WO2022102787A1

WO2022102787A1 - Method of producing astrocytes from pluripotent stem cells

Info

Publication number: WO2022102787A1
Application number: PCT/JP2021/042096
Authority: WO
Inventors: 治久井上; 孝之近藤
Original assignee: 国立大学法人京都大学
Priority date: 2020-11-16
Filing date: 2021-11-16
Publication date: 2022-05-19
Also published as: JPWO2022102787A1

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

How to make astrocytes from pluripotent stem cells

The present invention relates to a method for producing astrocytes from pluripotent stem cells. The present invention also relates to astrocytes obtained by this method. Furthermore, the present invention relates to a cell preparation containing the above astrocytes.

Astrocytes are the cell type with the largest number of cells in the brain. Unlike nerve cells, it does not show electrical activity, so it has long been thought to be a "glue" -like entity that only fills the area around nerve cells. However, it is now clear that astrocytes are cells that play a variety of functions essential for maintaining homeostasis of the brain, such as regulation of synaptic transmission, immune response, and regulation of cerebral blood flow including the blood-brain barrier. ing.

Efforts have already been made to induce differentiation of astrocytes from pluripotent stem cells in order to elucidate the physiological functions of astrocytes and analyze the pathophysiology of related diseases. There are also reports from multiple groups on how to induce astrocytes differentiation from human iPS cells.

First, a method of inducing astrocyte differentiation by culturing human pluripotent stem cells in the presence of a nerve differentiation-inducing factor and mimicking neurogenesis during the embryonic period was reported (Non-Patent Document 1). In this method, it takes several months (3 to 6 months) to obtain astrocytes because glial cells are generated after nerve cells are generated.

Subsequently, human pluripotent stem cells are forcibly expressed with transcription factors (eg, NFIA, or NFI1 and SOX9) that are considered to be relatively specific for astrocytes, thereby differentiating astrocytes in a shorter period of time. A method for inducing has been reported (for example, Non-Patent Document 2). Non-Patent Document 2 discloses that astrocytes were obtained in 4 to 7 weeks by forcibly expressing NFIA or NFIA + SOX9 in human pluripotent stem cells using a CRISPER / Cas9 gene editing system.

Furthermore, by transiently expressing the NFIA gene in neural stem cells derived from human pluripotent stem cells using compounds such as cytokines (specifically, for 5 days), it is possible to fate astrocytes. Reported (Non-Patent Document 3). Non-Patent Document 3 discloses that astrocytes were obtained in about 90 days by this method.

Thus, with the conventional methods, it generally took a long period of 4 weeks to several months to induce the differentiation of astrocytes. Furthermore, the differentiation efficiency is not always high, which makes it extremely difficult to analyze the physiological function and pathological condition of astrocytes, and to use them as cell preparations.

Japanese Unexamined Patent Publication No. 2019-58192

An object of the present invention is to provide a method for rapidly and highly efficiently producing astrocytes from pluripotent stem cells.

As a result of diligent studies to solve the above problems, the present inventors are required to apply the Neurogenin 2 gene (hereinafter referred to as NEUROG2 gene) to pluripotent stem cells for a short period of time (specifically, to induce differentiation of nerve cells). It was found that when forced expression was performed only for a period shorter than the period), a cell population consisting approximately of astrocytes was obtained only 18 days after the start of the expression, and the present invention was completed.

The NEUROG2 gene is a gene that can induce differentiation of the cells into cerebral cortical neurons with almost 100% efficiency by forcibly expressing them in mouse pluripotent stem cells for 3 days or longer and in human pluripotent stem cells for 5 days or longer. There is (Patent Document 1). The present inventor has found that the nerve cell population obtained by this method contains a very small amount of cells having a glial-like morphology. Then, the present invention was completed by examining in detail the relationship between the expression period of the NEUROG2 gene and the appearance frequency of the glial-like cells.

That is, the present invention includes the following.
[1] A method for producing astrocytes from pluripotent stem cells, wherein the following steps:
(1) A step of expressing the NEUROG2 gene in pluripotent stem cells for less than 3 days.
How to include.
[2] The method according to [1], wherein the expression period of the NEUROG2 gene is 8 hours or more.
[3] After the step (1),
(2) A step of culturing the pluripotent stem cells without inducing the expression of the NEUROG2 gene.
The method according to [1] or [2], which comprises.
[4] Any of [1] to [3], wherein the NEUROG2 gene is a gene regulated by an inducible promoter, and the step (1) is a step of activating the promoter to express NEUROG2. The method described.
[5] The method according to [4], wherein the inducible promoter is a drug-responsive promoter.
[6] The step (1) is a step of culturing the pluripotent stem cells in the presence of a drug that activates the drug-responsive promoter, and the step (2) is a step of culturing the pluripotent stem cells in the absence of the drug. The method according to [5], which is a step of culturing the pluripotent stem cells.
[7] The method according to [6], wherein the culture period of the step (2) is 10 to 20 days.
[8] The method according to [6], wherein the culture period of the step (2) is 10 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 cell 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 signaling.
[12] 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) [11]. ] The method described in.
[13] The method according to any one of [1] to [12], wherein the pluripotent stem cell is a human induced pluripotent stem cell.
[14] The method according to [13], wherein the human induced pluripotent stem cells are derived from a disease patient with an astrocyte abnormality.
[15] The astrocyte produced by the method according to any one of [1] to [14].
[16] A cell preparation containing the astrocyte according to [15] as an active ingredient.

According to the present invention, astrocytes can be produced from pluripotent stem cells in a significantly shorter period than before and with high efficiency close to 100%. The cells obtained by the method of the present invention can also be used as a cell preparation for the treatment of neurodegenerative diseases.

FIG. 1 shows a protocol for a method for producing astrocytes according to this embodiment. In FIG. 1, the arrow indicates the timeline. The 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. FIG. 2 shows immunostaining (green) of astrocyte marker GFAP on Day 12 and Day 18 against iAstrocytes produced from four types of NEUROG2-iPSC (HC1A, HC6B, N117E11, AD2S1) according to the method of FIG. A typical image obtained by performing nuclear staining (gray) with DAPI is shown. The image on the right side of Day 12 is a magnified view of the stained cells (shown in a square) observed in the image on the left side of Day 12. Similarly, the image on the right side of Day 18 is an enlarged view of the stained cells (shown in a square) observed in the image on the left side of Day 18. FIG. 3 shows various astrocyte markers (GFAP, S100B, AQP4) and housekeeping genes (GAPDH,) for iAstrocytes produced from various types of NEUROG2-iPSC using the method of FIG. It is a graph which shows the result of having analyzed the transcription level of ACTB). The horizontal axis in FIG. 3 represents the original iPS cell line name. For the graphs of all the markers in FIG. 3, the leftmost shows the expression level in cells in which HC6B derived from a healthy person was cultured in a DOX-containing medium for 5 days to induce differentiation into nerve cells (iN). FIG. 4 is a graph showing the results of analysis of the expression of nerve cell markers (TUBB3, MAP2, and SYN1) in the cells of FIG. FIG. 5 is a diagram showing a protocol for an experiment in which NEUROG2-iPSC is cultured in a medium containing DOX for 2 days (Condition A), 3 days (Condition B), 4 days (Condition C), or 5 days (Condition D). .. “NB” and “AM” are synonymous with FIG. FIG. 6 represents a typical image obtained by phase contrast microscopy for Day 8 cells obtained according to the protocol of FIG. Conditions A to D show the same culture conditions as in FIG. FIG. 7A represents a typical image obtained by immunostaining GFAP on iAstrocytes prepared from NEUROG2-HC1A according to the protocol of FIG. FIG. 7B is a chart showing the results (calcium oscillation) of measuring the intracellular Ca ion concentration over time in the iAstrocyte shown in FIG. 7A. In FIG. 7B, the vertical axis is the signal intensity indicating the Ca ion concentration. FIG. 8 is a chart showing the results of simultaneously recording the intracellular calcium ion concentration at 29 sites different from those in FIG. 7 (simultaneous recording at multiple points) in the culture system of FIG. 7. In FIG. 8, the horizontal axis represents time, and the vertical axis indicates the number assigned to each measurement site (ROI: Region of Interests). FIG. 9 shows a co-immunostochemical image of GFAP immunostaining and DAPI staining for iAstrocytes obtained from iPS cells (A266) derived from Alexander disease patients and iPS cells (HC1A) derived from healthy subjects using the method shown in FIG. Represents A-E) and CD44 immunostaining image (F-K). B is an enlarged image of the area surrounded by a square in A. Similarly, D and E are partially enlarged images of C. Also, H is a partially enlarged image of G, G is a partially enlarged image of F, K is a partially enlarged image of J, and J is a partially enlarged image of I. The scale bar represents 100 μm. FIG. 10 shows a Western blot showing the expression of GFAP protein in iAstrocyte. hc1 and hc6 indicate astrocytes (Day 18) produced under the conditions of Example 1 from HC1A and HC6B, which are iPS cells derived from healthy subjects, respectively. Arrows indicate bands of GFAP protein. FIG. 11 shows the effect of the expression period of the NEUROG2 gene on the induction efficiency of iAstrocyte. hc1 and hc6 are the same as in FIG. The ratio of iAstrocyte (= GFAP positive) to the total number of living cells (= DAPI positive) was calculated (n = 3). The error bar indicates the SD value.

<NEUROG2 gene>
In the present invention, the NEUROG2 gene means a gene generally known in the present art as the Neurogenin 2 gene. The official genetic symbol is NEUROG2. The structure of the nucleic acid encoding the NEUROG2 protein is exemplified in NCBI Accession Number: NM_024019 (human) or NM_009718 (mouse).

<Astrocyte>
In the present invention, astrocytes can be defined as cells expressing one or more astrocyte marker genes, and examples of the marker genes include GFAP, S100B, Aquaporine4 (AQP4) and the like. Furthermore, in addition to satisfying the above definition, cells in which calcium oscillation is observed can also be referred to as functionally mature astrocytes. Calcium oscillation in astrocytes is basically a spontaneous fluctuation of individual cells alone, but as it matures, it laterally adheres to surrounding astrocytes through intercellular adhesion or signal transduction by extracellular paracrine. Directional oscillation propagation occurs. It is known that this lateral signal propagation (oscillation propagation) forms a network and plays an important role in the regulation of nerve cell activity and the control of vasoconstriction.

Producing astrocytes in the present invention means obtaining a cell population containing astrocytes, preferably containing 50%, 60%, 70%, 80%, 90%, or 95% or more of astrocytes. Is to obtain an astrocyte population.

<Pluripotent stem cells>
In the present invention, the pluripotent stem cell is a stem cell having pluripotency capable of differentiating into a wide variety of cells existing in a living body and also having a proliferative ability, and is not particularly limited thereto. For example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells (GS cells), embryonic germ cells (EG cells), artificial pluripotent stems (iPS). ) Cells, cultured fibroblasts, pluripotent cells derived from bone marrow stem cells (Muse cells), etc. are included.

ES cells are pluripotent and self-replicating stem cells established from the inner cell mass of early mammalian embryos (eg, blastocysts) such as humans and mice. ES cells are embryo-derived stem cells derived from the inner cell mass of the scutellum vesicle, which is the embryo after the morula at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called polymorphism. It has the ability and the ability to proliferate by self-replication. Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is possible.

Sperm stem cells are testis-derived pluripotent stem cells, which are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various lineages of cells, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012).

Embryonic germ cells are cells with pluripotency similar to ES cells, which are established from primordial germ cells in the embryonic period.

Induced pluripotent stem (iPS) cells are made by introducing specific reprogramming factors into somatic cells in the form of DNA, RNA or protein, and are pluripotent and self-replicating proliferative bodies. Cell-derived artificial stem cells (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 release WO 2007/069666). Initialization factors include, for example, 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 and the like can be exemplified.

The embodiment in which iPS cells are used as pluripotent stem cells in the present invention is suitable, and the embodiment in which human iPS cells are used is even more suitable. Astrocytes produced from iPS cells derived from patients with diseases associated with astrocyte abnormalities (eg Alexander's disease) are useful as model cells for the disease.

As already described, astrocytes produced by the method of the present invention can be used as an active ingredient of a cell preparation. From this point of view, a cell preparation containing astrocytes produced from iPS cells of a healthy person or astrocytes produced from iPS cells modified to express a protein having a therapeutic effect is particularly suitable. Such a cell product will be described in more detail in the section <Cell product> below.

<Process (1)>
The step (1) of the present invention is a step of directing the differentiation of pluripotent stem cells into astrocytes. In step (1), the NEUROG2 gene is expressed in pluripotent stem cells for a period shorter than the period required for the direction of differentiation into nerve cells, thereby directing the differentiation into astrocytes. The period for expressing the NEUROG2 gene 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, still more preferably 24 hours or more and 60 hours or less, and particularly preferably 36 hours. Hours or more and 60 hours or less, most preferably 36 hours or more and 48 hours or less.

Forced expression of the NEUROG2 gene can be performed using a method known per se, and is not particularly limited. The NEUROG2 gene to be forcibly expressed may be either an endogenous NEUROG2 gene or an exogenous NEUROG2 gene, but an exogenous gene is preferable because of its ease of expression regulation. As one embodiment, for example, a vector such as a virus containing a nucleic acid encoding NEUROG2, a plasmid, an artificial chromosome, or the like may be expressed by introducing it into pluripotent stem cells using a technique such as lipofection, liposome, or microinjection. Further, in step (1), cells into which the exogenous NEUROG2 gene has already been introduced may be used.

Examples of the virus vector include a retro virus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a Sendai virus vector. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Further, as the plasmid vector, a plasmid for mammalian cells can be used.

In another embodiment, the above vector transposons before and after this expression cassette (gene expression unit including promoter, gene sequence and terminator) in order to excise the sequence encoding the NEUROG2 gene incorporated into the chromosome as needed. It may have a sequence. The transposon sequence is not particularly limited, but piggyBac is exemplified as a suitable one.

These vectors can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the NEUROG2 gene can be expressed.

In order to express the NEUROG2 gene in a pluripotent stem cell, the pluripotent stem cell may contain a "nucleic acid encoding NEUROG2" functionally conjugated to an inducible promoter. Thereby, the NEUROG2 gene can be expressed in pluripotent stem cells at a desired time. Examples of such an inducible promoter include a drug-responsive promoter, and a suitable example thereof is the Tet-on promoter (tetO sequence having seven consecutive tetcycline responses), which is one of the tetracycline-responsive promoters. CMV minimal promoter with sequence (TRE)). The promoter is a promoter that is activated by supplying tetracycline or a derivative thereof under the expression of a reverse tetracycline-regulated transactivator (rtTA: a fusion protein with 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 having a mode of expressing the activator. Doxycycline (DOX) can be preferably used as the derivative of the tetracycline.

Expression-inducing systems using drug-responsive promoters other than the above include an expression-inducing system using an estrogen-responsive promoter (eg WO2006 / 129735) and RheoSwitch mammalian-inducible using a promoter induced by RSL1. Expression system (New England Biolabs), Q-mate system using a promoter induced by cumate (Krackeler Scientific), or Cumate-induced expression system (National Research Council (NRC)), and ecdison responsive sequences. Examples thereof include a GenoStat-induced expression system (Upstate cell signaling solutions) using a promoter.

When an expression vector having an expression-inducing system based on a drug-responsive promoter as shown above (that is, a drug-responsive inducing vector) is used, a drug capable of inducing activation of the promoter (for example, the tetracycline responsiveness) is used. In the case of a vector containing a promoter, the expression of NEUROG2 can be maintained by continuing to add tetracycline or DOX) to the medium for a desired period of time. Then, by removing the drug from the medium (for example, replacing it with a medium containing no such drug), it is possible to stop the expression of the gene.

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

As the medium used in the step (1) in the present invention, only the basal medium or the basal medium to which the neurotrophic factor is added can be used. Such basic media include Neurobasal Medium (Life Technologies), Glassgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Medium (GMEM) medium. Medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and a mixed medium thereof are included.

The basal medium may contain serum or may be serum-free. If necessary, the medium may be, for example, B27 supplement (Invitrogen), B27Plus supplement (Invitrogen), Knockout Serum Replacement (KSR) (FBS serum substitute during ES cell culture), N2 supplement (Invitrogen), albumin, transferase. , Apotransferase, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. may contain one or more serum substitutes, lipids, amino acids, L-glutamine, Glutamax ( Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenic acid, progesterone and putresin, even if they contain one or more substances good.

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 preferably used as the basic medium.

The culture temperature in the 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 CO ₂ -containing air, and the CO ₂ concentration is preferable. Is about 2-5%.

In step (1), it is preferable that the culture vessel is coated with extracellular matrix, as in the conventional method. The extracellular matrix that can be used in the step (1) is not particularly limited as long as it is usually used for adhesive culture, and is, for example, poly-L-lysine, matrigel, vitronectin and a synthetic peptide derived from vitronectin, human type I. Examples thereof include collagen-like recombinant peptides, gelatin, laminin, collagen, and fibronectin.

<Process (2)>
The step (2) is a step in which the cells that have been directed to differentiate into astrocytes in the step (1) differentiate into astrocytes. Specifically, it is a step of culturing the cells without substantially expressing NEUROG2 (for example, without inducing the expression of NEUROG2). The culture of step (2) may be carried out either in vivo or under conditions in the medium (in vitro). From the viewpoint of ease of controlling the differentiation rate, the in vitro culture step is preferable.

As the medium of the step (2), a medium containing a factor that promotes WNT signaling can be preferably used. Factors that promote WNT signaling promote differentiation into astrocytes, and the addition of such factors can increase the efficiency of astrocyte production. Factors that promote WNT signaling that can be suitably used in the present invention include, but are limited to, BMP4 (bone morphogenetic protein 4), CNTF (Ciliary neurotrophic factor), and FBS (fetal bovine serum). It's not something.

The medium of step (2) may contain serum, and Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferase, It may contain one or more serum substitutes exemplified by apotransferase, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol and the like. In the present invention, it is a preferred embodiment to include a serum substitute, preferably a serum substitute for nerve cells such as N2 supplements.

Furthermore, the medium of step (2) is Glutamax (Invitrogen), lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts. , Serenic acid, progesterone and putrecin and the like may also contain one or more substances.

When the step (2) is performed in vitro, subculture may be performed so as to have a low cell density. An example of such low cell density may be a cell density of 2 × 10 ⁴ to 10 × 10 ⁴ cells / cm ² per 48-well plate, preferably 5 × 10 ⁴ cells / cm ² cells per 48-well plate. Density. Culturing at low cell densities is preferred as it can reduce the likelihood that neurons will adhere to astrocytes and continue to survive (even under naturally non-adherable substrates).

In connection with this, when the step (2) is performed in vitro, the coating of the culture vessel may be gradually changed from one having a high affinity with cells to one having a low affinity with cells, and finally. May be uncoated. For example, first PMSC (PolyLlysine, Matrigel, Synthemax (Vitronectin-derived synthetic peptide, Corning), and Cellnest (human type I collagen-like recombinant peptide, Fujifilm)) coating (eg, Day1 to Day5), and then A gelatin-coated culture vessel (Day 6 to Day 12) may be used, and then an uncoated culture vessel may be used. The uncoated culture vessel may be used after Day11, Day12, Day13, and Day14.

The culture period of step (2) is preferably 10 to 20 days, more preferably 10 to 16 days.

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

Even when NEUROG2 is expressed in the step (1) for less than 3 days, preferably 2 days, and then administered in vivo without culturing in a medium (in vivo embodiment), astrocytes are generated in vivo. Is thought to occur. Therefore, such an in vivo aspect is also included in the present invention.

<IAstrocyte>
Further, the present invention provides astrocytes produced by the method of the present invention. The astrocyte may be referred to as induced Astrocyte (iAstrocyte) in order to distinguish it from naturally occurring astrocytes. Similarly, nerve cells that have been induced to differentiate from pluripotent stem cells may be referred to as induced neurons (iN).

The iAstrocyte according to the present invention is different from naturally-derived astrocytes in that a gene is introduced. Preferably, it is an iAstrocyte produced from human iPS cells.

<Cell product>
Furthermore, the present invention provides a cell preparation containing astrocyte (iAstrocyte) produced by the method of the present invention as an active ingredient. The cell preparation of the present invention containing iAstrocyte is effective for the treatment of neurodegenerative diseases, preferably neurodegenerative diseases associated with astrocyte abnormalities. Neurodegenerative diseases associated with astrosite abnormalities include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal cerebral degeneration, polyline atrophy, and progressive hepatic palsy. Examples include primary degenerative types of nerve cells such as frontotemporal dementia, Huntington's disease, and spastic vs. paralysis, and primary degenerative types of astrosite such as Alexander's disease. Of these, it can be particularly suitably used for the treatment of Alexander disease, Alzheimer's disease and ALS.

Alexander disease is a rare hereditary neurodegenerative disease characterized by the presence of rosental fibers composed of glial fibrous acidic protein (GFAP), αB-crystallin, heat shock protein, etc. in astrocytes. Abnormal aggregates consisting of mutant GFAP or overexpressed GFAP are thought to be involved in Alexander's pathology. Clinically, it is classified into cerebral dominant type (type 1), medulla oblongata / spinal cord dominant type (type 2), and intermediate type (type 3) based on clinical symptoms and MRI imaging findings. Missense mutations in GFAP or deletions or insertions of several bases have been observed in 97% of Alexander's diseases, and genetic testing has been used as a definitive diagnostic method in recent years. Research is progressing on the elucidation of the pathophysiology of Alexander disease, but it is still insufficient.

The cell preparation of the present invention can be used as a transplant therapeutic agent for treating the neurodegenerative disease. iAstrocyte is produced as a parenteral preparation such as an injection, a suspension, and an infusion by mixing with a pharmaceutically acceptable carrier according to conventional means. Pharmaceutically acceptable carriers that may be included in the parenteral preparation include, for example, isotonic solutions containing saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride solution, etc.). An aqueous solution for injection can be mentioned. The preparation may be, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalconium chloride, prokine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol). Etc.), preservatives, antioxidants, etc. may be blended. When formulating iAstrocyte as an aqueous suspension, add iAstrocyte to the above aqueous solution so that the content is 1 × 10 ⁵ to 1 × 10 ⁸ cells / ml, preferably 1 × 10 ⁶ to 1 × 10 ⁸ cells / ml. It may be suspended. iAstrocyte transplantation can be performed by injecting the above suspension into lesions such as the cerebrum, medulla oblongata, and spinal cord. The number of cells to be administered can be appropriately changed depending on the degree of lesion, etc., but for example, in the case of a patient with human Alexander disease, 1 × 10 ⁵ to 1 × 10 ⁸ cells, preferably 1 × 10 ⁶ to 1 × 10 ⁸ cells. Can be administered.

Further, as a different embodiment of the present invention, the cell preparation may contain cells immediately after undergoing the step (1) as an active ingredient. By injecting the cell preparation into lesions such as the cerebrum, medulla oblongata, and spinal cord according to the above-mentioned method, the cells immediately after the step (1) can be differentiated into astrocytes in vivo and exert a therapeutic effect. Be expected.

The above transplantation treatment and drug therapy can be used in combination. As a concomitant drug, for example, when the target disease is Alexander disease, it can be used in combination with an antiepileptic drug currently used as a symptomatic treatment for Alexander disease. Such combination drugs can be used, for example, in the dosage and administration route usually used for the treatment of Alexander's disease.

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

First, the cells used in this example and the main method will be described.
<Cell>
In this example, as pluripotent stem cells that induce differentiation into astrosites, an iPS cell line in which a human NEUROG2 gene controlled by a Tet-on promoter is introduced into iPS cells established from a healthy subject or patient using piggyBac transposon. (Hereinafter referred to as NEUROG2-iPSC) was used. In the NEUROG2-iPSC, the exogenous NEUROG2 gene is inserted into the genome. It should be noted that piggyBac transposon was used because the construct used in Patent Document 1 was available, and it has been confirmed that similar results can be obtained even with a transient expression system that is not inserted into the genome. Table 1 shows the iPS cell lines used to prepare NEUROG2-iPSC and their origins.

In this example, unless otherwise specified, NEUROG2-iPSC was cultured according to the conditions shown in FIG. 1 to produce astrocytes. In this embodiment, the start date of induction of differentiation into astrocytes, that is, the start date of induction of expression of NEUROG2 may be set to Day 0, and the number of days thereafter may be represented by Day.

The expression of NEUROG2 was induced by adding DOX to the medium at 2 μg / ml for 2 days from Day 0 to Day 2 unless otherwise specified. NB medium (Neurobasal plus medium containing 0.5% B27 plus supplement) is used during the expression induction period of NEUROG2, and AM medium (1% N2 supplement, 10 ng / ml BMP4, 10 ng / ml CNTF, 2% FBS) is used thereafter. DMEM / F12 medium containing) was used. In addition, Y-27632 was added for a predetermined period to 10 μM in order to reduce damage caused by medium replacement.

As the culture plate, a PMSC-coated 48-well or 96-well plate was used for Day 0 to Day 5, and a gelatin-coated 6-well plate was used for Day 5 to Day 12.

On Day 0, NEUROG2-iPSC was seeded at 30 × 10 ⁴ cells / cm ² per 48-well plate, and the first passage was performed at the timing of changing the type of culture plate (Day 5). On Day 5, seeds were seeded at 5 × 10 ⁴ cells / cm ² per 48-well plate. After that, in addition to the timing of changing the type of the culture plate (Day 12), passage was appropriately performed according to the rate of cell proliferation.

From Day 12 to Day 18, cells were collected for assay.

Example 1: Production of astrocytes NEUROG2-iPSC produced from three iPS cell lines (HC1A, HC6B, N117E11) derived from healthy subjects and one iPS cell line (AD2S1) derived from familial Alzheimer's disease patients. Was cultured under the conditions shown in FIG. 1 to induce differentiation into astrocytes. On Day 12 or Day 18, GFAP immunostaining (green) and nuclear staining (DAPI, gray) were performed. The results are shown in FIG. With either NEUROG2-iPSC, only some cells were GFAP-positive on Day 12, but almost all cells were GFAP-positive on Day 18 (95% or more). From this, it was clarified that when NEUROG2 was expressed in pluripotent stem cells for only 2 days, it rapidly differentiated into astrocytes from about 10 to 16 days after that. Furthermore, the GFAP-positive cells on Day 18 had a thicker cytoplasm and extended multiple protrusions than the GFAP-positive cells on Day 12, and were morphologically mature as astrocytes.
Therefore, when NEUROG2 is expressed in pluripotent stem cells for a period shorter than the period required for nerve cell differentiation induction (for example, 2 days), it takes only 18 days from the start of NEUROG2 expression induction, which is much shorter than before. , Almost all cells have been shown to differentiate into morphologically mature astrocytes. In this example, the astrocytes obtained by forced expression of NEUROG2 may be referred to as "iAstrocyte" hereafter.

Next, iAstrocytes were produced from NEUROG2-iPSC (Alex1-Alex3) derived from Alexander disease patients using the same method. In addition, iN was also produced from HC6B NEUROG2-iPSC as a negative control. In FIG. 1, the iN was produced by extending the period of culturing in a DOX-containing medium from 2 days to 5 days, and then culturing in a DOX-free NB medium (not AM medium). The results of collecting each cell on Day 12 and analyzing the transcription levels of the astrosite marker genes (GFAP, S100B, and AQP4) and the housekeeping genes (GAPDH and ACTB) are shown in FIG. The results of analyzing the transcription levels of MAP2 and SYN1) are shown in FIG. In FIGS. 3 and 4, the numerical value of the expression level on the vertical axis is a numerical value calibrated with the GAPDH expression level of FIG. 3 as 1.

As shown in FIG. 3, expression of none of the astosite markers was detected in iN, but expression of GFAP and S100B was observed in iAstrocyte regardless of the cell line. The expression level of AQP4 varied widely among cells, and in some cases, expression was not detected in iAstrocytes derived from Alexander disease patients (Alex12, Alex3). This result may be related to the pathophysiology of Alexander disease with astrocyte dysfunction. On the other hand, the expression levels of GAPDH and ACTB did not differ significantly between iN and iAstrocyte, or between iAstrocytes from different cell lines.

On the other hand, the expression of neuronal markers was very prominent in iN, but was very low in iAstrocyte (TUBB3, MAP2) or below the detection limit (SYN1) (Fig. 4).

From these results, it is only 12-18 by forced expression of NEUROG2 not only in pluripotent stem cells derived from healthy subjects but also in pluripotent stem cells derived from patients with neurodegenerative diseases caused by neurodegenerative and glia degeneration. It was shown that astrocytes can be induced to differentiate in days.

Example 2: Examination of NEUROG2 forced expression period (1)
We investigated the appropriate forced expression period of NEUROG2 to induce differentiation of pluripotent stem cells into astrocytes instead of neurons.
For NEUROG2-iPSC (HC1A, HC6B) derived from healthy subjects and NEUROG2-iPSC (AD2S1, AD2EL) derived from familial Alzheimer's disease patients, the period of culturing in DOX-containing medium was changed to 2, 3, 4, and 5 days. The cells were cultured until Day 8 (Fig. 5, Condition AD). A phase-contrast microscope observation was performed at the time of Day 8, and a typical phase-contrast microscope image obtained under each condition is shown in FIG.

In Condition A cultured in Dox-containing medium for 2 days, iN (cells extending neurites) was hardly found. On the other hand, iN was observed in Condition B with a culture period of 3 days in Dox-containing medium, the number of iN increased further in Condition C, and almost all cells extended long neurites in Condition D. It was iN.

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 in 2 days, it does not substantially differentiate into iN.

Therefore, it was clarified that the forced expression period of NEUROG2 suitable for inducing differentiation into astrocytes is less than 3 days, most preferably about 2 days.

Example 3: Examination of calcium oscillation Next, the functionality of astrocytes produced by the method according to the present invention was examined. In functionally mature astrocytes, a phenomenon (calcium oscillation) in which the Ca ion concentration in the astrocytes changes autonomously and regularly is observed, and neurotransmitter / regulatory factors such as glutamate, ATP, and serine are detected. It has been shown to regulate nearby astrocytes by releasing in a calcium concentration-dependent manner. Therefore, the presence or absence of calcium oscillation was analyzed as an index of the functional maturity of iAstrocyte.

For Day18 iAstrocyte (Fig. 7A) obtained by culturing in DOX medium for 2 days, the Fluo-8AM body was incorporated into the cells and the intracellular Ca ion concentration was continuously recorded as the upper and lower fluorescence intensities and measured over time. .. The results are shown in FIG. 7B. As shown in FIG. 7B, it was confirmed that the Ca ion concentration changed up and down autonomously and regularly in the iAstrocyte, and calcium oscillation occurred.

Subsequently, Fig. 8 shows the results of recording calcium oscillations at multiple points at the same time. The vertical axis of FIG. 8 represents the number assigned to each measurement site (ROI: Region of Interests). From this result, it can be seen that calcium oscillation occurs in various places in the iAstrocyte culture system. Furthermore, at the

measurement sites

5, 6, 8, 9, 11-14, and 16 (particularly, 5, 6, and 12), the calcium concentration is high for about 190 seconds from the start of measurement. It was observed that the rising timings (vertical red dotted line in FIG. 8) tended to match.

As described in the above section <Astrocytes>, astrocytes are known to form a network by propagating lateral calcium oscillations. As described above, it was also observed that calcium oscillations were synchronized between multiple cells in iAstrocytes produced by this differentiation induction method. This result indicates that a communication network is formed between iAstrocytes.

Therefore, it was clarified that the iAstrocyte produced by the method according to the present invention can spontaneously perform calcium oscillation and can also form an intercellular network via the oscillation. This indicates that the method according to the present invention can produce morphologically and functionally mature iAstrocytes.

Example 4: Production of cell model of neurodegenerative disease with astrocyte abnormality The presence or absence of GFAP-positive aggregates was examined for iAstrocytes produced from iPS cells (A266 strain) derived from Alexander disease patients. GFAP or CD44 was immunostained on Day 18 iAstrocytes produced from two types of NEUROG2-iPSC (A226, HC1A) by the method of Example 1. The results are shown in FIG.

No GFAP-positive intracellular aggregates were observed in iAstrocytes produced from healthy subject-derived iPSC (HC1A) (Fig. 9A, B). In contrast, in iAstrocytes produced from iPSC (A226) derived from Alexander disease patients, GFAP-positive intracellular aggregates were frequently observed (Fig. 9C-E). That is, it was shown that astrocytes produced from iPSCs derived from Alexander disease patients by the method according to the present invention frequently spontaneously generate GFAP-positive intracellular aggregates characteristic of Alexander disease patients. Since the GFAP-positive intracellular aggregate is considered to contribute significantly to the onset of Alexander disease, it is considered that the astrocytes produced by the method according to the present invention can be a cell model of Alexander disease. ..

CD44 is a surface antigen marker expressed in glial progenitor cells and astrocytes. Whether it is an iAstrocyte (Fig. 9F-H) manufactured from a healthy person-derived iPSC (HC1A) or an iAstrocyte (Fig. 9I-K) manufactured from an Alexander disease patient-derived iPSC (A226), CD44 is present on the membrane surface. It was found to be distributed. This result suggests that astrocyte lineage differentiation is progressing in both iAstrocyte derived from healthy subjects and iAstrocyte derived from Alexander disease patients.

Therefore, it was shown that the production method according to the present invention can produce model cells for diseases associated with astrocyte abnormalities (for example, Alexander disease).

Example 5: Expression of GFAP protein in iAstrocyte It was confirmed by Western blot analysis that iAstrocyte expressed GFAP protein.
Proteins were extracted (0.5 μg / μL) from cells on Day 18 of iAstrocytes produced under the conditions of Example 1 from healthy person-derived iPS cells HC1A and HC6B according to a conventional method, and a glass capillary electrophoresis device Wes (Protein simple) was used. Analyzed using. As the antibody, an antibody prepared using the full length of the human GFAP recombinant protein as an immunogen was used. The results are shown in FIG. It was confirmed that all iAstrocytes express GFAP.

Example 6: Examination of NEUROG2 forced expression period (2)
We investigated the forced expression period of NEUROG2 required to induce differentiation of pluripotent stem cells into astrocytes.
From HC1A and HC6B, which are iPS cells derived from healthy subjects, iAstrocytes were induced by shaking the culture period in a DOX-containing medium from 8 hours to 72 hours in the same manner as in Example 2. On Day 18, GFAP immunostaining and DAPI nuclear staining were performed, and the ratio of iAstrocyte (= GFAP positive) to the total number of living cells (= DAPI positive) was calculated. The results are shown in FIG. It was clarified that astrocyte induction is possible even with expression at 8 hours. It was found that about 48 hours is optimal, and that the induction efficiency may decrease after 48 hours. It was suggested that the induction efficiency was particularly high in about 36-60 hours. The GFAP positive rate was substantially 0% when NEUROG2 was not forcibly expressed (data not shown).

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 than before and with high efficiency close to 100%. The astrocytes produced by the method of the present invention are useful as model cells for analyzing the physiological function and role of astrocytes in pathological conditions, and as therapeutic agents (cell preparations) for diseases associated with astrocyte abnormalities.

This application is based on Japanese Patent Application No. 2020-190575 filed in Japan on November 16, 2020, the entire contents of which are incorporated herein by reference.

Claims

A method for producing astrocytes from pluripotent stem cells, the following steps:
(1) A step of expressing the NEUROG2 gene in pluripotent stem cells for less than 3 days.
How to include.
The method according to claim 1, wherein the expression period of the NEUROG2 gene is 8 hours or more.
After the step (1),
(2) A step of culturing the pluripotent stem cells without inducing the expression of the NEUROG2 gene.
The method according to claim 1 or 2, wherein the method comprises.
The method according to any one of claims 1 to 3, wherein the NEUROG2 gene is a gene regulated by an inducible promoter, and the step (1) is a step of activating the promoter to express NEUROG2. ..
The method according to claim 4, wherein the inducible promoter is a drug-responsive promoter.
The step (1) is a step of culturing the pluripotent stem cells in the presence of a drug that activates the drug-responsive promoter, and the step (2) is a step of culturing the pluripotent stem cells in the absence of the drug. The process of culturing sexual stem cells,
The method according to claim 5.
The method according to claim 6, wherein the culture period of the step (2) is 10 to 20 days.
The method according to claim 6, wherein the culture period of the step (2) is 10 to 16 days.
The method according to any one of claims 1 to 8, wherein the NEUROG2 gene is a gene introduced into the pluripotent stem cell using a transposon.
The method according to claim 9, wherein the transposon is a piggyBac transposon.
The method according to any one of claims 6 to 10, wherein the step (2) is a step of culturing in the presence of a factor that promotes WNT signaling.
11. The factor according to claim 11, wherein the factor promoting 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). the method of.
The method according to any one of claims 1 to 12, wherein the pluripotent stem cell is a human induced pluripotent stem cell.
The method according to claim 13, wherein the human induced pluripotent stem cells are derived from a disease patient with an astrocyte abnormality.
Astrocyte manufactured by the method according to any one of claims 1 to 14.
A cell preparation containing the astrocyte according to claim 15 as an active ingredient. The