Method for selectively removing undifferentiated pluripotent stem cells

Abstract

The invention relates to a method for selectively removing undifferentiated pluripotent stem cells, which comprises the following steps of: firstly inducing stem cell differentiation, and then applying an effective amount of phthalide compound to remove the undifferentiated stem cells.

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] Stem cells refer to cells with the potential for self-renewal and differentiation. According to their differentiation potential from high to low, they are generally divided into totipotent stem cells, pluripotent stem cells, oligopotent stem cells, and unipotent stem cells.

[0003] Pluripotent stem cells refer to primitive cells that have not yet differentiated, have the ability of self-renewal, and can differentiate into cells of the three germ layers of the human body. According to their sources, they can be divided into embryonic stem cells and induced pluripotent stem cells. Among them, embryonic stem cells come from the inner cell mass of the human blastocyst embryo and can differentiate into various somatic cells. In addition, by forcibly expressing specific genes and proteins or using chemical small molecules in somatic cells, induced pluripotent stem cells can be generated through cell reprogramming and have similar properties and differentiation potential to 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 simulate embryonic development, enabling us to further understand the process of human development. Applied to disease research, the pluripotent stem cells of patients can be transformed into various diseased cells, which helps to explore the unknown process and pathogenic mechanism 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 isolated 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 committed to applying the above-mentioned pluripotent stem cells and oligopotent stem cells to cell transplantation therapy for various diseases. Currently, it is expected to apply differentiated nerve cells, glial cells, ocular cells, cardiomyocytes, blood cells, islet cells, and mesenchymal stem cells to Parkinson's disease, spinal cord injury, macular degeneration, corneal transplantation, myocardial infarction, tumor immunotherapy, diabetes, and autoimmune diseases, and many of them have entered clinical trials.

[0007] When applying stem cells to transplantation applications, if there are residual undifferentiated pluripotent stem cells during the specific somatic cell differentiation process, teratomas may be produced after transplantation into a living body, and the carcinogenic risk may be increased. In order to remove undifferentiated pluripotent stem cells from the transplanted cell population, current methods mainly use flow cytometry to exclude cells with pluripotent stem cell-specific surface antigens, or collect cells with specific somatic cell surface antigens to avoid the residual of undifferentiated pluripotent stem cells. Although this method can eliminate the residual of undifferentiated pluripotent stem cells, it consumes a lot of time, cost and labor, and the genes, cell properties and cell health of some specific somatic cells may change after sorting. In recent years, some papers have reported that early treatment with quercetin can reduce the residual of undifferentiated pluripotent stem cells.

[0008] In summary, in order to reduce the risks of stem cell applications in clinical cell transplantation therapy and improve their application safety, there is an urgent need in this field to develop methods for selectively removing undifferentiated pluripotent stem cells. This is an important and key development focus for reducing the carcinogenicity of undifferentiated pluripotent stem cells. Summary of the Invention

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

[0010] In one aspect of the present disclosure, the phthalide compound is selected from at least one of the group consisting of n-butylidenephthalide, 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-fluoryl-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. In another aspect of the present disclosure, the phthalide compound includes n-butylidenephthalide, n-butylphthalide, tetrachlorophthalide and phenolphthalein. In another aspect of the present disclosure, the phthalide compound is n-butylidenephthalide.

[0011] In one aspect of the present disclosure, the effective amount of the phthalide compound is administered during the differentiation of the pluripotent stem cells. During the differentiation 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 population do not include somatic cells. In another aspect of the present disclosure, the effective amount of the phthalide compound is administered after somatic cells in the cell population.

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

[0013] In one aspect of the present disclosure, the treatment time of 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.

[0015] Through the treatment with an effective amount of the phthalide compound, the present disclosure selectively removes undifferentiated pluripotent stem cells in the induced differentiated pluripotent stem cells and retains the cells with completed differentiation, so as to effectively reduce the risk of subsequent applications of the differentiated pluripotent stem cells and improve the safety of their applications. Brief Description of the Drawings

[0016] Figure 1A and 1B are cell morphology diagrams of human induced pluripotent stem cells (iPSCs) treated with different concentrations of n-butylidenephthalide.

[0017] Figure 2 are cell morphology diagrams of embryoid bodies of human iPSCs treated with different concentrations of n-butylidenephthalide.

[0018] Figure 3A and 3B are cell morphology diagrams of human iPSCs differentiated into neural stem cells and then treated with different concentrations of n-butylidenephthalide.

[0019] Figure 4A is an immunofluorescence staining diagram for confirming the differentiation degree of human iPSCs differentiated by the SFEB suspension method on the 25th day, and it can be seen that they have been differentiated into neuron cells (i.e., nerve somatic cells). Figure 4B and 4C are cell morphology diagrams of human iPSCs differentiated into neuron cells and then treated with different concentrations of n-butylidenephthalide.

[0020] Figure 5A and 5BStatistical chart of the calcium ion change amount generated by neurotransmitters in differentiated neurons after being treated with n-butylidenephthalide. The groups include the response of cells cultured without n-butylidenephthalide treatment (Ctrl) to neurotransmitters; the response of cells cultured with 500 μM n-butylidenephthalide for 120 hours on the 10th day of differentiation (NPC-BP500) to neurotransmitters; and the response of cells cultured with 500 μM n-butylidenephthalide for 120 hours on the 25th day of differentiation (neuron-BP500) to neurotransmitters. The statistical results show that there is no significant difference in the calcium ion change amount in neurons after KCl and glutamate stimulation among the groups. The above tests were all carried out when cultured to the 38th day of differentiation (d38).

[0021] Figure 6 Immunofluorescence staining images of Oct-4 and N-cadherin (Ncad) in differentiating nerve cells treated with n-butylidenephthalide.

[0022] Figure 7 Flow chart of the identification test for differentiating dopamine neurons.

[0023] Figures 8A to 8M Results images of the identification test for differentiating dopamine neurons. Figures 8A to 8C Cell morphology images at different differentiation stages; Figures 8D to 8F Immunofluorescence staining images for identifying specific proteins of neural stem cells and dopamine progenitor cells; Figures 8G to 8J Immunofluorescence staining images for identifying differentiated cells with dopamine neuron-specific antigens from the 18th to 28th day of differentiation; Figure 8K Bar chart for testing dopamine secretion ability; Figure 8L and 8M Membrane current and membrane potential images for testing nerve electrophysiological functions respectively.

[0024] Figure 9A and 9B Images of the motor ability test and brain tissue immunofluorescence staining of Parkinson's disease rats transplanted with dopamine neurons treated with n-butylidenephthalide respectively.

[0025] Figure 10 Survival rate comparison chart of Parkinson's disease rats in the dopamine neuron transplantation group (preBP-DA) and the control group (Medium). Detailed implementation methods

[0026] The technical solutions described in the embodiments of the present invention will be described more clearly and completely below. Obviously, the described embodiments are only a part of the numerous embodiments covered by 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. Other embodiments obtained by those of ordinary skill in the art without creative efforts, such as modifications, changes, substitutions of certain elements or combinations thereof, etc., are all included in the scope of the present invention.

[0027] Further note that in the singular forms "a" and "the" described herein, unless clearly limited to one referent, they are meant to include a plurality of referents. Additionally, unless the context clearly indicates otherwise, the term "or" can be used interchangeably with the term "and / or".

[0028] The term "about" as described herein refers to an error or range of the described numerical value, numerical range or ratio within 20% of that numerical value, numerical range or ratio, preferably within 10%, and more preferably within 5% floating. The quantified numerical values described herein are approximate values, meaning that they can also be inferred if the term "about" is not used. The numerical ranges described herein cover all numerical values falling within that numerical range. For example, the numerical range of 50 μM to 800 μM covers numerical values such as 50 μM, 50.01 μM, 50.1 μM, 50.5 μM, etc., and also covers all sub-ranges falling within that numerical range. The sub-ranges are enclosed by the respective numerical values falling within that numerical range. For example, the numerical range of 50 μM to 800 μM covers sub-ranges such as 50 μM to 700 μM, 88 μM to 650 μM, 166 μM to 521 μM, etc.

[0029] The terms "comprising", "including", "containing", "having", etc. as described herein mean that there is a certain element (such as a component or a step, etc.) in the objects, methods, uses, etc. of the present invention, and unless the context clearly indicates otherwise, those unrecorded and unspecified elements also exist in an open-ended manner in the objects, methods, uses of the present invention, whether necessary or not. That is, those unrecorded and unspecified elements are not restrictively excluded.

[0030] The term "stem cells" as described herein refers to cells having the potential for self-renewal and differentiation into somatic cells, including totipotent stem cells, pluripotent stem cells, oligopotent stem cells, and unipotent stem cells. Stem cells can be stem cells isolated from a cell population containing stem cells (such as embryonic stem cells or mesenchymal stem cells), or can be stem cells generated by induced reprogramming of somatic cells (such as induced pluripotent stem cells).

[0031] As used herein, "removing undifferentiated collected pluripotent stem cells" refers to removing pluripotent stem cells from a cell population of oligopotent stem cells, unipotent stem cells, and / or somatic cells after at least a portion of the collected pluripotent stem cells have differentiated into oligopotent stem cells, unipotent stem cells, and / or somatic cells. In one aspect of the present disclosure, the pluripotent stem cells can be, but are not limited to, embryonic stem cells or induced pluripotent stem cells; and the oligopotent stem cells can be, but are not limited to, neural stem cells, mesenchymal stem cells, or hematopoietic stem cells.

[0032] As used herein, the term "effective amount" refers to the dose of a compound administered that produces the desired effect, i.e., the dose of a phthalide compound administered that produces the effect of selectively removing undifferentiated pluripotent stem cells.

[0033] The present disclosure relates to a method for selectively removing undifferentiated pluripotent stem cells, which includes: collecting pluripotent stem cells; inducing the collected pluripotent stem cells to differentiate 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 compound to the cell population to selectively remove the undifferentiated collected pluripotent stem cells.

[0034] In some specific embodiments of the present disclosure, the method of the present disclosure cultures and subcultures the pluripotent stem cells before collecting the pluripotent stem cells.

[0035] The treatment methods for inducing the differentiation of pluripotent stem cells are well-known to those of ordinary skill in the art. A non-limiting example of the treatment method for inducing the differentiation of pluripotent stem cells is to transfer the pluripotent stem cells into a suitable differentiation culture medium environment, which causes the pluripotent stem cells to differentiate into a specific cell morphology. For example, transferring pluripotent stem cells into a neural induction medium (Neural induction medium; NI medium, Gibco) and culturing with specific protein and small molecule compositions such as basic FGF, SB431542, CHIR99021, etc. can cause the pluripotent stem cells to differentiate into neural stem cells.

[0036] 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, propylidenephthalide, 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-hydroxy-6-methoxyphthalide, 3-(1,2-dimethyl-3-indolyl)phthalide, phenolphthalein, ligustilide, and sedanolide. In some other specific embodiments of the present disclosure, the phthalide compound is n-butylidenephthalide, butylphthalide, tetrachlorophthalide, and phenolphthalein. In an exemplary embodiment, the present disclosure illustrates the effect of selectively removing undifferentiated stem cells with n-butylidenephthalide.

[0037] In a specific embodiment of the present disclosure, the effective amount of the phthalide compound is administered during the differentiation of the pluripotent stem cells to achieve the effect of selectively removing undifferentiated pluripotent stem cells. Among them, the period of pluripotent stem cell differentiation can be the period when pluripotent stem cells differentiate into oligopotent stem cells, and the differentiated cells in the cell population at this time may not yet include somatic cells; or the period when the oligopotent stem cells differentiated from the pluripotent stem cells differentiate into somatic cells. In another aspect of the present disclosure, the effective amount of the phthalide compound is administered after the somatic cells differentiated from the pluripotent stem cells are completed to achieve the effect of selectively removing undifferentiated pluripotent stem cells. Those of ordinary skill in the art can easily determine the differentiation period when pluripotent stem cells differentiate into unspecified oligopotent stem cells or somatic cells and the time point after the differentiation is completed based on well-known cell surface antigens.

[0038] In a 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 optionally selected as the maximum or minimum value to derive a numerical range.

[0039] In a specific embodiment of the present disclosure, the treatment time of the effective amount of the phthalide compound is about 1 to 6 days. In other specific embodiments of the present disclosure, the treatment time of the effective amount of the phthalide compound is about 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, or 6 days; the above-mentioned multiple numerical endpoints can be optionally selected as the maximum or minimum value to derive a numerical range.

[0040] In a specific embodiment of the present disclosure, differentiated stem cells are treated with 100 μM phthalide compound for 6 days to selectively remove undifferentiated stem cells.

[0041] The present disclosure also relates to the use of phthalide compounds for selectively removing undifferentiated pluripotent stem cells.

[0042] The following specific specific examples are used to further illustrate the present invention, but it should not be regarded as a limitation to the scope of the present invention.

[0043] Materials and Methods

[0044] Culture and Differentiation of Human Pluripotent Stem Cells

[0045] 1. Culture of iPSCs in Essential 8 without feeder layer : Human iPSCs were cultured in Essential 8 medium (Gibco) and in culture dishes surface-treated with basement membrane matrix (Matrigel). Subculture was performed when the cells reached 70% - 80% confluence after about 3 to 5 days. When subculturing, the cells were first rinsed twice with PBS until the cell edges slightly curled up, and then treated with cell dissociation reagent (Accutase) for 1 to 5 minutes to make most of the cells round up. Then, Accutase was diluted with DMEM / F12, DMEM medium was added, and the cells were evenly scraped up with a cell scraper and mechanically dispersed into appropriate clump sizes. They were plated at an appropriate ratio (about 1:5 to 1:10) and 10 μM Y27632 was added.

[0046] 2. Adherent differentiation process of human iPSC dopamine neurons (CHSF-DA differentiation method) : Human iPSCs cultured without feeder layers were subcultured at a ratio of 1:10 and then induced to differentiate into dopamine neurons. During the differentiation process, from day 1 to day 12, a neural induction medium composed of DMEM / F12 (2:1) and N2 supplement (N2 supplement) was used, and dopamine precursor cell induction factors basic fibroblast growth factor (FGF-basic; also known as FGF-2 or bFGF) (10 mg / mL), SB431542 (2 μM), CHIR99021 (7.5 μM), SAG (1 μM), and LDN193189 (0.2 μM) were added for induction. On day 13 of differentiation, the cells were switched to neuron medium (Neurobasal medium and N2 / B27 supplement), and SAG (0.5 μM) was added during days 13 to 18. SAG was removed on day 18, and 100 μM n-butylphthalide was added from day 23 to day 28 for a total of 6 days. Subsequently, the cells were continuously cultured with Neurobasal medium and N2 / B27 supplement for experiments. During the above period, the culture medium was changed every 2 days, and proteins and small molecule reagents were re-added when changing the medium.

[0047] 3. SFEB (serum free embryoid body) neural differentiation method of human iPSCs : The procedure for differentiating iPSCs into nerve cells was carried out in four steps. The first three steps were the SFEB suspension method, and the suspension sphere cells were cultured for a total of 35 days. The fourth step was cell adhesion growth for 3 days:

[0048] Step 1: iPSCs aggregated into suspension embryoid bodies (EBs). This step was the starting step of differentiation. iPSCs were treated with 1 mg / mL Dispase II until the edges curled up, then rinsed 3 times with PBS. The iPSCs were collected with a scraper, visually mixed and dispersed with a pipette into appropriate clump sizes, and suspended in Essential 6 medium containing RevitaCell and then transferred to a 6 cm 2Aseptic non-adherent culture dish, cultured in suspension for 2 days, and the culture medium was changed daily during this period.

[0049] Step 2: Transfer the EBs to a 15 mL centrifuge tube, let the cells sediment at room temperature, aspirate the supernatant, and then add neural induction medium (Neural induction medium; NI medium, Gibco) for suspension culture for 2 days. At this time, add basic FGF (10 ng / mL), SB431542 (10 μM), and CHIR99021 (3 μM), and observe under the microscope every day.

[0050] Step 3: Replace the neural induction medium in Step 2 with Neurobasal medium (NB medium, Gibco), suspend the culture of the cells in Step 2, and continuously add basic FGF (10 ng / mL). The culture medium needs to be updated every two days during this stage, and the total culture time is 31 days.

[0051] Step 4: Attach the cells in Step 3 to a culture dish surface-treated with 1% basement membrane matrix or laminin / ornithine. The culture medium is Neurobasal medium plus basic FGF (10 ng / mL), and the cells grow from the center of the cell colony outwards. Subsequently, continue to culture for 3 days until nerve fibers grow and then conduct experiments.

[0052] 4. Adherent differentiation process of human iPSC neural stem cells (neural stem cell adherent differentiation method) : Human iPSCs cultured without feeder layer were subcultured and adhered at a ratio of 1:10 and then induced to differentiate into neural stem cells. During the differentiation process, a neural induction medium composed of DMEM / F12 (2:1) and N2 supplement was used, and induction factors basic FGF (10 mg / mL), SB431542 (2 μM), and CHIR99021 (3 μM) were added for induction, and the culture medium was updated every two days. When replacing, proteins and small molecule reagents were re-added. The culture time of this neural stem cell adherent differentiation method is up to 7 days.

[0053] Application method of n-butylidenephthalide (N-BP)

[0054] a. Drug effect and concentration test : Dilute n-butylidenephthalide with different concentrations and administer it to cells (final concentration ranging from 50 to 750 μM). The cell types administered include: human iPSCs in culture, human iPSCs aggregated into SFEB for one day, 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 adherent neural differentiation, etc. The administered concentration and time are as described in the examples.

[0055] b. Application of n-butylphthalide to the preparation process of dopamine (DA) neurons: Add 100 μM of n-butylphthalide from day 23 to day 28 during the CHSF-DA differentiation process for a total of 6 days.

[0056] Cell identification and functional evaluation

[0057] a. Immunofluorescence staining method : Treat a chamber slide with 1% basement membrane matrix for 3 to 4 hours, remove the basement membrane matrix for later use; after dissociating the cells into smaller cell clumps with enzymes, seed them on the chamber slide. After culturing for several days until the cells adhere and spread out to present a rosette-like neural tube cell morphology, perform identification staining for neural stem cells and various neuronal progenitor cells. When performing immunocytochemistry, first remove the culture medium, gently wash with normal temperature or 37 °C PBS 2 to 3 times, add 200 μl of 4% paraformaldehyde to the cells, incubate at room temperature for 20 minutes for cell fixation and then remove, gently wash with PBS 3 times, 5 minutes each time and then remove, add 200 μl of 99% methanol or 0.1 to 0.3% Triton and incubate at 4 °C for 5 to 10 minutes for cell membrane perforation, remove and let it volatilize, gently wash with PBS 3 times, 5 minutes each time, add 5% horse serum and incubate at room temperature for 1 hour for blocking, remove the 5% horse serum, add the primary antibody, the primary antibody is prepared in 3% horse serum and its concentration is prepared according to the required concentration of the antibody. After overnight incubation of the primary antibody, remove the primary antibody, gently wash with PBST (PBS and Tween 20) 3 times, 5 minutes each time, add the secondary antibody and operate in the dark. The secondary antibody is prepared in PBS at a concentration of 1:500, and the secondary antibody acts for 1 hour at room temperature in the dark. Remove the secondary antibody, gently wash with PBST 3 times, 5 minutes each time, perform nuclear staining, add 200 μl of DAPI (1 μg / mL) to the cells, react at room temperature in the dark for 10 minutes, remove DAPI, gently wash with PBST 2 times, 5 minutes each time, remove PBST, add PBS to keep the cells in a moist state, disassemble the chamber slide and seal it with a long coverslip and mounting medium, store in the dark at 4 °C, and observe under a fluorescence microscope for subsequent analysis.

[0058] b. Whole-cell patch clamp neuroelectrophysiological test :

[0059] Buffer solution:

[0060] 1. Artificial cerebrospinal fluid (aCSF): NaCl 127 mM / KCl 3 mM / NaHCO 3 26 mM / NaH 2 PO 4 1.25 mM / CaCl 2 2 mM / MgSO 4 1 mM / D-glucose 10 mM, adjust the pH to 7.45.

[0061] 2. Pipette solution: 140 mM K - gluconate / 10 mM NaCl / 0.5 mM EGTA / 10 mM HEPES / 3 mM ATP - Mg / 0.4 mM GTP, pH adjusted to 7.3.

[0062] Recording: Neurons differentiated from iPSCs were adhered to cover slips and transferred to a recording chamber containing artificial cerebrospinal fluid (95% O 2 + 5% CO 2 ). Electrodes were made of glass capillary with an inner diameter of 1.5 mm / 1.0 mm (World Precision Instruments PG52151 - 4), pulled by a Sutter P - 97 puller (Sutter instrument, Novato, CA) and ground by a MF - 830 microforge (Narishige, Tokyo, Japan), and filled with pipette solution. Neuronal electrophysiology was recorded using an Axoclamp 200B (Axon Instruments, Union city, CA). Membrane current changes were recorded in voltage - clamp mode. Action potentials were recorded in current - clamp mode. Cell stimulation and recording were performed as follows:

[0063] 1. The cell potential was maintained at - 60 mV, gradually increased from - 80 mV by 10 mV every 400 ms to + 40 mV, and sodium and potassium ion currents were detected.

[0064] 2. The current was gradually increased from - 60 pA to + 120 pA in steps of 20 pA and action potentials were recorded.

[0065] c. Analysis of dopamine secreted by cells by ELISA sandwich quantification method : Differentiated neurons were dissociated with Accutase and passaged at a density of 4x10 5 in a 6 - well culture plate. After stimulation with KCl, the culture medium was collected every 24 or 48 hours, centrifuged at 1000 rpm for five minutes, and immediately stored at - 80 °C in a refrigerator. The culture medium (or concentrated culture medium) was quantified by ELISA (Beckman Counter).

[0066] d. Calcium ion imaging analysis: Cells were seeded on 10-mm-diameter round coverslips coated with Geltrex and cultured in NB medium supplemented with RevitaCell and Compound E for 3 days. 1 μM Fluo-4 was prepared in physiological buffer. The coverslips with seeded cells were transferred to the 1 μM Fluo-4 solution and cultured at 37 °C for 40 minutes. Then they were transferred to physiological buffer and cultured at 37 °C for 20 minutes. The coverslips with seeded cells were transferred to a calcium imaging chamber for perfusion imaging preparation. Perfusion imaging was performed with physiological buffer for 30 seconds, then switched to 60 mM KCl perfusion for 1 minute, and then perfused with physiological buffer for 5 minutes. Then it was switched to 1 mM L-glutamate perfusion for 1 minute, and then perfused with physiological buffer for 5 minutes. Images were taken with a microscope (Nikon ECLIPSE Ti2-E) and analyzed with NIS-Elements AR software.

[0067] Parkinson's disease assessment

[0068] Animal transplantation experiment : 6-OHDA was injected into the striatum of animals using a stereotaxic apparatus to damage substantia nigra dopamine neurons and form a physiological response similar to human Parkinson's disease. One month after injecting 6-OHDA to induce dopamine nerve injury in rats, methamphetamine hydrochloride (2 mg / Kg) was injected subcutaneously, and the number of rotations of the rats was recorded using a rotator for a total of 60 minutes; when the rotation rate of the rats exceeded 300 / hr, it was determined that the induction of dopamine nerve injury was successful. Behavioral tests were performed three days before the surgery, and on the 30th day after the induction of the surgery, pluripotent stem cells were differentiated into precursor dopamine neurons and injected in 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; and TB, 0; 2x10 5 / μl, 2 μl / site), and behavioral tests (rotator) were performed at the 1st, 2nd, 3rd, 4th, 5th, and 6th months. The animals were sacrificed at the 26th week and tissues were taken for subsequent IF and IHC related analysis experiments.

[0069] Example 1

[0070] Human induced pluripotent stem cells (iPSCs) were collected and cultured in Essential 8 without feeder layer. After 24 hours of treatment with different concentrations of n-butylidenephthalide (100 μM, 200 μM, 500 μM, and 750 μM), the cell morphology was as Figure 1AAs shown, it can be observed that the number of human iPSC cells treated with 100 μM of n-butylidenephthalide was significantly less than that of the control group (Ctrl) and the DMSO control group. Human iPSCs treated with n-butylidenephthalide at concentrations above 500 μM were unable to adhere and survive. After 48 hours of treatment with different concentrations of n-butylidenephthalide, the cell morphology was as Figure 1B As shown, it can be observed that the number of human iPSC cells treated with 100 μM of n-butylidenephthalide was significantly less than that of the control group (Ctrl) and the DMSO control group. In the 200 μM treatment group, obvious floating and death of cells occurred. No viable cells were observed in human iPSCs treated with n-butylidenephthalide at concentrations above 500 μM.

[0071] Example 2

[0072] The cultured and collected iPSCs were aggregated into spheres by the SFEB suspension method and suspended in the culture medium to simulate the three-dimensional structure of general pluripotent cell differentiation and transplantation. After only one day of suspension differentiation to form intact spheres (embryoid bodies), different concentrations of n-butylidenephthalide (100 μM, 200 μM, and 500 μM) were given. After 24 hours, in Figure 2 it was observable that all the suspended spheres treated with 500 μM of n-butylidenephthalide dispersed and died. After trypan blue staining, it was found that the number of viable cells decreased after treatment with 200 μM, and no viable cells were observed after treatment with 500 μM.

[0073] Example 3

[0074] The collected human iPSCs were aggregated into spheres by the SFEB suspension method and differentiated into neural stem cells. During the differentiation process, a neural induction culture medium composed of DMEM / F12 (2:1) was used, and the neural induction factors basic FGF / SB431542 / CHIR99021 were added for neural stem cell induction. After 120 hours of adding different concentrations of n-butylidenephthalide (50 μM, 100 μM, 200 μM, and 500 μM) starting from the 10th day of suspension differentiation, as Figure 3A shown, no cell death or morphological changes were observed in the suspended spheres. To confirm whether the neural stem cells treated with n-butylidenephthalide still had the ability to differentiate into mature neurons, the suspended spheres treated with different concentrations of n-butylidenephthalide were cultured until the 38th day of differentiation according to the SFEB neural differentiation method, and immunofluorescence staining analysis was used to analyze whether they expressed the neuron-specific transcription protein NeuN and the neurofilament (NF). The experimental results showed that the neural stem cells treated with n-butylidenephthalide were all as Figure 3B shown, could normally differentiate into mature neurons and express NeuN (green fluorescence) and NF (red fluorescence) and presented a typical neurofilament morphology.

[0075] Example 4

[0076] To confirm whether n-butylidenephthalide is harmful to differentiated somatic cells, neurospheres differentiated by the SFEB suspension method for 25 days were collected. As Figure 4A shown, cultured by the SFEB suspension method until day 22, and then prematurely transferred to adherent culture in step 4 of the SFEB neural differentiation method for 3 days. After immunofluorescence staining, it was found that the cells expressed the mature neuron marker proteins NeuN (green fluorescence) and NF (red fluorescence), indicating that the cells differentiated by the SFEB suspension method for 25 days were neuron cells, that is, differentiated neural somatic cells. The neurosphere of neuron cells differentiated to day 25 was cultured with different concentrations of n-butylidenephthalide (50 μM, 100 μM, 200 μM, and 500 μM) for 120 hours. As Figure 4B shown, no damage to the spheres or cell dispersion and death were found. And as Figure 4C shown, the mature neurons that continued to be differentiated by the SFEB neural differentiation method to day 38 expressed NeuN (green fluorescence) and NF (red fluorescence) and showed a typical nerve fiber morphology.

[0077] Example 5

[0078] Cells cultured without n-butylidenephthalide treatment (Ctrl); cells cultured with 500 μM n-butylidenephthalide for 120 hours at day 10 of differentiation (NPC-BP500); and cells cultured with 500 μM n-butylidenephthalide for 120 hours at day 25 of differentiation (neuron-BP500). According to the SFEB neural differentiation method, they were first cultured until adherent at day 35 of differentiation, and then cultured for another 3 days after adherent. The neural cells differentiated by the SFEB neural differentiation method were tested by a calcium imaging system for the change in calcium ion flow across the cell membrane after stimulation with KCl and glutamate. As Figure 5A and 5B shown, it was found that all three groups of neurons (Ctrl, NPC-BP500, neuron-BP500) showed obvious responses to potassium ions and neurotransmitters, and there was no significant difference statistically, indicating that the neural cells treated with 500 μM n-butylidenephthalide still retained normal neuroelectrophysiological functions.

[0079] Example 6

[0080] To differentiate the collected human iPSCs into neurons by the adherent differentiation method of neural stem cells, during the differentiation process, a neural induction culture medium composed of DMEM / F12 (2:1) and N2 supplement was used, and the neural induction factors basic FGF / SB431542 / CHIR99021 were added for neural stem cell induction. As Figure 6As shown, on the fifth day of differentiation, when approximately half of the cells expressed the pluripotent stem cell-specific protein Oct-4 and the other half expressed the neural stem cell protein N-cadherin, after adding 50 μM, 100 μM, and 200 μM of n-butylidenephthalide for 24 hours, the results of immunofluorescence staining analysis showed that as the concentration of n-butylidenephthalide increased, the proportion of undifferentiated pluripotent stem cells expressing Oct-4 decreased significantly.

[0081] Example 7

[0082] The adherent differentiation process of dopamine neurons from human iPSCs collected (CHSF-DA differentiation method) was carried out. The identification test process of dopamine neurons during differentiation was as Figure 7 shown. The process is briefly described as follows: On the 12th day of differentiation, the neural stem cell-specific proteins sox-1 and N-cadherin, 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; on the 18th to 28th days of differentiation, the mature dopamine neuron proteins TH and Nurr1, and the midbrain ventral dopamine neuron protein Aldh1A1 were identified; and on the 35th day of differentiation, dopamine in the neuron culture medium was tested by ELISA antibody method to confirm the dopamine secretion ability of dopamine neurons, and the single cell patch-clamp was used to measure its neuroelectrophysiological function. The results showed in Figures 8A to 8M .

[0083] Figures 8A to 8C showed the morphological changes of cells during differentiation. On the 4th day of differentiation ( Figure 8A ) the cells still had the typical iPSC morphology. On the 11th day ( Figure 8B ) the cells turned into a rosettes-like stacked morphology of neural stem cells. On the 17th day of differentiation ( Figure 8C ) many neuron-like fiber structures could be observed. Figures 8D to 8F showed that on the 12th day of differentiation, the vast majority of cells expressed both neural stem cell and dopamine progenitor cell-specific proteins, and expressed a variety of dopamine neuron-specific proteins on the 18th to 28th days of differentiation ( Figures 8G to 8J ). The neural function of dopamine neurons was identified on the 35th day of differentiation, Figure 8K showed that only after KCl stimulation, the neurons could secrete dopamine into the culture medium; Figure 8L showed that after different voltage stimulations, typical inward and outward feedback currents were generated on the neuron surface; Figure 8MThe feedback potential difference generated by neurons after different current stimulations is shown. The above results show that the dopamine neurons generated through this differentiation process are no different from typical dopamine neurons in terms of morphology, specific protein expression, neuroelectrophysiological responses, and dopamine secretion function. Therefore, the addition of n-butylphthalide does not cause abnormalities in the specific protein expression and nerve function of dopamine neurons.

[0084] Example 8

[0085] 6-OHDA was used to damage the unilateral midbrain dopamine neurons in rats to establish a rat model of Parkinson's disease. After the rats with successfully induced Parkinson's disease were evenly divided into two groups according to the average number of unilateral rotations, a craniotomy was performed 6 weeks after 6-OHDA. One group (preBP-DA, 21 rats in total) was injected with dopamine neurons differentiated for 28 days and treated with n-butylphthalide for 6 days, and the other group (blank injection solution, 8 rats in total) was injected with an equal volume of injection solution without cells. Cyclosporine A (15 mg / kg) was given daily after transplantation to inhibit immune rejection. The 21 preBP-DA rats were divided into a specimen group and a behavioral test group. Among them, 4 rats were sacrificed at 2, 4, and 8 weeks after cell transplantation in 12 rats (specimen group), and brain specimens were taken for tissue staining to calibrate the differentiation and survival of transplanted cells; the remaining 9 rats (behavioral test group) were subjected to a rotator behavior test at 4, 8, 12, 16, 20, and 24 weeks after cell transplantation, and their natural survival rate was recorded until 26 weeks. The 9 rats in the behavioral test group were all sacrificed at 26 weeks after cell transplantation to take brain specimens for analyzing the survival and maturation of transplanted cells.

[0086] Figure 9B The results of immunofluorescence staining of brain slices of rats at 2, 4, and 8 weeks after transplantation with TH (red, calibrating dopamine neurons), STEM121 (green, calibrating human cells), and DAPI (blue, calibrating cell nuclei). Figure 9B The right brain side is the side of the brain 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 without experimental treatment. Comparing the results of immunofluorescence staining on both the left and right brain sides shows that the dopamine neurons treated with n-butylphthalide can survive 2 weeks after being transplanted into the rat brain, can proliferate between 2 and 4 weeks and begin to mature into dopamine neurons in small amounts, and can mature into dopamine neurons in large amounts and distribute throughout the striatum between 4 and 8 weeks. In addition, Figure 9A shows the improvement of the motor ability of the Parkinson's disease rats transplanted with the above dopamine neurons.

[0087] Figure 10The survival rate was statistically analyzed by the number of surviving rats / total number (%) every 4 weeks. It was shown that in the dopamine neuron transplantation group treated with n-butylphthalide, the survival rate of rats at 24 weeks after transplantation of the transplanted cells was not lower than that of the blank injection control group. And Table 1 showed that no tumor or teratoma-like tissue was observed in the brain sections of the cell transplantation animals, indicating that there was no risk of tumor formation or any observable harmful risk after transplantation of the dopamine neurons treated with n-butylphthalide.

[0088] Comprehensively Figure 9A 、 Figure 9B and Figure 10 The results of showed that after intracerebral transplantation of the cells treated with n-butylphthalide, there was no risk of tumor formation or detectable adverse effects on the animals, and it had therapeutic effects.

[0089] Table 1

[0090]

[0091] Although some specific embodiments of the present invention have been described in detail above, those of ordinary skill in the art can make various modifications and changes to the illustrated embodiments without substantially departing from the teachings and advantages of the present invention. Therefore, such modifications and changes should still be included within the scope of the present invention as set forth in the appended claims.

Claims

1. A method for selectively removing undifferentiated pluripotent stem cells, comprising: collecting pluripotent stem cells; Inducing the collected pluripotent stem cells to differentiate to obtain a cell population having differentiated cells, wherein the cell population further includes undifferentiated collected pluripotent stem cells; as well as An effective amount of a phthalide compound is administered to the cell population to selectively remove the undifferentiated collected pluripotent stem cells.

2. The method according to claim 1, wherein: The phthalide compound is selected from at least one of 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 according to claim 1, wherein: The differentiated cells include at least one selected from the group consisting of oligopotent stem cells, unipotent stem cells and somatic cells.

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

5. The method according to claim 1, wherein: The effective amount of the phthalide compound is administered after the cells are present in the cell population.

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

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

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

9. The method according to claim 1, wherein: The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

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