Method for producing schwann progenitor cells and method for producing schwann cells

Abstract

The present disclosure provides: a method for producing Schwann progenitor cells efficiently or with high purity from pluripotent stem cells; or a method for producing mature Schwann cells having enhanced expression of nerve growth factor (NGF) and having a neuranagenesis effect in a living body. The present disclosure provides a method for producing Schwann precursor cells, the method comprising: obtaining, from pluripotent stem cells having sizes that are controlled, a cell cluster of the pluripotent stem cells; culturing the cell cluster to differentiate the cell cluster into a neural rosette; and obtaining Schwann precursor cells from the neural rosette.

Description

Method for producing Schwann precursor cells and method for producing Schwann cells

[0001] The present invention relates to a method for producing Schwann precursor cells, a method for producing Schwann cells, and the like.

[0002] Schwann cells are neuroglial cells in the peripheral nervous system that surround the axons of nerve cells and are involved in maintaining the nerve cells and regenerating and repairing the axons. Schwann cells, including their progenitor cells, are valuable for commercialization as a means of treating nerve disorders for which there are few therapeutic agents, such as entrapment neuropathy, spinal cord injury, and peripheral nerve injury. Patent Document 1 and Non-Patent Document 1 disclose methods for producing Schwann progenitor cells from human-derived pluripotent stem cells.

[0003] International Publication No. 2018 / 135907

[0004] Han-Seop Kim et al., “Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair” Stem Cell Reports, 8, 1714-1726, 2017.

[0005] However, conventional techniques have difficulty in obtaining the quantity of human-derived Schwann cells required for therapeutic applications. In addition, Schwann progenitor cells and Schwann cells are difficult to culture for production, making it difficult to obtain highly purified Schwann progenitor cells or Schwann cells. In other words, in relation to the production of Schwann progenitor cells required for Schwann cell production, a method for efficiently producing Schwann progenitor cells from pluripotent stem cells is desired. Furthermore, a method for producing highly purified Schwann progenitor cells from pluripotent stem cells is desired.

[0006] Therefore, there is a need for a method for efficiently producing highly pure Schwann progenitor cells from pluripotent stem cells. Naturally, it would be of great value if a method for efficiently producing highly pure Schwann progenitor cells were provided. In addition, there is a need for a method for producing high-performance (mature) Schwann cells with enhanced expression of nerve growth factor (NGF), which is involved in the maintenance of nerve cells and the regeneration and repair of axons. Furthermore, if Schwann cells capable of promoting nerve regeneration in vivo could be produced from pluripotent stem cells, they would be promising as pharmaceuticals.

[0007] Therefore, the present invention aims to provide a method for producing Schwann precursor cells that can solve any of the above problems, a method for producing neural rosettes that can be used in said production method, and a method for producing Schwann cells.

[0008] As a result of extensive research to solve the above problems, the inventors discovered that neural rosettes can be obtained efficiently by using cell masses of size-controlled pluripotent stem cells, that highly pure Schwann precursor cells can be obtained by processing neural rosettes in a specified manner, and that culturing Schwann cells under specified conditions enhances the expression of NGF, resulting in the production of mature Schwann cells that have nerve regeneration effects in the body, and thus completed the present invention.

[0009] That is, the present disclosure includes the following embodiments. [1] A method for producing Schwann precursor cells, comprising obtaining cell clusters of size-controlled pluripotent stem cells from pluripotent stem cells, culturing the cell clusters to differentiate them into neural rosettes, and obtaining Schwann precursor cells from the neural rosettes. [2] The method according to [1], wherein obtaining cell clusters of size-controlled pluripotent stem cells comprises culturing the pluripotent stem cells to form pluripotent stem cell colonies to obtain cell clusters, and selecting cell clusters having a specific size from the obtained cell clusters. [3] The method according to [2], wherein selecting cell clusters having a specific size comprises filtering the obtained cell clusters. [4] The method according to [3], wherein the filtering is performed using a filter having a pore size of 40 μm and a filter having a pore size of 70 μm, and the cell clusters having the specific size are cell clusters that pass through the filter having a pore size of 70 μm but do not pass through the filter having a pore size of 40 μm. [5] The manufacturing method according to any one of [1] to [4], wherein obtaining the Schwann precursor cells comprises treating the neural rosettes with a cell detachment solution containing collagenase for 4 minutes or less, and culturing the detached cells in a Schwann precursor cell induction medium. [6] The manufacturing method according to [5], wherein the cell detachment solution further contains a protease other than collagenase and a calcium salt. [7] A manufacturing method for Schwann cells with enhanced NGF expression, comprising producing Schwann precursor cells by the manufacturing method according to any one of [1] to [6], and culturing the Schwann precursor cells or Schwann cells obtained from the Schwann precursor cells in the presence of a substance that increases intracellular cAMP concentration, thereby obtaining Schwann cells with enhanced NGF expression. [8] The manufacturing method according to [7], wherein the substance that increases intracellular cAMP concentration is dibutyryl cAMP. [9] The manufacturing method according to [7] or [8], wherein the obtained Schwann cells with enhanced NGF expression promote nerve regeneration.

[10] A method for producing Schwann cells, comprising producing Schwann precursor cells by the production method according to any one of [1] to [6], and culturing the Schwann precursor cells or Schwann cells obtained from the Schwann precursor cells in the presence of a substance that increases intracellular cAMP concentration.

[11] The production method according to

[10] , wherein the produced Schwann cells promote nerve regeneration in an animal sciatic nerve crush injury model.

[12] A method for producing neural rosettes, comprising obtaining size-controlled cell clusters of pluripotent stem cells from pluripotent stem cells, and culturing the cell clusters to differentiate into neural rosettes.

[13] A method for producing Schwann precursor cells, comprising treating neural rosettes with a cell detachment solution for 4 minutes or less, and culturing the detached cells in a Schwann precursor cell induction medium, wherein the cell detachment solution contains collagenase.

[14] A method for producing Schwann cells with enhanced NGF expression, comprising culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases intracellular cAMP concentration.

[15] A method for producing Schwann cells that promote nerve regeneration, comprising culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases intracellular cAMP concentration.

[0010] According to the present invention, it is possible to provide a method for producing Schwann precursor cells that can solve any of the above problems, a method for producing neural rosettes that can be used in said production method, and a method for producing Schwann cells.

[0011] Human iPS cell clusters prepared to different sizes. Sizes were determined using the following filters: A: 40 μm or less, B: 40 μm-70 μm, C: 70 μm or more. Differentiation into neural rosettes from human iPS cell clusters of different sizes was induced (7-day culture). Cell cluster sizes were determined using the following filters: A: 40 μm or less, B: 40 μm-70 μm, C: 70 μm or more. Figures show the results of 1-2-day culture of cells isolated from human iPS cell-derived neural rosettes treated with different cell detachment solutions. Images A-D are phase-contrast microscopy images taken after 1 day of culture. Images E-H are fluorescent immunostained images of cells cultured for 2 days after treatment, fixed, and immunostained for SOX10. Light gray indicates SOX10, and dark gray indicates cell nuclei. (A. Phase contrast microscope image of cells cultured for 1 day after treatment with Accutase for 1 minute. B. Phase contrast microscope image of cells cultured for 1 day after treatment with Accutase for 5 minutes. C. Phase contrast microscope image of cells cultured for 1 day after treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for 1 minute. D. Phase contrast microscope image of cells cultured for 1 day after treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for 5 minutes. E. Fluorescent immunostaining image of SOX10-positive cells cultured for 2 days after treatment with Accutase for 1 minute. F. Phase contrast microscope image of cells cultured for 5 minutes after treatment with Accutase. A. Fluorescent immunostaining image of SOX10-positive cells cultured for two days after treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for one minute. B. Fluorescent immunostaining image of SOX10-positive cells cultured for two days after treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for five minutes. C. Fluorescent immunostaining image of SOX10-positive cells cultured for two days after treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for five minutes. In A-D, cells indicated by arrows or circled are presumed to be cells other than Schwann progenitor cells. D. db-cAMP treatment promotes NGF production in Schwann cells. E. db-cAMP-treated Schwann cells promote neurite outgrowth in mouse neurons after addition of culture supernatant. F. db-cAMP-treated Schwann cells administered to rats showed recovery of walking function as assessed by three-dimensional motion analysis. F. db-cAMP-treated Schwann cells administered to rats showed recovery of compound muscle action potential as assessed by electrophysiological analysis.1 shows the restoration of myelinated axon density, as assessed by light microscopic analysis, myelinated axon diameter, and myelin sheath thickness, as assessed by electron microscopic analysis, in peripheral nerves of rats administered db-cAMP-treated Schwann cells. 1 shows the restoration of skeletal muscle-related proteins in rats administered db-cAMP-treated Schwann cells.

[0012] Below, we will explain in detail the form for implementing the present invention (hereinafter referred to as the ``present embodiment''), but the present invention is not limited to this and various modifications are possible within the scope of the gist of the present invention.

[0013] One aspect of the present disclosure relates to a method for producing Schwann precursor cells, which allows Schwann precursor cells to be obtained with high efficiency from pluripotent stem cells. Another aspect of the present disclosure relates to a method for producing Schwann precursor cells, which allows highly pure Schwann precursor cells to be obtained from pluripotent stem cells. Another aspect of the present disclosure relates to a method for producing Schwann precursor cells, which allows highly pure Schwann precursor cells to be obtained with high efficiency from pluripotent stem cells. Another aspect of the present disclosure relates to a method for producing neural rosettes that can be used in the method for producing Schwann precursor cells of one aspect of the present disclosure. Another aspect of the present disclosure relates to a method for producing Schwann cells using the Schwann precursor cells of one aspect of the present disclosure. Another aspect of the present disclosure relates to a method for producing Schwann cells, which allows Schwann cells with enhanced NGF expression to be obtained from pluripotent stem cells. Another aspect of the present disclosure relates to a method for producing Schwann cells, which allows Schwann cells that promote nerve regeneration to be obtained from pluripotent stem cells.

[0014] The method for producing Schwann progenitor cells in this embodiment includes obtaining size-controlled pluripotent stem cell cell clusters from pluripotent stem cells, culturing these cell clusters to differentiate them into neural rosettes, and obtaining Schwann progenitor cells from these neural rosettes.

[0015] Another aspect of the method for producing Schwann progenitor cells of this embodiment includes treating neural rosettes with a cell detachment solution containing collagenase for 4 minutes or less, and culturing the detached cells in a Schwann progenitor cell induction medium.

[0016] The method for producing neural rosettes in this embodiment includes obtaining size-controlled cell clusters of pluripotent stem cells from pluripotent stem cells, and culturing the cell clusters to differentiate them into neural rosettes.

[0017] The method for producing Schwann cells in this embodiment includes obtaining size-controlled cell masses of pluripotent stem cells from pluripotent stem cells, culturing the cell masses to differentiate them into neural rosettes, obtaining Schwann progenitor cells from the neural rosettes, and culturing the Schwann progenitor cells or Schwann cells obtained from the Schwann progenitor cells in the presence of a substance that increases the intracellular cAMP concentration, thereby obtaining Schwann cells with enhanced NGF expression.

[0018] Another aspect of the method for producing Schwann cells of this embodiment includes culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases the intracellular cAMP concentration to obtain Schwann cells with enhanced NGF expression.

[0019] Another aspect of the method for producing Schwann cells of this embodiment includes obtaining size-controlled cell masses of pluripotent stem cells from pluripotent stem cells, culturing the cell masses to differentiate them into neural rosettes, obtaining Schwann progenitor cells from the neural rosettes, and culturing the Schwann progenitor cells or Schwann cells obtained from the Schwann progenitor cells in the presence of a substance that increases the intracellular cAMP concentration to obtain Schwann cells that promote nerve regeneration.

[0020] Another aspect of the method for producing Schwann cells of this embodiment includes culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases intracellular cAMP concentration to obtain Schwann cells that promote nerve regeneration.

[0021] The method for producing Schwann precursor cells or Schwann cells of this embodiment may be a production method in which the steps of the above production methods are combined in any order.

[0022] The pluripotent stem cells used in the production methods of this embodiment are not particularly limited as long as they have the function of pluripotent stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and iPS cells. Furthermore, the pluripotent stem cells may be derived from mammals, such as rodents such as mice and rats, or primates such as monkeys and humans, and may be human pluripotent stem cells. The pluripotent stem cells used in the production methods of this embodiment may be human iPS cells. Each step in each of the above production methods will be described in detail below.

[0023] [Preparation of Size-Controlled Pluripotent Stem Cell Clusters] In one embodiment, the production method of this embodiment includes a step of obtaining size-controlled pluripotent stem cell clusters from pluripotent stem cells (hereinafter referred to as a "cell cluster preparation step"). "Obtaining size-controlled pluripotent stem cell clusters" means that the cell cluster preparation step is intended to control the size of pluripotent stem cell clusters. The cell cluster preparation step may be, for example, a step in which culture conditions are specified to obtain cell clusters of a predetermined size in a pluripotent stem cell culture step, or a step in which pluripotent stem cells are cultured to obtain pluripotent stem cell clusters, which may be a mixture of various sizes, and then cell clusters of a predetermined size are separated or selected to obtain cell clusters of a predetermined size. The step in which culture conditions are specified to obtain cell clusters of a predetermined size may be, for example, a step in which a culture area for pluripotent stem cells is spatially limited to obtain cell clusters of a size corresponding to the culture area.

[0024] Thus, by preparing size-controlled pluripotent stem cell clusters from pluripotent stem cells and then using the size-controlled pluripotent stem cell clusters to generate neural rosettes, neural rosettes can be generated efficiently. After extensive research, the inventors discovered that using cell clusters that are too small reduces the survival rate of colonies in cell culture, while using cell clusters that are too large reduces the rate of differentiation into neural rosettes. Therefore, the inventors speculate that generating neural rosettes using cell clusters from pluripotent stem cells regulated to a predetermined size can adjust the balance between the survival rate of colonies in cell culture and the efficiency of differentiation into neural rosettes, thereby enabling efficient generation of neural rosettes. However, the present invention is not limited by this speculation.

[0025] The cell cluster preparation step preferably includes, for example, culturing pluripotent stem cells to form pluripotent stem cell colonies to obtain cell clusters, and selecting cell clusters having a specific size from the obtained cell clusters. By selecting cell clusters having a specific size after culture, the size of the cell clusters can be easily controlled.

[0026] The process of culturing pluripotent stem cells to obtain cell clusters of pluripotent stem cells involves culturing pluripotent stem cells to form colonies of pluripotent stem cells while maintaining the function of the pluripotent stem cells, thereby obtaining cell clusters. Examples of such processes include subculturing cells on a cell scaffold. Prior to subculturing on the cell scaffold, cells may be cultured in a cell-free scaffold culture system. In culturing pluripotent stem cells, in order to obtain colonies of pluripotent stem cells, it is preferable to culture them in a culture system that does not contain differentiation-inducing factors for differentiation into neural rosettes or Schwann progenitor cells, as described below. Examples of cell scaffold materials include feeder cells such as mouse embryonic fibroblasts (MEFs), collagen, laminin and laminin fragments, fibronectin and fibronectin fragments, and gelatin.

[0027] The process of culturing pluripotent stem cells to form pluripotent stem cell colonies and obtain cell masses may, for example, be a process of seeding pluripotent stem cells maintained and cultured in a cell-free scaffold culture system onto a cell scaffold and culturing them for a total of 7 days or more through one or more passages. The number of passages may be 1 or more, or 2 or more, or 6 or less, or 5 or less. The number of days of culture on the cell scaffold may be, for example, 4 to 15 days, or 7 to 12 days. A known culture medium may be used for the culture system. After culturing, pluripotent stem cell colonies are detached from the cell scaffold using a cell detachment solution to obtain a cell mass suspension.

[0028] The step of selecting cell clusters having a specific size is a step of selecting or separating cell clusters having a specific size from a population of cell clusters that may contain cell clusters of various sizes. In this step, cell clusters may be selected using any method that can select or separate cell clusters having a specific size. Such methods include, for example, filtering.

[0029] The step of selecting cell clusters having a specific size preferably includes filtering the cell clusters obtained by culturing pluripotent stem cells. The filter used for filtering is not particularly limited as long as it is used for filtering cells. In this specification, the term "filter" refers to a component having a large number of pores with a specific pore size and configured to allow only objects of a specific size or smaller to pass through.

[0030] Filtering of cell clusters may be performed so as to obtain cell clusters of a predetermined size by removing cell clusters that are too small and cell clusters that are too large. Filtering of cell clusters may be performed by using a filter with a predetermined pore size to obtain cell clusters that do not pass through the filter to remove cell clusters that are too small, and then using a filter with a predetermined pore size to obtain cell clusters that pass through the filter to remove cell clusters that are too large, or vice versa. Furthermore, each treatment using a filter with a predetermined pore size may be performed multiple times.

[0031] Although not particularly limited, filtering of cell clusters may be performed using, for example, a filter having a pore size of 40 μm and a filter having a pore size of 70 μm, and cell clusters that pass through the filter having a pore size of 70 μm but not the filter having a pore size of 40 μm may be selected as cell clusters having a specific size. More specifically, filtering cell clusters obtained by culturing pluripotent stem cells may include (1) obtaining cell clusters from colonies formed by culturing cells and filtering the obtained cell clusters using a filter having a pore size of 40 μm, and (2) filtering the cell clusters that did not pass through the filter having a pore size of 40 μm after (1) using a filter having a pore size of 70 μm, and cell clusters that passed through the filter having a pore size of 70 μm in (2) may be selected as cell clusters having a specific size. However, the specific values ​​of the pore size of the above filters are merely examples, and the pore size may be adjusted to increase the probability of neural rosette appearance in the process of producing neural rosettes using cell clusters of pluripotent stem cells described below. For example, the pore size of the filter used in (1) above may be 20 μm, 25 μm, 30 μm, 35 μm, 45 μm, or 50 μm, or within a range having these upper and lower limits (e.g., 20 to 50 μm, 30 to 50 μm), and the pore size of the filter used in (2) above may be 50 μm, 55 μm, 60 μm, 65 μm, 75 μm, 80 μm, 85 μm, or 90 μm, or within a range having these upper and lower limits (e.g., 50 to 90 μm, 60 to 80 μm).

[0032] The average size of the size-controlled cell clusters of pluripotent stem cells obtained by the cell cluster preparation step may be, for example, 30 μm or more and 200 μm or less, preferably 70 μm or more and 160 μm or less, and more preferably 90 μm or more and 140 μm or less after passing through a filter. The average size may be any combination of the upper and lower limits described above within the above range. The average size of the cell clusters may be, for example, the average value of the major axis length and the minor axis length for three or more cell clusters in an image of the cell clusters observed under a microscope.

[0033] [Neural Rosette Production] In one embodiment, the production method of this embodiment includes a step of culturing cell clusters of size-controlled pluripotent stem cells and differentiating them into neural rosettes (hereinafter referred to as the "neural rosette differentiation step"). In this step, neural rosettes can be produced efficiently by using cell clusters of size-controlled pluripotent stem cells to produce neural rosettes. The efficient production of neural rosettes in this embodiment leads to an efficient method for producing Schwann precursor cells. The cell clusters of size-controlled pluripotent stem cells may be cell clusters produced by the cell cluster production step described above. A neural rosette is a cell population with a rosette-like shape that contains neural progenitor cells capable of differentiating into various neural cells. Whether a pluripotent stem cell colony has differentiated into a neural rosette can be confirmed by observing the shape of the colony using a microscope or by analyzing the gene or protein expression of PAX7, Sox9, and nestin.

[0034] The neural rosette differentiation step involves culturing size-controlled cell clusters of pluripotent stem cells in a neural rosette differentiation-inducing medium. This culture may be performed by placing the cell clusters on an extracellular matrix such as Matrigel. The neural rosette differentiation-inducing medium may contain a differentiation-inducing factor for neural rosettes, and may be prepared with reference to, for example, Han-Seop Kim et al., "Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair," Stem Cell Reports, 8, 1714-1726, 2017. Examples of differentiation-inducing factors for neural rosettes include Noggin and Wnt.

[0035] The neural rosette differentiation step may be a step of culturing the cells for, for example, 3 days or more, 4 days or more, 5 days or more, or 6 days or more while appropriately changing the neural rosette differentiation-inducing medium until a rosette-like cell population appears. The number of days of culture may be 15 days or less, 12 days or less, 10 days or less, or 8 days or less. The number of days of culture may be, for example, 5 days or more and 10 days or less.

[0036] [Preparation of Schwann Progenitor Cells] In the manufacturing method of this embodiment, Schwann progenitor cells can be obtained from the neural rosettes obtained above. For example, a method for obtaining Schwann progenitor cells from neural rosettes includes culturing optionally pretreated neural rosettes in a Schwann progenitor cell induction medium. Specifically, a method may be used in which neural rosettes, optionally treated with a cell detachment solution, are cultured in a Schwann cell induction medium. The cell detachment solution may include an enzyme having protease activity and / or collagenase activity. The Schwann progenitor cell induction medium described below may be used. A method for obtaining Schwann progenitor cells from neural rosettes includes the method described in Han-Seop Kim et al., "Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair," Stem Cell Reports, 8, 1714-1726, 2017 (Non-Patent Document 1).

[0037] The manufacturing method of this embodiment may include a step (hereinafter referred to as the "Schwann progenitor cell differentiation step") that includes treating neural rosettes with a cell detachment solution containing collagenase for 4 minutes or less and culturing the detached cells in a Schwann progenitor cell induction medium. In this step, highly purified Schwann progenitor cells can be produced by treating the neural rosettes for a short period of time with the cell detachment solution containing collagenase. The neural rosettes may be those produced by the neural rosette differentiation step described above. Schwann progenitor cells are precursor cells of Schwann cells that can differentiate into neural crest-derived Schwann cells and are SOX10- and S100B-positive cells. Whether or not Schwann progenitor cells have been obtained can be confirmed by observing the cell morphology using a microscope or by immunostaining for SOX10 and S100B.

[0038] Thus, highly purified Schwann progenitor cells can be produced by treating neural rosettes for a short period of time with a cell detachment solution containing collagenase and culturing the detached cells in Schwann progenitor cell induction medium. The inventors speculate that by treating neural rosettes for a short period of time with a cell detachment solution with higher collagenase activity than ordinary cell detachment solutions, it is possible to selectively detach only cells that differentiate into Schwann progenitor cells, thereby producing highly purified Schwann progenitor cells. However, the present invention is not limited by this speculation.

[0039] "Highly pure Schwann precursor cells can be produced" means that a cell population containing 80% or more Schwann precursor cells in terms of the number of cells can be obtained. The Schwann precursor cell differentiation step may be a step of obtaining a cell population containing 85% or more, 90% or more, or 95% or more Schwann precursor cells in terms of the number of cells. The upper limit of the above percentage is not particularly limited and may be, for example, 100%, 99%, 98%, or 95%. The Schwann precursor cell differentiation step may be a step of obtaining a cell population containing 80% or more, 85% or more, 90% or more, or 95% or more SOX10-positive cells in terms of the number of cells. The upper limit of the above percentage is not particularly limited and may be, for example, 100%, 99%, 98%, or 95%.

[0040] The cell detachment solution for treating neural rosettes contains collagenase, which selectively detaches cells that differentiate into Schwann progenitor cells or Schwann progenitor cells. The collagenase contained in the cell detachment solution may be a mixture of multiple types of collagenase, but preferably contains collagenase I.

[0041] The cell detachment solution may contain components other than collagenase that can be contained in a cell detachment solution, such as enzymes other than collagenase, salts that contribute to enzyme activity, and salts that contribute to intercellular bonding.

[0042] Examples of enzymes other than collagenase include proteolytic enzymes other than collagenase, such as enzymes with protease and collagenase activity. Examples of such enzymes include Accutase manufactured by Innovative Cell Technologies. Accutase is a mixture of enzymes with protease and collagenase activity.

[0043] The salt that contributes to the activity of the enzyme may be a salt containing a metal ion that binds to or interacts with the enzyme to improve or inhibit the activity of the enzyme, but is preferably a salt containing a metal ion that improves the activity of the enzyme, more preferably a salt containing a metal ion that improves the activity of collagenase, and even more preferably a salt containing a metal ion that improves the activity of collagenase I. Examples of such salts include calcium salts, magnesium salts, zinc salts, cobalt salts, nickel salts, manganese salts, and iron salts, with calcium salts being preferred. The counter ion of the metal ion is not particularly limited as long as it is an ion that dissolves the salt and is acceptable to cells, and examples include chloride ions, nitrate ions, and sulfate ions.

[0044] The salt that contributes to intercellular bonding may be, for example, a salt containing a metal ion that improves or suppresses the adhesive strength of intercellular adhesion molecules, but is preferably a salt containing a metal ion that improves the adhesive strength of intercellular adhesion molecules. A preferred example of such a salt is a calcium salt. The counter ion of the metal ion is not particularly limited as long as it dissolves the salt and is acceptable to cells, and examples include chloride ions, nitrate ions, and sulfate ions.

[0045] The cell detachment solution preferably contains, for example, collagenase, a protease other than collagenase, and a calcium salt, more preferably collagenase I, a protease other than collagenase, and a calcium salt, and even more preferably collagenase I, Accutase, and a calcium salt. Instead of Accutase, a commercially available cell detachment solution typically used alone may be used. It is believed that by adding collagenase or collagenase I and a calcium salt to a cell detachment solution typically used alone, a cell detachment solution can be obtained that maintains intercellular junctions while exhibiting higher collagenase activity than the cell detachment solution. Such a cell detachment solution can particularly favorably select cells that differentiate into Schwann progenitor cells or Schwann progenitor cells.

[0046] Collagenase is preferably added to the cell detachment solution to a concentration of, for example, 100 U / mL to 300 U / mL, or 120 U / mL to 250 U / mL. Furthermore, when the cell detachment solution contains a cell detachment solution typically used alone (e.g., Accutase) or a calcium salt, the cell detachment solution typically used alone (e.g., Accutase) is preferably added to a dilution of, for example, 0.1 to 0.7 times, or 0.2 to 0.5 times, and the calcium salt is preferably added to a concentration of, for example, 0.1 mM to 10 mM, or 0.3 mM to 3.0 mM.

[0047] The treatment time for neural rosettes with the cell detachment solution is 4 minutes or less, preferably 3 minutes or less, and more preferably 2 minutes or less. The treatment time may be, for example, 1 minute or more. The treatment time may be, for example, 1 minute to 2 minutes, 1 minute to 3 minutes, or 1 minute to 4 minutes.

[0048] The Schwann progenitor cell differentiation step involves treating neural rosettes with a cell detachment solution and then culturing the detached cells in a Schwann progenitor cell induction medium to differentiate them into Schwann progenitor cells. This culture may be performed, for example, by seeding cells detached from neural rosettes onto a cell scaffold material. The Schwann progenitor cell induction medium may contain a differentiation-inducing factor for Schwann progenitor cells, and may be prepared, for example, with reference to Han-Seop Kim et al., "Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair," Stem Cell Reports, 8, 1714-1726, 2017 (Non-Patent Document 1). Examples of differentiation-inducing factors for Schwann progenitor cells include neuregulin 1 (NRG1). The Schwann progenitor cell induction medium may be, for example, a neural rosette induction medium supplemented with a differentiation-inducing factor for Schwann progenitor cells.

[0049] Culturing in a Schwann precursor cell induction medium may be a process of culturing the cells for, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, or 11 days or more, while appropriately changing the Schwann precursor cell induction medium, until Schwann precursor cells appear. The number of culture days may be 20 days or less, 18 days or less, 16 days or less, or 15 days or less. Typically, a change in cell shape is observed within about one day of culturing in the Schwann precursor cell induction medium, and SOX10, a marker for Schwann precursor cells, tends to be expressed by the second day.

[0050] During the culture step in Schwann precursor cell induction medium, the cultured cells may be briefly treated with a cell detachment solution containing collagenase during the culture to increase the purity of Schwann precursor cells. Alternatively, after culture in Schwann precursor cell induction medium, the cultured cells may be briefly treated with a cell detachment solution containing collagenase to increase the purity of Schwann precursor cells. The cell detachment solution used may be the same as the cell detachment solution used to treat neural rosettes, and the preferred embodiments are the same. The treatment time with the cell detachment solution is preferably 4 minutes or less, more preferably 3 minutes or less, and even more preferably 2 minutes or less. The treatment time may be, for example, 1 minute or more. The treatment time may be, for example, 1 minute to 2 minutes, 1 minute to 3 minutes, or 1 minute to 4 minutes.

[0051] [Preparation of Schwann Cells] The production method of this embodiment may include a step of culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases intracellular cAMP concentration (hereinafter referred to as the "Schwann cell preparation step"). In this step, mature Schwann cells can be produced by culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases intracellular cAMP concentration. The mature Schwann cells may be Schwann cells in which NGF expression is enhanced (NGF production is promoted), or may be Schwann cells that promote nerve regeneration in vivo. The amount of NGF produced can be measured, for example, by ELISA.

[0052] Substances that increase the intracellular cAMP concentration include cell-permeable cAMP analogs, substances that promote the conversion of ATP to cAMP in cells, and substances that inhibit enzymes that promote the decomposition of cAMP. Substances that promote the conversion of ATP to cAMP include adenyl cyclase activators, and substances that inhibit enzymes that promote the decomposition of cAMP include phosphodiesterase inhibitors. As a substance that increases the intracellular cAMP concentration, cell-permeable cAMP analogs are preferred.

[0053] Cell-permeable cAMP analogs are compounds in which a functional group that improves cell permeability is bound to cyclic adenosine monophosphate (cAMP). Examples of such compounds include 8-Br-cAMP, dibutyryl cAMP (db-cAMP), 8-CPT-cAMP, and Sp-cAMPS, with dibutyryl cAMP being preferred.

[0054] The cells may be cultured, for example, on a cell scaffold material, using a Schwann cell culture medium supplemented with a substance that increases intracellular cAMP concentration. The Schwann cell culture medium may be, for example, a medium containing a differentiation-inducing factor for Schwann cells. An example of the differentiation-inducing factor for Schwann cells is neuregulin 1 (NRG1). The cell culture period in the presence of a cell-permeable cAMP analog may be, for example, 3 to 32 days, 5 to 28 days, or 6 to 24 days.

[0055] The cells subjected to the Schwann cell preparation step may be Schwann precursor cells, Schwann cells, or cells obtained by pre-culture of Schwann precursor cells in a medium that does not contain a substance that increases the intracellular cAMP concentration. The pre-cultured cells may be Schwann cells or Schwann precursor cells, but are typically Schwann cells. The Schwann precursor cells subjected to the Schwann cell preparation step may be Schwann precursor cells obtained in the Schwann precursor cell differentiation step described above.

[0056] The Schwann cell preparation step may include culturing the Schwann cells in the presence of a substance that increases intracellular cAMP concentration during the process of obtaining Schwann cells from Schwann precursor cells. In the Schwann cell preparation step, in the step of culturing Schwann precursor cells to obtain Schwann cells, a substance that increases intracellular cAMP concentration may be added at any time during the culture period. After Schwann cells are obtained by culturing Schwann precursor cells, a substance that increases intracellular cAMP concentration may be added.

[0057] The Schwann cell preparation step may include obtaining Schwann cells from Schwann precursor cells and culturing the Schwann cells in the presence of a substance that increases intracellular cAMP concentration. The Schwann cell preparation step may also include culturing Schwann precursor cells in the presence of a substance that increases intracellular cAMP concentration, or may include culturing Schwann precursor cells and Schwann cells in the presence of a substance that increases intracellular cAMP concentration in a mixture of the cells. Obtaining Schwann cells from Schwann precursor cells may involve culturing the Schwann precursor cells in a medium that does not contain a substance that increases intracellular cAMP concentration, or adding a substance that increases intracellular cAMP concentration to the Schwann cells and culturing them in the presence of the substance.

[0058] The Schwann cell preparation step may include culturing Schwann precursor cells, for example, on a cell scaffold material using a medium that does not contain a substance that increases intracellular cAMP concentration, before culturing them in the presence of a substance that increases intracellular cAMP concentration. Examples of such a medium include a medium containing NRG1 and at least one, at least two, at least three, or four of PDGF-BB, forskolin, and all-trans retinoic acid (ATRA), and may be the medium described in Han-Seop Kim et al., "Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair," Stem Cell Reports, 8, 1714-1726, 2017. (Non-Patent Document 1). The culture period for such culturing may be, for example, 3 to 12 days, or 4 to 8 days.

[0059] By producing Schwann cells with enhanced NGF production in this way, it is expected that sufficient quantities of cells with the nerve repair ability necessary for administering Schwann cells to humans or animals as a therapeutic method can be produced. As will be shown in the Examples below, Schwann cells with enhanced NGF production promote nerve regeneration.

[0060] Nerve growth factors such as NGF have a neurite outgrowth effect on peripheral nerves. As will be shown in the Examples below, Schwann cells obtained by culturing in the presence of a substance that increases the intracellular cAMP concentration can promote neurite outgrowth in nerve cells of animals, including humans.

[0061] Furthermore, as shown in the Examples below, Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can promote in vivo nerve regeneration in animals, including humans. These Schwann cells may have enhanced expression of NGF. The Schwann cells thus obtained can restore neuronal functions, such as motor function, in animals, including humans.

[0062] The motor function restored by Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration may be walking function. Walking function may be evaluated, for example, by three-dimensional motion analysis. For example, in rats, walking function may be evaluated by the angle of attack (AoA) measured by the method described in the Examples below. Since AoA is known to fluctuate in peripheral neuropathy (Wang et al. PLoS One. 2018. 13(12): e0208985), recovery of walking function may be confirmed by a decrease in AoA.

[0063] The neuronal function restored by Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can be the compound muscle action potential. The muscle potential is the action potential generated when muscle cells contract. The change in the weak electric field generated within the muscle tissue, plotted on the vertical axis and the passage of time on the horizontal axis, is called an electromyogram. The waveform displayed on the electromyogram is the sum of all action potentials at the time of reaching the electrode, and is called the compound muscle action potential. For example, in rats, the compound muscle action potential is measured using the method described in the Examples below. It is known that the compound muscle action potential decreases when peripheral nerve damage occurs (Kavlak et al. J. Phys. Ther. Sci. 2014). Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can restore the compound muscle action potential (increase in the number of significant waveforms detected) measured by electrophysiological analysis in animals, including humans.

[0064] Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can improve the density, diameter, and myelin sheath thickness of myelinated axons in peripheral nerves. It has been reported that the density, diameter, and myelin sheath thickness of myelinated axons in peripheral nerves decrease when peripheral nerve damage occurs (Wang et al. PLoS One. 2018. 13(12): e0208985). Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can restore (improve) the density of myelinated axons measured by light microscopic analysis of peripheral nerves, and the diameter and myelin sheath thickness measured by electron microscopic analysis of myelinated axons in animals, including humans.

[0065] Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can increase skeletal muscle-related proteins and / or mature NMJs (neuromuscular junctions). The expression level of skeletal muscle-related proteins can be confirmed by myosin heavy chain 1 (MYH1), a fast-twitch muscle marker. Mature NMJs (neuromuscular junctions) can also be confirmed by acetylcholine receptor ε (ACHRE). It has been reported that MYH1 and ACHRE decrease during muscle atrophy and denervation (Wang et al. Biosci Rep. 2020; Daneshvar et al. Am J Physiol Cell Physiol. 2020). Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration can be expected to restore (increase) the decrease in skeletal muscle-related proteins and / or mature NMJs (neuromuscular junctions) in animals, including humans.

[0066] Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration may be administered to animals, including humans, and may promote nerve regeneration in vivo after administration. The animals to which Schwann cells are administered are not particularly limited and may be mammals, such as rodents such as mice and rats, or primates such as monkeys and humans. The animals to which Schwann cells are administered may be animals with nerve cell damage. Examples of animal models with nerve cell damage include models of sciatic nerve crush injury. Schwann cells cultured in the presence of a substance that increases intracellular cAMP concentration may promote nerve regeneration in models of sciatic nerve crush injury. The method of administering Schwann cells is not particularly limited, and they may be administered directly to the site of nerve damage or its vicinity. Examples of such methods include direct seeding at the site of damage or its vicinity, and injection.

[0067] As described above, the production method of this embodiment can produce Schwann progenitor cells with high efficiency or high purity. Furthermore, the production method of this embodiment can produce Schwann cells with high NGF production ability. Furthermore, the production method of this embodiment can produce Schwann cells that promote nerve regeneration in vivo.

[0068] The production method of this embodiment may be useful for the development of regenerative medicine (cell therapy) for peripheral neuropathy and central neuropathy. The production method of this embodiment may be useful for producing pathological models of Schwann progenitor cells and Schwann cells associated with peripheral neuropathy and peripheral nerve-related diseases, and for the development of pharmaceuticals using these. The production method of this embodiment may provide Schwann progenitor cells and Schwann cells derived from human pluripotent stem cells that can be applied to cell medicines for central injuries and diseases such as spinal cord injury, cerebral infarction, and brain injury, and peripheral neuropathy injuries and diseases such as carpal tunnel syndrome, peripheral nerve injury, and neuropathic pain. The production method of this embodiment may be useful for drug discovery using pathological models of Charcot-Marie-Tooth disease, diabetic neuropathy, atopic dermatitis, anticancer drug-induced peripheral neuropathy, etc.

[0069] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to the following examples.

[0070] [Preparation of neural rosettes] Neural rosettes were prepared from human iPS cells by the following procedure.

[0071] (Preparation of iPS cell-derived cell aggregates) Human iPS cells maintained and cultured in a feeder-free culture system containing StemFit AK02N (Ajinomoto Co., Inc.) medium / iMatrix-511 (Nippi Corporation) culture substrate were detached using a cell detachment solution (5.5 mM EDTA-containing TrypLE Select (Thermo Fisher Scientific, Inc.)) and collected as a single-cell cell suspension. These cells were plated at 7 × 10 cells on mitomycin C-treated mouse embryonic fibroblasts (MEFs) on a culture dish using StemFit AK02N medium containing 10 μM Y-27632. ５ Cells were seeded.

[0072] The next day, the culture supernatant was removed, and the cells were left on the MEFs. The medium was replaced with Primate ES Cell Medium (ReproCell, Inc.) containing 5 ng / mL FGF-2, a medium for maintaining human iPS cells. The cells were then cultured for three days, with daily medium changes. Human iPS cells expanded into colonies on MEFs were detached using CTK solution, a cell detachment solution, and pipetting was performed to reduce the cell clump size to 70-200 μm. The recovered cell clumps were seeded onto MEFs at a split ratio of 1:4 using Primate ES Cell Medium (ReproCell) containing 5 ng / mL FGF-2, and cultured for three days with daily medium changes. The same passage was repeated once.

[0073] Next, human iPS cells expanded on MEFs were detached with CTK solution and pipetted to reduce the cell clump size to 70-200 μm. The collected cell clumps were centrifuged (50 × g, 2 minutes) in Primate ES Cell Medium. After centrifugation, the supernatant was removed, and the human iPS cell clumps were suspended in neural rosette induction medium (serum-free medium containing N2, B27, SB431542, and CHIR-99021).

[0074] This cell suspension was passed through a 40 μm filter, and the cell clumps that passed through this filter were designated as "cell clumps of 40 μm or less." The cell clumps on the mesh of the 40 μm filter were collected, and the collected cell clumps were passed through a 70 μm filter. The cell clumps that passed through this filter were designated as "40-70 μm cell clumps." The cell clumps on the mesh of the 70 μm filter were collected, and the collected cell clumps were designated as "cell clumps of 70 μm or more."

[0075] Each cell cluster was observed under a phase-contrast microscope and recorded as an image. The results are shown in Figure 1. From Figure 1, the average size of the cell clusters shown in Figure 1B that did not pass through the 70 µm filter after passing through the filter was 130 µm. The average size of the cell clusters was calculated from the average length of the major axis and the minor axis in the image of the cell clusters observed under a microscope.

[0076] (Differentiation into neural rosettes) Cell clusters prepared to different sizes were seeded on Matrigel (BD Biosciences) using neural rosette induction medium. Culture was continued for 5 to 8 days, with medium changes every two days until day 4, and daily thereafter, until the appearance of rosette-like cell colonies. The morphology of cell colonies on day 7 of culture was observed using a phase-contrast microscope and images were recorded. The results are shown in Figure 2. As shown in Figure 2, in colonies formed using human iPS cell clusters of 40 μm or less and 70 μm or more, neural rosettes either failed to form (Figures 2A and 2C) or numerous cell populations other than neural rosettes appeared (Figure 2C). In contrast, numerous neural rosettes were observed in colonies formed using human iPS cell clusters of 40-70 μm (Figure 2B).

[0077] [Generation of Schwann Progenitor Cells] Human pluripotent stem cell-derived neural rosettes were treated with a cell detachment solution for 1 or 5 minutes. The cell detachment solution used was either Accutase (Innovative Cell Technologies) or a cell detachment solution consisting of Accutase, collagenase I, and calcium chloride (140 U / mL collagenase I, 0.3x Accutase, 0.7 mM calcium chloride). Cells treated under each condition were suspended in Schwann progenitor cell induction medium (neuregulin 1 (NRG1) added to the above neural rosette induction medium). These cells were seeded on Matrigel and cultured for 4 days, with medium changes every 2 days. Some cells were subjected to immunostaining as described below after 2 days of culture.

[0078] After culturing, the purity of Schwann progenitor cells was evaluated by immunostaining for SOX10, a Schwann progenitor cell marker. Specifically, the cells were fixed with 4% paraformaldehyde, permeabilized, and blocked, and then treated with SOX10 antibody (1:500) at 4°C overnight. After washing, the cells were treated with CF647donkey anti-rabbit antibody (1:1000) at room temperature for 1 hour. After washing, the cells were stained with DAPI and observed under a fluorescent microscope to calculate the rate of SOX10-positive cells. Images observed under a fluorescent microscope are shown in Figure 3.

[0079] As shown in Figure 3, treatment with Accutase for 1 minute and 5 minutes resulted in a high proportion of cells other than Schwann precursor cells (Figures 3A and 3B). On the other hand, treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for 1 minute resulted in a cell population consisting essentially of cells presumed to be Schwann precursor cells (Figure 3C). Treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for 5 minutes resulted in cells presumed to be other than Schwann precursor cells (Figure 3D). Immunostaining revealed that the percentages of SOX10-positive cells were 70% and 44% after treatment with Accutase for 1 minute (Figure 3E) and 5 minutes (Figure 3F), respectively. The percentages of SOX10-positive cells were 93% and 68%, after treatment with a cell detachment solution containing Accutase, collagenase I, and calcium chloride for 1 minute (Figure 3G) and 5 minutes (Figure 3H), respectively. Similarly, highly pure Schwann precursor cells were obtained when the cells were treated for 2 minutes with a cell detachment solution containing Accutase, collagenase I, and calcium chloride.

[0080] [Preparation of NGF-enhanced Schwann cells and evaluation of nerve regeneration effect] After treatment with the cell detachment solution, human iPS cell-derived Schwann progenitor cells cultured in Schwann progenitor cell induction medium were placed on Matrigel at 0.6-6 × 10 ４ cells / cm ２The cells were seeded at 100°C and cultured for one day in Schwann progenitor cell induction medium. The medium was then replaced with low-glucose DMEM (Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific)), 1% fetal bovine serum (FBS), and GlutaMAX. Neuregulin 1 (NRG1), PDGF-BB, forskolin, and all-trans retinoic acid (ATRA) were added and cultured for four days. Human iPS cell-derived Schwann cells were then generated by culturing the cells for two days in low-glucose DMEM, 1% FBS, GlutaMAX, NRG1, and PDGF-BB. The resulting Schwann cells were cultured in a medium consisting of low-glucose DMEM, 1% FBS, GlutaMAX, and NRG1 supplemented with dibutyryl-cAMP (db-cAMP) to generate Schwann cells with high NGF production. As a control, Schwann cells were also generated by culturing without the addition of db-cAMP.

[0081] NGF in the culture supernatant of Schwann cells treated with db-cAMP and those not treated with db-cAMP was quantitatively evaluated by ELISA. The results are shown in Figure 4. Figure 4 shows that Schwann cells with high NGF production ability were obtained by db-cAMP treatment.

[0082] Next, we investigated whether db-cAMP-treated Schwann cells have a neurite outgrowth effect in nerve regeneration. Mouse neuroblastoma Neuro2a cells (JCRB Cell Bank), a model cell for neurite outgrowth, were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS). 1 × 10 cells were cultured in a neurite outgrowth assay. ４Neuro2a cells were plated in 12-well plates for 24 hours, and the medium was replaced with (A) Schwann progenitor cell induction medium without db-cAMP, (B) Schwann progenitor cell induction medium with 100 μM db-cAMP, (C) culture supernatant of Schwann cells not treated with db-cAMP, or (D) culture supernatant of Schwann cells treated with db-cAMP. Neurite outgrowth was quantified using ImageJ software from phase-contrast images taken with an IX73 inverted microscope (Olympus). The results are shown in Figure 5. (A)-(D) are phase-contrast images, and (F) represents the average length of the longest neurites treated with each of the media in (A)-(D). Data represent the mean ± standard error (n = 20 per group). *p < 0.05 and **p < 0.01 (Tukey's test). Figure 5 shows that when Neuro2a cells were cultured in the culture supernatant (D) of mature Schwann cells treated with db-cAMP, the length of their processes extended longer than when cultured in (C) the culture supernatant of immature Schwann cells not treated with db-cAMP, (A) Schwann precursor cell induction medium without db-cAMP, or (B) Schwann precursor cell induction medium with 100 μM db-cAMP.

[0083] Next, we examined whether db-cAMP-treated Schwann cells promote nerve regeneration in vivo. In the experiment, male rats with crush injury to the right sciatic nerve were used as a sciatic nerve crush injury model. A suspension of db-cAMP-treated mature Schwann cells or immature Schwann cells not treated with db-cAMP was transplanted by injection (2.5 × 10 cells) into the vicinity of the sciatic nerve crush site of F344 rnu / rnu male rats. ５ Eighteen F344 rnu / rnu male rats were randomly divided into three groups: group A (administered immature Schwann cells, n = 6), group B (administered mature Schwann cells, n = 6), and control group (administered Dulbecco's PBS [D-PBS] instead of Schwann cells, n = 6).

[0084] To evaluate the behavioral recovery of rats administered db-cAMP-treated Schwann cells, we measured the movement characteristics of the right hindlimb while the rats were walking on a treadmill 28 days after surgery using a three-dimensional motion analyzer. Specifically, the posterior superior iliac spine, greater trochanter, knee joint, ankle joint, and fifth metatarsophalangeal joint were marked with colored hemispherical plastic markers, and the toes were marked with acrylic resin ink. After filming the rats' treadmill walking, we analyzed the positions of the six landmarks and measured the angle of attack (AoA), which is the angle between the three landmarks (ankle joint, fifth metatarsophalangeal joint, and toe) just before the foot hit the ground during the swing phase. The results are shown in Figure 6. The mean angle of attack (AoA) was 14.43±9.06° in the control group, 12.73±5.52° in group A (administered immature Schwann cells), and 7.35±5.04° in group B (administered mature Schwann cells) (n=6 per group). Group B had the smallest mean AoA, and its range of motion was within the normal range (AoA range of 1-10), demonstrating the best toe extension.

[0085] To confirm that newly elongated axons in rats receiving db-cAMP-treated Schwann cells reached the neuromuscular junction of the innervated muscles, we performed electrophysiological analysis of the compound motor action potential (CMAP) of the adductor muscles of the foot on days 14, 21, and 28 after surgery. Specifically, a stimulating needle electrode 1 was inserted behind the apex of the greater trochanter on the crushed side of the sciatic nerve, a stimulating needle electrode 2 was inserted into the popliteal fossa, and a lead electrode was inserted into the foot. The induced potentials were then measured using an electrical stimulator and analyzed using the measuring electrodes. The results are shown in Figure 7. Significant waveforms were detected only in the group receiving transplants of db-cAMP-treated mature Schwann cells, indicating that the target muscles were more strongly reinnervated.

[0086] In addition, the density of myelinated axons in the peripheral nerves of rats receiving db-cAMP-treated Schwann cells was analyzed by light microscopy, and the diameter and thickness of myelinated axons were analyzed by electron microscopy. Specifically, on day 28 after surgery, semi-thin sections of tissue surrounding the crush injury in each transplantation group were stained with toluidine blue, and phase-contrast images were obtained to calculate the density of myelinated axons. Similarly, ultrathin sections of tissue surrounding the crush injury were observed by electron microscopy, and the diameter and thickness of myelinated axons were quantitatively evaluated. The results are shown in Figure 8. The density of myelinated axons was highest in the group receiving db-cAMP-treated mature Schwann cells, and was significantly higher than that of the control group (Figures 8(A)-(D)). Furthermore, the diameter of myelinated axons in the db-cAMP-treated mature Schwann cell transplant group was significantly larger than that in the control group in which no cells were transplanted and in the group in which immature Schwann cells were transplanted without db-cAMP treatment, and the thickness of the myelin sheath was also the largest in the mature Schwann cell transplant group (Figure 8(E)-(I)).

[0087] We also analyzed skeletal muscle-related proteins in rats administered db-cAMP-treated Schwann cells. Specifically, to analyze the effect of nerve regeneration on muscle organization, we analyzed the expression levels of the neuromuscular junction marker acetylcholine receptor ε (ACHR) and the fast-twitch myosin heavy chain myosin heavy chain 1 (MYH1) in the tibialis anterior muscle by Western blotting. The results are shown in Figure 9. MYH1 and ACHR were expressed at the highest levels in the db-cAMP-treated mature Schwann cell transplant group, and MYH1 expression was significantly higher in the db-cAMP-treated mature Schwann cell transplant group than in the control group (p<0.05). These results suggest that group B showed the most rapid reinnervation of the tibialis anterior muscle and the least severe muscle atrophy. These results also clearly demonstrate that db-cAMP-treated mature Schwann cells have a significant therapeutic effect in a nerve crush injury model.

Claims

1. A method for producing Schwann progenitor cells, comprising obtaining a size-controlled cell mass of pluripotent stem cells from pluripotent stem cells, culturing the cell mass to differentiate into neural rosettes, and obtaining Schwann progenitor cells from the neural rosettes.

2. The method of claim 1, wherein obtaining the size-controlled cell mass of pluripotent stem cells comprises: culturing the pluripotent stem cells to form colonies of pluripotent stem cells to obtain cell masses; and selecting cell masses having a specific size from the obtained cell masses.

3. The method according to claim 2, wherein selecting cell clusters having a particular size comprises filtering the obtained cell clusters.

4. The manufacturing method according to claim 3, wherein the filtering is performed using a filter having a pore size of 40 μm and a filter having a pore size of 70 μm, and the cell mass having the specific size is a cell mass that passes through the filter having a pore size of 70 μm and does not pass through the filter having a pore size of 40 μm.

5. The method of claim 1, wherein obtaining the Schwann progenitor cells comprises: treating the neural rosettes with a cell detachment solution containing collagenase for 4 minutes or less; and culturing the detached cells in a Schwann progenitor cell induction medium.

6. The method according to claim 5, wherein the cell detachment solution further contains a protease other than collagenase, and a calcium salt.

7. A method for producing Schwann cells with enhanced NGF expression, comprising: producing Schwann precursor cells by the method according to any one of claims 1 to 6; and culturing the Schwann precursor cells or Schwann cells obtained from the Schwann precursor cells in the presence of a substance that increases intracellular cAMP concentration to obtain Schwann cells with enhanced NGF expression.

8. The method according to claim 7, wherein the substance that increases the intracellular cAMP concentration is dibutyryl cAMP.

9. The method according to claim 7, wherein the obtained Schwann cells in which expression of NGF is enhanced promote nerve regeneration.

10. A method for producing Schwann cells, comprising: producing Schwann precursor cells by the production method described in any one of claims 1 to 6; and culturing the Schwann precursor cells or Schwann cells obtained from the Schwann precursor cells in the presence of a substance that increases intracellular cAMP concentration.

11. The method according to claim 10, wherein the produced Schwann cells promote nerve regeneration in an animal sciatic nerve crush injury model.

12. A method for producing neural rosettes, comprising: obtaining a size-controlled cell mass of pluripotent stem cells from pluripotent stem cells; and culturing the cell mass to differentiate into neural rosettes.

13. A method for producing Schwann precursor cells, comprising: treating neural rosettes with a cell detachment solution for 4 minutes or less; and culturing the detached cells in a Schwann precursor cell induction medium, wherein the cell detachment solution contains collagenase.

14. A method for producing Schwann cells in which NGF expression is enhanced, comprising culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases the intracellular cAMP concentration.

15. A method for producing Schwann cells that promote nerve regeneration, comprising culturing Schwann precursor cells or Schwann cells in the presence of a substance that increases intracellular cAMP concentration.

Images