Method for selective removal of undifferentiated pluripotent stem cells

Abstract

To provide a method for selective removal of undifferentiated pluripotent stem cells.SOLUTION: A method includes inducing differentiation of stem cells and then administering an effective amount of phthalide to remove undifferentiated stem cells.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] The present invention relates to a method for culturing stem cells, and particularly to a method for selectively removing undifferentiated stem cells.

Background Art

[0002] A stem cell refers to a cell having self-renewal ability and differentiation ability. Generally, from the higher to the lower differentiation ability, it is classified into totipotent stem cells, pluripotent stem cells, oligopotent stem cells, and unipotent stem cells.

[0003] A pluripotent stem cell refers to a primitive cell that has not yet differentiated, has self-renewal ability, and can differentiate into three types of germ layer cells in the human body. Depending on its origin, it can be classified into embryonic stem cells and induced pluripotent stem cells. Among them, embryonic stem cells are derived from the inner cell mass of a human blastocyst embryo and can differentiate into various somatic cells. Furthermore, by delivering specific genes and proteins to somatic cells and forcing their expression, or by using chemical small molecules, induced pluripotent stem cells are generated by cell reprogramming, and they have properties and differentiation abilities similar to those of embryonic stem cells.

[0004] Pluripotent stem cells have the ability to differentiate into various somatic cells, can be cultured and proliferated in large quantities in vitro, and can be induced into specific somatic cells, organs, and tissues. The in vitro cell differentiation process of pluripotent stem cells can mimic embryonic development and enable us to better understand the process of human development. When applied to disease research, pluripotent stem cells of patients can be converted into various diseased cells, which is useful for studying unknown processes and pathogenesis of diseases and is applied to drug development and precision medicine.

[0005] Compared with pluripotent stem cells, oligopotent stem cells have a lower differentiation potential but still have the ability to differentiate into somatic cells. Oligopotent stem cells can be obtained by separating them from adults, and common types include mesenchymal stem cells such as umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental mesenchymal stem cells, amniotic fluid mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose mesenchymal stem cells.

[0006] Many scholars are attempting to apply the above-mentioned pluripotent stem cells and oligopotent stem cells to cell transplantation therapy for various diseases. Currently, it is expected to apply differentiated nervous system cells, glial cells, eye cells, cardiomyocytes, blood cells, pancreatic islet cells, and mesenchymal stem cells, etc. to Parkinson's disease, spinal cord injury, macular lesions, corneal transplantation, myocardial infarction, tumor immunotherapy, diabetes, and autoimmune diseases, and many of them are in clinical trials.

[0007] When applying stem cells to transplantation, in the process of performing specific somatic cell differentiation, if undifferentiated pluripotent stem cells remain, there is a possibility of teratoma formation after transplantation into the living body, which improves the cancer risk. In order to remove undifferentiated pluripotent stem cells in the transplanted cell population, current methods mainly exclude cells with pluripotent stem cell-specific surface antigens or collect cells with specific somatic cell surface antigens by sorting with flow cytometry to avoid the remaining of undifferentiated pluripotent stem cells. Although this method can eliminate the remaining of undifferentiated pluripotent stem cells, the time, cost, and labor cost are high, and the genes, cell characteristics, and cell health status, etc. after sorting some specific somatic cells may change. In recent years, it has been reported that early treatment with Quercetin can reduce the remaining of undifferentiated pluripotent stem cells. Summary of the Invention Problems to be Solved by the Invention

[0008] As described above, in order to reduce the risks associated with applying stem cells to clinical cell transplantation therapy and improve the safety of such applications, there remains a need in the art to develop methods for selectively removing undifferentiated pluripotent stem cells. This is an important and essential development focus for reducing the carcinogenicity of undifferentiated pluripotent stem cells.

Means for Solving the Problem

[0009] The present disclosure relates to a method for selectively removing undifferentiated pluripotent stem cells, which includes collecting pluripotent stem cells, inducing the differentiation of the collected pluripotent stem cells to obtain a cell population having differentiated cells (wherein the cell population further includes the undifferentiated collected pluripotent stem cells), and administering an effective amount of a phthalide-based compound to the cell population to selectively remove the undifferentiated collected pluripotent stem cells.

[0010] In one aspect of the present disclosure, the phthalide-based compound is at least one selected from the group consisting of n-butenyl phthalide, methyl phthalide, 7-methyl phthalide, ethyl phthalide, n-propenyl phthalide, n-butyl phthalide, 3-bromophthalide, 5-bromophthalide, 5-chlorophthalide, 6-chlorophthalide, 3,4-dichlorophthalide, tetrachlorophthalide, 3-hydroxy-3-trifluoromethyl phthalide, 3-methyl-3-(1-naphthyl) phthalide, 3-(5-fluoro-1-naphthyl) phthalide, 4-amino-3-hydroxy phthalide, 5-carboxy phthalide, 5-cyano phthalide, 7-methoxy phthalide, 7-hydroxy-6-methoxy phthalide, 3-(1,2-dimethyl-3-indolyl) phthalide, phenolphthalein, ligustilide and sedanolide. In another aspect of the present disclosure, the phthalide-based compound includes n-butenyl phthalide, n-butyl phthalide, tetrachlorophthalide and phenolphthalein. In another aspect of the present disclosure, the phthalide-based compound is n-butenyl phthalide.

[0011] In one aspect of the present disclosure, the effective amount of the phthalide compound is administered during the differentiation period of the pluripotent stem cells. During the differentiation period of the pluripotent stem cells, the differentiated cells include at least one selected from the group consisting of oligopotent stem cells, unipotent stem cells, and somatic cells. In one aspect of the present disclosure, the differentiated cells in the cell group do not include somatic cells. In another aspect of the present disclosure, after somatic cells are included in the cell group, the effective amount of the phthalide compound is administered.

[0012] In one aspect of the present disclosure, the concentration of the effective amount of the phthalide compound is 10 μM to 1000 μM. In another aspect of the present disclosure, the concentration of the effective amount of the phthalide compound is 50 μM to 800 μM.

[0013] In one aspect of the present disclosure, the treatment time with the effective amount of the phthalide compound is 1 to 6 days.

[0014] In one aspect of the present disclosure, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

Advantages of the Invention

[0015] The present disclosure selectively removes undifferentiated pluripotent stem cells in pluripotent stem cells induced to differentiate by treatment with an effective amount of a phthalide compound, retains differentiated cells, effectively reduces the risk of subsequent application of the differentiated pluripotent stem cells, and improves the safety of its application.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Modes for Carrying Out the Invention

[0017] The following further describes the technical solutions described in the embodiments of the present invention more clearly and completely. Obviously, the described embodiments are only a part of many embodiments included in the present disclosure, and are not intended to limit the scope of the present invention. The present invention can also be implemented or applied in similar or different embodiments. Modifying, changing, or substituting other embodiments obtained by those skilled in the art without creative efforts, such as modifying an element or its combination, etc., are all included in the scope of the present invention.

[0018] Furthermore, it should be noted that the singular forms "one" and "the foregoing" described in this specification mean including a plurality of designated objects unless clearly limited to only one designated object. Also, the term "or" is used interchangeably with the term "and / or" unless specifically stated otherwise in the context.

[0019] As used herein, the term "about" means that the error or range of a numerical value, numerical range or ratio varies within 20%, preferably within 10%, more preferably within 5% of the said numerical value, numerical range or ratio. The quantified values described herein are approximate values, which means that even if the term "about" is not used, it can be inferred. The numerical ranges described herein include all numerical values falling within the said numerical ranges. For example, a numerical range of 50 μM to 800 μM includes numerical values such as 50 μM, 50.01 μM, 50.1 μM, 50.5 μM, etc., and also includes all sub-ranges falling within the said numerical ranges. The sub-ranges are surrounded by each numerical value falling within the said numerical range. For example, a numerical range of 50 μM to 800 μM includes sub-ranges such as 50 μM to 700 μM, 88 μM to 650 μM, 166 μM to 521 μM, etc.

[0020] As used herein, the terms "comprise", "comprising", "include", "including", "have", etc. refer to the presence of an element (e.g., an element or a step, etc.) in the objects, methods, uses, etc. of the present invention. Also, unless clearly indicated in the context, elements that are not described and specified are also open to existence in the objects, methods, uses of the present invention, regardless of whether they are necessary or not. That is, elements that are not described and specified are not restrictively excluded.

[0021] As used herein, the term "stem cells" refers to cells having the ability of self-renewal and the ability to differentiate into somatic cells, including totipotent stem cells, pluripotent stem cells, oligopotent stem cells, and unipotent stem cells. The stem cells may be stem cells separated from a cell population containing stem cells (e.g., embryonic stem cells or mesenchymal stem cells), or may be stem cells generated by inducing and reprogramming somatic cells (e.g., induced pluripotent stem cells).

[0022] As used herein, "removing undifferentiated collected pluripotent stem cells" refers to differentiating at least a part of the collected pluripotent stem cells into oligopotent stem cells, unipotent stem cells, and / or somatic cells, and then removing the pluripotent stem cells in the cell population of the oligopotent stem cells, unipotent stem cells, and / or somatic cells. In one aspect of the present disclosure, the pluripotent stem cells may be, but are not limited to, embryonic stem cells or induced pluripotent stem cells. Also, the oligopotent stem cells may be, but are not limited to, neural stem cells, mesenchymal stem cells, or hematopoietic stem cells.

[0023] As used herein, the term "effective amount" refers to the dosage at which the administered compound produces the desired effect, i.e., the dosage at which the administered phthalide compound produces the effect of selectively removing undifferentiated pluripotent stem cells.

[0024] The present disclosure relates to a method for selectively removing undifferentiated pluripotent stem cells, comprising collecting pluripotent stem cells, inducing differentiation of the collected pluripotent stem cells to obtain a cell population having differentiated cells (wherein the cell population further comprises the undifferentiated collected pluripotent stem cells), and administering an effective amount of a phthalide compound to the cell population to selectively remove the undifferentiated collected pluripotent stem cells.

[0025] In some specific embodiments of the present disclosure, the method of the present disclosure cultures and passages the pluripotent stem cells before collecting the pluripotent stem cells.

[0026] Methods for inducing the differentiation of pluripotent stem cells are well-known to those skilled in the art. Non-limiting examples of methods for inducing the differentiation of pluripotent stem cells include transferring pluripotent stem cells to an appropriate differentiation medium environment, in which the pluripotent stem cells are differentiated into a specific cell type. For example, by transferring the pluripotent stem cells to a neural induction medium (Neural induction medium; NI medium, Gibco) and adding specific proteins and small molecule compositions such as basic FGF, SB431542, and CHIR99021 and culturing them, the pluripotent stem cells can be differentiated into neural stem cells.

[0027] In some specific embodiments of the present disclosure, the phthalide compound may be at least one of the group consisting of n-butylidenephthalide, methylphthalide, 7-methylphthalide, ethylphthalide, n-propylidenephthalide, n-butylphthalide, 3-bromophthalide, 5-bromophthalide, 5-chlorophthalide, 6-chlorophthalide, 3,4-dichlorophthalide, tetrachlorophthalide, 3-hydroxy-3-trifluoromethylphthalide, 3-methyl-3-(1-naphthyl)phthalide, 3-(5-fluoro-1-naphthyl)phthalide, 4-amino-3-hydroxyphthalide, 5-carboxyphthalide, 5-cyanophthalide, 7-methoxylphthalide, 7-hydorxy-6-methoxyphthalide, 3-(1,2-dimethyl-3-indolyl)phthalide, phenolphthalein, ligustilide and sedanolide. In another specific embodiment of the present disclosure, the phthalide compound is n-butylidenephthalide, n-butylphthalide, tetrachlorophthalide and phenolphthalein.In one exemplary embodiment of the present disclosure, the effect of selectively removing undifferentiated stem cells is described with n-butenyl phthalide.

[0028] In one specific embodiment of the present disclosure, the effective amount of the phthalide compound is applied during the period of differentiating the pluripotent stem cells to achieve the effect of selectively removing undifferentiated pluripotent stem cells. Among them, the period of differentiating the pluripotent stem cells may be the period of differentiating the pluripotent stem cells into oligopotent stem cells, and the differentiated cells in the cell population at this time may not yet contain somatic cells. Alternatively, it may be the period of differentiating the oligopotent stem cells differentiated from the pluripotent stem cells into somatic cells. In another aspect of the present disclosure, the effective amount of the phthalide compound is applied to the somatic cells after the differentiation of the pluripotent stem cells is completed to achieve the effect of selectively removing undifferentiated pluripotent stem cells. Those skilled in the art can easily determine the differentiation period and the time point after the completion of differentiation for differentiating pluripotent stem cells into unspecified oligopotent stem cells or somatic cells based on conventionally known cell surface antigens.

[0029] In one specific embodiment of the present disclosure, the concentration of the effective amount of the phthalide compound is about 10 μM to 1000 μM. In other specific embodiments of the present disclosure, the concentration of the effective amount of the phthalide compound is about 10 μM to 800 μM, about 50 μM to 800 μM, about 50 μM to 750 μM, or about 50 μM to 500 μM. In some specific embodiments of the present disclosure, the concentration of the effective amount of the phthalide compound is about 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750 μM, and 800 μM. The above-mentioned multiple numerical endpoints can be arbitrarily selected as the maximum value or the minimum value for deriving the numerical range.

[0030] In one specific embodiment of the present disclosure, the treatment time with the effective amount of the phthalide compound is about 1 to 6 days. In other specific embodiments of the present disclosure, the treatment time with the effective amount of the phthalide compound is about 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, or 6 days. Any of the above-mentioned multiple numerical endpoints can be arbitrarily selected as the maximum value or the minimum value for deriving a numerical range.

[0031] In one specific embodiment of the present disclosure, differentiated stem cells are treated with a 100 μM phthalide compound for 6 days to selectively remove undifferentiated stem cells.

[0032] The present disclosure also relates to the use of a phthalide compound for selectively removing undifferentiated pluripotent stem cells.

Examples

[0033] The present invention will be further described below by specific specific examples, but the present invention is not limited thereto. Materials and Methods

[0034] Culture and Differentiation of Human Pluripotent Stem Cells

[0035] 1. Culture of iPSCs in Essential8 (serum-free): Human iPSCs were cultured in a culture dish surface-treated with Essential8 medium (Gibco) and basement membrane matrix (matrigel). Subculture was performed when the cells reached 70-80% confluence in about 3-5 days. At the time of subculture, first, the cells were washed twice with PBS until the cell edges were slightly lifted, and then treated with a cell dissociation reagent (Accutase) for 1-5 minutes until most of the cells became round. After that, Accutase was diluted with DMEM / F12, DMEM medium was added, and the cells were scraped with a cell scraper with a uniform force, mechanically dispersed to an appropriate pellet size, plated at an appropriate ratio (about 1:5 - 1:10), and 10 μM Y27632 was added.

[0036] 2. Human iPSCs Dopamine Neuron Adhesion Differentiation Process (CHSF-DA Differentiation Method): After subculturing and adhering human iPSCs cultured without a feeder layer at a ratio of 1:10, dopamine neuron differentiation was performed. During the differentiation process, from day 1 to day 12, a neural induction culture medium composed of DMEM / F12 (2:1) and N2 supplement was used, and furthermore, basic FGF (FGF-basic, also called FGF-2 or bFGF) (10 mg / mL), SB431542 (2 μM), CHIR99021 (7.5 μM), SAG (1 μM), and LDN193189 (0.2 μM), which are dopamine precursor cell induction factors, were added for induction. On day 13 of differentiation, the cells were cultured in a neuron culture medium (Neurobasal medium and N2 / B27 supplement). SAG (0.5 μM) was added between day 13 and day 18, and SAG was removed on day 18. 100 μM of n-butylenephthalide was added from day 23 to day 28 for a total of 6 days. Subsequently, the cells were continuously cultured in Neurobasal medium and N2 / B27 supplement for the experiment. During the above period, the culture medium was changed every 2 days, and proteins and small molecule reagents were newly added when changing the medium.

[0037] 3. SFEB (Serum Free Embryoid Body) Neural Differentiation Method of Human iPSCs: The operation procedure for differentiating iPSCs into nerve cells was carried out in four steps. The first three steps were the SFEB suspension method, which involved culturing spheroid cells in suspension for a total of 35 days, and the fourth step was to let the cells adhere and grow for 3 days.

[0038] Step 1: Aggregate iPSCs into suspended embryoid bodies (EBs). This step is the differentiation initiation step. After treating iPSCs with 1 mg / mL of Dispase II until the edges are rolled up, wash them 3 times with PBS, collect the iPSCs with a scraper, mix them by visual pipetting to disperse them into appropriate clump sizes (pipetting), add Essential6 medium containing RevitaCell and suspend them, and then transfer them to a 6 cm2 Transfer it to a sterile non - adherent type culture dish and perform suspension culture for 2 days, during which the culture medium was changed daily.

[0039] Step 2: Transfer the EBs to a 15 mL centrifuge tube, sediment the cells at room temperature, aspirate and remove the supernatant, then add a neural induction medium (Neural induction medium; NI medium, Gibco) and perform suspension culture for 2 days. At this point, basic FGF (10 ng / mL), SB431542 (10 μM) and CHIR99021 (3 μM) were added and observed daily under a microscope.

[0040] Step 3: Replace the neural induction medium in Step 2 with a Neurobasal medium (NB medium, Gibco), suspend - culture the cells in Step 2, continuously add basic FGF (10 ng / mL), and renew the culture medium once every 2 days at this stage. The cells were cultured for a total of 31 days.

[0041] Step 4: Adhere the cells in Step 3 to a culture plate surface - treated with 1% basement membrane matrix or laminin / ornithine. The culture medium is the Neurobasal medium added with basic FGF (10 ng / mL). The cells grew from the center of the cell colony to the outside. Then, after continuous culture for 3 days until nerve fibers grew, the experiment was conducted.

[0042] 4. Adhesion and differentiation process of human iPSCs into neural stem cells (method for adhesion and differentiation of neural stem cells): After sub - culturing and adhering human iPSCs cultured without a feeder layer at a ratio of 1:10, the differentiation of neural stem cells was carried out. During the differentiation process, a neural induction medium composed of DMEM / F12 (2:1) and N2 supplement was used, and furthermore, the inductive factors basic FGF (10 mg / mL), SB431542 (2 μM), CHIR99021 (3 μM) were added for induction. The culture medium was renewed once every 2 days, and proteins and small - molecule reagents were newly added when replacing. The adhesion and differentiation method of this neural stem cell had a maximum culture time of 7 days.

[0043] Method for administering n-butenyl phthalide (N-BP)

[0044] a. Drug action and concentration test: n-butenyl phthalide was diluted at various concentrations and applied to cells (the final concentration was between 50 and 750 μM). The types of cells to which it was applied included human iPSCs in culture, human iPSCs aggregated for one day in SFEB, neural stem cells on the 10th day of SFEB differentiation, neuron cells on the 25th day of SFEB differentiation, cells on the 5th day of neural differentiation by the adhesion method, etc. The concentration and time of application were as described in the examples.

[0045] b. Apply n-butenyl phthalide to the dopamine (DA) neuron production flow: In the CHSF-DA differentiation flow, 100 μM of n-butenyl phthalide was added from the 23rd day to the 28th day, for a total of 6 days.

[0046] Cell identification and function evaluation

[0047] a. Immunofluorescence staining method: After surface-treating a chamber slide with 1% basement membrane matrix for 3 - 4 hours, the basement membrane matrix was removed and it was prepared for use. After dispersing the cells into small aggregated cells with an enzyme, they were seeded onto the chamber slide and cultured for several days until the cells adhered and extended outward, presenting the morphology of rosette-shaped neural tube-like cells. Immediately after that, identification staining of neural stem cells and various neuronal progenitor cells was performed. When performing immunocytochemistry staining, first, the culture medium was removed, and the cells were gently washed 2 - 3 times with PBS at room temperature or 37°C. Then, 200 μl of 4% paraformaldehyde was added to the cells and allowed to act at room temperature for 20 minutes to fix the cells. After that, it was removed, and the cells were gently washed 3 times with PBS, removing each time after 5 minutes. Then, 200 μl of 99% methanol or 0.1 - 0.3% Triton was added and allowed to act at 4°C for 5 - 10 minutes to perforate the cell membrane. After removing and volatilizing it, the cells were gently washed 3 times with PBS, each time for 5 minutes. Then, 5% horse serum was added and allowed to act at room temperature for 1 hour for blocking. After removing the 5% horse serum, the primary antibody was added. The primary antibody was prepared in 3% horse serum, and its concentration was adjusted according to the required concentration of the antibody. After incubating the primary antibody overnight, the primary antibody was removed, and the cells were gently washed 3 times with PBST (PBS and Tween 20), each time for 5 minutes. After adding the secondary antibody, a light-shielding operation was performed. The secondary antibody was prepared in PBS, and the concentration was 1:500. The action time of the secondary antibody was 1 hour of light-shielding action at room temperature. After removing the secondary antibody, the cells were gently washed 3 times with PBST, each time for 5 minutes. Then, cell nucleus staining was performed. 200 μl of DAPI (1 μg / mL) was taken and added to the cells, and a light-shielding reaction was carried out at room temperature for 10 minutes. After removing the DAPI, the cells were gently washed 2 times with PBST, each time for 5 minutes. After removing the PBST, PBS was added to keep the cells in a moist state. The chamber slide was removed, sealed with a long coverslip and a sealing solution, stored in the dark at 4°C, and observed with a fluorescence microscope for subsequent analysis.

[0048] b. Whole-cell patch-clamp neuroelectrophysiological test:

[0049] Buffer solution:

[0050] 1. Artificial cerebrospinal fluid (aCSF): 127 mM of NaCl / 3 mM of KCl / 26 mM of NaHCO3 / 1.25 mM of NaH2PO4 / 2 mM of CaCl2 / 1 mM of MgSO4 / 10 mM of D - glucose, pH adjusted to 7.45.

[0051] 2. Pipette solution: 140 mM of potassium gluconate / 10 mM of NaCl / 0.5 mM of EGTA / 10 mM of HEPES / 3 mM of ATP - Mg / 0.4 mM of GTP, pH adjusted to 7.3.

[0052] Recording: iPSC - differentiated neurons were adhered to a cover glass and replaced in a recording chamber containing artificial cerebrospinal fluid (95% O2 + 5% CO2). Electrodes were pulled using a Sutter P - 97 puller (Sutter instrument, Novato, CA) with a 1.5 mm / 1.0 mm inner diameter capillary glass (World Precision Instruments PG52151 - 4), polished with an MF - 830 microforge (Narishige, Tokyo, Japan), and filled with pipette solution. For neuroelectrophysiology, recordings were made with an Axoclamp 200B (Axon Instruments, Union city, CA). Changes in cell membrane current were recorded in voltage - clamp mode. Action potentials of nerve activity were recorded in current - clamp mode. Cell stimulation and recording were performed as follows. 1. The cell potential was maintained at - 60 mV, and increased by 10 mV every 400 ms step - by - step from - 80 mV to + 40 mV to detect sodium - potassium ion currents. 2. The current was increased by 20 pA each from - 60 pA to 120 pA to record action potentials.

[0053] c. Analyze dopamine secreted from cells by ELISA sandwich quantification method: Differentiated neurons were dispersed with Accutase, 4×105 Subcultured in a 6-well culture dish at the density of , stimulated with KCl, and after collecting the culture medium every 24 or 48 hours, centrifuged at 1000 rpm for 5 minutes and then quickly stored in a -80 °C refrigerator. The culture medium (or the concentrated culture medium) was quantitatively analyzed by ELISA (Beckman Counter).

[0054] d. Calcium ion image analysis: Cells were seeded on a 10-mm-diameter circular cover glass coated with Geltrex, and NB culture medium containing RevitaCell and Compound E was added and cultured for 3 days. 1 μM Fluo-4 was prepared in a physiological buffer. The cover glass seeded with cells was transferred to a 1 μM Fluo-4 solution and incubated at 37 °C for 40 minutes. Then it was transferred to the physiological buffer and incubated at 37 °C for 20 minutes. The cover glass seeded with cells was transferred to a calcium imaging chamber in preparation for perfusion imaging. Perfusion imaging was performed with the physiological buffer for 30 seconds, switched to 60 mM KCl and perfused for 1 minute, and further perfused with the physiological buffer for 5 minutes. Then it was switched to 1 mM L-glutamic acid and perfused for 1 minute, and further perfused with the physiological buffer for 5 minutes. Images were taken with a microscope (Nikon ECLIPSE Ti2-E) and analyzed with NIS-Elements AR software.

[0055] Evaluation of Parkinson's disease

[0056] Animal transplantation experiment: Using a positioning and fixing device, 6-OHDA was injected into the striatum of animals to destroy substantia nigra dopamine neurons, resulting in a physiological reaction similar to human Parkinson's disease. After inducing dopamine nerve injury in rats by injecting 6-OHDA for 1 month, methamphetamine hydrochloride (2 mg / Kg) was injected subcutaneously, and a rotometer was used to record the rotation speed of the rats. The total duration was 60 minutes. When the rotation speed of the rats exceeded 300 rotations / hr, it was determined that the induction of dopamine nerve injury was successful. An action test was conducted 3 days before the induction surgery, and on the 30th day after the induction surgery, pluripotent stem cells were differentiated into precursor dopamine neurons and injected into the front of the substantia nigra region (i.e., at the striatum, from bregma, A, +1.0, L, -3.0, V, -5.0 and -4.0, TB, 0; 2×10 5 / μl, 2 μl / site point), and action tests (rotometer) were conducted at the 1st, 2nd, 3rd, 4th, 5th, and 6th months. The animals were sacrificed at the 26th week, tissues were collected, and subsequent IF and IHC-related analysis experiments were performed.

[0057] (Example 1) Human induced pluripotent stem cells (iPSCs) were collected and cultured in Essential8 (without feeder layer). After treatment with different concentrations of n-butylenephthalide (100 μM, 200 μM, 500 μM, and 750 μM) for 24 hours, as shown in Figure 1A, the number of human iPSCs treated with 100 μM n-butylenephthalide was significantly less than that in the control group (Ctrl) and the DMSO control group. It was observed that human iPSCs treated with n-butylenephthalide at 500 μM or above were unable to adhere and survive. After treatment with different concentrations of n-butylenephthalide for 48 hours, as shown in Figure 1B, the number of human iPSCs treated with 100 μM n-butylenephthalide was significantly less than that in the control group (Ctrl) and the DMSO control group. The cells in the 200 μM treatment group were clearly floating and dead, and it was observed that there was no cell survival in human iPSCs treated with n-butylenephthalide at 500 μM or above.

[0058] (Example 2) The cultured and collected iPSCs were aggregated into spheres by the SFEB suspension method, suspended in a culture medium, mimicking the three-dimensional structure of the differentiation and transplantation of general pluripotent cells, suspended and differentiated for only 1 day, and aggregated into spheres (embryoid bodies) with a complete structure. Immediately afterwards, different concentrations of n-butylenyl phthalide (100 μM, 200 μM, and 500 μM) were applied for 24 hours. As shown in Figure 2, it was observed that all the suspended spheres treated with 500 μM of n-butylenyl phthalide were dispersed and died. After staining with trypan blue, it was found that the number of live cells decreased when treated with 200 μM, and no viable cells could be observed when treated with 500 μM.

[0059] (Example 3) The collected human iPSCs were aggregated into spheres by the SFEB suspension method and suspended and differentiated into neural stem cells. During the differentiation process, after inducing neural stem cells using a neural induction culture medium composed of DMEM / F12 (2:1) and adding the neural induction factors basic FGF / SB431542 / CHIR99021, different concentrations of n-butylenyl phthalide (50 μM, 100 μM, 200 μM, and 500 μM) were added on the 10th day of suspended differentiation. After 120 hours, as shown in Figure 3A, no cell death or morphological changes were observed in the suspended spheres. To confirm whether the neural stem cells treated with n-butylenyl phthalide still had the ability to differentiate into mature neurons, the suspended spheres treated with different concentrations of n-butylenyl phthalide were cultured until the 38th day of differentiation by the SFEB neural differentiation method, and immunofluorescence staining analysis was used to analyze whether they expressed the neuron-specific transcription protein NeuN and neurofilament (NF). The experimental results showed that all the neural stem cells treated with n-butylenyl phthalide could normally differentiate into mature neurons as shown in Figure 3B, expressed NeuN (green fluorescence) and NF (red fluorescence), and showed a typical nerve fiber morphology.

[0060] (Example 4) To confirm whether n-butylenyl phthalide has toxicity to differentiated somatic cells, neuron cell spheres differentiated until day 25 by the SFEB suspension method were collected. As shown in Fig. 4A, after culturing until day 22 by the SFEB suspension method and then transferring to adherent culture in step 4 of the SFEB neural differentiation method earlier for 3 days, it was found by immunofluorescence staining that mature neuron marker proteins NeuN (green fluorescence) and NF (red fluorescence) were expressed, indicating that the cells differentiated until day 25 by the SFEB suspension method were neuron cells, that is, differentiated neural somatic cells. Different concentrations of n-butylenyl phthalide (50 μM, 100 μM, 200 μM, and 500 μM) were added to the neuron cell spheres differentiated until day 25 and cultured for 120 hours. As shown in Fig. 4B, it was not found that the spheres were damaged or the cells were dispersed and died. Also, as shown in Fig. 4C, the mature neurons differentiated until day 38 by the SFEB neural differentiation method continued to express NeuN (green fluorescence) and NF (red fluorescence) and showed a typical nerve fiber morphology.

[0061] (Example 5) For cells cultured without n-butylenyl phthalide treatment (Ctrl), cells cultured with 500 μM of n-butylenyl phthalide for 120 hours (NPC-BP500) on day 10 of differentiation, and cells cultured with 500 μM of n-butylenyl phthalide for 120 hours (neuron-BP500) on day 25 of differentiation, they were cultured until day 35 of differentiation by the SFEB neural differentiation method and adhered, and after adhesion, they were cultured for another 3 days. For the nerve cells that completed differentiation by the SFEB neural differentiation method, the change amount of the flow of cell membrane calcium ions after stimulation with KCl and glutamate was measured by a calcium ion imaging system. As shown in Figs. 5A and 5B, all three groups of neurons (Ctrl, NPC-BP500, neuron-BP500) showed significant responses to potassium ions and neurotransmitters, and there was no statistically significant difference, indicating that the nervous system cells after treatment with 500 μM of n-butylenyl phthalide still had normal nerve electrophysiological functions.

[0062] (Example 6) To induce neuronal differentiation in the collected human iPSCs by the neural stem cell adhesion differentiation method, during the differentiation process, a neural induction culture medium composed of DMEM / F12 (2:1) and N2 supplement was used, and basic FGF / SB431542 / CHIR99021, which are neural induction factors, was added to induce neural stem cells. As shown in Figure 6, on the 5th day of differentiation, when about half of the cells expressed the pluripotent stem cell-specific protein Oct-4 and the other half of the cells expressed the neural stem cell protein N-cadherin, 50 μM, 100 μM, and 200 μM of n-butenyl phthalide were added. After 24 hours, the results of immunofluorescence staining analysis showed that as the concentration of n-butenyl phthalide increased, the proportion of undifferentiated pluripotent stem cells expressing Oct-4 decreased significantly.

[0063] (Example 7) The dopamine neuron adhesion differentiation process (CHSF-DA differentiation method) of the collected human iPSCs was performed. The flow of the dopamine neuron identification test during differentiation is shown in Figure 7. A brief description of the flow is as follows. On the 12th day of differentiation, the neural stem cell-specific proteins sox-1 and N-cadherin, and the dopamine progenitor cell-specific proteins FOXA2, Lmx1A, and Corin were identified by immunofluorescence staining to determine whether the neural stem cells on the 12th day of differentiation were dopamine neuron progenitor cells. From the 18th day to the 28th day of differentiation, the mature dopamine neurons TH and Nurr1, and the ventral midbrain dopamine neuron Aldh1A1 were identified. Also, on the 35th day of differentiation, dopamine in the neuron culture medium was tested by the ELISA antibody method to confirm the dopamine secretion ability of dopamine neurons, and its neuroelectrophysiological function was measured using the single cell patch-clamp. The results are shown in Figures 8A - 8M.

[0064] Figures 8A - 8C show the changes in cell morphology during the differentiation process. The cells on the 4th day of differentiation (Figure 8A) still have a typical iPSC morphology. The cells on the 11th day (Figure 8B) change to a stacked form with a rosette - like shape of neural stem cells. On the 17th day of differentiation (Figure 8C), many nerve fiber - like structures are observed. As shown in Figures 8D - 8F, on the 12th day of differentiation, most cells co - express neural stem cell and dopamine progenitor cell - specific proteins. Also, on the 18th - 28th days of differentiation (Figures 8G - 8J), multiple dopamine neuron - specific proteins are expressed. For dopamine neurons, the neural function was identified on the 35th day of differentiation. Figure 8K shows that neurons can secrete dopamine into the culture medium only after KCl stimulation. Figure 8L shows that typical inward and outward feedback currents occur on the neuron surface after stimulation at different voltages. Figure 8M shows that a feedback potential difference occurs in neurons after stimulation with different currents. The above results indicate that the dopamine neurons generated by this differentiation flow have no differences in morphology, specific protein expression, neuroelectrophysiological responses, and dopamine secretion function compared with typical dopamine neurons. Therefore, the addition of n - butenylphthalide did not cause abnormalities in the specific protein expression and neural function of dopamine neurons.

[0065] (Example 8) The midbrain dopamine neurons on one side of the rats were destroyed with 6-OHDA to establish a Parkinson's disease rat model. For the rats in which Parkinson's disease was successfully induced, they were divided into two groups on average based on the number of rotations on one side. After 6 weeks after 6-OHDA, a craniotomy was performed. For one group (preBP-DA, a total of 21 rats), dopamine neurons differentiated for 28 days and treated with n-butylenephthalide for 6 days were implanted. For the other group (blank injection solution, a total of 8 rats), an equal volume of injection solution without cells was implanted. After transplantation, cyclosporine A 15 mg / kg was administered daily to suppress the immune rejection reaction. The 21 preBP-DA rats were divided into a specimen group and a behavioral test group. Among them, 12 rats (specimen group) were sacrificed 4 rats each at 2 weeks, 4 weeks, and 8 weeks after cell transplantation, and brain specimens were taken for tissue staining to label the differentiation and survival of the transplanted cells. The remaining 9 rats (behavioral test group) were subjected to a rotameter behavioral test at 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks after cell transplantation, and their natural survival rates were recorded up to 26 weeks. All 9 rats in the behavioral test group were sacrificed 26 weeks after cell transplantation, and brain specimens were taken to analyze the survival and maturation status of the transplanted cells.

[0066] Figure 9B shows the results of immunofluorescence staining of rat brain sections at 2 weeks, 4 weeks, and 8 weeks after transplantation with TH (red, labeling dopamine neurons), STEM121 (green, labeling human cells), and DAPI (blue, labeling cell nuclei). In Figure 9B, the right brain side is the brain side where the original dopamine neurons of the rat were removed by 6-OHDA and human cells were transplanted, and the left brain side is the healthy and normal brain side that has not been treated in the experiment. When comparing the results of immunofluorescence staining on both the left and right brain sides, it was found that the dopamine neurons treated with n-butylenephthalide could survive when transplanted into the rat brain at 2 weeks, could proliferate from 2 to 4 weeks, and a small amount began to mature into dopamine neurons. From 4 to 8 weeks, a large amount matured into dopamine neurons and were distributed throughout the striatum. In addition, Figure 9A shows the improvement of the motor ability of the Parkinson's disease rats transplanted with the above dopamine neurons.

[0067] Figure 10 shows the survival rate statistically analyzed as the number of surviving rats / total number (%) every four weeks, comparing the dopamine neuron transplantation group treated with n-butenyl phthalide with the blank injection control group. The survival rate of rats 24 weeks after cell transplantation was not lower than that of the blank injection control group, and Table 1 shows that no tumors or teratoma-like tissues were observed in the brain sections of cell-transplanted animals. This indicates that dopamine neurons treated with n-butenyl phthalide do not develop tumors or any observable harmful risks after transplantation.

[0068] Summarizing the results of FIGS. 9A, 9B and 10, it shows that after brain transplantation of cells treated with n-butenyl phthalide, there are no tumor risks and detectable adverse effects on animals, and it has a therapeutic effect.

[0069]

Table 1

[0070] Although several specific embodiments of the present invention have been described in detail above, those skilled in the art can make various modifications and changes to the shown embodiments without substantially departing from the teachings and advantages of the present invention. Therefore, such modifications and changes should be included within the scope of the present invention described in the appended claims.

Claims

1. A method for selectively removing undifferentiated pluripotent stem cells, comprising: harvesting pluripotent stem cells; Inducing differentiation of the collected pluripotent stem cells to obtain a cell population having differentiated cells, wherein the cell population further comprises the collected pluripotent stem cells that are undifferentiated; and treating the cell population with an effective amount of a phthalide compound to selectively remove the undifferentiated harvested pluripotent stem cells; The method includes:

2. The method according to claim 1, wherein the phthalide compound is at least one selected from the group consisting of n-butenylphthalide, methylphthalide, 7-methylphthalide, ethylphthalide, n-propenylphthalide, n-butylphthalide, 3-bromophthalide, 5-bromophthalide, 5-chlorophthalide, 6-chlorophthalide, 3,4-dichlorophthalide, tetrachlorophthalide, 3-hydroxy-3-trifluoromethylphthalide, 3-methyl-3-(1-naphthyl)phthalide, 3-(5-fluoro-1-naphthyl)phthalide, 4-amino-3-hydroxyphthalide, 5-carboxyphthalide, 5-cyanophthalide, 7-methoxyphthalide, 7-hydroxy-6-methoxyphthalide, 3-(1,2-dimethyl-3-indolyl)phthalide, phenolphthalein, ligustilide, and sedanolide.

3. The method of claim 1 , wherein the differentiated cells comprise at least one selected from the group consisting of oligopotent stem cells, unipotent stem cells and somatic cells.

4. The method of claim 1 , wherein the differentiated cells in the cell population do not include somatic cells.

5. 2. The method of claim 1, wherein the effective amount of a phthalide compound is administered after the population of cells includes somatic cells.

6. 2. The method according to claim 1, wherein the concentration of the effective amount of the phthalide compound is 10 μM to 1000 μM.

7. 7. The method according to claim 6, wherein the effective amount of the phthalide compound is at a concentration of 50 μM to 800 μM.

8. 2. The method according to claim 1, wherein the treatment time with the effective amount of a phthalide compound is from 1 to 6 days.

9. The method of claim 1 , wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

Images