Method for producing oligodendrocyte progenitor cells

Abstract

The present disclosure is directed to a method for producing oligodendrocyte progenitor cells (OPCs). In addition, a method for treating demyelinating diseases using oligodendrocyte progenitor cells (OPCs) is also disclosed.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 345,251, filed May 24, 2022, and U.S. Provisional Patent Application No. 63 / 356,536, filed Jun. 29, 2022, the contents of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Oligodendrocyte progenitor cells (OPCs) are resident glial cells in the central nervous system (CNS) that are highly motile and readily differentiate into mature oligodendrocytes that ensheathe axons throughout their lifespan. Newly emerging cell replacement therapies harness OPCs in the treatment of demyelinated conditions where endogenous OPCs are dysfunctional. Myelin sheaths are important structures of the nervous system, and demyelinating diseases can cause cognitive, memory, and motor function impairments that can severely affect a patient's quality of life. The derivation of OPCs from induced pluripotent stem cells (iPSCs) provides a promising platform with allogeneic potential. However, major hurdles exist in scaling up the production of oligodendrocyte lineage cells from Petri dishes to the clinical scale (estimated to be more than 10 8 cells per demyelinated lesion) and in still having a prolonged time frame compared to most neuronal differentiations.

Prior Art Documents

Non - Patent Literature

[0003]

Non - Patent Literature 1

Summary of the Invention

Means for Solving the Problems

[0004] In one aspect, the present disclosure provides a method for producing oligodendrocyte progenitor cells (OPCs), comprising the steps of: obtaining induced pluripotent stem cells (iPSCs) from a subject; differentiating the iPSCs into neural progenitor cells (NPCs); culturing the NPCs in suspension culture in a bioreactor with agitation to form oligospheres; dissociating the oligospheres into single oligodendrocyte progenitor cells (OPCs) using enzymatic treatment and mechanical agitation; and cryopreserving the single OPCs in a step - wise freezing process.

[0005] In some embodiments, the bioreactor is an impeller - driven DASbox mini - bioreactor. In some embodiments, the DASbox mini - bioreactor has a rotational speed of about 100 rpm (rotation per minute) for about 1 hour, followed by about 400 rpm for about 2 hours.

[0006] In some embodiments, agitation by the impeller - driven DASbox mini - bioreactor leads to a high post - thaw cell viability after cryopreservation.

[0007] In some embodiments, the enzymatic treatment comprises 1x AccuMax and 2x TrypLE Select diluted in HBSS (Hank's Balanced Salt Solution) or 4x TrypLE Select diluted in HBSS. In some embodiments, the enzymatic treatment does not include the addition of exogenous DNase I.

[0008] In some embodiments, NPCs are differentiated in suspension culture for about 60 days. In some embodiments, OPCs express the lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 by about day 60. In some embodiments, NPCs are differentiated in suspension culture for about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, about 60 days, about 65 days, about 70 days, about 75 days, about 80 days, about 85 days, about 90 days, about 95 days, about 100 days, about 105 days, about 110 days, about 115 days or about 120 days.

[0009] In another aspect, the present disclosure provides a method of treating a demyelinating disease or disorder, the method comprising administering oligodendrocyte progenitor cells (OPCs) into the central nervous system of a subject to be treated, enabling the administered oligodendrocyte progenitor cells (OPCs) to engraft in the central nervous system, and thereby restoring a function supported by mature oligodendrocytes in addition to the OPCs. In another aspect, the present disclosure provides a method of treating a demyelinating disease, the method comprising administering oligodendrocyte progenitor cells (OPCs) into the central nervous system of a subject to be treated, enabling the administered oligodendrocyte progenitor cells (OPCs) to engraft in the central nervous system, and thereby restoring the expression of myelin basic protein (MBP).

[0010] In some embodiments, the expandable differentiation platform produces functional oligodendrocyte progenitor cells (OPCs), and the OPCs are produced in vitro within about 60 days. In some embodiments, the OPCs are differentiated from neural progenitor cells (NPCs).

[0011] In some embodiments, in an expandable differentiation platform, NPCs are differentiated in suspension culture to form oligospheres. In some embodiments, the suspension culture is performed in an impeller-driven mini bioreactor.

[0012] In some embodiments, the oligospheres generated in the impeller-driven mini bioreactor allow for an expansion of about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, and about 10-fold in cell yield and physical homogeneity of the spheres.

[0013] In some embodiments, OPCs express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 by about day 60.

[0014] In some embodiments, lactate dehydrogenase release serves as an indicator of cell health during sphere dissociation.

[0015] In one aspect, the present disclosure provides oligodendrocyte progenitor cells (OPCs) produced by a method comprising obtaining induced pluripotent stem cells (iPSCs) from a subject, differentiating the iPSCs into neural progenitor cells (NPCs), culturing the NPCs in suspension culture in a bioreactor with agitation to form oligospheres, dissociating the oligospheres into single oligodendrocyte progenitor cells (OPCs) using enzymatic treatment and mechanical agitation, and cryopreserving the single OPCs in a stepwise cryopreservation process.

[0016] In another aspect, the present disclosure provides oligodendrocyte progenitor cells (OPCs) that express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 by about day 60 of in vitro differentiation of neural progenitor cells (NPCs) in suspension culture in a bioreactor.

[0017] In another aspect, the present disclosure provides oligodendrocyte progenitor cells (OPCs) modulated by WNT.

[0018] In some embodiments, oligodendrocyte progenitor cells (OPCs) are SOX10 and O4 positive at about day 60.

[0019] In some embodiments, the percentage of SOX10 and O4 positive cells of oligodendrocyte progenitor cells (OPCs) at day 60 increases to over 50% with the addition of a WNT modulator from about day 40 to about day 60.

[0020] The patent or application documents contain color drawings. Copies of this patent or patent application publication that include color drawings are provided by the Patent Office upon request and payment of the necessary fee.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0022] Definition Unless defined otherwise, all scientific and technical terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] As used herein, the term "about" refers to a variation of about + / - 10% from a given value.

[0024] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to, a human, non-human primate, and rodent, etc., that is the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject.

[0025] As used herein, the term "non-human animal" refers to all non-human animals, including but not limited to, vertebrates such as rodents, non-human primates, ovine animals, bovine animals, reptiles, lagomorphs, porcine animals, caprine animals, equine animals, canine animals, feline animals, birds, etc.

[0026] As used herein, the term "cell culture" refers to any in vitro culture of cells. Continuous cell lines (e.g., those having an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro are included in this term.

[0027] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within the artificial environment. The in vitro environment can consist of, but is not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur within the natural environment.

[0028] The term "pluripotent stem cell" or "PSC", as used herein, has its ordinary meaning in the art, i.e., a self-renewing cell capable of differentiating into endodermal, ectodermal, and mesodermal cells. In some embodiments, the PSC is a human PSC. PSCs include embryonic stem cells (ESCs) and induced pluripotent stem cells (the "iPS cells" or "iPSCs"). The terms ES cells and iPS cells have their ordinary meanings in the art.

[0029] The terms "treat", "treatment", or "treating", as used herein, refer to therapeutic measures that cure a diagnosed pathological disease or disorder, restore regenerative function, slow down, reduce the symptoms thereof, and / or halt its progression. Thus, those in need of treatment include those already having the disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder if it exhibits complete, partial, permanent, or temporary alleviation or elimination of any symptom associated with the disease or disorder.

[0030] As used herein, the phrase "administering" refers to the physical introduction of an agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for oligodendrocyte progenitor cells (OPCs) prepared by the methods disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral route of administration", as used herein, means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepithelial, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Routes that are not parenteral include oral, topical, epidermal, or mucosal routes of administration, such as oral, intranasal, vaginal, rectal, sublingual, or topical routes. Administration can also be carried out, for example, once, multiple times, and / or over one or more periods.

[0031] The term "oligodendrocyte progenitor cell (OPC)", as used herein, refers to a subtype of glial cells that cause myelin regeneration. OPCs represent a highly proliferative cell population that is resident in the adult mammalian and human central nervous system (CNS). OPCs can be used as a treatment for demyelinating conditions including spinal cord injury, stroke, Parkinson's disease, multiple sclerosis, cerebral palsy, in addition to white matter dystrophies (such as Krabbe disease, Canavan disease), neuromyelitis optica spectrum (including NMO, TM) and radiation-induced brain injury (RBI).

[0032] The term "neural progenitor cell (NPC)", as used herein, refers to a CNS progenitor cell that gives rise to many, if not all, of the glial and neuronal cell types that exist in the CNS. NPCs do not generate non-neuronal cells, such as immune system cells, which also exist in the CNS. NPCs can be generated in vitro by differentiating embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0033] The term "suspension culture", as used herein, refers to a type of cell culture in which single cells or small aggregates of cells are enabled to function and multiply in a stirred growth medium, thereby forming a suspension.

[0034] The term "bioreactor", as used herein, refers to any manufactured device or system that supports a biologically active environment. A bioreactor is a vessel or tank in which whole cells or cell-free enzymes convert raw materials into biochemical products and / or more desirable by-products. Bioreactors can be operated batchwise or continuously, aerobically or anaerobically, and with pure or mixed cultures. In some embodiments, the bioreactor is an impeller-driven DASbox mini bioreactor. In other embodiments, the bioreactor is a Thermo-mixer C or a Miltenyi gentleMACS octo-dissociator.

[0035] As used herein, the term "central nervous system" refers to the spinal cord, brain, and cerebrospinal fluid (CSF).

[0036] As used herein, the term "oligosphere" refers to an aggregate of oligodendrocyte progenitor cells.

[0037] As used herein, the term "enzyme" refers to a biological molecule (typically a protein) that significantly accelerates substantially all the rates of chemical reactions without itself being consumed or permanently altered. In some embodiments, the enzyme mixture acts as a dissociating agent and helps dissociate the oligosphere into single oligodendrocyte progenitor cells (OPCs).

[0038] As used herein, the term "dissociation" refers to the process of separating a cell mass into single cells. In some embodiments, the cell mass is an aggregate of oligodendrocyte progenitor cells (OPCs) or an oligosphere.

[0039] As used herein, the term "cryopreservation" refers to the process of preserving cell organelles, cells, tissues, or any other biological construct by cooling a sample to a very low temperature. In some embodiments, cryopreservation is a stepwise freezing process.

[0040] As used herein, the term "lineage marker" refers to characteristic molecules for a cell lineage, such as cell surface markers, mRNA, microRNA, or intracellular and secreted proteins. In some embodiments, oligodendrocyte progenitor cells (OPCs) express the lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2.

[0041] The term "demyelinating disease" or "demyelinating disorder", as used herein, refers to any condition that results in damage to the protective covering (myelin sheath) that surrounds nerve fibers in the brain, optic nerve, and spinal cord. When the myelin sheath is damaged, nerve impulses slow down or even stop, causing neurological problems. Examples of demyelinating diseases are multiple sclerosis, Parkinson's disease, Guillain-Barré syndrome, stroke, etc.

[0042] The term "myelin basic protein (MBP)", as used herein, refers to the major protein component of myelin and is produced by oligodendrocytes. MBP is released into the extracellular matrix after shear injury (i.e., diffuse axonal injury) to white matter tracts.

[0043] The term "cell", as used herein, refers to the basic membrane-bound unit that contains the fundamental molecules of life and from which all living organisms are made.

[0044] The term "expandable differentiation platform", as used herein, refers to the process of differentiating neural progenitor cells (NPCs) in suspension culture in a bioreactor with agitation to form oligospheres.

[0045] The term "closed system", as used herein, refers to a system that has equipment designed and operated so that the product is not exposed to the indoor environment.

[0046] The term "ataxia" as used herein refers to a group of neurological conditions. There are several types of ataxia, including ataxia telangiectasia (AT), episodic ataxia, Friedreich's ataxia, multiple system atrophy (MSA), and spinocerebellar ataxia. This condition occurs when the cerebellum is damaged. Ataxia as used herein also refers to a group of disorders that affect co-ordination, balance, and speech. Any part of the body can be affected, but people with ataxia often have difficulty with balance and walking, speech, and / or swallowing.

[0047] Overview The present disclosure describes a method of manufacturing oligodendrocyte progenitor cells (OPCs) using an expandable differentiation platform that enables the production of functional OPCs in vitro within about 60 days. Figure 1 depicts a protocol by which oligodendrocyte progenitor cells are differentiated from human induced pluripotent stem cells (iPSCs). Figure 1 also depicts (B) the lactate concentration in the spent medium measured daily during the adherent culture period. The data shown are from multiple representative runs. (C) On day 12, adherent cells, namely neural progenitor cells (NPCs), are enzymatically dissociated prior to seeding into suspension culture. The cell yield (millions / cm 2 ) is plotted against the vessel type. CS, CellStack with a surface area of 636 cm 2 .

[0048] Generation of neural progenitor cells (NPCs) from pluripotent stem cells In some embodiments, the first phase of the disclosed method is the dual SMAD inhibition method (Chambers S.M, Craft C.A, Papapetrou E.P, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275-280. Epub 2009 Mar 1. Erratum: Nat Biotechnol. 2009 May;27(5):485.), a well-established method for inducing neural progenitor cells from pluripotent stem cells, to induce neural progenitor cells (NPCs) from pluripotent stem cells. According to the present disclosure, as shown in FIG. 2, the dual SMAD inhibition method linked to the activation of Sonic Hedgehog signaling induced robust co-expression of OLIG2 and NKX2.2 in up to about 80% of neural progenitor cells (NPCs) on day 12 of monolayer differentiation treatment.

[0049] Differentiation of neural progenitor cells (NPCs) into oligospheres In some embodiments, these NPCs are then transferred, as shown in FIG. 3A, to suspension culture to form oligospheres either in static culture or in an impeller-driven mini-bioreactor for the remainder of the differentiation. The oligospheres generated in the bioreactor enabled an approximately 4-fold expansion in cell yield and improved the physical homogeneity of the spheres. Stirring produced more uniform sphere size and shape and increased SOX9+ / NKX2-2+ expression as shown by flow cytometry (FIG. 3B).

[0050] Immunofluorescent staining of sphere sections on day 20 (D20) showed differences in sphere organization and SOX9 and NKX2.2 expression (Figure 3B). By day 60, OPCs expressed the lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2. Cells generated by static culture and bioreactor shared significant similarities in protein and gene expression. Static and stirred spheres showed similar gene expression patterns for neural and glial progenitor cell markers when assayed by RT-qPCR panel (Figure 3B). Notably, OPCs generated in bioreactor exhibited enhanced extracellular contacts leading to differential enzyme selection during dissociation. Thus, comparison of static and stirred bioprocesses reveals several surprising and unexpected advantages of stirring, including uniform sphere formation enabling reduction of variability in culture, and higher expression of key oligodendrocyte lineage markers as assayed by flow, immunofluorescent staining, and RT-qPCR. Stirred culture in bioreactor allows higher initial seeding density than static culture, which enables higher yields in cell product harvest and further scalability to larger bioreactors. In some embodiments, the process is referred to as an expandable differentiation platform.

[0051] Dissociation of oligospheres into single oligodendrocyte progenitor cells (OPCs) The present disclosure describes a dissociation procedure that is expandable, time-efficient, and has improved batch-to-batch consistency with reduced operator manipulation.

[0052] The dissociation procedure combines enzymatic treatment and mechanical agitation. Additionally, the method eliminates the need for exogenous DNase I during enzymatic treatment. Together, the method results in high viable cell yields and post-thaw cell viability exceeding 70% benchmark.

[0053] In some embodiments, different types of stirrers were tested, including but not limited to DASbox Mini Bioreactor, Thermo Mixer C (Eppendorf), or Octo Dissociator (Miltenyi). All three provided tunability with respect to rotational speed and heating (37 °C) capabilities.

[0054] In some embodiments, the improved extensibility relies on two components, the source of the enzyme and the container used to perform dissociation, and both components need to be extensible for this procedure to function.

[0055] In some embodiments, different types of enzyme mixtures / dissociating agents were used, including but not limited to Miltenyi Neurosphere Dissociation Kit (P), Accutase (1x), AccuMax, 10x TrypLE Select, 2x TrypLE Select following Accutase, 1x AccuMax + 2x TrypLE Select diluted in HBSS (Hanks' Balanced Salt Solution), or 4x TrypLE Select. In some embodiments, the enzyme treatment includes 1x AccuMax and 2x TrypLE Select diluted in HBSS or 4x TrypLE Select diluted in HBSS. The efficiency of enzymatic dissociation depends on a sufficient enzyme-to-cell mass ratio. In some embodiments, the enzyme-to-cell mass ratio is volume-based. For example, a sufficient ratio of cells (spheres):enzyme is 1:20 to 1:100. In some embodiments, the ratio is 1:25 to 1:90, 1:30 to 1:80, 1:40 to 1:70, 1:50 to 1:60. In some embodiments, the DASbox Mini Bioreactor enables the supply of enzymes in an extensible amount. The DASbox Mini Bioreactor holds a maximum of 250 mL of solution in each unit.

[0056] In some embodiments, the dissociation method using the DASbox mini bioreactor results in a significantly reduced elapsed time. The mini bioreactor of the present disclosure enables a 3-hour procedure that can process oligosphere cultures of up to about 800 mL or larger. In some embodiments, cell yield is defined as the number of viable single cells per milliliter of the original culture volume. The cell yield using the DASbox mini bioreactor according to the present disclosure is about 1.3 million / mL, about 1.35 million / mL, about 1.4 million / mL, about 1.45 million / mL, about 1.5 million / mL, about 1.55 million / mL, about 1.59 million / mL, about 1.6 million / mL, about 1.65 million / mL or about 1.69 million / mL.

[0057] In some embodiments, the enzymatic treatment does not require the addition of exogenous DNase I into the dissociation mixture. Conventionally, an issue in the dissociation of any type of spheroid is the release of genomic DNA into the dissociation mixture, which is due to various degrees of degraded cell membranes or cell death during the treatment. As a result, the viscosity of the dissociation mixture increases and cell clumping can occur. Adding exogenous DNase I into the dissociation mixture is a common mitigation measure recognized in the art. However, the use of DNase I introduces foreign biological substances into the cell product. Complete elimination of DNase I after the procedure is done either by dilution via extensive washing that increases operator handling, or heat inactivation at 65°C which is mostly incompatible with most cells. The method disclosed herein completely eliminates the need for DNase I.

[0058] In some embodiments, lactate dehydrogenase release serves as an indicator of cell health during spheroid dissociation.

[0059] Cryopreservation Cryopreservation is a process that maintains biological samples in a hypothermic suspended animation state for any reasonable period of time and is used to preserve the fine structure of cells. The freezing behavior of cells can be altered in the presence of cryoprotective substances (also called cryoprotectants), which affect the rates of water transport, nucleation, and ice crystal growth. In some embodiments, a stepwise freezing process (controlled-rate freezer) was used to cryopreserve single oligodendrocyte progenitor cells (OPCs). The use of a CRF (controlled-rate freezer) ensures batch-to-batch reproducibility. The type of cryoprotectant does not itself play a critical role. In some embodiments, the cryoprotectant is StemCell Banker. In some embodiments, the cryoprotectant is FresRS (FreSR™-S Single Cell Freezing Medium for ES / iPS Cells | STEMCELL Technologies) or BamBanker (BAMBANKER™ & BAMBANKER™ Direct | [Cell Culture]Products | Laboratory Chemicals - FUJIFILM Wako Chemicals U.S.A. Corporation).

[0060] Treatment Methods of treating demyelinating diseases and disorders using a cell replacement therapy approach are disclosed herein. Demyelination describes the loss of myelin with relative preservation of axons. This results from diseases that damage the myelin sheath or the cells that form them. Oligodendrocyte progenitor cells (OPCs) are generated from induced pluripotent stem cells derived from healthy subjects. PSC-derived oligodendrocyte progenitor cells (OPCs) are administered into the central nervous system of the subject to be treated. FIGS. 7A-7C show that, according to the present disclosure, PSC-derived OPCs successfully engrafted in the dysmyelinated mouse brain and matured into myelin basic protein (MBP)-expressing oligodendrocytes. Significantly, the mature oligodendrocytes successfully restored the expression of myelin basic protein (MBP). The present disclosure is further illustrated by the following non-limiting examples.

Example

[0061] (Example 1: Dissociation protocol for oligospheres) Neural progenitor cells (NPCs) were differentiated in spinner-driven DASbox mini-bioreactors for 60 days in suspension culture to generate oligospheres. The oligospheres were then allowed to settle for 30 minutes. The suspension culture medium was pumped out and a dissociation reagent / enzyme mix (1x AccuMax and 2x TrypLE Select diluted in HBSS (Hank's balanced salt solution) or 4x TrypLE Select diluted in HBSS) was added. The dissociation reagent / enzyme mix did not contain exogenous DNase I. The final bioreactor volume was 200 mL. Next, the impeller of the DASbox mini-bioreactor was started at 100 rpm at 37 °C for 1 hour. After 1 hour, the impeller speed was increased to 400 rpm at 37 °C for 2 hours. 1 mL samples were taken every 30 minutes for cell counting and metabolite measurements. After 3 hours, the impeller was stopped when the oligospheres had dissociated into single oligodendrocyte progenitor cells (OPCs). Next, the entire 200 mL cell suspension volume was taken and passed through a 70 μm filter into a 250 mL centrifuge tube. It was then centrifuged at 200 g for 10 minutes. The supernatant was aspirated and the pellet was resuspended in 40 mL of E6 medium supplemented with Y27632 (1:2000). If there were aggregates, it was passed through another 70 μm filter. Next, the final cell count was performed in 40 mL using three replicate 1 mL samples.

[0062] (Example 2: Cryopreservation protocol) After dissociating oligodendrocyte progenitor cells (OPCs) into single cells, they were immediately resuspended in a cryoprotectant solution, aliquoted, and slowly frozen for storage in liquid nitrogen. After recording the average cell count as described in Example 1, the cells were centrifuged at 200 g for 10 minutes and resuspended in a cryoprotectant solution (StemCell Banker) at 5 million or 10 million / mL in pre-cooled and labeled 1 mL cryotubes. These cryotubes / aliquots were transferred to a controlled rate freezer (CRF) on ice and the program was started. After the program was completed, the aliquot was immediately transferred to dry ice and then into liquid nitrogen tank storage. The CRF freezing program is a controlled stepwise freezing process designed specifically for mammalian cells and for batch-to-batch reproducibility. This program is designed with 6 steps for the sample to go from room temperature to -130 °C. In some embodiments, the sample goes from room temperature to -125 °C. In some embodiments, the sample goes from room temperature to -120 °C. In some embodiments, the sample goes from room temperature to -115 °C. In some embodiments, the sample goes from room temperature to -110 °C. In some embodiments, the sample goes from room temperature to -105 °C. In some embodiments, the sample goes from room temperature to -100 °C. In some embodiments, the sample goes from room temperature to -95 °C. In some embodiments, the sample goes from room temperature to -90 °C. In some embodiments, the sample goes from room temperature to -40 °C. In some embodiments, the sample goes from room temperature to -15 °C. In some embodiments, the sample goes from room temperature to -12 °C. In some embodiments, the sample goes from room temperature to -10 °C. The key component is the first 10 minutes of rapid cooling to 0 °C. This ensures that every batch of cells reaches 0 °C within a tightly controlled time frame and thus the sample temperature is synchronized before the next cooling period begins.

[0063] (Example 3: Mouse ataxia model) The effectiveness of OPCs was evaluated in Shiverer mice, a myelin-deficient mouse model. OPCs were transplanted into the brainstem and cerebellum of Shiverer mice (regions with large volumes of white matter and important for motor coordination). The OPCs rescued ataxic gait compared to vehicle controls (Figure 8), which is a key phenotype observed in clinical populations. Furthermore, the rescue in ataxic gait correlated with functional remyelination of the cerebellum as measured by the presence of myelin basic protein (MBP) expression, which is absent in Shiverer mice (data not shown). As a readout of functional myelination, conduction velocity was measured across major axon bundles. Taken together, these data demonstrate that OPCs are sufficient to rescue molecular, functional, and subsequent behavioral phenotypes in myelin-deficient mice, suggesting that OPCs provide a treatment option for demyelinating disorders where existing therapies are limited.

Claims

1. A method for producing oligodendrocyte progenitor cells (OPCs), comprising: obtaining induced pluripotent stem cells (iPSCs) from a subject; differentiating the iPSCs into neural progenitor cells (NPCs); culturing the NPCs in suspension culture in a bioreactor with agitation to form oligospheres; dissociating the oligospheres into single oligodendrocyte progenitor cells (OPCs) using enzymatic treatment and mechanical agitation; and cryopreserving the single OPCs in a stepwise freezing process A method comprising the above steps.

2. The method according to claim 1, wherein the bioreactor is an impeller-driven DASbox mini bioreactor.

3. The method according to claim 2, wherein the DASbox mini bioreactor has a rotational speed of about 100 revolutions per minute (rpm) for about 1 hour, followed by about 400 rpm for about 2 hours.

4. The method according to claim 1, wherein the agitation by the impeller-driven DASbox mini bioreactor leads to a high post-thaw cell viability after cryopreservation.

5. The method according to claim 1, wherein the enzymatic treatment comprises 1x AccuMax diluted in Hank's Balanced Salt Solution (HBSS) and 2x TrypLE Select or 4x TrypLE Select diluted in HBSS.

6. The method according to claim 1, wherein the enzymatic treatment does not include exogenous DNase I treatment.

7. The method according to claim 1, wherein the NPCs are differentiated in the suspension culture for about 60 days.

8. The method according to claim 1, wherein the OPCs express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 by day 60.

9. A method for treating a demyelinating disease, comprising: administering the oligodendrocyte progenitor cells (OPCs) defined in claim 1 into the central nervous system of a subject to be treated; enabling the administered oligodendrocyte progenitor cells (OPCs) to engraft in the central nervous system; and thereby, restoring the expression of myelin basic protein (MBP) A method comprising the above steps.

10. An expandable differentiation platform for producing functional oligodendrocyte progenitor cells (OPCs), wherein the OPCs are produced in vitro within about 60 days. The expandable differentiation platform.

11. The expandable differentiation platform according to claim 10, wherein the OPC is differentiated from neural progenitor cells (NPC).

12. The expandable differentiation platform according to claim 10, wherein the NPC is differentiated in suspension culture to form oligospheres.

13. The expandable differentiation platform according to claim 10, wherein the suspension culture is performed in an impeller-driven mini bioreactor.

14. The expandable differentiation platform according to claim 10, wherein the oligospheres generated in the impeller-driven mini bioreactor enable an approximately 4-fold expansion in cell yield and physical homogeneity of the spheres.

15. The expandable differentiation platform according to claim 10, wherein the OPC expresses lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 by day 60.

16. The expandable differentiation platform according to claim 10, wherein lactate dehydrogenase release serves as an indicator of cell health during sphere dissociation.

17. The expandable differentiation platform according to claim 10, which is a closed system.

18. A method of obtaining induced pluripotent stem cells (iPSC) from a subject; Differentiating the iPSC into neural progenitor cells (NPC); Culturing the NPC in suspension culture in a bioreactor with agitation to form oligospheres; Dissociating the oligospheres into single oligodendrocyte progenitor cells (OPC) using enzymatic treatment and mechanical agitation; and Cryopreserving the single OPC in a stepwise freezing process An oligodendrocyte progenitor cell (OPC) produced by a method comprising.

19. An oligodendrocyte progenitor cell (OPC) that expresses lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 by approximately day 60 of in vitro differentiation of neural progenitor cells (NPC) in suspension culture in a bioreactor.

20. The oligodendrocyte progenitor cell (OPC) according to claim 19, which is modulated by WNT.

21. The oligodendrocyte progenitor cell (OPC) according to claim 19, which is SOX10 and O4 positive at day 60.

22. The oligodendrocyte progenitor cell (OPC) according to claim 21, wherein the percentage of SOX10- and O4-positive cells on day 60 increases to more than 50% with the addition of a WNT modulator from about day 40 to about day 60.

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