Method for producing clinically usable schwann precursor cells, and method for producing schwann cells

Abstract

The present disclosure provides: a new method for producing clinically usable Schwann precursor cells; and a new method for producing Schwann cells. The present disclosure provides a method for producing Schwann precursor cells, the method comprising: culturing human pluripotent stem cells on a substrate in which a culture region is spatially limited, to obtain a cell cluster of human pluripotent stem cells having a controlled size; culturing the cell cluster and differentiating the same into a neural rosette; and obtaining Schwann precursor cells from the neural rosette.

Description

Method for producing clinically usable Schwann progenitor cells, and method for producing Schwann cells

[0001] This invention relates to a method for producing clinically usable Schwann progenitor cells, and to a method for producing Schwann cells, etc.

[0002] Schwann cells are glial cells of the peripheral nervous system that surround the axons of nerve cells and are involved in the maintenance of nerve cells and the regeneration and repair of axons. Schwann cells, including their progenitor cells, have value as a means of treatment for nerve disorders for which there are few therapeutic drugs, such as compressive neuropathy, spinal cord injury, and peripheral nerve injury. Patent document 1 and non-patent document 1 disclose a method 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 technologies use heterologous animal-derived components, including mouse fibroblasts and mouse-derived component-containing Matrigel, in the process of producing Schwann progenitor cells and Schwann cells from human pluripotent stem cells. However, considering the use of human pluripotent stem cells, Schwann progenitor cells, and Schwann cells for medical applications such as regenerative medicine, or similar applications, it is desirable to eliminate the use of heterologous animal-derived components. In other words, there is a need for a method to produce Schwann progenitor cells and Schwann cells from human pluripotent stem cells without using heterologous animal-derived components.

[0006] Therefore, the present invention aims to provide a novel method for producing clinically usable Schwann progenitor cells and a novel method for producing Schwann cells that solve the above problems.

[0007] As a result of diligent research to solve the above problems, the present inventors have completed an invention relating to a novel method for producing Schwann progenitor cells and a novel method for producing Schwann cells.

[0008] In other words, the present disclosure includes the following embodiments: [1] A method for producing Schwann progenitor cells, comprising: culturing human pluripotent stem cells on a substrate in which the culture area is spatially restricted to obtain a cell aggregate of human pluripotent stem cells of controlled size; culturing the cell aggregate to differentiate it into a neural rosette; and obtaining Schwann progenitor cells from the neural rosette. [2] The method according to [1], wherein the substrate is a low-adsorption well plate. [3] The method according to [1] or [2], wherein differentiating it into a neural rosette involves culturing the cell aggregate on a scaffold coated with an adhesion molecule acceptable in a method for producing clinical cells. [4] The method according to any one of [1] to [3], wherein obtaining Schwann progenitor cells involves culturing the neural rosette on a scaffold coated with an adhesion molecule acceptable in a method for producing clinical cells; enzymatically treating the cultured neural rosette; and culturing the cells detached by the enzymatic treatment in a Schwann progenitor cell induction medium. [5] A method for producing Schwann cells, comprising culturing the cells detached by the enzyme treatment in the Schwann progenitor cell induction medium, wherein the culture is performed at least twice. [6] A method for producing Schwann cells, wherein the proportion of SOX10-positive and S100B-positive cells in the obtained Schwann progenitor cells is 50% or more. [7] A method for producing Schwann cells, wherein the adhesion molecule is laminin or a fragment thereof. [8] A method for producing Schwann cells, wherein the adhesion molecule used for culturing the neural rosette is laminin 511, laminin 521, or a fragment of either thereof. [9] A method for producing Schwann cells, comprising producing Schwann progenitor cells by a method for producing Schwann progenitor cells according to any one of items [1] to [8], and culturing the Schwann progenitor cells on a scaffold coated with an adhesion molecule acceptable in a method for producing clinical cells, wherein the culture is performed in a culture medium that is acceptable in a method for producing clinical cells and does not contain non-human animal-derived components.

[10] The method for producing the Schwann progenitor cells according to [9], wherein the adhesion molecule used for culturing the Schwann progenitor cells is laminin or a fragment thereof.

[11] The method for producing the Schwann progenitor cells according to [9] or

[10] , wherein the adhesion molecule used for culturing the Schwann progenitor cells is laminin 221, laminin 411, laminin 511, laminin 521, or a fragment of any of these.

[12] The method for producing the Schwann cells according to any one of [9] to

[11] , wherein the Schwann cells produced produce at least one selected from the group consisting of NGF, GDNF, CCL2, TIMP-1, TIMP-2, OSTEOPONTIN, ANGIOGENIN, VEGF, PDGF-BB, SDF-1, and IGF-BP-2, or express at least one gene selected from the group consisting of LAMA2, LAMA4, and LAMC1.

[13] A pharmaceutical product comprising Schwann cells, wherein the Schwann cells are produced by the method for producing the Schwann cells according to any one of [9] to

[12] .

[0009] According to the present invention, it is possible to provide either a novel method for producing Schwann progenitor cells that can solve the above problems, or a novel method for producing Schwann cells.

[0010] Phase-contrast micrograph of cell aggregates derived from human embryonic stem (ES) cell line A, cultured for 24 hours in a low-adsorption 96-well culture plate. Scale bar is 500 μm. Phase-contrast micrograph of cell aggregates derived from human iPS cell line, cultured for 24 hours in a low-adsorption 96-well culture plate. Scale bar is 500 μm. Phase-contrast micrograph of cell aggregates derived from human ES cell line A, cultured for 24 hours in a culture dish coated with laminin or fragments thereof. Scale bar is 500 μm. Phase-contrast micrograph of cell aggregates derived from human iPS cell line, cultured for 24 hours in a culture dish coated with laminin or fragments thereof. Scale bar is 500 μm. Immunofluorescence staining images for SOX10, βIII-tubulin, or DAPI of a cell population containing neural rosettes derived from human ES cell line A, cultured for 3 days in Schwann progenitor cell induction medium. This report shows the changes in the percentage of cells positive for both SOX10 and S100B in human ES cell line A, human ES cell line B, and human iPS cell line cells, which were repeatedly passed using Schwann progenitor cell induction medium. It also shows the results of flow cytometry analysis of the percentage of MBP-positive cells in human ES cell line A and B cell populations. Furthermore, it presents the results of quantitative reverse transcription PCR measurement of MBP and MPZ gene expression levels in human ES cell line A and human iPS cell line populations. Finally, it shows the results of ELISA measurement of NGF and GDNF production in human ES cell line A and human iPS cell line populations. The results of cytokine array measurement in human ES cell line A are also presented. Lastly, it shows the results of quantitative reverse transcription PCR measurement of LAMA2, LAMA4, and LAMC1 in human ES cell line A and human iPS cell line populations. This study demonstrates the effect of neurite extension using the culture supernatant of Schwann cells derived from human ES cell line A and Schwann cells derived from human iPS cell lines.

[0011] The following describes in detail an embodiment for carrying out the present invention (hereinafter referred to as "this embodiment"), but the present invention is not limited thereto, and various modifications are possible without departing from its essence.

[0012] The method for producing Schwann progenitor cells according to this embodiment includes: culturing human pluripotent stem cells on a substrate in which the culture area is spatially restricted to obtain a cell aggregate of human pluripotent stem cells with controlled size; culturing such cell aggregate to differentiate it into a neural rosette; and obtaining Schwann progenitor cells from such neural rosette.

[0013] The method for producing Schwann cells according to this embodiment includes culturing Schwann progenitor cells on a scaffold coated with adhesion molecules acceptable in the production of clinical cells, and such cultivation is carried out in a culture medium that does not contain any non-human animal-derived components.

[0014] This embodiment may allow for the efficient acquisition of Schwann progenitor cells from pluripotent stem cells. Furthermore, this embodiment may allow for the acquisition of high-purity Schwann progenitor cells from pluripotent stem cells. This embodiment also relates to a method for producing Schwann cells using the Schwann progenitor cells of this embodiment.

[0015] The pluripotent stem cells used in the manufacturing method of this embodiment are not particularly limited as long as they are cells that have the function of human pluripotent stem cells. For example, embryonic stem cells (ES cells) and iPS cells can be used as human pluripotent stem cells. The pluripotent stem cells used in the manufacturing method of this embodiment may be human ES cells or human iPS cells. The steps in each of the above manufacturing methods will be described in detail below.

[0016] [Preparation of cell aggregates of pluripotent stem cells with controlled size] The manufacturing method of this embodiment includes a step (hereinafter referred to as the "cell aggregate preparation step") in which human pluripotent stem cells are cultured on a substrate in which the culture area is spatially restricted in order to obtain cell aggregates of human pluripotent stem cells with controlled size. "Obtaining cell aggregates of pluripotent stem cells with controlled size" means that the size of the cell aggregates of pluripotent stem cells is intended to be controlled in the cell aggregate preparation step. "Culturing pluripotent stem cells on a substrate in which the culture area is spatially restricted" means that the culture area of ​​the pluripotent stem cells is spatially limited in order to obtain cell aggregates of a size appropriate to that culture area.

[0017] By creating cell aggregates of pluripotent stem cells with controlled size from pluripotent stem cells, and then using these cell aggregates to create neural rosettes, neural rosettes can be efficiently produced. Through diligent research, the inventors have discovered that using cell aggregates that are too small reduces the survival rate of colonies in cell culture, while using cell aggregates that are too large reduces the probability of differentiation into neural rosettes. Therefore, the inventors hypothesize that by using cell aggregates of pluripotent stem cells with a controlled size to create neural rosettes, the balance between the survival rate of colonies in cell culture and the differentiation efficiency into neural rosettes can be adjusted, thus enabling efficient neural rosette production. However, the present invention is not limited by this hypothesis. Furthermore, by culturing human pluripotent stem cells on a substrate with a spatially restricted culture area, cell aggregates of human pluripotent stem cells with controlled size can be efficiently obtained.

[0018] Culturing human pluripotent stem cells on a substrate with spatially restricted culture areas involves culturing human pluripotent stem cells on a culture plate having a specific culture area or a specific culture volume. Such culture plates include well plates. The number of wells in the culture plate is not particularly limited, and examples include 48, 96, and 384, etc., and preferably 96. The shape of each well of the culture plate in plan view is not particularly limited, and examples include a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom surface of the well of the culture plate is not particularly limited, and examples include a flat bottom, a round bottom (U-shaped), a spindle, a V-shaped, etc., and preferably a U-shaped, a spindle, a V-shaped, and more preferably a U-shaped. The culture plate is preferably a 96-well U-bottom well plate. Other culture plates include culture devices with fine holes on the bottom surface, culture devices made by arranging a large number of small circular cell adhesion regions on the bottom surface of the container and treating the outside to be a cell non-adhesion region, etc. The material of the culture plate is not particularly limited, and examples include resins, metals, and inorganic materials, and preferably resins. The culture plate is preferably low-adsorbent to cells. A culture plate being low-adsorbent means that the surface of the culture plate is treated so that non-specific adsorption between cells and the culture plate is suppressed. Such treatments include coating treatments, surface shape treatments, etc.

[0019] The culture plate has a well bottom area of 0.05 cm ２ , ２ , ２ , ２ , ２ , ２ , ２ , 0.1 cm ２ , 0.5 cm ２ , 1.0 cm ２ , 1.5 cm ２ , 2.0 cm ２ , 2.5 cm ２ , 3.0 cm ２ , 3.5 cm ２ , 4.0 cm ２ , 4.5 cm ２ , 5.0 cm ２ , 5.5 cm ２ , 6.0 cm ２ , 6.5 cm [[ID=​​​​２ 8.5 cm ２ 9.0 cm ２ 9.5 cm ２ 10.0 cm ２ , or a range with these as the upper and lower limits (for example, 0.1 cm) ２ ~10.0cm ２ , 1.5 cm ２ ~5.0cm ２ The culture plate may have well depths of 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, 2.0 cm, 2.1 cm, 2.2 cm, 2.3 cm, 2.4 cm, 2.5 cm, or a range with these as the upper and lower limits (for example, 0.5 cm to 2.5 cm, 1.0 cm to 2.0 cm). If the bottom of the culture plate is rounded, the spherical radius (reciprocal of curvature) when the rounded bottom is considered as a sphere may be 0.1 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, 2.0 cm, 2.1 cm, 2.2 cm, 2.3 cm, 2.4 cm, 2.5 cm, or a range with these as the upper and lower limits (for example, 0.1 cm to 2.5 cm, 0.5 cm to 2.0 cm).

[0020] When culturing human pluripotent stem cells on a culture plate, preferably 0.1 to 100 × 10⁶ of cells should be used per well. ４ Cells are sown, more preferably 0.5 to 50 × 10 ４ Cells are sown, more preferably 1 to 10 × 10 ４ Cells are seeded. After seeding, the cells are subjected to centrifugation before cultivation begins, which allows for cultivation on a substrate with a suitably spatially restricted culture area. The centrifugation conditions may be, for example, 190 × g for 1 to 5 minutes.

[0021] The cell aggregate preparation step may be a step of culturing human pluripotent stem cells to form colonies of human pluripotent stem cells while maintaining the function of the human pluripotent stem cells, thereby obtaining a cell aggregate. In culturing human 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 later, in order to obtain colonies of human pluripotent stem cells. Known culture media and culture conditions may be used for the culture system. Examples of culture media for human pluripotent stem cells in the cell aggregate preparation process include StemFitAK03N (manufactured by Ajinomoto Healthy Supply Co., Ltd.), Essential 8 MEDIUM (manufactured by Thermo Fisher Scientific), ESSENTIAL 8 FLEX MEDIUM (manufactured by Thermo Fisher Scientific), mTeSR Plus-cGMP (manufactured by STEMCELL Technologies), StemMACS PSC-Brew XF (manufactured by Millenny Biotec), and Nutri Stem hPSC XF (manufactured by Sartorius GmbH).

[0022] The cell aggregate preparation step may include, for example, passing human pluripotent stem cells one or more times on a substrate with a spatially restricted culture area for a total of seven days or more. The number of passages may be one or more, two or more, six or fewer, or five or fewer. After culture, a cell aggregate suspension can be obtained by recovering the colonies of human pluripotent stem cells from the substrate. The culture period on the substrate with a spatially restricted culture area in the cell aggregate preparation step is preferably 12 hours to 7 days, more preferably 18 hours to 3 days, and even more preferably 22 hours to 38 hours.

[0023] The average size of the cell aggregates of human pluripotent stem cells obtained by the cell aggregate preparation process, which have controlled size, may be, for example, 150 μm or more and 2000 μm or less, preferably 200 μm or more and 1500 μm or less, and more preferably 250 μm or more and 1000 μm or less. Such an average size may be a range obtained by arbitrarily combining the upper and lower limits described above within the above range. The average size of the cell aggregates may be, for example, the average value of the length of the long axis and the length of the short axis in images of the cell aggregates observed under a microscope, averaged over three or more cell aggregates.

[0024] Human pluripotent stem cells may be cultured on a substrate with an unrestricted culture area as a pre-process before being cultured on a substrate with a spatially restricted culture area. Such a pre-process may include, for example, subculturing cells on a cell scaffold. Before subculturing on a cell scaffold, culture may be performed in a culture system that does not use a cell scaffold. In this pre-process as well, in order to obtain colonies of human 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 will be described later. In this embodiment, the cell scaffold is preferably a scaffold coated with adhesion molecules acceptable in the method for producing clinical cells. Adhesion molecules acceptable in the method for producing clinical cells will be described later.

[0025] [Preparation of Neural Rosettes] The manufacturing method of this embodiment includes a step (hereinafter referred to as the "neural rosette differentiation step") in which a cell aggregate of human pluripotent stem cells with controlled size is cultured and differentiated into a neural rosette. In this step, by producing a neural rosette using a cell aggregate of pluripotent stem cells with controlled size, neural rosettes can be produced efficiently. By efficiently producing neural rosettes, this embodiment leads to an efficient method for producing Schwann progenitor cells. A neural rosette is a cell population having a rosette-like shape that contains neural progenitor cells that have the ability to differentiate into various nerve cells. Whether a colony of pluripotent stem cells has differentiated into a neural rosette can be confirmed by observing the shape of the colony using a microscope, or by gene or protein expression analysis of PAX6, PAX7, SOX9, SOX10, and nestin.

[0026] The neural rosette differentiation step involves culturing cell aggregates of pluripotent stem cells of controlled size in neural rosette differentiation induction medium. Such culture may be carried out by placing the cell aggregates on a cell scaffold. The cell scaffold is preferably coated with adhesion molecules acceptable in the production of clinical cells. Examples of such adhesion molecules include collagen, laminin and laminin fragments, fibronectin and fibronectin fragments, and gelatin. When fragments are used as adhesion molecules, these fragments are preferably those that retain cell adhesion factor (integrin) binding activity. The cell scaffold used in culturing the cell aggregate is preferably coated with adhesion molecules acceptable in the production of clinical cells, preferably laminin or laminin fragments. The adhesion molecules acceptable in the production of clinical cells are more preferably laminin (e.g., laminin 511 or laminin 521) or fragments of either of these. Commercially available cell scaffolds may be used. Furthermore, in the neural rosette differentiation process, it is preferable to culture the cells in a flat culture dish, for example, where the culture area is not spatially restricted.

[0027] The neural rosette differentiation induction medium may be any medium containing a differentiation induction factor for inducing differentiation into neural rosettes. For example, it may be prepared by referring 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). More specifically, the differentiation induction medium for neural progenitor cells described in the examples can be mentioned. Examples of the differentiation induction factor for neural rosettes include Noggin and Wnt.

[0028] In the neural rosette differentiation step, before culturing the cell mass of pluripotent stem cells with controlled size in the neural rosette differentiation induction medium, it may be pre-cultured using a culture system that does not contain a differentiation induction factor for differentiating into neural rosettes or Schwann precursor cells, for example, the same medium as used in the above cell mass preparation step. Also in this pre-culture, it is preferably cultured on a scaffold coated with an adhesion molecule acceptable in the method for producing clinical cells. The culture period of the pre-culture is preferably 12 hours or more and 3 days or less, more preferably 18 hours or more and 2 days or less, and even more preferably 22 hours or more and 38 hours or less.

[0029] The neural rosette differentiation step may be a step of culturing for, for example, 3 days or more, 4 days or more, 5 days or more, or 6 days or more until a rosette-like cell population appears while appropriately replacing the neural rosette differentiation induction medium. The number of culture days may be 15 days or less, 12 days or less, 10 days or less, or 8 days or less. The number of culture days may be, for example, 5 days or more and 10 days or less. After culturing, a suspension of neural rosettes may be obtained by detaching the neural rosettes from the cell scaffold with a cell detachment solution, or the step of preparing Schwann precursor cells, which will be described in detail below, may be carried out while keeping them on the cell scaffold. Examples of the cell detachment solution include those containing an enzyme having protease activity and / or collagenase activity.

[0030] [Preparation of Schwann Precursor Cells] The production method of this embodiment includes a step of obtaining Schwann precursor cells from the neural rosette obtained above (hereinafter referred to as "Schwann precursor cell acquisition step"). Regarding the method of obtaining Schwann precursor cells from neural rosettes, reference may be made to 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).

[0031] The Schwann precursor cell acquisition step is not particularly limited as long as Schwann precursor cells can be obtained from neural rosettes. For example, a method of culturing optionally pretreated neural rosettes in a Schwann precursor cell induction medium, a method of culturing cells detached from neural rosettes in a Schwann precursor cell induction medium, etc. can be mentioned. Schwann precursor cells are precursor cells of Schwann cells that can differentiate into Schwann cells derived from the neural crest, and are cells that are positive for SOX10 and / or S100B. Whether Schwann precursor cells are obtained can be confirmed not only by observing the cell shape using a microscope or the like, but also by performing immunostaining for SOX10 and / or S100B.

[0032] The Schwann progenitor cell acquisition step preferably includes culturing nerve rosettes on a scaffold coated with adhesion molecules acceptable in the method for producing clinical cells, enzymatically treating the cultured nerve rosettes, and culturing the cells detached by the enzymatic treatment in Schwann progenitor cell induction medium. Note that a portion of the Schwann progenitor cell acquisition step may be carried out continuously with the nerve rosette differentiation step described above. For example, after a rosette-like cell population appears in the cell aggregate culture in the nerve rosette differentiation step, the culture of nerve rosettes in the Schwann progenitor cell acquisition step may be carried out by continuing to culture the rosette-like cell population while maintaining it on the cell scaffold. After a rosette-like cell population appears in the cell aggregate culture in the nerve rosette differentiation step, the Schwann progenitor cell acquisition step may be started by replacing the culture medium with the Schwann progenitor cell induction medium described later.

[0033] If the Schwann progenitor cell acquisition process includes culturing nerve rosettes on a scaffold coated with adhesion molecules acceptable in the method for producing clinical cells, and enzymatically treating the cultured nerve rosettes, the culture period for culturing the nerve rosettes on the scaffold is not particularly limited. However, this process may involve culturing for, for example, one day or more, two days or more, or three days or more, while appropriately changing the culture medium, until cell migration from the nerve rosettes is confirmed. The culture period may be 10 days or less, 8 days or less, 7 days or less, or 5 days or less. The culture period may be, for example, one day or more and five days or less. The culture period may be changed to the Schwann progenitor cell induction medium described later, and then cultured for the specified number of days.

[0034] In the process of obtaining Schwann progenitor cells, the scaffold for culturing nerve rosettes is preferably a scaffold coated with an adhesion molecule acceptable in the method of producing clinical cells, and more preferably a scaffold coated with laminin (e.g., laminin 511 or laminin 521) or a fragment thereof. Among these fragments, fragments that retain cell adhesion factor (integrin) binding activity are preferred. The scaffold for culturing nerve rosettes may be the same scaffold as the scaffold for culturing cell aggregates to obtain nerve rosettes. Schwann progenitor cell induction medium may be used as the culture medium. Examples of Schwann progenitor cell induction medium are described later.

[0035] By culturing nerve rosettes on a scaffold coated with adhesion molecules acceptable in the method for producing clinical cells, cells including Schwann progenitor cells can migrate. To collect these migrated cells, the cultured nerve rosettes may be enzymatically treated. Subsequently, the cells detached by the enzymatic treatment can be collected and cultured in Schwann progenitor cell induction medium. Through this process, high-purity Schwann progenitor cells can be produced.

[0036] Enzymatic treatment of nerve rosettes may be carried out by treating the nerve rosettes with a cell detachment solution containing enzymes. The cell detachment solution may contain, for example, collagenase and may selectively detach cells that differentiate into Schwann progenitor cells, or Schwann progenitor cells. The collagenase contained in the cell detachment solution may be a mixture of several types of collagenase, but preferably contains collagenase I.

[0037] The cell detachment fluid may contain components other than collagenase that may be present in the cell detachment fluid. Such components include enzymes other than collagenase, salts that contribute to enzyme activity, and salts that contribute to intercellular bonding.

[0038] Examples of enzymes other than collagenase include other proteolytic enzymes, which include enzymes possessing both protease and collagenase activity. One such enzyme is Accutase, manufactured by Innovative Cell Technologies. Accutase is a mixture of enzymes possessing both protease and collagenase activity.

[0039] The salt contributing 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 its activity, but it is preferable that the salt contains 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 counterion of the metal ion is not particularly limited as long as the salt dissolves and the ion is acceptable to the cell, and examples include chloride ions, nitrate ions, and sulfate ions.

[0040] The salt contributing to intercellular bonding may be, for example, a salt containing a metal ion that improves or inhibits the adhesion of intercellular adhesion molecules, but it is preferable that it is a salt containing a metal ion that improves the adhesion of intercellular adhesion molecules. As such a salt, calcium salts are preferred. The counterion of the metal ion is not particularly limited as long as the salt dissolves and the ion is tolerable by the cells, and examples include chloride ions, nitrate ions, and sulfate ions.

[0041] A commercially available cell detachment solution may be used as the cell detachment solution.

[0042] The time for treating nerve rosettes with cell decomposition solution is not particularly limited, but is, for example, 1 to 30 minutes, preferably 2 to 20 minutes, and more preferably 2 to 10 minutes.

[0043] Next, the purity of Schwann progenitor cells in the recovered cell population can be increased by culturing the detached and recovered cells in Schwann progenitor cell induction medium. Such cultivation may be carried out, for example, by seeding the cells detached from the neural rosette onto a cell scaffold. The Schwann progenitor cell induction medium may be any medium containing differentiation-inducing factors for Schwann progenitor cells, but it may be prepared by referring 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. (Non-Patent Literature 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 medium obtained by adding differentiation-inducing factors for Schwann progenitor cells to the neural rosette differentiation induction medium used in the neural rosette differentiation process.

[0044] Such culture can increase the purity of Schwann progenitor cells by subculturing. Subculturing should be carried out until the purity of the Schwann progenitor cells reaches the required level, but it is preferable to carry it out at least twice. Culture may be carried out 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 progenitor cell induction medium. The culture period may be 60 days or less, 50 days or less, 40 days or less, 30 days or less, or 20 days or less.

[0045] The cell scaffold used when culturing the recovered cells is not particularly limited, but it is preferable that the scaffold be coated with an adhesion molecule acceptable in the method for producing clinical cells. Examples of adhesion molecules are as described above, but are preferably laminin (e.g., laminin 511 or laminin 521) or fragments thereof.

[0046] The cell population obtained in this manner may contain high-purity Schwann progenitor cells. In the obtained cell population, the proportion of SOX10-positive and S100B-positive cells may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, respectively. The upper limit of the above proportions is not particularly limited and may be, for example, 100%, 99%, 98%, or 95%.

[0047] [Preparation of Schwann Cells] The production method of this embodiment may include a step of culturing Schwann progenitor cells on a scaffold coated with adhesion molecules acceptable in the production of clinical cells (hereinafter referred to as the "Schwann cell production step"). Here, the culture is carried out in a culture medium that does not contain any non-human animal-derived components. In this step, by using a culture medium that does not contain any non-human animal-derived components and culturing on a scaffold coated with adhesion molecules acceptable in the production of clinical cells, clinically suitable Schwann cells can be produced.

[0048] The Schwann cells produced may be Schwann cells in which the production of NGF and / or GDNF is promoted, and may be Schwann cells that promote nerve regeneration in vivo. Furthermore, the Schwann cells produced may produce at least one substance selected from the group consisting of NGF, GDNF, CCL2, TIMP-1, TIMP-2, OSTEOPONTIN, ANGIOGENIN, VEGF, PDGF-BB, SDF-1, and IGF-BP-2, and the production of these substances may be promoted, and LAMA2, LAMA4, and / or LAMC1 may be expressed, and the expression of these genes may be enhanced. The production amounts of NGF, GDNF, CCL2, TIMP-1, TIMP-2, and OSTEOPONTIN can be measured, for example, by ELISA, and the production amounts of ANGIOGENIN, VEGF, PDGF-BB, SDF-1, and IGF-BP-2 can be measured, for example, by a cytokine array. The expression levels of LAMA2, LAMA4, and LAMC1 can be measured, for example, by quantitative reverse transcription PCR. Whether Schwann cells have been obtained can also be confirmed by the expression of MBP (Myelin basic protein). In the Schwann cells obtained by the production method of this embodiment, the percentage of MBP-expressing cells may be 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. The upper limit of the above percentage is not particularly limited and may be, for example, 100%, 99%, 98%, or 95%.

[0049] The culture medium used in the Schwann cell production process is not particularly limited, as long as it does not contain animal-derived components other than those from humans. Examples of such animal-derived components include fetal bovine serum (FBS). Therefore, the culture medium does not contain fetal bovine serum. Preferably, the culture medium contains human serum or a substitute thereof. Examples of human serum substitutes include recombinant proteins such as albumin. The culture medium may also contain differentiation-inducing factors for Schwann cells. Examples of differentiation-inducing factors for Schwann cells include neuregulin 1 (NRG1), and the culture medium may contain at least one selected from the group consisting of retinoic acid, platelet-derived growth factor-BB (PDGF-BB), forskolin, and dibutyryl cAMP (db-cAMP).

[0050] The scaffold used for culturing Schwann progenitor cells to obtain Schwann cells is preferably a scaffold coated with an adhesion molecule acceptable in the method for producing clinical cells, more preferably a scaffold coated with laminin (e.g., laminin 111, laminin 211, laminin 221, laminin 411, laminin 511, or laminin 521) or a fragment thereof, and even more preferably a scaffold coated with laminin 221, laminin 411, laminin 511, laminin 521, or a fragment thereof. Among these fragments, fragments that retain cell adhesion factor (integrin) binding activity are preferred.

[0051] The culture period in the Schwann cell production process may be, for example, 3 to 32 days, 5 to 28 days, or 6 to 24 days.

[0052] The cells used in the Schwann cell production process may be Schwann progenitor cells, and may be Schwann progenitor cells obtained by the production method described in [Production of Schwann Progenitor Cells] above.

[0053] Thus, in this embodiment, by producing Schwann cells in which the production of NGF and / or GDNF is promoted, it is expected that a sufficient amount of Schwann cells with the nerve repair capacity necessary for the treatment of diseases in humans and animals can be produced. Furthermore, since the produced Schwann cells produce at least one selected from the group consisting of NGF, GDNF, CCL2, TIMP-1, TIMP-2, OSTEOPONTIN, ANGIOGENIN, VEGF, PDGF-BB, SDF-1, and IGF-BP-2, or express at least one gene selected from the group consisting of LAMA2, LAMA4, and LAMC1, they can be used to treat at least one of the diseases selected from the group consisting of diseases treated by promoting tissue regeneration and diseases that may cause inflammation or fibrosis.

[0054] Nerve growth factors such as NGF have a neurite outgrowth effect on peripheral nerves. As shown in the examples described below, Schwann cells obtained by the manufacturing method of this embodiment can promote neurite outgrowth in nerve cells of animals, including humans.

[0055] Furthermore, Schwann cells obtained by the manufacturing method of this embodiment can promote nerve regeneration in vivo in animals, including humans. Such Schwann cells may have enhanced expression of NGF and GDNF. Schwann cells obtained in this way can restore nerve-related functions, such as motor function, in animals, including humans.

[0056] The motor function restored by Schwann cells obtained by the manufacturing method of this embodiment may be walking function. Walking function may be evaluated, for example, by three-dimensional motion analysis.

[0057] The function involving nerve cells restored by the Schwann cells obtained by the manufacturing method of this embodiment may be the compound muscle action potential. An electromyogram (EMG) is an action potential generated when a muscle cell contracts. 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 EMG. The waveform represented in the EMG is the sum of all action potentials at the point they reach the electrodes, and this is called the compound muscle action potential.

[0058] Schwann cells obtained by the manufacturing method of this embodiment 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 obtained by the manufacturing method of this embodiment can restore (improve) the density of myelinated axons measured by optical microscopy analysis of peripheral nerves, and the diameter and myelin sheath thickness measured by electron microscopy analysis of myelinated axons in animals including humans.

[0059] Schwann cells obtained by the manufacturing method of this embodiment may increase skeletal muscle-related proteins and / or mature NMJs (neuromuscular junctions). The expression level of skeletal muscle-related proteins may be confirmed by myosin heavy chain 1 (MYH1), a fast-twitch muscle marker. Mature NMJs may be confirmed by acetylcholine receptor ε (ACHRE). It has been reported that MYH1 and ACHRE decrease when muscle atrophy or denervation occurs (Wang et al. Biosci Rep. 2020; Daneshvar et al. Am J Physiol Cell Physiol. 2020). Schwann cells obtained by the manufacturing method of this embodiment can be expected to restore (increase) the decrease in skeletal muscle-related proteins and / or mature NMJs (neuromuscular junctions) in animals, including humans.

[0060] Schwann cells obtained by the manufacturing method of this embodiment 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 like mice and rats, or primates such as monkeys and humans. The method of administering Schwann cells is not particularly limited, but may be administered directly to or near the site of nerve cell damage. Such methods include direct seeding at or near the site of damage, and injection.

[0061] Accordingly, this disclosure also provides cell therapies containing Schwann cells, and in particular Schwann cells produced by the manufacturing method of this embodiment. Such Schwann cells produce the substances described above or express the genes described above, and are therefore useful in the treatment of diseases that require these substances. The pharmaceuticals of this embodiment may optionally contain pharmacoagulants that are pharmacologically acceptable. The dosage of the pharmaceuticals is appropriately determined considering the purpose, the severity of the disease, the patient's age, weight, sex, medical history, and the type of other active ingredients.

[0062] Furthermore, this disclosure also provides a therapeutic method, which includes administering Schwann cells to a subject in need of treatment. Such a therapeutic method is useful in treating diseases that require the substances described above, as it produces the substances described above or expresses the genes described above.

[0063] The diseases targeted by the pharmaceutical and therapeutic methods of this embodiment are not particularly limited as long as they can be treated by the administration of Schwann cells, but examples include peripheral neuropathy and central neuropathy (central injuries such as spinal cord injury, cerebral infarction and brain injury, peripheral neuropathy such as carpal tunnel syndrome, peripheral nerve injury and neuropathic pain, Charcot-Marie-Tooth disease, diabetic neuropathy, anticancer drug-induced peripheral neuropathy, etc.). Diseases that can be treated by promoting tissue regeneration, and diseases that may cause inflammation or fibrosis, are also examples of diseases.

[0064] As described above, this embodiment provides a novel method for producing Schwann progenitor cells and a novel method for producing Schwann cells.

[0065] The manufacturing method of this embodiment may be useful in the development of regenerative medicine (cell therapy) for peripheral and central neuropathy. The manufacturing method of this embodiment may be useful in the creation of disease models of Schwann progenitor cells and Schwann cells related to peripheral neuropathy and peripheral nerve-related diseases, and in the development of pharmaceuticals using these. The manufacturing method of this embodiment can provide human pluripotent stem cell-derived Schwann progenitor cells and Schwann cells that can be applied to cell therapies for central nervous system injuries such as spinal cord injury, cerebral infarction and brain injury, and peripheral neuropathy such as carpal tunnel syndrome, peripheral nerve injury, and neuropathic pain. The manufacturing method of this embodiment may be useful in drug discovery using disease models such as Charcot-Marie-Tooth disease, diabetic neuropathy, atopic dermatitis, and anticancer drug-induced peripheral neuropathy.

[0066] The present invention will be described more specifically below with reference to examples and comparative examples. The present invention is not limited in any way by the following examples.

[0067] [Preparation of Schwann progenitor cells] Schwann progenitor cells were prepared from human ES cell lines and human iPS cell lines using the following procedure. Two types of human ES cell lines (strain A and strain B) were used. Human ES cell lines and human iPS cell lines were placed in 1 to 10 × 10⁶ wells of a low-adsorption 96-well culture plate using human pluripotent stem cell medium containing Y-27632 (StemFitAK03N, Ajinomoto Healthy Supply Co., Ltd.). ４ Seeds were sown in cells / well. After centrifugation (190 x g, 4°C, 3 minutes), the seeds were sown at 37°C / 5% CO2. ２ Cell aggregates of human pluripotent stem cells with controlled size were obtained by culturing them in an incubator for 24 hours. Figure 1A shows the cell aggregates obtained from human ES cell line A, and Figure 1B shows the cell aggregates obtained from human iPS cell line, as observed by a phase-contrast microscope after culturing.

[0068] Subsequently, the obtained cell aggregates were transferred to the surface of a culture dish coated with laminin (laminin 511 or laminin 521) or fragments thereof. Figure 2A shows the culture results of cell aggregates obtained from human ES cell line A, and Figure 2B shows the culture results of cell aggregates obtained from human iPS cell line, as observed by a phase-contrast microscope after 24 hours of culture. After 24 hours of culture, the culture medium was replaced with a differentiation induction medium for neural progenitor cells (Advanced DMEM-F12 (Thermo Fisher Scientific) / Neurobasal medium, B-27 Supplement (Thermo Fisher Scientific), N-2 Supplement (Thermo Fisher Scientific), human serum albumin solution (Japan Blood Products Organization), GlutaMAX Supplement (Thermo Fisher Scientific), CT99021 (Fujifilm Wako Pure Chemical Industries, Ltd.), SB431542 (Fujifilm Wako Pure Chemical Industries, Ltd.)), and the cells were cultured for 7 days to induce a cell population containing neural rosettes. Subsequently, the cells were cultured for 3 days in Schwann progenitor cell induction medium (the above differentiation induction medium for neural progenitor cells with the addition of Neureglin 1 (NRG1)), and cell migration from the neural rosettes was confirmed. Figure 3 shows the results of immunofluorescence staining of nerve rosettes obtained by culturing two human ES cell lines (line A and line B). Immunofluorescence staining was performed using SOX10 (green), a Schwann progenitor cell marker; βIII-tubulin (red), a neuronal cell marker; and nuclear staining (DAPI: blue).

[0069] Cell populations containing nerve rosettes were cultured for 3 days using the method described above. Cells that migrated from the nerve rosettes were detached by enzymatic treatment (Accutase (Innovative Cell Technologies)) and collected. The collected cells were seeded on the surface of a culture dish coated with laminin (laminin 511 or laminin 521) or fragments thereof and cultured for 14 days. To purify the Schwann progenitor cells, the cells were repeatedly passed using the Schwann progenitor cell induction medium described above. Cells were collected after each passage (P2, P3, P4), and the percentage of cells positive for both SOX10 and S100B, which are Schwann progenitor cell markers, was analyzed by flow cytometry. The analysis results for cells derived from human ES cell lines A and B, and cells derived from human iPS cell lines are shown in Figure 4. In all results, cells that had been passaged four or more times had a percentage of cells positive for both SOX10 and S100B of 90% or more.

[0070] [Differentiation into Schwann cells] The obtained Schwann progenitor cells were seeded on the surface of a culture dish coated with laminin (laminin 221, laminin 411, laminin 511, or laminin 521) or fragments thereof. After 24 hours, the cells were cultured for 7 days in a Schwann cell differentiation induction medium (human serum, GlutaMAX Supplement, Forskolin, retinoic acid, PDGF-BB, NRG1-containing medium). Subsequently, the cells were cultured for 7 days in mature Schwann cell medium (human serum, GlutaMAX Supplement, Forskolin, NRG1, Dibutyryl-cAMP).

[0071] Cultured cells were collected, and the percentage of MBP-positive cells, a mature Schwann cell marker, was analyzed by flow cytometry in cells derived from human ES cell lines A and B. The percentage of MBP-positive cells in the produced Schwann cells ranged from 74% to 88% (Figure 5). In addition, the gene expression levels of MBP and MPZ (Myelin protein zero) were measured by quantitative reverse transcription PCR in cells derived from human ES cell line A and human iPS cell lines. MBP expression levels were higher in human ES cell line A cells than in human iPS cell-derived cells, but MPZ expression levels were almost the same in both groups (Figure 6).

[0072] Furthermore, NGF and GDNF were quantified using ELISA in the supernatant of the culture medium after harvesting cells from the human ES cell line A and the human iPS cell line. In the supernatant of the human ES cell line A cells, the NGF concentration was 15 pg / 10 ４ Cells and GDNF concentrations were 1.1 pg / 10 ４ It was cells. In the supernatant of human iPS cell line-derived cells, the concentration of NGF was 18 pg / 10 ４ Cells and GDNF concentrations are 1.0 pg / 10 ４ The cells were found to be cells (Figure 7). Similarly, CCL2, TIMP-1, TIMP-2, and Osteopontin were quantified using ELISA in the supernatant of the culture medium after cell harvesting from the human ES cell strain A described above. The results are shown in the table below. It was found that all of these substances are produced by Schwann cells.

[0073]

[0074] Furthermore, ANGIOGENIN, VEGF, PDGF-BB, SDF-1, and IGF-BP-2 were quantified using a cytokine array in the supernatant of the culture medium after cell harvesting from the human ES cell strain A described above. The results are shown in Figure 8. It was found that all of these substances are produced by Schwann cells. In addition, the gene expression levels of LAMA2, LAMA4, and LAMC1 were measured by quantitative reverse transcription PCR in cells derived from the human ES cell strain A and cells derived from human iPS cells. The expression levels of LAMA2 and LAMA4 were higher in human iPS cell line-derived cells than in human ES cell strain A-derived cells, but the expression level of LAMC1 was almost the same in both (Figure 9). It was found that all of these genes are expressed in Schwann cells.

[0075] [Evaluation of the nerve regeneration effect of Schwann cells] Neuro2a cells (JCRB Cell Bank), a model cell for process formation, were cultured in the supernatant of the culture medium after harvesting cells derived from the manufactured human ES cell line A and human iPS cell line, and the neurites were measured. The control was cultured in the differentiation induction medium for Schwann cells used above. The measurement results are shown in Figure 10. Figure 10 shows the average length of the longest neurites cultured in each medium. The data represents the mean ± standard error (n=30 for each group). *p < 0.001 (Dunnet test). From Figure 10, it was shown that when cultured in the culture supernatant of the manufactured human pluripotent stem cell-derived Schwann cells, the length of the processes of Neuro2a cells was extended more than when cultured in the differentiation induction medium for Schwann cells.

Claims

1. A method for producing Schwann progenitor cells, comprising: culturing human pluripotent stem cells on a substrate in which the culture area is spatially restricted to obtain a cell aggregate of human pluripotent stem cells with controlled size; culturing the cell aggregate to differentiate it into a neural rosette; and obtaining Schwann progenitor cells from the neural rosette.

2. The manufacturing method according to claim 1, wherein the substrate is a low-adsorption well plate.

3. The method for producing the cells according to claim 1, wherein differentiation into the neural rosette is performed by culturing the cell mass on a scaffold coated with an adhesion molecule acceptable in a method for producing clinical cells.

4. The method for obtaining the Schwann progenitor cells according to claim 1, comprising: culturing the nerve rosette on a scaffold coated with an adhesion molecule acceptable in a method for producing clinical cells; enzymatically treating the cultured nerve rosette; and culturing the cells detached by the enzymatic treatment in a Schwann progenitor cell induction medium.

5. The manufacturing method according to claim 4, wherein culturing the cells detached by the enzyme treatment in the Schwann progenitor cell induction medium comprises at least two subculturings.

6. The method for producing Schwann progenitor cells according to any one of claims 1 to 5, wherein the proportion of SOX10-positive and S100B-positive cells in the obtained Schwann progenitor cells is 50% or more.

7. The manufacturing method according to any one of claims 3 to 5, wherein the adhesive molecule is laminin or a fragment thereof.

8. The manufacturing method according to claim 4 or 5, wherein the adhesion molecule used for culturing the nerve rosette is laminin 511, laminin 521, or a fragment of either thereof.

9. A method for producing Schwann cells, comprising: producing Schwann progenitor cells by a manufacturing method described in any one of claims 1 to 5; and culturing the Schwann progenitor cells on a scaffold coated with an adhesion molecule acceptable in a method for producing clinical cells, wherein the culturing is carried out in a culture medium that is acceptable in a method for producing clinical cells and does not contain any non-human animal-derived components.

10. The manufacturing method according to claim 9, wherein the adhesion molecule used for culturing the Schwann progenitor cells is laminin or a fragment thereof.

11. The manufacturing method according to claim 9, wherein the adhesion molecule used for culturing the Schwann progenitor cells is laminin 221, laminin 411, laminin 511, laminin 521, or a fragment of any of these.

12. The method for producing Schwann cells according to claim 9, wherein the Schwann cells produced produce at least one selected from the group consisting of NGF, GDNF, CCL2, TIMP-1, TIMP-2, OSTEOPONTIN, ANGIOGENIN, VEGF, PDGF-BB, SDF-1, and IGF-BP-2, or express at least one gene selected from the group consisting of LAMA2, LAMA4, and LAMC1.

13. A pharmaceutical product comprising Schwann cells, wherein the Schwann cells are produced by the manufacturing method described in claim 9.

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