Patent Application
20250188411
This application claims priority to Taiwanese Application No. 112148328, filed on Dec. 12, 2023, the entire contents of which are incorporated herein by reference.
The present disclosure relates to a method for culturing stem cells, particularly a method for selectively removing undifferentiated stem cells.
Stem cells are cells with the capacity for self-renewal and differentiation. Based on differentiation potential, stem cells are generally categorized from highest to lowest into totipotent stem cells, pluripotent stem cells, oligopotent stem cells, and unipotent stem cells.
Pluripotent stem cells refer to undifferentiated cells that have the ability to self-renew and can differentiate into cells from the three germ layers of the human body. These pluripotent stem cells can be classified into embryonic stem cells and induced pluripotent stem cells based on their origin. Embryonic stem cells are derived from the inner cell mass of human blastocyst-stage embryos and can differentiate into various types of somatic cells. Induced pluripotent stem cells, which possess similar properties and differentiation potential as embryonic stem cells, can be generated by reprogramming somatic cells through the delivery of specific genes and proteins or the use of small chemical molecules.
Pluripotent stem cells have the capacity to differentiate into a variety of somatic cells, can be cultured and expanded in large quantities in vitro, and can be induced to form specific somatic cells, organs, and tissues. The in vitro differentiation process of pluripotent stem cells can simulate embryonic development, enhancing our understanding of human developmental processes. In disease research, pluripotent stem cells derived from patients can be transformed into various types of pathological cells, aiding in the exploration of unknown disease processes and pathogenic mechanisms, as well as in drug development and precision medicine.
Compared to pluripotent stem cells, oligopotent stem cells have a lower differentiation potential but still retain the ability to differentiate into somatic cells. Oligopotent stem cells can be isolated from adults, with common types including mesenchymal stem cells derived from umbilical cord, umbilical cord blood, placenta, amniotic fluid, bone marrow, and adipose tissue.
Many researchers focused on applying pluripotent and oligopotent stem cells in the cell transplantation treatment of various diseases. It is anticipated that differentiated cells, such as neural cells, glial cells, ocular cells, cardiomyocytes, blood cells, islet cells, and mesenchymal stem cells, can be used in the treatment of Parkinson's disease, spinal cord injury, macular degeneration, corneal transplantation, myocardial infarction, tumor immunotherapy, diabetes, and autoimmune diseases, with several of these applications already in clinical trials.
During the differentiation process of specific somatic cells for transplantation, if any residual undifferentiated pluripotent stem cells remain, there is a risk of teratoma formation after transplantation into a living organism, which increases the potential for cancer. Current methods for removing undifferentiated pluripotent stem cells from transplanted cell populations primarily involve flow cytometry sorting to exclude cells with pluripotent stem cell-specific surface antigens or selecting cells with specific somatic cell surface antigens to avoid the presence of undifferentiated pluripotent stem cells. Although this method can reduce the residual undifferentiated pluripotent stem cells, it is time-consuming, costly, and labor-intensive, and the genetic, cellular properties, and health of certain specific somatic cells may be altered after sorting. Recent studies have reported that early treatment with quercetin can reduce the presence of residual undifferentiated pluripotent stem cells.
In summary, to reduce the risks associated with the use of stem cells in clinical cell transplantation treatments and to improve the safety of the applications of stem cells, there is an urgent need to develop methods for selectively removing undifferentiated pluripotent stem cells, which is a crucial and essential point for reducing the carcinogenic potential of undifferentiated pluripotent stem cells.
The present disclosure relates to a method for selectively removing undifferentiated pluripotent stem cells. The method includes: collecting pluripotent stem cells; inducing differentiation of the collected pluripotent stem cells to obtain a cell population comprising differentiated cells and undifferentiated pluripotent stem cells; and administering an effective amount of a phthalide compound to the cell population to selectively remove the undifferentiated collected stem cells in the cell population.
In one aspect of the present disclosure, the phthalide compound is selected from the group consisting of n-butylidenephthalide, 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-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 serdanolide. In another aspect of the present disclosure, the phthalide compound include n-butylidenephthalide, n-butyl phthalide, tetrachlorophthalide, and phenolphthalein. In yet another aspect, the phthalide compound is n-butylidenephthalide.
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 period, 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, the differentiated cells in the cell population do not include somatic cells. In another aspect, the effective amount of the phthalide compound is administered after somatic cells are present in the cell population.
In one aspect of the present disclosure, an concentration of the effective amount of the phthalide compound is from 10 μM to 1000 μM. In another aspect, the concentration of the effective amount of the phthalide compound is from 50 μM to 800 μM.
In one aspect of the present disclosure, the effective amount of the phthalide compound is administered for 1 to 6 days.
In one aspect of the present disclosure, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
The present disclosure effectively reduces the risk associated with the subsequent use of differentiated pluripotent stem cells and improves the safety of their applications by selectively removing undifferentiated pluripotent stem cells from a population of induced differentiated pluripotent stem cells while retaining the differentiated cells through treatment with an effective amount of the phthalide compound.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The technical solutions set forth in the embodiments of the present disclosure will be described more clearly and completely below. It is apparent that the described embodiments are only some of the many possible embodiments encompassed by the present disclosure and are not intended to limit the scope of the present disclosure. The present disclosure may also be practiced or used in similar or different embodiments. Modifications, changes, substitutions of certain elements or combinations thereof, and other implementations that can be made by those skilled in the art without creative effort are all included within the scope of the present disclosure.
Further, it should be noted that the singular forms “a,” “an,” and “the,” as used herein, are intended to include plural referents unless explicitly limited to a single referent. Additionally, the term “or” is used interchangeably with the term “and/or” unless the context clearly indicates otherwise.
As used herein, the term “about” refers to a variance or range within 20%, preferably within 10%, and more preferably within 5% of the stated numerical value, numerical range, or ratio. The quantitative values stated herein are approximations and are intended to be inferred even when the term “about” is not explicitly used. The numerical ranges stated herein encompass all values within those ranges. For example, the range of 50 μM to 800 μM includes values such as 50 μM, 50.01 μM, 50.1 μM, and 50.5 μM, and also includes all sub-ranges within the stated range, such as 50 μM to 700 μM, 88 μM to 650 μM, and 166μ M to 521 μM.
The terms “including,” “comprising,” “containing,” and “having,” as used herein, refer to the inclusion of specified elements (such as components or steps) in the objects, methods, or uses of the present disclosure. Unless the context clearly indicates otherwise, these terms are intended to be open-ended, meaning that unspecified elements may also be present in the objects, methods, and uses of the present disclosure, whether or not they are necessary. Therefore, the exclusion of unspecified elements is not implied by these terms.
The term “stem cells,” as used herein, refers to cells with 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 may be isolated from a cell population containing stem cells (such as embryonic stem cells or mesenchymal stem cells) or produced by the reprogramming of somatic cells (such as induced pluripotent stem cells).
The term “removal of undifferentiated collected pluripotent stem cells,” as used herein, refers to the removal of undifferentiated pluripotent stem cells from a cell population 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 embodiment of the present disclosure, the pluripotent stem cells may be, but are not limited to, embryonic stem cells or induced pluripotent stem cells; the oligopotent stem cells may be, but are not limited to, neural stem cells, mesenchymal stem cells, or hematopoietic stem cells.
The term “effective amount,” as used herein, refers to the dosage of a compound that produces the desired effect, specifically, the dosage of a phthalide compound that results in the selective removal of undifferentiated pluripotent stem cells.
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 comprising differentiated cells, wherein the cell population further comprises 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.
In some embodiments of the present disclosure, the method involves culturing and subculturing the pluripotent stem cells before collection.
Methods for inducing the differentiation of pluripotent stem cells are well known to those skilled in the art. A non-limiting example of methods for inducing differentiation of pluripotent stem cells involves transferring the pluripotent stem cells into an appropriate differentiation medium which promotes differentiation of the pluripotent stem cells into specific cell types. For instance, pluripotent stem cells can be differentiated into neural stem cells by culturing in a neural induction medium (NI medium, Gibco) with the addition of specific proteins and small molecules such as basic FGF, SB431542, and CHIR99021.
In some embodiments of the present disclosure, the phthalide compound may be selected from 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-methoxyphthalide, 7-hydroxy-6-methoxyphthalide, 3-(1,2-dimethyl-3-indolyl) phthalide, phenolphthalein, ligustilide, and sedanolide. In other embodiments of the present disclosure, the phthalide compound may be n-butylidenephthalide, n-butyl phthalide, tetrachlorophthalide, or phenolphthalein. In an exemplary embodiment, n-butylidenephthalide is used to illustrate the effect of selective removal of undifferentiated stem cells.
In one embodiment of the present disclosure, the effective amount of the phthalide compound is administered during a differentiation period of the pluripotent stem cells to selectively remove undifferentiated pluripotent stem cells. The differentiation period may be the differentiation of pluripotent stem cells into oligopotent stem cells, during which the differentiated cells in the cell population may not yet include somatic cells, or the differentiation of oligopotent stem cells into somatic cells. In another embodiment of the present disclosure, the effective amount of the phthalide compound is administered after the differentiation of pluripotent stem cells into somatic cells to achieve selective removal of undifferentiated pluripotent stem cells. Those skilled in the art can easily determine the appropriate differentiation period of the pluripotent stem cells differentiating into unspecific oligopotent stem cells or somatic cells and a time point of completion of differentiation based on cell surface antigens known in the art.
In one embodiment of the present disclosure, the concentration of the effective amount of the phthalide compound is about 10 μM to 1000 μM. In other 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 800μ, about 50μ M to 750μ, or about 50μ M to 500 μM. In some 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, 350μ, 400 μM, 450 μM, 500μ, 550 μM, 600 μM, 650μ, 700 μM, 750 μM, or 800 μM. The aforementioned values can be optionally selected as the maximum or minimum to derive a numerical range.
In one embodiment of the present disclosure, the effective amount of the phthalide compound is administered for approximately 1 to 6 days. In other embodiments of the present disclosure, the effective amount of the phthalide compound is administered for about 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, or 144 hours; the aforementioned numerical endpoints can be selected as either the maximum or minimum value to derive a numerical range.
In another embodiment of the present disclosure, the differentiated stem cells are treated with 100 μM of the phthalide compound for 6 days to selectively remove undifferentiated stem cell.
The present disclosure also relates to an use of the phthalide compounds for the selective removal of undifferentiated pluripotent stem cells.
The present disclosure will be further illustrated with specific examples which should not be considered as limitation to the scope of the present disclosure.
1. Culture of iPSCs in Essential 8 Feeder-Free Culture Medium: Human iPSCs were cultured in Essential 8 medium (Gibco) in culture dishes treated with basement membrane matrix (Matrigel). Subculture was performed when the cells reached 70% to 80% confluence, approximately 3 to 5 days. During subculture, the cells were rinsed twice with PBS until the edges began to lift, followed by treatment with a cell dissociation regent (Accutase) for 1 to 5 minutes to round up most of the cells. The cell dissociation regent was then diluted with DMEM/F12, and DMEM medium was added. The cells were scraped with even force by a cell scraper, dispersed into cell mass with appropriate size by mechanical force, and plated at an appropriate ratio (about 1:5 to 1:10) with 10 μM of Y27632 added.
2. Dopaminergic Neuron Adherent Differentiation of Human iPSCs (CHSF-DA Differentiation Method): Human iPSCs, cultured in feeder-free environment, were subcultured at a 1:10 ratio for dopaminergic neurons differentiation. For induction, a neural induction medium composed of DMEM/F12 (2:1) and N2 supplement were used and dopaminergic precursor cell-inducing factors basic FGF (FGF-basic, as 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 in days 1 to 12 of the differentiation process. On day 13 of the differentiation process, the medium was switched to a neuron culture medium (Neurobasal medium and N2/B27 supplements), with SAG (0.5 μM) added from days 13 to 18. SAG was removed on day 18, and 100 μM of n-butylidenephthalide was added on days 23 to 28 for a total of 6 days. The cells were subsequently cultured in Neurobasal medium with N2/B27 supplements, the medium was changed every 2 days during the differentiation process with proteins and small molecules re-added.
3. Neural Differentiation of Human iPSCs Using the Serum-Free Embryoid Body (SFEB) Method: The differentiation of iPSCs into neural cells was performed in four steps. The first three steps involved SFEB suspension, in which suspended spheroid cells were cultured for 35 days, and the fourth step is a 3-day cell adhesion growth:
Step 1: iPSCs were aggregated into suspended embryoid bodies (EBs), which is an initial step of differentiation. iPSCs were treated with 1 mg/mL Dispase II until the edges rolled up, then rinsed three times with PBS. The iPSCs were collected by a cell scraper and dispersed into cell mass with appropriate size by visual inspection of pipet. After suspended in Essential 6 medium containing RevitaCell, and the iPSCs were transferred to a sterile, non-adherent 6 cm2 culture plate for a 2-day suspension culture, with daily medium changing.
Step 2: The EBs were transferred to a 15 mL centrifuge tube for settlement at room temperature, and the EBs were cultured in suspension for 2 days by adding neural induction medium (NI medium, Gibco) after removal of the supernatant. Basic FGF (10 ng/mL), SB431542 (10 μM), and CHIR99021 (3 μM) were added, with daily observations under a microscope.
Step 3: The neural induction medium in Step 2 was replaced with Neurobasal medium (NB medium, Gibco), and the cells from Step 2 were cultured in suspension with continuous addition of basic FGF (10 ng/mL). The medium was changed every two days in this stage, with a total culture period of 31 days.
Step 4: The cells were attached to a culture plate coated with 1% basement membrane matrix or laminin/ornithine. The culture medium was Neurobasal medium with basic FGF (10 ng/mL). The cells grow outward from the center of the colony, and after 3 days of continued culture, the cells were ready for experimentation.
4. Neural Stem Cell Adhesion Differentiation of Human iPSCs (Neural Stem Cell Adhesion Differentiation Method): Human iPSCs, cultured in a feeder-free environment, were subcultured at a 1:10 ratio for neural stem cell differentiation. The differentiation was induced by using a neural induction medium composed of DMEM/F12 (2:1) and N2 supplement, with the addition of basic FGF (10 mg/mL), SB431542 (2 μM), and CHIR99021 (3 μM). The medium was changed every two days with proteins and small molecules re-added. Culture time for this neural stem cell adhesion differentiation is up to 7 days.
Administration of n-Butylidenephthalide (N-BP)
a. Drug Efficacy and Concentration Testing: n-Butylidenephthalide was diluted to different concentrations and administered to cells at final concentrations ranging from 50 to 750 μM. The types of administered cells include cultured human iPSCs, human iPSCs aggregated into SFEBs for one day, neural stem cells on day 10 of SFEB differentiation, neurons on day 25 of SFEB differentiation, and cells on day 5 of neural differentiation by the adhesion. The concentrations and treatment durations were as described in the Examples.
b. Application of n-Butylidenephthalide in Dopaminergic Neuron Preparation: 100 μM of n-Butylidenephthalide was added from days 23 to 28 during the CHSF-DA differentiation process for a total of 6 days.
a. Immunofluorescence Staining Method: Chamber slides were surface-treated with 1% basement membrane matrix for 3 to 4 hours, and the slides were set aside after the matrix was removed. Cells were enzymatically dissociated into smaller clumps and seeded onto the chamber slides. After a few days of culture until the cells adhered and extended outward to present a rosette-like neural tube morphology, identification and staining for neural stem cell and various neuronal precursor cell markers are performed. The culture medium was removed before immunofluorescence staining, and the cells were gently washed 2 to 3 times with PBS at room temperature or 37° C. Adding 200 μL of 4% paraformaldehyde to the cell, the cells were fixed after 20 minutes at room temperature. After removing the 4% paraformaldehyde, the cells were gently washed three times PBS, each for 5 minutes, and PBS was then removed. Adding 200 μL of 99% methanol or 0.1 to 0.3% Triton for 5 to 10 minutes at 4° C. to perforate the cell membranes. After removal and evaporation of methanol or Triton, the cells were gently washed three times with PBS, 5 minutes each time. Adding 5% horse serum at room temperature for 1 hour to mask the cells, and then adding primary antibodies prepared in 3% horse serum (the concentration thereof is prepared based on the required concentration of the antibody) after removal of 5% horse serum. After overnight conjugation of the primary antibody, the primary antibody was removed, and the cells were washed three times with PBST (PBS with Tween 20) for 5 minutes each. Adding the secondary antibody with an action time of 1 hour at room temperature in the dark, and the secondary antibody is prepared in PBS at a concentration of 1:500. The secondary antibody was removed, and the cells were gently washed three times with PBST for 5 minutes each time. Adding 200 μL of DAPI (1 μg/mL) to the cells for 10 minutes at room temperature in the dark to perform nuclear staining. After removal of DAPI, the cells were washed twice with PBST for 5 minutes each time. After removing the PBST, PBS was added to keep the cells moist. The chamber slides were removed, and the cells were mounted with a long coverslip and mounting medium. The slides were stored in the dark at 4° C. for subsequent analysis under a fluorescence microscope.
b. Whole-Cell Patch-Clamp Neuroelectrophysiological Testing:
1. Artificial cerebrospinal fluid (aCSF): NaCl 127 mM/KCl 3 mM/NaHCO326 mM/NaH2PO4 1.25 mM/CaCl2) 2 mM/MgSO4 1 mM/D-Glucose 10 mM, pH was adjusted to 7.45.
2. Pipette Solution: The pipette solution consists of K gluconate 140 mM/NaCl 10 mM/EGTA 0.5 mM/HEPES 10 mM/ATP-Mg 3 mM/and GTP 0.4 mM, pH was adjusted to 7.3.
Recording: Differentiated neurons derived from iPSCs were attached to coverslips and placed in a recording chamber containing artificial cerebrospinal fluid (95% O2+5% CO2). The electrodes used were made from 1.5 mm/1.0 mm inner diameter capillary glass (World Precision Instruments PG52151-4), drawn using a Sutter P-97 puller (Sutter Instrument, Novato, CA) and polished with an MF-830 microforge (Narishige, Tokyo, Japan). The electrodes were filled with pipette solution. Neuronal electrophysiology was recorded using an Axoclamp 200B (Axon Instruments, Union City, CA). Membrane currents were recorded in voltage clamp mode, while action potentials were recorded in current clamp mode. The following steps were used for cell stimulation and recording:
c. Quantitative Analysis of Dopamine Secretion Using ELISA Sandwich Method: Differentiated neurons were dissociated using Accutase, and subcultured at a density of 4×105 cells per well in a 6-well plate. Following stimulation with KCl, the culture medium was collected every 24 or 48 hours, centrifuged at 1000 rpm for five minutes, and the supernatant was rapidly stored at −80° C. The culture medium (or concentrated culture medium) was then subjected to ELISA quantification (Beckman Coulter).
d. Calcium Ion Imaging Analysis: Cells were seeded onto 10 mm diameter coverslips coated with Geltrex and cultured in NB medium supplemented with RevitaCell and Compound E for 3 days. A 1 μM solution of Fluo-4 was prepared in physiological buffer. The coverslips were incubated in the Fluo-4 solution at 37° C. for 40 minutes, followed by incubation in physiological buffer at 37° C. for 20 minutes. The coverslips were then transferred to a calcium imaging chamber for perfusion imaging. The cells were perfused with physiological buffer for 30 seconds, then switched to 60 mM KCl perfusion for one minute, followed by physiological buffer perfusion for five minutes. Subsequently, the cells were perfused with 1 mM L-glutamic acid for one minute, followed by physiological buffer perfusion for five minutes. Images were captured using a Nikon ECLIPSE Ti2-E microscope and analyzed with NIS-Elements AR software.
Animal transplantation experiment: A stereotaxic instrument was used to inject 6-OHDA into the striatum of rats to destroy dopamine neurons in the substantia nigra, thereby inducing a physiological response similar to human Parkinson's disease. One month after 6-OHDA injection and dopamine neuron damage induction, the rats were injected subcutaneously with methamphetamine hydrochloride (2 mg/kg). The number of rotations was recorded using a rotameter for a total of 60 minutes; rats with more than 300 rotations per hour were considered to have successfully induced dopamine neuron damage. Behavioral tests were conducted three days before surgery, and on day 30 post-induction, pluripotent stem cells were differentiated into precursor dopamine neurons and injected into the anterior substantia nigra (striatum) at the following coordinates: bregma: A, +1.0; L, −3.0; V, −5.0 and −4.0; TB, 0; with 2×105 cells/μL, 2 L/site. Behavioral testing was performed at 1, 2, 3, 4, 5, and 6 months using a rotometer. At week 26, the animals were sacrificed, and tissues were collected for subsequent immunofluorescence (IF) and immunohistochemistry (IHC) analysis.
Human induced pluripotent stem cells (iPSCs) were collected and cultured in Essential 8 medium without a 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 shown in
Cultured and collected iPSCs were aggregated into spheres using the SFEB suspension method and suspended in culture medium to simulate the three-dimensional structure of general pluripotent cell differentiation and transplantation. After only one day of suspension differentiation, the cells aggregated into structurally complete spheres (embryoid bodies). After 24 hours of treatment with different concentrations of n-butylidenephthalide (100 μM, 200 μM, and 500 μM), it was observed in
Collected human iPSCs were aggregated into spheres using the SFEB suspension method and differentiated into neural stem cells. During the differentiation process, the cells were cultured in a neural induction medium composed of DMEM/F12 (2:1) with the addition of basic FGF, SB431542, and CHIR99021 as neural induction factors. Starting on day 10 of suspension differentiation, different concentrations of n-butylidenephthalide (50 μM, 100 μM, 200 μM, and 500 μM) were added for 120 hours. As shown in
To confirm whether n-butylidenephthalide was harmful to differentiated somatic cells, neuronal cell spheroids differentiated up to day 25 using the SFEB suspension method were collected. As shown in
Three sets of cells were prepared: one set of cells cultured without n-butylidenephthalide treatment (Ctrl), another set cultured with 500 μM n-butylidenephthalide for 120 hours on day 10 of differentiation (NPC-BP500), and a third set cultured with 500 μM n-butylidenephthalide for 120 hours on day 25 of differentiation (Neuron-BP500). These cells were initially cultured using the SFEB neural differentiation method until day 35 of differentiation, at which point they were attached and cultured for an additional three days. The nerve cells, differentiated using the SFEB neural differentiation method, were tested using a calcium ion imaging system to observe changes in calcium ion flow across the cell membrane after stimulation with KCl and glutamic acid. As shown in
The collected human iPSCs were differentiated into neurons using the neural stem cell attachment differentiation method. During differentiation, a neural induction culture medium composed of DMEM/F12 (2:1) and N2 supplement was used, along with the neural induction factors basic FGF, SB431542, and CHIR99021. As shown in
The dopamine neuron attachment differentiation process (CHSF-DA differentiation method) was applied to the collected human iPSCs. The test procedure for identifying differentiating dopamine neurons was outlined in
A rat model of Parkinson's disease was established by destroying unilateral midbrain dopamine neurons using 6-OHDA. After successfully inducing Parkinson's disease, the rats were evenly divided into two groups based on the average number of unilateral rotations. Brain surgery was performed six weeks post-6-OHDA, with one group (preBP-DA, 21 rats) receiving dopamine neurons differentiated for 28 days and treated with n-butylidenephthalide for six days, while the other group (blank injection, eight rats) received an equal volume of injection solution without cells. Post-transplantation, Cyclosporine A (15 mg/kg) was administered daily to suppress immune rejection. The 21 preBP-DA rats were divided into a specimen group and a behavior test group. The specimen group (12 rats) was sacrificed at 2, 4, and 8 weeks post-cell transplantation, with brain specimens collected for tissue staining to determine the differentiation and survival of the transplanted cells. The remaining nine rats (behavior test group) underwent rotameter behavioral tests at 4, 8, 12, 16, 20, and 24 weeks post-cell transplantation, with natural survival rates recorded up to 26 weeks. All nine rats in the behavior test group were sacrificed 26 weeks post-cell transplantation, and brain specimens were collected to analyze the survival and maturation of the transplanted cells.
The combined results from
Although the present disclosure has been described in detail through some specific embodiments above, various modifications and changes may be made to the illustrated embodiments by those skilled in the art without substantially departing from the teachings and advantages of the present disclosure. Therefore, such modifications and changes should still be considered within the scope of the present disclosure as defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 112148328 | Dec 2023 | TW | national |
