and excipients.

[0066] As used herein, the term "therapeutic agent" can refer to the cells of the present disclosure that can account for a biological effect in a subject. Depending on the aspect of the present disclosure, the "therapeutic agent" can refer to the oligodendrocyte precursor cells of the present disclosure. Alternatively, the "therapeutic agent" can refer to one or more factors secreted by the oligodendrocyte precursor cells of the present disclosure.

[0067] As used herein, the terms "carrier", "pharmaceutical acceptable carrier" and "biologically acceptable carrier" may be used interchangeably and refer to a diluent or carrier substance that does not cause significant adverse effects or irritation in a subject and does not abolish the biological activity or effect of a therapeutic agent. In some embodiments, a pharmaceutical acceptable carrier can include dimethyl sulfoxide (DMSO). In other embodiments, a pharmaceutical acceptable carrier does not include dimethyl sulfoxide. The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a therapeutic agent.

[0068] One or more therapeutic agents of the present disclosure may be administered as a component of a hydrogel, such as those described in U.S. Patent Application No. 14 / 275,795, filed May 12, 2014, and U.S. Patent Nos. 8,324,184 and 7,928,069.

[0069] The composition according to the present disclosure can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservative.The composition can contain compounding agents, such as suspending agents, stabilizing agents and / or dispersing agents.In some embodiments, the composition can be formulated to be compatible with cryopreservation.

[0070] The composition according to the present disclosure may be formulated for administration via injection into the spinal cord of a subject. The composition may also be formulated for direct injection into the spinal cord of a subject. The composition may be formulated for administration via implantation or other delivery methods. In some embodiments, the composition according to the present disclosure may be formulated for intracerebral, intraventricular, intrathecal, intranasal or intracisternal administration to a subject. In some embodiments, the composition according to the present disclosure may be formulated for administration via injection directly into or directly adjacent to the infarct cavity in the brain of a subject. In some embodiments, the composition according to the present disclosure may be formulated for administration via implantation. In some embodiments, the composition according to the present disclosure may be formulated for administration via other suitable delivery methods. In some embodiments, the composition according to the present disclosure may be formulated as a solution.

[0071] In one embodiment, a composition according to the present disclosure comprises about 1×10 6 ~Approx. 5×10 8 Cells / ml, e.g., approximately 1 x 10 6 cells / ml, e.g., approximately 2 x 10 6 cells / ml, e.g., approximately 3 x 10 6 cells / ml, e.g., approximately 4 x 10 6 cells / ml, e.g., approximately 5 x 10 6 cells / ml, e.g., approximately 6 x 10 6 cells / ml, e.g., approximately 7 x 10 6 cells / ml, e.g., approximately 8 x 10 6 cells / ml, e.g., approximately 9 x 10 6 Cells / ml, e.g., approximately 1 x 10 7 cells / ml, e.g., approximately 2 x 10 7 cells / ml, e.g., approximately 3 x 10 7 cells / ml, e.g., approximately 4 x 10 7 cells / ml, e.g., approximately 5 x 10 7 cells / ml, e.g., approximately 6 x 10 7 cells / ml, e.g., approximately 7 x 10 7 cells / ml, e.g., approximately 8 x 10 7cells / ml, e.g., approximately 9 x 10 7 Cells / ml, e.g., approximately 1 x 10 8 cells / ml, e.g., approximately 2 x 10 8 cells / ml, e.g., approximately 3 x 10 8 cells / ml, e.g., approximately 4 x 10 8 cells / milliliter, or for example, approximately 5 x 10 8 In one embodiment, the composition according to the present disclosure can include about 1×10 cells / milliliter. 8 ~Approx. 5×10 8 Cells / ml, e.g., approximately 1 x 10 8 ~Approx. 4×10 8 cells / ml, e.g., approximately 2 x 10 8 ~Approx. 5×10 8 Cells / ml, e.g., approximately 1 x 10 8 ~Approx. 3×10 8 cells / ml, e.g., approximately 2 x 10 8 ~Approx. 4×10 8 cells / milliliter, or for example, approximately 3 x 10 8 ~Approx. 5×10 8 In yet another embodiment, the composition according to the present disclosure can comprise about 1×10 cells / milliliter. 7 ~Approx. 1×10 8 cells / ml, e.g., approximately 2 x 10 7 ~Approx. 9×10 7 cells / ml, e.g., approximately 3 x 10 7 ~Approx. 8×10 7 cells / ml, e.g., approximately 4 x 10 7 ~Approx. 7×10 7 cells / milliliter, or for example, approximately 5 x 10 7 ~about 6×10 7 In one embodiment, the composition according to the present disclosure can include about 1×10 cells / milliliter. 6 ~Approx. 1×10 7 cells / ml, e.g., approximately 2 x 10 6 ~Approx. 9×10 6 cells / ml, e.g., approximately 3 x 10 6 ~Approx. 8×10 6 cells / ml, e.g., approximately 4 x 106 ~Approx. 7×10 6 cells / milliliter, or for example, approximately 5 x 10 6 ~about 6×10 6 In yet another embodiment, the composition according to the present disclosure comprises at least about 1×10 cells / milliliter. 6 cells / milliliter, e.g., at least about 2 x 10 6 cells / ml, e.g., at least about 3 x 10 6 cells / ml, e.g., at least about 4 x 10 6 cells / milliliter, e.g., at least about 5 x 10 6 cells / milliliter, e.g., at least about 6 x 10 6 cells / milliliter, e.g., at least about 7 x 10 6 cells / milliliter, e.g., at least about 8 x 10 6 cells / milliliter, e.g., at least about 9 x 10 6 cells / milliliter, e.g., at least about 1 x 10 7 cells / milliliter, e.g., at least about 2 x 10 7 cells / ml, e.g., at least about 3 x 10 7 cells / ml, e.g., at least about 4 x 10 7 cells / milliliter or, for example, at least about 5 x 10 7 In one embodiment, the composition according to the present disclosure can include up to about 1×10 cells / milliliter. 8 More than 2 x 10 cells, e.g. 8 More than 3 x 10 cells / ml 8 More than 4 x 10 cells / ml 8 More than 5 x 10 cells / ml 8 cells / ml or more, or for example up to about 6 x 10 8 It may contain cells / milliliter.

[0072] In one embodiment, a composition according to the present disclosure comprises about 4×10 7 ~Approx. 2×10 8 It may contain cells / milliliter.

[0073] In yet another embodiment, the composition according to the present disclosure may have a volume in the range of about 10 microliters to about 5 milliliters, such as about 20 microliters, for example about 30 microliters, such as about 40 microliters, for example about 50 microliters, such as about 60 microliters, for example about 70 microliters, such as about 80 microliters, for example about 90 microliters, such as about 100 microliters, for example about 200 microliters, such as about 300 microliters, for example about 400 microliters, such as about 500 microliters, for example about 600 microliters, such as about 700 microliters, for example about 800 microliters, such as about 900 microliters, for example about 1 milliliter, such as about 1.5 milliliters, for example about 2 milliliters, such as about 2.5 milliliters, for example about 3 milliliters, such as about 3.5 milliliters, for example about 4 milliliters, or for example about 4.5 milliliters. In one embodiment, the composition according to the present disclosure can have a volume in the range of about 10 microliters to about 100 microliters, for example, about 20 microliters to about 90 microliters, for example, about 30 microliters to about 80 microliters, for example, about 40 microliters to about 70 microliters, or for example, about 50 microliters to about 60 microliters. In another embodiment, the composition according to the present disclosure can have a volume in the range of about 100 microliters to about 1 milliliter, for example, about 200 microliters to about 900 microliters, for example, about 300 microliters to about 800 microliters, for example, about 400 microliters to about 700 microliters, or for example, about 500 microliters to about 600 microliters. In yet another embodiment, the composition according to the present disclosure can have a volume in the range of about 1 milliliter to about 5 milliliters, for example, about 2 milliliters to about 5 milliliters, for example, about 1 milliliter to about 4 milliliters, for example, about 1 milliliter to about 3 milliliters, for example, about 2 milliliters to about 4 milliliters, or for example, about 3 milliliters to about 5 milliliters. In one embodiment, a composition according to the present disclosure can have a volume of about 20 microliters to about 500 microliters.In another embodiment, the composition according to the present disclosure can have a volume of about 50 microliters to about 100 microliters. In yet another embodiment, the composition according to the present disclosure can have a volume of about 50 microliters to about 200 microliters. In another embodiment, the composition according to the present disclosure can have a volume of about 20 microliters to about 400 microliters.

[0074] In some embodiments, the present disclosure provides a container that contains a composition that includes a population of OPCs induced according to one or more methods of the present disclosure.In some embodiments, the container can be configured for cryopreservation.In some embodiments, the container can be configured for administration to a subject in need thereof.In some embodiments, the container can be a pre-filled syringe.

[0075] For general principles in pharmaceutical formulations, the reader is referred to Allogeneic Stem Cell Transplantation, Lazarus and Laughlin Eds. Springer Science+Business Media LLC 2010; and Hematopoietic Stem Cell Therapy, ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The choice of cell excipient and any accompanying components of the composition is adapted according to the route and device used for administration. In some embodiments, the composition may also include or be accompanied by one or more other components that promote engraftment or functional mobilization of enriched target cells. Suitable components may include matrix proteins that support or promote adhesion of target cell types or promote vascularization of the implanted tissue.

[0076] Uses of the cells of the present disclosure In various embodiments described herein, the present disclosure provides a method of using a cell population comprising OPCs derived from pluripotent stem cells to improve one or more neurological functions in a subject in need of treatment. In one embodiment, a method is provided for using OPCs derived from pluripotent stem cells in the treatment of acute spinal cord injury. In another embodiment, a method is provided for using OPCs derived from pluripotent stem cells in the treatment of other traumatic CNS injuries. In another embodiment, a method is provided for using OPCs derived from pluripotent stem cells in the treatment of non-traumatic CNS disorders or conditions. In one embodiment, the cell population according to the present disclosure can be injected or implanted into a subject in need thereof.

[0077] In some embodiments, methods are provided for using pluripotent stem cell derived OPCs in the treatment of conditions requiring myelin repair or remyelination. The following are non-limiting examples of conditions, diseases and conditions requiring myelin repair or remyelination: multiple sclerosis, leukodystrophies, Guillain-Barre syndrome, Charcot-Marie-Tooth neuropathy, Tay-Sachs disease, Niemann-Pick disease, Gaucher disease and Hurler syndrome. Other conditions that result in demyelination include, but are not limited to, inflammation, stroke, immune disorders, metabolic disorders and nutritional deficiencies (such as vitamin B12 deficiency). The OPCs of the present disclosure can also be used for myelin repair or remyelination in traumatic injuries that result in loss of myelination, such as acute spinal cord injury.

[0078] OPCs are administered in a manner that allows OPCs to transplant or migrate to the intended tissue site and reconstitute or regenerate the functionally deficient area. Administration of cells can be achieved by any method known in the art. For example, cells can be administered directly surgically to the organ or tissue that requires cell transplantation. Alternatively, non-invasive procedures can be used to administer cells to subjects. Non-limiting examples of non-invasive delivery methods include the use of syringes and / or catheters to deliver cells to the organ or tissue that requires cell therapy.

[0079] Subjects receiving the OPCs of the present disclosure may be treated to reduce immune rejection of the transplanted cells. Contemplated methods include administration of traditional immunosuppressants such as, for example, tacrolimus, cyclosporine A (Dunn et al., Drugs 61:1957, 2001), or using matched populations of cells derived from pluripotent stem cells to induce immune tolerance (WO 02 / 44343; U.S. Patent No. 6,280,718; WO 03 / 050251). Alternatively, a combination of an anti-inflammatory drug (such as prednisone) and an immunosuppressant may be used. The OPCs of the present invention may be provided in the form of a pharmaceutical composition comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration.

[0080] Use in the treatment of CNS traumatic injury. In certain embodiments, the cell population according to the present disclosure is capable of engrafting at the site of spinal cord injury following implantation of a composition comprising the cell population at the site of spinal cord injury.

[0081] In some embodiments, the cell population according to the present disclosure can remain in the spinal cord injury site of a subject for about 180 days or more after implantation of a dose of the composition at the spinal cord injury site.In other embodiments, the cell population according to the present disclosure can remain in the spinal cord injury site of a subject for about 2 years or more after implantation of a dose of the composition at the spinal cord injury site.In further embodiments, the cell population according to the present disclosure can remain in the spinal cord injury site of a subject for about 3 years or more after implantation of a dose of the composition at the spinal cord injury site.In still further embodiments, the cell population according to the present disclosure can remain in the spinal cord injury site of a subject for about 4 years or more after implantation of a dose of the composition at the spinal cord injury site.

[0082] In some embodiments, the cell composition of the present disclosure can reduce spinal cord injury-induced parenchymal hollowing in a subject.In some embodiments, lesion volume is reduced by forming tissue matrix at spinal cord injury site.In some embodiments, the cell of the present disclosure can form tissue matrix at spinal cord injury site within about 180 days.In some embodiments, the subject in which injury-induced parenchymal hollowing is reduced is a human.

[0083] In some embodiments, the cell population according to the present disclosure can reduce the volume of injury-induced central nervous system parenchymal hollowing in a subject within about 12 months. In some embodiments, the cell population according to the present disclosure can reduce the volume of injury-induced central nervous system parenchymal hollowing in a subject within about 6 months, within about 5 months, or less than about 4 months. In some embodiments, the subject is a human.

[0084] In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the central nervous system of a subject in need thereof to one or more second locations. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a spinal cord of a subject to a diseased tissue in the brain of a subject. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the spinal cord of a subject to a second location in a diseased tissue in the spinal cord of a subject. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the brain of a subject to a second location in a diseased tissue in the brain of a subject. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the brain of a subject to a diseased tissue in the spinal cord of a subject. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the spinal cord of a subject to a second location in a diseased tissue in the spinal cord of a subject, and to one or more locations in one or more diseased tissues in the brain of a subject. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the brain of a subject to a second location in a diseased tissue in the brain of the subject, and to one or more locations in one or more diseased tissues in the spinal cord of the subject.

[0085] In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location to one or more second locations of one or more diseased tissues in the central nervous system of a subject in less than about 150 days, such as less than about 100 days, such as less than about 50 days, or such as less than about 10 days. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location to one or more second locations of one or more diseased tissues in the central nervous system of a subject in less than about 180 days. EXAMPLES

[0086] Examples 1-8 describe a first-in-human Phase 1 safety clinical trial of human pluripotent stem cell derived oligodendrocyte precursor cells (LCTOPC1) that have mechanistic properties to support survival and potential repair of critical cellular components and structures at the site of SCI. Example 9 describes a Phase 1 / 2a dose escalation study of human pluripotent stem cell derived oligodendrocyte precursor cells (AST-OPC1) for use in subacute cervical SCI.

[0087] Example 1 - Patients and Methods Study design: The study design was an open-label, multicenter study. A single dose of 2 × 10^6 LCTOPC1 was injected within 7 to 14 days after SCI. Subjects receiving LCTOPC1 also received tacrolimus to prevent rejection. Subjects were followed per protocol for 15 years after administration of LCTOPC1.

[0088] Study Participants. Male or female participants aged 18-65 years with acute traumatic spinal cord injury were eligible to participate in the study. Because this was a first-in-human study with a risk of neurological deterioration, inclusion was limited to neurologically complete injuries (American Spinal Cord Injury Association Impairment Scale A) with a single neurological impairment (NLI) between levels T3 and T10, and no preserved motor function (i.e., partial functional residual areas) below this single neurological level for less than 5 levels. These inclusion criteria were selected to minimize loss of function if neurological deterioration occurred.

[0089] Post-stabilization magnetic resonance imaging (MRI) was used to confirm the presence of a single spinal cord lesion, with sufficient visualization of the spinal cord for 30 mm above and below the site of injury to allow for safety monitoring after injection. Participants had to be eligible for an elective surgical procedure to inject LCTOPC1 7–14 days after SCI.

[0090] The study was a phase 1 multicenter, nonrandomized, single-arm, allocation interventional clinical trial. Participants were enrolled from one of seven centers in the United States. The study was registered (NCT01217008) and the primary endpoint was safety, measured by the frequency and severity of adverse events related to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or the concomitant immunosuppression administered. Secondary endpoints were neurological function, measured by sensory scores and lower limb motor scores on the ISNCSCI examination. Eligibility criteria are summarized in Supplementary Table 1. Participants have been followed by protocol for in-person visits for a total of 5 years and annual telephone visits for an additional 10 years. Figure 1 provides the overall study scheme of the clinical trial.

[0091] The LCTOPC1 product is a cell population containing a mixture of oligodendrocyte precursor cells and other characterized cell types obtained after directed differentiation of undifferentiated human embryonic stem cells. The initial characterization of the LCTOPC1 population was reported by Nistor et al. 2005, who showed that these cells could differentiate into oligodendrocyte precursor cells. Subsequent studies demonstrated that oligodendrocyte precursor cells survived after delivery to the site of spinal cord injury in an acute incomplete rat contusion injury model. The cells showed evidence of remyelination of denuded axons, resulting in tissue sparing at the contusion site. When delivered during the acute injury period, the cells resulted in improved locomotor function as measured by standardized behavioral tests. Preclinical studies in rats and mice have demonstrated that the intended clinical, cryopreserved, human equivalent dose formulation of LCTOPC1 can survive and migrate after injection into the SCI site, produce neurotrophic factors to support cell survival, provide remyelination capacity to support denuded axons, and result in tissue sparing at the SCI contusion site. Additionally, studies have demonstrated that the cells do not generate teratomas or result in increased pain in injured animals.

[0092] This Phase 1 clinical trial was monitored by the FDA, the Institutional Data and Safety Monitoring Board (DSMB), the SCI clinical organization, the Surgery and Outcomes Steering Committee, the internal and external ethics committees, the internal and clinical trial site stem cell research oversight committees, and the IRBs of each participating clinical trial site. As a first-in-human study, the study design considered the need to minimize risk to participants, so individuals with complete SCI located between high thoracic neurological sites T3-T11 were selected for the intervention. This was an open-label, open-label, non-randomized, non-placebo-controlled study to establish the safety of intraparenchymal injections of LCTPOC1 and to determine changes in neurological function.

[0093] Determining the long-term safety of stem cell therapeutics is a crucial step in allowing future trials to investigate novel stem cell therapeutics or combination therapies. Ten years after implantation, there have been no medical or neurological complications indicating that implantation of LCTOPC1 is unsafe. Specifically, there have been no serious adverse events (SAEs) related to the procedure, cell implantation or immunosuppression. Additionally, there have been no significant changes in neurological function. Safety data from this first-in-human study supported progression to clinical trials for individuals with cervical spinal cord injuries.

[0094] The starting material for the generation of AST-OPC1 is the H1 master cell bank generated from the H1 uhESC line obtained at the University of Wisconsin in 1998. Compositional analysis of LCTOPC1 by immunocytochemistry and flow cytometry indicates that the cell population is composed primarily of neural lineage cells of oligodendrocyte precursor phenotype. In this safety study, the intended route of administration for LCTOPC1 was direct injection of 2x10^6 live LCTOPC1 cells into the spinal cord at a level 5mm caudal to the site of injury.

[0095] The rationale for selecting this dose was based on preclinical studies in rats and mice, as well as dose extrapolation to humans using two methods: (1) comparing the relative size of human and rat spinal cords and (2) assessing tumorigenicity in terms of absolute number of cells injected. Additionally, the rationale for selecting this delivery route was based on results from nonclinical pharmacology studies that showed that LCTOPC1 has properties that support the repair of pathology in spinal cord lesions. Results from these studies also demonstrated that improved locomotor recovery was accompanied by robust LCTOPC1 cell survival at the lesion site.

[0096] Spinal cord injuries located between T3 and T11 neurological levels, as assessed by the ISNCSCI, were selected as the target of intervention. The primary objective of this first-in-human study was to establish the safety of intraparenchymal injection of hESC-derived oligodendrocyte precursor cells into the spinal cord of individuals 7-14 days after injury. Secondary endpoints in this trial were ISNCSCI examinations that allowed for the identification of motor and sensory changes in any of 13 scheduled in-person evaluations within the first 5 years after injection.

[0097] Ten years after implantation, there have been no medical or neurological complications indicating that cell implantation is unsafe. Specifically, there have been no serious adverse events (SAEs) related to the procedure, cell implantation, or immunosuppression. This report summarizes the first 10 years of data from this important clinical trial, including early postoperative events, in-person follow-up out to 5 years, and concludes with data from telephone follow-up to date.

[0098] Investigational Product, Dose Formulation, Dose, and Mode of Administration. LCTOPC1 is a cell population containing a mixture of oligodendrocyte precursor cells and other characterized cell types, derived following differentiation of undifferentiated human embryonic stem cells (hESCs) from the H1 stem cell line, created at the University of Wisconsin in 1998.

[0099] Compositional analysis of LCTOPC1 by immunocytochemistry and flow cytometry indicates that the cell population is composed primarily of neural lineage cells of oligodendrocyte precursor phenotype. In this safety study, the intended route of administration for LCTOPC1 was direct injection of 2x10^6 live LCTOPC1 cells into the spinal cord at a level 5mm caudal to the site of injury.

[0100] LCTOPC1 is a cryopreserved cell therapy product. At the time of cryopreservation, each vial contains 7.5 x 10 cells in 1.2 mL of cryoprotectant solution. 6 Each LCTOPC1 sample contained viable cells. LCTOPC1 was supplied in 2.0 mL cryovials, transported in the vapor phase of liquid nitrogen to the clinical site, and stored under the same conditions at the site. Prior to administration, LCTOPC1 was thawed and prepared by qualified study personnel trained in the preparation of study drugs.

[0101] Participants received 2 × 10 6 Patients received a single dose of 2 × 10 live LCTOPC1 cells, total volume per dose = 50 μL. The rationale for selecting this dose was based on preclinical studies in rats and mice, and dose extrapolation to humans using two methods: (1) comparing the relative size of human and rat spinal cords, and (2) assessing the risk of tumor formation in terms of the absolute number of cells injected. At that point in the development of LCTOPC1, 6 This was the highest dose that could be administered into the injured rat spinal cord, and rats were the largest animals available to meet the number of animals required for IND feasibility studies for this novel product. Therefore, to be conservative, we chose 2 × 10 cells, the highest dose tested in rats. 6 cells were used as the dose for the Phase 1 trial. To prevent rejection of this allogeneic cell-based product, participants who received LCTOPC1 were also given tacrolimus.

[0102] The intended route of administration for LCTOPC1 was intraparenchymal at a depth of 6 mm, in the midline, and 5 mm caudal to the site of injury as determined by MRI, as modeled in preclinical studies. Caudal injection was chosen as a precaution to avoid the possibility of direct tissue damage above the level of injury. Based on preclinical studies, injected cells were expected to migrate rostrally throughout the injury site. LCTOPC1 was administered into the spinal cord in a dedicated surgical procedure 7–14 days after injury. This time frame was chosen based on results from animal studies suggesting poor graft survival for implantation within the first 7 days of injury, while at the same time attempting to maximize potential neuroprotective and remyelination effects. A custom-designed syringe positioning device was utilized to assist the neurosurgeon in the controlled delivery of cells.

[0103] Tacrolimus immunosuppression. Immunosuppression with tacrolimus was initiated 6–12 hours after injection of LCTOPC1. If participants were unable to take oral medication, tacrolimus was administered intravenously at a starting dose of 0.01 mg / kg / day and given as a continuous intravenous infusion. Participants were switched to oral tacrolimus whenever possible. The starting dose of oral tacrolimus was 0.03 mg / kg / day, divided into two daily doses. Tacrolimus doses were adjusted to achieve target whole blood trough levels of 3–7 ng / mL.

[0104] On day 46, the tacrolimus dose was reduced by 50% (rounded to the nearest 0.5 mg, as this was the smallest capsule size available). On day 53, the tacrolimus dose was reduced by another 50% (rounded to the nearest 0.5 mg). If the rounded total daily dose was 0.5 mg or less, participants received 0.5 mg once per day until tacrolimus was discontinued. Tacrolimus was discontinued on day 60. The tacrolimus dose was reduced if trough blood levels were >7 ng / mL. In addition, all ISNCSCI examination forms were reviewed by an expert to assess whether there were any changes in neurological function that could be related to the tapering and / or discontinuation of tacrolimus.

[0105] Follow-up and evaluation. An overview of study visits for the 1-year protocol follow-up (CP35A007) and the 2-15-year protocol follow-up (CP35A008) is provided in the study schema (Figure 1). Because this was the first clinical trial of hESC-derived cells, numerous study visits and long-term follow-up were necessary. For the 1-year protocol, three study visits were required before product administration and 13 in the first year after study administration. For the long-term protocol, annual visits were required in years 2-5. After the annual visit in year 5, follow-up was performed by telephone questioning (Figure 4) and in-person evaluation, as appropriate. Telephone evaluation included documentation of all new medications taken for longer than 30 days, hospitalizations, and documentation of AEs and SAEs.

[0106] Safety Evaluation. The primary endpoint of the Phase 1 clinical trial was safety as measured by the frequency and severity of adverse events (AEs) within 1 year of LCTOPC1 injections related to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or the concomitant immunosuppression administered. Safety evaluations included physical examination, vital signs, ISNCSCI neurological examination, pain questionnaire, electrocardiogram, MRI, laboratory tests for hematology and blood chemistry, laboratory tests for immunosuppression safety monitoring (whole blood trough levels of tacrolimus, serum levels of creatinine, potassium, magnesium, phosphate, ionized calcium, aspartate aminotransferase, alanine aminotransferase, and total bilirubin), concomitant medications, and AEs.

[0107] Definition of Adverse Events. AEs were listed by system organ class and by preferred term within the system organ class according to the Medical Dictionary for Regulatory Activities (MedDRA®) Version 10. An AE was any untoward medical occurrence in a study participant after the participant signed the informed consent form through the participant's last study visit, regardless of whether the event was considered drug-related. Standard FDA criteria were used to assess the severity of AEs and characterization of serious adverse events (SAEs).

[0108] The relationship of an AE to the study drug was determined by the investigator at each site and classified as "possibly related" based on the following criteria: (1) the AE was reasonably related in time to LCTOPC1 exposure, the injection procedure used to administer LCTOPC1, and / or the concomitant immunosuppression administered, and (2) the AE could be equally well explained by exposure to the study product or by factors or causes other than exposure to the study product. Adverse events were monitored by the external medical monitor, the sponsor medical monitor, and the DSMB.

[0109] Neurological assessment. The secondary outcome was neurological function, including measurement of sensory and lower extremity motor scores. Neurological function was assessed using the ISNCSCI test for motor and sensory testing and for the designation of the American Spinal Cord Injury Association (ASIA) Impairment Scale (AIS).

[0110] Exploratory endpoints. Pain assessment was performed using the International Spinal Cord Injury Pain Basic Data Set. A series of three questions was added to assess allodynia. These questions encompassed the presence and severity of pain elicited or increased by brushing, pressure, or contact with cold. Information on analgesics was collected as part of the assessment of concomitant medications. Potential exploratory endpoints regarding recovery of neurological function were the University of Alabama at Birmingham Index of Motor Recovery (UAB-IMR), Spinal Cord Independence Measure (SCIM), and assessment of bowel and bladder function.

[0111] Lumbar puncture. After receiving general anesthesia, but before LCTOPC1 injection and on day 60 after injection, a lumbar puncture was performed to obtain 10 mL of cerebrospinal fluid (CSF). The volume required by the individual study site was sent to the hospital laboratory for the following tests: white blood cell count, glucose, total protein, oligoclonal banding, myelin basic protein and immunoglobulin G index. Additionally, CSF was evaluated by the sponsor to evaluate the immune response to LCTOPC1.

[0112] Magnetic resonance imaging. Screening / baseline MRIs were obtained 3–5 days (days −3 to −5) prior to injection of LCTOPC1, but no later than 4 days after SCI. Screening / baseline MRIs included the entire brain, cerebellum, and spinal cord, with and without contrast (gadolinium diethylenetriaminepentaacetic acid [Gd-DTPA]). Repeat MRIs of the thorax without contrast were subsequently obtained if surgery for LCTOPC1 injection was delayed beyond 3 days. Follow-up MRIs of the spinal cord and cerebellum, with and without contrast (Gd-DTPA), were obtained at days 7, 60, 120, and 270 after injection. MRIs of the entire central nervous system, with and without contrast (Gd-DTPA), were obtained at days 30, 90, 180, and 365, and annually from years 2 to 5. Image acquisition protocols were standardized. Image review was centralized and standardized by an independent radiologist, DD, at Radiology Imaging Associates Denver.

[0113] HLA typing and immunological monitoring. LCTOPC1 cells do not express human leukocyte antigen (HLA) class II and are resistant to NK cell lysis. However, one concern regarding the safety and potential efficacy of LCTOPC1 was the possibility of allogeneic rejection by the host's immune system. Immunosuppression was minimized for periods up to 60 days and administered below typical long-term maintenance therapy levels used for solid organ transplantation. Peripheral blood and cerebrospinal fluid (CSF) samples from participants injected with LSTOPC1 were collected according to protocol. To evaluate for allogeneic cell rejection and immunological monitoring, lumbar punctures to obtain 10 mL of CSF were performed after undergoing general anesthesia, but before LCTOPC1 injection and on day 60 after injection. The following assessments were performed in the hospital laboratory: white blood cell count, glucose, total protein, oligoclonal banding, myelin basic protein and immunoglobulin G index. Peripheral blood was examined for the presence of antibodies specific for donor-specific HLA molecules on LCTOPC1 and to detect T cell-mediated responses to LCTOPC1. Additionally, CSF was evaluated by the sponsor to further evaluate immune responses to LCTOPC1 and the presence of LCTOPC1 (day 60) using a PCR-based assay.

[0114] Statistical methods: Descriptive analysis was used due to the small sample size, open-label and non-randomized study design. The primary and secondary endpoints of the study will be presented descriptively in tables, figures and text format.

[0115] result Study participants. The first participant was implanted in the winter of 2010, and the last participant was enrolled in the winter of 2011. Eleven individuals with SCI were screened for enrollment, and six were ineligible at screening: four because of MRI artifacts that prevented adequate spinal cord visualization, one based on ISNCSCI testing (NLI T1), and one because of elevated liver enzymes. A total of five individuals with SCI underwent LCTOPC1 at three study sites. Figure 2 provides a Consolidated Standards for Reporting Clinical Trials (CONSORT) flow diagram. In this study, the most common mechanism of injury was motor vehicle related for four of the five participants, and a fall was the cause of injury in one. Four of the five participants enrolled were male. Cohort age ranged from 21 to 32 years (Table 1).

[0116] TIFF2024505051000002.tif40149Abbreviations: BMI = body mass index. kg = kilograms. m = meters. Table 1. Demographic and baseline disease characteristics - all treated subjects

[0117] Participant Follow-up. At the time of this publication, all participants are in their 10th year of follow-up. In accordance with the FDA, the trial was structured to begin with an in-person assessment at 5 years, followed by telephone interviews from years 6 through 15. During the first 5 years of the study, 24 of the 25 in-person annual visits were completed. One participant did not attend the year 5 in-person visit but participated in the scheduled telephone follow-up. From year 6 to present, 21 of the 21 annual telephone interviews have been completed. One participant has completed 10 years of follow-up, and four participants have participated in the 10-year follow-up interview.

[0118] Primary endpoint: Evaluation of safety. All SAEs and AEs (related and unrelated to treatment, cell implantation, or immunosuppression) are summarized in Table 2 and described below.

[0119] (Table 2) TIFF2024505051000003.tif112128N=number of subjects in the safety analysis population or number of subjects with each event category;n=number of subjects in each category;%=n*100 / N

[0120] Serious adverse events related to the procedure, cell implant, or immunosuppression. There were no SAEs related to the procedure, cell implant, or immunosuppression. MRI scans of the entire central nervous system had no findings of clinical concern up to 5 years after injection in any participant. During long-term telephone follow-up, participants denied having fever of unknown origin, had no changes in sensation in the chest, arms, or legs (other than as described below), and no participant had been diagnosed with any type of cancer. No participants died during the study. Safety events were monitored by the DSMB and no discontinuation rules were invoked.

[0121] Serious adverse events not related to the procedure, cell implant, or immunosuppression. Three participants reported four SAEs not related to the procedure, cell implant, or immunosuppression. These SAEs included a urinary tract infection (UTI) and subsequent transient autonomic dysreflexia in one individual, and pyelonephritis and mood disorders in two different individuals.

[0122] Adverse events classified by grade. During the course of the study, 25 AEs were judged by the investigators to be possibly related to LCTOPC1 (grade 1 / mild [n=9], grade 2 / moderate [n=15] and grade 3 / severe [n=1]). A grade 3 AE was described as a burning sensation in the trunk and lower extremities that was first reported at day 57 post-injection with grade 1 severity and increased to grade 3 severity at day 90 post-injection. This neuropathic pain resulted in three further grade 2 severity AEs and was ongoing through the 9th year of follow-up. Grade 2 AEs included surgical site pain, hypomagnesemia, urinary tract infection, vaginal yeast infection, vomiting, upper back pain, shoulder pain, pain with range of motion, and autonomic discomfort during catheter placement that was relieved after treatment with lidocaine. Grade 1 AEs included hypomagnesemia, urinary tract infection, fever, headache, nausea and spasticity.

[0123] Adverse events categorized by association with procedure, cell implantation, or immunosuppression. Nine of the 25 associated adverse events were considered probably specifically related to the injection procedure. Eight of the nine were grade 1 or 2 in severity, and one was grade 3. AEs were primarily transient postoperative pain, with one case of fever and one case of urinary tract infection. There were no AEs attributable to the cell implantation. Additionally, the immunosuppressive regimen was well tolerated, with all five participants completing the regimen according to protocol. Sixteen of the 25 adverse events were considered probably specifically related to immunosuppression. Seven grade 1 AEs and nine grade 2 AEs were judged probably specifically related to tacrolimus. These AEs were primarily known common adverse reactions to tacrolimus (nausea / vomiting, low magnesium levels, infections). Of the reported infections, one of seven was a vaginal yeast infection and six of seven infections were urinary tract, which are common complications of SCI.

[0124] Adverse events not related to procedure, cell implantation, or immunosuppression. At year 6, one participant reported increased frequency and intensity of muscle spasms due to functional electrical stimulation (FES) cycling. This participant reported resolution of these symptoms between years 7 and 9 and is currently not using any medications for muscle spasms. At year 9, another individual underwent outpatient testing after developing a deep vein thrombosis (DVT).

[0125] Secondary Outcome: Neurological Assessment. After discharge from acute inpatient rehabilitation, participants continued to be assessed in person throughout the first 5 years after implantation according to the schedule shown in Figure 1. Of note, between baseline and year 5, participants' annual in-person assessments included at least 13 ISNCSCI exams. All participants had an ASIA Impairment Score (AIS) grade of A at the time of enrollment in the study, and all participants maintained the same impairment grade. The highest single NLI and the lowest NLI registered in the study were T3 and T8, respectively. Only individuals with T3 NLI improved to T4, and a sensory ZPP of T4 initially on both sides was noted to improve to T5 on the left and T6 on the right at the 1-year follow-up. In total, 3 of 5 participants experienced an improvement of at least 1 level of ZPP. All participants began and ended the 5-year in-person ISNCSCI examination with intact upper limb motor function, with an upper limb motor score (UEMS) of 50 out of 50 and a lower limb motor score (LEMS) of 0 out of 50 (Table 3). Over the course of the 5-year in-person follow-up, sensory testing did not change substantially. Figure 3 provides a schematic of each patient's motor and sensory function at baseline and 5 years after LCTOPC1 administration.

[0126] Table 3: ISNCSCI baseline, 1 year after transplantation, and 5 years after transplantation TIFF2024505051000004.tif77151TSS=Total Sensory Score; *=Participant was unable to comply Table 3 presents the total sensory score (TSS), upper limb motor score (UEMS), lower limb motor score (LEMS), sensory neurological impairment level (NLI), motor NLI, sensory partial function residual area (ZPP), motor ZPP and ASIA impairment score (AIS) grades at baseline, 1 year and 5 years. All five individuals were AIS grade A at enrollment and there was no conversion to AIS B. The highest single NLI and the lowest NLI registered in the study were T3 and T8, respectively. Only the individual with T3 NLI improved to T4 and a sensory ZPP of T4 initially on both sides was noted to improve to T5 on the left and T6 on the right at 1 year follow-up. In total, three of the five participants experienced an improvement of at least one level of ZPP. ND = not determinable.

[0127] MRI findings. Participants did not have evidence of enlarging cysts, enlarging masses, spinal cord injury related to the injection procedure, intramedullary hemorrhage, CSF leaks, epidural abscesses or infections, inflammatory lesions in the spinal cord, obstruction of CSF flow, or masses in the ventricular system. No adverse neurologic changes or evidence of adverse changes on MRI were reported during tapering or after tacrolimus discontinuation.

[0128] Immune monitoring. LSTOPC1 is an allogeneic cell therapy and is potentially susceptible to rejection by the recipient immune system. As a baseline evaluation, HLA class I and class II molecular typing was performed for 10 alleles of donor LCTOPC1 cells and peripheral blood cells of each of the five recipients. The potential development of a cellular immune response against LCTOPC1 was evaluated and none of the serum samples from the five recipients showed evidence of a T cell-mediated response against LCTOPC1, even after cessation of tacrolimus immunosuppression. Furthermore, CSF samples obtained by lumbar puncture showed no signs of antibody or T cell responses against LCTOPC1.

[0129] Consideration In January 2009, Nature reported that LCTOPC1 was entering "the world's first clinical trial of a therapy generated by human embryonic stem cells." At the time, pharmaceutical research in acute SCI was considered a relatively recent development. Although clinical trials of hESC-derived cell lines had never been evaluated in any context before, other procedures for intraparenchymal injection of cell products (e.g., activated autologous macrophages) into the spinal cord have been evaluated, providing a partial roadmap for research based on LCTOPC1.

[0130] We found that 2 × 10 6 We present the primary and secondary endpoints of the five participants who received allogeneic hESC-derived oligodendrocyte precursor cells. Primary results from the first 10 years of follow-up establish the safety and feasibility of intraparenchymal LCTOPC1 injections. The injection procedure and low-dose immunosuppressive regimen were well tolerated. To date, all five participants who received LCTOPC1 show no evidence of neurological deterioration or adverse findings on MRI scans.

[0131] Although the study was not designed to evaluate efficacy, animal studies of LCTOPC1 resulted in improved motor function through remyelination and mechanisms that appeared to represent neuroprotection, suppression of inflammation, promotion of axonal regeneration, and / or homeostasis. Proposed mechanisms of locomotor function improvement included remyelination and expression of neurotrophic factors. Despite promising results in animals, limited signs of functional recovery in various human trials may be related to the relative severity of human injury compared with preclinical studies using incomplete contusion injuries, suggesting that subsequent trials using incomplete injuries may demonstrate recovery more similar to that seen in animal models. Although the study did not demonstrate significant recovery, no participants showed evidence of neurological deterioration on the ISNCSCI examination throughout 5 years of in-person follow-up or 10 years of self-reported neurological function. ISNCSCI total motor and total sensory scores remained stable over time. No unexpected SAEs related to LCTOPC1 were reported at 98% follow-up visits (45 of 46 annual visits) for participants throughout the first 10 years of the clinical trial.

[0132] Neuropathic pain in response to LCTOPC1 after remyelination or neurotrophic factors was assessed using the International Spinal Cord Injury Pain Basic Data Set and a series of three questions to assess allodynia. Neuropathic above-injury pain and below-injury pain often develop during the first few months after SCI, and by 1 year the prevalence of neuropathic pain approaches 60%. The prevalence of pain in this study is consistent with the natural history of neuropathic pain. One participant experienced neuropathic pain reported as a burning sensation in the trunk and lower extremities, possibly related to LCTOPC1, that persisted at long-term follow-up. The pain reported by this participant is consistent with two of the major categories of pain that are common after SCI: neuropathic pain at the level of injury (termed above-injury pain) and neuropathic pain that occurs diffusely below the level of injury (termed below-injury pain). Unfortunately, despite attempts at pain management, in affected individuals, both supra- and sub-injury neuropathic pain is often severe and persistent for at least 5 years after SCI. Furthermore, 40-50% of individuals with these types of pain report their pain as severe or unbearable. This small, open-label study does not allow us to determine causal relationships between LCTOPC1 and changes in the incidence of long-term neuropathic pain.

[0133] Serial MRI studies did not demonstrate the formation of ectopic tissue and / or teratomas. In addition to the absence of space-occupying lesions, the natural history of chronic SCI MRI studies suggest that cavitary lesions were identifiable in 58% of individuals undergoing thoracic level cell testing. MRI results during the long-term follow-up period for LCTOPC1 were particularly significant, as 80% of individuals showed T2 signal changes consistent with the formation of a tissue matrix at the injury site. Although the specimen size is limited, these findings suggest that LCTOPC1 cells may have either persistent engraftment and / or induced long-term changes that limited cavitation at the injury site. SCI is a relatively rare condition, with the potential population of AIS A injuries at T3–T11 representing less than 20% of acute SCIs in the United States (NSCISC National Spinal Cord Injury Statistical Center, University of Alabama at Birmingham, 2011 Annual Statistical Report-Complete Public Version).

[0134] conclusion The LCTOPC1 thoracic SCI clinical trial is one of the longest running clinical trials in the hESC field. This study provides important first-in-human positive safety data for future hESC-derived therapies. Although we cannot exclude the possibility of future adverse events, the experience in this study provides evidence that these treatments can be well tolerated and event-free for up to 10 years. In addition, this report supports participants' willingness to participate in long-term follow-up and sets a benchmark for corporate sponsors to commit to data collection beyond their immediate financial interests. Based on the safety profile of LCTOPC1 obtained in this study, a cervical dose escalation study has been initiated.

[0135] Stem Cell Materials and Methods The following examples provide exemplary methods for obtaining cells that can be used in the methods and uses described herein. Further methods, materials and uses can be found, for example, in International Publication No. WO2020 / 154533, the entire contents of which are incorporated herein by reference.

[0136] Example 2 - Culture and expansion of undifferentiated human embryonic stem cells. Undifferentiated human embryonic stem cells (uhESCs) from a working cell bank (WCB) generated from the H1 line (WA01; Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7) were cultured in complete mTeSR™-1 medium (Stem Cell Technologies #85850) on tissue culture treated polystyrene T-75 culture flasks (Corning #431082) coated with recombinant human laminin 521 (rhLn-521, Corning #354224). The medium was changed completely every day until the cells reached approximately 80-90% confluency, and then uhESCs were passaged using ReLeSR™ reagent (Stem Cell Technologies #05872). 2 flasks were seeded with ReLeSR™ dissociated uhESC cells and daily medium changes were resumed 2 days after seeding. Cultured uhESCs from WCB were expanded in this manner for 2-5 passages, depending on the experiment, prior to differentiation into neuroectodermal progenitor cells as described in Example 3.

[0137] Example 3 - Methods for differentiating human embryonic stem cells into neuroectodermal progenitor cells with a dorsal spinal cord progenitor phenotype. Expanded uhESCs were seeded onto rhLn-521-coated vessels and cultured until they reached 40–70% confluency, at which point differentiation was initiated. Days 0-3: Differentiation was initiated by complete removal of mTeSR™-1 medium and addition of glial progenitor medium (GPM; consisting of DMEM / F12 (Gibco catalog number 10565-018) supplemented with 2% B27 supplement (Gibco catalog number 17504-044) and 0.04 μg / mL tri-iodo-thyronine (Sigma-Aldrich catalog number T5516-1MG)) supplemented with 10 μM MAPK / ERK inhibitor, PD0325901 (PD; Sigma-Aldrich catalog number PZ0162), 2 μM BMP signaling inhibitor, dorsomorphin (Dorso; Sigma-Aldrich catalog number P5499) and 1 μM retinoic acid (RA; Sigma-Aldrich catalog number R2625). This medium was replenished daily. Days 4-6: On day 4, culture medium was switched to GPM supplemented with 1 μM RA and 150 μM ascorbic acid (Sigma-Aldrich Catalog No. A4544) and replenished daily. Day 7: On day 7, cells were harvested for expansion and further differentiation into glial progenitor cells as described in Example 4. A portion of cells was collected for analysis by quantitative PCR (qPCR; as described in Example 7), flow cytometry (as described in Example 6), and, if available, a separate well plate reserved for analysis was prepared for immunocytochemistry (ICC) (as described in Example 6). On day 7, these cells showed marker expression consistent with dorsal spinal cord progenitor cells (Table 4).

[0138] (Table 4) qPCR analysis of pluripotent and neuroectodermal progenitor cell (NPC) gene markers in H1 uhESCs differentiated into NPCs using different combinations of small molecule inhibitors. TIFF2024505051000005.tif84148

[0139] Example 4 - Methods for differentiating human embryonic stem cells into glial lineage cells. Days 7-13: Differentiation of uhESCs into neuroectodermal progenitor cells, specifically neuroectodermal progenitor cells of dorsal phenotype, was performed as described in Example 3. On day 7, cells were dissociated using TrypLE™ Select (Thermo Fisher, Catalog No. A12859-01), counted, and cultured at 2.7×10 in GPM supplemented with 20 ng / mL human basic fibroblast growth factor (hbFGF, Thermo Fisher, Catalog No. PHG0263), 10 ng / mL epidermal growth factor (EGF, Thermo Fisher, Catalog No. PHG0311), and 10 μM Rho kinase inhibitor (RI, Tocris Catalog No. 1254). 4 cells / cm 2 Cells were seeded onto rhLn-521-coated vessels at a seeding density of 1.83 × 10 cells / mL. Culture medium was replenished daily by aspirating spent medium and replacing the spent medium with fresh GPM+hbFGF+EGF. Days 14-21: On day 14, cells were dissociated using TrypLE™ Select, counted, resuspended in GPM+hbFGF+EGF+RI, and plated at 1.83 × 10 cells / mL in either PBS-0.1L or PBS-0.5L Mini Bioreactor Systems (PBS Biotech). 6 The cells were reseeded in dynamic suspension culture at a density of 10 ...

[0140] Example 5 - Methods for differentiating human embryonic stem cells into oligodendrocyte precursor cells. Days 21-42: The glial-restricted progenitor cells obtained in Example 4 were further differentiated into oligodendrocyte precursor cells (OPCs). The differentiation protocol for days 0-20 was carried out as described in Examples 3 and 4. On day 21, the aggregates were transferred from dynamic suspension to a culture vessel coated with rhLn-521. For example, starting with a 1x PBS-0.1L Mini Bioreactor with a total volume of 60 mL, 60 mL of culture was split onto two T75 flasks with a capacity of 30 mL each. Thereafter, the cells were fed every other day with GPM supplemented with 20 ng / mL EGF and 10 ng / mL platelet-derived growth factor AA (PDGFAA; PeproTech, Cat. No. AF-100-13A). Every 7 days (i.e., days 28 and 35), the cells were dissociated with TrypLE™ Select, counted, and cultured at 4 × 10 4 live cells / cm 2 The cells were reseeded onto new rhLn-521-coated culture vessels at a seeding density of 1000 μg / ml. Differentiated cells were harvested on day 42. Cells were detached from the vessels using TrypLE™ Select, counted, and reconstituted in CryoStor10 (BioLife Solutions, Catalog No. 210102) prior to cryopreservation. A portion of the cells was collected for analysis by flow cytometry (Example 6), ICC (Example 6), and qPCR (Example 7). By day 42, differentiated cells expressed markers characteristic of OPCs as measured by the three analytical methods (Table 4).

[0141] Example 6 - Characterization of differentiated cell populations by immunocytochemistry and flow cytometry. Flow cytometry and immunocytochemistry (ICC) can be used to detect and characterize different aspects of protein marker expression in cell populations. Flow cytometry can be used to quantify the percentage of individual cells in a population that exhibit a given protein marker profile, whereas ICC provides additional information about the subcellular localization of each protein marker and can be applied to single cells or cell aggregates. By using either or both of these protein profiling approaches, we followed the differentiation of human embryonic stem cells into neuroectodermal progenitor cells, glial progenitor cells and oligodendrocyte progenitor cells according to the method of the present disclosure. For human embryonic stem cells differentiated into neuroectodermal progenitor cells and glial progenitor cells, protein marker expression in differentiated day 7 and day 21 cells was characterized by ICC. Adherent cells and cell aggregates were fixed in 4% paraformaldehyde (PFA) for 30 minutes at room temperature (RT). Fixed cells and aggregates were washed with phosphate-buffered saline (PBS) and then fixed aggregates were sequentially placed in increasing concentrations of sucrose solutions (10%, 20% and 30% weight / volume) for 30 min at RT, 30 min at RT and overnight at 4°C, respectively. After sucrose replacement, aggregates were embedded in Tissue-Tek Optimal Cutting Temperature (OCT) solution (Sakura Finetek USA #4583) and frozen at -80°C. OCT-embedded aggregates were warmed to -20°C and cut into 30 μm sections using a cryostat (model CM3050S, Leica Biosystems, Buffalo Grove, IL, USA) and mounted on poly-L-lysine (Sigma-Aldrich #P4707)-coated glass slides.

[0142] To perform immunocytochemical staining, the fixed adherent cells and slide-mounted aggregate sections were permeabilized and blocked in a blocking solution consisting of 0.1% Triton™X-100 / 2% normal goat serum / 1% bovine serum albumin in PBS for 2 hours at room temperature (RT). After permeabilization and blocking, the adherent cells and aggregate sections were incubated overnight at 4°C in a blocking solution without Triton™X-100 and containing primary antibodies specific for protein markers of interest, including PAX6 (BD Pharmingen#561462 or BioLegend#901301) for detecting neuroectodermal progenitor cells, and AP2 (Developmental Studies Hybridoma Bank-DSHB#3B5), PAX3 (DSHB#Pax3) and PAX7 (DSHB#Pax7) for detecting dorsal spinal cord progenitor cells. Adherent cells and aggregate sections were then washed three times with PBS and then incubated with secondary antibodies specific for the selected primary antibodies and 4',6-diamidino-2-phenylindole (DAPI) counterstain in blocking solution without Triton™ X-100 for 1 h at RT protected from light. Adherent cells and aggregate sections were washed three times with PBS and imaged using an IN Cell Analyzer 2000 (GE Healthcare, Pittsburgh, PA, USA).

[0143] After 7 days of differentiation, adherent cell populations from two representative experiments expressed PAX6, a protein marker characteristic of neuroectodermal progenitors, and also expressed the dorsal spinal cord progenitor markers AP2, PAX3, and PAX7.

[0144] Aggregates were sectioned and stained for the dorsal progenitor markers AP2, PAX3, and PAX7, and the pan-neuronal progenitor marker PAX6. These early progenitors were still present at day 21, but a distinct glial population expressing the oligodendrocyte progenitor marker NG2 was also present.

[0145] Human embryonic stem cells differentiated into oligodendrocyte precursor cells by day 42 were characterized for protein marker expression in the resulting single cell populations by both flow cytometry and ICC. To characterize protein marker expression in oligodendrocyte precursor cells by ICC, slide-mounted aggregate sections were stained as described above, except that permeabilization was performed with 100% methanol for 2 min at RT, and the blocking solution consisted of 10% fetal bovine serum in PBS.

[0146] Based on the ICC data for oligodendrocyte precursors at day 42, single cell populations from two representative experiments expressed the oligodendrocyte precursor marker NG2 and had reduced expression of the dorsal spinal cord precursor marker AP2.

[0147] To quantify cell surface markers by flow cytometry on day 42, cells were thawed in thawing medium (10% fetal bovine serum in DMEM medium), centrifuged, and resuspended in staining buffer (2% fetal bovine serum / 0.05% sodium azide in PBS). Cells were incubated on ice for 30 minutes with primary antibodies specific for markers of interest, including NG2 (Invitrogen#37-2300), PDGFRα (BD Biosciences#563575), GD3 (Millipore#MAB2053), A2B5 (BD#563775), CD49f (Millipore#CBL458P), EpCAM (Dako#M080401-2), and CLDN6 (Thermo Fisher#MA5-24076), as well as their isotype controls. To remove unbound antibodies, cells were washed with staining buffer, and in the case of unconjugated antibodies, cells were then incubated with the appropriate fluorophore-conjugated secondary antibody on ice for 30 minutes. Cells were washed and then propidium iodide was added to distinguish dead cells. In some cases, cells were cultured overnight at 37°C / 5%CO2 in tissue culture vessels coated with Matrigel (Corning #356231) to recover protein markers that were sensitive to the harvesting procedure on day 42 described in Example 5, and then harvested with TrypLE™ Select (Thermo Fisher #A12859-01) and stained for flow cytometry analysis as described above. All cells were analyzed on an Attune NxT (Thermo Fisher, Waltham, MA, USA) flow cytometer. To calculate the percentage of cells expressing a given protein marker, dead cells were gated for staining with propidium iodide, and the number of live cells bound to the corresponding antibody was expressed as a percentage of the total number of cells analyzed after correcting for the number of cells that showed non-specific binding to the isotype control antibody.

[0148] Table 5 shows representative flow cytometry data for day 42 oligodendrocyte precursor cells generated according to the method described in Example 5. As shown for two representative runs, a high percentage of cells in the resulting cell population expressed characteristic oligodendrocyte markers, including NG2 and PDGFRα. Additionally, non-OPC markers were minimally detected in the resulting population, including the neural progenitor / epithelial marker CD49f and the epithelial markers CLDN6 and EpCAM.

[0149] Table 5: Representative flow cytometry data for oligodendrocyte precursor cells produced by methods according to the present disclosure. TIFF2024505051000006.tif24152

[0150] Cell populations generated by the methods described in this disclosure are currently in clinical trials to treat spinal cord injury and resulted in a higher percentage of cells positive for the oligodendrocyte progenitor marker NG2 and reduced expression of non-OPC markers CD49f, CLDN6 and EpCAM when compared to OPCs generated using alternative methods (Priest CA, Manley NC, Denham J, Wirth ED 3rd, Lebkowski JS. Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury. Regen Med. 2015 Nov;10(8):939-58; Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929).

[0151] Example 7 - Characterization of differentiated cell populations by gene expression profiling. Gene expression profiling can be used to characterize the cell phenotype of the starting pluripotent cell population and each stage of differentiation, including the generation of neuroectodermal, glial and oligodendrocyte progenitors. Gene expression profiling includes both global transcriptome profiling using methods such as microarrays and RNA-seq, and targeted gene profiling using methods of increased sensitivity such as quantitative real-time PCR (qPCR). To perform gene expression profiling, cells were lysed in Qiagen RLT Lysis Buffer (Qiagen#79216) and RNA was purified using Qiagen RNeasy Mini Kit (Qiagen#74106) according to the manufacturer's guidelines. For qPCR-based analysis, the purified RNA was then converted to cDNA according to standard methods using Invitrogen Superscript IV VILO Mastermix (Thermo Fisher Scientific#11756050) according to the manufacturer's guidelines. The relative expression levels of the target genes and reference housekeeping genes were then quantified using gene-specific primer-probe sets (Applied Biosystems Taqman Gene Expression Assays, Thermo Fisher Scientific #4331182) according to the manufacturer's guidelines. To determine the relative expression levels of a given set of target genes, PCR reactions were performed on an ABI 7900HT Real-Time Sequence Detection System (Applied Biosystems), BioMark HD System (Fluidigm) or equivalent. Each target gene was normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0152] Table 6 shows qPCR results from two representative experiments measuring expression of pluripotency genes, neuroectodermal progenitor genes, glial progenitor genes, dorsal spinal cord progenitor genes, ventral spinal cord progenitor genes and oligodendrocyte progenitor genes in cell populations generated by the methods according to the present disclosure. RNA samples were collected at the following time points: before differentiation (day 0), after differentiation into neuroectodermal progenitor cells (day 7), after differentiation into glial progenitor cells (day 21) and after differentiation into oligodendrocyte progenitor cells (day 42). RNA samples were processed for qPCR using the methods described above. A selected panel of genes indicative of each differentiation state was quantified, including three pluripotency genes (NANOG, LIN28A, SOX2), three neuroectodermal progenitor genes (PAX6, HES5, ZBTB16), three glial progenitor genes (CACGN4, DCC, FABP7) and three oligodendrocyte progenitor genes (CSPG4, PDGFRα, DCN). For each gene, the average of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1) was used to calculate normalized ΔCT values ​​and the fold expression relative to baseline (expression below the limit of quantification) was calculated using the ΔΔCT method.

[0153] Table 6: qPCR analysis of gene markers for pluripotency, neuroectodermal progenitor cells (NPCs), dorsal spinal cord progenitor cells, ventral spinal cord progenitor cells, glial progenitor cells (GPCs) and oligodendrocyte progenitor cells (OPCs) in H1 uhESCs differentiated into OPCs according to the present disclosure. TIFF2024505051000007.tif135162

[0154] Referring to Table 6, differentiation of uhESCs for 7 days according to methods of the present disclosure resulted in a gene expression profile consistent with neuroectodermal progenitor cells, including downregulation of NANOG and expression of LIN28A, SOX2, PAX6, HES5 and ZBTB16.

[0155] Furthermore, neuroectodermal progenitor cells generated after 7 days of differentiation displayed a phenotype consistent with dorsal spinal cord progenitor cells based on expression of dorsal markers TFAP2A (also known as AP2), PAX3, and PAX7. As further evidence of a dorsal spinal cord progenitor phenotype, the resulting neuroectodermal progenitor cells did not express the ventral spinal cord progenitor markers OLIG2 or NKX2-2, the expression of which requires activation of the Sonic Hedgehog signaling pathway.

[0156] After 21 days of differentiation, the resulting cell population displayed a gene expression profile consistent with glial progenitors, including downregulation of pluripotency and neuroectodermal progenitor markers and induction of CACNG4, DCC (also known as the netrin receptor) and FABP7. As further evidence of a glial progenitor phenotype, the resulting day 21 cells displayed sustained expression of HES5, which, in addition to its expression in neuroectodermal / neural progenitors, has also been shown to promote the switch from neural to glial progenitors in the developing mammalian central nervous system. Furthermore, the resulting day 21 glial progenitor cells displayed sustained expression of dorsal spinal cord precursor markers TFAP2A, PAX3 and PAX7, providing further evidence of derivation from dorsally patterned neural progenitors.

[0157] After 42 days of differentiation according to the methods described in this disclosure, the resulting cell population expressed markers consistent with oligodendrocyte precursor cells, including downregulation of both earlier lineage markers and dorsal spinal cord progenitor markers, and induction of CSPG4 (also known as NG2), PDGFRα and DCN.

[0158] Example 8 - Differentiation of human embryonic stem cells into dorsal neuroectodermal progenitor cells using alternative small molecule inhibitors of MAPK / ERK and BMP signaling. In addition to the small molecule inhibitors used in Example 3 (PD0325901 and dorsomorphin), alternative small molecule inhibitors of MAPK / ERK and BMP signaling were tested for their ability to differentiate human embryonic stem cells into dorsal neuroectodermal progenitor cells. Table 7 lists the alternative small molecule inhibitors that were tested. Each condition was tested in duplicate wells of a 6-well tissue culture plate.

[0159] Table 7. Small molecule inhibitors used to differentiate human embryonic stem cells into dorsal neuroectodermal progenitor cells. TIFF2024505051000008.tif90160

[0160] On day 7 of differentiation, cells were harvested and processed for RNA extraction and gene expression profiling by qPCR as described in Example 7. For each gene, normalized ΔCT values ​​were calculated compared to the average of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and fold expression relative to baseline (expression below the limit of quantification) was calculated using the ΔΔCT method. Table 6 shows the average fold expression values ​​(relative to baseline) for the biological duplicates of each small molecule combination. With reference to Table 7, differentiation of uhESCs for 7 days with each of the tested small molecule combinations resulted in downregulation of the pluripotency marker NANOG, as well as a similar degree of sustained expression or induction of genes associated with neuroectodermal progenitor phenotype, including LIN28A, SOX2, PAX6, HES5, and ZBTB16. Furthermore, each of the small molecule combinations tested resulted in a dorsal spinal cord progenitor cell phenotype, based on the lack of expression of dorsal markers TFAP2A, PAX3, and PAX7, and ventral markers OLIG2 and NKX2-2.

[0161] To obtain a more comprehensive comparison of the day 7 cell phenotypes obtained after treatment with each small molecule combination, Fluidigm qPCR was performed using a panel of 96 genes consisting of known markers for pluripotency, neuroectodermal progenitors, neural tube patterning, glial progenitors, oligodendrocyte progenitors, neural crest cells, neurons, astrocytes, pericytes, Schwann cells and epithelial cells. Comparison of the day 7 cell phenotypes for each alternative small molecule combination with the cell phenotypes generated by treatment with PD0325901 + Dorsomorphin by regression plots of normalized ΔCT values ​​indicated that a similar overall cell phenotype could be achieved with each of the small molecule combinations tested. Taken together, the results shown in Table 8 confirm that various combinations of (i) MAPK / ERK inhibitors, with (ii) BMP signaling inhibitors, in the absence of (iii) SHH signaling activators, can be used to differentiate uhESCs into dorsal neuroectodermal progenitor cells, and further into glial and oligodendrocyte progenitor cells, using the methods of the present disclosure.

[0162] (Table 8) qPCR analysis of pluripotency and neuroectodermal progenitor cell (NPC) gene markers in H1 uhESCs differentiated into NPCs using different combinations of small molecule inhibitors. TIFF2024505051000009.tif97160

[0163] Table 9. Inclusion and Exclusion Criteria TIFF2024505051000010.tif173158

[0164] Table 10. Schedule of events from screening to day 90 TIFF2024505051000011.tif211162

[0165] (Table 11) Schedule of events from days 120 to 365. TIFF2024505051000012.tif91158ALT=alanine aminotransferase;AST=aspartate aminotransferase;Gd-DTPA=gadolinium diethylenetriaminepentaacetate 1. If a participant's participation ends between the 60th and 365th day, 2. Rapid testing. 3. Complete Inspection 4. SCIM was collected as a questionnaire (as opposed to behavioral observation). 5. MRI was performed with and without Gd-DTPA contrast, including the spinal cord and cerebellum. 6. MRI included the brain in addition to general imaging of the spinal cord and cerebellum, with and without Gd-DTP. 7. Serum albumin, alkaline phosphatase, blood urea nitrogen, total bilirubin, chloride, serum creatinine, glucose,

[0166] Example 9 - Phase 1 / 2a dose escalation study of AST-OPC1 in subjects with subacute cervical spinal cord injury Study design. The study design was an open-label, alternating dose-escalation, cross-sequential, multicenter study. Subjects with subacute cervical spinal cord injury (SCI) were administered three sequential, ascending doses of AST-OPC1 at a single time point, 21–42 days after injury. Subjects were given AST-OPC1 by direct intraspinal injection using a Syringe Positioning Device (SPD). To prevent rejection of the engraftment, low-dose tacrolimus was initiated 6–12 hours after the intraspinal injection, continued for 46 days, tapered from day 46 to day 60, and discontinued on day 60. Subjects were followed for 1 year after administration of AST-OPC1 under this protocol. Male or female subjects, aged 18–69 years at the time of consent, with sensorimotor complete traumatic SCI (American Spinal Injury Association Impairment Scale A) or sensorimotor incomplete traumatic SCI (American Spinal Injury Association Impairment Scale B). Subjects had a single neurological injury (NLI) of C-5 to C-7 or a C4 NLI with an upper extremity motor score (HEMS) ≥ 1. A single spinal cord lesion was present on magnetic resonance imaging (MRI) scan after stabilization, providing adequate visualization of the spinal cord injury site and lesion margins to allow for safety monitoring after injection. Subjects were able to participate in an elective surgical procedure for injection of AST-OPC1 21-42 days after SCI. Subjects received 2 × 10 6 , 1×10 7 or 2×10 7 Patients received a single dose of live AST-OPC1 cells from either M08D1A or M22D1A and were administered 21–42 days after SCI. A Syringe Positioning Device was used to deliver the product into the spinal cord during surgery. The AST-OPC1 batch numbers used in this study were M08D1A, M22D1A, and M25D1A. AST-OPC1 was administered as a single dose and followed up for one year. A schematic of the planned study schedule and subject screening and treatment is provided in Figure 6 and Figure 7, respectively. A schematic of the final open-label, alternating dose escalation, multicenter Phase 1 / 2a clinical trial is shown in Figure 8. An overview of the trial visits for the one-year protocol is shown in the study summary in Figure 9.

[0167] Neurological examination was completed using the standardized International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) test for motor and sensory testing and for designation of the American Spinal Cord Injury Association Impairment Scale. The ISNCSCI is used for efficacy in terms of improved motor function of the limbs, improved sensory function, and / or neurological decline. Safety assessments included physical examination, vital signs, ISNCSCI neurological examination, pain questionnaire (International Spinal Cord Injury Pain Basic Data Set, ISCIP BDS), electrocardiogram (ECG), magnetic resonance imaging (MRI), laboratory tests, concomitant medications, and adverse events (AEs). At each scheduled visit, ISNCSCI results were enumerated for each subject and analyzed by changes in motor level and motor scores, as well as other exploratory assessments of arm / hand function, self-care ability, and overall voluntary activity. Adverse events were listed by organ system class and by preferred terms within organ systems class according to the Medical Dictionary for Regulatory Activities (MedDRA®) Version 18.0. Statistical analysis of safety data was performed using descriptive statistics including AE incidence, severity and association with AST-OPC1, association with injection procedure for product administration, and concomitant immunosuppression with tacrolimus. The number of clinical laboratory evaluations (hematology, clinical chemistry) below, within, or above the usual clinical laboratory reference range were summarized for each analyte at scheduled study visits. Vital signs were summarized by calculating the mean, standard deviation, median, and range of values ​​at each of the time points specified by the protocol.

[0168] Results: Twenty-six subjects were enrolled in the study. Twenty-five subjects received AST-OPC1 at five study sites. All 25 subjects completed one year of follow-up. The 25 subjects who received AST-OPC1 ranged in age from 18 to 62 years, 21 subjects were male and 4 were female, and the majority were Caucasian (22 subjects). Motor vehicle accidents were the cause of SCI in eight subjects. None of the 25 treated subjects showed evidence of unexpected neurological deterioration on ISNCSCI examination after completing one year of follow-up. Safety data indicate that AST-OPC1 can be safely administered to subjects in the subacute phase after cervical SCI. The injection procedure and low-dose temporary immunosuppression regimen were well tolerated. The 25 subjects who received AST-OPC1 completed one year of follow-up and did not show evidence of neurological deterioration or adverse findings on MRI scans.

[0169] Table 12 below shows the AST-OPC1 dose cohorts and injection preparations used in this study. AST-OPC1 is a cryopreserved cell population containing a mixture of oligodendrocyte precursor cells and other characterized cell types obtained following differentiation of undifferentiated human embryonic stem cells. At the time of cryopreservation, each vial contained 7.5×10 6 The cryopreservation medium contained viable cells. The components of the cryopreservation medium were as follows: Glial Progenitor Medium (GPM)-86% (v / v) [98% DMEM / F12 with GlutaMAX supplement, 1.9% B-27 supplement and 0.1% T3]; 25% Human Serum Albumin (HSA)-3.6% (v / v); 1M HEPES-0.9% (v / v); DMSO-9.5% (v / v).

[0170] Table 12. Dose cohorts and preparation of injections TIFF2024505051000013.tif38164

[0171] Dose selection and timing: 2×10 6The first proposed dose of cells was evaluated for safety in a previous thoracic SCI study. To establish the absence of complications due to the injection procedure, cohort 1 of this study will receive 2 × 10 cells. 6 The first dose (2 × 10 cells in 50 μL) was used again. 6 cells) to the second dose (1 × 10 in 50 μL 7 The increase to 1000 cells (1 × 10 in 50 μL) involved only increasing the concentration of AST-OPC1 such that both of these doses were delivered by a single injection in a volume of 50 μL. Thus, the neurosurgeon consulted on this study considered this initial dose escalation to be a very small step in terms of the potential risks associated with the injection procedure. Furthermore, the second dose (1 × 10 in 50 μL) was increased to 1 × 10 in 50 μL. 7 The safety of administering 1 × 10 cells (cells) was demonstrated in uninjured porcine cervical spinal cords at C6. This study confirmed the minimal expected tissue damage associated with injection into the uninjured spinal cord, and no evidence of efflux or spreading of cells via the CSF was observed. The third dose was 1 × 10 cells in 50 µL at a second site within the lesion in a manner similar to that used for rodent safety studies. 7 represents an additional injection of cells. This dose is within a 6- to 12-fold safety margin compared to the highest dose tested in the rat safety study, especially with respect to the total volume injected.

[0172] LCTOPC1 was supplied to clinical sites in sterile, single-dose, disposable, 2.0 mL Corning™ cryovials. At the time of cryopreservation, each vial typically contained 7.5×10 6 Each cryopreserved preparation contained viable cells. The components of the cryopreservation medium were: 1) Glial Progenitor Medium (GPM)-86% (v / v) [98% DMEM / F12 with GlutaMAX supplement, 1.9% B-27 supplement and 0.1% T3]; 2) 25% Human Serum Albumin (HSA)-3.6% (v / v); 3) 1M HEPES-0.9% (v / v); 4) DMSO-9.5% (v / v). The cryopreserved preparations were thawed, washed, resuspended in injection medium and loaded into injection syringes at the clinical facility.

[0173] LCTOPC1 is a cell population containing a mixture of oligodendrocyte precursor cells (OPCs) and other characterized cell types obtained after differentiation of undifferentiated human embryonic stem cells (uhESCs). LCTOPC1 formulations (DPs) are manufactured by a continuous process. The harvested LCTOPC1 drug substance is a transient intermediate that is immediately formulated into LCTOPC1 DPs, vialed and cryopreserved without the use of a storage step. Compositional analysis of LCTOPC1 by immunocytochemistry (ICC), flow cytometry and quantitative polymerase chain reaction (qPCR) shows that the cell population is composed mainly of neural lineage cells of oligodendrocyte precursor phenotype. Other neural lineage cells, i.e. astrocytes and neurons, are present at low frequencies. The only non-neuronal cells detected in the population are epithelial cells. Mesodermal and endodermal lineage cells as well as uhESCs are usually below the quantification or detection limit of the assay.

[0174] The subacute phase of SCI is hypothesized to be the optimal time window for administering AST-OPC1. This phase occurs early enough to avoid early damage that would cause apoptosis of endogenous oligodendrocytes and to allow AST-OPC1 cells to migrate to denuded axons before extensive glial scarring occurs. This hypothesis is supported by studies in rodent models of SCI that showed functional benefits when AST-OPC1 or other similar preparations were injected 7 days after SCI, but not when the interval between injury and injection was more than 8 weeks (Keirstead 2005, Karimi-Abdolrezaee 2006). Since natural functional recovery in rats with contusion SCI begins to reach a plateau approximately 6 weeks after injury, injection of AST-OPC1 at day 7 corresponds to approximately 1 / 6 (17%) of the time elapsed between injury and the onset of the recovery plateau.

[0175] Given that the rate of spontaneous recovery typically begins to plateau approximately 6 months after SCI, the subacute phase of SCI is likely to be much longer in humans (Fawcett 2007). Extrapolating from preclinical efficacy data in rodents, injection of AST-OPC1 corresponds to approximately 30 days after injury, which is one-sixth of the time to onset of recovery plateau in humans.

[0176] The original dosing window of 14-30 days was chosen to avoid the early bleeding and inflammation that occurs after SCI, as well as the scar tissue formation that occurs in the chronic phase of SCI. This window was also based on preclinical data available before the start of this clinical trial. However, dedicated preclinical studies have been conducted that suggest that the optimal dosing window in human subjects may be extended up to 60 days after SCI.

[0177] A review of the new preclinical data was conducted by a panel including the investigator and several SCI experts to determine whether the dosing window should be adjusted. Additionally, the planned inclusion of subjects with C4 NLI was taken into account during the review. The final recommendation indicated that a dosing window of 21–42 days after SCI would still be considered within the subacute period, although a longer time would be allowed to allow subjects to be medically stable before undergoing elective surgery for AST-OPC1 injection. Therefore, AST-OPC1 was administered to subjects 21–42 days after SCI in this study.

[0178] Tacrolimus Administration. Tacrolimus immunosuppression was initiated 6–12 hours after injection of AST-OPC1. If the subject was unable to take oral medication, tacrolimus was administered intravenously at a starting dose of 0.01 mg / kg / day and given as a continuous intravenous infusion. As soon as oral dosing was possible, subjects were switched to oral tacrolimus. The starting dose of oral tacrolimus was 0.03 mg / kg / day, divided into two daily doses. Tacrolimus doses were adjusted to achieve a target whole blood trough level of 3–7 ng / ml. This target range was slightly below the typical range for long-term maintenance therapy after solid organ transplantation and was chosen based on the low allogeneic reactogenicity of AST-OPC1. On day 46, the tacrolimus dose was reduced by 50% (rounded to the nearest 0.5 mg, as this was the smallest capsule size available). On day 53, the tacrolimus dose was further reduced by 50% (rounded to the nearest 0.5 mg). If the rounded total daily dose was 0.5 mg or less, the subject was given 0.5 mg once per day until tacrolimus was discontinued. If the rounded total daily dose was 0.5 mg or less, the participant was given 0.5 mg once per day until tacrolimus was discontinued. Tacrolimus was discontinued on day 60 (Figure 2). The tacrolimus dose was reduced if trough blood levels were >7 ng / mL. In addition, all ISNCSCI examination forms were reviewed by an expert to assess whether there was any change in neurological function that could be related to the tapering and / or discontinuation of tacrolimus. Tacrolimus was discontinued if any of the following occurred: infection, uncontrolled fever, elevated liver function tests, elevated serum creatinine, seizures, or tacrolimus-induced thrombotic thrombocytopenic purpura. Tacrolimus was discontinued on day 60.

[0179] The schedule of assessments and procedures to be performed from screening through day 365 is shown in Table 13 below.

[0180] (Table 13) Event Schedule TIFF2024505051000014.tif182165Abbreviations: CSF, cerebrospinal fluid; GRASSP, Graded Redefined Assessment of Strength, Sensibility and Prehension; ISNCSCI, International Standards for Neurologic Classification of Spinal Cord Injury; MRI, magnetic resonance imaging; SCIM, Spinal Cord Independence Measure. 1 ISNCSCI test (may be performed on days -3 to -1 if screening ISNCSCI was not performed on day -3) 2 MRI of cervical spine and brain at screening and day 365: MRI of cervical spine only at days 7, 30, and 180 3 Vitals on days -2 and -1 4 Injections may be performed on the day of surgery. 5 May be performed as early as day -4 if necessary to accommodate clinical site staffing 6 Tacrolimus blood levels on day 3 will be obtained + / - 1 day

[0181] Efficacy Assessment. Neurological examinations were performed using the standardized International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) examination for motor and sensory testing and for designation of the American Spinal Cord Injury Association Impairment Scale. For the entire intention-to-treat analysis population, upper extremity motor scores (0-50) and changes from baseline were summarized with N, mean, standard deviation, 95% confidence interval, median, minimum and maximum. Positive changes are considered improvement and negative changes are considered deterioration.

[0182] Motor and sensory levels at each visit and the change in motor / sensory level from baseline were defined as follows: "Level change" is defined as the number of levels the motor / sensory level changed from baseline. Positive numbers represent a change in the caudal direction, which is considered an improvement. Negative numbers represent a change in the rostral direction, which is considered a deterioration.

[0183] Changes in motor and sensory levels were summarized as follows: percentage of subjects with elevated motor levels on either side of the body relative to baseline; percentage of subjects with no change in motor level on either side of the body relative to baseline; percentage of subjects with a motor level improvement of 1 on at least one side of the body relative to baseline; percentage of subjects with a motor level improvement of 1 on both sides of the body relative to baseline; percentage of subjects with a motor level improvement of 2 or greater on at least one side of the body relative to baseline.

[0184] Efficacy Results. Efficacy of the study was measured by change in ISNCSCI Upper Extremity Motor Score (UEMS) and change in motor level from baseline to 12 months after injection of AST-OPC1. A total of 22 subjects were part of the intention-to-treat population (Cohorts 2-5). The mean UEMS of the 22 subjects who completed the Day 365 visit was 28.4 (min: 7, max: 46), a mean change from baseline of 8.9 points. There were 3 subjects (2004, 2007, 2008) who showed some improvement in the Lower Extremity Motor Score (LEMS) that did not correlate with the level of improvement in the UEMS. Compared to baseline, a total of 7 of the 22 subjects (31.8%) achieved a motor level improvement of 2 on at least one side of the body at the Day 365 visit, and 21 of the 22 subjects (95.5%) achieved a motor level improvement of 1 on at least one side of the body at the Day 365 visit. The percentage of subjects with motor level improvement is shown in Table 14 below.

[0185] Table 14: Percentage improvement in physical activity level from baseline. TIFF2024505051000015.tif45164

[0186] Study endpoints. The primary endpoint of the study was safety as measured by the frequency and severity of adverse events (AEs) and serious adverse events (SAEs) within 1 year of LCTOPC1 injections, related to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or concomitant immunosuppression administration. Measures to assess safety included physical examination, vital signs, electrocardiogram (ECG), neurological examination, ISNCSCI examination, magnetic resonance imaging (MRI) scan, pain questionnaire, concomitant medications, AEs, and laboratory tests for hematology, blood chemistry, and immunosuppression safety monitoring. Secondary endpoints were neurological function as measured by the International Standards for Classification of Spinal Cord Injury (ISNCSCI). The ISNCSCI is a highly reproducible research and clinical assessment of neurological impairment for individuals with SCI and has been used as a tool to evaluate the efficacy of acute SCI clinical interventions (Rupp PMID:34108832; Marino 2008 PMID:18581663). To maximize inter- and intra-rater reliability of the neurological assessments, a half-day training session, supervised by an outside expert and including testing of individuals with SCI, was required as part of each center's initiation visit. During the first year of the study, ISNCSCI testing was performed 30, 60, 90, 180, 270, and 365 days after injection of LCTOPC1. The follow-up visit at day 365 was pre-specified as the time point for secondary outcome measures.

[0187] Statistical analysis of efficacy. Efficacy outcomes were assessed for neurological function by characterizing upper extremity motor scores and motor levels on the ISNCSCI test (point estimates and 95% confidence intervals) at 30, 60, 90, 180, 270, and 365 days after LCTOPC1 injection. The baseline ISNCSCI assessment was defined as the baseline visit that occurred 24-48 hours before injection. For the overall intention-to-treat population, upper extremity motor scores and changes from baseline were summarized by participant, mean, standard deviation, 95% confidence interval, median, minimum, and maximum.

[0188] Statistical Analysis of Safety. The collection period for adverse events (AEs) began when participants signed the informed consent form and ended after 365 days of observation. Statistical analysis of AEs that started after the date and time of LCTOPC1 injection or that started before LCTOPC1 injection and worsened after administration of the investigational product. AEs were listed by system organ class (SOC) and by preferred term (PT) within system organ class according to Medical Dictionary for Regulatory Activities (MedDRA®) Version 18 and as reported by participants. A top-line summary of AEs with number of events, number of participants and percentage of participants for each category was listed by cohort and overall. Categories of possible relationships included LCTOPC1, injection procedure and tacrolimus. Total AEs, related events, grade ≥ 3 events and serious events were tabulated.

[0189] Table 15. Summary of relevant adverse events in all treated participants TIFF2024505051000016.tif130170Abbreviations: NK, unknown; AE, adverse event; SAE, serious adverse event. *Paresthesia resolved at 2-year follow-up. **One cerebrospinal fluid leak required lumbar drainage. Participant returned to rehabilitation on day 22.

[0190] Discussion. Safety data from the present study suggest that AST-OPC1 may be safely administered to participants in the subacute phase after cervical SCI. The injection procedure and the low-dose temporary immunosuppression regimen were well tolerated. None of the 25 participants who received LCTOPC1 showed evidence of neurological deterioration. There were no SAEs reported as directly related to LCTOPC1, and the evaluation of AEs did not indicate an increased incidence of commonly reported SCI complications such as urinary tract infections, muscle spasms or neuropathic pain (Sezer 2015). In the present study, which included participants with cervical C4–C7 AIS-A and AIS-B, at 1-year follow-up, 24 / 25 (96%) of participants had recovered ≥1 level of motor function on at least one side of the body, and 8 / 25 (32%) of participants had recovered ≥2 levels of motor function on at least one side of the body. Improvement in motor levels of 2 can transform a person's functional ability from needing complete assistance for activities of daily living to nearly independent (Whiteneck et al., 1999). Safety and neurological recovery data from both thoracic and cervical studies provided evidence that hESC-derived treatments can be safely delivered into the spinal cord.

[0191] Example 10 - Decorin secretion as a potency assay for OPC1. Treatment with recombinant decorin in a rat model of spinal cord injury (SCI) has been shown to inhibit inflammation and glial scar formation and may promote axonal growth across the injury border after acute SCI (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). Decorin has been shown to inhibit acute scarring and wound cavitation and induce dissolution of mature scar tissue in a dorsal column lesion SCI model system in adult rats (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). DFL cavitation treatment with recombinant decorin inhibits inflammation and scar deposition during the acute and subacute phases of the CNS injury response in a rat model of SCI and also contributes to dissolution of mature scar after SCI (Esmaeli, Berry et al. 2014, Ahmed, Bansal et al. 2014). OPC1 treatment in non-clinical models of SCI demonstrated results similar to those seen in the published studies above.

[0192] OPC1 cells have been shown to produce large amounts of decorin. Results seen in OPC1 animal studies have demonstrated anatomical outcomes very similar to those seen in the studies mentioned above. Thus, the anatomical effects observed in preclinical efficacy studies of OPC1 transplantation into SCI lesions may be due, at least in part, to the secretion of decorin.

[0193] While previous preclinical studies have established an optimal window of 14-60 days post-injury for OPC1 implantation to achieve maximal efficacy, decorin has been shown to have an effect on dissolving mature scars after SCI (Esmaeli, Berry et al., 2014; Ahmed, Bansal et al., 2014), providing evidence of a potential role for OPC1 cells in the treatment of chronic SCI subjects.

[0194] Introduction Decorin is a naturally occurring extracellular small leucine-rich proteoglycan TGF-β1 / 2 antagonist that regulates diverse cellular functions through interactions with components of the extracellular matrix (ECM) and plays several important roles in the cellular response to spinal cord injury. Therefore, in vitro decorin secretion was developed and qualified as a potency assay for OPC1.

[0195] Briefly, OPC1 formulation cells are thawed and cultured for 48 hours, then the medium is collected and the secreted decorin concentration is measured by ELISA assay.

[0196] Preliminary molecular analysis and initial ELISA revealed that decorin is not secreted by H1 hESCs and that its expression is gradually activated during OPC1 differentiation, with the highest levels of secreted protein detected in the formulation. This, together with the scientific literature demonstrating biological activities including inhibition of scarring at injury sites and stimulation of axonal growth through spinal cord injury (SCI) sites, supports decorin as a suitable potent candidate.

[0197] Decorin (secreted) is useful as a potency indicator for OPC1 cells by describing their ability to regulate SCI tissue remodeling through attenuating deleterious processes.

[0198] Decorin is a biological regulator secreted by OPC1 The OPC1 preparation is a cryopreserved cell population containing oligodendrocyte precursor cells and other characterized cell types obtained following differentiation of H1 human embryonic stem cells (hESCs). OPC1 has been shown to have three potential repair functions that address the complex pathology observed at the SCI injury site. These activities of OPC1 include the production of neurotrophic factors, stimulation of angiogenesis and induction of remyelination of denuded axons, all of which are crucial for the survival, regrowth and conduction of nerve impulses through axons at the injury site. One potential pathway by which OPCs overcome inhibitory factors at the injury site is the uptake of NG2 in response to retinoic acid. +(Neuronal-glial antigen 2, CSPG4) This could be decorin upregulation as a cellular response (Goncalves, Wu et al. 2019).

[0199] It is important to note that decorin secretion is acquired by OPC1 cells as part of their maturation during the differentiation process, a feature that allows for easy and quantifiable measurement of decorin as a potential potency marker for OPC1.

[0200] Decorin secretion was approximately 25 ng of decorin per day. Therefore, the current threshold for the decorin potency assay was defined as 25 ng / ml.

[0201] Decorin has been shown to be effective in reducing scarring when produced endogenously by different cell types at the site of injury or given exogenously in recombinant form in non-clinical models of SCI, as described below.

[0202] Therefore, active de novo secretion of endogenous decorin by OPC1s while they are implanted in the SCI cavity may be a key factor to ensure successful treatment and therefore a good efficacy marker.

[0203] Spinal cord injury The majority of traumatic SCIs result in contusion or compression of the spinal cord. The mechanical injury in these cases (primary injury) triggers a cascade of molecular and cellular changes collectively referred to as secondary injury (Kakulas 1999). Some of the pathological changes associated with secondary injury include petechial hemorrhages that progress to hemorrhagic necrosis, free radical-induced lipid peroxidation, elevated intracellular calcium leading to activation of neutral proteases, accumulation of extracellular potassium, accumulation of excitatory amino acids, and ischemia (Anderson 1993, Hulsebosch 2002). Traumatic demyelination also begins within hours after injury (Kakulas 1999).

[0204] The cellular response to SCI is generally thought to consist of three phases: an acute hemorrhagic phase, during which hematogenous inflammatory cells infiltrate the wound; a subacute phase, during which scarring is initiated from astrocytes interacting with infiltrating meningeal fibroblasts to produce a glial border around the wound cavity with a core of extracellular matrix (ECM) proteins, revascularization is also initiated, and axonal growth is arrested at the wound edge; and a fixed or chronic phase, during which ECM deposits are remodeled by proteases to establish a mature contractile scar.

[0205] Superimposed on the cellular response is progressive wound cavitation, resulting in progressive cystic expansion of astrocyte-free spaces filled with proteoglycans and macrophages and bordered by proteoglycan-rich neuropil causing secondary destruction of axons.

[0206] Cellular and extracellular composition of spinal cord injury scar tissue Traumatic SCI triggers a complex cascade of events that culminates in the formation of a scar composed of multiple cell types as well as extracellular and non-neuronal components. In the late acute injury phase (0–72 h), cell death and injury result in the release of numerous cellular and blood-derived damage-associated molecular patterns (DAMPs). These are potent activating and inflammatory stimuli for interstitial cells, astrocytes, NG2+ OPCs and microglia. Fibroblast-like cells proliferate from perivascular origins during this acute phase. Activated cells increase deposition of ECM molecules such as chondroitin sulfate proteoglycans (CSPGs) and interstitial-derived matrix. Circulating immune response cells (neutrophils, monocytes) are recruited and their relative expression of cytokines, chemokines and matrix metalloproteinases is of a mixed cellular phenotype (pro-inflammatory and pro-resolving). Over time, the injury microenvironment becomes increasingly pro-inflammatory. In chronic SCI scars, monocyte-derived macrophages / microglia adopt a predominantly pro-inflammatory phenotype. Rather than entering a resolution phase, responding innate immune cells present DAMPs to circulating adaptive immune cells and pathology spreads. Hypertrophy of reactive astrocytes, upregulated expression of intermediate filament-associated proteins and secretion of matrix CSPGs occurs. Scar-forming reactive astrocytes organize into barrier-like structures that separate remaining tissue from the central region of inflammation and fibrosis where wound healing cannot undergo resolution. In most mammalian species, chronic cystic spaces develop. Wallerian degeneration of damaged axonal processes contributes to the continued extracellular deposition of axonal and myelin debris, which is inefficiently processed by immune cells and, together with CSPGs, acts to inhibit neuronal regeneration and neuroplasticity (Bradbury and Burnside 2019).

[0207] Function of decorin in spinal cord injury OPC1 clinical application targets the subacute phase, 21-60 days after SCI. Therefore, it is assumed that transplantation of OPC1 takes place during the transition from the acute to chronic phase, in an inflammatory active environment. Therefore, the ability of OPC1 to actively secrete decorin, which can potentially reduce ongoing negative cues, may be useful for the therapeutic activity of OPC1.

[0208] Decorin has been shown to (1) attenuate both TGF-β1 / 2 receptor activation and signaling via downstream SMADs (a family of intracellular proteins that mediate signaling by members of the TGF-β superfamily), which mediate transcriptional activation of ECM production; (2) bind to type I collagen fibrils and inhibit fibrillogenesis; (3) form an activity-blocking complex with connective tissue growth factor (CTGF); (4) bind to fibronectin and inhibit cell adhesion and fibroblast migration; (5) suppress inflammation, CSPG / laminin / fibronectin-rich scar formation, and astrocytes; 6) stimulate microglia to secrete plasminogen / plasmin (whose activity is regulated by PAI-1), which then modulates the matrix metalloproteinase (MMP):tissue inhibitor of MMP (TIMP) ratio in the wound to initiate fibrolytic degradation of ECM that supports remodeling during the fixed phase of acute scarring; and (7) inhibit CNS scarring through several mechanisms, including binding to epidermal growth factor receptor (EGFR), hepatocyte growth factor (Met) receptor, and toll-like receptors to regulate angiogenesis and inflammatory responses (Esmaeili, Berry et al. 2014; Gubbiotti, Vallet et al. 2016).

[0209] Recombinant human decorin (galacolin) was investigated as a potential treatment for macular degeneration, diabetic retinopathy and diabetic macular edema (Nastase, Janicova et al. 2018). In US Patent No. 9,061,047, the authors propose using galacolin to prevent or reduce scar formation by administration to patients with neurological conditions, including central nervous system injury and / or disease. Formulations of decorin in eye drops were reported as an anti-scarring agent that could replace corneal transplants (Hill, Moakes et al. 2018).

[0210] Decorin promotes axonal regeneration directly by inhibiting the production of scar-derived growth inhibitory ligands (Esmaeili, Berry et al. 2014) and indirectly by (1) plasmin activation of neurotrophins (Davies, Tang et al. 2006); (2) disinhibiting axonal growth cone progression by digesting CSPGs and CNS myelin inhibitors through plasmin and plasmin-induced activation of MMPs (Minor, Tang et al. 2008); and (3) suppressing EGFR activity in growth cones, thereby potentially preventing CSPG / CNS myelin-mediated growth cone collapse.

[0211] Treatment with recombinant decorin in a rat model of SCI supports therapeutic potential Recombinant human decorin (rh-decorin) has been shown to inhibit inflammation, glial scar formation and CSPG expression and may promote axonal growth across the injury border after acute spinal cord injury (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). These data indicate that decorin treatment in animal models initiated immediately after spinal cord injury inhibits inflammatory cell infiltration, scar deposition and cavitation mediated by TGF-β1 / 2 and subsequently regulates ECM remodeling during the fixation phase by both induction of MMP and tissue plasminogen activator (tPA) activity and suppression of TIMP and PAI-1. Furthermore, in decorin-treated mature scars with reduced acute titers of TGF-β1 / 2, scar lysis appears to be induced by fibrinolytic activity mediated by MMP / tPA and enhanced by reduced levels of TIMP and PAI-1 activity.

[0212] Decorin inhibited acute scarring (fibrosis) and wound cavitation and induced dissolution of mature scar tissue (fibrolysis) in a dorsal column lesion (DFL) SCI model system in adult rats (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). DFL cavitation treatment with recombinant decorin inhibits inflammation and scar deposition during the acute and subacute phases of the CNS injury response in a rat model of SCI and also contributes to dissolution of mature scars after SCI. Decorin treatment of spinal cord injury (Esmaeili, Berry et al. 2014, Ahmed, Bansal et al. 2014).

[0213] Furthermore, decorin promoted axonal regrowth in both acute and chronic experiments: in both cases, axons were absent in PBS-treated DFL but present in decorin-treated DFL.

[0214] OPC1 treatment in non-clinical models of SCI demonstrates similar outcomes to those seen with decorin treatment In five studies of OPC1 transplantation in rodent models of SCI (Table 16), a statistically significant reduction in cavitation area was observed in animals treated with OPC1 compared to animals injected with control vehicles (HBSS or IsoLyte + human serum albumin). In these studies, axonal regrowth through the SCI lesion was seen in all animals treated with OPC1 but not in control animals. The tabulated results of these studies are shown below. The rat model of SCI injury used closely mimics the injury and outcomes seen in humans after contusion or crush injury of the cervical spinal cord. For the rat model, the treatment time window after SCI injury is defined as follows: 1-7 days: acute; 7-14 days: subacute; >14 days: chronic.

[0215] Table 16. Summary of preclinical studies showing similar results seen with decorin treatment. TIFF2024505051000017.tif114166

[0216] Staining for the presence of myelinated axonal fibers performed in the first four studies listed above showed the presence of myelinated axonal fibers traversing the lesion area in animals treated with OPC1 but not in control treated animals.

[0217] The results observed in OPC1 animal studies, as described above, demonstrate anatomical outcomes very similar to those seen in studies in which decorin-injected implants were implanted into animals with spinal cord injury (Wu, Li et al. 2013; Ahmed, Bansal et al. 2014). Thus, the anatomical effects observed in preclinical efficacy studies of OPC1 transplantation into SCI lesions may be due, at least in part, to the secretion of decorin.

[0218] summary As mentioned above, the current knowledge of the positive role that decorin plays in attenuating damage in the SCI cavity as well as the results of non-clinical studies in decorin-treated and OPC1-treated models of SCI justify the use of decorin as a potency indicator for OPC1. The ability of OPC1 cells to produce and secrete decorin is predicted to be one of the key therapeutic effects of OPC1 by positively regulating scarring and axonal regrowth inhibition processes. Therefore, we plan to use a qualified decorin assay to be included in a panel of release tests for the new and improved OPC1 manufacturing process.

[0219] References for Example 10: TIFF2024505051000018.tif209165

[0220] Example 11 - Comparability of GPOR OPC1 cells and LTCOPC1 OPC1 cells Some of the processes for making cells and OPC1 products, such as some of the processes used in the clinical trials described in the Examples herein, are referred to as Geron processes and Geron cells or GPOR. The new process described in this and other Examples herein is used to manufacture LCTOPC1 products. The LCTOPC1 process described herein is the process used for the current GMP manufacturing of cells for clinical use. This Example provides data supporting the comparability between these manufacturing processes.

[0221] List of abbreviations TIFF2024505051000019.tif196101

[0222] LCTOPC1 (OPC1), previously called GRNOPC1 and then AST-OPC1, is a population of oligodendrocyte precursor cells derived from the H1 hESC line that is intended for one-time administration for the treatment of subacute spinal cord injury (SCI). Preclinical studies have shown that OPC1 produces neurotrophic factors, migrates within the spinal cord parenchyma, stimulates angiogenesis, and induces remyelination of denuded axons, all of which are essential functions of oligodendrocyte precursor cells and are important for axonal survival, regrowth, and function.

[0223] Clinical evaluation of LCTOPC1 was initiated by Geron Corporation in 2010. The first clinical trial was conducted at 2 × 10 6 A Phase 1 safety study (NCT01217008) in which low doses of OPC1 cells were injected into the lesion site of subjects with subacute neurologically complete thoracic spinal cord T3-T11 injuries. A total of five subjects out of eight planned received OPC1 as part of the initial Phase 1 CP35A007 safety study from October 2010 to November 2011.

[0224] In 2014, a Phase 1 / 2a study (NCT02302157) dose escalation of OPC1 in subjects with subacute sensorimotor complete (ASIA Impairment Scale A) and single neurological level (SNL) from C5 to C7 cervical spinal cord injury was initiated, and the study was subsequently expanded to patients with C4 neurological level (NLI) if the upper extremity motor score (UEMS) was ≥ 1, changing the dosing window from 14–30 days to 21–42 days after spinal cord injury. A total of 25 subjects across five cohorts were enrolled in the AST-OPC-01 study and received a single dose of OPC1 cells delivered by intraparenchymal injection into the spinal cord injury site using a Syringe Positioning Device during a dedicated surgical procedure. Enrollment in the AST-OPC-01 study was completed in December 2017 and reported in December 2020.

[0225] Briefly, the origin of the new Master Cell Bank (MCB) is H1 bank lot number MCBH101. MCBH101 was produced by Geron directly from the H1 Original Cell Bank (OCB) in 2009. MCBH101 was produced in feeder-free conditions using defined raw materials, new culture systems and harvesting procedures, and cryopreserved by a cryopreservation process customized for hESCs. Furthermore, we optimized the method for evaluating H1 hESC pluripotency. A new WCB was derived from the new MCB and expanded for four passages in tissue culture while maintaining hESC characteristics, and then cryopreserved. The WCB provides the starting material for LCTOPC1 production.

[0226] The purpose of this example is also to present the scientific data generated during the development of the LCTOPC1 CMC program. Information provided includes preliminary comparability results based on improved manufacturing process R&D runs, comparability between GPOR and LCT R&D manufactured material, introduction of newly proposed potency assays, and LCTOPC1 development plans for re-examination of the safety status of OPC1 based on GPOR in vivo data and re-analysis of GPOR lots utilizing improved analytical methods.

[0227] Rationale for process improvement OPC1 is an investigational drug studied in Phase 1 and Phase 1 / 2a spinal cord injury clinical trials using OPC1 clinical lots manufactured by Geron Inc. Geron's (GPOR) manufacturing process was initially developed in the early 2000s. At that time, defined cell therapy grade reagents and materials were not widely available. Thus, stage 1 of the manufacturing process involved the growth of H1 embryonic cells on Matrigel™, an animal-derived extracellular matrix (ECM), collagenase, and manual scraping of the culture dish surface for harvesting, passaging, and expansion of H1 embryonic stem cells.

[0228] Moreover, the GPOR manufacturing process was based on a poorly controlled differentiation process driven by three inducer molecules. Most of the differentiation process occurred in cell aggregates starting directly from pluripotent H1 cells in the form of embryoid bodies (day 0-26, Figure 10), which have a strong susceptibility to spontaneous differentiation. From day 27 onwards, differentiation was completed on an adhesive surface coated with Matrigel™ for the expansion proliferation and maturation of oligodendrocyte precursor cells. The GPOR manufacturing process had low yields and key quality attributes defined by purity / impurity / non-target cell population markers showed limited reproducibility. Moreover, the final cryopreserved product required thawing, washing and formulation preparation before administration.

[0229] The development of an improved manufacturing process focused on a more controlled and directed differentiation of H1 into OPCs, guided by a specific sequence of growth factors and small molecules to inhibit or direct the differentiation pathway, using cell therapy grade materials where possible (as detailed in Figure 11). Additionally, the new process shortens the extremely long aggregation phase used by Geron from 26 days intact from a pluripotent cell state (which is prone to spontaneous aberrant differentiation) to 7 days after 14 days of monolayer directed differentiation of H1 cells into neuroectoderm, lowering the chance of spontaneous differentiation during the aggregation phase. A comparison of the GPOR and LCTOPC1 differentiation processes is summarized in Figure 10. The biological rationale for the improved differentiation process inducer and inhibitor signaling sequences is described in Figure 11. Additionally, new in-process controls (IPCs) were added to better monitor and characterize the differentiation process, as detailed in Figure 12.

[0230] The materials used for the production of OPC1 cells (both the original GPOR and the modified process) are summarized in Table 17.

[0231] Table 17: Materials used during production of OPC cells (GPOR and LCTOPC1 processes). TIFF2024505051000020.tif71153

[0232] Overview of LCTOPC1 manufacturing process Stage I-H1 Expansion Thaw pluripotent H1 cells and culture them in mTeSR Plus medium on laminin-coated vessels for 12-15 days. Passage the cells and expand them using the non-enzymatic reagent ReLeSR (as described for MCB and WCB hESC culture).

[0233] During expansion, cells are evaluated morphologically and at the end of the third passage (before the start of the differentiation process) hESC pluripotency is assessed by flow cytometry-based analysis.

[0234] Stage II-H1 differentiation to OPC1 From differentiation day 0 until the end of the process, cells are cultured in glial progenitor medium (GPM), which is DMEM / F-12 supplemented with B27 and T3.

[0235] Day 0-7 - On day 0, once the H1 cultures have reached the required criteria defined by lactate concentration and cell morphology, the differentiation process is initiated by changing the medium for the expanded pluripotent hESCs cultured on laminin-coated vessels as follows: On days 0-3, supplement the GPM medium with retinoic acid (RA), dorsomorphin and PD0325901 to direct the differentiation process towards the neuroectoderm pathway (Kudoh, Wilson et al. 2002). Dorsomorphin inhibits bone morphogenetic protein (BMP) signaling (SMAD pathway) and thus inhibits mesoderm and trophoblast cell differentiation (Li and Parast 2014). PD0325901 inhibits downstream bFGF signaling in MEK1 and MEK2, inhibiting pluripotency and endoderm differentiation (Sui, Bouwens et al. 2013). In summary, inhibition of pluripotency, endoderm, mesoderm and trophoblast cell formation, in addition to activation of the RA signaling pathway, promotes neural tube (neurectoderm) formation (Watabe and Miyazono 2009, Sui, Bouwens et al. 2013, Li and Parast 2014, Patthey and Gunhaga 2014, Janesick, Wu et al. 2015). On days 4-7, cultures are supplemented with retinoic acid and ascorbic acid to continue neuroectoderm differentiation induction (Duester 2008).

[0236] Days 7-14 - On day 7, cells are enzymatically harvested using TrypLE Select and then plated as monolayer cultures on laminin-coated vessels from day 7 to day 14 and cultured in GPM supplemented with rhEGF, hsFGF and ROCK inhibitor (ROCK inhibitor for the first 48 hours only) (Hu, Du et al. 2009, Patthey and Gunhaga 2014, Zheng, Li at al. 2018).

[0237] Days 14–21 - On day 14, to promote neuronal body (NB) aggregate formation, cells are enzymatically harvested using TrypLE Select and cultured for a week as dynamic suspension cultures in GPM supplemented with ROCK inhibitor (first 48 h), as well as rhEGF and hs-rhFGF throughout the entire period.

[0238] Days 21-42 - On day 21, aggregates are reseeded as adherent cultures on laminin-coated vessels in GPM supplemented with rhEGF and PDGF (Ota and Ito 2006; Koch, Lehal et al. 2013), then on day 28, cells are enzymatically harvested using TrypLE Select and expanded as adherent cultures on laminin-coated vessels in GPM supplemented with rhEGF and PDGF until days 35-42 for final expansion and maturation, with enzymatic passage approximately every 7 days using TrypLE Select.

[0239] At the end of the expansion process, OPC1 cells are harvested using TrypLE Select and cryopreserved in CryoStor® 10 (CS10; BioLife Solutions, Inc.) cryopreservation solution as a Thaw-and-Inject (TAI) formulation. The flow of the LCTOPC1 production process is shown in Figure 12.

[0240] As shown in Figure 12, in-process control tests are performed at all critical steps during the differentiation process of hESCs to OPC1. Biomarker protein and mRNA expression are assessed using multicolor flow cytometry (FCM) and qPCR methods (respectively). Cells are tested for expression of OPC1, epithelial, mesodermal, astrocytic and neuronal biomarkers as well as residual hESCs. Additionally, viability, cell yield and metabolic activity (e.g., lactate) are assessed during the process. Lactate concentration is used as an indicator of differentiation initiation starting on day 0 and as a surrogate for cell counting to determine the surface area required for aggregate seeding for pre-OPC generation and expansion proliferation on day 21.

[0241] Proposed CMC Comparability Test OPC1 will be manufactured according to the improved process, released according to revised release parameters, and stored frozen. LCTOPC1 DP will be compared to Geron's manufactured representative batches and characterized with the updated analytical methods used for the release of OPC1 manufactured via the new process. Plans will include testing of additional markers identified in addition to the attributes used as release criteria for GPOR. Proposals for comparison are based on the quality attributes that characterize the formulation as described in Table 18.

[0242] Side-by-side comparisons of LCTOPC1 batches with GPOR OPC1 batches will be based on statistical analyses that calculate expected ranges for quantitative measurements of quality attributes from the GPOR OPC1 batch. Values ​​of these quality attributes measured in the LCTOPC1 batches will be evaluated in relation to expected ranges for the quality attributes tested. Comparability data analyses are expected to establish reproducible release criteria for the LCTOPC1 process and demonstrate that LCTOPC1 has low batch-to-batch variability.

[0243] Quality attributes to be tested will include viability, identity / purity, impurities / non-target populations, genetic profiling and functional / potency assays for 2-3 representative GPOR and LCT OPC1 batches each.

[0244] Suggested comparability quality attributes are: Viability - a critical quality attribute of any live cell preparation; Identity / Purity - assessment of characteristic oligodendrocyte precursor protein markers: NG2, GD3, PDGFRα and PDGRFβ; Non-target cell populations / impurities - (i) residual H1 hESC from starting material - human embryonic stem cell protein markers TRA-1-60 and Oct-4 as potential sources of teratogens, combined in a multi-color flow cytometry test. TRA-1-60 and Oct-4 are commonly known and used as markers for embryonic stem cells and were previously used as OPC1 release criteria, and (ii) evaluation of potential aberrant differentiation pathways (epithelial cell protein markers: keratin 7, claudin-6, EpCAM and CD49f, known epithelial markers, mesodermal cell protein markers CXCR4 and CD56 as mesodermal and cartilage markers, astrocyte protein marker GFAP as known astrocyte marker, neuronal phenotype cell protein marker b-tubulin 3 as known neuronal cell marker, mesenchymal cell mRNA to induce epithelial-mesenchymal transition). OLR1, FOXA2 as a known endoderm marker, SOX17 and AFP as endoderm cell mRNA markers, new data indicates that nestin is not specific to OPCs but rather a marker for NSCs and other cell types, and α-actinin does not include the nestin and α-actinin properties previously used, as it can be effectively replaced by the CXCR4 / CD56 combination; CXCR4 is expressed in definitive endoderm and mesoderm); in vitro cell function / potency - (i) Decorin secretion - a small secreted cell matrix proteoglycan as a potency indicator for OPC1. Decorin expressed by neurons and astrocytes within the central nervous system attenuates scar tissue formation, inhibits cavitation and promotes wound healing. The detailed rationale of decorin secretion as a proposed potency assay is discussed in Example 10. (ii) Migration of OPC1 cells in response to platelet-derived growth factor-BB (PDGF-BB), which is important for cell motility at the injury site to ensure the broadest anatomical coverage from a single injection location within the spine.(iii) Development of a new potency test to evaluate OPC1 maturation and myelination potential. This assay is based on the essential function of OPC1 cells to remyelinate denuded axons. In this assay, OPC1 cells are thawed and seeded in a specific medium in a 3D tissue culture environment (e.g., Matrigel or nanofiber tubes) that should induce OPC1 maturation into oligodendrocytes (OLs).

[0245] The 3D environmental nanotubes mimic denuded axons to induce myelination activity by OPC1 cells in a simple in vitro setting. The assay measures secretion of proteins related to OL function (MBP and decorin) and tests OL protein marker (MBP, O4, MAG, MOG) expression by immunocytochemistry. The assay is currently being developed as a proof of concept (POC) and will be established if proven sufficiently robust.

[0246] The proposed test panels to be used to test GPOR OPC1 and LCTOPC1 along with the proposed release criteria for LCTOPC1 can be seen in Table 18 below.

[0247] (Table 18) Proposed test panels and rationale used for process development, release, and comparability. TIFF2024505051000021.tif250170

[0248] Preliminary comparability of R&D LCTOPC1 batches from representative runs is shown in Tables 19-23 below.

[0249] Table 19. Comparability data from representative GPOR OPC1 and LCTOPC1 R&D runs - OPC1 identity profile by flow cytometry. TIFF2024505051000022.tif64166*Cells are seeded on Matrigel overnight prior to MG-FCM analysis.GPOR OPC1 formulation and freezing reduces NG-2 and PDGFR-α expression. 1 Clinical batch

[0250] Table 20. Comparability data from representative GPOR and LCTOPC1 R&D runs - non-target / impurity cell population profiles by flow cytometry. TIFF2024505051000023.tif61166 1 Clinical batch

[0251] Table 21. Comparability data from representative GPOR OPC1 and LCTOPC1 R&D runs - hESC survival by flow cytometry. TIFF2024505051000024.tif28166 1 Clinical batch

[0252] Table 22. Comparability data from representative GPOR OPC1 and LCTOPC1 R&D runs - Non-targeted / impurity cell population gene profile (compared to GPOR OPC1 M08) by qPCR. TIFF2024505051000025.tif59166*ND-Not Detected 1 Clinical batch

[0253] Table 23: Comparability data from representative GPOR OPC1 and LCT OPC1 R&D runs - in vitro function as determined by decorin secretion and migration assays. TIFF2024505051000026.tif32166 1 Clinical batch

[0254] Summary of evaluation of ectopic tissue formation as a release criterion for OPC1 preparations This analysis revealed certain correlations between available data from GLP tox testing of Geron GMP batches and purity and expression levels of impurity / non-target cell population specific markers.

[0255] Retest results and analysis demonstrated a correlation between impurities and batch failures in vivo, indicating that LCTOPC1, characterized by significantly lower levels of impurities and a high purity profile, is significantly less likely to form cysts in vivo than GPOR OPC1.

[0256] summary Preliminary comparability data of representative GPOR and LCTOPC1 batches demonstrates that GPOR OPC1 and R&D LCTOPC1 exhibit similar OPC1 identity / purity profile profiles, with the exception of the PDGFR-α biomarker, which is higher in R&D LCTOPC1 and may be due to the improved OPC1 manufacturing process (directed differentiation). LCTOPC1 has lower levels of impurities from epithelial, astrocyte and neuronal non-target cells compared to GPOR OPC1. Both LCTOPC1 and GPOR OPC1 have very rare detectable residual hESCs (detected by %TRA-1-60+ / Oct4+), but GPOR OPC1 has a higher percentage of pluripotent cells compared to LCTOPC1, as demonstrated by the population of cells exhibiting TRA-1-60+ / Oct4- and CD49f+. LCTOPC1 has lower levels of endodermal and mesenchymal non-target cell gene expression compared to GPOR OPC1. LCTOPC1 and GPOR OPC1 demonstrate similar decorin secretion and migration capabilities.

[0257] Conclusion: The data accumulated to date indicate that LCTOPC1 preparations, manufactured using improved processes, generated from a defined cell banking system with a highly reproducible and tightly controlled differentiation process, and monitored using modern analytical methods, exhibit essential quality characteristics similar to GPOR OPC1, but with the advantage of a higher overall expression of purity markers and a lower expression of impurity / non-target population markers, thus potentially increasing the safety profile of LCTOPC1 preparations.

[0258] References for Example 11: TIFF2024505051000027.tif172158

[0259] All references mentioned herein are incorporated by reference in their entirety for all teachings therein.

[0260] Although the present disclosure has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes can be made and equivalents substituted for elements thereof without departing from the scope of the present disclosure. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope. Therefore, the present disclosure is not intended to be limited to the particular embodiment disclosed as the best mode contemplated for carrying out the disclosure, but rather, the present disclosure is intended to include all aspects within the scope and spirit of the appended claims.

[0261] Aspects Embodiment P1. A method of cell therapy comprising administering to a subject an OPC composition to improve one or more neurological functions in the subject. Embodiment P2. The method of embodiment P1, wherein said population of OPC cells is injected or implanted into said subject. Embodiment P3 The method of embodiment P1 or P2, wherein said subject has neurological damage due to spinal cord injury, stroke or multiple sclerosis.

[0262] Aspect 1. 1. A method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising: administering to the subject a first dose of a composition comprising human pluripotent stem cell derived oligodendrocyte precursor cells (OPCs); and Optionally, administering two or more doses of the composition. A method comprising: Aspect 2. The method of embodiment 1, further comprising the step of administering a second dose of the composition to the subject. Aspect 3. The method of embodiment 1, further comprising the step of administering to said subject a third dose of said composition. Aspect 4. The method of any one of embodiments 1-3, wherein each administration comprises injecting the composition into the spinal cord of the subject. Aspect 5. The method of any one of aspects 1 to 4, wherein the SCI is a subacute cervical SCI. Aspect 6. The method of any one of aspects 1 to 4, wherein the SCI is a chronic cervical SCI. Aspect 7. The method according to any one of aspects 1 to 4, wherein the SCI is a subacute thoracic SCI. Aspect 8. The method according to any one of aspects 1 to 4, wherein said SCI is a chronic thoracic SCI. Aspect 9. The first dose, second dose, and / or third dose of the composition is about 1×10 6 ~Approx. 3×10 7 2. The method of any one of the preceding aspects, comprising the OPC cells. Aspect 10. The first dose of the composition is about 2×10 6 2. The method of any one of the preceding aspects, comprising the OPC cells. Aspect 11. The first dose or the second dose of the composition is about 1×10 7 2. The method of any one of the preceding aspects, comprising the OPC cells. Aspect 12. The second dose or the third dose of the composition is about 2×10 7 2. The method of any one of the preceding aspects, comprising the OPC cells. Aspect 13. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 45 days after the SCI. Aspect 14. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 90 days after the SCI. Aspect 15. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 75 days after the SCI. Aspect 16. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 60 days after the SCI. Aspect 17. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 30 days after the SCI. Aspect 18. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 75 days after the SCI. Aspect 19. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 60 days after the SCI. Aspect 20. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 40 days after the SCI. Aspect 21. The method of any one of the preceding aspects, wherein each of the first dose, second dose, and third dose of the composition is administered from about 14 days after the SCI to the lifetime of the subject. Aspect 22. 22. The method of any one of aspects 2 to 21, wherein the injection is performed in the caudal half of the site of the SCI. Aspect 23. The method of embodiment 22, wherein said injection is about 6 mm into said spinal cord of said subject. Aspect 24. The method of embodiment 22, wherein said injection is about 5 mm into said spinal cord of said subject. Aspect 25. 1. A method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising: administering to said subject a dose of a composition comprising human pluripotent stem cell derived oligodendrocyte precursor cells (OPCs). A method comprising: Aspect 26. The dose of the composition is about 1×10 6 ~Approx. 3×10 7 The method of embodiment 25, comprising the OPC cells. Aspect 27. The dose of the composition is about 2×10 6 The method of embodiment 26, comprising the OPC cells. Aspect 28. 28. The method of any one of aspects 25-27, wherein said administering of said composition comprises injecting said composition into the spinal cord of said subject. Aspect 29. 29. The method of any one of aspects 25-28, wherein the composition is administered about 7 days to about 14 days after the SCI. Aspect 30. 30. The method of any one of aspects 25 to 29, wherein the injection is performed in the caudal half of the site of the SCI. Aspect 31. The method of any one of aspects 25 to 30, wherein the injection is about 6 mm into the spinal cord of the subject. Aspect 32. The method of any one of aspects 25 to 30, wherein the injection is about 5 mm into the spinal cord of the subject. Aspect 33. 33. The method of any one of aspects 25 to 32, wherein the SCI is a subacute thoracic SCI. Aspect 34. 33. The method of any one of aspects 25 to 32, wherein the SCI is a chronic thoracic SCI. Aspect 35. 33. The method of any one of aspects 25 to 32, wherein the SCI is a subacute cervical SCI. Aspect 36. 33. The method of any one of aspects 25 to 32, wherein the SCI is a chronic cervical SCI. Aspect 37. The method of any one of the preceding embodiments, wherein improving one or more neurological functions comprises improving ISNCSCI test Upper Extremity Motor Score (UEMS). Aspect 38. The method of embodiment 37, wherein said amelioration of UEMS occurs within about 6 months, within about 12 months, within about 18 months, within about 24 months, or more after injection. Aspect 39. The method of embodiment 37 or 38, wherein said improvement is an increase in UEMS of at least 10% compared to baseline. Aspect 40. The method of any one of the preceding embodiments, wherein improving one or more neurological functions comprises improving Lower Extremity Motor Score (LEMS). Aspect 41. The method of embodiment 40, wherein said amelioration of LEMS occurs within about 6 months, within about 12 months, within about 18 months, within about 24 months, or more after injection. Aspect 42. The method of embodiment 37 or 38, wherein said improvement is an improvement in at least one motor level. Aspect 43. The method of embodiment 37 or 38, wherein said improvement is an improvement in at least two motor levels. Aspect 44. The method of any one of aspects 37-43, wherein the improvement is on one side of the subject's body. Aspect 45. The method of any one of aspects 37-43, wherein the improvement is on both sides of the subject's body. Aspect 46. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 14 days to about 90 days after said SCI. Aspect 47. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 14 days to about 75 days after said SCI. Aspect 48. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 14 days to about 60 days after said SCI. Aspect 49. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 14 days to about 30 days after said SCI. Aspect 50. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 20 days to about 75 days after said SCI. Aspect 51. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 20 days to about 60 days after said SCI. Aspect 52. The method of any one of the preceding aspects, wherein said dose of said composition is administered about 20 days to about 40 days after said SCI. Aspect 53. The method of any one of the preceding aspects, wherein said dose of said composition is administered from about 14 days after said SCI to the lifetime of the subject. Aspect 54. 1. A cell population comprising an increased percentage of cells positive for the oligodendrocyte precursor marker NG2 and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM, said cell population being characterized by the following method: Culturing undifferentiated human embryonic stem cells (uhESCs) in glial progenitor medium containing a MAPK / ERK inhibitor, a BMP signaling inhibitor, and retinoic acid to obtain glial-restricted cells; Differentiation of said glial restricted cells into oligodendrocyte precursor cells (OPCs) with an increased percentage of cells positive for the oligodendrocyte precursor marker NG2 and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM. A cell population prepared according to Aspect 55. The cell population of embodiment 54 for use in treating a thoracic spinal cord injury (SCI) in a subject. Aspect 56. 56. The cell population of embodiment 55, wherein said thoracic SCI is subacute thoracic SCI. Aspect 57. 56. The cell population of embodiment 55, wherein said thoracic SCI is chronic thoracic SCI. Aspect 58. The cell population of embodiment 54 for use in treating a cervical spinal cord injury (SCI) in a subject. Aspect 59. The cell population of embodiment 58, wherein said cervical SCI is subacute cervical SCI. Aspect 60. The cell population of embodiment 58, wherein said cervical SCI is chronic cervical SCI. Aspect 61. The cell population of any one of aspects 54-60, wherein the composition is administered to the subject by injection after the SCI. Aspect 62. 62. The cell population of embodiment 61, wherein the injection is performed in the caudal half of the site of the SCI. Aspect 63. 62. The cell population of embodiment 61, wherein said injection is about 6 mm into said spinal cord of said subject. Aspect 64. 62. The cell population of embodiment 61, wherein said injection is about 5 mm into said spinal cord of said subject. Aspect 65. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 14 days to about 90 days after said SCI. Aspect 66. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 14 days to about 75 days after said SCI. Aspect 67. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 14 days to about 60 days after said SCI. Aspect 68. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 14 days to about 30 days after said SCI. Aspect 69. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 20 days to about 75 days after said SCI. Aspect 70. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 20 days to about 60 days after said SCI. Aspect 71. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs about 20 days to about 40 days after said SCI. Aspect 72. 65. The cell population of any one of aspects 54 to 64, wherein said injection occurs from about 14 days after said SCI to the lifetime of said subject. Aspect 73. 1. A method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising: administering to the subject a first dose of the cell population of embodiment 54; administering a second dose of the cell population to the subject; and Optionally, administering a third dose of said cell population to said subject. A method comprising: Aspect 74. 74. The method of embodiment 73, wherein the SCI is a subacute cervical SCI. Aspect 75. 74. The method of embodiment 73, wherein the SCI is a chronic cervical SCI. Aspect 76. 74. The method of embodiment 73, wherein said SCI is a subacute thoracic SCI. Aspect 77. 74. The method of embodiment 73, wherein the SCI is a chronic thoracic SCI. Aspect 78. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 90 days after the SCI. Aspect 79. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 75 days after the SCI. Aspect 80. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 60 days after the SCI. Aspect 81. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 30 days after the SCI. Aspect 82. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 75 days after the SCI. Aspect 83. 78. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 60 days after the SCI. Aspect 84. 78. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 40 days after the SCI. Aspect 85. 78. The method of any one of aspects 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered from about 14 days after the SCI to the lifetime of the subject.

Claims

1. 1. A composition comprising human pluripotent stem cell derived oligodendrocyte precursor cells (OPCs) for use in a method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising: the method comprising administering a first dose of the composition or two or more doses of the composition; The composition.

2. The composition of claim 1, wherein the method further comprises administering a second dose of the composition to the subject.

3. The composition described in claim 1, wherein the method further comprises the step of administering a third dose of the composition to the subject.

4. The composition of any one of claims 1 to 3, wherein each administration comprises injecting the composition into the spinal cord of the subject.

5. The composition of any one of claims 1 to 3, wherein the SCI is a subacute cervical SCI.

6. The composition of any one of claims 1 to 3, wherein the SCI is a chronic cervical SCI.

7. The composition of any one of claims 1 to 3, wherein the SCI is subacute thoracic SCI.

8. The composition of any one of claims 1 to 3, wherein the SCI is a chronic thoracic SCI.

9. The first dose, second dose, and / or third dose of the composition is about 1×10 6 ~Approx. 3×10 7 4. The composition of claim 1, comprising OPC cells.

10. The first dose of the composition is about 2×10 6 2. The composition of claim 1, comprising OPC cells.

11. The first dose or the second dose of the composition is about 1×10 7 4. The composition of claim 3, comprising OPC cells.

12. The second dose or the third dose of the composition is about 2×10 7 4. The composition of claim 3, comprising OPC cells.

13. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 45 days after the SCI.

14. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 90 days after the SCI.

15. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 75 days after the SCI.

16. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 60 days after the SCI.

17. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 30 days after the SCI.

18. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 75 days after the SCI.

19. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 60 days after the SCI.

20. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 40 days after the SCI.

21. The composition of claim 3, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered from about 14 days after the SCI to the survival period of the subject.

22. The composition of claim 4, wherein the injection is performed in the caudal half of the site of the SCI.

23. 23. The composition of claim 22, wherein the injection is about 6 mm into the spinal cord of the subject.

24. 23. The composition of claim 22, wherein the injection is about 5 mm into the spinal cord of the subject.

25. 1. A composition comprising human pluripotent stem cell derived oligodendrocyte precursor cells (OPCs) for use in a method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising: The method includes administering a dose of the composition to the subject. The composition.

26. The dose of the composition is about 1×10 6 ~Approx. 3×10 7 26. The composition of claim 25, comprising OPC cells.

27. The dose of the composition is about 2×10 6 27. The composition of claim 26, comprising OPC cells.

28. 28. The composition of any one of claims 25 to 27, wherein said administering of said composition comprises injecting said composition into the spinal cord of said subject.

29. The composition described in claim 25, wherein, in the method, the composition is administered about 7 days to about 14 days after the SCI.

30. 29. The composition of claim 28, wherein the injection is performed in the caudal half of the site of the SCI.

31. 30. The composition of claim 28, wherein the injection is about 6 mm into the spinal cord of the subject.

32. 30. The composition of claim 28, wherein the injection is about 5 mm into the spinal cord of the subject.

33. 26. The composition of claim 25, wherein the SCI is a subacute thoracic SCI.

34. 26. The composition of claim 25, wherein the SCI is a chronic thoracic SCI.

35. 26. The composition of claim 25, wherein the SCI is a subacute cervical SCI.

36. 26. The composition of claim 25, wherein the SCI is a chronic cervical SCI.

37. 26. The composition of claim 25, wherein improving one or more neurological functions comprises improving ISNCSCI test upper extremity motor score (UEMS).

38. 38. The composition of claim 37, wherein said amelioration of UEMS occurs within about 6 months, within about 12 months, within about 18 months, within about 24 months, or more after injection.

39. The composition of claim 37 or 38, wherein the improvement is an increase in UEMS of at least 10% compared to baseline.

40. 26. The composition of claim 25, wherein improving one or more neurological functions comprises improving Lower Extremity Motor Score (LEMS).

41. 41. The composition of claim 40, wherein said amelioration of LEMS occurs within about 6 months, within about 12 months, within about 18 months, within about 24 months, or more after injection.

42. 39. The composition of claim 37 or 38, wherein the improvement is an improvement in at least one motor level.

43. 39. The composition of claim 37 or 38, wherein the improvement is an improvement in at least two motor levels.

44. 38. The composition of claim 37, wherein the improvement is on one side of the subject's body.

45. 38. The composition of claim 37, wherein the improvement is on both sides of the subject's body.

46. The composition described in claim 25, wherein in the method, the dose of the composition is administered about 14 days to about 90 days after the SCI.

47. The composition described in claim 25, wherein in the method, the dose of the composition is administered about 14 days to about 75 days after the SCI.

48. The composition described in claim 25, wherein in the method, the dose of the composition is administered about 14 days to about 60 days after the SCI.

49. The composition described in claim 25, wherein in the method, the dose of the composition is administered about 14 days to about 30 days after the SCI.

50. The composition of claim 25, wherein said dose of said composition is administered about 20 days to about 75 days after said SCI.

51. The composition described in claim 25, wherein in the method, the dose of the composition is administered about 20 days to about 60 days after the SCI.

52. The composition described in claim 25, wherein, in the method, the dose of the composition is administered about 20 days to about 40 days after the SCI.

53. The composition of claim 25, wherein in the method, the dose of the composition is administered from about 14 days after the SCI to the lifetime of the subject.

54. 1. A cell population comprising an increased percentage of cells positive for the oligodendrocyte precursor marker NG2 and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM, said cell population being characterized by the following method: (i) culturing undifferentiated human embryonic stem cells (uhESCs) in glial progenitor medium containing a MAPK / ERK inhibitor, a BMP signaling inhibitor, and retinoic acid to obtain glial-restricted cells; (iii) differentiating said glial restricted cells into oligodendrocyte precursor cells (OPCs) that have an increased proportion of cells positive for the oligodendrocyte precursor marker NG2 and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM. A cell population prepared according to

55. 55. The cell population of claim 54, for use in treating a thoracic spinal cord injury (SCI) in a subject.

56. 56. The cell population of claim 55, wherein said thoracic SCI is subacute thoracic SCI.

57. 56. The cell population of claim 55, wherein said thoracic SCI is chronic thoracic SCI.

58. 55. The cell population of claim 54, for use in treating a cervical spinal cord injury (SCI) in a subject.

59. 59. The cell population of claim 58, wherein said cervical SCI is subacute cervical SCI.

60. 59. The cell population of claim 58, wherein said cervical SCI is chronic cervical SCI.

61. A cell population as described in any one of claims 54 to 60 for use in a method for treating SCI in a subject, the method comprising a step of administering the cell population by injection after the SCI.

62. The cell population of claim 61, wherein the injection is performed in the caudal half of the site of the SCI.

63. The cell population of claim 61, wherein said injection is about 6 mm into said spinal cord of said subject.

64. The cell population of claim 61, wherein said injection is about 5 mm into said spinal cord of said subject.

65. The cell population of claim 54, wherein said injection occurs about 14 days to about 90 days after said SCI.

66. The cell population of claim 54, wherein said injection occurs about 14 days to about 75 days after said SCI.

67. The cell population of claim 54, wherein said injection occurs about 14 days to about 60 days after said SCI.

68. The cell population of claim 54, wherein said injection occurs about 14 days to about 30 days after said SCI.

69. The cell population of claim 54, wherein said injection occurs about 20 days to about 75 days after said SCI.

70. The cell population of claim 54, wherein said injection occurs about 20 days to about 60 days after said SCI.

71. The cell population of claim 54, wherein said injection occurs about 20 to about 40 days after said SCI.

72. The cell population of claim 54, wherein said injection occurs from about 14 days after said SCI to the life span of said subject.

73. 55. A composition comprising the cell population of claim 54 for use in a method for improving one or more neurological functions in a subject having a spinal cord injury (SCI), comprising: The method further comprising: administering a first dose of the composition to the subject; administering a second dose of the composition to the subject; and administering a third dose of the composition to the subject. Including, The composition.

74. 74. The composition of claim 73, wherein the SCI is a subacute cervical SCI.

75. 74. The composition of claim 73, wherein the SCI is a chronic cervical SCI.

76. 74. The composition of claim 73, wherein the SCI is a subacute thoracic SCI.

77. 74. The composition of claim 73, wherein the SCI is a chronic thoracic SCI.

78. A composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered from about 14 days to about 90 days after the SCI.

79. A composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 75 days after the SCI.

80. A composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 60 days after the SCI.

81. A composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 14 days to about 30 days after the SCI.

82. The composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 75 days after the SCI.

83. The composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 60 days after the SCI.

84. A composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered about 20 days to about 40 days after the SCI.

85. A composition described in any one of claims 73 to 77, wherein in the method, each of the first dose, second dose, and third dose of the composition is administered from about 14 days after the SCI to the survival period of the subject.

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