Method for preparing oligodendrocyte progenitor cells

Abstract

The present disclosure relates to methods of producing oligodendrocyte progenitor cells (OPCs). In addition, methods of treating demyelinating disease using oligodendrocyte progenitor cells (OPCs) are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 345,251 filed on May 24, 2022 and U.S. Provisional Patent Application No. 63 / 356,536 filed on June 29, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] background

[0004] Oligodendrocyte progenitor cells (OPCs) are resident glial cells in the central nervous system (CNS) that are highly mobile and readily differentiate into axon-wrapping mature oligodendrocytes throughout their lifespan. Emerging cell replacement therapies utilize OPCs to treat demyelinating conditions in which endogenous OPCs are dysfunctional. Myelin is an essential structure of the nervous system, and demyelinating diseases can lead to cognitive, memory, and motor dysfunction, severely impacting patients’ quality of life. Derivation of OPCs from induced pluripotent stem cells (iPSCs) provides a promising platform with allogeneic capabilities. However, translating the production of oligodendrocyte lineage cells from the culture dish to the clinical scale (estimated to be >10 per demyelinating lesion) is a challenging task. 8 cells) remains a major obstacle and the time frame is prolonged compared to most neuronal differentiations. Summary of the invention

[0005] In one aspect, the present disclosure provides 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 stirred bioreactor to form oligospheres; dissociating the oligospheres into single oligodendrocyte progenitor cells (OPCs) using enzymatic treatment and mechanical agitation; and cryopreserving single OPCs in a step-freezing process.

[0006] In some embodiments, the bioreactor is an impeller driven DASbox micro bioreactor. In some embodiments, the DASbox micro bioreactor has a rotation speed of 100 rpm (revolutions per minute) for about 1 hour, then 400 rpm for about 2 hours.

[0007] In some embodiments, agitation of the impeller-driven DASbox micro-bioreactor results in high post-thaw cell viability after cryopreservation.

[0008] In some embodiments, the enzyme treatment comprises 1x AccuMax and 2x TrypLE Select diluted in HBSS or 4x TrypLE Select diluted in HBSS (Hanks Balanced Salt Solution). In some embodiments, the enzyme treatment does not include the addition of exogenous DNase I.

[0009] In some embodiments, NPCs differentiate in suspension culture for about 60 days. In some embodiments, OPCs express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 at about day 60. In some embodiments, NPCs differentiate 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.

[0010] On the other hand, the present disclosure provides a method for treating a demyelinating disease or condition, comprising administering oligodendrocyte progenitor cells (OPC) to the central nervous system of a subject to be treated, allowing the administered oligodendrocyte progenitor cells (OPC) to be implanted in the central nervous system, thereby restoring the functions supported by the OPC and mature oligodendrocytes. On the other hand, the present disclosure provides a method for treating a demyelinating disease, comprising administering oligodendrocyte progenitor cells (OPC) to the central nervous system of a subject to be treated, allowing the administered oligodendrocyte progenitor cells (OPC) to be implanted in the central nervous system, thereby restoring the expression of myelin basic protein (MBP).

[0011] In some embodiments, the scalable differentiation platform produces functional oligodendrocyte progenitor cells (OPCs), wherein OPCs are produced in about 60 days in vitro. In some embodiments, OPCs are differentiated from neural progenitor cells (NPCs).

[0012] In some embodiments, NPCs are differentiated in suspension culture to form oligospheres in a scalable differentiation platform. In some embodiments, the suspension culture is in an impeller-driven microbioreactor.

[0013] In some embodiments, oligospheres produced in an impeller-driven microbioreactor allow for about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, and about 10-fold expansion of cell yield and physical uniformity of the spheres.

[0014] In some embodiments, the OPCs express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 at about day 60.

[0015] In some embodiments, lactate dehydrogenase release is used as an indicator of cell health during spheroid dissociation.

[0016] In one aspect, the present disclosure provides oligodendrocyte progenitor cells (OPCs) produced by the following method, the 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 stirred bioreactor 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-freezing process.

[0017] In another aspect, the present disclosure provides oligodendrocyte progenitor cells (OPCs), wherein the OPCs express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 about 60 days after in vitro differentiation of neural progenitor cells (NPCs) cultured in suspension in a bioreactor.

[0018] In another aspect, the present disclosure provides oligodendrocyte progenitor cells (OPCs), wherein the OPCs are regulated by WNT.

[0019] In some embodiments, the oligodendrocyte precursor cells (OPCs) are positive for SOX10 and O4 at about 60 days.

[0020] In some embodiments, after addition of a WNT modulator between about 40 days to about 60 days, the percentage of oligodendrocyte progenitor cells (OPCs) that are SOX10 and O4 positive increases to >50% at day 60. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains color drawings. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] Figures 1A-1E (A) Schematic diagram depicting the differentiation scheme of oligodendrocyte progenitors from human induced pluripotent stem cells (iPSCs). (B) Lactate concentration in spent culture medium was measured daily during the adherent culture phase. Data shown are from multiple representative runs. (C) At day 12, adherent cells, i.e., neural progenitor cells (NPCs), were enzymatically dissociated before being seeded into suspension cultures. Cell yield (millions / cm 2 ) plotted against vessel type. CS, surface area 636 cm 2 (D) The slope of the lactate curve from day 0 to 12 was plotted against the total cell yield on day 12 and showed a positive correlation. (E) A correlation was found between the seeding density on day 0, the slope of the lactate curve, and the total cell yield on day 12.

[0024] Figure 2 .Differentiation day 12 monolayers were characterized for expression of characteristic neural progenitor markers using flow cytometry, RT-qPCR, and immunofluorescence. Day 12 cells expressed ventralization markers OLIG2 and NKX2.2 by flow cytometry and RT-qPCR. HOXB4 expression indicates ventral characteristics of the spinal cord. Scale bar 100 μm.

[0025] Figure 3A-3B .(A) Suspension cells were cultured in fixed vessels or in an agitated format in a bioreactor. (B) Agitation produces more uniform spheroid size and shape. Flow cytometry analysis shows SOX9+ / NKX2-2+ dual expression. Immunofluorescence staining of day 20 (D20) spheroid sections shows differences in spheroid organization as indicated by SOX9 and NKX2.2 expression. Dissociated spheroids at day 60 (D60) were stained by flow and IF O4. Static and agitated spheroids show similar gene expression patterns of neural and glial progenitor cell markers as determined by RT-qPCR panels. Scale bars, 200 μm in brightfield images and 100 μm in fluorescent images.

[0026] Figure 4A-4B .The percentage (%) of OPCs committed to oligodendrocyte fate at day 60 is regulated by WNT (wingless / integrated) signaling. Co-expression of SOX10 and O4 antigens indicates the fate commitment of OPCs to the oligodendrocyte lineage. After the addition of WNT modulators between days 40 and 60 (B), the percentage of SOX10 and O4 positive cells increased to >50% at day 60 compared to those without any manipulation (A). WNT modulators (agonists) were added at 3uM.

[0027] Figure 5 .Dissociation and cryopreservation of oligodendrocyte progenitor cell (OPC) cell products. Spheres were enzymatically dissociated into a single cell suspension through a 70 μm filter. Dissociation progress was monitored by tracking cell concentration over time, while cell health was monitored by lactate dehydrogenase (LDH) release as an indicator of cell death. Data shown are technical replicates from one representative experiment. Single cells were cryopreserved using a controlled rate freezing procedure and transferred to liquid nitrogen for long-term storage. The stirred bioprocess supported higher seeding densities than static cultures and translated into significantly higher cell yields by day 60. After thawing, oligodendrocyte progenitor cell (OPC) cell products were comparable between static and stirred workflows. Individual data points in the bar graph represent 2-3 separate experiments, each with 2-3 technical replicates.

[0028] Figure 6Single-cell transcriptomes of resting and agitated spheroids were compared on day 20 of culture. Unbiased single-cell RNA sequencing revealed that both culture methods induced NKX2-2 ventral fate neuronal (MNX1) and glial progenitor cell (SOX9) populations.

[0029] Figures 7A-7C . Day 60 cells dissociated from resting cultures were transplanted into P13 MBP (myelin basic protein) deficient shiver mice (Rag2 / ll2 KO) in an immunocompromised genetic background. (B) Schematic diagram of OPC injection. (C) Representative images of OPCs integrated in the corpus callosum (left) and cerebellum (right). Human cells express STEM121 (red) and mature oligodendrocytes restore expression of the myelin protein MBP (green). Scale bar 200 μm.

[0030] Figure 8 Comparison of the effects of OPCs with vehicle in ataxic mice. OPCs rescued ataxic gait compared to vehicle controls.

[0031] DETAILED DESCRIPTION OF THE DISCLOSURE

[0032] definition

[0033] Unless defined otherwise, 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 invention belongs.

[0034] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value.

[0035] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to human subjects.

[0036] As used herein, the term "non-human animals" refers to all non-human animals, including but not limited to vertebrates, such as rodents, non-human primates, sheep, cattle, ruminants, lagomorphs, pigs, goats, horses, dogs, cats, birds, etc.

[0037] As used herein, the term "cell culture" refers to any in vitro cell culture. The term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro.

[0038] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment. An 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 a natural environment.

[0039] As used herein, the term "pluripotent stem cell" or "PSC" has its usual meaning in the art, i.e., a self-replicating cell with the ability to develop into endoderm, ectoderm, and mesoderm cells. In some embodiments, the PSC is a human PSC. PSCs include embryonic stem cells (ESCs) and induced pluripotent stem cells ("iPS cells" or "iPSCs"). The terms ES cells and iPS cells have their usual meaning in the art.

[0040] As used herein, terms such as "treating" or "treatment" or "to treat" refer to therapeutic measures that cure, restore regenerative function, slow down, alleviate the symptoms of a diagnosed pathological disease or condition, and / or prevent its progression. Therefore, those in need of treatment include those already suffering from the condition. In certain embodiments, a subject's disease or condition is successfully "treated" if the subject shows, for example, complete, partial, permanent or temporary relief or elimination of any symptoms associated with the disease or condition.

[0041] As used herein, the phrase "administer" refers to any of various methods and delivery systems known to those skilled in the art that medicament is physically introduced into a subject. The exemplary administration route of oligodendrocyte progenitor cells (OPC) prepared by the method disclosed herein includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion. As used herein, the term "parenteral administration route" means the mode of administration except 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, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Non-parenteral routes include oral, topical, epidermal or mucosal administration routes, such as oral, intranasal, vaginal, rectal, sublingual or local. Administration can also be performed, for example, once, repeatedly and / or in one or more extended time periods.

[0042] As used herein, the term "oligodendrocyte progenitor cell (OPC)" refers to a subtype of glial cells responsible for remyelination. OPC represents a highly proliferative cell population resident in the CNS (central nervous system) of adult mammals and humans. OPC can be used to treat spinal cord injury, stroke, Parkinson's disease, multiple sclerosis, cerebral palsy and demyelinating disorders, including leukodystrophy (e.g. Krabbe disease, Canavan disease), neuritic myelitis spectrum (including NMO, TM) and radiation-induced brain injury (RBI).

[0043] As used herein, the term "neural progenitor cell (NPC)" refers to a progenitor cell of the CNS that produces many, if not all, of the glial and neuronal cell types that populate the CNS. NPCs do not produce non-neural cells that are also present in the CNS, such as immune system cells. NPCs can be produced in vitro by differentiating embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0044] As used herein, the term "suspension culture" refers to a cell culture in which single cells or small aggregates of cells are allowed to function and proliferate in an agitated growth medium, thereby forming a suspension.

[0045] As used herein, the term "bioreactor" refers to any manufactured device or system that supports a biologically active environment. A bioreactor is a container or tank in which whole cells or cell-free enzymes convert feedstocks into biochemical products and / or less desirable byproducts. A bioreactor can be operated as a batch reactor or continuously, aerobically or anaerobically, and in pure or mixed cultures. In some embodiments, the bioreactor is an impeller-driven DASbox microbioreactor. In other embodiments, the bioreactor is a Thermomixer C or a Miltenyi gentleMACS octo-dissociator. As used herein, the term "central nervous system" refers to the spinal cord, brain, and cerebrospinal fluid (CSF).

[0046] As used herein, the term "oligosphere" refers to an aggregate of oligodendrocyte progenitor cells.

[0047] As used herein, the term "enzyme" refers to a biological molecule (usually a protein) that significantly speeds up the rate of almost any chemical reaction without being consumed or permanently altered itself. In some embodiments, the enzyme mixture acts as a dissociating agent and helps dissociate oligospheres into single oligodendrocyte progenitor cells (OPCs).

[0048] As used herein, the term "dissociation" refers to the process by which a cell cluster is separated into single cells. In some embodiments, the cell cluster is an aggregate of oligodendrocyte precursor cells (OPCs) or oligospheres.

[0049] As used herein, the term "cryopreservation" refers to the process of preserving organelles, cells, tissues, or any other biological structures by cooling a sample to very low temperatures. In some embodiments, cryopreservation is a step-freezing process.

[0050] As used herein, the term "lineage marker" refers to a characteristic molecule of a cell lineage, such as a cell surface marker, mRNA, microRNA, or internal and secreted protein. In some embodiments, oligodendrocyte progenitor cells (OPCs) express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2.

[0051] As used herein, the term "demyelinating disease" or "demyelinating disorder" refers to any condition that causes damage to the protective covering (myelin sheath) around nerve fibers in the brain, optic nerves, and spinal cord. When the myelin sheath is damaged, nerve impulses slow down or even stop, leading to neurological problems. Examples of demyelinating diseases are multiple sclerosis, Parkinson's disease, Guillain-Barre syndrome, stroke, etc.

[0052] As used herein, the term "myelin basic protein (MBP)" refers to the major protein component of the myelin sheath and is produced by oligodendrocytes. MBP is released into the extracellular matrix following shear injury of white matter tracts (ie, diffuse axonal injury).

[0053] As used herein, the term "cell" refers to the fundamental membrane-bound unit that contains the basic molecules of life and of which all living organisms are composed.

[0054] As used herein, the term "scalable differentiation platform" refers to a method for differentiating neural progenitor cells (NPCs) to form oligospheres in suspension culture in a stirred bioreactor.

[0055] As used herein, the term "closed system" refers to a system having equipment that is designed and operated so that the product is not exposed to the indoor environment.

[0056] As used herein, the term "ataxia" refers to a group of neurological disorders. There are several types of ataxia, including: ataxia telangiectasia (AT), paroxysmal ataxia, Friedreich's ataxia, multiple system atrophy (MSA), and spinocerebellar ataxia. This condition occurs when the cerebellum is damaged. Ataxia also refers to a group of disorders that affect coordination, balance, and speech in this article. Any part of the body can be affected, but people with ataxia usually have trouble with balance and walking, speaking, and / or swallowing.

[0057] Overview

[0058] The present disclosure describes a method for producing oligodendrocyte progenitor cells (OPCs) using a scalable differentiation platform that is capable of producing functional OPCs in vitro within approximately 60 days. Figure 1 depicts a scheme for differentiating oligodendrocyte progenitor cells from human induced pluripotent stem cells (iPSCs). Figure 1 also describes (B) daily measurement of lactate concentration in spent culture medium during the adherent culture phase. The data shown are from multiple representative runs. (C) On day 12, adherent cells, i.e., neural progenitor cells (NPCs), were enzymatically dissociated before being inoculated into suspension cultures. The cell yield (million / cm 2 ) plotted against vessel type. CS, surface area 636 cm 2 of cell piles.

[0059] Generation of Neural Progenitor Cells (NPCs) from Pluripotent Stem Cells

[0060] In some embodiments, the first stage of the disclosed method is to derive neural progenitor cells (NPCs) from pluripotent stem cells by a dual SMAD inhibition method (Chambers SM, Craft CA, Papapetrou EP, 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. Erratumin: Nat Biotechnol. 2009 May; 27(5): 485.), which is a recognized method for deriving neural progenitor cells from pluripotent stem cells. According to the present disclosure, the dual SMAD inhibition method is combined with the activation of sonic hedgehog signaling to induce robust co-expression of OLIG2 and NKX2.2 in up to about 80% of neural progenitor cells (NPCs) on day 12 of the monolayer differentiation process, such as Figure 2 shown.

[0061] Differentiation of Neural Progenitor Cells (NPCs) into Oligospheres

[0062] In some embodiments, these NPCs are then moved to suspension culture to form oligospheres in static culture or impeller-driven microbioreactors for the remainder of the differentiation, as shown in FIG3A . Oligospheres produced in bioreactors allow for approximately 4-fold expansion of cell yield and improved physical uniformity of the spheres. Agitation produces more uniform sphere size and shape and increases SOX9+ / NKX2-2+ expression, as shown by flow cytometry ( FIG3B ).

[0063] Immunofluorescence staining of day 20 (D20) spheroid sections showed differences in spheroid organization and expression of SOX9 and NKX2.2 (Figure 3B). By day 60, OPCs expressed lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2. Cells produced by static culture and bioreactors had significant similarities in protein and gene expression. As determined by the RT-qPCR panel, static spheroids and agitated spheroids showed similar gene expression patterns for neural and glial progenitor cell markers (Figure 3B). Notably, OPCs produced in bioreactors exhibited enhanced extracellular contacts, which led to different enzymatic selection during dissociation. Therefore, comparison of static and agitated bioprocesses revealed several surprising and unexpected advantages of agitation, including uniform spheroid formation, allowing for reduced variability in culture and higher expression of key oligodendrocyte lineage markers, as determined by flow cytometry, immunofluorescence staining, and RT-qPCR. Stirred culture in a bioreactor allows for higher initial inoculation densities than static culture, which enables higher yields when harvesting cell products, allowing further expansion to larger bioreactors. In some embodiments, this process is referred to as a scalable differentiation platform.

[0064] Dissociation of oligospheres into single oligodendrocyte progenitor cells (OPCs)

[0065] The present disclosure describes a dissociation procedure that is scalable, time efficient, and reduces operator handling and improves batch-to-batch consistency.

[0066] The dissociation procedure combines enzymatic treatment and mechanical agitation. In addition, the present method eliminates the need for exogenous DNase I during enzymatic treatment. Overall, the current method results in a high yield of viable cells, and post-thaw cell viability exceeds the benchmark of 70%.

[0067] In some embodiments, different types of stirrers were tested, including but not limited to DASbox microbioreactor, Thermo Mixer C (Eppendorf), or Octo Dissociator (Miltenyi). All three offer adjustable speed and heating (37°C) capabilities.

[0068] In some embodiments, improved scalability relies on two components, an enzyme source and a vessel for performing the dissociation, and both components need to be scalable for the procedure to work.

[0069] In some embodiments, different types of enzyme mixtures / dissociation agents are used, including but not limited to Miltenyi Neurosphere Dissociation Kit (P), Accutase (1x), AccuMax, 10x TrypLE Select. After the Accutase reagent, there is 2xTrypLE Select, 1xAccuMax plus 2xTrypLE Select or 4x TrypLE Select diluted in HBSS (Hanks Balanced Salt Solution). 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 the enzymatic hydrolysis depends on a sufficient enzyme to cell mass ratio. In some embodiments, the enzyme to cell mass ratio is based on volume. For example, the ratio of sufficient 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 micro-bioreactor allows the supply of enzymes in scalable quantities. The DASbox micro bioreactor can hold up to 250 mL of solution per unit.

[0070] In some embodiments, the dissociation method using the DASbox micro-bioreactor can significantly shorten the time process. The micro-bioreactor disclosed in the present invention realizes a 3-hour procedure that can process oligosphere cultures of up to about 800 mL or more. In some embodiments, the cell yield is defined as the number of live single cells per milliliter of original culture volume. The cell yield using the DASbox micro-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.

[0071] In some embodiments, the enzyme treatment does not require the addition of exogenous DNase I to the dissociation mixture. Conventionally, the problem when dissociating any type of spheroids is that genomic DNA is released into the dissociation mixture due to varying degrees of damaged cell membranes or cell death during the process. As a result, the viscosity of the dissociation mixture increases, and cell clumps may occur. Adding exogenous DNase I to 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. The complete elimination of DNase I after this procedure is either diluted by extensive washing, which increases the operator's handling, or by heat inactivation at 65°C, which is incompatible with most cells. The method disclosed herein completely eliminates the need for DNase I.

[0072] In some embodiments, lactate dehydrogenase release is used as an indicator of cell health during spheroid dissociation.

[0073] Cryopreservation

[0074] Cryopreservation is the process of keeping a biological sample in a state of suspended animation for any considerable period of time at low temperatures, for preserving the fine structure of the cell. In the presence of a cryoprotectant (also referred to as a cryoprotectant), the freezing behavior of the cell can be changed, which affects the rate of water transport, nucleation and ice crystal growth. In some embodiments, a single oligodendrocyte progenitor cell (OPC) is cryopreserved using a step-by-step freezing process (controlled rate freezer). The use of CRF (controlled rate freezer) ensures batch-to-batch reproducibility. The type of cryoprotectant itself does not play an important role. In some embodiments, the cryoprotectant is a stem cell bank. In some embodiments, the cryoprotectant is FresRS (FresRS for ES / iPS cells). TM Single Cell Freezing Medium | STEMCELL Technologies) or BamBanker (BAMBANKER TM &BAMBANKER TM Direct|[Cell Culture] Products|Laboratory Chemicals-FUJIFILM Wako Chemicals USA Corporation).

[0075] treat

[0076] Disclosed herein are methods of using cell replacement therapy to treat demyelinating diseases and conditions. Demyelination refers to the loss of myelin sheaths with relative preservation of axons. This is caused by disease that damages the myelin sheath or the cells that form the myelin sheath. Oligodendrocyte progenitor cells (OPCs) are generated from induced pluripotent stem cells derived from healthy subjects. PSC-derived oligodendrocyte progenitor cells (OPCs) are administered to the central nervous system of the subject to be treated. According to the present disclosure, Figures 7A-7C PSC-derived OPCs were successfully transplanted and matured into oligodendrocytes expressing myelin basic protein (MBP) in the brain of hypomyelinated mice. Notably, mature oligodendrocytes successfully restored the expression of myelin basic protein (MBP). The present disclosure is further illustrated by the following non-limiting examples.

[0077] Example

[0078] Embodiment 1:

[0079] Oligosphere dissociation protocol

[0080] Neural progenitor cells (NPC) were differentiated in suspension culture in an impeller-driven DASbox micro-bioreactor for 60 days to produce oligospheres. The oligospheres were then settled for 30 minutes. The suspension culture medium was pumped out and a dissociation reagent / enzyme mixture (1xAccuMax and 2x TrypLE Select diluted in HBSS (Hanks balanced salt solution) or 4xTrypLE Select diluted in HBSS) was added. The dissociation reagent / enzyme mixture does not include exogenous DNA enzyme I. The final bioreactor volume is 200mL. The impeller of the DASbox micro-bioreactor was then turned on at 100rpm for 1 hour at 37°C. After 1 hour, the impeller speed was increased to 400rpm at 37°C for 2 hours. 1mL samples were collected every 30 minutes for cell counting and metabolite measurement. After 3 hours, when the oligospheres were dissociated into single oligodendrocyte progenitor cells (OPCs), the impeller was stopped. Then all 200mL of cell suspension volume was taken out and passed through a 70μm filter and placed in a 250mL centrifuge tube. Then centrifuge 200g x 10min. Aspirate the supernatant and resuspend the pellet in 40mL E6 medium supplemented with Y27632 (1:2000). If there are aggregates, pass them through another 70μm filter. Then use 3 replicates of 1mL samples in 40mL for a final cell count.

[0081] Embodiment 2:

[0082] Cryopreservation protocol

[0083] After oligodendrocyte progenitor cells (OPC) are dissociated into single cells, they are immediately resuspended in a cryoprotectant solution, aliquoted, and slowly frozen and stored in liquid nitrogen. After recording the average cell count as described in Example 1, the cells are centrifuged at 200g x 10min and resuspended in a cryoprotectant solution (StemCell Banker) in a 1mL cryovial precooled and labeled at 5 million or 10 million / mL. These cryovials / aliquots are placed on ice and placed in a controlled rate freezer (CRF), and the program is started. After the program is completed, the aliquots are immediately transferred to dry ice and then transferred to a liquid nitrogen tank for storage. The CRF freezing program is a controlled step-by-step freezing process designed specifically for mammalian cells with batch-to-batch reproducibility. The program is designed to run samples from room temperature to -130°C in six steps. In some embodiments, the sample is run from room temperature to -125°C. In some embodiments, the sample is run from room temperature to -120°C. In some embodiments, the sample is run from room temperature to -115°C. In some embodiments, the samples are run from room temperature to -110°C. In some embodiments, the samples are run from room temperature to -105°C. In some embodiments, the samples are run from room temperature to -100°C. In some embodiments, the samples are run from room temperature to -95°C. In some embodiments, the samples are run from room temperature to -90°C. In some embodiments, the samples are run from room temperature to -40°C. In some embodiments, the samples are run from room temperature to -15°C. In some embodiments, the samples are run from room temperature to -120°C. In some embodiments, the samples are run from room temperature to -10°C. A key component is the rapid cooling to 0°C in the first 10 minutes. This ensures that each batch of batteries reaches 0°C within a tightly controlled time frame, so the sample temperatures are synchronized before the next cooling stage begins.

[0084] Embodiment 3:

[0085] Mouse ataxia model

[0086] The efficacy of OPCs was evaluated in Shiverer mice, a mouse model of hypomyelination. OPCs were transplanted into the brainstem and cerebellum (an area with a large amount of white matter that is important for motor coordination) of Shiverer mice. Compared with vehicle controls, OPCs rescued ataxic gait ( Figure 8 ), which is a key phenotype observed in clinical populations. In addition, rescue of ataxic gait was associated with functional remyelination of the cerebellum, as measured by myelin basic protein (MBP) expression, which was absent in shiver mice (data not shown). Conduction velocity of major axonal tracts was measured as a readout of functional myelination. Together, these data demonstrate that OPCs are sufficient to rescue the molecular, functional, and subsequent behavioral phenotypes in hypomyelinated mice, suggesting that OPCs offer a therapeutic option for demyelinating diseases for which existing treatments are limited.

Claims

1. A method for generating oligodendrocyte progenitor cells (OPCs), comprising: Obtaining induced pluripotent stem cells (iPSCs) from a subject; differentiating the iPSCs into neural progenitor cells (NPCs); NPCs were grown in suspension culture in stirred bioreactors to form oligospheres; dissociating oligospheres into single oligodendrocyte progenitor cells (OPCs) using enzymatic treatment and mechanical agitation; and Single OPCs were cryopreserved during a step-freezing procedure.

2. The method of claim 1, wherein the bioreactor is an impeller-driven DASbox microbioreactor.

3. The method of claim 2, wherein the rotation speed of the DASbox micro-bioreactor is 100 rpm (revolutions per minute) for about 1 hour and then 400 rpm for about 2 hours.

4. The method of claim 1, wherein agitation of the impeller-driven DASbox micro-bioreactor results in high post-thaw cell viability after cryopreservation.

5. The method of claim 1, wherein the enzyme treatment comprises 1x AccuMax and 2xTrypLE Select diluted in HBSS or 4xTrypLE Select diluted in HBSS (Hanks Balanced Salt Solution). The method according to claim 1 , wherein the enzyme treatment does not include DNase I treatment. The method of claim 1 , wherein the NPCs are differentiated in the suspension culture for about 60 days.

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

9. Methods for treating demyelinating diseases, comprising: administering the oligodendrocyte progenitor cells (OPCs) of claim 1 into the central nervous system of a subject to be treated; allowing the administered oligodendrocyte progenitor cells (OPCs) to engraft in the central nervous system; therefore Restoration of myelin basic protein (MBP) expression.

10. A scalable differentiation platform for generating functional oligodendrocyte progenitor cells (OPCs), wherein the OPCs are generated in vitro within about 60 days.

11. The scalable differentiation platform of claim 10, wherein the OPCs are differentiated from neural progenitor cells (NPCs).

12. The scalable differentiation platform of claim 10, wherein the NPCs are differentiated in suspension culture to form oligospheres.

13. The scalable differentiation platform of claim 10, wherein the suspension culture is in an impeller-driven microbioreactor.

14. The scalable differentiation platform of claim 10, wherein the oligospheres generated in the impeller-driven microbioreactor allow for a 4-fold expansion of cell yield and physical uniformity of the spheres.

15. The scalable differentiation platform of claim 10, wherein the OPCs express lineage markers CD9, O4, SOX10, OLIG2, and NKX2.2 within about 60 days.

16. The scalable differentiation platform of claim 10, wherein lactate dehydrogenase release is used as an indicator of cell health during spheroid dissociation.

17. The scalable differentiated platform of claim 10, wherein the scalable differentiated platform is a closed system.

18. Oligodendrocyte progenitor cells (OPCs) produced by the following method, the method comprising: Obtaining induced pluripotent stem cells (iPSCs) from a subject; Differentiate iPSCs into neural progenitor cells (NPCs); NPCs were grown in suspension culture in stirred bioreactors to form oligospheres; dissociating oligospheres into single oligodendrocyte progenitor cells (OPCs) using enzymatic treatment and mechanical agitation; and Single OPCs were cryopreserved during a step-freezing procedure.

19. Oligodendrocyte progenitor cells (OPCs), wherein the OPCs express lineage markers CD9, O4, SOX10, OLIG2 and NKX2.2 about 60 days after in vitro differentiation from neural progenitor cells (NPCs) in suspension culture in a bioreactor.

20. The oligodendrocyte progenitor cell of claim 19, wherein the OPC is regulated by WNT.

21. The oligodendrocyte progenitor cell (OPC) of claim 19, wherein the OPC is positive for SOX10 and O4 at about 60 days.

22. The oligodendrocyte progenitor cell (OPC) of claim 21, wherein by adding a WNT modulator between about 40 days to about 60 days, the percentage of SOX10 and O4 positive cells is increased to >50% on day 60.

Images