Compositions and methods for treating spinal cord injury

Abstract

Provided herein are methods, compositions, and devices for treating neurological disorders and diseases, including spinal cord injury.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 304,405, filed January 28, 2022, and U.S. Provisional Application No. 63 / 327,253, filed April 4, 2022, which are incorporated by reference in their entireties herein.

[0002] Sequence Listing Reference This application contains an electronically submitted Sequence Listing, which is incorporated herein by reference in its entirety. The Sequence Listing can be found in the file entitled "058125-646001WO_SL_ST26.xml", created on Jan. 27, 2023, and is approximately 3.43 KB in size. [Background technology]

[0003] 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 lose mobility and are often wheelchair bound, in addition to numerous medical complications. It is estimated that more than 12,000 Americans suffer from spinal cord injury (SCI) each year, with approximately 1.3 million people living with spinal cord injury in the United States. Traumatic SCI most commonly occurs in people in their 20s and 30s, resulting in severe and permanent disability in young, previously healthy individuals. In addition to impaired limb function, individuals with SCI suffer from bowel and bladder dysfunction, decreased sensation, spasticity, autonomic reflex disorders, 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 be life threatening. The life expectancy of a person who suffers from a cervical spinal cord injury at age 20 years is 20-25 years shorter than a person of the same age without SCI (NSCISC Spinal Cord Injury Facts and Figures 2013). To date, there are no therapies approved by the US Food and Drug Administration (FDA) to induce neurological recovery after spinal cord injury (SCI). Several interventions have been studied in clinical trials, including glucocorticoids, modulation of voltage-gated channels, tetracycline antibiotics, and cell-based therapies, but so far none have met key enrollment endpoints.

[0004] The clinical impact of spinal cord injury varies depending on the site and extent of the injury. Permanent destruction of the nervous system below the level of injury not only results in loss of limb muscle control and the protective role of temperature and pain sensation, but also affects 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 lead to a cascade of secondary problems, such as bedsores and urinary tract infections, which were rapidly fatal before modern medicine. Spinal cord injury often results in the loss of the unconscious control mechanisms that maintain an appropriate level of excitability in the neural circuits of the spinal cord. As a result, spinal motor neurons can become spontaneously hyperactive, causing debilitating stiffness and uncontrollable muscle spasms or contractures. This hyperactivity can also cause unpleasant sensations in the sensory system, such as chronic neuropathic pain and paresthesia, numbness, tingling, pain, and burning. In a recent opinion survey of patients with spinal cord injury, regaining walking function was not the most desired function that patients wanted to regain, and in many cases, alleviating the aftereffects of spontaneous hyperactivity was given the highest priority (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 (Non-Patent Document 2)).

[0005] A need exists for treating spinal cord injuries and associated pathologies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] NSCISC Spinal Cord Injury Facts and Figures 2013 [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):334-8 Summary of the Invention

[0007] overview The examples and embodiments presented herein describe human embryonic stem cell (hESC) derived cells for the treatment of spinal cord injury (SCI), as detailed herein.

[0008] For example, oligodendrocyte precursor cell (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, an OPC cell population according to the present disclosure can be injected, implanted, or otherwise delivered to a subject in need thereof. In one embodiment, a cell population according to the present disclosure can be implanted, or otherwise delivered to a subject in need thereof for the treatment of spinal cord injury, stroke, or multiple sclerosis.

[0009] 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 lines of hESC.AST-OPC1 (formerly 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).

[0010] Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells in the central nervous system (CNS) that originate in the ventricular region of the brain and the spinal cord and migrate throughout the developing CNS before maturing into oligodendrocytes. Mature oligodendrocytes generate the myelin sheath that insulates nerve axons and remyelinate CNS lesions where myelin sheaths have been lost. Oligodendrocytes also contribute to neuroprotection through other mechanisms, such as 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 most progenitor cells, OPCs remain abundant in the adult CNS and retain the capacity to generate new oligodendrocytes. Thus, OPCs, and the mature oligodendrocytes derived from OPCs, are important therapeutic targets for demyelinating and dysmyelinating disorders (such as multiple sclerosis, adrenoleukodystrophy, and adrenomyeloneuropathy), 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)).

[0011] The OPC composition 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.

[0012] In certain embodiments, the OPC1 composition is administered after the subject has suffered a traumatic spinal cord injury. In some embodiments, the OPC1 composition is administered 14-90 days after the spinal cord injury, e.g., 14-75 days after the spinal cord injury, e.g., 14-60 days after the spinal cord injury, e.g., 14-30 days after the injury, e.g., 20-75 days after the injury, e.g., 20-60 days after the injury, and e.g., 20-40 days after the injury. In certain embodiments, the OPC1 composition is administered about 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10 0, 51, 52, 53, 54, 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days later. In certain embodiments, the OPC1 composition is administered between day 14 and the life of the subject.

[0013] Methods and compositions for obtaining cell populations comprising dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells are described 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.

[0014] In one aspect, a method of improving one or more neurological functions in a subject having a spinal cord injury (SCI) is provided, the method 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. In some embodiments, the first dose of the composition is administered using a parenchymal spinal delivery (PSD) system. In some embodiments, the PSD is a system as described herein and shown, for example, in Figures 39-41. See also U.S. Publication No. 2015 / 0224331 (incorporated herein by reference in its entirety for all devices, methods, etc. described therein).

[0015] 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 to the spinal cord of the subject, for example, by injection. In some embodiments, each administration comprises delivering two or more portions of a dose. In some embodiments, the second dose of the composition is administered using a parenchymal spinal delivery (PSD) system, such as those systems described herein and shown, for example, in Figures 39-41. See also U.S. Publication No. 2015 / 0224331 (incorporated herein by reference in its entirety for all devices, methods, etc. described therein).

[0016] In some embodiments, the third dose of the composition using a parenchymal spinal delivery (PSD) system is as shown in Figures 39-41.

[0017] 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 is about 1×10 6 ~Approx. 3×10 7In 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. 7 The OPC cells are included. In some embodiments, the first, second, and third doses of the composition are each administered about 20 to about 45 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 14 to about 90 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 14 to about 75 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 14 to about 60 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 14 to about 30 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 20 to about 75 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 20 to about 60 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 20 to about 40 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 14 days after SCI to within the life of the subject. In some embodiments, the injection is administered in the caudal half of the epicenter 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.

[0018] In another aspect, there is provided a method of improving one or more neurological functions in a subject having a spinal cord injury (SCI), the method comprising administering to the subject a dose of a composition comprising oligodendrocyte precursor cells (OPCs) derived from human pluripotent stem cells.

[0019] In some embodiments, the dose of the composition is about 1×10 6 ~Approx. 3×10 7 In some embodiments, the dose of the composition comprises about 2×10 OPC cells. 6OPC cells. In some embodiments, administering the composition includes injecting, implanting, or otherwise delivering the composition to the spinal cord of the subject. In some embodiments, the dose of the composition is administered about 7 to about 14 days after the SCI. In some embodiments, the injection is performed in the caudal half of the epicenter 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, the improvement in one or more neurological functions includes an improvement in ISNCSCI Upper Extremity Motor Score (UEMS). In some embodiments, the improvement in UEMS occurs within about 6 months, about 12 months, about 18 months, 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, the improvement in one or more neurological functions includes an improvement in a lower extremity motor score (LEMS). In some embodiments, the improvement in the LEMS occurs within about 6 months, about 12 months, about 18 months, about 24 months, or more after the 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 an improvement in one side of the subject's body. In some embodiments, the improvement is an improvement in both sides of the subject's body. In some embodiments, the dose of the composition is administered about 14 to about 90 days after the SCI. In some embodiments, the dose of the composition is administered about 14 to about 75 days after the SCI. In some embodiments, the dose of the composition is administered about 14 to about 60 days after the SCI. In some embodiments, the dose of the composition is administered about 14 to about 30 days after the SCI. In some embodiments, the dose of the composition is administered about 20 to about 75 days after the SCI. In some embodiments, the dose of the composition is administered about 20 to about 60 days after the SCI.In some embodiments, the dose of the composition is administered from about 20 to about 40 days after the SCI, hi some embodiments, the dose of the composition is administered from about 14 days after the SCI to within the life of the subject.

[0020] In another aspect, a cell population is provided having an increased percentage of oligodendrocyte progenitor marker NG2 positive cells and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM, the cell population being 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 progenitor cells (OPCs) having an increased percentage of oligodendrocyte progenitor marker NG2 positive cells and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM.

[0021] 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 method. In some embodiments, the composition is administered by injection to the subject after SCI. In some embodiments, the injection is administered in the caudal half of the epicenter 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 to about 90 days after the SCI. In some embodiments, the injection is administered about 14 to about 75 days after the SCI. In some embodiments, the injection is administered about 14 to about 60 days after the SCI. In some embodiments, the injection is administered from about 14 to about 30 days after SCI. In some embodiments, the injection is administered from about 20 to about 75 days after SCI. In some embodiments, the injection is administered from about 20 to about 60 days after SCI. In some embodiments, the injection is administered from about 20 to about 40 days after SCI. In some embodiments, the injection is administered from about 14 days after SCI to within the subject's lifetime.

[0022] In another aspect, a method of improving one or more neurological functions in a subject having a spinal cord injury (SCI) is provided, the method 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.

[0023] 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 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 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 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 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 to about 75 days after the SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 20 to about 60 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 20 to about 40 days after SCI. In some embodiments, the first, second, and third doses of the composition are each administered about 14 days after SCI through the lifetime of the subject.

[0024] In some embodiments, the composition is administered using a parenchymal spinal delivery (PSD) system, which in some embodiments includes a device that includes a system as shown in any of Figures 39-41.

[0025] In one embodiment, the PSD system comprises: (a) a frame having an elongated body and a plurality of holders extending therefrom; (b) a plurality of first magnets, each fixed to a holder; (c) a tube having a first end and a second end, the tube being slidably disposed within a through hole disposed in each of the holders and each of the first magnets; (d) a plurality of second magnets secured to an exterior surface of the tube; and (e) a needle secured to a first end of the tube; An apparatus comprising: Includes.

[0026] In one embodiment, each of the first magnets and each of the second magnets are arranged such that the north pole of one of the first magnets faces the north pole of one of the second magnets, or the south pole of one of the first magnets faces the south pole of one of the second magnets, thereby providing a magnetic repulsion force that suspends the tube.

[0027] In some embodiments, the PSD system further comprises a reservoir in fluid communication with the needle, hi some embodiments, the reservoir contains a composition described herein.

[0028] In certain embodiments, the PSD system further comprises a digital microinjector configured to control the flow of the composition through the needle.

[0029] In some embodiments, the device includes a stopper adjacent to the needle, hi some embodiments, the stopper prevents the needle from advancing further into the spinal cord.

[0030] In some embodiments, the subject is not removed from the ventilator during administration.

[0031] In one embodiment, administering the composition comprises: (i) positioning a PSD system over the spinal cord of a subject; (ii) lowering the needle into the spinal cord; and (iii) delivering a dose of the composition to the spinal cord; Includes.

[0032] In one embodiment, the needle and tube of the device are suspended by magnetic repulsion within the device to compensate for spinal pulsation.

[0033] In one embodiment, step (ii) comprises lowering the needle until the stopper rests on the spinal cord.

[0034] In some embodiments, a parenchymal spinal delivery (PSD) system is provided, the system comprising: (i) a device as shown in any of Figures 39-41 or described herein, comprising a needle; and (ii) a reservoir in fluid communication with the needle, the reservoir containing a composition described herein.

[0035] In an embodiment, the apparatus comprises: (a) a frame having an elongated body and a plurality of holders extending therefrom; (b) a plurality of first magnets, each fixed to a holder; (c) a tube having a first end and a second end, the tube being slidably disposed within the through holes disposed in each of the holders and each of the first magnets; (d) a plurality of second magnets secured to an exterior surface of the tube; and (e) a needle secured to a first end of the tube; Includes.

[0036] In an embodiment, each of the first magnets and each of the second magnets are arranged such that the north pole of one of the first magnets faces the north pole of one of the second magnets, or the south pole of one of the first magnets faces the south pole of one of the second magnets, thereby providing a magnetic repulsion force for suspending the tube. In an embodiment, the system includes a microinjector or microinjection pump configured to control the flow of the composition through the needle. In an embodiment, the device includes an XYZ manipulator for manipulating the position of the needle. In an embodiment, the device includes a stopper adjacent to the needle. In an embodiment, the stopper prevents the needle from advancing further into the spinal cord. [Brief description of the drawings]

[0037] Embodiments will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0038] [Figure 1]1 is a schematic timeline of Phase 1 clinical trials. [Diagram 2] Overview of patient screening, treatment, and follow-up during Phase 1 clinical trials (CONSORT flow diagram). [Figure 3-1] International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) screening and 5-year follow-up (*1 ISNSCI performed at 4 years). White in the figure indicates normal movement and / or sensation, left-to-right hatching indicates absence of movement and / or sensation, and right-to-left hatching indicates presence but abnormal sensation. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4] This is an example of a survey administered in a longitudinal protocol, which required annual visits from years 2 to 5. After the annual visit in year 5, follow-up was performed with annual telephone surveys. [Diagram 5] This is an outline of the research topic. [Figure 6] 1 is a schematic timeline of clinical trials. [Figure 7] FIG. 1 is a schematic diagram of patient screening and treatment during clinical trials. [Figure 8] FIG. 1 is a schematic diagram of the cohort structure and enrollment progress of a clinical trial consistent with the implementation of the present disclosure. [Figure 9] 1 is a schematic timeline of another Phase 1 clinical trial consistent with the implementation of the present disclosure. [Figure 10] 1 is an overview of two exemplary cell manufacturing processes consistent with the practice of the present disclosure. [Figure 11] 1 is a flow chart outlining the signaling sequence of a cell differentiation process consistent with the practice of the present disclosure. [Figure 12] 1 is a flowchart of a manufacturing process flow consistent with the practice of the present disclosure. [Figure 13]Schematic of SCI treatment goals. Loss of motor skills is the primary feature of spinal cord injury. More severe injuries result in more widespread disabilities. Improved motor function, especially of the upper limbs, offers significant benefits for self-care and reduces care costs. The goal of cell therapy is to provide additional function of the arm, hand, and fingers, improving independence and quality of life. [Figure 14] Schematic diagram illustrating OPC1 cells for SCI. Replacing oligodendrocytes may provide additional function of the upper limbs and fingers, improving the patient's quality of life. OPC1 consists of OPCs (oligodendrocyte precursor cells). OPCs are the precursor cells of oligodendrocytes, the myelin-forming cells of the central nervous system, which provide insulation to nerve axons in the form of myelin sheaths. Myelin sheaths are necessary for neurons to function properly. OPC1 cells are injected into the spinal cord at the site of injury. [Figure 15-1] FIG. 1 is a schematic diagram of the three major physiologically relevant functional activities of OPC1 cells. [Figure 15-2] See description of Figure 15-1. [Figure 16] 13 is an image showing prevention of cavity formation by OPC1 cells. [Figure 17] Image showing OPC1: hESC-derived oligodendrocyte precursor cells (OPC). OPC1 is a cryopreserved allogeneic cell population. It is derived from an NIH-registered human embryonic stem cell line (hESC). It is a characterized cell composition consisting of oligodendrocyte precursor cells, neural precursor cells, low frequency mature neural cells, and other characterized rare cell types. OPC1 has three identified functions: producing neurotrophic factors, inducing remyelination, and inducing angiogenesis. OPC1 can be used for "off-the-shelf" administration. Its first indication is spinal cord injury. Its efficacy could be expanded to other neurodegenerative diseases. [Figure 18]FIG. 1 is a schematic diagram showing the OPC1 injection procedure. Injections are performed using a table-mounted syringe positioning device (SPD). The compositions are administered by direct intraparenchymal injection into the spinal cord lesion. The dose consists of a single 50 μL injection for both the 2M and 10M doses, and two injections for the 20M dose. There are no intraoperative complications. [Figure 19-1] An image showing the international standards for the neurological classification of spinal cord injury. [Figure 19-2] See description of Figure 19-1. [Figure 20] FIG. 1 is a schematic diagram showing a chest exam schema. [Figure 21] 1 is an image showing adverse events and serious adverse events from the chest study. [Figure 22] 1 is an image showing serious adverse events from the chest study. [Figure 23] Table showing the chest study ISNCSCI study, baseline, 1 year and 5 years. Table shows total sensory score (TSS), upper limb motor score (UEMS), lower limb motor score (LEMS), neurological impairment level (NLI), motor NLI, partially residual sensory function (ZPP), motor ZPP and ASIA Impairment Score (AIS) grade at baseline, 1 year and 5 years. All 5 participants had AIS grade A at enrollment and none converted to AIS B. The highest and lowest NLI enrolled in the study were T3 and T8, respectively. Only the individual with T3 NLI improved to T4 and the sensory ZPP, initially T4 on both sides, was noted to have improved to T5 on the left and T6 on the right at 1 year follow-up. In total, 3 of 5 participants experienced at least 1 level improvement in ZPP. ND=not measurable. [Figure 24-1] 1 is an image showing the sensory results of the chest exam and a graphical representation of each patient's sensory function at baseline and 5 years after administration of OPC1, where white indicates normal movement and / or sensation, left to right hatching indicates absence of movement and / or sensation, and right to left hatching indicates presence but abnormal sensation. [Figure 24-2] See description of Figure 24-1. [Figure 24-3]See description of Figure 24-1. [Figure 25-1] An image showing the international standards for the neurological classification of spinal cord injury. [Figure 25-2] See description of Figure 25-1. [Figure 26] 2Image showing the benefits of improved physical activity levels, which translates into clinically meaningful improvements in self-care and reduced costs of care. [Figure 27] FIG. 1 is a schematic showing the study design for the cervical clinical trial. [Figure 28] Images showing 1-year follow-up of the cervical study. [Figure 29] FIG. 1 is a schematic diagram showing the cervical testing scheme. [Diagram 30] Image showing summary of adverse events in the neck study. The majority of adverse events in the neck study were mild to moderate. Eight of eleven adverse events related to tacrolimus were hypomagnesemia, and 20 were related to the injection procedure (nausea, vomiting, postoperative wound infection, autonomic dysreflexia, procedural pain, muscle pain, neck pain, headache, incision drainage, cerebrospinal fluid retention, and cerebrospinal fluid leak). [Diagram 31] Images showing motor function gain from cervical testing: 2 / 3 (68%) had 1 level and 1 / 4 (25%) had 2 levels. Regarding spontaneous recovery, Steeves et al. reported that "The percentage of people living with a cervical (C4-C7) sensorimotor complete (AIS A) SCI who spontaneously regained upper limb motor level 1 or >2 [right or left side, whichever is greater] at [48 weeks] postinjury was 68% at 1 level and 26% at 2 levels, respectively." [Diagram 32] FIG. 1 is a schematic diagram showing upper extremity motor score (UEMS) recovery in cohorts 2-5. [Diagram 33] 13 is an image showing neck test assessment of UEMS changes (12 months post-injection for key variables). [Diagram 34]Images showing analysis of patients with the least UEMS recovery from cervical testing. C4 or spinal cord compression occurred in 5 of the 7 worst patient outcomes, and both issues may be addressed in the next study. Two patients had spinal cord compression after OPC1 injection (17 and 23 on days 30 and 7, respectively). Patients 15, 23, and 18 had a C4 (highest / most severe) injury level at baseline. Patient 15 had a spinal cord hematoma at baseline and failed engraftment. [Diagram 35] Shows images of subject 15 (cohort 3) with a hematoma in the spinal cord, indicating that some lesions may be too severe for OPC1 cell survival. [Diagram 36] Shown are images of subject 17 (cohort 4) presenting with spinal cord compression on day 30 of the cervical study. [Figure 37] Schematic showing 12-month MRI results supporting persistent engraftment of OPC1 cells. Cavitation is estimated to occur in approximately 80% of spinal cord injury patients who meet the inclusion criteria for the cervical study. 96% (24 / 25) of participants underwent serial MRI scans after 12 months with no signs of lesion cavitation. The MRI results were consistent with the formation of a tissue matrix at the injury site, which is considered supporting evidence that OPC1 cells persistently engraft to prevent cavitation at the injury site (see Wirth et al. Exp Neurology 1995). [Figure 38] 1 is an image showing the original syringe positioning device in a cervical exam. [Figure 39] 1 is an image showing a parenchymal spinal delivery (PSD) system. [Diagram 40] 1 is an image showing an overview of the PSD system. [Diagram 41] 1 is an image showing an overview of the PSD system. [Diagram 42] 1 is a bar graph showing improvement in OPC1 production. [Diagram 43] 1 is a graph showing the movable range of a magnetic needle (DW-29002). [Diagram 44] 1 shows the magnetic needle test setup. [Diagram 45] 1 shows characteristic curves of repulsive load against displacement. A is a graph showing the repulsive force of a magnetic needle. B is a table showing the needle displacement. [Figure 46A] 1 shows a video analysis of magnetic needle deflection during simulated use in a porcine spine model, showing initial penetration. [Figure 46B] 13 shows a video analysis of magnetic needle deflection during simulated use in a porcine spine model, with final penetration shown. [Figure 46C] 1 shows a video analysis of magnetic needle deflection during simulated use in a porcine spine model. The needle is shown in a resting position. [Figure 46D] 13 shows a video analysis of magnetic needle deflection during simulated use in a porcine spine model, demonstrating respiratory displacement. [Figure 47] 1 is a graph showing force and deflection at the point of use. [Figure 48] 1 shows the initial fixture setup and loading of the magnetic needle assembly. [Figure 49] 1 shows the tightening screw that holds the syringe mount fastener. [Figure 50] 1 shows a magnetic needle assembly. [Figure 51] A test frame is shown. [Figure 52] 1 is a graph showing a load-displacement curve of sample B1. [Figure 53] 1 is a graph showing a load-displacement curve of sample B2. [Figure 54] 1 is a graph showing a load-displacement curve of sample B3. [Figure 55] 1 is a graph showing a load-displacement curve of sample B4. [Figure 56] 1 is a graph showing a load-displacement curve of sample B5. [Figure 57] 1 is a graph showing a load-displacement curve of sample B6. [Figure 58] 1 is a graph showing a load-displacement curve of sample B7. [Figure 59] 1 is a graph showing a load-displacement curve of sample B8. [Figure 60] 1 is a graph showing a load-displacement curve of sample B9. [Figure 61] 1 is a graph showing a load-displacement curve of sample B10. [Figure 62] 1 is a graph showing a load-displacement curve of sample B11. [Figure 63] 1 is a graph showing a load-displacement curve of sample B12. [Figure 64] 1 is a graph showing a load-displacement curve of sample B13. [Figure 65] 1 is a graph showing a load-displacement curve of sample B14. [Figure 66] 1 is a graph showing a load-displacement curve of sample B15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description Before describing the compositions and methods of the present invention, it is to be understood that the disclosure of the present invention is not limited to the specific processes, compositions, or methodologies described, as these may vary widely. It is also to be understood that the terms used in the description are merely for the purpose of describing the particular versions or embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. For example, features described with respect to one embodiment may be incorporated in other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. Thus, the present disclosure contemplates that some embodiments of the present disclosure may exclude or omit features or combinations of features described herein. Moreover, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, and do not depart from the present disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail in order not to unnecessarily obscure the present invention. It is not intended that any part of this specification be construed as disclaiming any portion of the full scope of the present invention. Accordingly, the following description is intended to describe certain aspects of the present disclosure, and is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description of the disclosure herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the disclosure.

[0041] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0042] Unless otherwise indicated by context, it is specifically intended that the various features of the present disclosure described herein can be used in any combination. Further, the present disclosure also contemplates that in some embodiments of the present disclosure, any feature or combination of features described herein can be excluded or omitted.

[0043] The methods disclosed herein may include one or more steps or acts to achieve the described method. The steps and / or acts of the methods 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 acts is required for the proper operation of the present embodiment, the order and / or use of specific steps and / or acts may be modified without departing from the scope of the present invention.

[0044] 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 as well, unless the context clearly indicates otherwise.

[0045] 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 absence of a combination when interpreted in the alternative ("or").

[0046] As used herein, the terms "about" and "approximately" 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 ±0.1% of the specified amount.

[0047] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y", and phrases such as "about X to Y" mean "about X to about Y".

[0048] 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 the specific differentiation protocol disclosed herein.

[0049] Compositional analysis of AST-OPC1 by immunocytochemistry (ICC), flow cytometry, and quantitative polymerase chain reaction (qPCR) indicates that the cell population consists 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 within the population are epithelial cells. Mesodermal, endodermal lineage cells, and uhESCs are generally below quantification or detection of the assay.

[0050] 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 normally express the characteristic markers nestin, NG2, and PDGF-Ra.

[0051] As used herein, the terms "treatment", "treat", "treated", or "treating" may refer to both therapeutic treatment or prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological condition, symptom, disorder, or disease, or to obtain a beneficial or desired clinical outcome. In some embodiments, the term may refer to both treatment and prevention. For purposes of this disclosure, beneficial or desired clinical outcomes may include, but are not limited to, one or more of the following: alleviation of symptoms, reduction in the extent of the condition, disorder, or disease, stabilization (i.e., no progression) of the condition, disorder, or disease state, delay in onset or slowing of progression of the condition, disorder, or disease, improvement of the condition, disorder, or disease state, and detectable or undetectable remission (partial or complete), or improvement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response. Treatment also includes prolonging survival compared to expected survival in the absence of treatment.

[0052] As used herein, the term "subject" includes, but is not limited to, humans, non-human primates, and non-human vertebrates, wild, domestic, and livestock animals, including any mammal, 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.

[0053] As used herein, "implantation" or "implantation" refers to administering a cell population to a target tissue using a suitable delivery technique (e.g., an injection device, an implantation device, or other delivery device). In some examples, the composition is administered using a parenchymal spinal delivery (PSD) system. In some examples, the composition is administered using a PSD system described herein. In some examples, the composition is administered using a PSD described in U.S. Publication No. 2015 / 0224331, the entirety of which is incorporated herein by reference.

[0054] As used herein, "engraftment" and "engrafting" refer to the incorporation of implanted tissue or cells (i.e., "implanted tissue" or "implanted cells") into the subject's body. The presence of the implanted tissue or cells at or near the implantation site 180 days or more after implantation indicates engraftment. In certain embodiments, imaging techniques (e.g., MRI imaging, etc.) can be used to detect the presence of the implanted tissue.

[0055] 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 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 other embodiments, the allogeneic cell population is derived from primate pluripotent (pPS) cells.

[0056] 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 macrophages, small bands of connective tissue, and blood vessels.

[0057] The terms "central nervous system" and "CNS," used interchangeably herein, refer to the complex of nervous tissue that controls one or more activities of the body, including, but not limited to, the brain and spinal cord of a vertebrate animal.

[0058] 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.

[0059] The term "chronic" as used herein includes, but is not limited to, conditions that occur in a subject for a period ranging from 90 days after injury to the life of the subject.

[0060] As used herein, the term "subacute" includes, but is not limited to, conditions that occur in a subject over a period between 14 and 90 days after injury.

[0061] Multiple pathologies are observed in the injured spinal cord due to the injury itself and the secondary effects of subsequent edema, hemorrhage, and inflammation (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 tissues 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 provide both neurotrophic factors and axonal myelination support, but are susceptible to cell death after SCI, making them 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 not only support both remaining and injured axons, but also remyelinate axons and 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).

[0062] Oligodendrocyte precursor cells (OPCs) are a subtype of glial cells in the central nervous system (CNS) that originate in the ventricular region of the brain and the spinal cord and migrate throughout the developing CNS to mature into oligodendrocytes. Mature oligodendrocytes generate myelin sheaths that insulate nerve axons and remyelinate CNS lesions where myelin sheaths have been lost. Oligodendrocytes also contribute to neuroprotection through other mechanisms, such as 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). In addition, OPCs are known to produce decorin. Decorin is a secreted factor that has been shown to inhibit scarring in the CNS (Esmaeili, Berry et al. 2014, Gubbiotti, Vallet et al. 2016). Unlike most progenitor cells, OPCs are abundant in the adult CNS and retain the ability to generate new oligodendrocytes. Thus, OPCs and OPC-derived mature oligodendrocytes are important therapeutic targets for demyelinating and dysmyelinating diseases (such as multiple sclerosis, adrenoleukodystrophy, and adrenomyeloneuropathy), 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)).

[0063] Proliferation and culture of undifferentiated pluripotent stem cells In certain embodiments, the present disclosure provides methods for producing large quantities of highly pure and characterized oligodendrocyte progenitor cells from pluripotent stem cells. Derivation of oligodendrocyte progenitor cells (OPCs) from pluripotent stem cells according to the methods of the present invention provides a renewable and expandable source of OPCs for many important therapeutic, research, development, and commercial purposes, including the treatment of acute spinal cord injury.

[0064] Methods for the proliferation and culture of undifferentiated pluripotent stem cells have been previously described. With regard to the tissue and cell culture of pluripotent stem cells, the reader may wish to refer to any of the numerous publications available in the art, such as, for example, 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).

[0065] In certain embodiments, the method can be performed on a pluripotent stem cell line. In other embodiments, the method can be performed on an embryonic stem cell line. In one embodiment, the method can 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 can be derived from an induced pluripotent stem cell (iPS) line. In another embodiment, the method can be performed on a primate pluripotent stem (pPS) cell line. In yet another embodiment, the undifferentiated stem cells can be obtained from a parthenogenetic embryo, which is an embryo that is stimulated to produce hESCs without fertilization.

[0066] In one embodiment, undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without the addition of 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 cell proliferation without differentiation (see, e.g., U.S. Pat. No. 6,800,480). In one embodiment, conditioned medium containing such factors can be used. Conditioned medium is obtained by culturing a medium containing 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). Medium can be conditioned by plating feeders in serum-free medium such as knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. Conditioned medium for 1-2 days can be further supplemented with bFGF and used to support pPS cell cultures for 1-2 days (see, e.g., WO01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0067] Alternatively, fresh or unconditioned media can be used that is supplemented with additional factors (e.g., fibroblast growth factor or forskolin) that promote the proliferation of cells in undifferentiated morphology. Non-limiting examples include basal media such as X-VIVOTM10 (Lonza, Walkersville, Md.) or QBSFTM-60 (Quality Biological Inc. Gaithersburg, Md.) supplemented with 40-80 ng / mL 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 media 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, e.g., hESCs, can be cultured in media containing bFGF and TGFP. Non-limiting example concentrations of bFGF include about 80 ng / ml. Non-limiting example concentrations of TGFP include about 0.5 ng / ml.

[0068] In one embodiment, the undifferentiated pluripotent cells can be cultured on a layer of feeder cells, which are generally 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 obtained by differentiating human embryonic stem cells into fibroblasts (see, e.g., WO01 / 51616). In one embodiment, human feeder cells that can 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).

[0069] A variety of solid surfaces can be used for culturing 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 culturing undifferentiated pluripotent cells can be made from 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 one or more acrylates. In one embodiment, the solid surface can be a three-dimensional shape. Non-limiting examples of three-dimensional solid surfaces are described, for example, in U.S. Patent Publication No. 2005 / 0031598.

[0070] In one embodiment, the undifferentiated stem cells can be grown on a growth substrate under feeder-free conditions. 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 subcultured using various methods, such as the use of collagenase, or manual scraping. In another embodiment, the undifferentiated stem cells can be subcultured using non-enzymatic means, such as 0.5 mM EDTA in PBS, or the use of ReLeSR™. In one embodiment, a plurality of 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 6.0×10 3 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 , for example, about 1.0 × 10 4 cells / cm 2 , for example, about 5.0 × 10 4 cells / cm 2 , for example, about 1.0 × 10 5 cells / cm 2 , or for example about 3.0 x 10 5 cells / cm 2 In another embodiment, the seeding density can be in the range of about 6.0×10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 - the growth surface may be in the range of, for example, about 6.0 x 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , or, for example, about 7.0 x 10 3 cells / cm2 ~Approx. 8.0×10 3 cells / cm 2 In yet another embodiment, the seeding density can be in the range of about 1.0×10 4 cells / cm 2 ~Approx. 1.0×10 5 cells / cm 2 -Growth surface, e.g., about 2.0 x 10 4 cells / cm 2 ~Approx. 9.0×10 4 cells / cm 2 , for example, about 3.0 x 10 4 cells / cm 2 ~Approx. 8.0×10 4 cells / cm 2 , for example, about 4.0 x 10 4 cells / cm 2 ~Approx. 7.0×10 4 cells / cm 2 , or, for example, about 5.0 x 10 4 cells / cm 2 ~Approx. 6.0×10 4 cells / cm 2 -growth surface area. In one embodiment, the seeding density is about 1.0 x 10 5 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 -Growth surface, e.g., about 1.0 x 10 5 cells / cm 2 ~Approx. 4.5×10 5 cells / cm 2 , for example, about 1.5 x 10 5 cells / cm 2 ~Approx. 4.0×10 5 cells / cm 2 , for example, about 2.0 x 10 5 cells / cm 2 ~Approx. 3.5×10 5 cells / cm 2 , or, for example, about 2.5 × 10 5 cells / cm 2 ~Approx. 3.0×10 5 cells / cm 2 - may be the extent of the growth surface.

[0071] Any of a variety of suitable cell culture and subculture techniques can be used to culture cells according to the invention. For example, in one embodiment, the culture medium can be changed at suitable time intervals. In one embodiment, starting about 2 days after subculture of the cells, the culture medium can be changed completely every day. In another embodiment, when the culture reaches about 90% colony coverage, a surrogate flask can be sacrificed and counted using one or more suitable reagents, such as collagenase IV and 0.05% trypsin-EDTA, successively to obtain a single cell suspension for quantification. In one embodiment, after subculture of a plurality of undifferentiated stem cells, the cells can be seeded on a suitable growth substrate (e.g., Matrigel® GFR) at a seeding density that allows the cells to reach confluence in a suitable period of time, such as about 3-10 days. In one embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and grown on a recombinant laminin matrix. In one embodiment, the 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.

[0072] In one embodiment, the seeding density is about 6.0×10 3 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 , for example, about 1.0 x 10 4 cells / cm 2 , for example, about 5.0 x 10 4 cells / cm 2 , for example, about 1.0 x 10 5 cells / cm 2 , or, for example, about 3.0 x 10 5 cells / cm 2 In another embodiment, the seeding density can be in the range of about 6.0×10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 -Growth surface, e.g., about 6.0 x 103 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , or, for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 8.0×10 3 cells / cm 2 In yet another embodiment, the seeding density can be in the range of about 1.0×10 4 cells / cm 2 ~Approx. 1.0×10 5 cells / cm 2 -Growth surface, e.g., about 2.0 x 10 4 cells / cm 2 ~Approx. 9.0×10 4 cells / cm 2 , for example, about 3.0 x 10 4 cells / cm 2 ~Approx. 8.0×10 4 cells / cm 2 , for example, about 4.0 x 10 4 cells / cm 2 ~Approx. 7.0×10 4 cells / cm 2 , or, for example, about 5.0 x 10 4 cells / cm 2 ~Approx. 6.0×10 4 cells / cm 2 -growth surface area. In one embodiment, the seeding density is about 1.0 x 10 5 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 -Growth surface, e.g., about 1.0 x 10 5 cells / cm 2 ~Approx. 4.5×10 5 cells / cm 2 , for example, about 1.5 x 10 5 cells / cm 2~Approx. 4.0×10 5 cells / cm 2 , for example, about 2.0 x 10 5 cells / cm 2 ~Approx. 3.5×10 5 cells / cm 2 , or, for example, about 2.5 × 10 5 cells / cm 2 ~Approx. 3.0×10 5 cells / cm 2 - may be the extent of the growth surface.

[0073] Oligodendrocyte precursor cell composition As discussed above, the present disclosure provides compositions comprising populations of oligodendrocyte progenitor cells (OPCs) for use in treating acute spinal cord injury and other related CNS conditions, as well as methods of making and using the same. In certain embodiments, the OPCs of the present disclosure are capable of producing and secreting one or more biological factors that can enhance neural repair.

[0074] In one embodiment, the cell population may have a common genetic background. In one embodiment, the cell population may be derived from one host. In one embodiment, the cell population may be derived from a pluripotent stem cell line. In another embodiment, the cell population may be derived from an embryonic stem cell line. In one embodiment, the cell population may be derived 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 be derived from an induced pluripotent stem cell (iPS) line. In one embodiment, the cell population may be derived from a subject in need thereof (e.g., the cell population may be derived from a subject in need of treatment). In yet another embodiment, the hESC line may be derived from a parthenogenetic embryo, which is an embryo that is stimulated to produce hESCs without fertilization.

[0075] In certain embodiments, OPCs of the disclosure express one or more markers selected from nestin, NG2, Olig1, and PDGF-Ra, hi certain embodiments, OPCs of the disclosure express all of the markers nestin, NG2, Olig1, and PDGF-Ra.

[0076] In certain embodiments, the OPCs of the present disclosure can secrete one or more biological factors. In certain embodiments, the one or more biological factors secreted by the OPCs of the present disclosure can promote, without limitation, 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 metalloproteases. In some embodiments, the OPCs can secrete one or more factors that inhibit cell death following spinal cord injury. In some embodiments, the OPCs can secrete one or more factors that are upregulated following cell injury and support the clearance of misfolded proteins.

[0077] 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 factors selected from clusterin, ApoE, TIMP1, and TIMP2. In yet further embodiments, OPCs are capable of producing and secreting all of MCP-1, clusterin, ApoE, TIMP1, and TIMP2.

[0078] In one embodiment, the biological factor may be secreted by the composition comprising the OPC population at a concentration of greater than about 50 pg / ml, such as greater than about 100 pg / ml, such as greater than about 200 pg / ml, such as greater than about 300 pg / ml, such as greater than about 400 pg / ml, such as greater than about 500 pg / ml, such as greater than about 1,000 pg / ml, such as greater than about 2,000 pg / ml, such as greater than about 3,000 pg / ml, such as greater than about 4,000 pg / ml, such as greater than about 5,000 pg / ml, such as greater than about 6,000 pg / ml, or such as greater than about 7,000 pg / ml.In certain embodiments, the biological agent has a concentration of from about 50 pg / ml to about 100,000 pg / ml, such as 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. l, for example about 750 pg / ml, for example about 800 pg / ml, for example about 850 pg / ml, for example about 900 pg / ml, for example about 1,000 pg / ml, for example about 1,500 pg / ml, for example about 2,000 pg / ml, for example about 2,500 pg / ml, for example about 3,000 pg / ml, for example about 3,500 pg / ml, for example about 4,000 pg / ml, for example about 4,500 pg / ml, for example about 5,000 pg / ml, for example about 5,500 pg / ml, for example About 6,000 pg / ml, for example, about 6,500 pg / ml, for example, about 7,000 pg / ml, for example, about 7,500 pg / ml, for example, about 8,000 pg / ml, for example, about 8,500 pg / ml, for example, about 9,000 pg / ml, for example, about 10,000 pg / ml, for example, about 15,000 pg / ml, for example, about 20,000 pg / ml, for example, about 25,000 pg / ml, for example, about 30,000 pg / ml, for example, about 35,000 pg / ml, for example, about 40,000 pg In some embodiments, the OPCs may be secreted by the composition comprising a cell population comprising OPCs at a concentration of about 10,000 pg / ml, such as about 45,000 pg / ml, for example, about 50,000 pg / ml, for example, about 55,000 pg / ml, such as 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.

[0079] In a specific 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. The OPC-containing cell population may be secreted by the composition comprising the cell population at a concentration of from 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.

[0080] In a particular embodiment, the biological agent is at a concentration of 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 / ml. In one embodiment, the OPC-containing cell population may be secreted by the composition comprising the cell population containing OPCs at a concentration in the range of about 10,000 pg / ml to about 60,000 pg / ml, for example, about 60,000 pg / ml to about 70,000 pg / ml, for example, about 70,000 pg / ml to about 80,000 pg / ml, for example, about 80,000 pg / ml to about 90,000 pg / ml, or, for example, about 90,000 pg / ml to about 100,000 pg / ml.

[0081] In some embodiments, clusterin may be secreted by a composition comprising a cell population comprising OPCs at a concentration of about 1,000 pg / ml to about 100,000 pg / ml. In certain embodiments, clusterin may be secreted by a composition comprising a cell population comprising OPCs at a concentration of about 10,000 pg / ml to about 50,000 pg / ml. In some embodiments, MCP-1 may be secreted by a composition comprising a cell population comprising OPCs at a concentration of about 500 pg / ml to about 50,000 pg / ml. In certain embodiments, MCP-1 may be secreted by a composition comprising a cell population 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 cell population 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 cell population 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 cell population comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In certain embodiments, TIMP1 may be secreted by a composition comprising a cell population 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 cell population comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In certain embodiments, TIMP2 may be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 500 pg / ml to about 5,000 pg / ml.

[0082] Pharmaceutical Compositions The OPCs of the present disclosure can be administered to a subject in need of treatment, such as SCI therapy. Alternatively, the cells of the present disclosure can be administered to a subject in need of SCI therapy as a pharmaceutical composition with a suitable carrier and / or using a delivery system.

[0083] As used herein, the term "pharmaceutical composition" refers to a preparation containing a therapeutic agent or agents in combination with other ingredients, such as physiologically suitable carriers and excipients.

[0084] As used herein, the term "therapeutic agent" may refer to the cells of the present disclosure that are responsible for a biological effect in a subject. Depending on the embodiment of the present disclosure, the "therapeutic agent" may refer to the oligodendrocyte precursor cells of the present disclosure. Alternatively, the "therapeutic agent" may refer to one or more factors secreted by the oligodendrocyte precursor cells of the present disclosure.

[0085] As used herein, the terms "carrier", "pharmaceutical acceptable carrier" and "biologically acceptable carrier" are used interchangeably and refer to a diluent or carrier substance that does not cause significant side effects or irritation in a subject and does not abolish the biological activity or effect of a therapeutic agent. In certain embodiments, a pharmaceutical acceptable carrier may 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.

[0086] The therapeutic agent or agents of the present disclosure can be administered as a component of a hydrogel, such as those described in U.S. patent application Ser. No. 14 / 275,795, filed May 12, 2014, and U.S. Patent Nos. 8,324,184 and 7,928,069.

[0087] The composition according to the present disclosure can be formulated for parenteral administration by injection, for example, 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 a preservative. The composition can include formulating agents, such as suspending agents, stabilizing agents, and / or dispersing agents. In certain embodiments, the composition can be formulated to be compatible with cryopreservation.

[0088] The compositions according to the present disclosure can be formulated for administration by injection into the spinal cord of a subject. The compositions may be formulated for direct injection into the spinal cord of a subject. The compositions can be formulated for administration by implantation or other delivery methods. In certain embodiments, the compositions according to the present disclosure can be formulated for intracerebral, intraventricular, intrathecal, intranasal, or intracapsular administration to a subject. In certain embodiments, the compositions according to the present disclosure can be formulated for administration by injection directly into or proximal to the infarct cavity in the brain of a subject. In certain embodiments, the compositions according to the present disclosure can be formulated for administration by implantation. In certain embodiments, the compositions according to the present disclosure can be formulated for administration by other suitable delivery methods. In certain embodiments, the compositions according to the present disclosure can be formulated as a solution.

[0089] In certain embodiments, compositions according to the present disclosure contain about 1×10 6 ~Approx. 5×10 8 cells per milliliter, e.g., approximately 1 x 10 6 cells per milliliter, e.g., approximately 2 x 10 6 cells per milliliter, e.g., about 3 x 10 6 cells per milliliter, e.g., about 4 x 10 6 cells per milliliter, e.g., about 5 x 10 6 cells per milliliter, e.g., about 6 x 10 6 cells per milliliter, e.g., about 7 x 10 6 cells per milliliter, e.g., about 8 x 10 6 cells per milliliter, e.g., about 9 x 10 6 cells per milliliter, e.g., approximately 1 x 10 7 cells per milliliter, e.g., approximately 2 x 10 7 cells per milliliter, e.g., about 3 x 10 7 cells per milliliter, e.g., about 4 x 10 6 cells per milliliter, e.g., about 5 x 10 7 cells per milliliter, e.g., about 6 x 107 cells per milliliter, e.g., about 7 x 10 7 cells per milliliter, e.g., about 8 x 10 7 cells per milliliter, e.g., about 9 x 10 7 cells per milliliter, e.g., approximately 1 x 10 8 cells per milliliter, e.g., approximately 2 x 10 8 cells per milliliter, e.g., about 3 x 10 8 cells per milliliter, e.g., about 4 x 10 8 cells, or for example, about 5 x 10 per milliliter 8 In certain embodiments, compositions according to the present disclosure may contain about 1 x 10 cells per milliliter. 8 ~Approx. 5×10 8 cells per milliliter, e.g., approximately 1 x 10 8 ~Approx. 4×10 8 cells per milliliter, e.g., approximately 2 x 10 8 ~Approx. 5×10 8 cells per milliliter, e.g., approximately 1 x 10 8 ~Approx. 3×10 8 cells per milliliter, e.g., approximately 2 x 10 8 ~Approx. 4×10 8 cells, or for example, about 3 x 10 per milliliter 8 ~Approx. 5×10 8 In yet another embodiment, the composition according to the present disclosure may comprise about 1 x 10 cells per milliliter. 7 ~Approx. 1×10 8 cells per milliliter, e.g., approximately 2 x 10 7 ~Approx. 9×10 7 cells per milliliter, e.g., about 3 x 10 7 ~Approx. 8×10 7 cells per milliliter, e.g., about 4 x 10 7 ~Approx. 7×10 7 cells, or for example, about 5 x 10 per milliliter 7 ~about 6×10 7In one embodiment, a composition according to the present disclosure may contain about 1 x 10 cells per milliliter. 6 ~Approx. 1×10 7 cells per milliliter, e.g., approximately 2 x 10 6 ~Approx. 9×10 6 cells per milliliter, e.g., about 3 x 10 6 ~Approx. 8×10 6 cells per milliliter, e.g., about 4 x 10 6 ~Approx. 7×10 6 cells, or for example, about 5 x 10 per milliliter 6 ~about 6×10 6 In yet another embodiment, a composition according to the present disclosure may comprise at least about 1 x 10 cells per milliliter. 6 cells, e.g., at least about 2 x 10 cells per milliliter 6 cells, e.g., at least about 3 x 10 cells per milliliter 6 cells, e.g., at least about 4 x 10 cells per milliliter 6 cells, e.g., at least about 5 x 10 cells per milliliter 6 cells, e.g., at least about 6 x 10 cells per milliliter 6 cells, e.g., at least about 7 x 10 cells per milliliter 6 cells, e.g., at least about 8 x 10 cells per milliliter 6 cells per milliliter, e.g., at least about 9 x 10 6 cells, e.g., at least about 1 x 10 cells per milliliter 7 cells, e.g., at least about 2 x 10 cells per milliliter 7 cells, e.g., at least about 3 x 10 cells per milliliter 7 cells, e.g., at least about 4 x 10 cells per milliliter 7 cells, or at least about 5 x 10 7 In one embodiment, the composition according to the present disclosure may comprise up to about 1 x 10 cells. 8 More than 10 cells, e.g. up to about 2 x 10 cells per milliliter 8More than 10 cells, e.g. up to about 3 x 10 cells per milliliter 8 More than 10 cells, e.g. up to about 4 x 10 cells per milliliter 8 More than 10 cells, e.g. up to about 5 x 10 cells per milliliter 8 10 cells or more, or for example up to about 6 x 10 cells per milliliter 8 It may comprise one or more cells.

[0090] In one embodiment, a composition according to the present disclosure has a concentration of about 4×10 per milliliter. 7 ~about 2×10 8 The cell may include cells.

[0091] 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, for example about 500 microliters, such as about 600 microliters, for example about 700 microliters, such as about 800 microliters, for example about 900 microliters, such as about 1 milliliter, for example about 1.5 milliliters, such as about 2 milliliters, for example about 2.5 milliliters, such as about 3 milliliters, for example about 3.5 milliliters, such as about 4 milliliters, or for example about 4.5 milliliters. In one embodiment, the composition according to the present disclosure may 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 may 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 may 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 may have a volume of about 20 microliters to about 500 microliters.In another embodiment, a composition according to the present disclosure may have a volume of about 50 microliters to about 100 microliters. In yet another embodiment, a composition according to the present disclosure may have a volume of about 50 microliters to about 200 microliters. In another embodiment, a composition according to the present disclosure may have a volume of about 20 microliters to about 400 microliters.

[0092] In certain embodiments, the present disclosure provides a container that comprises a composition that comprises a population of OPCs obtained according to one or more methods of the present disclosure.In certain embodiments, the container can be configured for cryopreservation.In certain embodiments, the container can be configured for administration to a subject that needs it.In certain embodiments, the container can be a pre-filled syringe.

[0093] For general principles of 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 excipients and accompanying components of the composition is 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 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.

[0094] Uses of the cells of the present disclosure In various embodiments described herein, the disclosure provides methods of using cell populations comprising pluripotent stem cell-derived OPCs to improve one or more neurological functions in a subject in need of treatment. In certain embodiments, methods of using pluripotent stem cell-derived OPCs in the treatment of acute spinal cord injury are provided. In other embodiments, methods of using pluripotent stem cell-derived OPCs in the treatment of other traumatic CNS injuries are provided. In other embodiments, methods of using pluripotent stem cell-derived OPCs in the treatment of non-traumatic CNS disorders or conditions are provided. In certain embodiments, cell populations according to the disclosure can be injected or implanted into a subject in need thereof.

[0095] In certain embodiments, methods of using pluripotent stem cell derived OPCs in the treatment of conditions requiring myelin repair or remyelination are provided. The following are non-limiting examples of conditions, diseases, and pathologies 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 cause 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 trauma resulting in loss of myelination, such as acute spinal cord injury.

[0096] The OPCs are administered in such a way that they can engraft or migrate to the desired tissue site and reconstitute or regenerate the dysfunctional area. The administration of cells can be accomplished by any method known in the art. For example, the cells can be administered directly surgically to the organ or tissue that requires cell transplantation. Alternatively, the cells can be administered to the subject using a non-invasive procedure. Non-limiting examples of non-invasive delivery methods include the use of a syringe and / or catheter to deliver cells to the organ or tissue that requires cell therapy.

[0097] Subjects receiving OPCs of the present disclosure can be treated to reduce immune rejection of the transplanted cells. Potential methods include, for example, administration of traditional immunosuppressants such as tacrolimus, cyclosporine A (Dunn et al., Drugs 61:1957, 2001), or induction of immune tolerance using matched populations 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 can be used. The OPCs of the present invention can be provided in the form of a pharmaceutical composition comprising an isotonic excipient prepared under sterile conditions sufficient for administration to humans.

[0098] Use in treating CNS trauma In certain embodiments, the cell populations according to the present disclosure can be engrafted at the site of spinal cord injury after a composition comprising the cell population is implanted at the site of spinal cord injury.

[0099] In certain embodiments, the cell population according to the present disclosure can remain in the spinal cord injury site of the subject for a period of about 180 days or more after a dose of the composition is implanted in 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 the subject for a period of about 2 years or more after a dose of the composition is implanted in 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 the subject for a period of about 3 years or more after a dose of the composition is implanted in the spinal cord injury site. In yet another embodiment, the cell population according to the present disclosure can remain in the spinal cord injury site of the subject for a period of about 4 years or more after a dose of the composition is implanted in the spinal cord injury site.

[0100] In certain embodiments, the cell composition of the present disclosure can reduce spinal cord injury-induced parenchymal hollowing in a subject. In certain embodiments, the formation of tissue matrix at the spinal cord injury site reduces injury volume. In certain embodiments, the cells of the present disclosure can form tissue matrix at the spinal cord injury site within about 180 days. In certain embodiments, the subject in which injury-induced parenchymal hollowing is reduced is a human.

[0101] In certain 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 certain 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 certain embodiments, the subject is a human.

[0102] In one embodiment, one or more cells from a cell population according to the present disclosure can be moved 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 can be moved from the 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 can be moved 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 can be moved 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 can be moved 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 can be moved 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 can migrate 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.

[0103] In one embodiment, one or more cells from a cell population according to the present disclosure can migrate from a first location to one or more second locations in one or more diseased tissues within 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 can migrate from a first location to one or more second locations in one or more diseased tissues within the central nervous system of a subject in less than about 180 days.

[0104] Parenchymal Spinal Delivery (PSD) System The compositions described herein can be administered using a PSD system, which includes a PSD device that uses a repulsive force generated by a micromagnet to create a spring effect that compensates for the pulsation of the spinal cord during intraspinal injection.In an embodiment, the PSD device is the device shown and described in PCT Patent Publication No. WO2014047540 (this document is incorporated by reference in its entirety for all devices, systems, methods, etc. described therein).

[0105] In one aspect, the PSD is as shown in any of Figures 39-41. In an embodiment, the PSD is as shown in any of Figures 39-41.

[0106] In one aspect, the PSD device includes a frame having an elongated body and a plurality of holders extending therefrom, a plurality of first magnets each secured to a holder, a tube having a first end and a second end (the tube being slidingly disposed within a through hole disposed in each of the holders and the first magnet), a plurality of second magnets secured to an exterior surface of the tube, and a needle secured to the first end of the tube. In some embodiments, the frame may be made of a non-corroding metal such as stainless steel. In some embodiments, the needle may range from about 27 to about 32 gauge. In some embodiments, each of the first magnets and each of the second magnets may be arranged such that the north pole of one of the first magnets faces the north pole of one of the second magnets or the south pole of one of the first magnets faces the south pole of one of the second magnets, thereby providing a magnetic repulsive force that suspends the tube.

[0107] In various embodiments, the frame includes two holders, each with a first magnet attached and a single second magnet secured to the tube, while in other embodiments, the frame includes two holders, each with a first magnet attached and two second magnets secured to the tube.

[0108] The device may further include a stop ring secured to a first end of the tube or a region near the first end of the tube. The device may further include a stop ring secured to a second end of the tube or a region near the second end of the tube. The device may further include a tube removably attached to the second end of the tube and configured to deliver a composition, such as a composition described herein, to the needle.

[0109] In some embodiments, a stop ring for the needle may be secured to the needle to provide the surgeon with a guide as to the maximum distance the needle should be lowered into the spinal cord during surgery. If present, the stop ring may be positioned along the length of the needle such that when the needle reaches a predetermined depth, the stop ring contacts the subject / patient.

[0110] In some embodiments, the stopper is placed about 1 mm to about 10 mm above the needle. In some embodiments, the stopper is placed about 1 mm to about 8 mm above the needle (e.g., from the sharp end of the needle). In some embodiments, the stopper is placed about 4 mm to about 8 mm above the needle. In some embodiments, the stopper is placed about 6 mm above the needle. The placement of the stopper prevents the needle from advancing further into the spinal cord. The stopper allows for a "floating" effect that compensates for spinal pulsation caused by breathing. This is a major advantage over other systems because it does not require the patient to be removed from the ventilator during injection.

[0111] In some embodiments, the system includes an XYZ manipulator for manipulating the position of the needle. In one embodiment, the device includes a microinjection pump connected to the needle via a flow channel (e.g., a tube) for delivering the composition to the needle. In some embodiments, the system includes a foot pedal for operating the pump. In some embodiments,

[0112] In another embodiment, a PSD system includes a PSD device as described herein and a reservoir in fluid communication with the needle, the reservoir containing a composition to be administered to a subject. The system may further include a digital microinjector configured to control the flow of the composition through the needle.

[0113] In various embodiments, the frame includes two holders, each holder having a first magnet attached thereto and one second magnet secured to the tube, while in other embodiments, the frame includes two holders, each holder having a first magnet attached thereto and two second magnets secured to the tube.

[0114] In yet another aspect, a method for compensating for spinal pulsation during administration of a composition to the spinal cord of a subject is provided. The method includes placing a PSD system on the spinal cord of a subject, lowering a needle into the spinal cord, and delivering a dose of the composition to the spinal cord. In some embodiments, the needle and tube of the device are suspended by magnetic repulsion within the device, thereby compensating for spinal pulsation. In various embodiments, the method further includes repeating each step at multiple sites along the spinal cord. The delivering step can include activating a digital microinjector configured to control the flow of the composition through the needle. The lowering step can include inserting the needle into the spinal parenchyma until a needle stop ring fixedly attached to the needle contacts the subject. Thus, the present invention also provides for the use of a PSD system as described herein to compensate for spinal pulsation during administration of a composition as described herein.

[0115] The novel PSD system device offers several advantages. It provides stability and control to eliminate platform / XYZ manipulator / injection needle movement. The pump and syringe are also not in the sterile field, allowing for precisely programmed administration rates. The device does not require breath hold. The device is attached directly to the patient and responds to the patient's respiratory movements. The magnetic needle stabilizes the micro-movements caused by heartbeat. The device is easy to use in the clinical setting because it is small, has few parts, is easily assembled before surgery, allows one-handed XYZ positioning, allows needle insertion at precise depth, is easy to clean and sterilize, and is compatible with OPC1 TAI formulations. Finally, the device does not require dose preparation the day before. EXAMPLES

[0116] Examples 1-8 describe a Phase 1 safety clinical trial of a first-in-human dose of human pluripotent stem cell-derived oligodendrocyte progenitor cells (LCTOPC1), which have mechanistic properties that support survival and potential repair of key 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 progenitor cells (AST-OPC1) for use in subacute cervical SCI.

[0117] 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 administered within 7–14 days after SCI. Subjects who received LCTOPC1 also received tacrolimus to prevent rejection. Subjects followed the protocol for 15 years after LCTOPC1 administration.

[0118] 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 at risk for neurological deterioration, inclusion was limited to those with injuries that were neurologically complete (American Spinal Injury Association Impairment Scale A), with a single neurological level of impairment (NLI) between levels T3-T10, and preservation of motor function not below a single neurological level of <5 (i.e., partial residual area). These inclusion criteria were selected to minimize loss of function in the event of neurological deterioration.

[0119] Magnetic resonance imaging (MRI) after stabilization was used to confirm the presence of a single spinal cord injury and to allow adequate visualization of the spinal cord 30 mm above and below the epicenter of the injury to allow safety monitoring after injection. Participants were required to be eligible for elective surgery to receive LCTOPC1 injections 7–14 days after SCI.

[0120] 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 is registered (NCT01217008), and the primary endpoint was safety, as measured by the frequency and severity of adverse events related to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or the concomitant immunosuppressants administered. Secondary endpoints were neurological function, as measured by the sensory and lower limb motor scores of the ISNCSCI examination. Eligibility criteria are summarized in Supplementary Table 1. Participants will be followed up with in-person visits for a total of 5 years according to the protocol, and with annual telephone visits for an additional 10 years. Figure 1 and the two following paragraphs show the overall study scheme of the clinical trial.

[0121] The objective of the thoracic study was to evaluate the safety of human pluripotent stem cell-derived oligodendrocyte progenitor cells (LCTOPC1) administered 7-14 days after injury to individuals with neurologically complete spinal cord injury (SCI) at T3-T11. The rationale for this first-in-human study was based on evidence that LCTOPC1 supports the survival and potential repair of key cellular components and structures at the SCI site. This was a multicenter, open-label, single-arm interventional clinical trial with n=5.

[0122] This method uses a syringe positioning device to deliver a 2 × 10 syringe caudal to the epicenter of the injury. 6 The study consisted of a single intraparenchymal injection of 100 OPC1 cells. Immunosuppression with tacrolimus was administered for a total of 60 days. Follow-up consisted of annual in-person visits and magnetic resonance imaging (MRI) examinations for 5 years after injection, and annual telephone questionnaires for 6 to 15 years after injection. The primary outcome was safety, as measured by the frequency and severity of adverse events related to the OPC1 injection, the injection procedure, and / or the co-administered immunosuppressants. Secondary outcomes were sensory score, as measured by the ISNCSCI test, and neurological function, as measured by the lower extremity motor score.

[0123] The LCTOPC1 product is a cell population containing a mixture of oligodendrocyte precursor cells and other characterized cell types obtained after induced differentiation of undifferentiated human embryonic stem cells. The initial characterization of the LCTOPC1 population was reported by Nistor et al. (2005), where it was shown that these cells can differentiate into oligodendrocyte precursor cells. Subsequent studies showed that oligodendrocyte precursor cells delivered to the site of spinal cord injury survived in an acute incomplete contusion model in rats. The cells spared tissue at the contusion site with evidence of remyelination of denuded axons. When delivered during the acute injury phase, the cells led to improved motor function as measured by standardized behavioral tests. Preclinical studies in rats and mice showed that the intended clinical cryopreserved human-equivalent dose of the LCTOPC1 formulation could survive, migrate, and produce neurotrophic factors to support cell survival, provide potential for remyelination to support denuded axons, and spare tissue at the SCI contusion site after injection into the SCI site. Furthermore, tests have shown that the cells do not produce teratomas or result in increased pain in injured animals.

[0124] This Phase 1 clinical trial was reviewed by the FDA, the Data Safety Monitoring Board (DSMB), the SCI clinical community, the Surgery and Outcomes Steering Committee, the internal and external ethics committees, the internal and clinical trial site stem cell research oversight boards, 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, and therefore individuals with complete SCI limited to thoracic nerve levels 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.

[0125] Determining the long-term safety of stem cell therapy is an important step to allow future trials to investigate novel stem cell therapies or combination therapies. Ten years after implantation, no medical or neurological complications have occurred that would indicate that LCTOPC1 implantation is unsafe. Specifically, no serious adverse events (SAEs) related to the procedure, cell implantation, or immunosuppression have occurred. Additionally, there have been no significant changes in neurological function. The safety data from this first-in-human study supported progression to clinical trials for individuals with cervical spinal cord injuries.

[0126] The starting material for the production 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 consists mainly of neural lineage cells of oligodendrocyte precursor phenotype. In this safety study, the route of administration of LCTOPC1 was assumed to be direct injection of 2x10^6 live LCTOPC1 cells into the spinal cord 5mm caudal to the lesion epicenter.

[0127] The choice of this dose was based on preclinical studies in rats and mice and extrapolation of the dose to humans using two methods: 1) comparing the relative size of the human and rat spinal cords, and 2) assessing tumorigenicity versus absolute number of injected cells. Additionally, the choice of this administration route was based on the results of preclinical pharmacology studies that showed that LCTOPC1 has properties that support the repair of spinal cord injury pathology. The results of these studies also showed that improved locomotor recovery was associated with robust survival of LCTOPC1 cells at the injury site.

[0128] Spinal cord injuries limited to the T3-T11 nerve levels as assessed by ISNCSCI were selected for intervention. The primary objective of this first-in-human study was to establish the safety of injecting hESC-derived oligodendrocyte progenitor cells into the spinal cord parenchyma of individuals between 7 and 14 days after injury. The secondary outcome measure for this study was ISNCSCI testing, which allowed for the identification of motor and sensory changes at any of 13 scheduled in-person evaluations within the first 5 years after injection.

[0129] Ten years after implantation, no medical or neurological complications have occurred indicating that cell implantation was unsafe. Specifically, no serious adverse events (SAEs) related to the procedure, cell implantation, or immunosuppression have occurred. This report reviews data from the first 10 years of this landmark clinical trial, including early postoperative events, in-person follow-up through five years, and concludes with telephone follow-up data to date.

[0130] Investigational Drug, Dose Formulation, Dosage and Method of Administration. LCTOPC1 is a cell population containing a mixture of oligodendrocyte precursor cells and other characterized cell types obtained following differentiation of undifferentiated human embryonic stem cells (hESCs) from the H1 stem cell line generated at the University of Wisconsin in 1998.

[0131] Compositional analysis of LCTOPC1 by immunocytochemistry and flow cytometry indicates that the cell population is composed mainly of neural lineage cells of oligodendrocyte precursor phenotype. In this safety study, the route of administration of LCTOPC1 was assumed to be direct injection of 2x10^6 live LCTOPC1 cells into the spinal cord 5 mm caudal to the lesion epicenter.

[0132] LCTOPC1 is a cryopreserved cell therapy product. When cryopreserved, each vial contains 7.5 x 10 cells in 1.2 mL of cryoprotectant solution. 6Each LCTOPC1 sample contained viable cells. LCTOPC1 was supplied in 2.0 mL cryovials, transported to the clinical site in the vapor phase of liquid nitrogen, and stored under the same conditions at the site. LCTOPC1 was thawed and prepared prior to administration by study personnel trained and qualified in study drug preparation.

[0133] Participants received 2 × 10 mAbs suspended in a total volume of 50 μL of Hank's Balanced Salt Solution (HBSS) per dose. 6 Patients received a single dose of 2 × 10 live LCTOPC1 cells. The choice of this dose was based on preclinical studies in rats and mice and extrapolation of the dose to humans using two methods: 1) comparing the relative size of the human and rat spinal cords, and 2) assessing the risk of tumor formation in relation to the absolute number of cells injected. During the development of LCTOPC1, patients received a dose of 2 × 10 6 This is the highest dose that can be administered to the injured spinal cord of rats, and rats were the largest animals available to meet the animal numbers required for IND feasibility studies of this novel product. Therefore, we conservatively chose to administer 2 × 10 cells, the highest dose tested in rats. 6 This was the dose used in the Phase 1 trial. Participants who received LCTOPC1 also received tacrolimus to prevent rejection of the allogeneic cell-based product.

[0134] The route of administration of LCTOPC1 was assumed to be intraparenchymal, 6 mm deep on the midline, 5 mm caudal to the epicenter of the injury identified by MRI, as modeled in preclinical studies. Injection into the tail was chosen with great care to avoid the possibility of direct damage to tissue above the level of injury. Based on preclinical studies, it was expected that the injected cells would migrate anteriorly throughout the injury site. LCTOPC1 was administered to the spinal cord in a dedicated surgical procedure 7-14 days after injury. This time frame was chosen to maximize the potential for neuroprotective and remyelination effects based on the results of animal studies suggesting poor survival of implants when implanted within 7 days after injury. A custom-designed syringe positioning device was utilized to assist the neurosurgeon in controlled delivery of the cells. In other examples, the route of administration includes administering a composition (e.g., including LCTOPC1) to a subject using a parenchymal spinal delivery (PSD) system, such as the PSD system described herein. In one embodiment, the composition is administered using the PSD system shown and described in U.S. Publication No. 2015 / 0224331, which is incorporated by reference herein in its entirety.

[0135] Tacrolimus immunosuppression. Tacrolimus immunosuppression was initiated 6–12 hours after injection of LCTOPC1. If participants were unable to take oral medications, tacrolimus was administered intravenously at a starting dose of 0.01 mg / kg / day 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, given in two divided doses per day. Tacrolimus doses were adjusted to achieve target whole blood trough levels of 3–7 ng / mL.

[0136] 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 daily until tacrolimus was discontinued. Tacrolimus was discontinued on day 60. The tacrolimus dose was reduced if trough blood concentrations exceeded 7 ng / mL. In addition, experts reviewed all ISNCSCI examination forms to assess whether there were any changes in neurological function that could be related to the tapering and / or discontinuation of tacrolimus.

[0137] Follow-up and evaluation. An overview of the study visits for the 1-year protocol follow-up (CP35A007) and the 2- to 15-year protocol follow-up (CP35A008) is shown in the study scheme (Figure 1). Because this was the first clinical trial of hESC-derived cells, numerous study visits and long-term follow-up were necessary. The 1-year protocol required 3 study visits before product administration and 13 study visits in the first year after study administration. The long-term protocol required annual visits in years 2 to 5. After the 5-year annual visit, follow-up was performed by annual telephone questionnaires (Figure 4) and in-person evaluations, as needed. The telephone evaluations included recording all new medications taken for longer than 30 days, hospitalizations, and recording of AEs and SAEs.

[0138] 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 after LCTOPC1 injection related to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or the concomitant immunosuppressants administered. Safety evaluations included physical examination, vital signs, ISNCSCI neurological examination, pain questionnaire, electrocardiogram, MRI, hematology and blood chemistry laboratory tests, immunosuppressant safety monitoring laboratory tests (whole blood trough values ​​of tacrolimus, serum values ​​of creatinine, potassium, magnesium, phosphate, ionized calcium, aspartate aminotransferase, alanine aminotransferase, and total bilirubin), concomitant medications, and AEs.

[0139] Definitions of Adverse Events. AEs were tabulated by system organ class and basic terms within system organ classes according to the Medical Dictionary for Regulatory Affairs (MedDRA®) version 10. An AE is any untoward medical occurrence, whether drug-related or not, that occurred in a study participant between the time the participant signed the informed consent form and the participant's last study visit. AE severity and characteristics of serious adverse events (SAEs) were assessed using standard FDA criteria.

[0140] The relationship of AEs to the study drug was determined by the investigator at each site and classified as "related" based on the following criteria: 1) the AE was reasonably related in time to exposure to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or concomitantly administered immunosuppressants, and 2) the AE was equally reasonably explainable by exposure to the study drug or by factors or causes other than exposure to the study drug. Adverse events were monitored by the external medical monitor, the sponsor medical monitor, and the DSMB.

[0141] Neurological Assessment. Secondary outcome measures were neurological function, including measurement of sensory and lower extremity motor scores. Neurological function was assessed using the ISNCSCI examination for motor and sensory testing, and the American Spinal Cord Injury Association (ASIA) Impairment Scale (AIS) designations.

[0142] Exploratory endpoints. Pain assessment was performed using the International Spinal Cord Injury Pain Basic Data Set. An additional set of three questions assessed allodynia. These questions covered the presence and severity of pain elicited or enhanced by brushing, pressure, or contact with cold. Information on analgesics was collected as part of the assessment of concomitant medications. Potential exploratory endpoints of neurological function recovery were the University of Alabama at Birmingham Index of Motor Recovery (UAB-IMR), Spinal Cord Independence Scale (SCIM), and assessment of bowel and urinary function.

[0143] Lumbar puncture. After receiving general anesthesia, but before LCTOPC1 injection, and 60 days after injection, a lumbar puncture was performed to obtain 10 mL of cerebrospinal fluid (CSF). The amount required at each 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. In addition, the sponsor evaluated the CSF to evaluate the immune response to LCTOPC1.

[0144] Magnetic resonance imaging. Screening / baseline MRIs were obtained 3–5 days prior to injection of LCTOPC1 (days −3 and −5), 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]). If surgery for LCTOPC1 injection was subsequently postponed for more than 3 days, MRIs of the thoracic spine were obtained again without contrast. Follow-up MRIs of the spinal cord and cerebellum were obtained with and without contrast (Gd-DTPA) at days 7, 60, 120, and 270 after injection. Complete central nervous system MRIs were obtained with and without contrast (Gd-DTPA) at days 30, 90, 180, and 365, and annually between years 2 and 5. Image acquisition protocols were standardized. Image review was centralized and standardized by an independent radiologist, DD, at Radiology Imaging Associates Denver.

[0145] 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 minimal in terms of duration to 60 days and administered at doses below typical long-term maintenance therapy levels used for solid organ transplantation. Peripheral blood and cerebrospinal fluid (CSF) samples were collected according to protocol from participants injected with LSTOPC1. After receiving general anesthesia, but before LCTOPC1 injection, and on day 60 after injection, lumbar puncture was performed to collect 10 mL of CSF to evaluate allogeneic cell rejection and immunological monitoring. In the hospital laboratory: evaluation of white blood cell count, glucose, total protein, oligoclonal banding, myelin basic protein, and immunoglobulin G index were performed. Peripheral blood was tested to confirm the presence of antibodies specific for donor-specific HLA molecules on LCTOPC1 and to detect T cell-mediated responses to LCTOPC1. Additionally, the sponsor evaluated CSF to further evaluate the immune response to LCTOPC1 and the presence of LCTOPC1 (day 60) using a PCR-based assay.

[0146] 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 this study are presented narratively in tables, figures, and text format.

[0147] result Study participants. The first participant underwent implantation in the winter of 2010, and the last participant was enrolled in the winter of 2011. Eleven patients with SCI were screened for enrollment, and six failed screening. Four were because MRI artifacts prevented proper visualization of the spinal cord, one was based on ISNCSCI testing (NLI T1), and one was due to elevated liver enzymes. A total of five patients with SCI underwent LCTOPC1 at the three study sites. Figure 2 shows a flow diagram of the Consolidated Criteria for Reporting Clinical Trials (CONSORT). In this study, the most common mechanism of injury was motor vehicle related in four of five patients, and a fall was the cause of injury in one patient. Four of the five patients enrolled were male. The age of the cohort ranged from 21 to 32 years (Table 1).

[0148] [Table 1]

[0149] Participant follow-up. At the time of publication, all participants were in their 10-year follow-up. In accordance with an agreement with the FDA, the study was structured to begin with an in-person assessment at 5 years and be conducted by telephone interview from years 6 to 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 in-person visit at year 5 but did participate in the scheduled telephone follow-up. From years 6 to present, 21 of the 21 annual telephone interviews were completed. One participant has completed the 10-year follow-up, and four participants are in their 10-year follow-up interview.

[0150] 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.

[0151] [Table 2]

[0152] Serious adverse events related to treatment, cell implantation, or immunosuppression. There were no SAEs related to treatment, cell implantation, or immunosuppression. There were no clinically concerning findings on MRI scans of the entire central nervous system in any participant for 5 years after injection. During long-term telephone follow-up, participants denied fever of unknown origin, no abnormal sensations in the chest, arms, or legs (other than those described below), and no participant was diagnosed with any type of cancer. No participant died during the study. Safety events were monitored by the DSMB and no pause rules were invoked.

[0153] Serious adverse events not related to treatment, cell implantation, or immunosuppression. Three participants reported four SAEs not related to treatment, cell implantation, or immunosuppression. These SAEs included a urinary tract infection (UTI) followed by transient autonomic dysreflexia in one patient, and pyelonephritis and mood disorders in two different patients.

[0154] Adverse events classified by grade. During the study, there were 25 AEs considered by the investigator 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 a burning sensation in the trunk and legs that was first reported at day 57 post-injection with grade 1 severity, increasing to grade 3 severity at day 90 post-injection. This neuropathic pain led to three additional AEs of grade 2 severity that continued through the 9th year of follow-up. Grade 2 AEs included surgical site pain, hypomagnesemia, urinary tract infection, vaginal fungal infection, vomiting, upper back pain, shoulder pain, range of motion pain, and autonomic discomfort during catheter insertion (relieved after treatment with lidocaine). Grade 1 AEs included hypomagnesemia, urinary tract infection, pyrexia, headache, nausea, and spasticity.

[0155] Adverse events categorized by their relationship to the procedure, cell implantation, or immunosuppression. Nine of the 25 associated adverse events were judged to be specifically related to the injection procedure. Eight of the nine were grade 1 or 2 in severity, with one being grade 3. AEs were primarily transient postoperative pain, one fever, and one urinary tract infection. No AEs were attributable to 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 judged to be specifically related to immunosuppression. Seven grade 1 and nine grade 2 AEs were judged to be specifically related to tacrolimus. These AEs were primarily known side effects commonly associated with tacrolimus (nausea / vomiting, decreased magnesium levels, infections). Of the reported infections, one of seven was a vaginal fungal infection and six of seven was a urinary tract infection, which are common complications of SCI.

[0156] Adverse events not related to the procedure, cell implantation, or immunosuppression. In 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 in years 7-9 and is currently not using medications for muscle spasms. In year 9, another individual underwent outpatient testing after developing a deep vein thrombosis (DVT).

[0157] Secondary outcome measures: neurological assessment. After discharge from acute inpatient rehabilitation and throughout the first 5 years after implantation, participants continued to be assessed in person according to the schedule shown in Figure 1. Of note, participants' annual in-person assessments included at least 13 ISNCSCI examinations from baseline through year 5. All participants' ASIA Impairment Score (AIS) grade was A at study entry, and all participants maintained the same disability grade. The highest and lowest NLI enrolled in the study were T3 and T8, respectively. Only individuals with T3 NLI improved to T4, and sensory ZPP was initially bilateral T4, improving to T5 on the left and T6 on the right at 1-year follow-up. In total, 3 of 5 participants experienced at least 1 level of improvement in ZPP. All participants had normal upper limb motor function at the beginning and end of the 5-year in-person ISNCSCI testing, 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). Sensory testing was virtually unchanged throughout the 5 years of in-person follow-up. Figures 3 and 23 show a graphical representation of motor and sensory function for each patient at baseline and 5 years after LCTOPC1 administration.

[0158] [Table 3]

[0159] Table 3 shows the total sensory score (TSS), upper limb motor score (UEMS), lower limb motor score (LEMS), sensory neurological impairment level (NLI), motor NLI, sensory partial 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 none converted to AIS B. The highest single NLI and lowest NLI enrolled in the study were T3 and T8, respectively. Only the individual with T3 NLI improved to T4, and the sensory ZPP was initially bilateral T4, improving to T5 on the left and T6 on the right at 1 year follow-up. In total, three of the five participants experienced at least one level of improvement in ZPP. ND = not measurable.

[0160] MRI findings. No participants had 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 evidence of adverse neurologic or MRI changes was reported during tapering or after discontinuation of tacrolimus.

[0161] Immune monitoring. LSTOPC1 is an allogeneic cell therapy and is potentially susceptible to rejection by the recipient's 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 from each of the five recipients. The potential development of a cellular immune response against LCTOPC1 was evaluated, but serum samples from none of 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.

[0162] 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 hESC-derived cell lines had never been evaluated in clinical trials in any setting, other procedures in which cell products (e.g., activated autologous macrophages) were injected into the spinal cord parenchyma had been evaluated, providing a partial roadmap for research based on LCTOPC1.

[0163] 2 × 10 within 7–14 days after injury 6 Primary and secondary outcome measures of the five participants who received allogeneic hESC-derived oligodendrocyte precursor cells are presented. 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 immunosuppression regimen were well tolerated. To date, all five participants who received LCTOPC1 have shown no evidence of neurological deterioration or adverse findings on MRI scans.

[0164] Although the study was not designed to evaluate efficacy, animal studies of LCTOPC1 improved motor function through remyelination, as well as mechanisms that may represent neuroprotection, inhibition 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 studies may be related to the relative severity of injury in humans compared with preclinical studies with incomplete contusion, suggesting that subsequent studies with incomplete injury may demonstrate recovery closer to that seen in animal models. Although the study did not show 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 remain stable over time. Throughout the first 10 years of the clinical trial, no unexpected SAEs related to LCTOPC1 were reported in 98% of participants followed up (45 of 46 annual visits).

[0165] Neuropathic pain due to remyelination or neurotrophic factors relative to LCTOPC1 was assessed using the International Spinal Cord Injury Pain Basic Data Set and a set of three questions to assess allodynia. Neuropathic high-injury and low-injury pain often develop during the first few months after SCI, and by one 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 the two major pain categories that are common after SCI: neuropathic pain at the level of injury (termed neuropathic high-injury pain) and diffuse neuropathic pain below the level of injury (termed neuropathic low-injury pain). Unfortunately for affected individuals, neuropathic pain above and below the injury is often severe and persistent for at least 5 years after SCI, despite attempts at pain management. Furthermore, 40-50% of individuals with these types of pain report the pain as severe or unbearable. In this small, open-label study, it is not possible to determine a causal relationship between LCTOPC1 and changes in the incidence of long-term neuropathic pain.

[0166] Serial MRI studies did not show 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 suggests that cavitary lesions are identifiable in 58% of individuals undergoing thoracic level cell testing. MRI results during long-term follow-up of LCTOPC1 were particularly significant as 80% of individuals showed T2 signal changes consistent with the formation of tissue matrix at the injury site. Although the sample size is limited, these findings suggest that LCTOPC1 cells may persistently engraft and / or induce long-term changes that limit cavitation at the injury site. SCI is a relatively rare condition, with the potential population of T3-T11 AIS A injuries representing less than 20% of acute SCI in the United States (NSCISC National Spinal Cord Injury Statistical Center, University of Alabama at Birmingham, 2011 Annual Statistical Report - Full Publication).

[0167] conclusion The LCTOPC1 thoracic SCI clinical trial is one of the longest running clinical trials in the hESC field. This study provides first-in-human positive safety data that are important for future hESC-derived therapies. Although we cannot exclude the possibility of adverse events in the future, our experience in this study provides evidence that these therapies are well tolerated and free of adverse events for up to 10 years. Furthermore, this report supports participants' willingness to participate in long-term follow-up and sets a standard for corporate sponsors to commit to data collection beyond immediate financial gain. Based on the safety profile of LCTOPC1 obtained in this study, we have initiated a cervical dose escalation study.

[0168] 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 are described, for example, in WO / 2020 / 154533, which is incorporated herein by reference in its entirety.

[0169] 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 coated with recombinant human laminin-521 (rhLn-521, Corning #354224) and cultured in complete mTeSR™-1 medium (Stem Cell Technologies #85850) on tissue culture treated polystyrene T-75 culture flasks (Corning #431082). The medium was changed completely every day until the cells reached approximately 80-90% confluency, after which uhESCs were passaged using ReLeSR™ reagent (Stem Cell Technologies #05872). uhESC cells cultured in ReLeSR™ were transferred onto fresh rhLn-521-coated 225 cm 2 The WCB-derived cultured uhESCs were expanded in this manner for 2-5 passages depending on the experiment and then differentiated into neuroectodermal progenitor cells as described in Example 3.

[0170] 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 completely removing mTeSR™-1 medium and adding glial progenitor medium (GPM; consisting of DMEM / F12 (Gibco catalog no. 10565-018) supplemented with 2% B27 supplement (Gibco catalog no. 17504-044) and 0.04 μg / mL triiodothyronine (Sigma-Aldrich catalog no. T5516-1MG)) supplemented with 10 μM MAPK / ERK inhibitor PD0325901 (PD; Sigma-Aldrich catalog no. PZ0162), 2 μM BMP signaling inhibitor Dorsomorphin (Dorso; Sigma-Aldrich catalog no. P5499), and 1 μM retinoic acid (RA; Sigma-Aldrich catalog no. 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, expanded, and further differentiated into glial progenitor cells as described in Example 4. A subset of cells was collected and analyzed by quantitative PCR (qPCR; described in Example 7), flow cytometry (described in Example 6), and, if available, a separate well plate set aside for analysis was prepared for immunocytochemistry (ICC) (described in Example 6). On day 7, these cells showed marker expression consistent with dorsal spinal cord progenitor cells (Table 4).

[0171] [Table 4]

[0172] Example 4 – Methods for differentiating human embryonic stem cells into glial lineage cells Days 7-13: Differentiation of uhESCs into neuroectodermal progenitors, specifically the dorsal phenotype, was performed as described in Example 3. On day 7, cells were detached 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 with fresh GPM+hbFGF+EGF. Days 14-21: On day 14, cells were detached 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 ...

[0173] 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 from days 0 to 20 was carried out as described in Examples 3 and 4. On day 21, the aggregates were transferred from dynamic suspension to rhLn-521-coated culture vessels. For example, starting with a 1x PBS-0.1L Mini Bioreactor with a total volume of 60 mL, 60 mL of culture was split into 2x T75 flasks with a volume 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 detached with TrypLE™ Select, counted, and transferred to a new rhLn-521-coated culture vessel at 4x10 4 live cells / cm 2 The cells were replated at a seeding density of 100-200 μg / ml. Differentiated cells were harvested on day 42. Cells were detached from the vessel using TrypLE™ Select, counted, and reconstituted in CryoStor10 (BioLife Solutions, Catalog No. 210102) prior to cryopreservation. A subset of cells was harvested 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).

[0174] Example 6 –Characterization of differentiated cell populations by immunocytochemistry and flow cytometry Flow cytometry and immunocytochemistry (ICC) can be used to detect and characterize various aspects of protein marker expression within a cell population. Flow cytometry can be used to quantify the percentage of individual cells within a population that exhibit a given protein marker characteristic, while ICC provides additional information regarding the subcellular localization of each protein marker and can be applied to single cells or cell aggregates. Using either or both of these protein profiling approaches, human embryonic stem cell differentiation into neuroectodermal progenitor cells, glial progenitor cells, and oligodendrocyte progenitor cells was tracked according to the methods 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 placed in increasing concentrations of sucrose solutions (10%, 20%, and 30% weight / volume) for 30 min at room temperature, 30 min at room temperature, and overnight at 4° C. 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.

[0175] For immunocytochemical staining, fixed adherent cells and slide-mounted aggregate sections were permeabilized and blocked for 2 hours at room temperature (RT) in blocking solution consisting of 0.1% Triton™ X-100 / 2% normal goat serum / 1% bovine serum albumin in PBS. After permeabilization and blocking, adherent cells and aggregate sections were incubated overnight at 4° C. in blocking solution without Triton™ X-100 and containing primary antibodies specific for protein markers of interest, including PAX6 (BD Pharmingen #561462 or BioLegend #901301) to detect neuroectodermal progenitor cells, and AP2 (Developmental Studies Hybridoma Bank-DSHB #3B5), PAX3 (DSHB #Pax3), and PAX7 (DSHB #Pax7) to detect dorsal spinal cord progenitor cells. The adherent cells and aggregate pieces were then washed three times with PBS, incubated in the presence of secondary antibodies specific for the selected primary antibodies, and counterstained with 4',6-diamidino-2-phenylindole (DAPI) in blocking solution without Triton™ X-100 for 1 h at room temperature in the dark. The adherent cells and aggregate pieces were washed three times with PBS and imaged using an IN Cell Analyzer2000 (GE Healthcare, Pittsburgh, PA, USA).

[0176] 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.

[0177] 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 precursor marker NG2 was also present.

[0178] Human embryonic stem cells differentiated into oligodendrocyte progenitors 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 progenitor cells by ICC, slide-mounted aggregate sections were stained as above, except permeabilization was performed with 100% methanol for 2 min at room temperature and blocking solution consisted of 10% fetal bovine serum in PBS.

[0179] Based on ICC data for oligodendrocyte progenitors at day 42, single cell populations from two representative experiments expressed the oligodendrocyte progenitor marker NG2 and had reduced expression of the dorsal spinal cord progenitor marker AP2.

[0180] 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 for 30 minutes on ice with primary antibodies specific for the 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), and their isotype controls. The cells were washed with staining buffer to remove unbound antibody, and in the case of unconjugated antibodies, the cells were then incubated with the appropriate fluorophore-conjugated secondary antibody on ice for 30 minutes. The cells were washed and then propidium iodide was added to distinguish dead cells. In some cases, the 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 with an Attune NxT (Thermo Fisher, Waltham, MA, USA) flow cytometer. To calculate the percentage of cells expressing a given protein marker, dead cells stained with propidium iodine were gated 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.

[0181] Table 5 shows representative flow cytometry data for day 42 oligodendrocyte precursor cells generated according to the method described in Example 5. As shown in two representative runs, the majority of cells in the resulting cell populations expressed characteristic oligodendrocyte markers, including NG2 and PDGFRα. Additionally, non-OPC markers, including neural progenitor / epithelial marker CD49f and epithelial markers CLDN6 and EpCAM, were minimally detected within the resulting populations.

[0182] [Table 5]

[0183] Cell populations generated by the methodology described herein are currently in clinical trials for the treatment of spinal cord injury and showed a higher percentage of cells positive for the oligodendrocyte progenitor marker NG2 and reduced expression of non-OPC markers CD49f, CLDN6, and EpCAM 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).

[0184] Example 7 –Characterization of differentiated cell populations by gene expression profiling Gene expression profiling can be used to determine the cellular phenotypic characteristics of the starting pluripotent cell population as well as each stage of differentiation, including the generation of neuroectodermal progenitors, glial progenitors, 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 with increased sensitivity such as quantitative real-time PCR (qPCR). To perform gene expression profiling, cells were lysed with 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, 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. Gene-specific primer-probe sets (Applied Biosystems Taqman Gene Expression Assays, Thermo Fisher Scientific #4331182) were then used to quantify the relative expression levels of the target genes and reference housekeeping genes according to the manufacturer's guidelines. To measure 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 system. Each target gene was normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0185] Table 6 shows qPCR results of 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 of the present disclosure. RNA samples were collected 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, a normalized ΔCT value was calculated using 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.

[0186] [Table 6]

[0187] Referring to Table 6, differentiation of uhESCs for 7 days according to methods according to 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.

[0188] 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, which require activation of the Sonic Hedgehog signaling pathway for expression.

[0189] After 21 days of differentiation, the resulting cell population displayed gene expression characteristics consistent with glial progenitors, including downregulation of pluripotency and neuroectodermal progenitor markers, as well as 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 showed sustained expression of HES5, which, in addition to its expression in neuroectodermal / neuronal progenitors, has also been shown to promote switching from neural progenitors to glial progenitors in the developing mammalian central nervous system. Furthermore, the resulting day 21 glial progenitor cells showed sustained expression of dorsal spinal cord progenitor markers TFAP2A, PAX3, and PAX7, providing further evidence of derivation from dorsally patterned neural progenitors.

[0190] After differentiation for 42 days according to the methods described herein, the resulting cell population expressed markers consistent with oligodendrocyte precursor cells, including downregulation of both early lineage and dorsal spinal cord progenitor markers, as well as induction of CSPG4 (also known as NG2), PDGFRα, and DCN.

[0191] 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.

[0192] [Table 7]

[0193] 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, ΔCT values ​​normalized to the average of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1) were calculated, and fold expression relative to baseline (expression below the limit of quantification) was calculated using the ΔΔCT method. Table 6 shows the average (relative to baseline) fold expression values ​​of biological duplicates for each small molecule combination. With reference to Table 7, 7-day differentiation of uhESCs with each small molecule combination tested resulted in downregulation of the pluripotency marker NANOG, as well as comparable sustained expression or induction of genes associated with neuroectodermal progenitor phenotype, including LIN28A, SOX2, PAX6, HES5, and ZBTB16. Furthermore, each small molecule combination tested resulted in a dorsal spinal cord progenitor phenotype based on the expression of dorsal markers TFAP2A, PAX3, and PAX7, and the lack of expression of ventral markers OLIG2 and NKX2-2.

[0194] To obtain a more comprehensive comparison of the cell phenotypes at day 7 after treatment with each small molecule combination, Fluidigm qPCR was performed using a 96-gene panel consisting of known markers of pluripotency, neuroectodermal progenitors, neural tube patterning, glial progenitors, oligodendrocyte progenitors, neural crest cells, neurons, astrocytes, pericytes, Schwann cells, and epithelial cells. Comparison of the cell phenotypes generated by treatment with PD0325901 + Dorsomorphin with the cell phenotypes at day 7 of each alternative small molecule combination by regression plot of normalized ΔCT values ​​showed that similar overall cell phenotypes could be achieved with each small molecule combination tested. Taken together, the results shown in Table 8 support that the methods of the present disclosure can be used to differentiate uhESCs into dorsal neuroectodermal progenitors and further into glial progenitors and oligodendrocyte progenitors using various combinations of (i) MAPK / ERK inhibitors and (ii) BMP signaling inhibitors in the absence of (iii) SHH signaling activators.

[0195] [Table 8]

[0196] [Table 9] TIFF2025504662000011.tif163166

[0197] [Table 10] TIFF2025504662000013.tif2291651. If the injection date is postponed, repeat the following baseline tests and procedures on the new Day 1: brief physical exam, vital signs, ISNCSCI exam, hematology, blood chemistry panel 1, concomitant medications, and adverse events. 2. If a participant's participation ended between days 0 and 60, all tests and procedures scheduled for the day 60 visit were collected, if possible, at the last visit before study termination. If a participant's participation ended between days 60 and 365, all tests and procedures scheduled for day 365 were collected. 3. Informed consent was collected regarding treatment and long-term follow-up. 4. Full inspection. 5. Rapid testing. 6. Rectal examination solely to determine the completeness of the injury. 7. A single pre-taperation ISNCSCI test was performed between days 42 and 46. Additional ISNCSCI tests were performed twice weekly between days 46 and 60 and once weekly between days 60 and 90. Participants who demonstrated better than expected neurological improvement by day 46 were 8. The SCIM was collected as a questionnaire (versus observation of activities). 9. A screening / baseline MRI was obtained ≥4 days after SCI between days -5 and -3. This MRI included the entire brain, cerebellum, and spinal cord with and without Gd-DTPA contrast. If the AST-OPC1 injection procedure was delayed for ≥3 days thereafter, a repeat MRI of the T-spine was performed without Gd-DTPA contrast. 10. MRI included the spinal cord and cerebellum with and without Gd-DTPA contrast. 11. MRI included routine images of the spinal cord and cerebellum as well as the brain with and without Gd-DTPA contrast. 12. Blood chemistry panel 1: Serum albumin, alkaline phosphatase, blood urea nitrogen, total bilirubin, chloride, serum creatinine, glucose, potassium, total serum protein, sodium, AST, ALT. 13. Blood chemistry panel 2: Serum creatinine, potassium, magnesium, phosphate, and calcium iodide were obtained daily for 1 week after initiation of tacrolimus treatment, twice weekly from days 7-30, and once weekly from days 31-60. 14. Blood chemistry panel 3: Serum AST, ALT, and total bilirubin were measured 1 week after starting tacrolimus treatment and then weekly until day 60. 15. The same blood sample may be used for both purposes if there is an overlap with a routine study visit (Day 1, 7, 30, or 60). For example, panel 1 consisted of serum albumin, alkaline phosphatase, blood urea nitrogen, total bilirubin, chloride, serum creatinine, glucose, potassium, total serum protein, sodium, and AST. 16. Serum creatinine, potassium, magnesium, phosphate, ionized calcium, AST, ALT, and total bilirubin were obtained on day 60 regardless of the time elapsed since the previous chemistry panel was obtained. 17. Whole blood concentrations (if intravenous) or trough concentrations (if oral) of tacrolimus were measured within 3 days of starting treatment and then twice weekly until day 30. Trough blood concentrations were obtained if tacrolimus administration was changed from intravenous to oral or if the dose was adjusted. 18. Tacrolimus whole blood trough concentrations were measured weekly from days 30 to 60. If the dose was adjusted during this period (other than the scheduled taper from days 46 to 60), trough blood concentrations were obtained within 3 days, after which weekly blood concentration monitoring was resumed until day 60. 19. Tacrolimus blood concentrations and all serum samples were obtained on day 60, regardless of the time since the last blood draw. 20. Additional blood samples for immune response monitoring were collected weekly on days 45 to 60 and days 60 to 90. If there was an overlap with the day 60 or day 90 visit, the same blood sample could be used for both purposes. 21. In preparation for surgery, pharmacologic DVT prophylaxis may be discontinued on day -1, depending on the prophylaxis used and the standard of care in clinical practice. 22. Pharmacological DVT prophylaxis was resumed after the AST-OPC1 implantation procedure depending on the prophylaxis used and the standard of care in the clinical setting. 23. AST was started 6–12 hours after injection of OPC1. 24. On day 46, the tacrolimus dose was reduced by 50% (rounded to the nearest 0.5 mg because the smallest capsule size available was 0.5 mg). On day 53, the tacrolimus dose was reduced 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. 25. CSF collection on day 0 was performed via LP after anesthesia but before surgical incision. 26. Individual testing facilities sent the necessary amounts to the hospital laboratory for the following tests: white blood cell count, glucose, total protein, oligoclonal banding, myelin basic protein, and immunoglobulin G index. The remainder of the samples were processed and sent to an outside laboratory.

[0198] [Table 11]

[0199] 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 successive ascending doses of AST-OPC1 at a single time point between days 21 and 42 after injury. Subjects received AST-OPC1 by direct intraspinal injection using a syringe positioning device (SPD). To prevent rejection of the implant, low-dose tacrolimus was initiated 6–12 hours after intraspinal injection, continued for 46 days, tapered between days 46 and 60, and discontinued on day 60. Subjects were followed for 1 year after administration of AST-OPC1 according to this protocol. Male or female subjects, aged 18–69 years at the time of consent, suffered from sensorimotor complete traumatic SCI (American Spinal Cord Injury Association Impairment Scale A) or sensorimotor incomplete traumatic SCI (American Spinal Cord Injury Association Impairment Scale B). Subjects had a single neurological injury high level (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) scans after stabilization, with sufficient visualization of the spinal cord injury epicenter and lesion border to allow for safety monitoring after injection. Subjects were able to participate in elective surgery to be injected with AST-OPC1 21–42 days after SCI. Subjects were able to participate in elective surgery to be injected with AST-OPC1 21–42 days after SCI, with AST-OPC1 2 × 10 6 , 1×10 7 , or 2 × 10 7 Patients received a single dose of either AST-OPC1 live cells via injection. The product was delivered to the spinal cord during surgery using a syringe positioning device. The AST-OPC1 batch numbers used in this study were M08D1A, M22D1A, and M25D1A. A single dose of AST-OPC1 was administered with one-year follow-up. The planned study timeline and subject screening and treatment are outlined in Figures 6 and 7, respectively. The final open-label, alternating dose escalation, multicenter Phase 1 / 2a clinical trial is outlined in Figure 8. The study visits for the one-year protocol are outlined in the study scheme in Figure 9.

[0200] Neurological examinations were completed 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. The ISNCSCI is used to examine efficacy in terms of improved limb motor function, 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). ISNCSCI results were enumerated for each subject at each scheduled visit 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 tabulated by organ system class and basic terms within organ systems class according to the Medical Dictionary for Regulatory Affairs (MedDRA®) version 18.0. Statistical analysis of safety data was performed using descriptive statistical methods including incidence, severity and association of AEs 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 normal clinical reference ranges were summarized for each analyte at scheduled study visits. Vital signs were summarized by calculating the mean, standard deviation, median, and range at each protocol-specified time point.

[0201] Results: Twenty-six subjects were enrolled in the study. Twenty-five subjects received AST-OPC1 at five study sites. All 25 subjects completed the one-year follow-up. The ages of the 25 subjects who received AST-OPC1 ranged from 18 to 62 years, 21 were male and 4 were female, and the majority were Caucasian (22 subjects). The cause of SCI in eight subjects was a motor vehicle accident. None of the 25 treated subjects showed evidence of unexpected neurological deterioration on ISNCSCI testing after completing the one-year 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 the low-dose temporary immunosuppression regimen were well tolerated. None of the 25 subjects who received AST-OPC1 completed the one-year follow-up and showed evidence of neurological deterioration or adverse findings on MRI scans.

[0202] 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 contains 7.5 x 10 cells in 1.2 mL of cryopreservation medium. 6 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).

[0203] [Table 12]

[0204] Dose selection and timing. The first proposed dose of 2 × 10 6 The cells were evaluated for safety in a previous thoracic SCI study, in which 2 × 10 cells were again administered in cohort 1.6 The first dose (2 × 10 cells in 50 μL) was used to demonstrate no complications from the injection procedure. 6 cells) to the second dose (1 x 10 in 50 µL) 7 The increase to AST-OPC1 only involved increasing the concentration of AST-OPC1, such that both of these doses were administered in a single injection of a volume of 50 µL. Thus, the neurosurgeons consulted for this study considered this initial dose increase to be a very small step with respect to the potential risks associated with the injection procedure. Furthermore, the second dose (1 × 10 in 50 µL) was administered in a single injection of AST-OPC1. 7 The safety of administering 1 × 10 cells (1 × 10 cells) in 50 μL to a second site within the lesion in a manner similar to that used in rodent safety studies was demonstrated at C6 in uninjured porcine cervical spinal cords. This study confirmed minimal tissue damage associated with injection into the uninjured spinal cord, and no evidence of efflux or cell spreading via the CSF was observed. The third dose was 1 × 10 cells in 50 μL administered to a second site within the lesion in a manner similar to that used in rodent safety studies. 7 This dose is within a 6- to 12-fold safety margin compared to the highest dose tested in safety studies in rats, especially with respect to the total volume injected.

[0205] LCTOPC1 was supplied to the clinical site in sterile, single-dose, single-use, 2.0 mL Corning™ cryovials. Upon cryopreservation, each vial typically contained 7.5 × 10 cells in 1.2 mL of cryopreservation medium. 6 Each sample contained 10000 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). Cryopreserved drugs were thawed, washed, resuspended in injection medium, and loaded into syringes at the clinical site.

[0206] 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 drug product (DP) is manufactured in a continuous process. The recovered LCTOPC1 drug substance is a transient intermediate that is immediately formulated into LCTOPC1 DP, 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 consists mainly of neural cells of oligodendrocyte precursor cell phenotype. Other neural cells, namely astrocytes and neurons, are present at low frequencies. The only non-neuronal cells detected in the population are epithelial cells. Mesodermal and endodermal cells, as well as uhESCs, are usually below the quantification or detection limits of the assay.

[0207] The subacute phase of SCI is hypothesized to be the optimal time window for administering AST-OPC1, since this avoids premature damage that would lead to apoptosis of endogenous oligodendrocytes and occurs early enough that AST-OPC1 cells can migrate to denuded axons before extensive glial scarring occurs. This hypothesis is supported by tests in rodent models of SCI, which have shown that injection of AST-OPC1 or other similar preparations 7 days after SCI confers a functional benefit, but not if the interval between injury and injection exceeds 8 weeks (Keirstead 2005, Karimi-Abdolrezaee 2006). Spontaneous functional recovery in rats with contusive SCI begins to plateau approximately 6 weeks after injury, so injecting AST-OPC1 on day 7 represents approximately 1 / 6 (17%) of the time elapsed between injury and the onset of the recovery plateau.

[0208] Given that the rate of spontaneous recovery usually begins to plateau at 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 at a time 1 / 6 of the time it takes for recovery to begin to plateau in humans would correspond to approximately 30 days after injury.

[0209] The initial dosing period of 14–30 days was chosen to avoid early bleeding and inflammation following SCI, as well as scar tissue formation during the chronic phase of SCI. This dosing period was also based on preclinical data available prior to the initiation of this clinical trial. However, dedicated preclinical studies have been performed, suggesting that the optimal dosing period in human subjects may be extended up to 60 days after SCI.

[0210] A review of the emerging preclinical data was conducted by a panel including the investigator and several SCI experts to determine whether the dosing period needed to 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 period of 21-42 days after SCI would still be considered within the subacute phase, while a longer time would be acceptable to allow subjects to be medically stable before undergoing elective surgery for AST-OPC1 injection. Therefore, in this study, AST-OPC1 was administered to subjects 21-42 days after SCI.

[0211] Tacrolimus Administration. Tacrolimus immunosuppression was initiated 6–12 hours after injection of AST-OPC1. If subjects were unable to take oral medications, tacrolimus was administered intravenously at a starting dose of 0.01 mg / kg / day as a continuous intravenous infusion. Subjects were switched to oral tacrolimus as soon as they were able to take medications orally. The starting dose of oral tacrolimus was 0.03 mg / kg / day, administered twice daily in divided 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 alloreactivity of AST-OPC1. On day 46, the tacrolimus dose was reduced by 50% (rounded to the nearest 0.5 mg, as 0.5 mg 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, subjects received 0.5 mg once daily until tacrolimus was discontinued. If the rounded total daily dose was 0.5 mg or less, subjects received 0.5 mg once daily until tacrolimus was discontinued. On day 60, tacrolimus was discontinued (Figure 2). If trough blood concentrations were greater than 7 ng / mL, the tacrolimus dose was reduced. In addition, an expert reviewed all ISNCSCI examination forms to assess whether there were any changes in neurological function that could be related to the dose reduction 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.

[0212] The schedule of assessments and procedures to be performed from screening through Day 365 is shown in Table 13 below.

[0213] [Table 13] TIFF2025504662000017.tif208169

[0214] 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. Upper extremity motor scores (0-50) and changes from baseline were summarized with N, mean, standard deviation, 95% confidence interval, median, minimum, and maximum for the entire intention-to-treat analysis population. Positive changes are considered improvement and negative changes are considered deterioration.

[0215] Motor and sensory levels at each visit and 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. A positive number represents a change in the caudal direction, which is considered an improvement. A negative number represents a change in the rostral direction, which is considered a deterioration.

[0216] Changes in motor and sensory levels were tabulated as follows: percentage of subjects with increased motor levels on either side of the body compared to baseline; percentage of subjects with no change in motor levels on either side of the body compared to baseline; percentage of subjects with an improvement of 1 motor level on at least one side of the body compared to baseline; percentage of subjects with an improvement of 1 motor level on both sides of the body compared to baseline; percentage of subjects with an improvement of 2 or more motor levels on at least one side of the body compared to baseline.

[0217] Efficacy Results. Efficacy of the study was measured by change in upper extremity motor score (UEMS) of the ISNCSCI test from baseline to 12 months after injection of AST-OPC1, and change in motor level. A total of 22 subjects were part of the treatment population (cohorts 2-5). The mean UEMS of the 22 subjects who completed the 365th day 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 lower extremity motor score (LEMS) that did not correlate with the level of improvement in UEMS. Compared to baseline, a total of 7 of 22 subjects (31.8%) achieved an improvement of 2 motor levels on at least one side of the body at the 365th day visit, and 21 of 22 subjects (95.5%) achieved an improvement of 1 motor level on at least one side of the body at the 365th day visit. The percentage of subjects with improvement in motor level is shown in Table 14 below.

[0218] [Table 14]

[0219] 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 after LCTOPC1 injection related to LCTOPC1, the injection procedure used to administer LCTOPC1, and / or the administration of concomitant immunosuppressants. 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 immunosuppressant 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 led by an external expert and including testing of individuals with SCI was required as part of each center's initiation visit. In the first year of the study, ISNCSCI testing was performed 30, 60, 90, 180, 270, and 365 days after injection of LCTOPC1. The 365-day follow-up visit was pre-specified as the time point for secondary outcomes.

[0220] Statistical analysis of efficacy. Efficacy outcomes, neurological function, were assessed by characterizing upper extremity motor scores and motor levels (point estimates and 95% confidence intervals) on the ISNCSCI test at 30, 60, 90, 180, 270, and 365 days after LCTOPC1 injection. The baseline ISNCSCI assessment was defined as the baseline visit performed 24-48 hours before injection. Upper extremity motor scores and changes from baseline were summarized by participant, mean, standard deviation, 95% confidence interval, median, minimum, and maximum for the entire intention-to-treat population.

[0221] 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 began on or after the date and time of LCTOPC1 injection, or that began before LCTOPC1 injection and worsened after administration of study drug. AEs were aggregated by system organ class (SOC) and preferred terms (PTs) within system organ classes according to the Medical Dictionary for Regulatory Affairs (MedDRA®) version 18 and as reported by participants. Top-line summaries of AEs, including number of events, number of participants, and percentage of participants for each category, were aggregated by cohort and overall. Potentially relevant categories included LCTOPC1, injection procedure, and tacrolimus. Aggregations were generated for all AEs, related events, grade ≥3 events, and serious events.

[0222] [Table 15]

[0223] Summary of the cervical study. The overall safety profile of OPC1 was excellent, and immunosuppression with tacrolimus was well tolerated. MRI scans were consistent with very high sustained engraftment rates (96%) over the course of 1 year after injection. The majority of participants who received 10M or 20M OPC1 cells demonstrated motor recovery in the upper limbs. 21 of 22 participants in cohorts 2-5 improved by at least 1 motor level on at least one side. 7 of 22 participants in cohorts 2-5 improved by at least 2 motor levels on at least one side. Two issues that may negatively impact motor recovery (C4 NLI; postoperative spinal cord compression) could be addressed in future studies. These promising engraftment and motor recovery data merit further evaluation in studies incorporating rehabilitation periods utilizing novel strategies designed to enhance the potential of hESC-based therapies to promote functional recovery. Data from the cervical study will inform the design of future randomized trials regarding inclusion / exclusion criteria, dose, and timing of administration.

[0224] Discussion: Safety data from this study suggest that AST-OPC1 can 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. No SAEs were reported directly related to LCTOPC1, and evaluation of AEs did not show an increased incidence of commonly reported SCI complications, such as urinary tract infections, muscle spasms, or neuropathic pain (Sezer 2015). In this study of participants with cervical C4-C7 AIS-A and AIS-B, at 1-year follow-up, 24 / 25 (96%) participants had regained ≥1 level of motor function on at least one side of the body, and 8 / 25 (32%) participants had regained ≥2 levels of motor function on at least one side of the body. 2 Improvements in motor level can change a person's functional ability from needing total assistance to being nearly independent in activities of daily living (Whiteneck et al. 1999). Safety and neurological recovery data from both thoracic and cervical studies provide evidence that hESC-derived therapeutics can be safely delivered to the spinal cord.

[0225] Overall, following first-in-human trials, there have been several improvements in the production and quality of OPC1: a novel ready-to-inject formulation was developed, elimination of dose preparation was achieved, production scale was expanded 10-20 fold, product impurities were significantly reduced, functional activity was improved, 12 novel analytical and functional methods were developed, and all animal-based manufacturing reagents were eliminated.

[0226] 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 suppress inflammation and glial scar formation and may promote axonal growth across the injury interface after acute spinal cord injury (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). Decorin has been shown to suppress acute scar formation 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 suppresses 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 the dissolution of mature scar after SCI (Esmaeli, Berry et al. 2014, Ahmed, Bansal et al. 2014). OPC1 treatment in non-clinical models of SCI showed results similar to those seen in the published studies mentioned above.

[0227] OPC1 cells have been shown to produce large amounts of decorin. Results seen in OPC1 animal studies show very similar anatomical outcomes to those seen in the studies mentioned above. Therefore, the anatomical effects observed in preclinical efficacy studies of OPC1 transplantation into SCI injuries may be due, at least in part, to the secretion of decorin.

[0228] Previous preclinical studies have established that the optimal time for OPC1 implantation to exert its maximum effect is 14–60 days after injury, however decorin has been shown to be effective in dissolving mature scars after SCI (Esmaeli, Berry et al., 2014; Ahmed, Bansal et al., 2014), providing evidence for a potential role for OPC1 cells in the treatment of chronic SCI subjects.

[0229] Introduction Decorin is a natural 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, an in vitro decorin secretion assay was developed and qualified as a potency assay for OPC1.

[0230] Briefly, OPC1 drug cells are thawed and cultured for 48 hours, then the medium is harvested and the secreted decorin concentration is measured by ELISA assay.

[0231] Preliminary molecular analysis and initial ELISA revealed that decorin is not secreted by H1 hESCs, its expression is gradually turned on during OPC1 differentiation, and the highest levels of secreted protein were detected in the drug product. This, together with the scientific literature showing biological activities such as inhibition of scar formation at injury sites and stimulation of axonal growth through spinal cord injury (SCI) sites, supports decorin as a suitable efficacy candidate.

[0232] Decorin (secreted) is useful as a potency indicator of OPC1 cells by demonstrating their ability to regulate SCI tissue remodeling by attenuating deleterious processes.

[0233] Decorin is a biological regulator secreted by OPC1 The OPC1 preparation is a cryopreserved cell population containing oligodendrocyte progenitor 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 essential 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 induction of NG2 in response to retinoic acid. +(Neuronal-glial antigen 2, CSPG4) This could be an upregulation of decorin as a cellular response (Goncalves, Wu et al. 2019).

[0234] It is important to note that decorin secretion is acquired during the differentiation process as part of the maturation of OPC1 cells, a feature that allows easy and quantifiable measurement of decorin as a potential potent marker for OPC1.

[0235] 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.

[0236] Decorin has been shown to be effective in reducing scarring when produced endogenously by various cell types at the injury site or administered exogenously in recombinant form in preclinical models of SCI, as described below.

[0237] Therefore, active de novo secretion of endogenous decorin by OPC1 during implantation into the SCI cavity may be a key factor to ensure successful treatment and, therefore, may be a good efficacy marker.

[0238] Spinal cord injury The majority of traumatic SCIs result in contusion or compression of the spinal cord. The mechanical injury (primary injury) in these cases 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).

[0239] The cellular response to SCI is generally thought to consist of three phases: in the acute hemorrhagic phase, hematogenous inflammatory cells invade the wound; in the subacute phase, astrocytes interact with invading meningeal fibroblasts to form a glial border with a core of extracellular matrix (ECM) proteins around the wound cavity, revascularization is also initiated, and axonal growth ceases at the wound edge; and in the sclerotic or chronic phase, ECM deposits are remodeled by proteases to form a mature contracture scar.

[0240] The cellular response is superimposed on progressive wound cavitation, resulting in progressive cystic expansion of astrocyte-free cavities filled with proteoglycans and macrophages and bordered by proteoglycan-rich neutrophils that cause secondary destruction of axons.

[0241] Cellular and extracellular composition of spinal cord injury scar tissue Traumatic SCI triggers a complex cascade of events that ultimately leads to the formation of a scar composed of multiple cell types, as well as extracellular and non-neuronal components. During the acute phase (0–72 h), cell death and injury result in the release of numerous cell- and blood-derived damage-associated molecular patterns (DAMPs). These are potent activation and inflammatory stimuli for interstitial cells, astrocytes, NG2+ OPCs, and microglia. During this acute phase, fibroblast-like cells proliferate from perivascular sites. Activated cells increase the 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 (proinflammatory and prohealing). Over time, the injury microenvironment becomes increasingly proinflammatory. In the chronic SCI scar, monocyte-derived macrophages / microglia adopt a predominantly proinflammatory phenotype. Rather than entering a resolution phase, responding innate immune cells present DAMPs to circulating adaptive immune cells and the disease spreads. Reactive astrocyte hypertrophy, upregulated expression of intermediate filament-associated proteins, and secretion of matrix CSPGs occur. Scar-forming reactive astrocytes organize into barrier-like structures that separate remaining tissue from the central area of ​​inflammation and fibrosis where wound healing fails to heal. In most mammalian species, chronic cystic cavities 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 acts together with CSPGs to inhibit neuroregeneration and neuroplasticity (Bradbury and Burnside 2019).

[0242] Function of decorin in spinal cord injury The clinical application of OPC1 targets the subacute phase, 21-60 days after SCI. Therefore, it is assumed that transplantation of OPC1 will take place during the transition from the acute to chronic phase, in an environment of active inflammation. Therefore, the ability of OPC1 to actively secrete decorin, which can potentially reduce ongoing negative cues, may be useful for its therapeutic activity.

[0243] Decorin (1) attenuates 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) that mediate transcriptional activation of ECM production; (2) binds to type I collagen fibrils and inhibits fibrillogenesis; (3) forms an active inhibitory complex with connective tissue growth factor (CTGF); (4) binds to fibronectin and inhibits cell adhesion and migration of fibroblasts; and (5) inhibits inflammation, CSPG / laminin / fibronectin-rich scar formation, and proliferation by astrocytes, microglia, and macrophages. (6) stimulate microglia to secrete plasminogen / plasmin (the activity of which is regulated by PAI-1), which in turn regulates the matrix metalloproteinase (MMP):tissue inhibitor of MMP (TIMP) ratio in the wound to initiate fibrolytic degradation of the ECM underlying remodeling during the consolidation 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).

[0244] Recombinant human decorin (Galacorin) was investigated as a potential treatment for macular degeneration, diabetic retinopathy, and diabetic macular edema (Nastase, Janicova et al. 2018). In patent US9061047B2, the authors propose the use of Galacorin to prevent or reduce scar formation by administering Galacorin to patients with neurological pathologies, including damage and / or disease of the central nervous system. Formulation of Decorin as eye drops has been reported as a scar formation prevention agent that could be an alternative to corneal transplants (Hill, Moakes et al. 2018).

[0245] Decorin promotes axonal regeneration directly by inhibiting scar-derived production of growth inhibitory ligands (Esmaeili, Berry et al. 2014), and indirectly by (1) plasmin-mediated activation of neurotrophins (Davies, Tang et al. 2006), (2) disinhibiting axonal growth cone advancement and plasmin-induced MMP activation through plasmin-mediated digestion of CSPGs and CNS myelin inhibitors (Minor, Tang et al. 2008), and (3) inhibiting EGFR activity in growth cones, thereby potentially preventing CSPG / CNS myelin-mediated growth cone collapse.

[0246] Treatment with recombinant decorin in a rat model of SCI supports therapeutic potential It has been shown that recombinant human decorin (rh-decorin) may inhibit inflammation, glial scar formation, and CSPG expression and promote axonal growth across the injury interface after acute spinal cord injury (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). These data indicate that decorin treatment initiated in animal models immediately after spinal cord injury inhibits TGF-β1 / 2-mediated inflammatory cell invasion, scar deposition, and cavitation, and then during the consolidation phase, controls ECM remodeling 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-β31 / 2, scar dissolution appears to be induced by MMP / tPA-mediated fibrinolytic activity and promoted by reduced TIMP and PAI-1 activity.

[0247] Decorin inhibited acute scar formation (fibrosis) and wound cavitation and induced dissolution of mature scar tissue (fibrolysis) in a dorsal column lesion (DFL) SCI model system in adult rat spinal cord (Wu, Li et al. 2013, Ahmed, Bansal et al. 2014). DFL cavitation treatment with recombinant decorin inhibited inflammation and scar deposition during the acute and subacute phases of the CNS injury response in a rat model of SCI and also contributed to dissolution of mature scar tissue after SCI. Decorin treatment of spinal cord injury (Esmaeili, Berry et al. 2014, Ahmed, Bansal et al. 2014).

[0248] Furthermore, decorin promoted axonal regeneration in both acute and chronic experiments: in both cases, axons were absent in PBS-treated DFLs, but present in decorin-treated DFLs.

[0249] OPC1 treatment in preclinical models of SCI shows results similar to those seen with decorin treatment In five studies (Table 16) of OPC1 transplantation into rodent models of SCI, a statistically significant reduction in cavitation area was observed in OPC1-treated animals compared to animals injected with control vehicles (HBSS or IsoLyte + human serum albumin). In these studies, axonal regeneration through the SCI lesion was observed in all OPC1-treated animals, but not in control animals. The tabular results of these studies are shown below. The rat model of SCI injury used closely emulates the damage 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 days 1-7: acute, days 7-14: subacute, and days 14 and beyond: chronic. In other examples, the route of administration includes implanting the composition (e.g., including OPC1) into the subject using a PSD system, such as the PSD system described herein. In other examples, the route of administration includes implanting a composition (e.g., comprising OPC1) into a subject using a PSD as shown and described in U.S. Publication No. 2015 / 0224331, the entire contents of which are incorporated by reference herein.

[0250] [Table 16]

[0251] Staining for the presence of myelinated axonal fibers performed in the first four studies above showed the presence of myelinated axonal fibers crossing the lesion area in OPC1-treated animals but not in control-treated animals.

[0252] The results observed in OPC1 animal studies show anatomical results very similar to those observed in studies in which decorin-infused implants were implanted in animals with spinal cord injuries as described above (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 at least partially due to the secretion of decorin.

[0253] summary As mentioned above, the current knowledge of the positive role that decorin plays in reducing damage in the SCI cavity and the results of preclinical studies in decorin-treated and OPC1-treated SCI models 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 major therapeutic effects of OPC1 by positively regulating scar formation and axonal regeneration inhibitory processes. Therefore, it is intended to use a qualified decorin assay for inclusion in a panel of release tests of novel and improved OPC1 production processes.

[0254] References for Example 10: Ahmed,Z.,D.Bansal,K.Tizzard,S.Surey,M.Esmaeili,A.M.Gonzalez,M.Berry and A.Logan(2014).“Decorin blocks scarring and cystic cavitation in acute and induces scar dissolution in chronic spinal cord wounds.”Neurobiol Dis 64:163-176. Bradbury,E.J.and E.R.Burnside(2019).“Moving beyond the glial scar for spinal cord repair.”Nat Commun 10(1):3879. Davies,J.E.,X.Tang,J.C.Bournat and S.J.Davies(2006).“Decorin promotes plasminogen / plasmin expression within acute spinal cord injuries and by adult microglia in vitro.”J Neurotrauma 23(3-4):397-408. Esmaeili,M.,M.Berry,A.Logan and Z.Ahmed(2014).“Decorin treatment of spinal cord injury.”Neural Regen Res 9(18):1653-1656. Goncalves,M.B.,Y.Wu,E.Clarke,J.Grist,C.Hobbs,D.Trigo,J.Jack and J.P.T.Corcoran(2019).“Regulation of Myelination by Exosome Associated Retinoic Acid Release from NG2-Positive Cells.”J Neurosci 39(16):3013-3027. Gubbiotti,M.A.,S.D.Vallet,S.Ricard-Blum and R.V.Iozzo(2016).“Decorin interacting network:A comprehensive analysis of decorin-binding partners and their versatile functions.”Matrix Biol 55:7-21. Hill,L.J.,R.J.A.Moakes,C.Vareechon,G.Butt,A.Ng,K.Brock,G.Chouhan,R.C.Vincent,S.Abbondante,R.L.Williams,N.M.Barnes,E.Pearlman,G.R.Wallace,S.Rauz,A.Logan and L.M.Grover(2018).“Sustained release of decorin to the eye surface enables scarless corneal regeneration.” NPJ Regen Med 3:23. Minor, K., X. Tang, G. Kahrilas, SJArchibald, JEDavies and SJDavies(2008).“Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons.” Neurobiol Dis 32(1):88-95. Nastase,MV,A.Janicova,H.Roedig,LTHsieh,M.Wygrecka and L.Schaefer(2018).“Small Leucine-Rich Proteoglycans in Renal Inflammation:Two Sides of the Coin.” J Histochem Cytochem 66(4):261-272. Wu, L., J. Li, L. Chen, H. Zhang, L. Yuan and SJDavies(2013).

[0255] Figure 11 - GPOR OPC1 Converter and LTCOPC1 OPC1 Converter Interface Some of the processes for producing cells and OPC1 products, such as those used in the clinical trials described in the Examples herein, are referred to as the Geron Process and Geron Cells or GPOR. The novel process described in this Example and other Examples herein is used to produce LCTOPC1 products. The LCTOPC1 process described herein is the process used for current GMP production of cells for clinical use. This Example provides data supporting comparability between these manufacturing processes.

[0256] List of Abbreviations ASIA American Spinal Cord Injury Association Functional Disorders bFGF Basic FGF BMP Bone morphogenetic protein CMC Chemistry, Manufacturing and Quality Control CS10 CryoStor 10 DP formulation ECM Extracellular Matrix FCM Flow cytometry GMP Good Manufacturing Practice GPM Glial Progenitor Cell Medium GPOR Geron Recording Process hESC human embryonic stem cells hsFGF Heat-stable FGF IPC in-process control MCB Master Cell Bank NB nerve body NLI Neurological injury level OCB Original Cell Bank OL Oligodendrocyte POC Proof of Concept QC quality control RA Retinoic Acid R&D Research and Development rhEGF Recombinant human EGF RL Risk Level RMAT Advanced Regenerative Medicine Therapy SCI Spinal Cord Injury TAI thawing and injection UEMS Upper Extremity Motor Score WCB Working Cell Bank

[0257] LCTOPC1 (OPC1), previously termed GRNOPC1 and subsequently AST-OPC1, is an oligodendrocyte progenitor cell population derived from the H1 hESC line intended for one-time administration for the treatment of subacute spinal cord injury (SCI). In preclinical studies, OPC1 has been shown to produce neurotrophic factors, migrate to the spinal cord parenchyma, stimulate angiogenesis, and induce remyelination of denuded axons, all essential functions of oligodendrocyte progenitor cells and important for axonal survival, regeneration, and function.

[0258] Clinical evaluation of LCTOPC1 was initiated by Geron Corporation in 2010. The first clinical trial was a Phase 1 safety study (NCT01217008) in which 2 × 10 6 A total of five subjects out of a planned eight received OPC1 cells from October 2010 to November 2011 as part of the original Phase 1 CP35A007 safety trial.

[0259] In 2014, we initiated a dose escalation phase 1 / 2a study (NCT02302157) of OPC1 in subjects with subacute sensorimotor complete impairment (American Spinal Cord Injury Association Impairment Scale A (ASIA Impairment Scale A)), single neurologically significant (SNL) C5-C7 cervical spinal cord injury, and subsequently expanded the study to patients with C4 neurologically significant (NLI) if their upper extremity motor score (UEMS) was ≥1, and changed the dosing period from 14-30 days to 21-42 days after spinal cord injury. The AST-OPC-01 study enrolled a total of 25 subjects across five cohorts who received a single dose of OPC1 cells delivered by intraparenchymal injection at the site of spinal cord injury using a syringe positioning device during a dedicated surgical procedure. Enrollment for the AST-OPC-01 study was completed in December 2017 and reported in December 2020.

[0260] Briefly, the origin of the new Master Cell Bank (MCB) is H1 bank lot number MCBH101. MCBH101 was produced directly from the H1 Original Cell Bank (OCB) by Geron in 2009. It was produced in a feeder-free state using well-defined raw materials, a new culture system and harvesting procedures, and cryopreserved by a cryopreservation process customized for hESCs. Furthermore, we optimized the method to evaluate the pluripotency of H1 hESCs. A new WCB was derived from the new MCB, expanded in tissue culture for four passages while maintaining hESC properties, and then cryopreserved. The WCB will be the starting material for LCTOPC1 production.

[0261] 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 comparative results based on R&D runs of improved manufacturing processes, comparability of GPOR and LCT R&D manufacturing materials, introduction of proposed new potency assays, review of the safety status of OPC1 based on GPOR in vivo data, and LCTOPC1 development plans for reanalysis of GPOR lots using improved analytical methods.

[0262] Rationale for process improvement OPC1 is an investigational drug that has been tested 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 originally developed in the early 2000s. At that time, well-defined cell therapy grade reagents and materials were not widely available. Therefore, stage 1 of the manufacturing process included 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.

[0263] Moreover, the manufacturing process of GPOR was based on a poorly controlled differentiation process driven by three guide molecules. The majority of the differentiation process occurred in cell aggregates starting directly from pluripotent H1 cells in the form of embryoid bodies (day 0-day 26, Figure 10), which are highly susceptible to spontaneous differentiation. From day 27 onwards, differentiation was completed on an adhesive surface coated with Matrigel™ for the proliferation and maturation of oligodendrocyte precursor cells. The GPOR manufacturing process had low yields and the main quality attributes defined by purity / impurity / non-target cell population markers had limited reproducibility. Moreover, the final cryopreserved product had to be thawed, washed and formulated before administration.

[0264] The development of the improved manufacturing process focused on a more controlled guided differentiation of H1 to OPC induced by a sequence of specific growth factors and small molecules that inhibit or induce the differentiation pathway, using cell therapy grade materials where possible (as detailed in Figure 11). Furthermore, this novel process shortens the long aggregation phase used by Geron from 26 days directly from a pluripotent cell state prone to spontaneous aberrant differentiation to 7 days after 14 days of monolayer guided differentiation of H1 cells to neuroectoderm, reducing the chance of spontaneous differentiation during the aggregation phase. The GPOR and its corresponding LCTOPC1 differentiation process are summarized in Figure 10. The biological basis of the signaling sequence of inducers and inhibitors of the improved differentiation process is shown in Figure 11. Furthermore, novel in-process controls (IPCs) were added to better monitor and characterize the differentiation process, as detailed in Figure 12.

[0265] The materials used for the production of OPC1 cells (both the original GPOR and the modified process) are summarized in Table 17.

[0266] [Table 17]

[0267] Overview of LCTOPC1 manufacturing process Stage I - H1 proliferation Pluripotent H1 cells are thawed and cultured on laminin-coated vessels in mTeSR Plus medium for 12-15 days. Cells are subcultured and expanded using the non-enzymatic reagent ReLeSR (as described for MCB and WCB hESC cultures).

[0268] During proliferation, cells are assessed morphologically and at the end of three passages (before the start of the differentiation process), hESC pluripotency is assessed based on flow cytometry.

[0269] Stage II - Differentiation of H1 to OPC1 From day 0 of differentiation until the end of the process, the cells are cultured in Glial Progenitor Medium (GPM), which is DMEM / F-12 supplemented with B27 and T3.

[0270] Days 0–7 - On day 0, when the H1 cultures reach the required criteria defined by lactate concentration and cell morphology, the differentiation process is initiated by changing the medium for expanded pluripotent hESCs cultured in laminin-coated vessels as follows: On days 0–3, the GPM medium is supplemented with retinoic acid (RA), dorsomorphin, and PD0325901 to direct the differentiation process towards the neuroectodermal pathway (Kudoh, Wilson et al. 2002). Dorsomorphin inhibits bone morphogenetic protein (BMP) signaling (SMAD pathway) and thus inhibits mesodermal and trophoblast differentiation (Li and Parast 2014). PD0325901 inhibits downstream bFGF signaling at MEK1 and MEK2, inhibiting pluripotency and endoderm differentiation (Sui, Bouwens et al. 2013). In summary, RA promotes neural tube (neurectoderm) formation by activating the RA signaling pathway and inhibiting pluripotency, endoderm, mesoderm, and trophoblast 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, retinoic acid and ascorbic acid are added to the cultures to continue the induction of neuroectoderm differentiation (Duester 2008).

[0271] Days 7-14 - On day 7, cells are enzymatically harvested using TrypLE Select and then plated as monolayers on laminin-coated vessels from days 7 to 14 and cultured in GPM supplemented with rhEGF, hsFGF, and ROCK inhibitor (ROCK inhibitor only for the first 48 hours) (Hu, Du et al. 2009, Patthey and Gunhaga 2014, Zheng, Li at al. 2018).

[0272] Days 14–21 - On day 14, to promote neural body (NB) aggregate formation, cells are enzymatically harvested using TrypLE Select and cultured as dynamic suspension cultures for 1 week in GPM supplemented with ROCK inhibitor (first 48 h) as well as rhEGF and hs-rhFGF.

[0273] Days 21-42 - On day 21, aggregates are re-seeded 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 cultured as adherent cultures on laminin-coated vessels in GPM supplemented with rhEGF and PDGF from days 35-42 for terminal expansion and maturation, with enzymatic passage approximately every 7 days using TrypLE Select.

[0274] At the end of the expansion process, OPC1 cells are harvested using TrypLE Select and cryopreserved in CryoStor® 10 (CS10; BioLife Solutions, Inc.) cryopreservation medium as a ready to thaw and inject (TAI) formulation. The manufacturing process flow of LCTOPC1 is shown in FIG.

[0275] In-process control tests are performed at all critical steps during the differentiation process of hESCs to OPC1s, as shown in Figure 12. 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, astrocyte, 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 to initiate differentiation starting at day 0 and as a surrogate for cell counting to determine the surface area required for aggregate plating for pre-OPC generation and proliferation at day 21.

[0276] 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 representative batches manufactured by Geron and characterized using the updated analytical methods used to release OPC1 manufactured by the new process. This plan includes testing of attributes used as release criteria for GPOR as well as additional markers identified. The proposed comparison is based on quality attributes that characterize the drug product as described in Table 18.

[0277] A side-by-side comparison of LCTOPC1 and GPOR OPC1 batches will be performed based on statistical analysis to calculate the expected ranges for quantitative measurements of quality attributes from GPOR OPC1 batches. The values ​​of the quality attributes measured in the LCTOPC1 batches will be evaluated in relation to the expected ranges of the quality attributes tested. It is expected that the comparative data analysis will establish reproducible release criteria for the LCTOPC1 process and demonstrate low batch-to-batch variability for LCTOPC1.

[0278] Quality attributes to be tested include viability, identity / purity, impurities / non-target populations, gene profiling, and functional / potency assays of two to three representative GPOR and LCTOPC1 batches each.

[0279] The proposed comparability quality attributes are: Viability - a critical quality attribute of any live cell preparation; Identity / Purity - assessment of characteristic oligodendrocyte progenitor protein markers: NG2, GD3, PDGFRα, and PDGRFβ; Non-target cell populations / impurities - (i) residual H1 from the starting material; Combined with hESC-multicolor flow cytometry testing, human embryonic stem cell protein markers TRA-1-60 and Oct-4 as potential sources of teratogens (TRA-1-60 and Oct-4 are commonly known markers of embryonic stem cells and were previously used as OPC1 release criteria), and (ii) evaluation of potential aberrant differentiation pathways (protein markers for epithelial cells: known epithelial markers keratin 7, claudin 6, EpCAM, and CD49f, mesodermal cell protein markers CXCR4 and CD56 (known mesoderm and cartilage markers), astrocyte cell protein marker GFAP, known astrocyte marker, neural phenotype cell protein marker b-tubulin 3, known neural marker, mesenchymal cell mRNA to induce epithelial-mesenchymal transition). Endodermal cell mRNA markers OLR1, known endoderm markers FOXA2, SOX17, and AFP, as well as previously used nestin and α-actinin attributes, are not included, as new data indicates that nestin is not specific for OPCs, but rather a marker for NSCs and other cell types, and α-actinin is 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 of the central nervous system, attenuates scar tissue formation, inhibits cavitation, and promotes wound healing. The detailed rationale for decorin secretion as a proposed potency assay is discussed in Example 10. (ii) OPC1 cell migration in response to platelet-derived growth factor-BB (PDGF-BB), which is critical for cell movement at the injury site to ensure the widest anatomical coverage from a single injection location in the spine.(iii) Development of a novel potency test to evaluate the maturation and myelination potential of OPC1. This assay is based on the essential function of OPC1 cells, remyelination of 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), which should allow OPC1 to mature into oligodendrocytes (OLs).

[0280] The 3D nanotube environment mimics 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 expression of OL protein markers (MBP, O4, MAG, MOG) by immunocytochemistry. The assay is currently under development as a proof of concept (POC) and will be established if proven sufficiently robust.

[0281] The proposed test panels to be used to test GPOR OPC1 and LCTOPC1, as well as the proposed release criteria for LCTOPC1, are shown below in Table 18.

[0282] [Table 18] TIFF2025504662000023.tif156170

[0283] Preliminary comparability of R&D LCTOPC1 batches from representative runs is shown in Tables 19-23 below.

[0284] [Table 19]

[0285] [Table 20]

[0286] [Table 21]

[0287] [Table 22]

[0288] [Table 23]

[0289] Review of ectopic tissue formation as a release criterion for OPC1 preparations This analysis revealed a consistent correlation between available data from GLP toxicity testing of Geron GMP batches and purity and expression levels of impurity / non-target cell population specific markers.

[0290] Retest results and analysis showed a correlation between in vivo impurities and batch rejections, and indicated that LCTOPC1, characterized by significantly lower impurity levels and higher purity profiles, had a significantly lower potential for cyst formation in vivo than GPOR OPC1.

[0291] summary Preliminary comparability data of representative GPOR and LCTOPC1 batches shows that GPOR OPC1 and R&D LCTOPC1 display similar OPC1 identity / purity characteristics, except for the PDGFR-α biomarker (higher in R&D LCTOPC1, potentially due to improved OPC1 manufacturing process (directed differentiation)). LCTOPC1 has lower levels of impurities from epithelial cells, astrocytes, and neural non-target cells compared to GPOR OPC1. While both LCTOPC1 and GPOR OPC1 have little detectable residual hESCs (detected by % TRA-1-60+ / Oct4+), GPOR OPC1 has a higher percentage of pluripotent cells compared to LCTOPC1, as indicated by cell populations displaying TRA-1-60+ / Oct4- and CD49f+. LCTOPC1 has lower gene expression levels of endodermal and mesenchymal non-target cells compared to GPOR OPC1. LCTOPC1 and GPOR OPC1 exhibit similar decorin secretion and migration capabilities.

[0292] Conclusion: Data accumulated to date indicate that LCTOPC1 preparations manufactured with improved processes, generated from a cell banking system defined by a highly reproducible and tightly controlled differentiation process, and monitored with modern analytical methods, exhibit similar fundamental quality characteristics as GPOR OPC1, but benefit from an overall higher expression of purity markers and a lower expression of impurity / non-target population markers, thus potentially improving the safety profile of LCTOPC1 preparations.

[0293] References for Example 11: Duester, G. (2008). “Retinoic acid synthesis and signaling during early organogenesis.” Cell 134(6):921-931. Hu,B.Y.,Z.W.Du,X.J.Li,M.Ayala and S.C.Zhang(2009).“Human oligodendrocytes from embryonic stem cells:conserved SHH signaling networks and divergent FGF effects.”Development 136(9):1443-1452. Janesick,A.,S.C.Wu and B.Blumberg(2015).“Retinoic acid signaling and neuronal differentiation.”Cell Mol Life Sci 72(8):1559-1576. Koch,U.,R.Lehal and F.Radtke(2013).“Stem cells living with a Notch.”Development 140(4):689-704. Kudoh,T.,S.W.Wilson and I.B.Dawid(2002).“Distinct roles for Fgf,Wnt and retinoic acid in posteriorizing the neural ectoderm.”Development 129(18):4335-4346. Li,Y.and M.M.Parast(2014).“BMP4 regulation of human trophoblast development.”Int J Dev Biol 58(2-4):239-246. Ota,M.and K.Ito(2006).“BMP and FGF-2 regulate neurogenin-2 expression and the differentiation of sensory neurons and glia.”Dev Dyn 235(3):646-655. Patthey, C. and L. Gunhaga (2014). “Signaling pathways regulating ectodermal cell fate choices.” Exp Cell Res 321(1):11-16. Sui, L., L. Bouwens and JKMfopou (2013). “Signaling pathways during maintenance and definitive endoderm differentiation of embryonic stem cells.” Int J Dev Biol 57(1):1-12. Watabe, T. and K. Miyazono (2009). “Roles of TGF-beta family signaling in stem cell renewal and differentiation.” Cell Res 19(1):103-115. Zheng, W., Q. Li, C. Zhao, Y. Da, HLZhang and Z. Chen (2018). “Differentiation of Glial Cells From hiPSCs: Potential Applications in Neurological Diseases and Cell Replacement Therapy.” Front Cell Neurosci 12:239.

[0294] Example 12. Use of a Parenchymal Spinal Delivery (PSD) System Described herein is a parenchymal spinal delivery system (FIGS. 39-41) that enables a method of improving neurological function in a subject with spinal cord injury. In one embodiment, the method comprises administering to a subject one or more doses of a population of oligodendrocyte precursor cells using the PSD system shown in FIGS. 39-41.

[0295] In other embodiments, the oligodendrocyte progenitor cell population comprises an increased percentage of cells positive for the oligodendrocyte progenitor cell marker NRG2 (neuregulin 2) and decreased expression of non-OPC markers including CD49f, CLDN6, and / or EpCAM.

[0296] The PSD system includes a needle / channel assembly, a platform, an XYZ manipulator, a syringe, a microinjection pump, and a foot pedal (see FIG. 39).

[0297] In certain embodiments, the PSD system described herein provides special advantages. For example, the system provides stability and control by eliminating movement between the platform, the XYZ manipulator, and the injection needle. In addition, the system (or device) is attached directly to the patient and synchronized with the patient's respiratory movements, so there is no need to hold the breath, and the magnetic needle provides stabilization from micro-movements due to heartbeat. Other advantages are that the system is easy to use in a clinical environment, at least due to its small size and the small number of parts required, is easily assembled before surgery, allows one-handed XYZ positioning, provides precise depth insertion of the needle, is easy to clean and sterilize, is compatible with OPC1 TAI formulations, and does not require day-before administration preparation.

[0298] All references cited herein are incorporated by reference in their entirety for all content taught therein.

[0299] Although the present disclosure has been described with reference to specific embodiments, those skilled in the art will recognize that various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope. Therefore, it is not intended that the disclosure be limited to the particular embodiment disclosed as the best mode contemplated for carrying out the disclosure, but rather, the disclosure is intended to include all aspects within the scope and spirit of the appended claims.

[0300] Example 13. Performance of the magnetic needle in a PSD system The purpose of this test was to determine the magnetic repulsion characteristics over the full range of motion of the magnetic needle of the PSD system and the magnetic needle component of the flow path assembly. The scope of the following description is specific to the magnetic needle of the PSD system and the magnetic needle component of the flow path assembly. The magnetic needle repulsion was tested using a total of fifteen (15) magnetic needles. The magnetic repulsion and displacement of each sample was measured over the full range of motion of the magnetic needle (FIG. 43).

[0301] The magnetic needle assembly was mounted in a fixture as shown in Figure 44. Each sample was mounted in the test fixture so that the tip of the needle was pointing vertically upward and floating.

[0302] Results and Discussion: The magnetic repulsion force and displacement of each sample were measured to characterize the performance over the full range of motion of the magnetic needle. At the maximum range of motion, the average magnetic repulsion force of the levitating needle for the 15 samples tested was determined to be 63.15 (7.74) gf with a corresponding average displacement of 6.85 (0.50) mm.

[0303] All needles performed similarly, and by combining the data from all samples into a single curve, an average characteristic load (force) vs. displacement (movement) curve was generated. Video analysis was then performed on the simulated use of the magnetic needle in the porcine spinal cord model throughout the injection process. From this analysis, correlations were generated between magnetic force and deflection at specific points of use: (1) initial penetration, (2) final penetration, (3) resting position, and (4) breathing. See Figures 45A, 45B, and 46A-46D.

[0304] During insertion of the 6mm needle, a penetration force of 19.38gF was measured at 4.62mm of stroke. The insertion force gradually increased during the injection and at final penetration, a magnetic load of 30.43gF was measured with a needle travel of 5.51mm. After the needle was fully inserted to the stop mechanism, the needle drive pressure was reduced and the needle was allowed to rest until no visible signs of dura were observed. In the rest position, the magnetic needle has a magnetic force of 8.65gF and is at 0.90mm of stroke. From this rest position, respiratory motion increases the magnetic needle stroke up to a maximum of 2.62mm with a load of 10.49gF. See Figure 47 for an explanation of the magnetic needle performance at these defined use points.

[0305] The deflection of the magnetic needle measured during simulated breathing was approximately 1.72 mm, which is the difference between the resting and breathing positions. In real human patients, the dynamics of the spinal cord and dura were quantitatively analyzed, and it was reported that the pulsation amplitude of the spinal cord ranged from 0.01 to 0.84 mm, and that of the dura ranged from 0.01 to 0.38. It was found that the spinal cord and dura can move completely independently of each other due to breathing and heartbeat (Kimura et al. Eur Spine J 21:2450-2455 (2012)). Thus, when synchronized movement of the spinal cord and dura occurs, the movement can amount to a total of 1.22 mm. The total spinal cord movement value from the literature (1.22 mm) is comparable to the video analysis measurements (1.72 mm) in a porcine spine model.

[0306] Conclusions. The magnetic repulsion force of the magnetic needle of the PSD system was characterized over its entire range of motion and at specific use points. The magnetic repulsion force of the magnetic needle at the use points was found to be adequate and not excessive. During use, the magnetic needle does not damage tissue with excessive force and the needle maintains the proper position at each use point. The displacement and magnetic force of the floating magnetic needle are sufficient to maintain the proper needle position during breathing and cardiac rhythm. These movements measured from video analysis are comparable to those reported in the literature.

[0307] Example 14. Biodistribution of OPC1 cells using the PSD system A quantitative polymerase chain reaction (qPCR) assay, the LCT biodistribution assay, was used to measure the distribution of GPOR OPC1 or LCT OPC1 cells in tissues and cerebrospinal fluid (CSF) of Yucatan minipigs 2 days after administration of GPOR OPC1 or LCT OPC1 cells.

[0308] In this study, we analyzed Yucatan minipigs' spinal cord, brain (cerebrum, cerebellum, midbrain, medulla, pons), heart, liver, lungs, kidneys, spleen, and cerebrospinal fluid (240 unique specimens) collected 2 days after administration of GPOR OPC1 or LCT OPC1 cells.

[0309] The animal IDs used for the biodistribution study are 7149, 7145, 7141, 7152, 7195, 7189, 7196, 6858, 7265, and 7252, as shown in Table 25 below. [Table 24]

[0310] [Table 25]

[0311] Controls are as follows: positive control is nuclease free water spiked with 10000 pg of GPOR OPC1 human gDNA.

[0312] The negative control was 100 ng of naive Yucatan mini pig (pig ID 6751) spleen gDNA per well (no human gDNA). A no-template control consisting of nuclease-free water was also present.

[0313] The LCT biodistribution assessment assay is a probe-based qPCR-based assay designed to detect a 232 bp region of human Alu repeat sequences and measure the distribution of human GPOR OPC1 or LCT OPC1 cells in tissues and cerebrospinal fluid (CSF) of Yucatan minipigs after administration of GPOR OPC1 or LCT OPC1 cells. Alu sequences are a heterogeneous group of primate-specific short interspersed repetitive DNA elements with an estimated frequency of 500,000 to 1 million copies per genome. Due to their species specificity, small size, and high copy number, Alu elements are ideal targets for quantitative qPCR aimed at detecting human cells in tissues and body fluids from Yucatan minipigs.

[0314] This type of assay is an oligonucleotide probe-based assay, and the target gene or region is named Alu. The primer and probe sequences are shown in Table 26.

[0315] [Table 26]

[0316] An oligonucleotide probe (containing the fluorescent reporter dye FAM at the 5' end and the quencher dye IBFQ at the 3' end) specific for the Alu region between the forward and reverse primers was constructed (Table 26). The probe is intact, but the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye through fluorescence resonance energy transfer (FRET). If the target sequence Alu is present, the probe anneals to its specific complementary region and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended. Cleavage separates the reporter dye from the quencher dye, increasing the reporter dye signal. Additional reporter dye is cleaved from the probe with each cycle, increasing the fluorescence intensity proportional to the amount of amplicon generated. Reaction volumes are listed in Table 27.

[0317] [Table 27]

[0318] [Table 28]

[0319] DNA was extracted from tissues and body fluids and DNA concentrations were quantified using a Nanodrop.

[0320] The qPCR assay reactions were aimed at detecting human Alu Y DNA sequences. In addition to the samples, each plate contained a standard curve, a no template control (NTC), a negative control, and a positive control. Each of these standards and controls was run in triplicate reactions.

[0321] The acceptance criteria were as follows: correlation coefficient of the standard curve (R 2) must be 0.96 or greater. The qPCR reagent control (NTC) must be tested below the LOD of the assay. For the positive control, the difference in threshold cycle (Ct) values for at least two of the three sets of reactions used for quantification must be 1 Ct or less. The negative control must be tested below the LOD of the assay. For specimens within the quantification range, the difference in threshold cycle (Ct) values for at least two of the three sets of reactions used for quantification must be 1 Ct or less.

[0322] The results were evaluated as follows. If at least two of the Ct values for the three sets of reactions were indeterminate, or if the average Ct value of the specimen was greater than the average Ct value of the LOD, the specimen was reported as below the limit of detection (<LOD). If the quantified value of the specimen exceeded the upper limit of the assay range, it was reported as greater than 10,000 pg (>10,000). The quantified value of human gDNA was reported as pg per 100 ng of minipig DNA or pg per 40 μL of body fluid.

[0323] For CSF, the results were reported as the amount of human gDNA (pg) detected in the DNA extracted from 40 μL of CSF. If at least two of the Ct values for the three sets of reactions showed indeterminacy, or if the average Ct value of the specimen was greater than the average Ct value of the LOD (3.2 pg), the specimen was reported as below the limit of detection (<LOD). If the quantification of the specimen exceeded the upper limit of the assay range, it was reported as greater than 10,000 pg (>10,000). Three outliers were excluded from the analysis because the replicate Ct value had a difference greater than 1 from the other two replicate Ct values.

[0324] Conclusions: A column-based method was used to extract gDNA from various Yucatan minipig tissues and body fluids. A TaqMan-based qPCR assay was then used to measure the distribution of human GROP OPC1 or LCT OPC1 cells after transplantation into Yucatan minipig. qPCR results revealed that no human gDNA was detectable in peripheral organ gDNA (heart, kidney, liver, lung, and spleen) from pigs sacrificed 2 days after transplantation. The highest levels of human gDNA were detected across tissue types in spinal cord tissue 2 days after transplantation. Trace amounts were detected in some other central nervous system tissues (cerebrum, cerebellum, midbrain, medulla oblongata, pons, and CSF) in a few animals.

[0325] Example 15. Evaluation of the intraspinal injection procedure using the PSD system in adult Yucatan pigs: Neurological and spinal cord histopathological studies Introduction and Study Objectives Rationale for species selection. Rodent models are suitable for many aspects of cell therapeutics development, but to fully answer questions regarding the potential clinical distribution of injected cells, it is important to mimic as closely as possible the route of administration and the equipment used in clinical delivery to humans. In the specific case of intraparenchymal spinal injection of cellular test articles, an additional factor to model is the volume and directional flow of CSF that may "carry" the cells during the acute phase after implantation. Because the CSF volume in rodents is very small, this theoretical distribution route is not accurately reflected in rodent models.

[0326] Conversely, the size and pulsation of the adult pig spinal cord is similar to that of adult humans (i.e., dorsoventral movement of the spinal cord observed after dura opening; this is primarily caused by respiration). Therefore, the porcine model can be used to effectively test intraparenchymal spinal delivery systems, especially magnetic needles designed to eliminate the effects of spinal cord pulsation during intraparenchymal cell injections.

[0327] Overview of Experimental Procedure: The objective of this study is to monitor the acute safety, toxicity, and biodistribution of the investigational drug formulation, LCTOPC1 (and a previously processed version of the same formulation, designated GPOR-OPC1), following injection into the cervical spinal cord of immunosuppressed, non-injured Yucatan pigs using a custom microsurgical delivery device, the Parenchymal Spinal Delivery System. Additionally, the safety and feasibility of the administration procedure was evaluated by using periodic neurological evaluations in treated subjects.

[0328] All animals were fasted for 24 hours prior to vehicle or test article administration. Immunosuppressant administration was administered immediately prior to vehicle or test article administration and consisted of a single intramuscular dose of methylprednisolone (depo-medrol; 10 mg / kg) administered intraoperatively and a subcutaneous dose of tacrolimus microspheres (0.05 mg / kg / day; 12-day releasable formulation) administered intraoperatively. Animals sacrificed on study day 14 received a second dose of tacrolimus microspheres on postoperative day 10.

[0329] After a skin incision (3-6 inches long) in the neck (C4-7) of anesthetized animals, the spinal segments C4-C5 were exposed by a dorsal laminectomy. A stainless steel platform (parenchymal spinal delivery system) with muscle retractors and a firmly fixed XYZ manipulator was then placed over the exposed vertebrae. The dura was then incised to expose the dorsal root spinal cord entry. Animals were then intraparenchymal injected unilaterally with the highest clinically intended dose of test article (LCTOPC1; n=8, or GPOR-OPC1; n=8) or vehicle (Cryostor CS10; n=4). As a control, no injection was given to the contralateral side. Test article (1x10 in 100 μl vehicle) was injected unilaterally. 7 A total of two 100 μl injections containing 100 μl of 0.01% erythrocytes (0.01% erythrocytes) were delivered. After injection, the dura was closed with 6.0 Proline or similar non-absorbable sutures.

[0330] During the study, all animals were observed at least twice daily for morbidity, mortality, or injury and weighed at least twice weekly. Detailed neurological examinations (modified Tarlov scores) and observations were recorded daily until scheduled termination on days 2 or 14 after injection of test article or vehicle.

[0331] Blood samples for clinical pathology evaluation and serum analysis were collected from all animals prior to injection of test article or vehicle and prior to terminal necropsy. At the end of the study, necropsy examination was performed and cerebrospinal fluid (CSF) and tissues were collected for subsequent analysis of human DNA content. Histopathological examination of additional samples from collected tissues was performed. Unstained tissue sections from brain and spinal cord were used for histological confirmation of human cells by immunohistochemistry (IHC) staining. Engrafted cells were examined for viability, integration, phenotype, and signs of inappropriate proliferation or formation of ectopic structures. Immune cell infiltration into surrounding tissues was also evaluated to monitor signs of xenograft rejection.

[0332] The study was conducted in accordance with GLP guidance, standard operating procedures (SOPs), current International Conference on Harmonization (ICH) harmonized tripartite guidelines, and generally accepted procedures for testing of pharmaceutical compounds.

[0333] Test Design All 20 animals in this study were deemed suitable for enrollment based on a veterinary health screening and were weighed prior to allocation to the study. Using a simple randomization procedure, 12 male animals (weight range 20.4-26 kg) and 8 female animals (weight range 21-25.1 kg) were assigned to control and treatment groups as identified in Table 29 below.

[0334] [Table 29]

[0335] Animals were fasted on day -1 for surgery day 0. Immunosuppressant administration began on the day of test article or vehicle administration, and all animals received a single dose of tacrolimus microspheres intraoperatively, which provided a long-term release delivery of tacrolimus lasting approximately 12 days, and a single dose of methylprednisolone injected intraoperatively.

[0336] After incision of the cervical (C4-7) skin, the spinal cord was exposed by a dorsal laminectomy at C4-C5, and a parenchymal spinal delivery system was placed on the exposed vertebrae. Using this system, 1 × 10 7 GPOR-OPC1 (n=8), 1×10 7 Two injections of 100 μl containing LCTOPC1 (n=8), or Cryostor CS10 (n=4) were administered. The dura was closed using Proline or similar non-absorbable sutures. The incision site was closed at the anatomical layers. During survival, all animals were observed at least twice daily for morbidity, mortality, or injury-induced neurological deficits and were weighed at least twice weekly. Detailed examinations and observations were recorded daily until scheduled termination.

[0337] Necropsy, clinical pathology, and hematology and histopathology were performed at the end of the study. Cerebrospinal fluid and tissue samples were collected from each animal from the brain hemisphere ipsilateral to the site of test article injection, as well as samples from the brain stem, cerebellum, pons / medulla oblongata, hippocampus, forebrain, and olfactory bulb, and samples from the lung, liver, spleen, heart, testes, and kidney. All samples were flash frozen and shipped for evaluation of human cell persistence and distribution by quantitative polymerase chain reaction (qPCR). Biodistribution and histopathology analyses were performed on additional tissue samples taken from each of the brain regions (both hemispheres) and each of the peripheral organs listed above. Samples were examined for engrafted cell survival, integration, phenotype, and signs of inappropriate proliferation or formation of ectopic structures. Immune cell infiltration in the surrounding tissues was also evaluated to monitor for signs of xenograft rejection.

[0338] Experimental procedure The immunosuppressant tacrolimus was used as received from the supplier and was not adjusted for purity. The immunosuppressant was prepared as microspheres on each day of use by mixing 5 ml of saline (0.9% Sodium Chloride for Injection, USP) with the appropriate amount of tacrolimus microspheres and injecting sodium chloride. When not in use, the tacrolimus microspheres were stored at -20°C.

[0339] The immunosuppressant methylprednisolone (Depo-Medrol) was used as received from the supplier and was not adjusted for purity. Formulations were prepared at the required nominal concentrations of 40, 20, 10, 4, and 2 mg / mL on each day of use by mixing the appropriate amount of saline (0.9% Sodium Chloride for Injection, USP) with the appropriate amount of methylprednisolone. The prepared formulations were dispensed into sterile amber glass serum bottles under a laminar flow hood using aseptic technique and sterile equipment. The prepared formulations were stored at room temperature when not in use.

[0340] The vehicle, Cryostor CS10, was dispensed the day before or on the day of use.

[0341] Animals were placed in a volume of 100 μL at approximately 1 × 10 7 Animals received two injections containing approximately 1 x 10 GPOR-OPC1 cells in a volume of 100 μL each. 7 Each mouse received two injections containing LCTOPC1 cells.

[0342] Prior to surgery, all animals were fasted overnight. Animals were premedicated by IM injection of acepromazine (1.0 mg / kg), atropine (0.05 mg / kg), ketoprofen (3 mg / kg), and ceftiofur (2.2 mg / kg). An IV dose of cefazolin (25 mg / kg) was also administered. Anesthesia was induced with nitrous oxide (delivered by inhalation). Ocular lubricant was administered, an endotracheal tube was inserted, and general anesthesia was maintained with isoflurane (delivered by inhalation) delivered in oxygen with a precision vaporizer and rebreathing anesthesia circuit. The hair at the surgical site was re-clipped, and the shaved area was washed with chlorhexidine scrub and solution. LRS was administered intravenously during surgery at approximately 180-240 mL / h via catheter and infusion pump.

[0343] After a skin incision (3-6 inches long) in the neck (C4-7) of anesthetized animals, the spinal segments C4-C5 were exposed by a dorsal laminectomy. Thrombin-soaked gelfoam was used to control bleeding during surgery as necessary. The animals were artificially ventilated to maintain normal physiological levels of oxygen saturation and exhaled CO2. A stainless steel platform with muscle retractors and a securely attached XYZ manipulator (parenchymal spinal delivery system) was then positioned over the exposed vertebrae. The dura was then incised to expose the spinal dorsal root entry sites. The animals were then intraparenchymal injected unilaterally with the highest clinically intended dose of test article (LCTOPC1; n=8, or GPOR-OPC1; n=8) or vehicle (Cryostor CS10; n=4). As a control, no injection was given to the contralateral side. Test article (1×10 in 100 μl vehicle) was administered. 7 A total of two injections of 100 μl containing 100 μl of 0.1% erythrocytes (0.01 mg / mL) were delivered. After injection, the dura was closed with 6.0 Proline or similar non-absorbable suture. Fascia harvested from the surrounding incisions was placed over the closed dura at the laminectomy site. The muscle layer was closed with 2-0 PDSII. The skin was closed with surgical staples. Sodium chloride (NaCl) was used for irrigation, and bupivacaine (2 mg / kg) was injected into the incision.

[0344] Immunosuppressant administration was administered immediately prior to vehicle or test article administration and consisted of a single intramuscular dose of methylprednisolone (depo-medrol; 10 mg / kg) administered intraoperatively, and a subcutaneous dose of tacrolimus microspheres (0.05 mg / kg / day; 12-day releasable formulation) administered intraoperatively. Animals sacrificed on study day 14 received a second dose of tacrolimus microspheres on day 10 postoperatively.

[0345] Blood samples (approximately 5-7 mL) were collected from all animals ordered for pre-experimental testing and from all animals undergoing treatment prior to terminal necropsy for serum analysis. Animals were fasted prior to blood collection to coincide with fasting for clinical pathology evaluation.

[0346] At scheduled necropsy, necropsy examination was performed on all euthanized animals using procedures approved by a veterinary pathologist. Animals were euthanized under Telazol sedation with intravenous administration of sodium pentobarbital via the jugular vein, followed by transcardial perfusion with ice-cold saline.

[0347] Prior to transcardial perfusion, CSF was collected from the cisterna magna of euthanized animals, approximately 1.0–3.0 mL was collected and flash frozen in liquid nitrogen.

[0348] Animals were carefully inspected for external abnormalities such as masses. The skin was reflected through a ventral midline incision and any abnormalities identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were inspected for abnormalities and organs were removed and examined, and sample biopsies were placed in neutral buffered formalin if necessary.

[0349] Tissue samples (approximately 5 mm each) were taken from the hemisphere ipsilateral to the test article injection site to include samples from the brain stem, cerebellum, pons / medulla, hippocampus, forebrain, and olfactory bulb. 3 ) were harvested. Samples were flash frozen to preserve DNA integrity.

[0350] The remaining tissues retrieved according to the protocol were placed in 10% neutral buffered formalin for standard histological processing, as well as hematoxylin and eosin (H&E) staining for analysis by a certified veterinary pathologist. From each brain region harvested, an additional H&E slide and five unstained slides with staged sections (five staged sections spaced 50-100 micrometers apart) were processed and used for IHC. Ipsilateral H&E was compared to the contralateral side for possible trauma caused by retrieval of frozen specimens.

[0351] Peripheral tissue samples (approximately 5 mm) from the lung (left lobe), liver (left lateral lobe), spleen, heart (apex), and kidney (left) were 3 ) were harvested, flash frozen, and shipped on dry ice. Additional samples from each tissue were taken and stored in 10% neutral buffered formalin for standard histopathological processing and H&E staining for analysis by a board-certified veterinary pathologist.

[0352] Transverse (coronal, transverse) slices of the spinal cord (approximately 5 mm thick) were collected at approximately 5 cm intervals to cover the rostrocaudal extent of the spinal cord. The specimens were marked to maintain the rostrocaudal orientation and flash frozen. The remaining spinal cord was marked to maintain the rostrocaudal and dorsoventral orientation and placed in 10% neutral buffered formalin for standard histological processing and H&E staining for analysis by a pathologist. After transverse tissue trimming, the spinal cord tissue between the blocks surrounding the injection site was cut longitudinally / horizontally to cover the longitudinal extent of these spinal cord tissue pieces (approximately 4.5-5 cm in length each). To maintain consistency between all sections of the submitted spinal cord, the first 4 mm sections from the dorsal side of the spinal cord were made at the time of trimming, not at the time of microscopic sectioning, to ensure a flat surface for embedding. The remaining ventral portion was kept on a wet tissue. The rostrocaudal orientation of each section was maintained by placing the cranial end of each spinal cord section within each block / slide close to the labeled portion of the respective block / slide. To aid in maintaining the above specified orientation during sectioning, the spinal cord was embedded off-center within the block. Each sectioned spinal cord was trimmed further, if necessary, to fit appropriately into the tissue cassette, but the rostro-caudal orientation was maintained between all trimmed sections. Standard H&E staining and histopathological analysis were performed by a veterinary pathologist on each longitudinal section.

[0353] IHC detection of cells of human origin was achieved using a human nucleolus-specific antibody (Abcam, ref. ab190710). A chromogenic HRP polymer-based method was performed on a Discovery Ventana Ultra automated stainer (Roche Diagnostics). Slides of formalin-fixed paraffin-embedded human cell line (H441 cell) pellets were included as positive controls in each staining run to ensure that the procedure worked as intended. H441 slides that received an isotype control antibody instead of the human nucleolus antibody were used as negative controls. The investigator selected staining procedure #659 as the most suitable.

[0354] Negative and positive control samples (human cell pellets) were evaluated with each staining run. The two negative control samples showed no positive staining (scored "0"). The positive control sample showed strong staining (scored "5" using the following scale): 0 = no staining 1 = Minimal (very few positive cells) 2 = Mild (some positive cells) 3 = moderate (at least half of cells positive) 4 = prominent (most cells positive) 5 = severe (nearly all cells positive)

[0355] This scale was also used to evaluate the test tissues.

[0356] Real-time quantitative polymerase chain reaction (qPCR) analysis Analytical samples were collected from 20 pigs at 2 and 14 days after sacrifice. From each pig, the following tissues were collected for analysis: spinal cord tissue (13 tissues), CSF (1 sample), brain (5 tissues), and peripheral organs (heart, liver, kidney, spleen, lung) (5 tissues total); total number of tissues per pig (20) = 24. DNA was extracted from each tissue and detected by qPCR and quantified using serial dilutions of hu gDNA as standards.

[0357] Assay Description and Qualification A quantitative polymerase chain reaction (qPCR) assay was used to measure the distribution of test articles in GPOR-OPC1 and LCTOPC1 cells after administration to Yucatan minipigs. A qPCR-based assay was designed to detect specific regions of human Alu repeat sequences. The assay was used to measure the distribution of human GPOR-OPC1 or LCTOPC1 cells in tissues and cerebrospinal fluid (CSF) of Yucatan minipigs after administration of GPOR-OPC1 or LCTOPC1 cells. The assay was performed in accordance with the U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) regulations, standards, and guidelines (21 CFR Part 58). Specimen extraction (excluding CSF) yielded gDNA at a concentration ≥ 10ng / μL (CV 0.3%-3.5% and 260 / 280 ratio > 1.2). Negative extraction controls had a NanoDrop reading of less than 2.5ng / μL and / or had no discernible OD peak at 260nm. CSF was extracted volumetrically with 200 μL of starting material, regardless of DNA yield.

[0358] Sample preparation For tissues, the entire tissue piece received was homogenized and an aliquot was removed for DNA isolation. For CSF, a maximum of 200 μL was used for extraction. Specimens from each batch included untreated tissue or PBS, extracted in parallel with the test samples to monitor for contamination during the extraction process (NEC negative extraction control).

[0359] For tissues, the concentration of DNA purified from each tissue was measured by absorbance at 260 nm (A260) and then adjusted to 100 ng / μL with water. Ten microliters (1 μg DNA) were used for each qPCR. For samples with DNA concentrations less than 100 ng / μL and greater than or equal to 50 ng / μL, DNA was adjusted to 50 ng / μL and 20 μL (1 μg DNA) of DNA preparation was processed per reaction. Samples with DNA concentrations less than 50 ng / μL were processed using 20 μL per qPCR. CSF samples were processed using a volume of DNA equivalent to a maximum of 10 μL of biofluid analyzed in each reaction. For all reactions, the mass of DNA analyzed was recorded.

[0360] Real-time qPCR reaction qPCR amplification and fluorescence detection were performed using an ABI7900HT real-time PCR instrument. To monitor the presence of PCR inhibitors in each test sample DNA, a single reaction was prepared with up to 1 μg of DNA from each sample and spiked with 1 pg of human gDNA. In addition to the sample DNA, each test plate contained a set of standards, an untreated Yucatan pig gDNA negative control (0 copies), and a qPCR reagent control (NTC). Each extraction control was included in at least one run with the corresponding sample. These controls monitor for non-specific amplification of animal model gDNA during the extraction process, contamination of qPCR reagents, and possible contamination of sample DNA, respectively. All controls were performed in duplicate reactions.

[0361] hematological findings No significant differences in hematological parameters were noted in the groups terminated on day 2. One animal receiving GPOR-OPC1 cells had an increase in ALT above the healthy range, and one animal receiving LCTOPC1 had an increase in hematocrit above the healthy range. There were animals in each of the three groups that had mild increases in mean corpuscular hemoglobin concentration, which were considered procedure related. All other mean and individual values ​​were considered to be within acceptable ranges of biological and procedure related variation.

[0362] In the group terminated on day 14, moderate reactive lymphocytes were observed in one animal receiving GPOR-OPC1 and one animal receiving LCTOPC1. Another animal receiving GPOR-OPC1 had eosinophil levels increased above the healthy range. One animal receiving Cryostor CS10 showed low levels of neutrophils. There were animals in each of the three groups that yielded samples containing WBCs below the healthy range. Two animals receiving GPOR-OPC1 showed increased hematocrit levels and one showed a decrease in hematocrit. Additionally, animals in all three groups showed mild increases or decreases in red blood cells and hemoglobin, which were similar and considered related to the procedure. No other meaningful changes in hematological parameters were observed in any of the groups.

[0363] Microscopic evaluation of spinal cord and brain tissue Implant sites in animals receiving vehicle on days 2 and 14 (4 animals, 8 sites total) were free of cells consistent with human cells (H&E assessment).

[0364] In eight animals treated with the GPOR-OPC1 test article (four on day 2 and four on day 14) and a total of 16 implantation sites, cells consistent with human cells were identified in 7 / 16 sites, including: Day 2: Part 7 / 8 -Area 0 / 8 on day 14.

[0365] This total of seven sites included the seven sites identified during the initial H&E evaluation and did not include the site where putative human cells were identified during the initial H&E evaluation but no positive cells were seen by IHC (implant site 2, animal 7609).

[0366] For each of the eight animals: 3 / 8 animals had cells identified at both implantation sites (3 / 4 animals on day 2) 1 in 8 animals had cells identified at one implantation site (1 / 4 animals on day 2) Four out of eight animals had no identified cells at any site (4 / 4 animals on day 14).

[0367] In addition to the presence of human cells at the implantation site, major additional microscopic findings were: Mononuclear cell infiltrates (generally perivascular in day 2 samples, primarily scattered, high grade, and extending into grey and white matter in day 14 samples). Rare polymorphonuclear cells were observed within the mononuclear cell infiltrate but were considered a minor component and were not scored separately. · Changes in the spinal cord white matter (mainly minimal to mild axonal swelling and expansion of the myelin spaces around the axons). Acute bleeding (mainly in day 2 specimens and due to recent implantation procedures).

[0368] The frequency and severity of these additional changes was generally greater at the day 14 sites compared to the day 2 sites. Overall, there was no gross destruction of spinal cord tissue adjacent to the implantation site in any animal (e.g., there was no necrosis and / or cavitation).

[0369] Nearly all of the seven HuNu-positive sites (IHC assessed) had minimal evidence of cell destruction (characterized by either extracellular material consistent with positive staining or intracellular cytoplasmic staining consistent with macrophages, suggesting phagocytosis of human cell debris). This change was usually a minor component of the overall positive staining and was listed as either present or absent but was not graded. Evidence of cell destruction was further differentiated from a light brown pigment (cytoplasmic and extracellular) suggestive of hemosiderin.

[0370] Microscopic findings in spinal cord sections anterior and caudal to the transplant site were generally minimal and similar to those at the transplant site (eg, perivascular mononuclear cell infiltrates).

[0371] The microscopic findings in the brain were not interpreted as related to test article administration.

[0372] In the remaining 8 animals administered the LCTOPC1 test article (4 on day 2 and 4 on day 14) and in a total of 16 sites, cells consistent with human cells were identified in 8 of 16 sites (7 / 8 sites on day 2 and 1 / 8 sites on day 14 (the single site was implantation site 1 in animal 7266)).

[0373] This total of eight sites included six sites identified during the initial H&E evaluation and two sites that were reexamined after IHC data became available (implant site 1 in animal 7196 and implant site 1 in animal 7266).

[0374] Of the eight individual animals that received cells, 3 / 8 had cells identified at both implantation sites (3 / 4 animals on day 2) 2 / 8 had cells identified at one implantation site (1 / 4 animals on day 2 and 1 / 4 animals on day 14) · 3 / 8 had no identified cells at any site (3 / 4 animals on day 14).

[0375] In addition to the presence of human cells at the implantation site, the main additional microscopic findings were: Mononuclear cell infiltrates (largely perivascular in day 2 samples, primarily scattered, high grade, and extending into gray and white matter in day 14 samples) Rare polymorphonuclear cells were observed within the mononuclear cell infiltrate but were considered a minor component and were not scored separately Spinal cord white matter changes (mainly minimal to mild axonal swelling and expansion of the myelin spaces around the axons) Acute bleeding (mainly in day 2 specimens and due to recent implantation procedures).

[0376] The frequency and severity of these changes was generally greater at the day 14 sites compared to the day 2 sites.

[0377] Overall, there was no gross destruction of spinal cord tissue adjacent to the implantation site in any animal (e.g., no necrosis and / or cavitation). Furthermore, the incidence of mononuclear cell severity in vehicle-treated sites was generally low at both days 2 and 14. No changes were seen in the spinal cord white matter in control sites (e.g., no axonal swelling or dilation of myelin spaces).

[0378] The two sections in which no human cells were identified in the H&E sections contained HuNu-positive cells (implant site 1 in animal 7196 and implant site 1 in animal 7266). Subsequent reexamination of the H&E sections revealed very few cells consistent with either host macrophages or human cells (animal 7196, day 2), or individual positive cells were centrally located within pale tissue foci surrounded by larger foci of mononuclear cell infiltration (animal 7266, day 14).

[0379] In nearly all eight HuNu-positive sites, there was minimal evidence of cell destruction (characterized by either extracellular material consistent with positive staining or intracellular cytoplasmic staining consistent with macrophages, suggesting phagocytosis of human cell debris). This change was usually a minor component of the overall positive staining and was listed as either present or absent but was not graded. Furthermore, evidence of cell destruction was distinguished from a light brown pigment (cytoplasmic and extracellular) suggestive of hemosiderin.

[0380] Brain microscopic findings were rare in animals treated with LCTOPC1 and were considered unrelated to test article administration. Microscopic findings in spinal cord sections cranial and caudal to the transplant site were generally minimal and similar to findings at the transplant site (e.g., perivascular mononuclear cell infiltrates).

[0381] In summary, animals receiving either GPOR-OPC1 or LCTOPC1 cells (intraparenchymal) using the parenchymal spinal delivery system for injection had increased cell numbers and decreased other microscopic findings at the transplant site on day 2 compared to day 14. Identification of cells on H&E sections generally correlated well with HuNu positive IHC staining. Overall, there was no gross destruction of spinal cord tissue adjacent to the transplant site in any animal (e.g., no necrosis and / or cavitation was observed). No changes in spinal cord white matter were observed in control sites (e.g., no axonal swelling or myelin space expansion was observed).

[0382] Tissue collection and processing Immediately after euthanasia with sodium pentobarbital, and prior to exsanguination, approximately 2 mL of CSF was collected from the cisterna magna of the animals and flash-frozen in liquid nitrogen. Transverse (coronal, transverse) slices (approximately 5 mm thick) of the spinal cord were taken at approximately 5 cm intervals covering the rostrocaudal extent of the spinal cord. Samples were marked to maintain the rostrocaudal orientation and flash-frozen to preserve DNA integrity. Additional tissue samples (approximately 5 mm each) were taken from the hemisphere ipsilateral to the site of test article injection to include samples of the brainstem, cerebellum, pons / medulla oblongata, hippocampus, forebrain, and olfactory bulb. 3These samples were flash frozen to preserve the integrity of the DNA.

[0383] Identification of GPOR-OPC1 or LCTOPC1 in porcine peripheral tissues DNA was extracted from eight tissue and fluid levels submitted for analysis (spinal cord, brainstem, cerebellum, pons / medulla, hippocampus, forebrain, olfactory bulb, heart, liver, lung, kidney, spleen, and CSF) using standard methods. For each sample, qPCR amplification reactions with fluorescent detection were performed in duplicate to label nuclear human-specific Alu DNA repeat sequences. Values ​​were compared to a standard curve generated using known amounts of human genomic DNA (gDNA) spiked into Yucatan minipig spleen homogenates to quantify the number of human-specific Alu DNA repeat gene sequences in the test samples. Values ​​were reported numerically if they were within the quantification range of the assay (1 pg to 10,000 pg human Alu DNA / μg Yucatan minipig tissue), and values ​​outside that range were reported as "below the lower limit of detection" (LLD; <500 fg) or "not quantifiable" (NQ; 500 fg to 1 pg).

[0384] In peripheral tissues (Table 30), the highest values ​​were measured in the heart of two animals that received Cryostor (animal nos. 2001 and 2003), suggesting that the samples were contaminated with human DNA. Animal nos. 2001 and 2002 were both the first animals in the study to receive Cryostor and were necropsied, and measurable amounts of human DNA were detected in many of the peripheral tissue samples from these two animals. Of the animals that received GRNOPCl, one animal (no. 2005) had measurable levels of human DNA in the heart at day 2. Additionally, animals nos. 2010 and 2011 that received GRNOPCl and were included at day 14 had measurable levels of human DNA in multiple peripheral tissues (liver, lung, and spleen in both animals, testes and kidney in animal no. 2010) that were within the levels reported for HBSS animal nos. 2001 and 2003. Notably, all other animals that received GRNOPCl and were included at either end had little or no human DNA in peripheral tissues collected.

[0385] [Table 30]

[0386] Identification of GPOR-OPC1 or LCTOPC1 in porcine CSF, spinal cord, and brain tissues No human DNA was detected in any of the CSF samples collected.

[0387] The highest levels detected in harvested brain tissue were again measured in Animals #2001 and #2002, both of which received vehicle and were the first to be necropsied in the study at the day 2 time point. Most animals receiving GRNOPCl had measurable levels of human DNA in samples from the brainstem and pons / medulla (the two brain levels most proximal to the cervical spinal cord injection site). Histological examination of similar brain samples did not identify any human cells in the brains of any animals in the study, although it is possible that some of the GRNOPCl had penetrated into the subdural space near the injection site (as occurred in the procedural pilot study 1058-018 preceding this study) and been collected during tissue collection of the brainstem and pons / medulla samples. There was no histological evidence of intraparenchymal migration of GRNOPCl from the injection site to these levels of the caudal brain, and all of the GRNOPCl identified was in the cervical spinal cord region most proximal to the injection site.

[0388] Notably, however, all measured levels in these tissues from animals receiving GRNOPCl were lower than those reported for animal no. 2001, which received vehicle only.

[0389] [Table 31]

[0390] In the collected spinal cord samples, all four animals that received vehicle had measurable levels of human DNA at multiple levels in the rostrocaudal extent of the spinal cord, reflecting a signal not derived from GRNOPC1. In animals that received GRNOPC1, most of the higher levels of human DNA were measured in spinal cord section samples contained in the section 1-3 region (corresponding to the cervical spinal cord and including the injection site). More GRNOPC1 animals in the end day 2 group had measurable levels of human DNA in their spinal cord tissue than were assayed in the end day 14 group animals. Although GRNOPC1 is expected to be present in the cervical spinal cord sections (based on histological results and identification of GRNOPC1 using ISH), the origin of the human DNA detected at spinal cord levels distal to the injection site is unknown. It is highly unlikely that this reflects intraparenchymal migration, as migration of GRNOPCl would be expected (based on extensive data collected in rodents) in all segments between the injection site and distal segments, but this was not observed. As mentioned above, it is possible that some GRNOPCl entered the subdural space near the injection site and was collected during tissue collection of the distal spinal cord segments, but notably, human DNA was also measured in many of the most lumbar spinal cord segments collected from animals that had received GRNOPCl, but importantly, no human DNA was detected in the CSF, as would be expected in this event.

[0391] The highest level detected in the spinal cord was measured in spinal cord section 6 of animal number 2013 (day 14 GRNOPCl). Given that the value for this segment (298.33 pg human DNA per microgram minipig gDNA) is higher than that calculated for other animals for the spinal cord segment containing the injection site, and that 2-3 spinal cord segments between the injection site and spinal cord section 6 had little to no measurable levels of human DNA, it is unlikely that this value was solely due to the presence of GRNOPCl at this level of the spinal cord.

[0392] A qPCR assay was additionally used to provide quantitative information on the biodistribution of GPOR-OPCl after cervical administration. However, the qPCR data obtained from the study are equivocal due to obvious contamination of samples during tissue collection. Expression of human DNA in animals receiving only the Cryostor vehicle does not reflect the persistence of GPOR-OPCl.

[0393] Very few animals in the study had significant levels of human cells in the peripheral tissues collected. Indeed, this was likely limited to one or two animals that received GRNOPCl and were included at the end of day 14. A small number of human cell equivalents were calculated in the brainstem and pons / medulla of animals that received GRNOPCl at both day 2 and day 14, but the levels were lower than those calculated in animals that received vehicle. This assay confirms that the highest concentration of GRNOPCl remaining in the collected tissues occurred in the cervical spinal cord, with the levels being higher in animals terminated on day 2 than in animals terminated on day 14. In most animals that received GRNOPCl, human cell equivalents were calculated in the most lumbar part of the cervical spinal cord segments collected, but human DNA was also detected in tissue samples from the same level of the spinal cord in animals that received HBSS. This data does not support a widespread distribution of GRNOPCl after administration to the cervical spinal cord using clinical delivery methods.

[0394] Summary of results In this study, there was no effect on mortality with administration of GPOR-OPC1 or LCTOPC1, or with Cryostor CS10.

[0395] The viability of GPOR-OPC1 and LCTOPC1 cells was confirmed by IHC.

[0396] HuNu positive cells were identified in four of eight animals that received the GPOR-OPC1 test article, with all four animals having detectable cells on day 2.

[0397] HuNu positive cells were identified in five of eight animals that received the LCTOPC1 test article, with all four animals on day 2 having detectable cells.

[0398] No HuNu positive staining was observed at the implantation sites in vehicle-treated animals on days 2 and 14 (4 animals, total of 8 sites).

[0399] In all animals, test article cells were clustered into a single group at the injection site with no evident cell migration.

[0400] Body weight remained normal throughout the study and was not affected by GPOR-OPC1 or LCTOPC1 treatment.

[0401] No GPOR-OPC1- or LCTOPC1-related effects were observed on hematological parameters on day 2. Moderate increases in neutrophil counts were measured in animals in groups receiving GPOR-OPC1 or LCTOPC1 on day 14, suggesting an ongoing inflammatory response.

[0402] In addition to the presence of human cells at the transplant sites, the main additional microscopic findings were mononuclear cell infiltration, spinal cord white matter changes (mainly minimal to mild axonal swelling and expansion of peri-axonal myelin spaces), and acute hemorrhage (mainly in the day 2 samples, due to the recent transplant procedure). The incidence and severity of these additional changes was generally higher in the day 14 sites compared to the day 2 sites.

[0403] No microscopic findings in the brain, lungs, heart, spleen, liver, or kidneys were interpreted as related to test article administration. Microscopic findings in spinal cord sections anterior and caudal to the implantation site were generally minimal and similar to those at the implantation site.

[0404] The highest concentrations of persistent GPOR-OPCI and LCTOPC1 cells were detected at the level of the cervical spinal cord, as confirmed histologically using ISH. Additional tissue samples that may contain test article cells include the pons / medulla, brainstem, and most of the lumbar spinal cord sections collected. Human DNA levels detected in animals receiving test article cells were higher at 2 days post-injection than at 14 days. This data does not support widespread distribution of GPOR-OPCI or LCTOPC1 cells following administration to the cervical spinal cord.

[0405] conclusion Either GPOR-OPCl or LCTOPC1, respectively, was injected at 1 × 10 into eight non-injured, immunosuppressed Yucatan minipigs using a parenchymal spinal delivery system (Lineage Cell Therapeutics, Inc.). 7 The cells were administered at dose levels (2 separate injections) via intrathecal injection.

[0406] Four non-injured immunosuppressed Yucatan minipigs received Cryostor vehicle using a similar method. Animals in both treatment groups were designated for terminal necropsy on days 2 or 14. Overall, GPOR-OPCl and LCTOPC1 were well tolerated by all animals compared to Cryostor control.

[0407] Viability of GPOR-OPCl and LCTOPC1 cells was confirmed in all day 2 animals receiving the test article by H&E and IHC assessment. Human cells could be detected at one implantation site in one day 14 animal receiving the LCTOPC1 test article. Localized accumulation of GPOR-OPCl and LCTOPC1 was present in the dorsal to dorsolateral corner of spinal cord sections taken near the cervical injection level, but was not identified in other tissue sections taken along the entire rostrocaudal length of the spinal cord.

[0408] Neither GPOR-OPC1 nor LCTOPC1 was observed in the brain by H&E and IHC evaluation.

[0409] There was no evidence of malformed or ectopic tissue in the brain, spinal cord, or peripheral tissues harvested from any of the animals.

[0410] Administration of either GPOR-OPC1 or LCTOPC1 did not affect mortality and all animals survived to scheduled termination.

[0411] Evaluation of neuromotor function after spinal injection revealed no detectable impairment in any of the animals. Normal motor function continued for the duration of the study (2 or 14 days), and all animals had normal forelimb and hindlimb coordination and gait function. No spontaneous signs of pain (expressed by vocalization) were observed in any of the animals.

[0412] No systemic effects were observed following administration of GPOR-OPC1 or LCTOPC1.

[0413] Mild to moderate micropathologic findings in the spinal cord, including mononuclear cell infiltration, spinal cord white matter changes (mainly minimal to mild axonal swelling and expansion of peri-axonal myelin spaces), and acute hemorrhage (mainly in day 2 samples, attributable to the recent implantation procedure). The incidence and severity of these additional changes were generally higher in the day 14 sites compared to the day 2 sites. These were considered procedure related and not attributable to test article administration.

[0414] No significant differences were observed in body weight or in hematology, coagulation, or clinical chemistry findings.

[0415] Although sample contamination with human DNA was detected in the qPCR assay, the highest concentrations of persistent GRNOPCl occurred at the level of the cervical spinal cord. Additional tissue samples that may contain low levels of GRNOPCl included the pons / medulla, brainstem, and most of the lumbar spinal cord sections collected, although GRNOPCl was confirmed histologically only in the cervical spinal cord.

[0416] Overall, GPOR-OPCl and LCTOPC1 cells were well tolerated in all animals compared to Cryostor controls.

[0417] Embodiment Embodiment P1. 1. A cell therapy method comprising administering to a subject an OPC composition to improve one or more neurological functions in the subject.

[0418] Embodiment P2. The method of embodiment P1, wherein the OPC cell population is injected or implanted into the subject.

[0419] Embodiment P3. The method of embodiment P1 or P2, wherein the subject has neurological damage due to spinal cord injury, stroke, or multiple sclerosis.

[0420] Embodiment 1. 1. A method for improving one or more neurological functions in a subject having a spinal cord injury (SCI), the method 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. The method comprising:

[0421] Embodiment 2. 2. The method of embodiment 1, further comprising administering to the subject a second dose of the composition.

[0422] Embodiment 3. 2. The method of embodiment 1, further comprising administering to the subject a third dose of the composition.

[0423] Embodiment 4. The method of any one of embodiments 1-3, wherein each administration comprises injecting the composition into the spinal cord of the subject.

[0424] Embodiment 5. The method according to any one of embodiments 1 to 4, wherein the SCI is a subacute cervical SCI.

[0425] Embodiment 6. The method according to any one of embodiments 1 to 4, wherein the SCI is a chronic cervical SCI.

[0426] Embodiment 7. The method according to any one of embodiments 1 to 4, wherein the SCI is a subacute thoracic SCI.

[0427] Embodiment 8. The method according to any one of embodiments 1 to 4, wherein the SCI is a chronic thoracic SCI.

[0428] EMBODIMENT 9. The first dose, second dose, and / or third dose of the composition is about 1×10 6 ~Approx. 3×10 7 13. The method of any preceding embodiment, comprising OPC cells.

[0429] EMBODIMENT 10. The first dose of the composition is about 2×10 6 13. The method of any preceding embodiment, comprising OPC cells.

[0430] Embodiment 11. The first dose or the second dose of the composition is about 1×10 7 13. The method of any preceding embodiment, comprising OPC cells.

[0431] Embodiment 12. The second dose or the third dose of the composition is about 2×10 7 13. The method of any preceding embodiment, comprising OPC cells.

[0432] Embodiment 13. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 45 days after the SCI.

[0433] Embodiment 14. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 90 days after the SCI.

[0434] EMBODIMENT 15. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 75 days after the SCI.

[0435] EMBODIMENT 16. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 60 days after the SCI.

[0436] EMBODIMENT 17. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 30 days after the SCI.

[0437] EMBODIMENT 18. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 75 days after the SCI.

[0438] EMBODIMENT 19. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 60 days after the SCI.

[0439] EMBODIMENT 20. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 40 days after the SCI.

[0440] EMBODIMENT 21. The method of any of the preceding embodiments, wherein each of the first dose, second dose, and third dose of the composition is administered between about 14 days after the SCI and the lifetime of the subject.

[0441] EMBODIMENT 22. The method according to any one of embodiments 2 to 21, wherein the injection is performed in the caudal half of the central part of the SCI.

[0442] EMBODIMENT 23. 23. The method of embodiment 22, wherein the injection is about 6 mm into the spinal cord of the subject.

[0443] EMBODIMENT 24. 23. The method of embodiment 22, wherein the injection is about 5 mm into the spinal cord of the subject.

[0444] 25. 1. A method for improving one or more neurological functions in a subject having a spinal cord injury (SCI), the method comprising: administering to said subject a dose of a composition comprising human pluripotent stem cell derived oligodendrocyte precursor cells (OPCs). The improvement method includes the steps of:

[0445] 26. The dose of the composition is about 1×10 6 ~Approx. 3×10 7 26. The method of embodiment 25, comprising OPC cells.

[0446] EMBODIMENT 27. The dose of the composition is about 2×10 6 27. The method of embodiment 26, comprising OPC cells.

[0447] 28. 28. The method of any one of embodiments 25-27, wherein said administering of said composition comprises injecting said composition into the spinal cord of said subject.

[0448] 29. 29. The method of any one of embodiments 25-28, wherein the composition is administered about 7 to about 14 days after the SCI.

[0449] EMBODIMENT 30. The method according to any one of embodiments 25 to 29, wherein the injection is performed in the caudal half of the epicenter of the SCI.

[0450] EMBODIMENT 31. The method of any of embodiments 25-30, wherein the injection is about 6 mm into the spinal cord of the subject.

[0451] EMBODIMENT 32. The method of any of embodiments 25-30, wherein the injection is about 5 mm into the spinal cord of the subject.

[0452] EMBODIMENT 33. The method according to any one of embodiments 25 to 32, wherein the SCI is a subacute thoracic SCI.

[0453] EMBODIMENT 34. The method according to any one of embodiments 25 to 32, wherein the SCI is a chronic thoracic SCI.

[0454] EMBODIMENT 35. The method according to any one of embodiments 25 to 32, wherein the SCI is a subacute cervical SCI.

[0455] EMBODIMENT 36. The method according to any one of embodiments 25 to 32, wherein the SCI is a chronic cervical SCI.

[0456] 37. The method of any of the above embodiments, wherein the improvement in one or more neurological functions comprises an improvement in ISNCSCI upper extremity motor score (UEMS).

[0457] 38. 38. The method of embodiment 37, wherein said improvement in UEMS occurs within about 6 months, about 12 months, about 18 months, about 24 months, or more after injection.

[0458] 39. The method of embodiment 37 or 38, wherein said improvement is an increase in UEMS of at least 10% compared to baseline.

[0459] EMBODIMENT 40. The method of any of the above embodiments, wherein the improvement in one or more neurological functions comprises an improvement in Lower Extremity Motor Score (LEMS).

[0460] EMBODIMENT 41. The method of embodiment 40, wherein said improvement in LEMS occurs within about 6 months, about 12 months, about 18 months, about 24 months, or more after injection.

[0461] EMBODIMENT 42. The method of embodiment 37 or 38, wherein said improvement is an improvement in at least one motor level.

[0462] EMBODIMENT 43. The method of embodiment 37 or 38, wherein said improvement is an improvement of at least two motor levels.

[0463] EMBODIMENT 44. The method of any one of embodiments 37-43, wherein the improvement is in one side of the subject's body.

[0464] EMBODIMENT 45. The method of any one of embodiments 37-43, wherein the improvement is in both sides of the subject's body.

[0465] EMBODIMENT 46. The method of any preceding embodiment, wherein said dose of said composition is administered about 14 to about 90 days after said SCI.

[0466] EMBODIMENT 47. The method of any preceding embodiment, wherein said dose of said composition is administered about 14 to about 75 days after said SCI.

[0467] 48. The method of any preceding embodiment, wherein said dose of said composition is administered about 14 to about 60 days after said SCI.

[0468] 49. The method of any of the preceding embodiments, wherein said dose of said composition is administered about 14 to about 30 days after said SCI.

[0469] EMBODIMENT 50. The method of any preceding embodiment, wherein said dose of said composition is administered about 20 to about 75 days after said SCI.

[0470] EMBODIMENT 51. The method of any of the preceding embodiments, wherein said dose of said composition is administered about 20 to about 60 days after said SCI.

[0471] EMBODIMENT 52. The method of any preceding embodiment, wherein said dose of said composition is administered about 20 to about 40 days after said SCI.

[0472] EMBODIMENT 53. The method of any preceding embodiment, wherein said dose of said composition is administered from about 14 days after said SCI to the lifetime of the subject.

[0473] EMBODIMENT 54. 1. A cell population comprising an increased percentage of oligodendrocyte precursor cell marker NG2 positive cells and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM, said cell population being characterized by the following method: To obtain glial-restricted cells, undifferentiated human embryonic stem cells (uhESCs) were cultured in glial progenitor medium containing a MAPK / ERK inhibitor, a BMP signaling inhibitor, and retinoic acid. Differentiation of said glial restricted cells into oligodendrocyte progenitor cells (OPCs) with an increased percentage of oligodendrocyte progenitor marker NG2 positive cells and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM. The cell population prepared by:

[0474] EMBODIMENT 55. 55. The cell population of embodiment 54 for use in treating a thoracic spinal cord injury (SCI) in a subject.

[0475] EMBODIMENT 56. 56. The cell population of embodiment 55, wherein said thoracic SCI is subacute thoracic SCI.

[0476] EMBODIMENT 57. 56. The cell population of embodiment 55, wherein said thoracic SCI is chronic thoracic SCI.

[0477] EMBODIMENT 58. 55. The cell population of embodiment 54 for use in treating a cervical spinal cord injury (SCI) in a subject.

[0478] EMBODIMENT 59. The cell population of embodiment 58, wherein said cervical SCI is subacute cervical SCI.

[0479] 60. The cell population of embodiment 58, wherein said cervical SCI is chronic cervical SCI.

[0480] 61. The cell population of any of embodiments 54-60, wherein the composition is administered by injection to the subject after the SCI.

[0481] 62. The cell population of embodiment 61, wherein the injection is performed in the caudal half of the central part of the SCI.

[0482] 63. The cell population of embodiment 61, wherein the injection is about 6 mm into the spinal cord of the subject.

[0483] 64. The cell population of embodiment 61, wherein the injection is about 5 mm into the spinal cord of the subject.

[0484] 65. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 14 to about 90 days after said SCI.

[0485] 66. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 14 to about 75 days after said SCI.

[0486] 67. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 14 to about 60 days after said SCI.

[0487] 68. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 14 to about 30 days after said SCI.

[0488] 69. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 20 to about 75 days after said SCI.

[0489] EMBODIMENT 70. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 20 to about 60 days after said SCI.

[0490] EMBODIMENT 71. 65. The cell population of any of embodiments 54-64, wherein said injection occurs about 20 to about 40 days after said SCI.

[0491] EMBODIMENT 72. 65. The cell population of any of embodiments 54-64, wherein the injection occurs from about 14 days after the SCI to within the life of the subject.

[0492] EMBODIMENT 73. 1. A method for improving one or more neurological functions in a subject having a spinal cord injury (SCI), the method comprising: administering to the subject a first dose of the cell population of embodiment 54; administering to the subject a second dose of the cell population; and Optionally, administering a third dose of said cell population to said subject. The method comprising:

[0493] 74. The method of embodiment 73, wherein said SCI is a subacute cervical SCI.

[0494] 75. The method of embodiment 73, wherein said SCI is a chronic cervical SCI.

[0495] 76. The method of embodiment 73, wherein said SCI is a subacute thoracic SCI.

[0496] EMBODIMENT 77. The method of embodiment 73, wherein said SCI is chronic thoracic SCI.

[0497] 78. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 90 days after the SCI.

[0498] 79. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 75 days after the SCI.

[0499] 80. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 60 days after the SCI.

[0500] 81. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 14 to about 30 days after the SCI.

[0501] 82. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 75 days after the SCI.

[0502] 83. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 60 days after the SCI.

[0503] 84. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered about 20 to about 40 days after the SCI.

[0504] 85. 78. The method of any one of embodiments 73-77, wherein each of the first dose, second dose, and third dose of the composition is administered between about 14 days after the SCI and during the life of the subject.

Claims

1. 1. A method for improving one or more neurological functions in a subject having a spinal cord injury (SCI), the method 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. The method comprising:

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

3. 10. The method of claim 1, further comprising administering to the subject a third dose of the composition.

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

5. The method of any one of claims 1 to 4, wherein the SCI is subacute cervical SCI.

6. The method according to any one of claims 1 to 4, wherein the SCI is chronic cervical SCI.

7. The method according to any one of claims 1 to 4, wherein the SCI is subacute thoracic SCI.

8. The method according to any one of claims 1 to 4, wherein the SCI is chronic thoracic SCI.

9. The first dose, the second dose, and / or the third dose of the composition is about 1×10 ６ ~Approx. 3×10 ７ 2. The method of any one of the preceding claims, comprising OPC cells.

10. The first dose of the composition is about 2×10 ６ 2. The method of any one of the preceding claims, comprising OPC cells.

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 claims, comprising OPC cells.

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 claims, comprising OPC cells.

13. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 20 to about 45 days after the SCI.

14. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 14 to about 90 days after the SCI.

15. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 14 to about 75 days after the SCI.

16. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 14 to about 60 days after the SCI.

17. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 14 to about 30 days after the SCI.

18. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 20 to about 75 days after the SCI.

19. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 20 to about 60 days after the SCI.

20. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered about 20 to about 40 days after the SCI.

21. 2. The method of any one of the preceding claims, wherein each of the first, second, and third doses of the composition is administered between about 14 days after the SCI and during the subject's lifetime.

22. The method according to any one of claims 2 to 21, wherein the injection is performed in the caudal half of the central part of the SCI.

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

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

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

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

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

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

29. 29. The method of any one of claims 25-28, wherein the composition is administered about 7 to about 14 days after the SCI.

30. 30. The method of any one of claims 25 to 29, wherein the injection is performed in the caudal half of the central SCI.

31. 31. The method of any one of claims 25 to 30, wherein the injection is about 6 mm into the spinal cord of the subject.

32. 31. The method of any one of claims 25 to 30, wherein the injection is about 5 mm into the spinal cord of the subject.

33. The method of any one of claims 25 to 32, wherein the SCI is subacute thoracic SCI.

34. The method according to any one of claims 25 to 32, wherein the SCI is chronic thoracic SCI.

35. The method of any one of claims 25 to 32, wherein the SCI is subacute cervical SCI.

36. The method according to any one of claims 25 to 32, wherein the SCI is chronic cervical SCI.

37. 2. The method of any one of the above claims, wherein the improvement in one or more neurological functions comprises an improvement in ISNCSCI Upper Extremity Motor Score (UEMS).

38. 38. The method of claim 37, wherein the improvement in UEMS occurs within about 6 months, about 12 months, about 18 months, about 24 months, or more after injection.

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

40. 2. The method of any one of the above claims, wherein the improvement in one or more neurological functions comprises an improvement in Lower Extremity Motor Score (LEMS).

41. 41. The method of claim 40, wherein the improvement in LEMS occurs within about 6 months, about 12 months, about 18 months, about 24 months, or more after injection.

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

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

44. 44. The method of any one of claims 37 to 43, wherein the improvement is on one side of the subject's body.

45. 44. The method of any one of claims 37 to 43, wherein the improvement is in both sides of the subject's body.

46. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 14 to about 90 days after said SCI.

47. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 14 to about 75 days after said SCI.

48. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 14 to about 60 days after said SCI.

49. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 14 to about 30 days after said SCI.

50. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 20 to about 75 days after said SCI.

51. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 20 to about 60 days after said SCI.

52. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered about 20 to about 40 days after said SCI.

53. 2. The method of any one of the preceding claims, wherein said dose of said composition is administered from about 14 days after said SCI to the lifetime of the subject.

54. 1. A cell population comprising an increased percentage of oligodendrocyte progenitor cell marker NG2 positive cells 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; The cell population prepared by:

55. (iii) differentiating said glial restricted cells into oligodendrocyte progenitor cells (OPCs) having an increased proportion of cells positive for the oligodendrocyte progenitor marker NG2, and decreased expression of non-OPC markers CD49f, CLDN6, and EpCAM.

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

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

58. 56. The cell population of claim 55, wherein the thoracic SCI is chronic thoracic SCI.

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

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

61. 59. The cell population of claim 58, wherein the cervical SCI is chronic cervical SCI.

62. The cell population of any one of claims 54 to 60, wherein the composition is administered by injection to the subject following the SCI.

63. 62. The cell population of claim 61, wherein the injection is performed in the caudal half of the central area of ​​the SCI.

64. 62. The cell population of claim 61, wherein the injection is approximately 6 mm into the spinal cord of the subject.

65. 62. The cell population of claim 61, wherein the injection is about 5 mm into the spinal cord of the subject.

66. The cell population of any one of claims 54 to 64, wherein the injection occurs about 14 to about 90 days after the SCI.

67. The cell population of any one of claims 54 to 64, wherein the injection occurs about 14 to about 75 days after the SCI.

68. The cell population of any one of claims 54 to 64, wherein the injection occurs about 14 to about 60 days after the SCI.

69. The cell population of any one of claims 54 to 64, wherein the injection occurs about 14 to about 30 days after the SCI.

70. The cell population of any one of claims 54 to 64, wherein the injection occurs about 20 to about 75 days after the SCI.

71. The cell population of any one of claims 54 to 64, wherein the injection occurs about 20 to about 60 days after the SCI.

72. The cell population of any one of claims 54 to 64, wherein the injection occurs about 20 to about 40 days after the SCI.

73. The cell population of any one of claims 54 to 64, wherein the injection occurs from about 14 days after the SCI to during the life of the subject.

74. 1. A method for improving one or more neurological functions in a subject having a spinal cord injury (SCI), the method comprising: Administering a first dose of the cell population of claim 54 to the subject; administering to the subject a second dose of the cell population; and Optionally, administering a third dose of said cell population to said subject. The method comprising:

75. 74. The method of claim 73, wherein the SCI is subacute cervical SCI.

76. 74. The method of claim 73, wherein the SCI is chronic cervical SCI.

77. 74. The method of claim 73, wherein the SCI is subacute thoracic SCI.

78. 74. The method of claim 73, wherein the SCI is chronic thoracic SCI.

79. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 14 to about 90 days after the SCI.

80. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 14 to about 75 days after the SCI.

81. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 14 to about 60 days after the SCI.

82. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 14 to about 30 days after the SCI.

83. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 20 to about 75 days after the SCI.

84. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 20 to about 60 days after the SCI.

85. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered about 20 to about 40 days after the SCI.

86. 78. The method of any one of claims 73-77, wherein each of the first, second, and third doses of the composition is administered between about 14 days after the SCI and the lifetime of the subject.

87. 100. The method of any one of claims 1-53 or 74-86, wherein the composition is administered using a parenchymal spinal delivery (PSD) system.

88. 88. The method of claim 87, wherein the PSD system comprises an apparatus including a system as shown in any of Figures 39-41.

89. The PSD system comprises: (a) a frame having an elongated body and a plurality of holders extending therefrom; (b) a plurality of first magnets, each fixed to a holder; (c) a tube having a first end and a second end, the tube slidably disposed within a through hole disposed within each of the holders and each of the first magnets; (d) a plurality of second magnets secured to an exterior surface of the tube; and (e) a needle secured to the first end of the tube. An apparatus comprising:

88. The method of claim 87, comprising:

90. 90. The method of claim 88 or 89, wherein each of the first magnets and each of the second magnets are arranged such that the north pole of a first magnet faces the north pole of a second magnet or the south pole of a first magnet faces the south pole of a second magnet, thereby providing a magnetic repulsive force that suspends the tube.

91. 91. The method of any one of claims 88-90, wherein the PSD system further comprises a reservoir in fluid communication with the needle, the reservoir containing the composition.

92. 92. The method of claim 91, wherein the PSD system further comprises a digital microinjector configured to control the flow of the composition through the needle.

93. A method according to any one of claims 87 to 92, wherein the device comprises a stopper adjacent the needle.

94. 94. The method of claim 93, wherein the stopper prevents the needle from advancing further into the spinal cord.

95. 95. The method of any one of claims 87-94, wherein the subject is not removed from a ventilator during administration.

96. Administering the composition comprises (i) positioning the PSD system on the spinal cord of the subject; (ii) lowering the needle into the spinal cord; and (iii) delivering a dose of the composition to the spinal cord. The method of any one of claims 87 to 95, comprising:

97. 97. The method of claim 96, wherein the needle and tube of the device are suspended by magnetic repulsion within the device, thereby compensating for spinal pulsation.

98. 98. The method of claim 96 or 97, wherein step (ii) comprises lowering the needle until the stopper rests on the spinal cord.

99. (i) a device as shown in any of FIGS. 39-41, comprising a needle; and (ii) a reservoir in fluid communication with the needle, the reservoir containing a composition according to any one of claims 54 to 73. A parenchymal spinal delivery (PSD) system comprising:

100. The apparatus, (a) a frame having an elongated body and a plurality of holders extending therefrom; (b) a plurality of first magnets, each fixed to a holder; (c) a tube having a first end and a second end, the tube slidably disposed within a through hole disposed within each of the holders and each of the first magnets; (d) a plurality of second magnets secured to an exterior surface of the tube; and (e) the needle secured to the first end of the tube; 100. The system of claim 99, comprising:

101. 101. The system of claim 100, wherein each of the first magnets and each of the second magnets are arranged such that the north pole of a first magnet faces the north pole of a second magnet or the south pole of a first magnet faces the south pole of a second magnet, thereby providing a magnetic repulsive force that suspends the tube.

102. 102. The system of any one of claims 99 to 101, further comprising a microinjector or microinjection pump configured to control the flow of the composition through the needle.

103. The system of any one of claims 99 to 102, further comprising an XYZ manipulator for manipulating the position of the needle.

104. The system of any one of claims 99 to 103, further comprising a stopper adjacent the needle.

105. 105. The system of claim 104, wherein the stopper prevents the needle from advancing further into the spinal cord.

106. 1. A method for administering cells to the spinal cord of a patient, comprising: (i) placing the system of any one of claims 99 to 105 on the spinal cord of the subject; (ii) lowering the needle into the spinal cord; and (iii) delivering a dose of the composition to the spinal cord. The method comprising:

107. 107. The method of claim 106, wherein the needle and tube of the device are suspended by magnetic repulsion within the device, thereby compensating for spinal pulsation.

108. 108. The method of claim 106 or 107, wherein step (ii) comprises lowering the needle until the stopper rests on the spinal cord.

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