A method for producing astrocytes from pluripotent stem cells

Abstract

The present invention provides a method of producing astrocytes from pluripotent stem cells, said method comprising a step for expressing NEUROG2 gene in the pluripotent stem cells for less than three days.

Description

[Technical Field]

[0001] The present invention relates to a method for producing astrocytes from pluripotent stem cells. The present invention also relates to astrocytes obtained by said method. Furthermore, the present invention relates to a cell preparation comprising the above astrocytes. [Background technology]

[0002] Astrocytes are the most numerous cell type in the brain. Unlike nerve cells, they do not exhibit electrical activity, and for a long time, they were thought to be merely a "glue" surrounding nerve cells. However, it is now clear that astrocytes are cells that play a variety of roles indispensable for maintaining brain homeostasis, including synaptic transmission regulation, immune response, and regulation of cerebral blood flow, including the blood-brain barrier.

[0003] Efforts are already underway to induce the differentiation of astrocytes from pluripotent stem cells in order to elucidate the physiological functions of astrocytes and analyze the pathogenesis of related diseases. Several groups have also reported on methods for inducing the differentiation of astrocytes from human iPS cells.

[0004] First, a method was reported in which human pluripotent stem cells were cultured in the presence of neuronal differentiation-inducing factors to induce the differentiation of astrocytes by mimicking embryonic neurogenesis (Non-Patent Literature 1). In this method, glial cells are formed after nerve cells, so it takes several months (3-6 months) to obtain astrocytes.

[0005] Subsequently, a method for inducing astrocyte differentiation in a shorter period of time was reported by forcing the expression of transcription factors that are considered to have relatively high specificity for astrocytes (for example, NFIA, or NFI1 and SOX9) in human pluripotent stem cells (for example, Non-Patent Literature 2). Non-Patent Literature 2 discloses that astrocytes were obtained in 4 to 7 weeks by forcing the expression of NFIA or NFIA+SOX9 in human pluripotent stem cells using the CRISPER / Cas9 gene editing system.

[0006] Furthermore, it has been reported that neural stem cells induced from human pluripotent stem cells using compounds such as cytokines can be transiently expressed (specifically for 5 days) to be fate-assigned to astrocytes (Non-Patent Literature 3). Non-Patent Literature 3 discloses that astrocytes were obtained in approximately 90 days using this method.

[0007] Thus, conventional methods typically required a long period of time, ranging from four weeks to several months, to induce astrocyte differentiation. Furthermore, the differentiation efficiency was not always high, making it extremely difficult to analyze the physiological functions and pathological conditions of astrocytes, and even to utilize them as cell therapies. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2019-58192 [Non-patent literature]

[0009] [Non-Patent Document 1] Krencik R, et al., Nat. Biotechnol., 29 (6), 528-534, 2011 [Non-Patent Document 2] Xiang Li et al., Stem Cell Reports, 2018, Oct 9; 11(2), 998-1008 [Non-Patent Document 3] Tchieu J, et al., Nat Biotechnol. 37 (3), 267-275, 2019 [Overview of the project] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a method for rapidly and efficiently producing astrocytes from pluripotent stem cells. [Means for solving the problem]

[0011] The inventors of this invention conducted diligent research to solve the above problems and discovered that when the Neurogenin 2 gene (hereinafter referred to as the NEUROG2 gene) is forcibly expressed in pluripotent stem cells for a short period (specifically, for a period shorter than the period required to induce differentiation of nerve cells), a cell population consisting almost entirely of astrocytes can be obtained just 18 days after the start of such expression, thus completing the present invention.

[0012] The NEUROG2 gene is a gene that can induce differentiation of mouse pluripotent stem cells into cerebral cortical neurons with nearly 100% efficiency by forcing its expression for more than 3 days in mouse pluripotent stem cells and more than 5 days in human pluripotent stem cells (Patent Document 1). The inventors of the present invention have found that a very small amount of cells with glial-like morphology are included in the neuronal population obtained by this method. The inventors of the present invention have completed the present invention by conducting a detailed examination of the relationship between the expression period of the NEUROG2 gene and the frequency of appearance of these glial-like cells.

[0013] In other words, the present invention encompasses the following: [1] A method for producing astrocytes from pluripotent stem cells, comprising the following steps: (1) A process of expressing the NEUROG2 gene in pluripotent stem cells for less than 3 days. A method that includes this. [2] The method according to [1], wherein the expression period of the NEUROG2 gene is 8 hours or longer. [3] After step (1), (2) A step of culturing the pluripotent stem cells without inducing the expression of the NEUROG2 gene, A method of [1] or [2], including the method described in [1] or [2]. [4] The method according to any one of [1] to [3], wherein the NEUROG2 gene is a gene controlled by an inducible promoter, and step (1) is a step of activating the promoter to express NEUROG2. [5] The method according to [4], wherein the inducible promoter is a drug-responsive promoter. [6] The method according to [5], wherein step (1) is a step of culturing the pluripotent stem cells in the presence of a drug that activates the drug-responsive promoter, and step (2) is a step of culturing the pluripotent stem cells in the absence of the drug. [7] The method according to [6], wherein the culture period of step (2) is from 10 days to 20 days. [8] The method according to [6], wherein the culture period of step (2) is from 10 days to 16 days. [9] The method according to any one of [1] to [8], wherein the NEUROG2 gene is a gene introduced into the pluripotent stem cells using a transposon.

[10] The method according to [9], wherein the transposon is a piggyBac transposon.

[11] The method according to any one of [6] to

[10] , wherein step (2) is a step of culturing in the presence of a factor that promotes WNT signal transduction.

[12] The method according to

[11] , wherein the factor that promotes WNT signal transduction is one or more selected from the group consisting of BMP4 (bone morphogenetic protein 4), CNTF (Ciliary neurotrophic factor), and FBS (fetal bovine serum).

[13] The method according to any one of [1] to

[12] , wherein the pluripotent stem cells are human induced pluripotent stem cells.

[14] The method according to

[13] , wherein the human induced pluripotent stem cells are derived from a patient with a disease associated with astrocyte abnormalities.

[15] An astrocyte produced by the method according to any one of [1] to

[14] .

[16] A cell preparation comprising the astrocyte according to

[15] as an active ingredient.

Advantages of the Invention

[0014] According to the present invention, astrocytes can be produced from pluripotent stem cells in a significantly shorter period of time and with nearly 100% efficiency compared to conventional methods. Furthermore, cells obtained by the method of the present invention can be used as cell therapies for the treatment of neurodegenerative diseases. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows the protocol for the production of astrocytes according to this embodiment. In Figure 1, the arrows indicate the timeline. The culture medium conditions are shown above the timeline, and the culture plate conditions (culture size and coating conditions) are shown below the timeline. "NB" represents Neurobasal plus medium supplemented with B27 plus supplement, and "AM" represents DMEM / F12 medium supplemented with N2 supplement, BMP4, CNTF, and FBS. [Figure 2] Figure 2 shows representative images obtained on Day 12 and Day 18 of iAstrocytes prepared from four types of NEUROG2-iPSCs (HC1A, HC6B, N117E11, AD2S1) according to the method in Figure 1, by immunostaining with GFAP (green), an astrocyte marker, and nuclear staining with DAPI (gray). The image on the right of Day 12 shows a magnified view of the stained cells (indicated by the square) observed in the left image of Day 12. Similarly, the image on the right of Day 18 shows a magnified view of the stained cells (indicated by the square) observed in the left image of Day 18. [Figure 3]Figure 3 is a graph showing the results of analyzing the transcription levels of various astrocyte markers (GFAP, S100B, AQP4) and housekeeping genes (GAPDH, ACTB) at Day 12 in iAstrocytes produced from various types of NEUROG2-iPSCs using the method in Figure 1. The horizontal axis in Figure 3 represents the name of the original iPS cell line. For all marker graphs in Figure 3, the leftmost column shows the expression level in cells differentiated into nerve cells (iN) by culturing HC6B derived from healthy individuals in DOX-containing medium for 5 days. [Figure 4] Figure 4 is a graph showing the results of analyzing the expression of neuronal markers (TUBB3, MAP2, and SYN1) in the cells shown in Figure 3. [Figure 5] Figure 5 shows the experimental protocol for culturing NEUROG2-iPSCs in DOX-containing medium for 2 days (Condition A), 3 days (Condition B), 4 days (Condition C), or 5 days (Condition D). "NB" and "AM" are equivalent to those in Figure 1. [Figure 6] Figure 6 shows a typical image obtained by phase-contrast microscopy of cells obtained on Day 8 according to the protocol in Figure 5. Conditions A to D represent the same culture conditions as in Figure 5. [Figure 7] Figure 7A shows a typical image obtained by immunostaining with GFAP in iAstrocytes prepared from NEUROG2-HC1A according to the protocol in Figure 1. Figure 7B is a chart showing the intracellular Ca ion concentration (calcium oscillation) measured over time in the iAstrocytes shown in Figure 7A. In Figure 7B, the vertical axis represents the signal intensity indicating Ca ion concentration. [Figure 8] Figure 8 is a chart showing the results of simultaneously recording intracellular calcium ion concentrations at 29 different locations (multi-point simultaneous recording) in the culture system shown in Figure 7. In Figure 8, the horizontal axis represents time, and the vertical axis represents the number assigned to each measurement site (ROI: Region of Interest). [Figure 9]Figure 9 shows co-immunostaining images (A-E) of GFAP immunostaining and DAPI staining and CD44 immunostaining images (F-K) for iAstrocytes obtained from iPS cells derived from an Alexander disease patient (A266) and iPS cells derived from a healthy subject (HC1A) using the method of Figure 1. B is an enlarged image of the area surrounded by the square in A. Similarly, D and E are partial enlarged images of C. Also, H is a partial enlarged image of G, G is a partial enlarged image of F, K is a partial enlarged image of J, and J is a partial enlarged image of I. The scale bar represents 100 μm. [Figure 10] Figure 10 shows a Western blot image indicating the expression of GFAP protein in iAstrocytes. hc1 and hc6 represent astrocytes (Day 18) produced under the conditions of Example 1 from HC1A and HC6B, which are iPS cells derived from healthy subjects, respectively. The arrow indicates the band of GFAP protein. [Figure 11] Figure 11 shows the effect of the expression period of the NEUROG2 gene on the induction efficiency of iAstrocytes. hc1 and hc6 are the same as in Figure 10. The ratio of iAstrocytes (=GFAP positive) to the total number of live cells (=DAPI positive) was calculated (n = 3). The error bar indicates the SD value.

Modes for Carrying Out the Invention

[0016] <NEUROG2 gene> In the present invention, the NEUROG2 gene means the gene generally known in the art as the Neurogenin 2 gene. The official gene symbol is NEUROG2. The structure of the nucleic acid encoding the NEUROG2 protein is exemplified by the NCBI accession numbers: NM_024019 (human) or NM_009718 (mouse).

[0017] <Astrocyte> In this invention, an astrocyte can be defined as a cell expressing one or more astrocyte marker genes, such as GFAP, S100B, and Aquaporine4 (AQP4). Furthermore, cells that satisfy the above definition and exhibit calcium oscillations can also be called functionally mature astrocytes. Calcium oscillations in astrocytes are basically spontaneous fluctuations that occur within individual cells, but as they mature, lateral oscillation propagation occurs through cell-cell adhesion with surrounding astrocytes or through extracellular paracrine signaling. This lateral signal propagation (oscillation propagation) is known to form a network and play an important role in regulating neuronal activity and controlling vasoconstriction.

[0018] In the present invention, producing astrocytes means obtaining a cell population containing astrocytes, and preferably, obtaining a cell population containing 50%, 60%, 70%, 80%, 90%, or 95% or more astrocytes.

[0019] <Pluripotent stem cells> In the present invention, pluripotent stem cells are stem cells that possess pluripotency, enabling them to differentiate into a wide variety of cells present in living organisms, and also possess proliferative capacity. These include, but are not limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells).

[0020] ES cells are stem cells that possess pluripotency and the ability to proliferate through self-renewal, established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans and mice. ES cells are embryo-derived stem cells originating from the inner cell mass of the blastocyst, an embryo that has progressed from the 8-cell stage of a fertilized egg to the morula stage. They possess the ability to differentiate into any cell that makes up an adult, known as pluripotency, and the ability to proliferate through self-renewal. Human ES cell lines, such as WA01(H1) and WA09(H9), are available from the WiCell Research Institute, while KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0021] Spermatogonial stem cells are pluripotent stem cells derived from the testes and are the cells that originate from spermatogenesis. Like ES cells, these cells can be differentiated into various cell lineages and have properties such as being able to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012).

[0022] Embryonic germ cells are pluripotent cells, similar to ES cells, that are established from primordial germ cells during the embryonic stage.

[0023] Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells that possess differentiation pluripotency and the ability to proliferate through self-renewal, created by introducing specific reprogramming factors into somatic cells in the form of DNA, RNA, or protein (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International Publication WO 2007 / 069666). Examples of initialization factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1.

[0024] The use of iPS cells as pluripotent stem cells in the present invention is preferred, and the use of human iPS cells is even more preferred. Astrocytes produced from iPS cells derived from patients with diseases involving astrocyte abnormalities (e.g., Alexander disease) are useful as model cells for said disease.

[0025] As already mentioned, astrocytes produced by the method of the present invention can also be used as the active ingredient in cell therapies. From this viewpoint, cell therapies containing astrocytes produced from iPS cells of healthy individuals, or astrocytes produced from iPS cells modified to express therapeutically effective proteins, are particularly preferred. Such cell therapies will be described in more detail in the <Cell Therapies> section below.

[0026] <Process (1)> Step (1) of the present invention is a step of directing pluripotent stem cells toward differentiation into astrocytes. In step (1), the NEUROG2 gene is expressed in pluripotent stem cells for a period shorter than the period required for directing differentiation into nerve cells, thereby directing differentiation into astrocytes. The period for which the NEUROG2 gene is expressed may be less than 3 days, preferably 8 hours or more and less than 3 days, more preferably 8 hours or more and 60 hours or less, even more preferably 24 hours or more and 60 hours or less, particularly preferably 36 hours or more and 60 hours or less, and most preferably 36 hours or more and 48 hours or less.

[0027] The forced expression of the NEUROG2 gene can be carried out using methods that are already known and is not particularly limited. The NEUROG2 gene to be forced expression may be an endogenous NEUROG2 gene or an exogenous NEUROG2 gene, but an exogenous gene is preferred due to the ease of expression regulation. In one embodiment, for example, a vector such as a virus, plasmid, or artificial chromosome containing nucleic acid encoding NEUROG2 may be introduced into pluripotent stem cells using methods such as lipofection, liposomes, or microinjection to induce expression. Alternatively, in step (1), cells into which an exogenous NEUROG2 gene has already been introduced may be used.

[0028] Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Furthermore, mammalian cell plasmids can be used as plasmid vectors.

[0029] In another embodiment, the vector may have transposon sequences before and after the expression cassette (a gene expression unit including a promoter, gene sequence, and terminator) to excise, as necessary, the sequence encoding the NEUROG2 gene incorporated into the chromosome. The transposon sequence is not particularly limited, but piggyBac is a preferred example.

[0030] These vectors may include regulatory sequences such as promoters, enhancers, ribosome-binding sequences, terminators, and polyadenylation sites to enable the expression of the NEUROG2 gene.

[0031] To express the NEUROG2 gene in pluripotent stem cells, the pluripotent stem cells may contain a nucleic acid encoding NEUROG2 functionally conjugated to an inducible promoter. This allows the NEUROG2 gene to be expressed in the pluripotent stem cells at a desired time. Examples of such inducible promoters include drug-responsive promoters, and a preferred example is the Tet-on promoter (a CMV minimal promoter having a tetocycline response element (TRE) consisting of seven consecutive tetO sequences), which is a type of tetracycline-responsive promoter. This promoter is activated by the supply of tetracycline or a derivative thereof under the expression of a reverse tetracycline-regulating transactivator (rtTA: a fusion protein of reverse tetR (rTetR) and VP16AD). Therefore, when inducing the expression of the gene using a tetracycline-responsive promoter, it is preferable to use a vector that also expresses the activator. Doxycycline (DOX) can be suitably used as a derivative of tetracycline.

[0032] Other expression induction systems using drug-responsive promoters include those using estrogen-responsive promoters (e.g., WO2006 / 129735), the RheoSwitch mammalian inducible expression system using a promoter induced by RSL1 (New England Biolabs), the Q-mate system using a promoter induced by cumate (Krackeler Scientific), or the Cumate-inducible expression system (National Research Council (NRC)), and the GenoStat-inducible expression system using a promoter with an ecdysone-responsive sequence (Upstate cell signaling solutions).

[0033] When using an expression vector equipped with an expression induction system based on a drug-responsive promoter as described above (i.e., a drug-responsive induction vector), the expression of NEUROG2 can be maintained by continuously adding a drug capable of inducing activation of the promoter (for example, tetracycline or DOX in the case of a vector containing the tetracycline-responsive promoter) to the culture medium for a desired period of time. The expression of the gene can then be stopped by removing the drug from the culture medium (for example, by replacing it with a medium that does not contain the drug).

[0034] Therefore, when using the Tet-on promoter as a drug-responsive promoter, NEUROG2 can be expressed in pluripotent stem cells by adding DOX to the culture medium. The amount of DOX added here is not particularly limited, but is between 0.01 and 100 μg / ml, preferably between 0.1 and 10 μg / ml, and more preferably between 1 and 5 μg / ml.

[0035] In the present invention, the culture medium used in step (1) may be a basic medium only, or a basic medium to which neurotrophic factors have been added. Such basic media include Neurobasal Medium (Life Technologies), Glasgow's Minimal Essential Medium (GMEM), 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, and mixed media of these.

[0036] The basic culture medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as B27 supplement (Invitrogen), B27Plus supplement (Invitrogen), Knockout Serum Replacement (KSR) (serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenic acid, progesterone, and putrescine.

[0037] Of these, Neurobasal Medium containing B27 supplement, or a mixed medium of DMEM and F12 containing insulin, apotransferrin, selenic acid, progesterone, and putrescine can be suitably used as the basic medium.

[0038] The culture temperature in step (1) of the present invention 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 a CO2 concentration of preferably about 2 to 5%.

[0039] In step (1), it is preferable that the culture vessel is covered with an extracellular matrix, as in the standard method. The extracellular matrix that can be used in step (1) is not particularly limited as long as it is commonly used in adherent culture, and examples include poly-L-lysine, Matrigel, vivonectin and vivonectin-derived synthetic peptides, human type I collagen-like recombinant peptides, gelatin, laminin, collagen, and fibronectin.

[0040] <Process (2)> Step (2) is the step in which the cells that have been directed to differentiate into astrocytes in step (1) differentiate into astrocytes. Specifically, it is the step of culturing the cells without substantially expressing NEUROG2 (for example, without inducing NEUROG2 expression). The culturing in step (2) may be carried out either in vivo or in vitro. In vitro culturing is preferred because it makes it easier to control the differentiation rate.

[0041] For step (2), a culture medium containing factors that promote WNT signaling can be suitably used. Since factors that promote WNT signaling promote differentiation into astrocytes, the efficiency of astrocyte production can be increased by adding such factors. Examples of factors that promote WNT signaling that can be suitably used in the present invention include, but are not limited to, BMP4 (bone morphogenetic protein 4), CNTF (Ciliary neurotrophic factor), and FBS (fetal bovine serum).

[0042] The culture medium in step (2) may contain serum, and may also contain one or more serum substitutes, such as Knockout Serum Replacement (KSR) (a serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. In the present invention, it is a preferred embodiment to include a serum substitute, preferably a serum substitute for nerve cells such as an N2 supplement.

[0043] Furthermore, the culture medium in step (2) may also contain one or more substances such as Glutamax (Invitrogen), lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenic acid, progesterone, and putrescine.

[0044] When performing step (2) in vitro, subculturing may be performed to achieve a low cell density. An example of such a low cell density is 2 × 10⁶ per 48-well plate. 4 ~10×10 4 cells / cm 2 The cell density may be 5 × 10 per 48-well plate, preferably 5 × 10 4 cells / cm 2 This is the cell density. Culture at a low cell density is preferable because it can reduce the likelihood that nerve cells will adhere to astrocytes and survive (even in a substrate that is normally incompatible with adhesion).

[0045] In addition, when performing step (2) in vitro, the coating of the culture vessel may be changed in stages from one with high affinity to the cells to one with low affinity, and ultimately the vessel may be left uncoated. For example, initially, a PMSC (Poly L lysine, Matrigel, Synthemax (vitronectin-derived synthetic peptide, Corning), and Cellnest (human type I collagen-like recombinant peptide, Fujifilm)) coated culture vessel may be used (for example, Day 1 to Day 5), then a gelatin coated culture vessel may be used (Day 6 to Day 12), and thereafter an uncoated culture vessel may be used. An uncoated culture vessel may be used from Day 11, Day 12, Day 13, Day 14 onwards.

[0046] The incubation period in step (2) is preferably 10 to 20 days, and more preferably 10 to 16 days.

[0047] The culture temperature in step (2) is not particularly limited, but is about 30-40°C, preferably about 37°C. Furthermore, it is preferable that the culture is carried out in an atmosphere of CO2-containing air, and the CO2 concentration may be about 2-5%.

[0048] Furthermore, even if NEUROG2 is expressed for less than 3 days, preferably 2 days, in step (1) and then administered into vivo without culturing in a culture medium (in vivo configuration), it is believed that astrocyte generation will occur in vivo. Therefore, such in vivo configurations are also included in the present invention.

[0049] <iastrocyte> Furthermore, the present invention provides astrocytes produced by the method of the present invention. These astrocytes may be called induced astrocytes (iAstrocytes) to distinguish them from naturally occurring astrocytes. Similarly, nerve cells differentiated from pluripotent stem cells may be called induced neurons (iNs).

[0050] The iAstrocyte according to the present invention differs from naturally occurring astrocytes in that it is genetically modified. Preferably, it is an iAstrocyte produced from human iPS cells.

[0051] <Cell Therapy> Furthermore, the present invention provides a cell preparation containing astrocytes (iAstrocytes) produced by the method of the present invention as an active ingredient. The cell preparation of the present invention containing iAstrocytes is effective in treating neurodegenerative diseases, preferably neurodegenerative diseases accompanied by astrocyte abnormalities. Neurodegenerative diseases accompanied by astrocyte abnormalities include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, multiple system atrophy, progressive supranuclear palsy, frontotemporal dementia, Huntington's disease, spastic paraplegia, and other primary degenerative neuronal diseases, as well as Alexander disease and other primary degenerative astrocyte diseases. Of these, the cell preparation is particularly suitable for the treatment of Alexander disease, Alzheimer's disease, and ALS.

[0052] Alexander disease is a rare hereditary neurodegenerative disease characterized by the presence of Rosenthal fibers composed of glial fibrillary acidic protein (GFAP), αB-crystallin, heat shock protein, etc. within astrocytes. Abnormal aggregates consisting of mutant GFAP or overexpressed GFAP are thought to be involved in the pathogenesis of Alexander disease. Clinically, it is classified into a cerebral dominant type (type 1), a medulla oblongata / spinal cord dominant type (type 2), and an intermediate type (type 3) based on clinical symptoms and MRI findings. Missense mutations or deletion / insertion of several bases of GFAP are observed in 97% of Alexander disease cases, and in recent years, genetic testing has been used as a definitive diagnostic method. Although research on elucidating the pathogenesis of Alexander disease is ongoing, it is still not sufficient.

[0053] The cell preparation of the present invention can be used as a transplantation therapeutic agent for treating the neurodegenerative disease. iAstrocyte is manufactured as a parenteral preparation such as an injection, a suspension, a drip infusion, etc. by mixing it with a pharmaceutically acceptable carrier according to conventional means. Examples of pharmaceutically acceptable carriers that can be included in the parenteral preparation include aqueous liquids for injection such as physiological saline, isotonic solutions containing glucose and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride solution, etc.). The preparation may be formulated with, for example, a buffer (e.g., phosphate buffer, sodium acetate buffer), a pain reliever (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc. When formulating iAstrocyte as an aqueous suspension, iAstrocyte may be suspended in the above aqueous liquid to a concentration of 1×10 5 ~1×10 8 cells / ml, preferably 1×10 6 ~1×10 8 cells / ml. Transplantation of iAstrocyte can be performed by injecting the above suspension into the lesion site such as the cerebrum, medulla oblongata, spinal cord, etc. The number of cells to be administered can be appropriately changed depending on the degree of the lesion, etc. For example, in the case of a patient with human Alexander disease, 1×10 5 ~1×10 8 Cells, preferably 1 × 10 6 ~1 × 10 8 Cells can be administered.

[0054] Furthermore, in a different embodiment of the present invention, the cell preparation may contain cells immediately after step (1) as an active ingredient. By injecting the cell preparation into a lesion site such as the cerebrum, medulla oblongata, or spinal cord according to the method described above, it is expected that the cells immediately after step (1) will differentiate into astrocytes in vivo and produce a therapeutic effect.

[0055] The above transplantation treatment can be used in combination with drug therapy. For example, if the target disease is Alexander disease, it can be used in combination with antiepileptic drugs currently used as symptomatic treatment for Alexander disease. Such concomitant drugs can be used, for example, in the dosage and route of administration typically used for the treatment of Alexander disease.

[0056] The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples. [Examples]

[0057] First, the cells and main techniques used in this embodiment will be described. <cell> In this example, as pluripotent stem cells to be induced to differentiate into astrocytes, we used iPS cell lines (hereinafter referred to as NEUROG2-iPSCs) in which the human NEUROG2 gene, regulated by the Tet-on promoter, was introduced using a piggyBac transposon into iPS cells established from healthy individuals or patients. In the NEUROG2-iPSCs, the exogenous NEUROG2 gene is inserted into the genome. The piggyBac transposon was used because the construct used in Patent Document 1 was available, and we have confirmed that similar results can be obtained with transient expression systems that are not inserted into the genome. Table 1 shows the iPS cell lines used to create NEUROG2-iPSCs and their origins.

[0058] [Table 1]

[0059] In this example, unless otherwise specified, NEUROG2-iPSCs were cultured to produce astrocytes according to the conditions shown in Figure 1. In this example, the day on which differentiation induction into astrocytes began, i.e., the day on which NEUROG2 expression induction began, is designated as Day 0, and subsequent time elapsed is sometimes expressed in Days.

[0060] Unless otherwise specified, NEUROG2 expression induction was performed by adding DOX to the culture medium at a concentration of 2 μg / ml for two days, from Day 0 to Day 2. During the NEUROG2 expression induction period, NB medium (Neurobasal plus medium containing 0.5% B27 plus supplement) was used, and thereafter, AM medium (DMEM / F12 medium containing 1% N2 supplement, 10 ng / ml BMP4, 10 ng / ml CNTF, and 2% FBS) was used. In addition, to mitigate damage from medium changes, Y-27632 was added at a concentration of 10 μM for a specified period.

[0061] For culture plates, PMSC-coated 48-well or 96-well plates were used from Day 0 to Day 5, and gelatin-coated 6-well plates were used from Day 5 to Day 12.

[0062] On Day 0, NEUROG2-iPSC was administered in 30 x 10 units per 48-well plate. 4 cells / cm 2 The seeds were sown in this manner, and the first subculturing was performed at the time of changing the type of culture plate (Day 5). On Day 5, 5 × 10 per 48-well plate were used. 4 cells / cm 2 The seeds were seeded in this manner. Subsequently, in addition to changing the type of culture plate (Day 12), subculturing was performed as needed according to the rate of cell proliferation.

[0063] Cells were collected for assay purposes from Day 12 to Day 18.

[0064] Example 1: Production of astrocytes NEUROG2-iPSCs were prepared from three iPS cell lines derived from healthy individuals (HC1A, HC6B, N117E11) and one iPS cell line derived from a patient with familial Alzheimer's disease (AD2S1). These were cultured under the conditions described in Figure 1 and differentiated into astrocytes. Immunostaining for GFAP (green) and nuclear staining (DAPI, gray) were performed on Day 12 or Day 18. The results are shown in Figure 2. In all cases of NEUROG2-iPSCs used, only some cells were GFAP-positive on Day 12, but almost all cells became GFAP-positive (over 95%) on Day 18. This revealed that expressing NEUROG2 in pluripotent stem cells for only two days leads to rapid differentiation into astrocytes approximately 10 to 16 days later. Furthermore, GFAP-positive cells on Day 18 showed thicker cytoplasm and extended multiple protrusions compared to GFAP-positive cells on Day 12, indicating morphological maturation as astrocytes. Therefore, when NEUROG2 is expressed in pluripotent stem cells for a period shorter than that required for neuronal differentiation induction (for example, 2 days), it was shown that almost all cells differentiated into morphologically mature astrocytes in a remarkably short period of just 18 days from the start of NEUROG2 expression induction, which is significantly shorter than conventional methods. In this example, astrocytes obtained by forced expression of NEUROG2 may hereafter be referred to as "iAstrocytes".

[0065] Next, iAstrocytes were produced from NEUROG2-iPSCs (Alex1-Alex3) derived from Alexander disease patients using the same method. Furthermore, iNs were produced from HC6B NEUROG2-iPSCs as a negative control. These iNs were produced by extending the culture period in DOX-containing medium from 2 days to 5 days (as shown in Figure 1), and then culturing in DOX-free NB medium (instead of AM medium) thereafter. Each cell was harvested on Day 12, and the transcription levels of astrocyte marker genes (GFAP, S100B, and AQP4) and housekeeping genes (GAPDH and ACTB) were analyzed. The results are shown in Figure 3, and the transcription levels of neuronal marker genes (TUBB3, MAP2, and SYN1) were analyzed. In Figures 3 and 4, the numerical values ​​of the expression levels on the vertical axis are calibrated with the GAPDH expression level in Figure 3 set to 1.

[0066] As shown in Figure 3, no astrocyte markers were detected in iN cells, but GFAP and S100B were expressed in iAstrocytes from all cell lines. AQP4 expression levels varied considerably between cells, and in particular, expression was not detected in some iAstrocytes derived from Alexander disease patients (Alex12, Alex3). This result may be related to the pathogenesis of Alexander disease, which involves astrocyte dysfunction. On the other hand, there were no significant differences in GAPDH and ACTB expression levels between iN cells and iAstrocytes, nor between iAstrocytes from different cell lines.

[0067] In contrast, neuronal cell markers were very clearly expressed in iN cells, but were at very low levels (TUBB3, MAP2) or below the detection limit (SYN1) in iAstrocytes (Figure 4).

[0068] These results demonstrate that forced expression of NEUROG2 can induce astrocyte differentiation in as little as 12-18 days, not only in pluripotent stem cells derived from healthy individuals, but also in pluripotent stem cells derived from patients with neurodegenerative diseases caused by neurodegeneration and glial degeneration.

[0069] Example 2: Investigation of NEUROG2 forced expression period (1) We investigated the appropriate duration of forced expression of NEUROG2 to induce differentiation of pluripotent stem cells into astrocytes rather than neurons. NEUROG2-iPSCs from healthy individuals (HC1A, HC6B) and from patients with familial Alzheimer's disease (AD2S1, AD2EL) were cultured in DOX-containing medium for 2, 3, 4, and 5 days, up to Day 8 (Figure 5, Condition AD). Phase-contrast microscopy observations were performed on Day 8, and typical phase-contrast microscopy images obtained under each condition are shown in Figure 6.

[0070] In Condition A, cells cultured for 2 days in Dox-containing medium showed almost no iNs (cells extending neurites). In contrast, in Condition B, where cells were cultured for 3 days in Dox-containing medium, iNs were observed, and in Condition C, the number of iNs increased further. In Condition D, almost all cells were iNs with long neurites extended.

[0071] Therefore, it was shown that if the forced expression period of NEUROG2 is less than 3 days, the proportion of cells that differentiate into iN is sufficiently small, and even more so, at 2 days, cells do not differentiate into iN at all.

[0072] Therefore, it was found that the optimal forced expression period for NEUROG2 to induce differentiation into astrocytes is less than 3 days, and most preferably around 2 days.

[0073] Example 3: Investigation of calcium oscillation Next, we investigated the functionality of astrocytes produced by the method according to the present invention. Functionally mature astrocytes exhibit a phenomenon in which the Ca ion concentration within the astrocyte changes autonomously and regularly (calcium oscillation), and it has been shown that they regulate nearby synaptic transmission by releasing neurotransmitters and regulators such as glutamate, ATP, and serine in a calcium concentration-dependent manner. Therefore, we analyzed the presence or absence of calcium oscillation as an indicator of the functional maturity of iAstrocytes.

[0074] Day 18 iAstrocytes (Figure 7A) cultured in DOX medium for 2 days were subjected to Fluo-8AM uptake, and intracellular Ca ion concentration was continuously recorded as fluctuations in fluorescence intensity and measured over time. The results are shown in Figure 7B. As shown in Figure 7B, the Ca ion concentration within the iAstrocytes fluctuated autonomously and regularly, confirming that calcium oscillation was occurring.

[0075] Next, Figure 8 shows the results of simultaneous recording of calcium oscillations at multiple points. The vertical axis in Figure 8 represents the number assigned to each measurement site (ROI: Region of Interest). From these results, it can be seen that calcium oscillations occur at various locations in the iAstrocyte culture system. Furthermore, at measurement sites 5, 6, 8, 9, 11-14, and 16 (especially 5, 6, and 12), a tendency was observed for the timing of the increase in calcium concentration (vertical red dotted line in Figure 8) to coincide over approximately 190 seconds from the start of measurement.

[0076] As mentioned in the section on <Astrocytes> above, astrocytes are known to form networks through the propagation of lateral calcium oscillations. As described above, synchronized calcium oscillations were observed among multiple cells in iAstrocytes produced by this differentiation induction method. This result indicates that an information transmission network is formed among iAstrocytes.

[0077] Therefore, it was revealed that iAstrocytes produced by the method according to the present invention can spontaneously perform calcium oscillations and can also form intercellular networks via such oscillations. This indicates that morphologically and functionally mature iAstrocytes can be produced by the method according to the present invention.

[0078] Example 4: Production of a cell model of neurodegenerative disease with astrocyte abnormalities The presence or absence of GFAP-positive aggregates was investigated in iAstrocytes produced from iPS cells (A266 strain) derived from Alexander disease patients. Day 18 iAstrocytes produced from two types of NEUROG2-iPSCs (A226, HC1A) using the method in Example 1 were immunostained for GFAP or CD44. The results are shown in Figure 9.

[0079] iAstrocytes produced from iPSCs (HC1A) derived from healthy individuals did not show GFAP-positive intracellular aggregates (Figure 9A, B). In contrast, iAstrocytes produced from iPSCs (A226) derived from Alexander disease patients frequently showed GFAP-positive intracellular aggregates (Figure 9C-E). This indicates that astrocytes produced from iPSCs derived from Alexander disease patients using the method according to the present invention spontaneously and frequently produce GFAP-positive intracellular aggregates characteristic of Alexander disease patients. Since these GFAP-positive intracellular aggregates are thought to contribute significantly to the development of Alexander disease, astrocytes produced using the method according to the present invention are considered to be a potential cell model for Alexander disease.

[0080] CD44 is a surface antigen marker expressed in glial progenitor cells and astrocytes. CD44 was found to be distributed on the membrane surface in both iAstrocytes produced from healthy individuals (HC1A) (Figure 9F-H) and iAstrocytes produced from Alexander disease patients (A226) (Figure 9I-K). This result suggests that astrocyte lineage differentiation is advanced in both iAstrocytes from healthy individuals and those from Alexander disease patients.

[0081] Therefore, it has been demonstrated that the manufacturing method according to the present invention can produce model cells for diseases involving astrocyte abnormalities (e.g., Alexander disease).

[0082] Example 5: Expression of GFAP protein in iAstrocyte Western blot analysis also confirmed that iAstrocytes express GFAP protein. Proteins were extracted (0.5 μg / μL) from iAstrocytes produced from healthy donor-derived iPS cells HC1A and HC6B under the conditions of Example 1 at Day 18, according to a standard procedure, and analyzed using a Wes glass capillary electrophoresis system (Protein simple). The antibody used was prepared using full-length human GFAP recombinant protein as the immunogen. The results are shown in Figure 10. All iAstrocytes were confirmed to express GFAP.

[0083] Example 6: Investigation of NEUROG2 forced expression period (2) We investigated the required duration of forced expression of NEUROG2 to induce differentiation of pluripotent stem cells into astrocytes. iAstrocytes were induced from HC1A and HC6B iPS cells derived from healthy individuals, using the same method as in Example 2, by varying the culture period in DOX-containing medium from 8 to 72 hours. On Day 18, GFAP immunostaining and DAPI nuclear staining were performed, and the ratio of iAstrocytes (=GFAP positive) to the total number of viable cells (=DAPI positive) was calculated. The results are shown in Figure 11. It was revealed that astrocyte induction is possible even with 8 hours of expression. Approximately 48 hours was optimal, and it was observed that induction efficiency may decrease beyond 48 hours. It was suggested that induction efficiency was particularly high between 36 and 60 hours. The GFAP positivity rate was virtually 0% when NEUROG2 was not forcibly expressed (data not shown). [Industrial applicability]

[0084] According to the method of the present invention, morphologically and functionally mature astrocytes can be produced from pluripotent stem cells in a significantly shorter period of time and with nearly 100% efficiency compared to conventional methods. Astrocytes produced by the method of the present invention are useful as model cells for analyzing the physiological functions and pathological roles of astrocytes, and as therapeutic agents (cell preparations) for diseases involving astrocyte abnormalities.

[0085] This application is based on Japanese Patent Application No. 2020-190575, filed in Japan on November 16, 2020, the entirety of which is incorporated herein by reference.< / iastrocyte>

Claims

1. A method for producing astrocytes from pluripotent stem cells, comprising the following steps: (1) A process of expressing the NEUROG2 gene in pluripotent stem cells for more than 8 hours but less than 3 days. (2) A step of culturing the pluripotent stem cells for 10 days or more in the presence of a factor that promotes WNT signaling, without inducing the expression of the NEUROG2 gene. A method that includes this.

2. The method according to claim 1, wherein the NEUROG2 gene is a gene controlled by an inducible promoter, and step (1) is a step of activating the promoter to express NEUROG2.

3. The method according to claim 2, wherein the inductive promoter is a drug-responsive promoter.

4. The method according to claim 3, wherein step (1) is a step of culturing the pluripotent stem cells in the presence of a drug that activates the drug-responsive promoter, and step (2) is a step of culturing the pluripotent stem cells in the absence of the drug.

5. The method according to claim 4, wherein the culture period in step (2) is 10 to 20 days.

6. The method according to claim 4, wherein the culture period in step (2) is 10 to 16 days.

7. The method according to any one of claims 1 to 6, wherein the NEUROG2 gene is a gene introduced into the pluripotent stem cell using a transposon.

8. The method according to claim 7, wherein the transposon is a piggyBac transposon.

9. The method according to any one of claims 1 to 8, wherein the factor that promotes WNT signaling is one or more selected from the group consisting of BMP4 (bone morphogenetic protein 4), CNTF (Ciliary neurotrophic factor), and FBS (fetal bovine serum).

10. The method according to any one of claims 1 to 9, wherein the pluripotent stem cells are human induced pluripotent stem cells.

11. The method according to claim 10, wherein the human induced pluripotent stem cells are derived from a patient with a disease involving astrocyte abnormalities.

12. A method for producing a cell preparation, comprising the step of formulating astrocytes produced by the method according to any one of claims 1 to 11.

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