Cell aggregates, cell aggregate mixtures, and methods for producing the same

Abstract

An object of the present invention is to provide a cell aggregate containing dopamine-producing neural progenitor cells suitable for transplantation, a mixture thereof, and methods for producing the same. [Solution] The cell aggregate of the present invention contains FOXA2-positive or TUJ1-positive neural cells and contains 1,000 or more cells.

Description

[Technical Field]

[0001] The present invention relates to adherent cell populations such as cell aggregates, mixtures of such cell populations, and methods for producing them. [Background technology]

[0002] Parkinson's disease is a neurodegenerative disorder caused by the loss of dopamine-producing neurons in the substantia nigra of the midbrain, affecting approximately 4 million people worldwide. Treatments for Parkinson's disease include drug therapy with L-DOPA or dopamine agonists, stereotactic coagulation, deep electrical stimulation, and fetal midbrain transplantation. Fetal midbrain transplantation raises ethical concerns about the source tissue and carries a high risk of infection.

[0003] In recent years, a therapy using dopaminergic neurons or their precursor cells, i.e., dopaminergic neural progenitor cells, induced to differentiate from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), has been proposed (Non-Patent Document 1), and a method for producing such a therapy has been reported. Specifically, a method for producing dopaminergic neural progenitor cells has been proposed that includes a step of selecting cells suitable for transplantation using a factor (specifically, Corin or Lrtm1) that serves as a marker for dopaminergic neurons or dopaminergic neural progenitor cells (Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3). However, further improvements are required to ensure uniform quality by reducing the influence of lot differences and to increase production efficiency. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 34012 [Non-patent literature]

[0005] [Non-Patent Document 1] Wernig M, et al., Proc Natl Acad Sci US A. 2008, 105: 5856-5861 [Non-patent document 2] Doi D, et al., Stem Cell Reports.2014, 2: 337-350 [Non-patent document 3] Samata B, et al., Nature communication. 2016, 7: 1-11 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide adhesive cell populations such as cell aggregates of neural cells that are favorable in terms of size and shape, highly uniform cell aggregates or mixtures of cell populations containing such cell populations, and methods for producing them; specifically, cell aggregates containing dopamine-producing neural progenitor cells, highly uniform mixtures of such cell aggregates, and methods for producing them. [Means for solving the problem]

[0007] As a result of extensive research, the inventors have discovered that by suspending a plurality of cells in a continuous flow of liquid medium, separating the cells into target neural progenitor cells and other cells by allowing them to flow into separate continuous flows of liquid medium, selecting the target neural progenitor cells, and culturing these to produce cell aggregates containing neural cells, it is possible to obtain cell aggregates containing a number of neural cells suitable for human transplantation, which require appropriate management of the cell number and cell state, as well as a uniform mixture of such cell aggregates, and have completed the present invention.

[0008] That is, the present invention relates to the following. [1] containing FOXA2-positive or TUJ1-positive neural cells, Cell aggregates containing more than 1000 cells. [2] A cell aggregate described in [1], which contains FOXA2-positive or TUJ1-positive nervous system cells at approximately 70% or more of the total cell number. [3] The cell aggregate according to [1] or [2], in which cell death can be suppressed during culture. [4] The cell aggregate according to any one of [1] to [3], further having at least one feature selected from the following: (a1) The equivalent circle diameter is 100 μm to 2000 μm, (a2) The envelopment index is 0.5 or more. (a3) The Feret diameter ratio is 0.5 or more, and (a4) The circularity is 0.3 or more. [5] The cell aggregate according to any one of [1] to [4], which has no debris layer on its surface and has a clear boundary line under a microscope. [6] A mixture of multiple cell aggregates, the mixture containing 50% or more of the total number of cell aggregates being cell aggregates according to any one of [1] to [5]. [7] A mixture of cell aggregates described in [6], having a coefficient of variation of 15% or less in one or more indicators selected from the group consisting of circularity, minimum diameter, maximum diameter, vertical Feret diameter or horizontal Feret diameter, Feret diameter ratio, circle equivalent diameter, perimeter, area, and perimeter envelopment or area envelopment. [8] 1. A method for producing a mixture of adherent cell populations, comprising: (1) inducing differentiation of a plurality of stem cells in the presence of a first differentiation-inducing factor to obtain a plurality of cells including one or more neural progenitor cells at the first differentiation stage; (2) selectively separating neural progenitor cells at the first differentiation stage from the plurality of cells obtained in step (1), suspending the plurality of cells obtained in step (1) in a continuous flow of liquid medium; and identifying neural progenitor cells at a first differentiation stage and separating the neural progenitor cells at a first differentiation stage from the cells that are not at a first differentiation stage into continuous flows of separate liquid media; and (3) a step of culturing the neural progenitor cells at the first differentiation stage separated in step (2) in the presence of a second differentiation-inducing factor to obtain a mixture of adherent cell populations, A method for producing an adherent cell population mixture, the method comprising the steps of: (b1) containing neural cells in the second differentiation stage; and (b2) Contains 1,000 or more cells. [9] The method of producing a cell population having the characteristics (b1) and (b2) according to [8], wherein cell death can be suppressed during culture.

[10] The production method according to [9], wherein when the adherent cell population is cultured for 14 to 20 days, the number of cells at the end of the culture is 5% or more, preferably 10% or more, of the number of cells at the start of the culture.

[11] The method according to any one of [8] to

[10] , wherein the mixture of adherent cell populations is a mixture of cell aggregates.

[12] The production method according to

[11] , wherein the adherent cell population is a cell aggregate, and the cell aggregate having the characteristics (b1) and (b2) above has an equivalent circle diameter of 100 μm to 2000 μm.

[13] The method according to

[12] , wherein the adherent cell population having the characteristics (b1) and (b2) is a cell aggregate and further has the following characteristics: (b3) The degree of envelopment is 0.5 or more. (b4) The Feret diameter ratio is 0.5 or more, and (b5) The circularity is 0.3 or more.

[14] A manufacturing method according to any one of

[11] to

[13] , wherein the mixture of cell aggregates has a coefficient of variation of 15% or less in one or more indicators selected from the group consisting of circularity, minimum diameter, maximum diameter, vertical Feret diameter or horizontal Feret diameter, Feret diameter ratio, circle equivalent diameter, perimeter, area, and perimeter envelopment or area envelopment.

[15] The method according to any one of [8] to

[14] , wherein in step (2), neural progenitor cells at the first differentiation stage are separated using a microfluidic cell sorter.

[16] The method of any one of [8 to

[15] ], wherein in step (2), neural progenitor cells at the first differentiation stage are isolated in a closed system.

[17] The method according to any one of [8] to

[16] , wherein the stem cells are pluripotent stem cells.

[18] The method of any one of [8] to

[17] , wherein the neural progenitor cells in the first differentiation stage are neural progenitor cells destined to grow into the midbrain floor plate.

[19] The method of production described in

[18] , wherein the neural progenitor cells in the first differentiation stage are Corin and / or Lrtm1 positive cells.

[20] A manufacturing method according to any one of [8] to

[19] , wherein the neural cells in the second differentiation stage are neural cells positive for at least one marker selected from the group consisting of TUJ1, OTX2, FOXA2, LMX1A, LMX1B, EN1, Nurr1, PITX3, DAT, GIRK2 and TH. [twenty one] A manufacturing method described in

[20] , wherein the neural cells in the second differentiation stage are FOXA2-positive and TUJ1-positive dopamine-producing neural progenitor cells. [twenty two] A mixture of adherent cell populations obtained by the production method according to any one of [8] to

[21] . [twenty three] A method for producing an adherent cell population, comprising: A manufacturing method comprising a step of separating an adherent cell population having the characteristics (b1) and (b2) above from a mixture of adherent cell populations obtained by the manufacturing method described in any one of [8] to

[21] . [twenty four] An adherent cell population obtained by the production method described in

[23] . [twenty five] A pharmaceutical composition for transplantation comprising a cell aggregate described in any one of [1] to [5], a mixture of cell aggregates described in [6] or [7], a mixture of adherent cell populations described in

[22] , or an adherent cell population described in

[24] .

[26] A therapeutic agent for a disease requiring replenishment of nervous system cells, comprising a cell aggregate described in any one of [1] to [5], a mixture of cell aggregates described in [6] or [7], a mixture of adhesive cell populations described in

[22] , or an adhesive cell population described in

[24] .

[27] A method for treating a disease requiring replenishment of nervous system cells, comprising the step of transplanting into the central nervous system of a patient either a cell aggregate described in any one of [1] to [5], a mixture of cell aggregates described in [6] or [7], a mixture of adherent cell populations described in

[22] , or an adherent cell population described in

[24] . [Effects of the Invention]

[0009] The present invention can provide an adherent cell population, such as a cell aggregate of neural cells, that is favorable in terms of size and shape, a mixture of highly uniform adherent cell populations containing the cell population, and methods for producing them. The present invention makes it possible to achieve the uniformity of adherent cell populations, such as cell aggregates, required for pharmaceuticals, and to provide nervous system cells suitable for transplantation into humans, for example. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows a protocol for inducing differentiation of human iPS cells into dopamine-producing neural progenitor cells. [Figure 2] Figure 2 shows microscopic images (n=3) of cell aggregates (days 16, 20, 24, and 28) of suspension culture at the second differentiation stage for each cell group sorted using Jazz or Gigasort. [Figure 3]Figure 3 shows morphological images of cell aggregates observed under a digital microscope on day 28 after the start of differentiation induction. (A) shows the results of Jazz, and (B) shows the results of Gigasort. [Figure 4] Figure 4 shows a graph comparing the circle-equivalent diameter (A), envelopment degree (B), area (C), Feret diameter ratio (D), and circularity (E) of each cell aggregate in Figure 3 measured between Jazz (light gray) and Gigasort (dark gray). [Figure 5] Figure 5 shows a graph comparing the minimum diameter, perimeter, Feret diameter (horizontal), Feret diameter (vertical), Feret diameter ratio, envelopment degree (area), envelopment degree (perimeter) area, maximum diameter, circularity, and circle equivalent diameter of each cell aggregate in Figure 3, and calculating the coefficient of variation (CV value) between Jazz (light gray) and Gigasort (dark gray). [Figure 6] FIG. 6 shows immunostained images obtained using anti-FOXA2 antibody, anti-Nurr1 antibody, anti-TH antibody, and DAPI on day 28 after the start of differentiation induction. DETAILED DESCRIPTION OF THE INVENTION

[0011] I. Definition [Cell population] As used herein, an adherent cell population refers to a mass of cells formed by the adhesion of multiple cells to one another, and is a concept that includes three-dimensional adherent cell populations in which cells are biologically bonded (i.e., adhered) in three dimensions, and two-dimensional adherent cell populations in which cells are biologically bonded in two dimensions.

[0012] The three-dimensional adherent cell population is also called a cell aggregate, and is not particularly limited as long as it is a mass of cells forming a three-dimensional structure, and may be spherical or non-spherical. The cell aggregate in this specification is preferably a cell aggregate having a three-dimensional shape close to a sphere. A three-dimensional shape close to a sphere is a shape having a three-dimensional structure, and when projected onto a two-dimensional surface, it shows, for example, a circular or elliptical shape.

[0013] The two-dimensional adherent cell population is also called a cell sheet, and is not particularly limited as long as it is a monolayer or multilayer structure formed by the planar adhesion of monolayer or multilayer cells. The term "cell sheet" as used herein includes both those produced by adherent culture and those produced by non-adherent culture.

[0014] As used herein, a "mixture of adherent cell populations" or a "mixture of cell aggregates" refers to an embodiment (composition) in which two or more adherent cell populations or cell aggregates are present. The adherent cell populations or cell aggregates may be in any of the following states: suspended in a liquid medium such as a culture medium in a container, attached to the container, or settled to the bottom of the container. Frozen adherent cell populations or cell aggregates are also included in the mixture of adherent cell populations or cell aggregates used herein.

[0015] As used herein, cells (including cells in cell aggregates, cell sheets, cell populations, etc.) refer to mammalian cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) cells, and more preferably human cells.

[0016] [Nervous system cells] As used herein, the term "neural cells" refers to any type of nervous system cell, such as a central nervous system cell, or an autonomic nervous system cell, or a peripheral nervous system cell such as a motor nerve or sensory nerve cell, and includes nerve cells such as neurons, neural crest-derived cells, glial cells, oligodendrocytes, microglia, and their stem cells or progenitor cells.

[0017] As used herein, FOXA2-positive or TUJ1-positive neural cells are not particularly limited as long as they express FOXA2 or TUJ1 at detectable levels. Examples of such neural cells include neural stem cells, neural progenitor cells, neurons, ventral midbrain-derived neural progenitor cells, dopaminergic neural progenitor cells, dopaminergic neural cells, GABAergic neural progenitor cells, GABAergic neural cells, cholinergic neural progenitor cells, cholinergic neural cells, glutamatergic neural progenitor cells, glutamatergic neural cells, retinal cells (including photoreceptor cells, photoreceptor precursor cells, retinal pigment epithelial cells, etc.), and corneal cells.

[0018] Specifically, examples of FOXA2-positive, TUJ1-negative nervous system cells include neural stem cells, neural progenitor cells, and ventral midbrain-derived neural progenitor cells. Examples of FOXA2-negative, TUJ1-positive nervous system cells include GABAergic neuronal precursor cells, GABAergic neurons, cholinergic neuronal precursor cells, cholinergic neurons, glutamic acid neuronal precursor cells, glutamic acid neuronal cells, retinal cells (including photoreceptors, photoreceptor precursor cells, retinal pigment epithelial cells, etc.), and corneal cells. FOXA2-positive and TUJ1-positive nervous system cells include nerve cells such as dopamine-producing neural progenitor cells and dopamine-producing nerve cells.

[0019] In this specification, unless otherwise specified, dopamine-producing neural progenitor cells may include dopamine-producing nerve cells or dopaminergic neurons, etc. Dopamine-producing neural progenitor cells are FOXA2-positive and TUJ1-positive, and more preferably contain cells positive for one or more of OTX2, LMX1A, LMX1B, EN1, Nurr1, PITX3, DAT, GIRK2, and TH.

[0020] Other embodiments of the nervous system cells include nervous system cells that are positive for at least one of FOXA2, TUJ1, OTX2, LMX1A, LMX1B, EN1, Nurr1, PITX3, DAT, GIRK2, and TH.

[0021] Examples of human FOXA2 include polynucleotides represented by NCBI accession numbers NM_021784 and NM_153675 and proteins encoded thereby. Examples of human TUJ1 (neuron-specific class III beta-tubulin) include polynucleotides represented by NCBI accession numbers NM_006086 and NM_001197118 and proteins encoded thereby. Examples of human OTX2 include polynucleotides represented by NCBI accession numbers NM_021728, NM_172337, NM_001270523, NM_001270524, and NM_001270525, and proteins encoded thereby. Examples of human LMX1A include polynucleotides represented by NCBI accession numbers NM_001174069 and NM_177398, and proteins encoded thereby. Examples of human LMX1B include polynucleotides represented by NCBI accession numbers NM_002316, NM_001174146, and NM_001174147, and proteins encoded thereby. Examples of human EN1 include the polynucleotide represented by NCBI accession number NM_001426 and the protein encoded thereby. Examples of human Nurr1 include the polynucleotide represented by NCBI accession number NM_006186 and the protein encoded thereby. Examples of human PITX3 include the polynucleotide represented by NCBI accession number NM_005029 and the protein encoded thereby. Examples of human DAT (SLC6A3) include the polynucleotide represented by NCBI accession number NM_001044 and the protein encoded thereby. Examples of human GIRK2 (KCNJ6) include the polynucleotide represented by NCBI accession number NM_002240 and the protein encoded thereby. Examples of human TH include polynucleotides represented by NCBI accession numbers NM_000360, NM_199292, and NM_199293, and proteins encoded thereby.

[0022] [Neural progenitor cells] Neural progenitor cells refer to precursor cells that can be induced to differentiate into more differentiated neural cells. Neural progenitor cells can differentiate into any type of neural cell, including neurons, such as central nervous system cells, or peripheral nervous system cells such as autonomic nervous system cells or motor and sensory nervous system cells.

[0023] [Stem cells] As used herein, stem cells are cells that have both pluripotency (the ability to differentiate into multiple types of cells) and self-renewal, and can proliferate indefinitely. Examples of stem cells include embryonic stem cells (ES cells); pluripotent stem cells such as induced pluripotent stem cells (iPS cells) artificially produced by gene transfer or other methods from cells derived from bone marrow, blood, or skin (epidermis, dermis, or subcutaneous tissue); and somatic stem cells that exist in fat, hair follicles, brain, nerves, liver, pancreas, kidney, muscle, and other tissues and differentiate into multiple specific types of cells.

[0024] [Pluripotent stem cells] The pluripotent stem cells referred to in this specification are not particularly limited as long as they have the pluripotency to be able to differentiate into all cells present in the living body and also have the ability to proliferate. Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Pluripotent stem cells also include Muse cells (multi-lineage differentiating stress enduring cells) derived from mesenchymal stem cells (MSCs) and spermatogonial stem cells (GS cells) derived from germ cells (e.g., testes). Embryonic stem cells were first established in 1981 and have been used to generate knockout mice since 1989. Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell mass on feeder cells or in a medium containing LIF (leukemia inhibitory factor). Methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, and US6,280,718. Embryonic stem cells can be obtained from designated institutions or purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. The human embryonic stem cell Rx::GFP strain (derived from the KhES-1 strain) is available from RIKEN, a national research and development agency. The mouse embryonic stem cell EB5 cell line and D3 cell line are available from RIKEN, a national research and development agency, and ATCC, respectively.

[0025] Nuclear transfer embryonic stem cells (ntES cells), a type of embryonic stem cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg cell from which the nucleus has been removed. EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF, and bFGF (Cell, 70:841-847, 1992).

[0026] As used herein, "induced pluripotent stem cells" refer to cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specifically, these cells include differentiated somatic cells, such as fibroblasts or peripheral blood mononuclear cells, that have been reprogrammed to pluripotency by expressing a combination of multiple genes selected from a group of reprogramming genes, including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, Lin28, and Esrrb. Preferred combinations of reprogramming factors include (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466).

[0027] In 2006, Yamanaka et al. established induced pluripotent stem cells using mouse cells (Cell, 2006, 126(4), pp. 663-676). In 2007, induced pluripotent stem cells were also established using human fibroblasts, and they possess the same pluripotency and self-renewal capabilities as embryonic stem cells (Cell, 2007, 131(5), pp. 861-872; Science, 2007, 318(5858), pp. 1917-1920; Nat. Biotechnol., 2008, 26(1), pp. 101-106).

[0028] In addition to methods for producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be produced by methods for inducing induced pluripotent stem cells from somatic cells by adding chemical compounds, etc. (Science, 2013, 341, pp. 651-654).

[0029] It is also possible to obtain established induced pluripotent stem cells, such as human induced pluripotent stem cell lines established at Kyoto University, such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, which are available from Kyoto University. Examples of established induced pluripotent stem cells available from Kyoto University include Ff-I01 cells, Ff-I01s04 cells, QHJ-I01, and Ff-I14 cells.

[0030] Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells (PBMCs), T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

[0031] When producing induced pluripotent stem cells, if reprogramming is performed by expressing several types of genes, the means for expressing the genes is not particularly limited. Examples of such means include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, or adeno-associated viral vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors or episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, or electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, or electroporation method), and direct protein injection methods (e.g., needle method, lipofection method, or electroporation method).

[0032] Induced pluripotent stem cells can be produced in the presence or absence of feeder cells (feeder-free). When producing induced pluripotent stem cells in the presence of feeder cells, they can be produced in the presence of undifferentiated maintenance factors by known methods. The medium used to produce induced pluripotent stem cells in the absence of feeder cells is not particularly limited, and known maintenance media for embryonic stem cells and / or induced pluripotent stem cells, or media for establishing feeder-free induced pluripotent stem cells can be used. Examples of media for establishing feeder-free induced pluripotent stem cells include feeder-free media such as Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. When producing induced pluripotent stem cells, for example, induced pluripotent stem cells can be produced by introducing the four factors Oct3 / 4, Sox2, Klf4, and Myc into somatic cells in a feeder-free manner using a Sendai virus vector.

[0033] The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (e.g., mouse or rat) or primate (e.g., human or monkey) pluripotent stem cells, more preferably human or mouse pluripotent stem cells, and even more preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0034] [Differentiation-inducing factor] The differentiation-inducing factor refers to a factor that regulates intracellular signal transduction to induce differentiation of stem cells into neural cells (including neural progenitor cells at the first differentiation stage and neural cells at the second differentiation stage). Differentiation-inducing factors well known to those skilled in the art can be appropriately selected depending on the type of neural cell.

[0035] For example, examples of differentiation-inducing factors used to induce differentiation from pluripotent stem cells into Corin- and / or Lrtm1-positive cells include BMP inhibitors, TGFβ inhibitors, SHH signal stimulators, FGF8 and GSK3β inhibitors.

[0036] Furthermore, examples of differentiation-inducing factors used to induce differentiation from Corin and / or Lrtm1-positive cells into dopamine-producing neural progenitor cells include neurotrophic factors.

[0037] [BMP inhibitors] As used herein, the term "BMP inhibitor" is not particularly limited as long as it is a substance that inhibits signal transduction induced by BMP, and may be any of a nucleic acid, a protein, or a low-molecular-weight organic compound. Examples of BMP include BMP2, BMP4, BMP7, and GDF7. Examples of BMP inhibitors include substances that act directly on BMP (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMPs, and substances that inhibit physiological activity resulting from signal transduction mediated by BMP receptors. Examples of BMPRs include ALK2 and ALK3. Compounds well known to those skilled in the art can be used as BMP signaling pathway inhibitors, including proteinaceous inhibitors such as chordin, noggin, and follistatin, and dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904), and LDN193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline; 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline). LDN193189 is known as a BMPR (ALK2 / 3) inhibitor (hereinafter referred to as a BMPR inhibitor) and is commercially available, for example, in the form of its hydrochloride salt. Dorsomorphin and LDN193189 are available from Sigma-Aldrich and Stemgent, respectively. One or more of these may be appropriately selected and used as the BMP inhibitor.The BMP inhibitor used in the present invention may preferably be LDN193189.

[0038] [TGFβ inhibitor] As used herein, a TGFβ inhibitor refers to a substance that inhibits signal transduction that continues from the binding of TGFβ to its receptor to SMADs. The substance is not particularly limited as long as it inhibits the resulting signal transduction pathway, and may be any of a nucleic acid, a protein, or a low-molecular-weight organic compound. Examples of such substances include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGFβ (e.g., antisense oligonucleotides, siRNAs, etc.), substances that inhibit the binding of TGFβ receptors to TGFβ, and substances that inhibit physiological activities resulting from signal transduction via TGFβ receptors (e.g., TGFβ receptor inhibitors, Smad inhibitors, etc.). Examples of such substances include substances that inhibit binding to the ALK family receptor, or substances that inhibit phosphorylation of SMAD by the ALK family. Examples of such substances include Lefty-1 (NCBI accession numbers include mouse: NM_010094 and human: NM_020997), Lefty-2 (NCBI accession numbers include mouse: NM_177099 and human: NM_003240 and NM_001172425), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Examples include SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide) and A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), which are known as inhibitors of TGFβ receptor (ALK5) and Activin receptor (ALK4 / 7). One or more of these may be appropriately selected and used as the TGFβ inhibitor.The TGFβ inhibitor used in the present invention may more preferably be A83-01.

[0039] The SMAD signaling inhibitory activity of TGFβ inhibitors, BMP inhibitors, and the like can be determined by methods well known to those skilled in the art, for example, by detecting Smad phosphorylation by Western blotting (Mol Cancer Ther. (2004) 3, 737-45.).

[0040] [SHH signal stimulator] In this specification, the term "SHH (Sonic hedgehog) signal stimulator" is defined as a substance that induces disinhibition of Smoothened (Smo) and subsequent activation of Gli2, which is caused by binding of SHH to its receptor, Patched (Ptch1). Examples of such stimulators include proteins belonging to the Hedgehog family, specifically SHH or IHH (Indian Hedgehog), SHH receptors, SHH receptor agonists, Hh-Ag1.5 (Li, X., et al., Nature Biotechnology, 23, 215-221 (2005)), and Smoothened. Examples of such agonists include N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane (SAG), 20a-hydroxycholesterol, purmorphamine (PMA; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), and derivatives thereof (Stanton BZ, Peng LF., Mol. Biosyst. 6:44-54, 2010). One or more of these may be appropriately selected and used as the SHH signal stimulator.

[0041] Preferred SHH signal stimulators used in the present invention include SHH protein (Genbank accession numbers: NM_000193, NP_000184), palmorphamin, and SAG. Palmorphamin is a more preferred SHH signal stimulator used in the present invention.

[0042] [FGF8] As used herein, FGF8 is not particularly limited, but in the case of human FGF8, examples include the four splicing forms FGF8a, FGF8b, FGF8e, and FGF8f, with FGF8b being more preferred. FGF8 is commercially available from, for example, Wako Co., Ltd. and R&D Systems Co., Ltd. and can be easily used, but it may also be obtained by forced expression in cells using methods known to those skilled in the art.

[0043] [GSK3β inhibitor] In this specification, a GSK3β inhibitor is defined as a substance that inhibits the kinase activity of GSK3β protein (for example, the ability to phosphorylate β-catenin), and many such inhibitors are already known, including, for example, BIO (also known as GSK3β inhibitor IX; 6-bromoindirubin 3'-oxime), an indirubin derivative; SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), a maleimide derivative; and G Examples of such inhibitors include SK-3β inhibitor VII (4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQpSP-NH2 (SEQ ID NO: 1)), and the highly selective CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile). One or more of these may be appropriately selected and used as the GSK-3β inhibitor. These compounds are commercially available from, for example, Calbiochem and Biomol, and can be easily used. However, they may also be obtained from other sources or prepared by the user. The GSK-3β inhibitor used in the present invention may preferably be CHIR99021.

[0044] [Extracellular matrix] As used herein, the term "extracellular matrix" refers to a supramolecular structure present outside cells, and may be naturally occurring or artificial (recombinant). Examples include collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, and laminin, or fragments thereof. These extracellular matrices may be used in combination, and may be cell-derived preparations such as BD Matrigel™. Laminin or a fragment thereof is preferred. As used herein, laminin is a protein with a heterotrimeric structure containing one α-chain, one β-chain, and one γ-chain, and is an extracellular matrix protein with isoforms that differ in the composition of the subunit chains. Laminin has approximately 15 isoforms, consisting of heterotrimeric combinations of five α-chains, four β-chains, and three γ-chains. Examples of laminin used in the present invention include, but are not limited to, α chains such as α1, α2, α3, α4, or α5, β chains such as β1, β2, β3, or β4, and γ chains such as γ1, γ2, or γ3. The laminin used in the present invention is more preferably laminin 511, which consists of α5, β1, and γ1 (Nat Biotechnol 28, 611-615 (2010)).

[0045] In the present invention, laminin may be a fragment, and is not particularly limited as long as it has integrin-binding activity. For example, it may be an E8 fragment obtained by elastase digestion (EMBO J., 3:1463-1468, 1984; J. Cell Biol., 105:589-598, 1987). Therefore, in the present invention, a preferred example is laminin 511E8 (preferably human laminin 511E8) described in WO2011 / 043405, which is obtained by elastase digestion of laminin 511. Note that laminin E8 fragments, such as laminin 511E8, used in the present invention do not necessarily have to be the product of elastase digestion of laminin and may be recombinant. Laminin 511E8 is commercially available, for example, from Nippi Corporation.

[0046] To avoid contamination with unidentified components, the laminin or laminin fragment used in the present invention is preferably isolated.

[0047] [Neurotrophic Factors] As used herein, neurotrophic factors refer to ligands for membrane receptors that play an important role in the survival and maintenance of function of motor neurons, and examples thereof include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 / 5 (NT-4 / 5), neurotrophin 6 (NT-6), basic fibroblast growth factor (basic FGF), acidic fibroblast growth factor (acidic FGF), fibroblast growth factor-5 (FGF-5), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF 1), insulin-like growth factor 2 (IGF 2), glial cell line-derived neurotrophic factor (Glia cell line-derived neurotrophic factor), and the like. Examples of neurotrophic factors include cell line-derived neurotrophic factor (GDNF), TGF-β2, TGF-β3, interleukin 6 (IL-6), ciliary neurotrophic factor (CNTF), and LIF. One or more of these may be appropriately selected and used. In the present invention, preferred neurotrophic factors are factors selected from the group consisting of GDNF and BDNF. Neurotrophic factors are commercially available from, for example, Wako Co., Ltd. and R&D Systems Co., Ltd. and can be easily used, but they may also be obtained by forced expression in cells using methods known to those skilled in the art.

[0048] [ROCK inhibitor] In the present invention, the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), and examples thereof include Y-27632 (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, for example, Uenata et al., Nature 389: 990-994 (1997)), H-1152 (see, for example, Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, for example, Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2002)), and the like. (2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. In addition, other low molecular weight compounds are known as ROCK inhibitors, and such compounds or their derivatives can also be used in the present invention (see, for example, U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, 20030087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). One or more ROCK inhibitors can be used in the present invention. The ROCK inhibitor used in the present invention may preferably be Y-27632.

[0049] [Culture medium] The medium used for cell culture herein can be prepared using a medium commonly used for culturing animal cells as the basal medium, and examples of the basal medium include media that can be used for culturing animal cells, such as BME medium, BGJb medium, CMRL 1066 medium, Glasgow's Minimal Essential Medium (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, StemFit medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, Neurobasal medium, and mixtures thereof. The media used in each step of the production method of the present invention can be prepared from these basal media.

[0050] Herein, the medium used to culture a cell population containing pluripotent stem cells is preferably a medium containing factors for maintaining an undifferentiated state (undifferentiation maintenance medium) to suppress cell death of pluripotent stem cells. Furthermore, the medium used to culture a cell population containing pluripotent stem cells is preferably a feeder-free, serum-free medium. This medium can be prepared, for example, by adding factors for maintaining an undifferentiated state, serum substitutes, and appropriate nutrient sources to a basal medium. Specifically, this medium can be prepared by adding bFGF, KSR, non-essential amino acids (NEAA), L-glutamine, and 2-mercaptoethanol to DMEM / F12 medium.

[0051] As used herein, the term "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum. In the present invention, media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) are also included in the serum-free medium as long as they do not contain unconditioned or unpurified serum. The serum-free medium may contain a serum substitute. Examples of serum substitutes include those containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. Commercially available serum substitutes may also be used. Examples of such commercially available serum substitutes include KnockOut Serum Replacement (KSR), Chemically-defined Lipid Concentrated, Glutamax, B-27 Supplement, N2 Supplement, and ITS Supplement, all manufactured by Life Technologies (now ThermoFisher).

[0052] The serum-free medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like, as appropriate.

[0053] To avoid the complicated preparation process, serum-free media may be prepared by adding an appropriate amount of commercially available KSR (e.g., about 0.5% to about 30%, preferably about 1% to about 20%) (e.g., GMEM medium supplemented with about 8% KSR and concentrated chemically-defined lipids), or by adding an appropriate amount of commercially available B-27 (e.g., about 0.1 to 5%) to Neurobasal medium. Examples of media equivalent to KSR include those disclosed in JP 2001-508302 A.

[0054] The culture is preferably carried out in a serum-free medium, preferably a serum-free medium containing KSR or B-27, or a xeno-free medium. Here, "xeno-free" refers to conditions in which components derived from organisms other than the organism of the cells to be cultured are excluded.

[0055] As used herein, feeder cells refer to cells other than stem cells that are allowed to coexist with the stem cells when the stem cells are cultured. Examples of feeder cells include mouse fibroblasts (MEF, etc.), human fibroblasts, SNL cells, and STO cells. The feeder cells may be growth-inhibited feeder cells, and examples of growth-inhibiting treatment include treatment with a growth inhibitor (e.g., mitomycin C) or treatment with gamma ray irradiation or UV irradiation. However, in the present invention, it is preferable to culture in the absence of feeder cells (feeder-free).

[0056] As used herein, "feeder-free" refers to culturing in the absence of feeder cells. Examples of "feeder-free" include conditions in which no feeder cells as described above are added, or conditions in which feeder cells are substantially absent (for example, the ratio of feeder cells to the total number of cells is 3% or less, preferably 0.5% or less). Many synthetic feeder-free media that can be used to maintain undifferentiated cells have been developed and are commercially available, such as Essential 8 medium. Essential 8 medium is a DMEM / F12 medium supplemented with the following additives: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and a TGFβ inhibitor (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Examples of commercially available feeder-free media include Essential 8 (Life Technologies; now ThermoFisher), S-medium (DS Pharma Biomedical), StemPro (Life Technologies; now ThermoFisher), hESF9 (Proc Natl Acad Sci US A. 2008 Sep 9;105(36):13409-14), mTeSR1 (STEMCELL Technologies), mTeSR2 (STEMCELL Technologies), and TeSR-E8 (STEMCELL Technologies). Another feeder-free medium is StemFit (Ajinomoto Co.). By using these media in step (1) above, the present invention can be carried out easily.

[0057] In this specification, "medium containing substance X" and "in the presence of substance X" mean a medium to which exogenous substance X has been added, a medium containing exogenous substance X, or in the presence of exogenous substance X. In other words, when cells or tissues present in the medium endogenously express, secrete, or produce substance X, endogenous substance X is distinguished from exogenous substance X, and a medium that does not contain exogenous substance X does not fall under the category of "medium containing substance X," even if it contains endogenous substance X.

[0058] II. Cell aggregates and mixtures thereof One embodiment of the present invention is a cell aggregate containing FOXA2-positive or TUJ1-positive nervous system cells, with the number of cells per cell aggregate being 1000 or more. The mixture of cell aggregates is a mixture of multiple cell aggregates, and contains the cell aggregates of the present invention in an amount of 50% or more of the total number of cell aggregates.

[0059] The number of FOXA2-positive or TUJ1-positive neural cells in the cell aggregate is not particularly limited, as long as the function of the neural cells can be exerted when the cell aggregate or a derivative of the cell aggregate is transplanted into a living body, and depends on the type of neural cells, but is preferably at least about 70%, more preferably at least about 80%, and even more preferably at least about 90% of the total number of cells.

[0060] One embodiment of the present invention includes cell aggregates containing FOXA2-positive and TUJ1-positive nerve cells, with the number of cells per cell aggregate being 1000 or more.

[0061] When the neural cells are dopamine-producing neural progenitor cells, the cell aggregates of the present invention preferably contain FOXA2-positive and TUJ1-positive neural cells at approximately 50% or more of the total number of cells, more preferably approximately 70% or more, and even more preferably approximately 80% or more.

[0062] In one aspect of the present invention, the cell aggregate is characterized in that cell death can be suppressed during culture. Here, "cell death can be suppressed during culture" means that neuronal cell death, which normally occurs when cells are cultured at about 37°C in the presence of a differentiation-inducing factor or the like, can be suppressed.

[0063] For example, when cell aggregates are cultured at 37°C for 14 to 20 days in the presence of a differentiation-inducing factor, if the number of cells at the end of the culture is 5% or more, preferably 8% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 30% or more of the number of cells at the start of the culture, the cell aggregates can be determined to be capable of "suppressing cell death during culture."

[0064] In one embodiment of the present invention, the cell aggregate has at least one characteristic selected from the following (a1) to (a4): The cell aggregate may have all of the characteristics (a1) to (a4). (a1) The equivalent circle diameter is 100 μm to 2000 μm, (a2) The envelopment index is 0.5 or more. (a3) The Feret diameter ratio is 0.5 or more, and (a4) The circularity is 0.3 or more.

[0065] Here, the above (a1) to (a4) can be measured by capturing an image generated by parallel transmitted illumination from a direction perpendicular to the observation surface using a microscope or digital microscope with a camera, and analyzing the resulting figure (i.e., the figure formed when the cell aggregate is projected onto a plane).

[0066] Here, the equivalent circle diameter is the diameter of a circle having the same area as the area of ​​the above-mentioned figure. The equivalent circle diameter is preferably 100 μm to 1000 μm, more preferably 200 μm to 600 μm, preferably 300 μm to 600 μm, and even more preferably 450 μm to 600 μm.

[0067] The degree of envelopment represents the ratio of the perimeter or area between the above-mentioned figure and the convex polygon that envelops the figure. Specifically, the degree of envelopment includes the degree of envelopment of perimeter and the degree of envelopment of area, where the degree of envelopment of perimeter is the ratio of the perimeter of the figure to the perimeter of the enveloping figure, and the degree of envelopment of area is the ratio of the area of ​​the figure to the area of ​​the enveloping figure. The degree of envelopment is preferably 0.7 to 1.0, and more preferably 0.8 to 1.0.

[0068] The Feret diameter ratio is the ratio of the horizontal length of a rectangle circumscribing the above-mentioned figure to the vertical length perpendicular to the horizontal length, and is expressed as the ratio of the vertical length to the horizontal length. The Feret diameter ratio is preferably 0.6 to 1.0, and more preferably 0.7 to 1.0.

[0069] Circularity is 1 when the above figure is a perfect circle, and approaches 0 as it becomes thinner and longer. 4π × (area) ÷ (perimeter) 2 The circularity is preferably 0.5 to 1.0, and more preferably 0.7 to 1.0.

[0070] One embodiment of the cell aggregate of the present invention is a cell aggregate in which no debris layer is formed on the surface of the isolated cell aggregate and the boundary line of the cell aggregate is clearly visible under a microscope. The microscope used here is not particularly limited as long as it has a magnification of about 4 to 10 times and is well known to those skilled in the art, but a specific example is ThermoFisher EVOS XL.

[0071] An "isolated cell aggregate" refers to a cell aggregate that is not in contact with other cell aggregates and whose outer edge can be observed.

[0072] A debris layer is a structure that exists on the surface of a cell aggregate and is a continuous layer of particles (e.g., a group of dead cells) that can be observed as single particles. When a debris layer is formed on the surface of a cell aggregate, the boundary of the cell aggregate is less clear than in cell aggregates that do not have a debris layer or that have a small amount of debris layer.

[0073] A mixture of cell aggregates containing a plurality of the above-described cell aggregates of the present invention is also within the scope of the present invention. As used herein, a mixture of cell aggregates contains at least two or more, preferably five or more, cell aggregates of the present invention, and the cell aggregates of the present invention account for about 20% or more, preferably about 40% or more, more preferably about 50% or more, and particularly preferably 60% or more of the total number of cell aggregates. The mixture of cell aggregates may also contain minute cell groups of measurable size that exist in a satellite shape.

[0074] Here, "a minute group of cells existing in a satellite form" refers to a small group of cells that exists independently without being bound to a cell aggregate and that consists of multiple cells (for example, dead cells).

[0075] The mixture of cell aggregates of the present invention has good uniformity at least in terms of size and shape, and the coefficient of variation (CV value) for one or more of the indices selected from the group consisting of circularity, minimum diameter, perimeter, Feret diameter (vertical Feret diameter or horizontal Feret diameter), Feret diameter ratio, maximum diameter, envelopment (perimeter envelopment or area envelopment), area, and equivalent circle diameter is 15% or less, preferably 12% or less or 10% or less, more preferably 8% or less or 5% or less. Here, each indices can be measured using a microscope or digital microscope by capturing an image generated by parallel transmitted illumination from a direction perpendicular to the observation surface with a camera and analyzing the resulting pattern, but there are no limitations on the measurement method as long as it can be measured with the same level of accuracy as this method.

[0076] Here, the minimum diameter is the minimum distance between two parallel lines when the figure is sandwiched between the two lines. The minimum diameter of the cell aggregate of the present invention is, for example, 200 μm to 600 μm, preferably 300 μm to 600 μm, and more preferably 400 μm to 600 μm.

[0077] The perimeter is the length of the perimeter of a shape, i.e., the length of the perimeter of the shape formed when the cell aggregate is projected onto a plane. The perimeter of the cell aggregate of the present invention is, for example, 800 μm to 2700 μm, preferably 1600 μm to 2700 μm.

[0078] The Feret diameter (vertical Feret diameter or horizontal Feret diameter) is the vertical or horizontal length of a rectangle circumscribing a figure. In other words, the Feret diameter means the length of each side of a rectangle circumscribing a figure formed when a cell aggregate is projected onto a plane. The vertical or horizontal Feret diameter of the cell aggregate of the present invention is, for example, 200 μm to 800 μm, preferably 300 μm to 600 μm, and more preferably 400 μm to 800 μm.

[0079] The maximum diameter is the length at which the distance between any two points on the inner circumference of the figure is the longest. In other words, the maximum diameter means the length of the longest distance between any two points on the inner circumference of the figure formed when the cell aggregate is projected onto a plane. The maximum diameter of the cell aggregate of the present invention is, for example, 200 μm to 900 μm, preferably 300 μm to 600 μm, and more preferably 400 μm to 900 μm.

[0080] The area is the area of ​​a figure calculated two-dimensionally, that is, the area of ​​a figure formed when the cell aggregate is projected onto a plane. The area of ​​the cell aggregate of the present invention is, for example, 46,000 μm 2 ~278,000 μm 2 , preferably 165,000 μm 2 ~278,000 μm 2 is.

[0081] Each of the above-mentioned indices has multiple values ​​depending on the direction when the cell aggregate is projected onto a plane, but for convenience, a value measured in any direction may be used. Among the indices, the Feret diameter ratio, envelopment index, and circularity index show more uniform values ​​as the cell aggregate approaches a true sphere, i.e., the shape of the cell aggregate when projected onto a plane approaches a perfect circle.

[0082] III. Method for producing a mixture of adherent cell populations One aspect of the present invention is a method for producing a mixture of adherent cell populations containing neural cells, comprising the steps of: (1) inducing differentiation of a plurality of stem cells in the presence of a first differentiation-inducing factor to obtain a plurality of cells including one or more neural progenitor cells at the first differentiation stage; (2) selectively separating neural progenitor cells at a first differentiation stage from the plurality of cells obtained in step (1), the step comprising suspending the plurality of cells obtained in step (1) in a continuous flow of a liquid medium, identifying the neural progenitor cells at a first differentiation stage, and separating the neural progenitor cells at a first differentiation stage from the cells that are not at a first differentiation stage by allowing them to flow into separate continuous flows of the liquid medium; and (3) a step of culturing the neural progenitor cells at the first differentiation stage separated in step (2) in the presence of a second differentiation-inducing factor to obtain a mixture of adherent cell populations, The method includes a step in which the mixture of adherent cell populations contains adherent cell populations having the following characteristics (b1) and (b2) in an amount of 50% or more of the total number of adherent cell populations: (b1) contain neural cells in the second differentiation stage; (b2) Contains 1,000 or more cells.

[0083] [Step (1)] Step (1) is a step of inducing differentiation of a plurality of stem cells in the presence of a first differentiation-inducing factor to obtain a plurality of cells including one or more neural progenitor cells at the first differentiation stage. As used herein, neural progenitor cells at the first differentiation stage are not particularly limited as long as they are neural progenitor cells that correspond to an intermediate state when stem cells, preferably pluripotent stem cells, are induced to differentiate into neural cells at the second differentiation stage, and examples thereof include neural progenitor cells that can be differentiated into neurons.

[0084] Specific examples of neural progenitor cells include neural progenitor cells that are destined to the midbrain floor plate. Examples of neural progenitor cells that are destined to the midbrain floor plate include Corin- and / or Lrtm1-positive cells. These cells can be produced by methods well known to those skilled in the art.

[0085] The differentiation induction method for obtaining neural progenitor cells at the primary differentiation stage from stem cells can be any method known to those skilled in the art, depending on the type of neural progenitor cell. Specifically, the neural progenitor cells can be cultured in an appropriate medium in the presence of a primary differentiation-inducing factor known to those skilled in the art. Here, the primary differentiation-inducing factor refers to a factor that affects the differentiation state of cells (expression of differentiation-related transcription factors, genes, and proteins), and examples include low-molecular-weight compounds, proteins, protein peptide fragments, and physical factors such as carbon dioxide, oxygen partial pressure, or pressure. Specifically, methods using SMAD inhibitors (BMP inhibitors, TGFβ inhibitors), SHH signal stimulators, GSK3β inhibitors, and neurotrophic factors are known.

[0086] For example, in the case of neural progenitor cells destined to the midbrain floor plate, a known method described in Stem Cell Reports, vol. 2, 337-350, 2014 can be used.

[0087] As used herein, neural progenitor cells committed to the midbrain floor plate specifically include Corin and / or Lrtm1-positive cells, which are cells expressing Corin protein and / or Lrtm1 protein in amounts that can be recognized by anti-Corin antibody or anti-Lrtm1 antibody.

[0088] The method for inducing differentiation of stem cells will be specifically described below, taking as an example the case where neural progenitor cells in the first differentiation stage are neural progenitor cells containing Corin and / or Lrtm1-positive cells.

[0089] Differentiation of pluripotent stem cells into Corin- and / or Lrtm1-positive cells can be induced using a medium containing a first differentiation-inducing factor. Examples of the first differentiation-inducing factor include the above-mentioned BMP inhibitor, TGFβ inhibitor, SHH signal stimulator, FGF8, and GSK3β inhibitor. Differentiation of pluripotent stem cells into Corin- and / or Lrtm1-positive cells is preferably induced by the following multi-step process: (1a) a step of culturing pluripotent stem cells on an extracellular matrix in a medium containing a BMP inhibitor and a TGFβ inhibitor as an adherent culture; (1b) culturing the cells obtained in step (1a) in an adherent manner on an extracellular matrix in a medium containing a BMP inhibitor, a TGFβ inhibitor, an SHH signal stimulator, and FGF8; (1c) culturing the cells obtained in step (1b) in an adherent manner on an extracellular matrix in a medium containing a BMP inhibitor, a TGFβ inhibitor, an SHH signal stimulator, FGF8, and a GSK3β inhibitor; (1d) A step of culturing the cells obtained in the above step (1c) in an adherent manner on an extracellular matrix in a medium containing a BMP inhibitor and a GSK3β inhibitor.

[0090] The medium used here can be prepared using a medium used for culturing animal cells as the basal medium. Examples of basal media include GMEM medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), StemFit medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies; now ThermoFisher), and mixtures thereof. GMEM medium is preferred. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR), N2 Supplement, B-27 Supplement, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. It may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts. A preferred medium is GMEM medium containing KSR, 2-mercaptoethanol, non-essential amino acids, and pyruvate. Culture can be performed by adding to this medium, as appropriate, a reagent selected from the group consisting of a BMP inhibitor, a TGFβ inhibitor, an SHH signal stimulator, FGF8, and a GSK3β inhibitor. The composition of the medium may be adjusted or changed as appropriate during the culture.

[0091] Adhesion culture on an extracellular matrix can be performed by culturing using a culture vessel coated with the extracellular matrix. The coating treatment can be performed by placing a solution containing the extracellular matrix in the culture vessel and then removing the solution as appropriate.

[0092] Typically, the above step (1a) is carried out in a medium further containing a ROCK inhibitor. That is, step (1a) may be "a step of culturing pluripotent stem cells on an extracellular matrix as an adhesion medium in a medium containing a ROCK inhibitor, a BMP inhibitor, and a TGFβ inhibitor."

[0093] Regarding the culture conditions, the culture temperature is not particularly limited, but is preferably about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0094] The culture period is not particularly limited as long as it is a period during which Corin and / or Lrtm1-positive cells appear, but it is preferable to culture for a period during which the proportion of Corin and / or Lrtm1-positive cells in the cell population obtained after completion of step (1) above is 10% or more, and it is desirable to culture for at least 10 days, more preferably 12 to 16 days.

[0095] The multiple pluripotent stem cells may be dissociated from one another. Examples of methods for dissociating cells include mechanical dissociation and dissociation methods using a dissociation solution having both protease and collagenase activity (e.g., Accutase™ and Accumax™), or a dissociation solution having only collagenase activity. Preferably, a trypsin substitute (e.g., TrypLE CTS (Life Technologies; now ThermoFisher)) is used to dissociate human pluripotent stem cells. When cells are dissociated, it is desirable to add a ROCK inhibitor as appropriate after dissociation and culture the cells. When a ROCK inhibitor is added, it is sufficient to add the inhibitor and culture the cells for at least one day, more preferably for one day.

[0096] In one embodiment, human pluripotent stem cells (e.g., human iPS cells) may be cultured in an adherent manner in a serum-free medium containing bFGF and an SHH signal stimulator in the absence of feeder cells prior to step (1). The adherent culture is preferably carried out in a cell container whose surface is coated with laminin-511, the E8 fragment of laminin-511, or vitronectin. The adherent culture is preferably carried out using Essential 8, TeSR medium, mTeSR medium, mTeSR-E8 medium, or StemFit medium as the feeder-free medium, more preferably Essential 8 or StemFit medium (WO2017 / 183736).

[0097] [Step (2)] Step (2) involves suspending the plurality of cells obtained in step (1) in a continuous flow of liquid medium, identifying neural progenitor cells at a first differentiation stage, and separating the neural progenitor cells at a first differentiation stage from the cells that are not at that stage into separate continuous flows of liquid medium.

[0098] In the present invention, to selectively separate neural progenitor cells at the primary differentiation stage from the plurality of cells obtained in step (1), the neural progenitor cells are identified based on a specific marker. The marker used here is not particularly limited, and any marker well known to those skilled in the art can be used as appropriate. Examples of such markers include marker genes or proteins specifically expressed in neural progenitor cells at the primary differentiation stage, cell size, and cell density.

[0099] When a marker specifically expressed in the neural progenitor cells is used as an indicator, marker-positive cells can be separated using a substance that specifically binds to the marker and a cell sorter.

[0100] The marker can be a protein expressed on the surface of neural progenitor cells at the first differentiation stage of interest, and the substance that specifically binds to the marker can be an antibody or an aptamer, preferably an antibody or an antigen-binding fragment thereof.

[0101] The antibody may be polyclonal or monoclonal. These antibodies can be produced using techniques well known to those skilled in the art (Current Protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley and Sons, Sec. 11.12-11.13). Specifically, polyclonal antibodies can be obtained by expressing and purifying a marker protein, or an oligopeptide or glycolipid having a partial amino acid sequence of the marker, in Escherichia coli or a mammalian cell line, according to standard methods, and immunizing a non-human animal such as a rabbit with the purified protein. Monoclonal antibodies can be obtained from hybridoma cells prepared by cell fusion of spleen cells obtained from the immunized non-human animal with myeloma cells (Current Protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley and Sons, Sec. 11.4-11.11). Examples of antibody antigen-binding fragments include antibody portions (e.g., Fab fragments) or synthetic antibody fragments (e.g., single-chain Fv fragments, "ScFv"). Antibody fragments such as Fab and F(ab)2 fragments can also be produced by well-known genetic engineering methods.

[0102] For the purpose of recognizing or isolating cells expressing the marker, the binding substance may be bound or conjugated to a detectable substance such as, for example, a fluorescent label, a radioactive label, a chemiluminescent label, an enzyme, biotin or streptavidin, or to a substance that allows isolation and extraction, such as protein A, protein G, beads or magnetic beads.

[0103] The binding substance may also be indirectly labeled using various methods known to those skilled in the art, such as using a pre-labeled antibody (secondary antibody) that specifically binds to the antibody.

[0104] In this specification, aptamers that specifically bind to markers can be produced using techniques well known to those skilled in the art (SELEX (systematic evolution of ligand by exponential enrichment) method: Ellington, AD & Szostak, JW (1990) Nature, 346, 818-822., Tuerk, C. & Gold, L. (1990) Science, 249, 505-510).

[0105] When neural progenitor cells in the first differentiation stage are neural progenitor cells destined to the midbrain floor plate, Corin and / or Lrtm1 can be used as markers. The sequence of human Corin can be obtained under NCBI accession number NM_006587. Similarly, the sequence of human Lrtm1 can be obtained under NCBI accession number NM_020678. For example, antibodies against Corin can be produced by the methods described in WO2004 / 065599 and WO2006 / 009241, and antibodies against Lrtm1 can be produced by the method described in WO2013 / 015457.

[0106] The cell separation device used in step (2) includes a mechanism for suspending the multiple cells obtained in step (1) in a continuous flow of liquid medium, identifying neural progenitor cells in the first differentiation stage, and separating the neural progenitor cells in the first differentiation stage from other cells by allowing them to flow into separate continuous flows of liquid medium.

[0107] In this specification, the cell separation device (also referred to as a cell sorter) is a device for detecting indicators characteristic of neural progenitor cells at the primary differentiation stage, such as markers, and a device equipped with a liquid flow path that allows continuous liquid transfer without forming droplets. By using this cell separation device, cells can be separated in a continuous solution system without forming droplets.

[0108] The cell separation device herein is preferably a completely closed system. A specific example of such a cell separation device is the microchannel cell sorter described in Hulspas R et al., Cytotherapy. 2014 Oct;16(10):1384-9 (Hulspas reference). The cell separation device in this reference is a completely closed microchannel system, and can separate cells without forming droplets. Furthermore, the cell separation device is preferably a device capable of separating cells at high speed (e.g., at least 5,000 particles / second, processing a total of 10 million cells or more in a single run).

[0109] Specifically, the Cytonome Gigasort cell sorter can be used (see https: / / www.ncbi.nlm.nih.gov / pubmed / 25065635 (Hulspas reference) and http: / / www.cytonome.com / ). This cell sorter uses a completely closed microfluidic system, and by using air pressure to bend the flow path of the cells to be separated after they pass through a detection device such as a marker, cells can be separated in a continuous solution system that does not form droplets.

[0110] [Step (3)] Step (3) is a step of culturing the neural progenitor cells at the first differentiation stage separated in step (2) in the presence of a second differentiation-inducing factor to obtain a mixture of adherent cell populations, which contains adherent cell populations having the following characteristics (b1) and (b2) at a concentration of 50% or more of the total number of adherent cell populations: (b1) contain neural cells in the second differentiation stage; (b2) Contains 1,000 or more cells.

[0111] As used herein, neural cells at the second differentiation stage are cells that have progressed to a more advanced differentiation stage through continued culture after the selection in step (2), and include progenitor cells that are destined to differentiate into specific neural cells. Here, the neural cells at the second differentiation stage are not particularly limited as long as they are more differentiated than neural progenitor cells at the first differentiation stage, and the degree of differentiation depends on the target neural cells.

[0112] Neural cells in the secondary differentiation stage include neural cells that are positive for at least one, preferably at least two, and more preferably at least three of TUJ1, OTX2, FOXA2, LMX1A, LMX1B, En1, Nurr1, PITX3, DAT, GIRK2, and TH. One embodiment of neural cells in the secondary differentiation stage includes FOXA2-positive and / or TUJ1-positive cells.

[0113] Preferably, the neural cells in the second differentiation stage are ventral midbrain-derived neural cells, specifically, dopaminergic neural progenitor cells or dopaminergic neural cells. Preferably, the neural cells in the second differentiation stage are FOXA2-positive and TUJ1-positive dopaminergic neural progenitor cells.

[0114] The differentiation induction method for obtaining neural cells at the second differentiation stage from the cells obtained in step (2) can be any method known to those skilled in the art, depending on the type of neural cell of interest. Specifically, the cells can be cultured in an appropriate medium in the presence of a second differentiation-inducing factor known to those skilled in the art. Here, the second differentiation-inducing factor refers to a factor that affects the differentiation state of the cells (expression of differentiation-related transcription factors, genes, and proteins), and includes low-molecular-weight compounds, proteins, protein peptide fragments, and physical factors such as carbon dioxide, oxygen partial pressure, or pressure. For example, in the case of dopamine-producing neural progenitor cells, a known method described in Stem Cell Reports, Vol. 2, pp. 337-350, 2014 can be used.

[0115] The differentiation induction method will be specifically described below, taking as an example the case where the neural cells at the second differentiation stage are nerve cells including dopamine-producing neural progenitor cells. The medium used here can be prepared using a medium used for culturing animal cells as the basal medium. Examples of basal media include GMEM medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies; now ThermoFisher), and mixtures thereof. Neurobasal Medium is preferred. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 Supplement, B-27 Supplement, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. It may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and nucleic acids (e.g., dibutyryl cyclic AMP (dbcAMP)). A preferred medium is Neurobasal Medium, which contains B-27 Supplement, ascorbic acid, and dbcAMP. Neurotrophic factors can be added to this medium as appropriate for culture.

[0116] Differentiation induction can be performed in suspension culture. Here, suspension culture refers to culturing cells in a non-adherent state to a culture vessel. The culture vessel can be, but is not limited to, a culture vessel that has not been artificially treated to improve adhesion to the cells (e.g., coated with an extracellular matrix or the like), or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA), a nonionic surface-active polyol (Pluronic F-127, etc.), or a phospholipid-like structure (e.g., a water-soluble polymer (Lipidure) whose constituent unit is 2-methacryloyloxyethyl phosphorylcholine).

[0117] Regarding the culture conditions, the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0118] The culture period is not particularly limited as long as it is long enough for Foxa2-positive cells to appear, but it is desirable for the culture to be carried out for at least 7 days, more preferably 7 to 30 days, even more preferably 14 to 21 days, 14 to 20 days, 14 to 18 days, or 14 to 16 days, and most preferably 16 days.

[0119] It is desirable to culture the cells with the appropriate addition of a ROCK inhibitor. When a ROCK inhibitor is added, the cells should be cultured with the addition for at least one day, more preferably one day.

[0120] IV. Adherent Cell Populations and Mixtures Thereof By the method for producing a mixture of adherent cell populations, it is possible to produce a mixture of adherent cell populations containing adherent cell populations having the following characteristics (b1) and (b2) at a concentration of 50% or more of the total number of adherent cell populations: (b1) Neural cells in the secondary differentiation stage, (b2) Contains more than 1000 cells. Furthermore, an adherent cell population having the characteristics (b1) and (b2) above can be obtained by a method for producing an adherent cell population, which method comprises a step of separating an adherent cell population having the characteristics (b1) and (b2) above from a mixture of adherent cell populations obtained by the method for producing a mixture of adherent cell populations.

[0121] The mixture of adherent cell populations may be a mixture of three-dimensional adherent cell populations (i.e., a mixture of cell aggregates), or a mixture of two-dimensional monolayer or multilayer adherent cell populations (i.e., a cell sheet). In the case of a three-dimensional adherent cell population, the circle equivalent diameter is 100 μm to 2000 μm, preferably 100 μm to 1000 μm, more preferably 200 μm to 600 μm, and even more preferably 300 μm to 600 μm. The adherent cell population or a mixture thereof can be cultured to suppress cell death, and when the adherent cell population is cultured for 14 to 20 days, the number of cells at the end of the culture is 5% or more, preferably 8% or more, more preferably 10% or more, more preferably 15% or more, even more preferably 60% or more, and even more preferably about 100% of the number of cells at the start of the culture.

[0122] It is known that the change in cell number due to culture depends on the type of cell, and when the neural cells in the second differentiation stage are dopamine-producing neural progenitor cells, typically about 80% or more of the cells die. However, by using the production method of the present invention, when cultured for 14-20 days in the second differentiation stage, the number of cells at the end of the culture is 5% or more, preferably 8% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more of the number of cells at the start of the culture in the second differentiation stage, specifically, for example, 15% to 80%, or 15% to 50%.

[0123] On the other hand, when the neural cells in the second differentiation stage are neural stem cells, it is known that even if the cell number decreases temporarily, the cell number will usually recover after a period of 14-20 days of culture in the second differentiation stage. In the case of such neural cells, the cell number at the end of the culture is 80% or more, or approximately 100%, of the cell number at the start of the culture in the second differentiation stage.

[0124] One embodiment of the three-dimensional adherent cell population is a cell aggregate, which preferably further has the following characteristics: (b3) the envelopment index is 0.5 or more, preferably 0.7 to 1.0, and more preferably 0.8 to 1.0; (b4) the Feret diameter ratio is 0.5 or more, preferably 0.6 to 1.0, and more preferably 0.7 to 1.0; and (b5) The circularity is 0.3 or more, preferably 0.5 to 1.0, and more preferably 0.7 to 1.0.

[0125] In one preferred embodiment, the cell aggregate has the following characteristics: The equivalent circle diameter is 100 μm to 1000 μm, The envelopment degree is 0.8 to 1.0, The Feret diameter ratio is 0.7 to 1.0. Circularity is 0.7 to 1.0.

[0126] The cell aggregates further preferably have the following characteristics: In the resulting mixture of cell aggregates, the coefficient of variation for one or more indicators selected from the group consisting of circularity, minimum diameter, maximum diameter, vertical Feret diameter or horizontal Feret diameter, Feret diameter ratio, circle equivalent diameter, perimeter, area, and perimeter envelopment or area envelopment is 15% or less.

[0127] In the above-mentioned production method, the stem cells used as raw materials are not particularly limited as long as they are stem cells that can be differentiated into neural cells, but preferred examples include pluripotent stem cells, neural stem cells, mesenchymal stem cells, Muse cells, etc.

[0128] More preferably, stem cells include pluripotent stem cells, and even more preferably, ES cells or iPS cells.

[0129] The adherent cell population obtained by the above-described production method of the present invention is also within the scope of the present invention.

[0130] Furthermore, the neural progenitor cells obtained in step (2) of the above-mentioned production method are a non-adherent cell population, i.e., a mixture of isolated cells, that can be induced to differentiate into the cell aggregates or adherent cell population of the present invention by culturing them in the presence of a second differentiation-inducing factor. Such a cell mixture also falls within the scope of the present invention.

[0131] Specifically, examples include mixtures of cells that contain approximately 70% or more Corin or Lrtm1-positive cells and that can be induced to differentiate into the cell aggregates or adhesive cell populations of the present invention by culturing them in the presence of a second differentiation induction factor.

[0132] Cell aggregates of neural cells at the second differentiation stage of the present invention can be obtained by subjecting the mixture of cells to suspension culture. Furthermore, a monolayer cell sheet can be produced by subjecting the mixture of cells to adhesion culture, and such cell sheets are also within the scope of the present invention.

[0133] V. Pharmaceutical Compositions The cell aggregate or a mixture thereof, or the adherent cell population of the present invention is useful as a pharmaceutical composition for transplantation for patients suffering from a disease requiring the transplantation of nerve cells or nervous system cells that can differentiate into nerve cells, and can be used as a pharmaceutical, such as a therapeutic agent for a disease accompanied by the degeneration, damage, or dysfunction of nerve cells. In other words, a pharmaceutical composition comprising the cell aggregate or adherent cell population of the present invention and a pharmaceutically acceptable carrier also falls within the scope of the present invention.

[0134] Examples of diseases requiring neuronal transplantation or diseases accompanied by neuronal damage or dysfunction include spinal cord injury, motor neuron disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, Alzheimer's disease, retinitis pigmentosa, age-related macular degeneration, and Parkinson's syndrome, preferably Parkinson's disease.

[0135] One aspect of the present invention is a therapeutic agent for Parkinson's disease, which comprises a cell aggregate or a mixture thereof containing the dopamine-producing neural progenitor cells of the present invention, or an adhesive cell population. The number of dopamine-producing neural progenitor cells contained in the therapeutic agent for Parkinson's disease is not particularly limited as long as the graft can survive after administration. For example, the number of dopamine-producing neural progenitor cells per transplantation is 1.0 × 10 4 The amount of dopamine-producing neural progenitor cells may be increased or decreased depending on the symptoms and body size. Transplantation of dopamine-producing neural progenitor cells into the diseased area can be performed, for example, by the method described in Nature Neuroscience, 2, 1137 (1999) or N Engl J Med.; 344: 710-9 (2001).

[0136] Pharmaceutically acceptable carriers are not particularly limited as long as they are substances used to maintain cell viability, and substances well known to those skilled in the art can be used. Specifically, physiological aqueous solvents (such as physiological saline, buffer solutions, serum-free solutions, and culture media) can be used. If necessary, commonly used preservatives, stabilizers, reducing agents, isotonicity agents, and the like may be blended into pharmaceuticals containing tissues or cells to be transplanted in transplantation medicine.

[0137] The pharmaceutical composition of the present invention can be prepared as a cell suspension by suspending the cell aggregate or a mixture thereof, or the adherent cell population of the present invention in an appropriate physiological aqueous solvent. If necessary, a cryopreservative may be added to the cell suspension for cryopreservation, and the cell suspension may be thawed and washed at the time of use and then used for transplantation.

[0138] VI. Treatment method One aspect of the present invention is a method for treating a disease requiring replenishment of nervous system cells, comprising the step of transplanting the cell aggregate of the present invention or a mixture thereof, or an adherent cell population into a patient suffering from a disease requiring transplantation of nervous system cells.

[0139] In one embodiment of the present invention, the cell aggregates or mixtures thereof or adhesive cell populations containing dopaminergic neural progenitor cells obtained by the present invention can be administered to Parkinson's disease patients as a preparation, specifically a transplant preparation, by suspending the obtained dopaminergic neural progenitor cells in physiological saline or the like and transplanting the suspension into a region of the patient lacking dopaminergic neurons, such as the striatum.

[0140] VII.Transplantation Before transplantation, the cell aggregates of the present invention may be preserved in a medium necessary to maintain the viability of the cell aggregates. Examples of "medium necessary to maintain viability" include culture media, physiological buffer solutions, etc., but are not particularly limited as long as the cell population containing dopamine-producing neural progenitor cells survives, and those skilled in the art can select an appropriate medium. Examples of basal media include media prepared using media commonly used for culturing animal cells as basal media. Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, GMEM medium, Improved MEM Zinc Option medium, Neurobasal medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof, which can be used to culture animal cells.

[0141] Here, "engraftment" in this specification means that transplanted cells survive in the body for a long period of time (for example, 30 days or more, 60 days or more, 90 days or more) and adhere to and remain within the organ. As used herein, the term "functional engraftment" refers to a state in which transplanted cells have engrafted and are performing their inherent functions in vivo.

[0142] As used herein, the term "functional engraftment rate" refers to the percentage of cells that have achieved functional engraftment among transplanted cells. The functional engraftment rate of transplanted dopamine-producing neural progenitor cells can be determined, for example, by counting the number of TH-positive cells in the graft.

[0143] By transplanting the above-mentioned cell aggregates, the functional engraftment rate of the transplanted cells (including dopamine-producing neural progenitor cells and dopamine-producing neural progenitor cells induced after transplantation) is 0.1% or more, preferably 0.2% or more, more preferably 0.4% or more, even more preferably 0.5% or more, and even more preferably 0.6% or more.

[0144] In this specification, examples of mammals to be transplanted include humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, and monkeys, preferably rodents (e.g., mice and rats) or primates (e.g., humans and monkeys), and more preferably humans. [Example]

[0145] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0146] (Test 1) <Cells and cultures> The protocol for inducing differentiation of human iPS cells into dopamine-producing neural progenitor cells is shown in Figure 1. The culture conditions for the expansion culture until the initiation of differentiation induction (day -7 to 0), the first differentiation stage from the initiation of differentiation induction to day 12 (day 0 to 12), and the second differentiation stage from day 12 to 28 days after the initiation of differentiation induction (day 12 to 28) are also shown in Figure 1. Sorting was performed on day 12 after the initiation of differentiation induction.

[0147] Human iPS cells QHJ-I01 were obtained by introducing dominant-negative mutants of Oct3 / 4, Sox2, Klf4, L-MYC, LIN28, and p53 (Okita, K., et al. Stem Cells 31, 458-66, 2013) into human PBMCs using episomal vectors and were received from Professor Yamanaka et al. at Kyoto University. These iPS cells were cultured according to the method described by Miyazaki T et al., Nat Commun. 3:1236, 2012. Briefly, iPS cells were maintained in an undifferentiated maintenance medium (AK03N) containing FGF2 (bFGF) on a 6-well plate coated with Laminin 511E8.

[0148] The cell population obtained by maintaining and culturing iPS cells was dissociated using TrypLE CTS (Life Technologies) and plated at 5 × 10 cells per well on a 6-well plate coated with Laminin511E8 (iMatrix-511, Nippi). 6The cells were seeded and the medium was replaced with differentiation medium (differentiation induction started: day 0). The differentiation medium was basal medium A supplemented with 10 μM Y-27632 (WAKO), 0.1 μM LDN193189 (STEMGENT), and 0.5 μM A83-01 (WAKO). Basal medium A was GMEM (Invitrogen) containing 8% KSR (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 0.1 mM MEM non-essential amino acids (Invitrogen), and 0.1 mM 2-mercaptoethanol (WAKO). The following day (day 1), the medium was replaced with basal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM palmorphamin (WAKO), and 100 ng / mL FGF8 (WAKO). Two days later (day 3), the medium was replaced with basal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM palmorfamine, 100 ng / mL FGF8, and 3 μM CHIR99021 (WAKO). Four days later (day 7), the medium was replaced with basal medium A containing 0.1 μM LDN193189 and 3 μM CHIR99021. During this period, the medium was replaced once a day. On day 12 after the start of differentiation induction, cell sorting was performed using an anti-Corin antibody.

[0149] <Pre-sorting processing> After 5 days of culture in basal medium A containing 0.1 μM LDN193189 and 3 μM CHIR99021 (i.e., on day 12 after the start of differentiation induction), cells were dissociated using TrypLE CTS and suspended in Ca2+Mg2+-free HBSS (Invitrogen) containing 2% FBS, 30 μM Y-27632 (WAKO), 20 mM D-glucose, and 50 μg / ml penicillin / streptomycin. The above anti-Corin antibody was added, and the cells were incubated at 4°C for 20 minutes. Fluorescence-activated cell sorting (FACS) was performed to collect Corin-positive cells, which were then subjected to various analyses. The anti-Corin antibody was generated as follows: A gene sequence encoding a portion of the extracellular domain (amino acids 79-453) of the cynomolgus monkey Corin gene was introduced into 293E cells, and an extracellular domain fragment of the Corin protein was expressed and recovered. Mice were immunized with the recovered protein, and lymphocytes were then isolated and fused with myeloma cells. A clone reactive with Corin was selected from the fused cell population. The culture supernatant of this clone was fluorescently labeled and used as an anti-Corin monoclonal antibody. <Sorting> The cell sorter used for FACS was a stream-in-air model, FACSJazz™, manufactured by BD, or a microfluidic model, Gigasort, manufactured by Cytonome. Corin-positive cells were collected and subjected to various analyses. For FACSJazz™, the sorting conditions are a nozzle diameter of 100 μm, which is commonly used for neuronal cell sorting, and a sheath pressure of 29 PSI. For Gigasort, the sorting conditions are a channel inner diameter of approximately 200 μm, which is the manufacturer's standard, and a sheath pressure of 14-20 PSI.

[0150] <Suspension culture after sorting> The collected Corin-positive cells were transferred to a PrimeSurface 96U plate (Sumitomo Bakelite) at 20,000 cells / well and cultured in suspension in basal medium B (Neurobasal® medium (Invitrogen) supplemented with B-27™ Supplement minus vitamin A (Invitrogen), 20 ng / mL BDNF (WAKO), 10 ng / mL GDNF (WAKO), 200 mM Ascorbic acid (WAKO), and 0.4 mM dbcAMP (Sigma)). The initial medium was supplemented with 30 μM Y-27632, but half of the medium was replaced every three days without Y-27632. The cells were cultured in suspension for 16 days after sorting (end of differentiation induction: day 28) to induce differentiation into dopamine-producing neural progenitor cells. During this period, photographs of the floating culture cell aggregates were taken using a microscope every four days of culture. The images are shown in Figure 2.

[0151] In the cell aggregates sorted using Jazz, the size of the suspension-cultured cell aggregates remained unchanged from day 16 to day 28 (day 28) after the initiation of differentiation induction. In contrast, in the cell aggregates sorted using Gigasort, the diameter of the cell aggregates increased around day 20 (day 20) after the initiation of differentiation induction. Furthermore, on all days, cell aggregates sorted using Jazz contained more dead cells, debris, and satellite cell populations than cell aggregates sorted using Gigasort. For example, in the third aggregate from the left on day 16, small black particles (i.e., satellite cell populations) and debris surrounding the cell aggregates were observed in addition to the cell aggregates. In contrast, when using Gigasort, debris and satellite cell populations were significantly less. Observation of the cell aggregates sorted using Gigasort revealed clear boundaries of the cell aggregates, no debris layer around the cell aggregates sorted using Jazz, and no small satellite cell clusters. Furthermore, the diameter of the cell aggregates derived from Gigasort after day 24 was approximately 450 μm to 600 μm, which was larger than that of the cell aggregates derived from Jazz (the outer edge was unclear and the diameter of the cell aggregates excluding debris was approximately 350 μm to 400 μm).

[0152] <Cell counting> On day 28, the number of cell aggregates listed in Table 1 was collected from the 96-well U-bottom plate along with the medium using a micropipette and allowed to settle under its own weight. The supernatant medium was removed, and 1 mL of PBS was added. The cell aggregates were allowed to settle under their own weight. The supernatant was removed, and 1 mL of the enzyme solution from the neuronal cell dispersion kit was added and incubated in a 37°C water bath. The cells were pipetted every 10 minutes. 30 minutes after the start of incubation, 10 μL of the cell suspension was collected, mixed with 10 μL of trypan blue (Thermo Fisher Scientific), and injected into a hemocytometer. The cell number was counted under a microscope. The results are shown in the "Enzyme Solution" column in Table 1. The number of trypan blue-nonpositive cells divided by the total number of cells was calculated as the cell viability. The dispersion solution and removal solution from the neuronal cell dispersion kit were then added and the mixture was centrifuged. After removing the supernatant, the cells were resuspended in 1 mL of PBS, and 10 μL of the mixture was mixed with 10 μL of trypan blue (Thermo Fisher Scientific) and injected into a hemocytometer. The cell count was measured under a microscope. The results are shown in the "After Washing [Hemocytometer]" column of Table 1. The resuspended sample was also counted using an automated cell counter (Chemometec, NC-200). The results are shown in the "After Washing [NC-200]" column of Table 1.

[0153] [Table 1]

[0154] As shown in Table 1, the cell count per cell aggregate in the group sorted using Gigasort was approximately three times higher than that in the group sorted using Jazz, and the viability at the time of cell counting was 100% in all cases.

[0155] <Cell morphology measurement> On day 28, 48 cell aggregates were collected from the 96-well U-bottom plate along with the medium using a micropipette and transferred to a 6 cm low-attachment dish (Sumitomo Bakelite). The cell aggregates were photographed using transmitted illumination with a digital microscope (Keyence; VHX-5000), resulting in the images shown in Figure 3. Within the field of view, there were 47 cell aggregates selected by Gigasort (B), and 48 cell aggregates selected by Jazz (A).

[0156] The acquired images were analyzed using the digital microscope's built-in VHX-5000 (Ver. 1.3.2.4) software, and the circularity, minimum diameter, perimeter, Feret diameter (horizontal), Feret diameter (vertical), Feret diameter ratio, envelopment (area), maximum diameter, envelopment (perimeter), area, and equivalent circle diameter of the cell aggregates were measured (Figure 4). Figure 4 shows a graph comparing the equivalent circle diameter, envelopment, area, Feret diameter ratio, and circularity between Jazz (light gray) and Gigasort (dark gray). The standard deviation and coefficient of variation (CV) were also calculated from the acquired data. The CV values ​​are shown in Figure 5.

[0157] As shown in Figure 3, the cell aggregates sorted using Gigasort were visually larger than those sorted using Jazz. Furthermore, as shown in Figure 4, the cell aggregates sorted using Gigasort had larger equivalent circle diameters and areas than those sorted using Jazz, and the variation in the envelopment index, which indicates the presence of imperfections or protrusions and is an index of the smoothness of the periphery of the sphere, was significantly smaller.

[0158] These results indicate that by sorting cells using Gigasort, more cells can be kept alive with less damage, and the cell aggregates formed from these cells are larger, closer to perfect spheres, and have a smoother spherical shape.

[0159] Furthermore, when the coefficient of variation (CV) of each measured parameter was calculated, the CV values ​​of the cell aggregates in the group sorted using Gigasort were smaller than those of the cell aggregates in the group sorted using Jazz, for all parameters, including size (minimum diameter, perimeter, Feret diameter, Feret diameter ratio, maximum diameter, area, and equivalent circle diameter), sphericity (circularity), and surface condition (envelopment), as shown in Figure 5. In other words, the cell aggregates in the group sorted using Gigasort were found to be highly uniform.

[0160] <Flow cytometry analysis> On day 28, cells were dispersed after the addition of enzyme solution and centrifuged with the dispersion and removal solutions. The supernatant was removed, and the cells were resuspended in PBS. The cells were stained with Live / Dead reagent (Thermo Fisher Scientific), Foxa2 (R&D) / Alexa647-anti-goat (Thermo Fisher Scientific), Alexa488-Tuj1 (BD), Alexa647-Oct3 / 4 (BD), FITC-TRA2-49 (Millipore), PerCP-Cy5.5-Sox1 (BD), Alexa647-Pax6 (BD), and Alexa488-Ki67 (BD). The percentage of FOXA2-positive and TUJ1-positive cells, FOXA2-positive cells, or TUJ1-positive cells relative to the total cells in the cell suspension was calculated using a Gallios flow cytometer (Beckman Coulter) (Table 2). When either Jazz or Gigasort was used, the positive rates of the FOXA2 and / or TUJ1 markers were high, and the positive rates of the pluripotency markers OCT3 / 4 and / or TRA-2-49 were low.

[0161] [Table 2]

[0162] Table 2 shows that the positive rates of expressed genes in cells selected using Gigasort and then cultured for maturation were comparable to those in cells cultured using Jazz.

[0163] <Immunostaining> On day 28, 10 cell aggregates were collected from a 96-well U-bottom plate using a micropipette, along with the medium, and allowed to settle under its own weight. The supernatant medium was removed, and 1 mL of PBS was added and allowed to settle under its own weight. After removing the supernatant, the cell aggregates were fixed with PFA, embedded in OCT compound, and frozen. The cell aggregates were then sectioned at 10 μm using a cryostat (Leica) and mounted on glass slides. After blocking with blocking buffer (2% normal donkey serum, 0.3% Triton X100 / PBS), primary staining was performed with anti-Nurr1 mouse IgG antibody (Perseus Proteomics), anti-Foxa2 goat IgG antibody (R&D Systems), and anti-TH rabbit IgG antibody (Millipore). Secondary staining was performed with Alexa488-conjugated anti-mouse antibody, Alexa594-conjugated anti-goat antibody, Alexa647-conjugated anti-rabbit antibody, and DAPI (all Thermo Fisher Scientific). After staining, the sections were mounted with VECTASHIELD Hard set and observed under a confocal microscope (Olympus FV1200) (Fig. 6).

[0164] The cells selected using Gigasort and then cultured for maturation showed no significant difference in the expression levels of markers compared to the cells cultured using Jazz, indicating that the degree of differentiation was comparable. [Industrial Applicability]

[0165] The present invention is useful in regenerative medicine, particularly in the treatment of Parkinson's disease.

Claims

1. A mixture of cell aggregates, The present invention relates to a method for treating a tumor comprising administering to a subject therapies ... (a1) the equivalent circle diameter is 100 μm to 2000 μm, and (a2) The circularity is 0.5 or more. and a cell aggregate having: The coefficient of variation in the indexes of circularity, minimum diameter, maximum diameter, vertical Feret diameter or horizontal Feret diameter, Feret diameter ratio, circle equivalent diameter, perimeter, area, perimeter envelopment and area envelopment is 15% or less, Furthermore, the mixture has a coefficient of variation of 10% or less in the circularity and equivalent circle diameter indices.

2. The mixture of claim 1, wherein the cell aggregates contain FOXA2-positive or TUJ1-positive neural cells, and the cell aggregates contain 1,000 or more cells, and the neural cells account for 70% or more of the total number of cells.

3. The mixture according to claim 1 or 2, wherein the neural cells are dopamine-producing neural progenitor cells.

4. The mixture according to any one of claims 1 to 3, wherein the neural cells are derived from pluripotent stem cells.

5. The mixture according to any one of claims 1 to 4, wherein the cell aggregates containing 1000 or more cells comprise FOXA2-positive or TUJ1-positive nervous system cells and have at least one characteristic selected from the following: (a1) the equivalent circle diameter is 200 μm to 600 μm, and (a2) The circularity is 0.7 or more.

6. The mixture according to any one of claims 1 to 5, wherein the cell aggregate comprising 1000 or more cells, which comprises FOXA2-positive or TUJ1-positive neural cells, further has at least one characteristic selected from the following: (a3) The envelopment degree of the perimeter and / or area is 0.5 or more; and (a4) The Feret diameter ratio is 0.5 or more.

7. 7. The mixture of claim 6, wherein the cell aggregates comprising 1,000 or more cells contain FOXA2-positive or TUJ1-positive neural cells and have at least one characteristic selected from the following: (a3) The envelopment degree of the perimeter and / or area is 0.7 or more; and (a4) The Feret diameter ratio is 0.7 or more.

8. The mixture according to any one of claims 1 to 7, wherein the cell aggregates containing FOXA2-positive or TUJ1-positive nervous system cells and containing 1,000 or more cells are cell aggregates that do not have a debris layer on their surface and have clearly defined boundaries under a microscope.

9. The mixture according to any one of claims 1 to 8, comprising FOXA2-positive or TUJ1-positive neural cells, wherein the cell aggregates comprising 1,000 or more cells account for 50% or more of the total number of cell aggregates.

10. The mixture according to any one of claims 1 to 9, which can suppress cell death during culture.

11. A pharmaceutical composition for transplantation, comprising a mixture of cell aggregates according to any one of claims 1 to 10.

12. A therapeutic agent for a disease requiring replenishment of dopamine-producing neurons, comprising the mixture of cell aggregates according to any one of claims 1 to 10.

13. The therapeutic agent according to claim 12, wherein the disease is Parkinson's disease.

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