Pluripotent stem cell-derived oligodendrocyte progenitor cells for the treatment of spinal cord injury

Abstract

This invention provides a population of oligodendrocyte progenitor cells derived from pluripotent stem cells, and methods for their use in the treatment of spinal cord injury. [Solution] Approximately 10 x 10 pluripotent stem cells derived from pluripotent stem cells. 6 From 50 x 10 6 The use of a pharmaceutical composition comprising individual allogeneic human oligodendrocyte progenitor cells, wherein the use comprises administering the pharmaceutical composition to a human subject between 14 and 30 days after the subject sustains a traumatic cervical spinal cord injury, wherein at least 70% of the cells in the pharmaceutical composition express PDGF-Rα, the pharmaceutical composition is injected approximately 2 to 10 mm caudal to the site of the traumatic cervical spinal cord injury, and the subject's upper limb motor function improves by at least two levels, either unilaterally or bilaterally, within approximately 1 to 12 months after administration of the pharmaceutical composition, with the motor level ranging from 0 to 5 according to the International Standard for Neurological Classification of Spinal Cord Injuries (ISNCSCI) revised in 2011.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 394,226 filed on 14 September 2016; U.S. Provisional Application No. 62 / 449,580 filed on 23 January 2017; and U.S. Provisional Application No. 62 / 518,591 filed on 12 June 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] field This disclosure relates to the fields of stem cell biology and oligodendrocyte progenitor cells. More specifically, this disclosure relates to oligodendrocyte progenitor cell compositions and methods of using them. [Background technology]

[0003] background More than 12,000 Americans suffer spinal cord injuries (SCIs) each year, and it is estimated that approximately 1.3 million people in the United States have spinal cord injuries. Traumatic SCIs most commonly affect individuals in their 20s and 30s, resulting in a high level of permanent disability in young, previously healthy individuals. Individuals with SCIs not only have impaired limb function, but also suffer from impaired bowel and bladder function, numbness, spasticity, autonomic nervous system dysfunction, thrombosis, sexual dysfunction, increased infections, pressure ulcers, and chronic pain, each of which can have a significant impact on quality of life and, in some cases, can even be life-threatening. The average life expectancy of individuals who suffer a cervical spinal cord injury at age 20 is 20 to 25 years lower than that of individuals of similar age without SCIs (NSCISC Spinal Cord Injury Facts and Figures 2013).

[0004] The clinical effects of spinal cord injury vary depending on the location and extent of the injury. Nervous systems that may be permanently damaged at a lower level than the injury level are severely affected not only by the loss of control over limb muscles and their protective roles in body temperature and pain perception, but also by cardiovascular, respiratory, 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 lead to a range of secondary problems, such as pressure ulcers and urinary tract infections, which were rapidly fatal before modern medicine. Spinal cord injury often eliminates the unconscious control mechanisms that maintain appropriate levels of excitability in the neural circuits of the spinal cord. As a result, spinal motor neurons can become spontaneously hyperactive, leading to debilitating rigidity and uncontrollable muscle spasms or spasticity. This hyperactivity can also cause unpleasant sensations in the sensory system, including chronic neuropathic pain and paresthesia, numbness, tingling, pain, and burning. A recent questionnaire survey of spinal cord injury patients found that recovery of walking function was not the highest-ranking function these patients desired to recover, and in many cases, mitigation of the sequelae of spontaneous hyperactivity was the 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).

[0005] Due to the injury itself, as well as subsequent secondary effects such as edema, bleeding, and inflammation, several pathological conditions can be 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 conditions include axonal transection, demyelination, parenchymal cavitation, and the formation of ectopic 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 both neurotrophic factors and support for axonal myelination, are susceptible to cell death after spinal cord injury (SCI) and therefore are an important therapeutic target (Almad A, Sahinkaya FR, Mctigue DM. Oligodendrocyte fate after spinal cord injury. Neurotherapics 2011 8(2):262~73). Replacement of oligodendrocyte populations can support both residual and damaged axons and can remyelinate axons to promote electrical conduction (Cao Q, He Q, Wang Yet et 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).

[0006] AST-OPC1 is a population of oligodendrocyte progenitor cells (OPCs) produced from human embryonic stem cells (hESCs) using a specific differentiation protocol (Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia. 2005 Feb;49(3):385~96). AST-OPC1 is characterized by the expression of several molecules associated with oligodendrocyte precursors, including nestin and NG2. These cells are further characterized by their minimal expression or absence of markers known to be present in other cell types such as neurons, astrocytes, endoderm, mesoderm, and hESCs (Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. Neurosci. 2005 May 11;25(19):4694~705; Zhang YW, Denham J, Thies RS. Oligodendrocyte progenitor cells derived from human embryonic stem cells express neurotrophic factors. Stem Cells Dev. 2006 Dec;15(6):943~52).In vitro, AST-OPC1 also produces diffusive factors that support neurite outgrowth from sensory neurons (Zhang YW, Denham J, Thies RS. Oligodendrocyte progenitor cells derived from human embryonic stem cells express neurotrophic factors. Stem Cells Dev. 2006 Dec;15(6):943~52). [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Pluripotent stem cell-derived nerve cells have been used by researchers to treat CNS injury and impairment in animal models. However, obstacles remain in developing such therapies for clinical application in humans. To date, there are no marketed therapies that utilize differentiated cell populations derived from human pluripotent stem cells for the treatment of spinal cord injury or other neurological conditions requiring CNS repair and / or remyelination. [Means for solving the problem]

[0008] overview In various embodiments described herein, the disclosure provides, among other things, a population of oligodendrocyte progenitor cells (OPCs) derived from pluripotent stem cells, and methods of using them in the treatment of spinal cord injury.

[0009] In one embodiment, the disclosure provides a method for improving upper limb motor function in a human subject having a spinal cord injury, the method comprising administering to the subject a composition comprising a population of allogeneic human oligodendrocyte progenitor cells (OPCs). In certain embodiments, the allogeneic human OPCs can engraft at the site of the spinal cord injury. In certain embodiments, administering the composition comprises injecting the composition into the site of the spinal cord injury. In some embodiments, the composition is injected approximately 2 to 10 mm caudal to the center of the spinal cord injury. In further embodiments, the composition is injected approximately 5 mm caudal to the center of the spinal cord injury. In some embodiments, the subject has a cervical spinal cord injury. In other embodiments, the subject has a thoracic spinal cord injury.

[0010] In certain embodiments, the composition is administered after the subject has suffered a traumatic spinal cord injury. In some embodiments, the composition is administered 14 to 60 days after the spinal cord injury, for example, between 14 and 30 days after the injury, between 20 and 40 days after the injury, or between 40 and 60 days after the injury. In certain embodiments, the composition is administered approximately 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after the injury.

[0011] In certain embodiments, improvement in upper limb motor function in a subject may be measured as an increase or change from baseline in the subject's upper limb motor score (UEMS) after administration of a composition containing allogeneic human OPCs. In some embodiments, the subject's UEMS increases detectably within 30 to 400 days of administration of the composition. In some embodiments, the increase in the subject's UEMS is detectable and significant compared to any potential increase in UEMS in a control subject that was not administered the allogeneic human OPC population. In some embodiments, the subject's UEMS increases detectably within 30 days of administration of the composition. In some embodiments, the subject's UEMS increases detectably within 60 days of administration of the composition. In some embodiments, the subject's UEMS increases detectably within 90 days of administration of the composition. In some embodiments, the subject's UEMS increases detectably within 180 days of administration of the composition. In some embodiments, the subject's UEMS score increases detectably within 270 days of administration of the composition. In some embodiments, the subject's UEMS score increases detectably within 360 days of administration of the composition.

[0012] In certain embodiments, the target UEMS score continues to improve from the initial UEMS baseline measurement for a period of approximately 1 to 24 months after administration of the composition containing the allogeneic human OPC. In some embodiments, the target UEMS score improves over time after administration of the allogeneic human OPC composition, with baseline UEMS ≤ 3 months, UEMS ≤ 6 months, UEMS ≤ 9 months, and UEMS ≤ 12 months. In certain embodiments, the target UEMS score continues to improve up to 18 months after administration of the allogeneic human OPC composition, or even beyond. In certain embodiments, the target UEMS score continues to improve up to 24 months after administration of the allogeneic human OPC composition, or even beyond.

[0013] In certain embodiments, the improvement in the subject's UEMS score over a period of 1 to 24 months following administration of the allogeneic human OPC may range from about 1 to about 30 points, e.g., about 2 points, e.g., about 4 points, e.g., about 6 points, e.g., about 8 points, e.g., about 10 points, e.g., about 12 points, e.g., about 14 points, e.g., about 16 points, e.g., about 18 points, e.g., about 20 points, e.g., about 22 points, e.g., about 24 points, e.g., about 26 points, e.g., about 28 points, e.g., about 30 points. In some embodiments, the improvement in the subject's UEMS score over a period of 1 to 18 months following administration of the allogeneic human OPC may exceed 20 points.

[0014] In certain embodiments, improvement in upper limb motor function in a subject may be measured as improved motor level recovery (motor level as defined by the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI)). In some embodiments, the improvement in motor level in a subject is more significant than any potential improvement in motor level in a control subject that was not administered the allogeneic human OPC population. In some embodiments, the improvement in motor level in a subject may be at about one level approximately 1 to 12 months after administration of the allogeneic human OPC composition. In some embodiments, the improvement in motor level in a subject may be at about two levels approximately 1 to 12 months after administration of the allogeneic human OPC composition. In some embodiments, the improvement in motor level in a subject may be at a level greater than two levels approximately 1 to 12 months after administration of the allogeneic human OPC composition. In some embodiments, the subject's measured exercise level continues to improve from the initial baseline measurement over a period of approximately 1 to 24 months after administration of the allogeneic human OPC composition, for example, over approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the subject's measured exercise level continues to improve from the initial baseline measurement over a period of approximately 12 months after administration of the allogeneic human OPC composition. In some embodiments, the improvement in exercise level may be unilateral. In other embodiments, the improvement in exercise level may be bilateral.

[0015] In certain embodiments, improvement in the subject's upper limb motor function may be measured or evaluated using means other than UEMS or motor level recovery, including, but not limited to, various neurological tests and clinical impairment measurements such as GRASSP (Graded Redefined Assessment of Strength, Sensibility and Prehension). In certain embodiments, improvement in the subject's upper limb motor function may be measured indirectly, for example, by using MRI, or by evaluating the subject's functional independence using, for example, SCIM (Spinal Cord Independence Measure). Any means known in the art for detecting or evaluating motor function improvement may be used.

[0016] In certain embodiments, the method further comprises administering a low-dose immunosuppressant regimen to the subjects. In certain embodiments, the immunosuppressant regimen comprises oral administration of about 0.03 mg / kg / day of tacrolimus, adjusted to maintain a trough blood concentration of about 3–7 ng / mL until about 46 days after administration of the composition, followed by tapering, and discontinuation of the immunosuppressant about 60 days after administration of the composition containing a population of allogeneic OPCs.

[0017] In certain embodiments, the method comprises administering a composition comprising a population of allogeneic oligodendrocyte progenitor cells (OPCs), where the dose of the composition is approximately 2 × 10⁻⁶. 6 From one piece, approximately 50 x 10 6 It contains 10 AST-OPC1 units. In some embodiments, the dose of the composition is approximately 50 × 10 6 It contains 10 AST-OPC1 units. In some embodiments, the dose of the composition is approximately 40 × 10 6 It contains 10 AST-OPC1 units. In some embodiments, the dose of the composition is approximately 30 × 10 6 It contains 10 AST-OPC1 units. In some embodiments, the dose of the composition is approximately 20 × 10 6comprises a number of AST-OPC1. In some embodiments, the dosage of the composition is about 10×10 6 comprises a number of AST-OPC1. In some embodiments, the dosage of the composition is about 5×10 6 comprises a number of AST-OPC1. In some embodiments, the dosage of the composition is about 2×10 6 comprises a number of AST-OPC1.

[0018] In certain embodiments, the OPC can remain within the spinal cord injury site of the subject for a period of about 90 days or more after administration of the composition to the spinal cord injury site. In certain embodiments, the OPC can remain within the spinal cord injury site of the subject for a period of about 1 year or more after administration of the composition to the spinal cord injury site. In further embodiments, the OPC can remain within the spinal cord injury site of the subject for a period of about 2 years or more after administration of the composition to the spinal cord injury site. In further embodiments, the OPC can remain within the spinal cord injury site of the subject for a period of about 3 years or more after administration of the composition to the spinal cord injury site. In further embodiments, the OPC can remain within the spinal cord injury site of the subject for a period of about 4 years or more. In further embodiments, the OPC can remain within the spinal cord injury site of the subject for a period of about 5 years or more.

[0019] In additional embodiments, the present disclosure provides a container comprising a composition comprising a population of allogeneic human oligodendrocyte progenitor cells (OPC) that can improve upper limb motor function when administered to a human subject having a spinal cord injury. The OPC of the present disclosure can be derived from any type of human pluripotent stem cell. In certain embodiments, the population of OPC is the in vitro differentiated progeny of human embryonic stem cells (hESC). In other embodiments, the OPC is the in vitro differentiated progeny of pluripotent stem cells other than human embryonic stem cells, such as induced pluripotent stem cells (iPSC). In certain embodiments, the subject has a cervical spinal cord injury. In other embodiments, the subject has a thoracic spinal cord injury.

[0020] To fully understand the properties and advantages of the present invention, please refer to the following detailed description in conjunction with the attached drawings. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows the study design and schedule of the Phase 1 / 2a dose-escalation trial of AST-OPC1 in subjects with traumatic spinal cord injury. [Figure 2] Figure 2 shows the study design regarding the target cohort and AST-OPC1 dosage. 2M = Cohort subjects receive 2 × 10⁶ AST-OPC1 infusions; 10M = Cohort subjects receive 10 × 10⁶ AST-OPC1 infusions; 20M = Cohort subjects receive 20 × 10⁶ AST-OPC1 infusions. AIS A = American Spinal Injury Association (ASIA) Disability Scale (AIS) Grade A spinal cord injury, complete sensorimotor impairment. AIS B = American Spinal Injury Association (ASIA) Disability Scale (AIS) Grade B spinal cord injury, complete motor impairment, incomplete sensory impairment. See, for example, American Spinal Injury Association: International Criteria for Neurological Classification of Spinal Cord Injuries, revised 2000; Atlanta, Georgia, 2008 reprint. [Figure 3] Figure 3 shows the AST-OPC1 injection procedure. The injection was performed using a syringe positioning device (SPD) mounted on a table. Participants in Cohorts 1 and 2 received a single intraparenchymal injection of 50 μl into the spinal cord injury site. [Figure 4A] Figure 4A shows the upper limb motor function recovery data for Cohort 1 available in September 2016. Figure 4A: All subjects in Cohort 1 (2 × 10⁶ AST-OPC1 units) and Cohort 2 (10 × 10⁶ AST-OPC1 units) showed improved upper limb motor scores (UEMS) compared to baseline. The mean UEMS improvement at 90 days post-AST-OPC1 infusion was 5.0 points in Cohort 1 (N=3) and 9.5 points in Cohort 2 (N=4). [Figure 4B]Figure 4B shows upper limb motor function recovery data for Cohort 2 available as of September 2016. Figure 4B: 90 days after infusion, 50% of Cohort 2 subjects (2 out of 4) showed a one-step improvement in motor level, and 50% of subjects (2 out of 4) showed a two-step improvement in motor level on at least one side. Motor level was defined based on the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI; see Kirshblum, SC et al., International Standards for Neurological Classification of Spinal Cord Injury (2011 revision), The Journal of Spinal Cord Medicine, 2011 34(6), 535-546). For evaluation of UEMS and initial motor level / improvement of motor level, see Steeves JD et al., Degree of Spontaneous Motor Recovery After Traumatic Cervical Spinal Cord Injury, Spinal Cord 2011 49:257~265; and Steeves JD et al., Outcome Measures for Acute / Subacute Cervical Sensorimotor Complete (AIS-A) Spinal Cord Injury During a Phase 2 Clinical Trial, Top Spinal Cord Inj Rehabil 2012;18(1):1014. [Figure 5] Figure 5 shows the matching criteria used to generate closely matched historical comparisons from the EMSCI database. [Figure 6A] Figure 6A shows motor function recovery measured by the change in UEMS over time from baseline (+ / - SEM) in subjects of Cohort 2, compared to closely matched historical controls. Data are shown over a 12-month follow-up period. SEM = standard error of the mean. [Figure 6B] Figure 6B shows motor function recovery measured by the change in UEMS over time (+ / -SEM) from baseline in Cohort 2 subjects compared to Cohort 1 subjects and closely matched historical controls. Data are shown over a 12-month follow-up period. SEM = standard error of the mean. [Figure 7]Figure 7 shows the motor function recovery measured as the percentage of subjects in Cohort 2 who showed an improvement of two or more levels in motor function over a 12-month follow-up period. Subjects in Cohort 2 were compared to closely matched historical controls from the EMSCI database. [Modes for carrying out the invention]

[0022] Detailed explanation Before describing the compositions and methods, it should be understood that the specific processes, compositions, or methodologies described may vary and therefore this disclosure is not limited thereto. Furthermore, the terminology used in the description is intended solely to describe specific versions or embodiments and is not intended to limit the scope of the invention, which is limited only by the appended claims. For example, features illustrated in one embodiment may be incorporated into other embodiments, and features illustrated in a particular embodiment may be omitted from that embodiment. Thus, this disclosure is intended to show that in some embodiments of this disclosure, any features or combinations of features described herein may be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be obvious to those skilled in the art in light of this disclosure and do not constitute a departure from this disclosure. In other examples, well-known structures, interfaces, and processes are not described in detail in order to avoid unnecessarily obscuring the invention. Nothing in this specification is intended to be construed as negating any part of the entire scope of the invention. Therefore, the following description is intended to illustrate some specific aspects of the present disclosure and not to exhaustively identify all possible substitutions, combinations, and variations thereof.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. The terms used in describing this disclosure herein are for the sole purpose of describing specific embodiments and are not intended to limit this disclosure.

[0024] All publications, patent applications, patents, and other references cited herein are incorporated in their entirety by reference.

[0025] Unless the context indicates otherwise, the various features of the disclosure described herein are particularly intended to be used in any combination. Furthermore, in some embodiments of the disclosure, any feature or combination of features described herein may be excluded or omitted.

[0026] The methods disclosed herein may include one or more steps or actions to achieve the described methods. The steps and / or actions of the methods may be interchangeable with one another without departing from the scope of the invention. In other words, unless a particular order of steps or actions is required for the proper operation of the embodiments, the order and / or use of any particular steps and / or actions may be modified without departing from the scope of the invention.

[0027] When used in the description of this disclosure and in the appended claims, the singular forms “a,” “an,” and “the” are intended to also include the plural forms unless the context clearly indicates otherwise.

[0028] As used herein, “and / or” means and encompasses any possible combination of one or more of the related enumerated items, and, when interpreted as an alternative (“or”), means and encompasses the absence of any combination.

[0029] As used herein, the terms “about” and “approximately” when referring to measurable values ​​such as percentages, densities, and volumes mean that they include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified quantity.

[0030] When used herein, phrases such as "between X and Y" and "about between X and Y" should be interpreted as including X and Y. When used herein, phrases such as "about between X and Y" mean "about between X and about Y," and phrases such as "about X to Y" mean "about X to about Y."

[0031] The term "AST-OPC1" refers to a specific, characterized, in vitro differentiated cell population, comprising a mixture of oligodendrocyte progenitor cells (OPCs) and other characterized cell types, obtained from undifferentiated human embryonic stem cells (uhESCs) according to a specific differentiation protocol disclosed herein.

[0032] Compositional analysis of AST-OPC1 by immunocytochemistry (ICC), flow cytometry, and quantitative polymerase chain reaction (qPCR) demonstrates that the cell population consists primarily of oligodendrocyte-phenotypic nervous system cells. Other nervous system cells, namely astrocytes and neurons, are present at low frequencies. The only non-neuronal cells detected in the population are epithelial cells. Mesoderm, endoderm-derived cells, and uhESCs are typically below quantification or detection in the assay.

[0033] As used herein, the term “oligodendrocyte progenitor cell” (OPC) refers to a cell of the neuroectoderm / glial lineage committed to forming offspring, including mature oligodendrocytes. These cells typically express the characteristic markers NG2 and PDGF-Rα.

[0034] As used herein, the term “therapeutic dose” refers to a dose, drug regimen, or amount sufficient to produce the desired result.

[0035] As used herein, the terms “treatment,” “treat,” “treated,” or “treating” can refer to both therapeutic and preventive or prophylactic measures, the purpose of which is to prevent or slow (reduce) an undesirable physiological condition, symptom, disorder, or disease, or to obtain a beneficial or desired clinical outcome. In some embodiments, the term can refer to both treatment and prevention. For the purposes of this disclosure, a beneficial or desired clinical outcome may include, but is not limited to, one or more of the following: reduction of symptoms; reduction of the severity of a condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease; delay of the onset or progression of a condition, disorder, or disease; recovery of the condition, disorder, or disease; and remission (whether partial or whole), enhancement, or improvement of a condition, disorder, or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response. Treatment also includes extending survival compared to the expected survival without treatment.

[0036] As used herein, the term "subject" refers to a human or animal. In some embodiments, the term "subject" refers to a male. In some embodiments, the term "subject" refers to a female.

[0037] As used herein, the terms “implantation” or “transplantation” refer to the administration of a population of cells to a target tissue using appropriate delivery techniques (e.g., using an infusion device).

[0038] As used herein, “engraftment” and “engrafting” refer to the integration of transplanted tissue or cells (i.e., “graft tissue” or “graft cells”) into the target body. The presence of graft tissue or graft cells at or near the transplant site 180 days or more after transplantation indicates engraftment. In certain embodiments, imaging techniques (such as MRI imaging) may be used to detect the presence of graft tissue.

[0039] As used herein, “allogeneic” and “allogeneic-derived” refer to a population of cells that originate from a source other than the subject and are therefore not genetically identical to the subject. In certain embodiments, the allogeneic cell population is derived from cultured pluripotent stem cells. In certain 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 another embodiment, the allogeneic cell population is derived from primate pluripotent (pPS) cells.

[0040] The terms “central nervous system” and “CNS,” as used interchangeably in this specification, refer to a complex of nerve tissue that controls one or more bodily activities, including but not limited to the brain and spinal cord of vertebrates.

[0041] Proliferation and culture of undifferentiated pluripotent stem cells Methods for the proliferation and culture of undifferentiated pluripotent stem cells have been previously described. For information on tissue and cell culture of pluripotent stem cells, readers should refer to one of the numerous publications available in the art, such as *Teratocarcinomas and Embryonic Stem Cells: A Practical Approach* (E.J. Robertson, IRL Press Ltd., 1987); *Guide to Techniques in Mouse Development* (PM. Wasserman et al., Academic Press, 1993); *Embryonic Stem Cell Differentiation in Vitro* (MV. Wiles, Meth. Enzymol. 225:900, 1993); *Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy* (PD. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and R. Freshney, *Culture of Animal Cells*, Wiley-Liss, New York, 2000).

[0042] In certain embodiments, the method may be carried out with pluripotent stem cell lines. In other embodiments, the method may be carried out with embryonic stem cell lines. In one embodiment, the method may be carried out with multiple undifferentiated stem cells derived from H1, H7, H9, H13, or H14 cell lines. In another embodiment, the undifferentiated stem cells may be derived from induced pluripotent stem cell (iPS) lines. In yet another embodiment, the method may be carried out with primate pluripotent stem (pPS) cell lines. In yet another embodiment, the undifferentiated stem cells may be derived from parthenogenetic germ cells, which are embryos stimulated to produce hESCs without fertilization.

[0043] In one embodiment, undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without additional feeder cells (see, e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are typically supported by nutrient media containing factors that promote cell proliferation without differentiation (see, e.g., U.S. Patent No. 6,800,480). In one embodiment, a conditioned medium containing such factors may be used. The 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, telomereated mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Patent No. 6,642,048). The medium can be conditioned by plating feeders in serum-free medium such as knockout DMEM supplemented with 20% serum substitute and 4 ng / mL bFGF. The culture medium, after being acclimatized for 1-2 days, can be further supplemented with bFGF and used to support the culture of pPS cells for another 1-2 days (see, e.g., WO01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0044] Alternatively, fresh or unconditioned media supplemented with additional factors that promote cell proliferation in the undifferentiated form (e.g., fibroblast growth factor or forskolin) may be used. Non-limiting examples include basic media such as X-VIVO® 10 (Lonza, Walkersville, Maryland) or QBSF®-60 (Quality Biological Inc., Gathersburg, Maryland), supplemented with 40–80 ng / mL of bFGF 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 medium formulations have the advantage of supporting cell proliferation at 2–3 times the rate of other systems (see, e.g., WO03 / 020920). In one embodiment, undifferentiated pluripotent cells such as hESCs may be cultured in a medium containing bFGF and TGFβ. An unrestricted, exemplary concentration of bFGF is approximately 80 ng / ml. An unrestricted, exemplary concentration of TGFβ is approximately 0.5 ng / ml.

[0045] In one embodiment, undifferentiated pluripotent cells may 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 may, among other things, be derived from human or mouse sources. Human feeder cells can be isolated from various human tissues or induced via the differentiation of human embryonic stem cells into fibroblasts (see, e.g., WO01 / 51616). In one embodiment, the 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), prepuce 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).

[0046] Various 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 from a variety of materials, including, but not limited to, glass or plastics such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, Merinex, Thermanox, or combinations thereof. In one embodiment, a suitable surface may comprise one or more polymers, such as one or more acrylates. In one embodiment, the solid surface may have a three-dimensional shape. Non-limiting examples of three-dimensional solid surfaces are described, for example, in U.S. Patent Application Publication 2005 / 0031598.

[0047] In one embodiment, undifferentiated stem cells can be grown on a growth substrate under feeder-free conditions. In one embodiment, the growth substrate may be Matrigel® (e.g., Matrigel® or Matrigel® GFR), recombinant laminin, or vitronectin. In another embodiment, undifferentiated stem cells may be subcultured using various methods, such as using collagenase or manual scraping. In yet another embodiment, undifferentiated stem cells may be subcultured using non-enzymatic means, such as 0.5 mM EDTA in PBS, or using ReLeSR®. In one embodiment, multiple undifferentiated stem cells are seeded or subcultured 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 1 cm² of growth surface. 2 Around this area, the cells are approximately 6.0 × 10 3 pieces / cm 2 From cells approximately 5.0 × 10 5 pieces / cm 2 The range, for example, a cell of approximately 1.0 × 10 4 pieces / cm 2 For example, a cell of approximately 5.0 × 104 pieces / cm 2 For example, a cell of approximately 1.0 × 10⁻⁶ 5 pieces / cm 2 , or for example, cells approximately 3.0 × 10 5 pieces / cm 2 It is possible. In another embodiment, the seeding density is such that the growth surface is 1 cm 2 Around this area, the cells are approximately 6.0 × 10 3 pieces / cm 2 From cells approximately 1.0 × 10 4 pieces / cm 2 For example, a cell of approximately 6.0 × 10 3 pieces / cm 2 From cells approximately 9.0 × 10 3 pieces / cm 2 For example, a cell of approximately 7.0 × 10 3 pieces / cm 2 From cells approximately 1.0 × 10 4 pieces / cm 2 For example, a cell of approximately 7.0 × 10 3 pieces / cm 2 From cells approximately 9.0 × 10 3 pieces / cm 2 , or for example, cells approximately 7.0 × 10 3 pieces / cm 2 From cells approximately 8.0 × 10 3 pieces / cm 2 It can be in the range of . In yet another embodiment, the seeding density is 1 cm on the growing surface 2 Around this area, the cells are approximately 1.0 × 10 4 pieces / cm 2 From cells approximately 1.0 × 10 5 pieces / cm 2 For example, a cell of approximately 2.0 × 10⁻⁶ 4 pieces / cm 2 From cells approximately 9.0 × 10 4 pieces / cm 2 For example, a cell of approximately 3.0 × 10 4 pieces / cm 2 From approximately 8.0 x 10 4 pieces / cm 2 For example, a cell of approximately 4.0 × 10 4 pieces / cm 2 From cells approximately 7.0 × 10 4 pieces / cm 2 , or cells approximately 5.0 × 10 4From a single cell, approximately 6.0 × 10 4 pieces / cm 2 It may be in the range of . In one embodiment, the seeding density is 1 cm on the growth surface. 2 Around this area, the cells are approximately 1.0 × 10 5 pieces / cm 2 From cells approximately 5.0 × 10 5 pieces / cm 2 For example, a cell of approximately 1.0 × 10⁻⁶ 5 pieces / cm 2 From cells approximately 4.5 × 10 5 pieces / cm 2 For example, a cell of approximately 1.5 × 10⁻⁶ 5 pieces / cm 2 From cells approximately 4.0 × 10 5 pieces / cm 2 For example, a cell of approximately 2.0 × 10⁻⁶ 5 pieces / cm 2 From cells approximately 3.5 × 10 5 pieces / cm 2 , or cells approximately 2.5 × 10 5 pieces / cm 2 From cells approximately 3.0 × 10 5 pieces / cm 2 It could be within the range of.

[0048] 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 exchanged at appropriate time intervals. In one embodiment, the culture medium can be completely exchanged daily starting from about 2 days after subculture of the cells. In another embodiment, when the culture reaches about 90% confluence, one or more suitable reagents such as collagenase IV and 0.05% trypsin-EDTA are continuously used to achieve a single cell suspension for quantification, and the surrogate flasks can be sacrificed and counted. In one embodiment, a plurality of undifferentiated stem cells can be subcultured before seeding the cells on a suitable growth substrate (e.g., Matrigel® GFR) at a seeding density that allows the cells to reach confluence in an appropriate period, e.g., about 3 to 10 days. In one embodiment, undifferentiated stem cells can be subcultured using collagenase IV and grown on a recombinant laminin matrix. In one embodiment, undifferentiated stem cells can be subcultured using collagenase IV and grown on a Matrigel® matrix. In one embodiment, undifferentiated stem cells can be subcultured using ReLeSR™ and grown on a vitronectin matrix.

[0049] In one embodiment, the seeding density is about 6.0×10 2 cells per cm 3 of growth surface to about 5.0×10 2 cells per cm 5 of growth surface, for example about 1.0×10 2 cells / cm 4 ², for example about 5.0×10 2 cells / cm 4 ², for example about 1.0×10 2 cells / cm 5 ², or for example about 3.0×10 2 cells / cm 5 ². In another embodiment, the seeding density is about 6.0×10 2 cells per cm 2 of growth surface to about 6.0×10 3 cells / cm 2From cells approximately 1.0 × 10 4 pieces / cm 2 For example, a cell of approximately 6.0 × 10 3 pieces / cm 2 From cells approximately 9.0 × 10 3 pieces / cm 2 For example, a cell of approximately 7.0 × 10 3 pieces / cm 2 From cells approximately 1.0 × 10 4 pieces / cm 2 For example, a cell of approximately 7.0 × 10 3 pieces / cm 2 From cells approximately 9.0 × 10 3 pieces / cm 2 , or for example, cells approximately 7.0 × 10 3 pieces / cm 2 From cells approximately 8.0 × 10 3 pieces / cm 2 It can be in the range of . In yet another embodiment, the seeding density is 1 cm on the growing surface 2 Around this area, the cells are approximately 1.0 × 10 4 pieces / cm 2 From cells approximately 1.0 × 10 5 pieces / cm 2 For example, a cell of approximately 2.0 × 10⁻⁶ 4 pieces / cm 2 From cells approximately 9.0 × 10 4 pieces / cm 2 For example, a cell of approximately 3.0 × 10 4 pieces / cm 2 From cells approximately 8.0 × 10 4 pieces / cm 2 For example, a cell of approximately 4.0 × 10 4 pieces / cm 2 From cells approximately 7.0 × 10 4 pieces / cm 2 , or cells approximately 5.0 × 10 4 From a single cell, approximately 6.0 × 10 4 pieces / cm 2 It may be in the range of . In one embodiment, the seeding density is 1 cm on the growth surface. 2 Around this area, the cells are approximately 1.0 × 10 5 pieces / cm 2 From cells approximately 5.0 × 10 5 pieces / cm 2 For example, a cell of approximately 1.0 × 10⁻⁶ 5 pieces / cm2 From cells approximately 4.5 × 10 5 pieces / cm 2 For example, a cell of approximately 1.5 × 10⁻⁶ 5 pieces / cm 2 From cells approximately 4.0 × 10 5 pieces / cm 2 For example, a cell of approximately 2.0 × 10⁻⁶ 5 pieces / cm 2 From cells approximately 3.5 × 10 5 pieces / cm 2 , or cells approximately 2.5 × 10 5 pieces / cm 2 From cells approximately 3.0 × 10 5 pieces / cm 2 It could be within the range of.

[0050] Oligodendrocyte progenitor cell composition Methods for generating a large number of high-purity, characterized oligodendrocyte progenitor cells from pluripotent stem cells have been previously described, for example, in U.S. Patent Application No. 15 / 156,316 and Provisional Patent Application No. 62 / 315,454. Induction of oligodendrocyte progenitor cells (OPCs) from pluripotent stem cells provides a renewable and scalable source of OPCs for numerous important therapeutic, research, development, and commercial purposes, including the treatment of acute spinal cord injury.

[0051] In certain embodiments, a method for generating a high-purity population of oligodendrocyte progenitor cells from pluripotent stem cells may include, for example, a pretreatment step in which cells are incubated with one or more modulators of stem cell differentiation, as described in U.S. Provisional Patent Application No. 62 / 315,454 filed March 30, 2016, and International Patent Application No. PCT / US2017 / 024986 filed March 30, 2017.

[0052] In one embodiment, the cell population may have a common genetic background. In one embodiment, the cell population may originate from a single host. In one embodiment, the cell population may originate from a pluripotent stem cell line. In another embodiment, the cell population may originate from an embryonic stem cell line. In one embodiment, the cell population may originate from an hESC line. In one embodiment, the hESC line may be an H1, H7, H9, H13, or H14 cell line. In another embodiment, the cell population may originate from an induced pluripotent stem cell (iPS) line. In one embodiment, the cell population may originate from a subject that needs it (for example, the cell population may originate from a subject that needs treatment). In yet another embodiment, the hESC line may originate from parthenogenetic germ cells, which are embryos stimulated to produce hESCs without fertilization.

[0053] In certain embodiments, the OPCs of this disclosure express one or more markers selected from nestin, NG2, Olig1, and PDGF-Rα. In certain embodiments, the OPCs of this disclosure express all of the markers nestin, NG2, Olig1, and PDGF-Rα. In some embodiments, at least 70% of AST-OPC1 are positive for nestin expression. In some embodiments, at least 30% of AST-OPC1 are positive for NG2 expression. In some embodiments, at least 70% of AST-OPC1 are positive for Olig1 expression. In some embodiments, at least 70% of AST-OPC1 are positive for PDGF-Rα expression. Specific markers and various marker combinations expressed by the cell populations of this disclosure can be determined and quantified, for example, by flow cytometry.

[0054] Pharmaceutical composition OPCs may be administered on their own to subjects in need of treatment. Alternatively, the cells of this disclosure may be administered to subjects in need of treatment in a pharmaceutical composition mixed with a suitable carrier and / or using a delivery system.

[0055] As used herein, the term “pharmaceutical composition” refers to a preparation comprising a therapeutic agent in combination with other components such as physiologically appropriate carriers and excipients.

[0056] As used herein, the term “therapeutic agent” may refer to the cells of the Disclosure that describe the biological effect in a subject. Depending on the embodiment of the Disclosure, “therapeutic agent” may refer to the oligodendrocyte progenitor cells of the Disclosure. Alternatively, “therapeutic agent” may refer to one or more factors secreted by the oligodendrocyte progenitor cells of the Disclosure.

[0057] As used herein, the terms “carrier,” “pharmaceutically acceptable carrier,” and “biologically acceptable carrier” may be used interchangeably and may refer to a diluent or carrier substance that does not cause significant adverse effects or irritation in the subject and does not inhibit the biological activity or effect of the therapeutic agent. In certain embodiments, the pharmaceutically acceptable carrier may include dimethyl sulfoxide (DMSO). In other embodiments, the pharmaceutically acceptable carrier does not include dimethyl sulfoxide. The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the therapeutic agent.

[0058] The therapeutic agents disclosed herein may be administered as components of a hydrogel, such as those described in U.S. Patent Application No. 14 / 275,795, filed on 12 May 2014, and U.S. Patents No. 8,324,184 and No. 7,928,069.

[0059] Compositions according to this disclosure may be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion. Formulations for injection may be presented in preservative-containing unit dosing forms, for example, ampoules or multi-dose containers. Compositions may contain formulation agents such as suspending agents, stabilizers and / or dispersants. In certain embodiments, compositions may be formulated to be suitable for cryopreservation.

[0060] Compositions according to this disclosure may be formulated for administration by direct injection into the spinal cord of a subject. In certain embodiments, compositions according to this disclosure may be formulated for intracerebral, intraventricular, intrathecal, intranasal, or intracisional administration to a subject. In certain embodiments, compositions according to this disclosure may be formulated for administration by direct injection into or immediately adjacent to an infarct cavity within the brain of a subject. In certain embodiments, compositions according to this disclosure may be formulated for administration by implantation. In certain embodiments, compositions according to this disclosure may be formulated as a solution.

[0061] In certain embodiments, the composition according to this disclosure contains approximately 1 × 10 cells per milliliter. 6 From one piece, approximately 5 x 10 8 For example, about 1 x 10 cells per milliliter 6 For example, about 2 x 10 cells per milliliter 6 For example, about 3 x 10 cells per milliliter 6 For example, about 4 x 10 cells per milliliter 6 For example, about 5 x 10 cells per milliliter 6 For example, about 6 x 10 cells per milliliter 6 For example, about 7 x 10 cells per milliliter 6 For example, about 8 x 10 cells per milliliter 6 For example, about 9 x 10 cells per milliliter 6 For example, about 1 x 10 cells per milliliter 7 For example, about 2 x 10 cells per milliliter 7 For example, about 3 x 10 cells per milliliter 7 For example, about 4 x 10 cells per milliliter 7 For example, about 5 x 10 cells per milliliter 7 For example, about 6 x 10 cells per milliliter 7 For example, about 7 x 10 cells per milliliter 7 For example, about 8 x 10 cells per milliliter 7 For example, about 9 x 10 cells per milliliter 7For example, about 1 x 10 cells per milliliter 8 For example, about 2 x 10 cells per milliliter 8 For example, about 3 x 10 cells per milliliter 8 For example, about 4 x 10 cells per milliliter 8 Approximately 5 x 10 cells per milliliter, or for example, 1 milliliter. 8 It may contain approximately 1 × 10 cells per milliliter. In certain embodiments, the composition according to this disclosure contains approximately 1 × 10 cells per milliliter. 8 From approximately 5 x 10 8 For example, about 1 x 10 cells per milliliter 8 From approximately 4 x 10 8 For example, about 2 x 10 cells per milliliter 8 From approximately 5 x 10 8 For example, about 1 x 10 cells per milliliter 8 From approximately 3 x 10 8 For example, about 2 x 10 cells per milliliter 8 From approximately 4 x 10 8 Approximately 3 x 10 cells per milliliter, or for example, 1 milliliter. 8 From approximately 5 x 10 8 It may contain approximately 1 × 10 cells per milliliter. In yet another embodiment, the composition according to the present disclosure contains approximately 1 × 10 cells per milliliter. 7 From approximately 1 x 10 8 For example, about 2 x 10 cells per milliliter 7 From approximately 9 x 10 7 For example, about 3 x 10 cells per milliliter 7 From approximately 8 x 10 7 For example, about 4 x 10 cells per milliliter 7 From approximately 7 x 10 7 Approximately 5 x 10 cells per milliliter, or for example, 1 milliliter. 7 From approximately 6 x 10 7 It may contain approximately 1 × 10 cells per milliliter. In one embodiment, a composition according to this disclosure contains approximately 1 × 10 cells per milliliter. 6 From approximately 1 x 10 7 For example, about 2 x 10 cells per milliliter 6 From approximately 9 x 10 6 For example, about 3 x 10 cells per milliliter6 From approximately 8 x 10 6 For example, about 4 x 10 cells per milliliter 6 From approximately 7 x 10 6 Approximately 5 x 10 cells per milliliter, or for example, 1 milliliter. 6 From approximately 6 x 10 6 It may contain at least about 1 × 10 cells per milliliter. In yet another embodiment, the composition according to the present disclosure contains at least about 1 × 10 cells per milliliter. 6 For example, at least about 2 × 10 cells per milliliter. 6 For example, at least 3 × 10 cells per milliliter 6 For example, at least 4 × 10 cells per milliliter 6 For example, at least 5 × 10 cells per milliliter 6 For example, at least 6 × 10 cells per milliliter 6 For example, at least 7 × 10 cells per milliliter 6 For example, at least 8 × 10 cells per milliliter. 6 For example, at least 9 x 10 cells per milliliter. 6 For example, at least about 1 × 10 cells per milliliter. 7 For example, at least about 2 × 10 cells per milliliter. 7 For example, at least approximately 3 × 10 cells per milliliter. 7 For example, at least 4 × 10 cells per milliliter 7 Each cell, or for example, at least about 5 × 10 cells per milliliter. 7 It may contain a number of cells. In one embodiment, a composition according to this disclosure contains a maximum of about 1 × 10 cells. 8 One or more cells, for example, up to approximately 2 x 10 cells per milliliter. 8 One or more cells, for example, up to approximately 3 x 10 cells per milliliter. 8 One or more cells, for example, up to approximately 4 x 10 cells per milliliter. 8 One or more cells, for example, up to approximately 5 x 10 cells per milliliter. 8 One or more cells, or for example, up to approximately 6 x 10 cells per milliliter.8 It may include individuals.

[0062] In one embodiment, a composition according to this disclosure contains approximately 4 × 10 cells per milliliter. 7 From approximately 2 x 10 8 It may include individuals.

[0063] In yet another embodiment, a composition according to the present disclosure may have a volume ranging from about 10 microliters to about 5 milliliters, for example, about 20 microliters, for example, about 30 microliters, for example, about 40 microliters, for example, about 50 microliters, for example, about 60 microliters, for example, about 70 microliters, for example, about 80 microliters, for example, about 90 microliters, for example, about 100 microliters, for example, about 200 microliters, for example, about 300 microliters, for example, about 400 microliters, for example, about 500 microliters, for example, about 600 microliters, for example, about 700 microliters, for example, about 800 microliters, for example, about 900 microliters, for example, about 1 milliliter, for example, about 1.5 milliliters, for example, about 2 milliliters, for example, about 2.5 milliliters, for example, about 3 milliliters, for example, about 3.5 milliliters, for example, about 4 milliliters, or for example, about 4.5 milliliters. In one embodiment, a composition according to the Disclosure may have a volume ranging from 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, a composition according to the Disclosure may have a volume ranging from 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, a composition according to the disclosure may have a volume ranging from 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 disclosure may have a volume ranging from about 20 microliters to about 500 microliters.In another embodiment, a composition according to the disclosure may have a volume of about 50 microliters to about 100 microliters. In yet another embodiment, a composition according to the disclosure may have a volume of about 50 microliters to about 200 microliters. In yet another embodiment, a composition according to the disclosure may have a volume of about 20 microliters to about 400 microliters.

[0064] In certain embodiments, the Disclosure provides a container comprising a composition comprising a population of OPCs derived according to one or more methods of the Disclosure. In certain embodiments, the container may be formed for cryopreservation. In certain embodiments, the container may be formed for administration to subjects requiring it. In certain embodiments, the container may be a pre-filled syringe.

[0065] For general principles in pharmaceutical formulation, readers should refer to *Allogeneic Stem Cell Transplantation*, edited by Lazarus and Laughlin, Springer Science+ Business Media LLC, 2010; and *Hematopoietic Stem Cell Therapy*, EDBall, J. Lister, and P. Law, Churchill Livingstone, 2000. The selection of cell excipients and any accompanying elements of the composition will be adapted according to the route and device used for administration. In certain embodiments, the composition may also include, or be accompanied by, one or more other components that facilitate the engraftment or functional mobilization of enriched target cells. Suitable components may include matrix proteins that support or promote the adhesion of the target cell type or promote angiogenesis in the transplanted tissue.

[0066] Use of cells in this disclosure In various embodiments described herein, the Disclosure provides methods for using a cell population comprising pluripotent stem cell-derived OPCs to improve one or more neurological functions in a subject requiring treatment. In certain embodiments, methods for using pluripotent stem cell-derived OPCs in the treatment of traumatic spinal cord injury are provided. In other embodiments, methods for using pluripotent stem cell-derived OPCs in the treatment of other traumatic CNS injuries are provided. In other embodiments, methods for using pluripotent stem cell-derived OPCs in the treatment of non-traumatic CNS injuries or conditions are provided. In certain embodiments, cell populations according to the Disclosure may be injected or transplanted into a subject requiring it.

[0067] In certain 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 pathologies requiring myelin repair or remyelination: multiple sclerosis, leukodystrophy, Guillain-Barré syndrome, Charcot-Marie-Tooth neuropathy, Ty-Sachs disease, Niemann-Pick disease, Gaucher disease, and Hurler syndrome. Other conditions resulting 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 this disclosure may also be used for myelin repair or remyelination in traumatic injuries resulting in loss of myelination, such as acute spinal cord injury.

[0068] OPCs are administered in a manner that allows them to engraft or migrate to the target tissue site and reconstruct or regenerate functionally deficient areas. Cell administration can be achieved by any method known in the art. For example, cells may be surgically administered directly to the organ or tissue requiring cell transplantation. Alternatively, non-invasive procedures may be used to administer cells to a target. Non-limiting examples of non-invasive delivery methods include the use of syringes and / or catheters to deliver cells to the organ or tissue requiring cell therapy.

[0069] Subjects receiving OPC of this disclosure may be treated to reduce immune rejection of transplanted cells. Possible methods include, for example, the administration of conventional immunosuppressants such as tacrolimus, cyclosporine A (Dunn et al., Drugs 61:1957,2001), or the induction of immune tolerance using a matched population of pluripotent stem cell-derived cells (WO02 / 44343; U.S. Patent No. 6,280,718; WO03 / 050251). Alternatively, a combination of an anti-inflammatory agent (such as prednisone) and an immunosuppressant may also be used. The OPC of the present invention may be supplied in the form of a pharmaceutical composition comprising an isotonic excipient prepared under sufficiently sterile conditions for administration to humans.

[0070] Use in the treatment of CNS traumatic injury. In certain embodiments, a cell population according to this disclosure may be able to engraft at the spinal cord injury site after a composition containing the cell population is implanted at the spinal cord injury site.

[0071] In certain embodiments, a cell population according to this disclosure can remain in the spinal cord injury site for a period of about 90 days or more after implantation of a certain dose of the composition. In other embodiments, a cell population according to this disclosure can remain in the spinal cord injury site for a period of about 1 year or more after implantation of a certain dose of the composition. In further embodiments, a cell population according to this disclosure can remain in the spinal cord injury site for a period of about 2 years or more after implantation of a certain dose of the composition. In further embodiments, a cell population according to this disclosure can remain in the spinal cord injury site for a period of about 3 years or more after implantation of a certain dose of the composition. In further embodiments, a cell population according to this disclosure can remain in the spinal cord injury site for a period of about 4 years or more after implantation of a certain dose of the composition. In further embodiments, a cell population according to this disclosure can remain in the spinal cord injury site for a period of about 5 years or more after implantation of a certain dose of the composition.

[0072] In certain embodiments, cell compositions according to this disclosure can improve upper limb motor function in human subjects having spinal cord injury when administered to said subjects. In certain embodiments, the subjects have cervical spinal cord injury. In other embodiments, the subjects have thoracic spinal cord injury.

[0073] In one embodiment, the present disclosure provides a method for improving upper limb motor function in a human subject having a spinal cord injury, the method comprising administering to the subject a composition comprising a population of allogeneic human oligodendrocyte cells capable of engrafting at the site of the spinal cord injury. In certain embodiments, the administration of the composition comprises injecting the composition into the site of the spinal cord injury. In some embodiments, the composition is injected approximately 2 to 10 mm caudal to the center of the spinal cord injury. In further embodiments, the composition is injected approximately 5 mm caudal to the center of the spinal cord injury. In some embodiments, the subject has a cervical spinal cord injury. In other embodiments, the subject has a thoracic spinal cord injury.

[0074] In certain embodiments, subjects administered a composition comprising a population of allogeneic human oligodendrocyte cells according to the method of this disclosure obtain an improvement in upper limb motor function equivalent to at least one motor level (as defined based on the International Standard for Neurological Classification of Spinal Cord Injuries [ISNCSCI]). The improvement in function may be unilateral or bilateral. In other embodiments, subjects administered a composition comprising a population of allogeneic human oligodendrocyte cells according to the method of this disclosure obtain an improvement in upper limb motor function equivalent to at least two motor levels on either one or both sides. In certain embodiments, subjects obtain an improvement in upper limb motor function equivalent to at least one motor level on one side and at least two motor levels on the other side. In certain embodiments, subjects exhibit an improved upper limb motor score (UEMS) compared to the subject's baseline score before administration of the population of allogeneic human oligodendrocyte cells according to the method of this disclosure.

[0075] Additional embodiments

[0076] Further embodiments of this disclosure include:

[0077] 1. A method for improving upper limb motor function in a human subject with traumatic spinal cord injury, comprising administering to the subject a therapeutically effective amount of a composition containing a population of allogeneic human oligodendrocyte progenitor cells.

[0078] 2. The method according to 1, wherein administering the composition comprises injecting the composition into the site of spinal cord injury.

[0079] 3. The method according to 2, wherein the composition is injected approximately 2 to 10 mm caudal to the center of the spinal cord injury.

[0080] 4. The method according to 2, wherein the composition is injected approximately 5 mm caudal to the center of the spinal cord injury.

[0081] 5. A method according to any one of 1-4, wherein human oligodendrocyte progenitor cells can engraft at the site of spinal cord injury.

[0082] 6. The method according to any one of 1 to 5, wherein the composition is administered between 15 and 60 days after the subject suffers a traumatic spinal cord injury.

[0083] 7. The method according to 6, wherein the composition is administered to the subject between 20 and 40 days after the subject suffers a traumatic spinal cord injury.

[0084] 8. The method according to any one of 1 to 7, further comprising administering a low-dose immunosuppressant regimen to the subject.

[0085] 9. The composition is approximately 2 × 10 6 From 50 x 10 6 A method according to any one of 1 to 8, comprising AST-OPC1 cells.

[0086] 10. The composition is approximately 10 x 10 6 The method according to 9, comprising AST-OPC1 cells.

[0087] 11. The composition is approximately 20 x 10 6 The method according to 9, comprising AST-OPC1 cells.

[0088] 12. The method described in any one of 1 to 11, wherein the subject has a cervical spinal cord injury.

[0089] 13. The method according to any one of 1 to 12, wherein the motor function of the subject's upper limb improves by at least two levels of motor function approximately 1 to 12 months after administration of the composition.

[0090] 14. The method described in 13, wherein the improvement in the subject's motor level is bilateral.

[0091] 15. The method according to 13, wherein the improvement in the subject's motor level is unilateral.

[0092] 16. The method according to 13, wherein the motor function of the subject's upper limb improves by at least two levels of motor function within approximately three months after administration of the composition.

[0093] 17. The method according to 13, wherein the motor function of the subject's upper limb improves by at least two levels of motor function approximately 12 months after administration of the composition.

[0094] 18. The method according to any one of 1 to 17, wherein allogeneic human oligodendrocyte progenitor cells are offspring differentiated in vitro from pluripotent stem cells.

[0095] 19. The method according to 18, wherein the pluripotent stem cells are human embryonic stem cells.

[0096] 20. A pharmaceutical composition for use in improving upper limb motor function in human subjects with traumatic spinal cord injury, comprising a population of allogeneic human oligodendrocyte progenitor cells.

[0097] 21. The pharmaceutical composition according to 20, further comprising a bioacceptable carrier.

[0098] 22. The pharmaceutical composition according to 20-21, wherein allogeneic human oligodendrocyte progenitor cells are offspring differentiated in vitro from pluripotent stem cells.

[0099] 23. The pharmaceutical composition according to 22, wherein the pluripotent stem cells are human embryonic stem cells.

[0100] 24. A pharmaceutical composition for use in the treatment of traumatic spinal cord injury in human subjects, comprising a population of allogeneic human oligodendrocyte progenitor cells.

[0101] 25. The pharmaceutical composition according to 24, further comprising a bioacceptable carrier.

[0102] 26. The pharmaceutical composition according to 24-25, wherein allogeneic human oligodendrocyte progenitor cells are offspring differentiated in vitro from pluripotent stem cells.

[0103] 27. The pharmaceutical composition according to 26, wherein the pluripotent stem cells are human embryonic stem cells.

[0104] Examples The following examples are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent all or only experiments that have been conducted. [Examples]

[0105] Example 1: Phase 1 / 2a dose escalation study of AST-OPC1 in patients with complete motor impairment of C4-C7 cervical spinal cord injury. AST-OPC1 cells were generated by differentiation of WA01(H1)hESCs from a master cell bank (MCB), as described in U.S. Patent Application No. 15 / 136,316.

[0106] The initial clinical safety of AST-OPC1 was previously evaluated in a Phase 1 clinical trial enrolling patients with neurologically complete T3–T11 thoracic spinal cord injury (SCI). Based on promising 5-year safety data from the Phase 1 trial, a Phase 1 / 2a trial was initiated to evaluate the safety and activity of escalating doses of AST-OPC1 in patients with complete sensorimotor C5–C7 cervical spinal cord injury.

[0107] In the Phase 1 trial, five subjects received 2 × 10⁶ doses 7 to 14 days after injury. 6 Each patient received AST-OPC1. The Phase 1 / 2a trial involved 2 × 10 between 14 and 40 days after SCI. 6 pieces, 10×10 6 1 or 20 x 10 6 Participants are being enrolled in a continuous dosing cohort receiving individual doses of AST-OPC1, and enrollment is planned to continue. The Phase 1 / 2a trial design is shown in Figure 1; the cohort design is shown in Figure 2. Participants will be followed for one year under the primary trial protocol and will be followed for a further 14 years under the long-term follow-up protocol.

[0108] In both Phase 1 and Phase 1 / 2a trials, AST-OPC1 demonstrated a robust safety profile.

[0109] Cohort 1 (2 × 10) available in September 2016 6 Individual AST-OPC1) and Cohort 2 (10 × 10) 6 The results of initial upper limb motor function recovery for individual AST-OPC1 patients are shown in Figures 4A (UEMS) and 4B (Motor Level Recovery). The results of motor function recovery for cohorts 1 and 2 through a 12-month follow-up are shown in Figures 6A and 6B (UEMS) and Figure 7 (Motor Level Recovery). [Examples]

[0110] Example 2: Comparison of improvement in upper limb motor function in patients from cohorts 1 and 2 compared to matched historical controls. The improvement in motor function in subjects of cohorts 1 and 2 after administration of AST-OPC1 was compared to a closely matched historical group of traumatic SCI patients obtained from the EMSCI (European Multicenter Study about Spinal Cord Injury) database of over 3300 cases. The matching criteria used to generate closely matched historical control data are shown in Figure 5.

[0111] Comparative data from a 12-month follow-up study are shown in Figures 6A, 6B, and 7.

[0112] Figure 6A: Subjects of Cohort 2 measured by changes in UEMS over time (10 × 10 6 Motor function recovery in individuals with AST-OPC1 was favorable compared to closely matched historical controls, showing significant improvement by 3 months and continuously increasing throughout 12 months. Figure 6B: As expected, in cohort 1 subjects (2 × 10⁶ 6The exercise function recovery (UEMS) at individual doses of AST-OPC1 (the same dose used in the Phase 1 safety trial) was similar to that of matched historical controls, further supporting the safety of AST-OPC1. A comparison of exercise score improvements between Cohorts 1 and 2 compared to the EMSCI historical control group supports a dose-dependent effect of AST-OPC1 on exercise score recovery. Figure 7: Exercise function recovery in Cohort 2 subjects was measured as improvement in exercise level over time relative to baseline measurements throughout a 12-month follow-up. These improvements were compared to those of closely matched historical controls from the EMSCI database. By 6 months after AST-OPC1 administration, 33% (2 / 6) of Cohort 2 subjects achieved an improvement of at least two levels in exercise level on at least one side. By 12 months, 67% (4 / 6) of Cohort 2 subjects achieved an improvement of at least two levels in exercise level on at least one side. In comparison, 29% of closely matched historical controls achieved an improvement of at least two levels in motor function on one side by 12 months after SCI. The percentage of subjects in Cohort 2 who achieved an improvement of two levels or more in motor function was significantly higher than both the motor function recovery rate in closely matched historical controls (29%) and the recovery rate reported in the literature (26%, Steeves JD et al., Outcome Measures for Acute / Subacute Cervical Sensorimotor Complete (AIS-A) Spinal Cord Injury During a Phase 2 Clinical Trial, Top Spinal Cord Inj Rehabil 2012;18(1):1014).

Claims

1. A method for improving upper limb motor function in a human subject with traumatic spinal cord injury, comprising administering to the subject a therapeutically effective amount of a composition containing a population of allogeneic human oligodendrocyte progenitor cells.

2. The method according to claim 1, wherein administering the composition includes injecting the composition into the site of spinal cord injury.

3. The method according to claim 2, wherein the composition is injected approximately 2 to 10 mm caudal to the center of the spinal cord injury.

4. The method according to claim 3, wherein the composition is injected approximately 5 mm caudal to the center of the spinal cord injury.

5. The method according to claim 2, wherein human oligodendrocyte progenitor cells can engraft at the site of spinal cord injury.

6. The method according to claim 1, wherein the composition is administered to the subject between 15 and 60 days after the subject suffers a traumatic spinal cord injury.

7. The method according to claim 6, wherein the composition is administered to the subject between 20 and 40 days after the subject suffers a traumatic spinal cord injury.

8. The method according to claim 1, further comprising administering a low-dose immunosuppressant regimen to the subject.

9. The composition is approximately 2 x 10 ６ From 50 x 10 ６ The method according to claim 1, comprising AST-OPC1 cells.

10. The composition is approximately 10 x 10 ６ The method according to claim 9, comprising AST-OPC1 cells.

11. The composition is approximately 20 x 10 ６ The method according to claim 9, comprising AST-OPC1 cells.

12. The method according to claim 1, wherein the subject has a cervical spinal cord injury.

13. The method according to claim 1, wherein the motor function of the target upper limb improves by at least two levels of motor function approximately 1 to 12 months after administration of the composition.

14. The method according to claim 13, wherein the improvement in the target's exercise level is bilateral.

15. The method according to claim 13, wherein the improvement in the target's exercise level is unilateral.

16. The method according to claim 13, wherein the motor function of the upper limb of the subject improves by at least two levels of motor function within approximately three months after administration of the composition.

17. The method according to claim 13, wherein the motor function of the upper limb of the subject improves by at least two levels of motor function approximately 12 months after administration of the composition.

18. The method according to any one of claims 1 to 17, wherein the allogeneic human oligodendrocyte progenitor cells are offspring differentiated in vitro from pluripotent stem cells.

19. The method according to claim 18, wherein the pluripotent stem cells are human embryonic stem cells.

20. A pharmaceutical composition for use in improving upper limb motor function in human subjects with traumatic spinal cord injury, comprising a population of allogeneic human oligodendrocyte progenitor cells.

21. The pharmaceutical composition according to claim 20, further comprising a bioacceptable carrier.

22. The pharmaceutical composition according to claim 20, wherein the allogeneic human oligodendrocyte progenitor cells are offspring differentiated in vitro from pluripotent stem cells.

23. The pharmaceutical composition according to claim 22, wherein the pluripotent stem cells are human embryonic stem cells.

24. A pharmaceutical composition for use in the treatment of traumatic spinal cord injury in human subjects, comprising a population of allogeneic human oligodendrocyte progenitor cells.

25. The pharmaceutical composition according to claim 24, further comprising a bioacceptable carrier.

26. The pharmaceutical composition according to claim 24, wherein the allogeneic human oligodendrocyte progenitor cells are offspring differentiated in vitro from pluripotent stem cells.

27. The pharmaceutical composition according to claim 26, wherein the pluripotent stem cells are human embryonic stem cells.

Images