CN112553160A - Method and culture medium for chemically inducing cortical neurons - Google Patents
Method and culture medium for chemically inducing cortical neurons Download PDFInfo
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- CN112553160A CN112553160A CN202011562592.8A CN202011562592A CN112553160A CN 112553160 A CN112553160 A CN 112553160A CN 202011562592 A CN202011562592 A CN 202011562592A CN 112553160 A CN112553160 A CN 112553160A
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Abstract
The invention relates to the technical field of neuron induction, and particularly discloses a method and a culture medium for chemically inducing cortical neurons. The induction method has clear chemical components and high differentiation efficiency. Moreover, the obtained human cortical neurons have the characteristics of low immunogenicity, short differentiation time and stable electrophysiological activity. Compared with the current internationally popular induction method of the combination of serum and small molecules, the invention completely avoids the potential danger caused by the existence of animal-derived components in the cell culture process, is particularly suitable for the in vitro screening of nervous system disease drugs and the treatment of neurodegenerative diseases, and has great economic and social effects.
Description
Technical Field
The invention belongs to the technical field of neuron induction, and particularly relates to a method and a culture medium for chemically inducing cortical neurons.
Background
Ectoderm is the outermost layer formed during embryonic development, and ectoderm cells gradually differentiate into important systems such as brain, spinal cord, sensory organs, and the like, as organogenesis begins. The nervous system is an important system responsible for functions such as thinking, emotion, perception, and movement. Ectodermal cells are associated with the occurrence of various degenerative diseases, for example, neurodegenerative diseases caused by neuronal aging death are common aging diseases at present, the treatment and care cost of the diseases is extremely expensive, and no specific medicine can be effectively treated in the market. Neurodegenerative diseases include Amyotrophic Lateral Sclerosis (ALS), Parkinson Disease (PD), Alzheimer Disease (AD), etc. The difficulty in the study of neurodegenerative diseases and spinal cord injury is that nerve cells of the central nervous system are not regenerative, and the scarcity of in vitro disease models is a limiting factor in basic research, and such diseases are caused by irreversible damage to the central nerve. The nerve cells include different nerve cell types such as nerve stem cells, mature neurons, astrocytes and oligodendrocytes. Studies of different cell types can reveal the mechanisms of development and progression of the disease. The study of cellular differentiation during development is the basis of pathological studies. Understanding the differentiation and development process of normal cells and the genes regulating the differentiation of stem cells will help to understand the causes of disease formation and develop corresponding disease treatment strategies. Taking cortical nerve cells as an example, the gray matter of the cerebral cortex, which covers both hemispheres of the brain, is the material basis for higher neural activity, and is composed of neurons, nerve fibers, and glia. The pathological changes and injuries of cortical neurons related to cerebral apoplexy and epilepsy are important target targets of various central nervous system diseases.
At present, the traditional cell line is mostly used in the research and development of drugs in related fields, and the rodentia nerve cell line is used for drug screening, so that the normal reaction of human beings to drugs cannot be reflected, most cortical cells in the field of drug screening are derived from rodent primary cells at present, and obvious species differences exist between mice and human beings, which is also one of the reasons for deviation of drug screening; in addition, the source of the human cortical neuron cells is insufficient, the uniformity difference of the human neural stem cells from the donation of the remains or the source of the embryos is obvious, the limitation of ethical use exists, and the industrial grade requirement of high-throughput screening cannot be met; the above factors greatly restrict the accuracy and efficiency of the development of nervous system drugs.
In 2006, the Ministry of elongation in the mountain invented a "cocktail" method consisting of four transcription factors, OCT4, SOX2, KLF4 and c-Myc, which successfully reprograms terminally differentiated dermal fibroblasts into stem cells with differentiation pluripotency, called induced pluripotent stem cells (Cell, 2006, 124(4) pp.663-676; Cell, 2007, 131(5) pp.861-872). These stem cells have a differentiation potential similar to that of embryonic stem cells (embryonic stem cells), and are capable of forming the three germ layers most essential for human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells. This invention breaks through the ethical limitation of using human embryonic stem cells in medicine. Taking a nervous system as an example, in the field of regenerative medicine, at present, the induction of neural stem cells and neurons mostly adopts a Dual SMAD inhibition method (Chambers SM, et al., Nat Biotechnol, 2009, 27(3):275-80), and the neural stem cells obtained by the method can be differentiated into other types of neuronal cells, and the principle is to simulate a signal pathway in the early embryonic development by inhibiting BMP and TGFBeta pathways, so as to induce the generation of the neural stem cells. LDN-193189 and SB431542 are two widely used chemical small molecule inhibitors, which act on ALK2 and ALK3 in BMP4 pathway and ALK5 in TGFBeta pathway to achieve the formation of transplanted endoderm and mesoderm, thereby inducing ectoderm development and neurogenesis. In this way, induced neuronal cells can be obtained in the presence of a serous component (Knockout Serum Replacement) (Chambers SM, et al., Nat Biotechnol., 2013, 30(7): 715-. The research of the induced nerve cells provides a novel idea for the treatment and drug screening of neurodegenerative diseases.
Based on the technical problems in the prior art, there is a need to research a culture medium and an induction method for chemically inducing cortical neurons.
Disclosure of Invention
In view of the above, the present invention aims to provide a method and a culture medium for chemically inducing cortical neurons, which solve the potential risks caused by the presence of animal-derived components in the cell culture process in the prior art, and are therefore suitable for in vitro screening of nervous system disease drugs and treatment of neurodegenerative diseases.
In order to solve the problems, the scheme of the invention is as follows:
the invention provides application of an inhibitor, namely, Crenigacestat in inducing neural stem cells to differentiate into cortical neuron cells; the inhibitor Crenigacestat is used in an amount of 1-20 μ M, preferably 7.5 uM.
The invention also provides a cortical neuron induction culture medium, which comprises a serum-free basal culture medium and an inhibitor Crenicesstat, wherein the dosage of the inhibitor Crenicesstat is 1-20 mu M.
Further, the amount of the inhibitor, Crenigacestat, is 7.5 uM.
Further, the serum-free basal medium comprises vitamins, inorganic salts, small molecule inhibitors, serum substitutes and other components; the vitamins comprise 1ug/ml vitamin E, 1.2uM vitamin B12, and 64mg/L vitamin C; the inorganic salt comprises 1.2g/L sodium bicarbonate, 0.5g/L sodium chloride and 13.6 mu g/L sodium selenite; the small molecule inhibitor comprises 12.5uM LY2157299, 5uM JW 55; the other components comprise 12.5ug/ml D (+) -galactose, 6.3ng/ml progesterone, 23ug/ml putrescine and 20mg/L insulin; the serum substitute is one or more of serum albumin, transferrin and fatty acid, and the dosage is 100 ng/ml.
The invention also provides a method for inducing the pluripotent stem cells to be differentiated into the cortical neuron cells by using the cortical neuron induction culture medium, which comprises the following steps:
1. the pluripotent stem cells induce and differentiate the neural stem cells: performing single-layer adherent culture on the pluripotent stem cells in a serum-free basal culture medium, and inducing the culture medium to perform adherent culture to obtain neural stem cells which form a neural rosette and are uniformly arranged;
2. inducing and differentiating cortical neurons by neural stem cells: and (3) inducing and differentiating the neural stem cells obtained from the S1 by using a cortical neuron induction culture medium to obtain cortical neurons.
Further, in the above method, there is a basement membrane preparation in the adherent culture, which is one or more of a basement membrane glue, laminin and vitronectin.
Further, in the above method, the step 1 further comprises identifying neural stem cell markers Nestin, ZO-1, SOX2, PAX6 and Nestin to verify the induction result of the neural stem cells.
Further, in the above method, the step 2 further comprises identifying cortical neuron expression genes GAD1, GRID1, GRIN1, VGLUT1 and VGLUT2 to verify the differentiation induction result of the cortical neurons.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a human cortical neuron chemical induction system which does not use animal serum or contain animal-derived components, and the induction system has definite chemical components and high differentiation efficiency.
2. The human cortical neurons obtained by induction have the characteristics of low immunogenicity, short differentiation time and stable electrophysiological activity; compared with the current internationally popular induction method of combining serum and small molecules, the method not only greatly saves the production cost, but also shows great purity and yield advantages, completely avoids potential risks caused by the existence of animal-derived components in the cell culture process, and is particularly suitable for in vitro screening of nervous system disease drugs and treatment of neurodegenerative diseases, thereby having great economic and social effects.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram showing the results of molecular identification of cells obtained by the present invention using NouvNeu002 for chemical induction of neural stem cells.
FIG. 2 is a diagram showing the results of identifying the respective expressed genes when the human cortical neuron cell 7d is induced by the human neural stem cell of the present invention.
FIG. 3 shows the expression difference results of different concentrations of Crenigacestat on cortical neurons markers MAP2 and VGlut 1.
FIG. 4 is a schematic diagram showing the cell morphology change of the experimental group and the control group with different concentrations of Crenigastat in the invention within 24 hours of induction.
FIG. 5 is a schematic diagram showing the difference between the experimental group and the control group of different concentrations of Crenigacestat for apoptosis at 7 days of induction.
Detailed Description
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
The invention provides a serum-free basal medium which comprises vitamins, inorganic salts, small molecule inhibitors, serum substitutes and other components, wherein:
the vitamins comprise 1ug/ml vitamin E, 1.2uM vitamin B12, and 64mg/L vitamin C; other vitamins may also be selected as desired.
The inorganic salt comprises 1.2g/L sodium bicarbonate, 0.5g/L sodium chloride and 13.6 mu g/L sodium selenite;
the small molecule inhibitor comprises 12.5uM LY2157299, 5uM JW 55;
the serum substitute is one or more of serum albumin, transferrin and fatty acid, and the dosage is 100 ng/ml;
the other ingredients include 12.5ug/ml D (+) -galactose, 6.3ng/ml progesterone, 23ug/ml putrescine, 20mg/L insulin.
The invention also provides a culture medium for inducing differentiation of cortical neurons, which comprises the serum-free basal culture medium and a secretase inhibitor Crenigacestat, wherein the dosage of the secretase inhibitor Crenigacetat can be 1-20 mu M, and specifically can be 1 mu M, 5 mu M, 7.5uM, 10 mu M, 15 mu M and 20 mu M; preferably 7.5 uM.
The invention carries out single-layer adherent culture on the pluripotent stem cells in a serum-free culture medium, the serum-free culture medium does not contain components such as serum, BMP or TGF signal transduction pathway substances, and the like, and the serum-free neural induction culture medium is used for adherent culture for 20 days to obtain the neural stem cells which form the neural rosette and are uniformly arranged. So as to further use serum-free culture medium containing specific secretase inhibitor Crenigacestat to induce differentiation to obtain cortical neurons.
The serum-free medium in the present invention means that it does not contain serum isolated directly from blood. Serum is a clear liquid portion of plasma that is free of fibrinogen or blood cells and remains a liquid after blood coagulation. The serum-free medium may contain a serum substitute, and examples of the serum substitute include purified substances such as serum albumin, transferrin, fatty acids, and the like, which are well known in the art as substances that can substitute for serum.
Example 1 preparation of human neuronal cell Induction basal Medium NouvNeu-002 and cortical neuron Induction Medium NouvNeu-C
A human nerve cell induction basal medium is prepared according to the following formula, and is called NouvNeu-002 for short:
duchenne modified eagle's medium (DMEM medium), vitamin E (1ug/ml), vitamin B12(1.2uM), vitamin C (L-ascorbyl acid, 64mg/L), Progesterone (Progesterone, 6.3ng/ml), Putrescine (Putrescine, 23ug/ml), sodium bicarbonate (NaHCO)31.2g/L), Sodium Chloride (Sodium Chloride, 0.5g/L), Sodium selenite (13.6. mu.g/L), D (+) -galactose (D (+) -galactose, 12.5ug/ml), LY2157299(12.5uM), JW55(5uM), Insulin (Insulin, 20mg/L), recombinant human transferrin (100 ng/ml).
1-20. mu.M of Crenigacestat (optimum concentration is 7.5. mu.M) was added to NouvNeu-002 cell culture medium. Cortical neuron induction medium NouvNeu-C was formed.
Control experiments, referred to by Jue Wang et al (Brain Research,2016,1634.), cortical neuron induction using DAPT, were performed in N2B27 medium, hereinafter CK, and formulated as: 50% Duchenne modified eagle's F12 medium (DMEM-F12), 50% neural basal medium (Neurobasal), 0.1% N2 additive (N2 supplement), 2% B27 additive (B27supplement), and 10uM DAPT.
Example 2 Induction culture of human neural Stem cells
Human pluripotent stem cells include embryonic pluripotent stem cells such as the H9 cell line and human induced pluripotent stem cells. Wherein the human induced pluripotent stem cells are obtained by reprogramming CD34+ cells according to a reprogramming medium and a culture method of reprogramming induced pluripotent stem cells (ZL 201910050800.7).
Human pluripotent stem cells include embryonic pluripotent stem cells such as the H9 cell line and human induced pluripotent stem cells. Wherein the human-induced pluripotent stem cells are obtained by reprogramming CD34+ cells in accordance with "a reprogramming medium and a method for culturing the reprogramming-induced pluripotent stem cells" (patent publication No. CN 109628383A).
Human pluripotent stem cells T25 cell culture flasks were coated with matrigel (STEMCELL technologies), plated, and incubated in a 37 ℃ incubator for more than one hour. According to 1 × 106Cells were seeded in T25 flasks for expansion and passaging.
When performing nerve induction, coating a 6-hole culture plate with 50ug/ml polylysine (SIGMA, P6407), laying the plate, and incubating in a 37 deg.C incubator for more than 3 hr; then further coated with 5ug/ml Laminin (Laminin, I2020, SIGMAALDRICH), plated and incubated in a 37 ℃ incubator for more than 3 hours. When the pluripotent stem cells reached 70% coverage, they were digested with EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped with DMEM. After washing and centrifuging the cells, the ratio of the cells to the total volume of the cells is 2X 105The ratio of each flask was re-inoculated in a T25 plate. The induction was carried out using NouvNeu002, cultured at 37 ℃ in 5% CO2(Panasonic, MCO-18AC), medium was changed daily until the neural stem cell rosette formed.
Neural stem cells were digested with EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped using DMEM. After washing and centrifuging the cells, the ratio of the cells to the total volume of the cells is 2X 105The culture medium was re-inoculated into T25 culture plates coated with 50ug/ml polylysine and 5ug/ml laminin at a ratio per flask for further cultureOr differentiation. The culture conditions were 37 ℃ and 5% CO2(Panasonic，MCO-18AC)。
Taking the neural stem cells of the generation after the induction of the NouvNeu002 system is completed to carry out immunofluorescence staining identification: fixing the cells with 4% paraformaldehyde at room temperature for 40 minutes, and washing twice with DPBS buffer solution; then carrying out permeabilization treatment for 5 minutes by using 0.1% TritonX-100, and washing twice by using a DPBS buffer solution; cells were then incubated overnight at 4 ℃ with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then, the antibody diluted with the DPBS buffer was added, incubated at 37 ℃ for 2 hours, washed three times with the DPBS buffer, photographed, and photographed using a Leica DMi 8. Details of antibody use are shown in table 1. The results are shown in FIGS. 1a to 1d, wherein FIG. 1a shows the reconstitution of the structure of neural rosette after in vitro passaging using neural stem cells obtained from NouvNeu 002; FIG. 1b shows a neural stem cell obtained from NouvNeu002, the neural rosette structure of which expresses the neural stem cell marker Nestin; FIG. 1c shows a neural stem cell obtained from NouvNeu002, the structure of which neural rosette expresses neural rosette marker ZO-1; FIG. 1d shows the positioning assembly of FIGS. 1a-1 c; the above results show that NouvNeu002 can induce the formation of neural stem cells in vitro.
Table 1: antibodies for cellular immunofluorescence staining
The transcriptional changes from different marker genes during neurogenesis were detected using Q-PCR and compared between pluripotent stem cells and neural stem cells induced using NouvNeu 002. Total RNA extraction was performed using RNeasy Mini or Micro Kit (QIAGEN), and 1mg of RNA was used to synthesize cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-reactive PCR were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal reference. All data were analyzed using delta-Ct method. Each set of experiments was performed using three replicates and variance statistics were performed. The primer sequences used to identify the genes encoding the different cellular markers are shown in table 2. The results are shown in FIGS. 1e-1g, which indicate that the neural stem cells induced by NouvNeu002 express the neural stem cell markers.
TABLE 2 Gene primers for different markers in the induction of pluripotent stem cells into neural stem cells
| Gene | Primer and method for producing the same |
| SOX2-F | CATGCAGGTTGACACCGTTGG |
| SOX2-R | ATGGATTCTCGGCAGACTGATTCA |
| PAX6-F | TCTTTGCTTGGGAAATCCG |
| PAX6-R | CTGCCCGTTCAACATCCTTAG |
| Nestin-F | TCAAGATGTCCCTCAGCCTGGA |
| Nestin-R | TGGCACAGGTGTCTCAAGGGTAG |
| ACTIN-F | TCCCTGGAGAAGAGCTACGA |
| ACTIN-R | AGCACTGTGTTGGCGTACAG |
Example 3 Induction of human cortical neuronal cells
For the induction culture of the human cortical neuron cells, a cell culture flask of a first layer is coated with polylysine (Poly-L-ornithine solution, Sigma), the cell culture flask is placed in an incubator at 37 ℃ for incubation for more than 16 hours after being plated, a cell culture flask of a second layer is coated with laminin (Biolamin 521LN), and the cell culture flask is placed in the incubator at 37 ℃ for incubation for more than 2 hours after being plated. According to 4 x 104Per cm2The human neural stem cells are inoculated into culture flasks or well plates for passage. The cells were cultured with NouvNeu-C containing 1/5/10/20. mu.M Crenigacestat at 37 ℃ in 5% CO2The cell culture box is used for induction culture so as to screen the concentration of the Crenigacestat. Control group was performed by culturing the same batch of cells in the medium containing 10. mu.M DAPT.
Example 4 identification of human cortical neuronal cells
4.1 identification of Crenigacestat-induced cortical neuron expression genes
Q-PCR was used to detect transcriptional changes in different marker genes during differentiation from neural stem cells to neurons. Total RNA extraction was performed using induced cortical neurons obtained from NouvNeu and N2B27 systems using RNeasy Mini or Micro Kit (QIAGEN), and 1mg of RNA was synthesized as cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-reactive PCR were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal reference. All data were analyzed using delta-Ct method. Each set of experiments was performed using three replicates and variance statistics were performed. The primer sequences used to identify the genes encoding the different cellular markers are shown in table 3.
The analysis results are shown in FIG. 2, using Actin expression gene as internal reference and DAPT induced human cortical neurons as control. The results show that the transcriptional levels of the genes of Crenigacet-induced human cortical neurons GAD1, GRID1 and GRIN1 are not significantly different from those of DAPT-induced human cortical neurons, while the transcriptional levels of the genes of 1-10 mu M Crenigacet-induced human cortical neurons GRIA1, VGLUT1 and VGLUT2 are higher than those of DAPT-induced human cortical neurons of a control group, which indicates that the human cortical neurons obtained by induction of the invention are more mature than those of the control group.
Table 3: real-time fluorescent quantitative PCR primer sequence
4.2 identification of protein expressed by Crenigacestat-induced human cortical neurons
And identifying the markers of the human cortical neuron cells induced by the Crenigacestat by adopting an immunofluorescence experiment. Inducing the human cortical neuron cells on a 24-well plate containing a glass sheet according to the method of example 4, and washing twice with a DPBS buffer solution after the induction is finished; fixing the cells with 4% paraformaldehyde at room temperature for 10 minutes, and washing twice with DPBS buffer solution; then carrying out permeabilization treatment for 2 minutes by using precooled anhydrous methanol at 4 ℃, and washing twice by using a DPBS buffer solution; then cells were incubated overnight at 4 ℃ with DPBS buffer containing 10% bovine serum albumin; then adding an antibody diluted by DPBS buffer solution containing 2% bovine serum albumin, incubating for 1h at room temperature, and washing twice by using the DPBS buffer solution; then adding an antibody diluted by DPBS buffer solution containing 2% bovine serum albumin, incubating for 45min at room temperature in a dark place, and washing twice by using the DPBS buffer solution; finally, DAPI containing 5. mu.g/mL was added and incubated at room temperature for 2min, washed three times with DPBS buffer, and photographed. Details of antibody use are shown in table 4. The expression difference of the markers MAP2 and VGlut1 of the cortical neurons by different concentrations of Crenigacestat is identified, and the result shows that the expression of the markers MAP2 and VGlut1 of the cortical neurons is positively correlated with the concentration of the Crenigacestat. 3a-3c show marker expression of cortical neurons at 1uM Crenigacestat concentration; FIG. 3a shows VGlut1 expression under 1uM Crenigacestat concentration conditions; FIG. 3b shows the expression of MAP2 at a concentration of 1uM Crenigacestat; FIG. 3c shows the composite picture of FIG. 3a and FIG. 3 b; FIGS. 3d-3f show marker expression of cortical neurons at 5uM Crenigacestat concentration; FIG. 3d shows VGlut1 expression at a concentration of 5uM Crenigacestat; FIG. 3e shows MAP2 expression at a concentration of 5uM Crenigacestat; FIG. 3f shows the composite picture of FIG. 3d and FIG. 3 f; FIGS. 3g-3i show marker expression of cortical neurons at 10uM Crenigacestat concentration; FIG. 3g shows VGlut1 expression at a concentration of 10uM Crenigacestat; FIG. 3h shows MAP2 expression at a concentration of 10uM Crenigacestat; fig. 3i shows the composite picture of fig. 3g and fig. 3 h. The results show that cortical neuron marker expression is correlated with Crenigacestat concentration.
Table 4: antibody for immunofluorescence staining of cortical neuronal cell marker protein
4.3 identification of Crenigacestat-induced cortical neuron morphology
A High Content imaging System (Molecular devices) is adopted to observe morphological differences of cortical neurons in the process of differentiation induction of the Crenigacestat and the DAPT. A corresponding number of human neural stem cells were seeded in a coated 96-well plate according to the method of example 4, and induction culture was performed in a high content imaging system using NouvNeu-C medium containing 1/10/20. mu.M crenigacettat, four parallel wells per concentration, and the same batch of cells in the four parallel wells was cultured in a control group using DAPT medium containing 10. mu.M. Four fields of view were set up for each well, every 1h for a total of 48 h. The results are shown in fig. 4, and the results show that the method induces and obtains the human cortical neurons more quickly than the control group.
Example 5 Long-term apoptosis assay of Crenigacestat-induced human cortical neuronal cells
Q-PCR was used to detect transcriptional changes in different marker genes during differentiation from neural stem cells to neurons. The human Neural Stem Cells (NSC) obtained in example 2 and cells induced for 7 days in NouvNeu-C medium containing 0/1/7.5/15/20. mu.M Crenigaprocess tat were subjected to total RNA extraction using RNeasy Mini or Micro Kit (QIAGEN), and 1mg of RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-reactive PCR were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal reference. All data were analyzed using delta-Cthod. Each set of experiments was performed using three replicates and variance statistics were performed. The primer sequences used to identify the genes encoding the different cellular markers are shown in table 5.
The analysis result is shown in fig. 5, and the result shows that the human cortical neurons obtained by the induction of the invention have higher maturity than the control group, wherein the result shows that the marker of apoptosis is obviously increased when the concentration is higher than 7.5 uM.
Table 5: apoptosis detection primer used for long-term culture
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.
Claims (10)
1. The application of a secretase inhibitor Crenigacestat in inducing neural stem cells to differentiate into cortical neuron cells.
2. Use according to claim 1, characterized in that the inhibitor Crenigacestat is used in an amount of 1-20 μ M.
3. Use according to claim 1, characterized in that the inhibitor Crenigacestat is used in an amount of 7.5 μ M.
4. A cortical neuron induction culture medium is characterized by comprising a serum-free basal culture medium and an inhibitor Crenicesstat, wherein the dosage of the inhibitor Crenicesstat is 1-20 mu M.
5. The culture medium according to claim 4, wherein the inhibitor Crenigacestat is used in an amount of 7.5. mu.M.
6. The culture medium of claim 4, wherein the serum-free basal medium comprises vitamins, inorganic salts, small molecule inhibitors, serum replacement, and other components;
the vitamins comprise 1ug/ml vitamin E, 1.2uM vitamin B12, and 64mg/L vitamin C;
the inorganic salt comprises 1.2g/L sodium bicarbonate, 0.5g/L sodium chloride and 13.6 mu g/L sodium selenite;
the small molecule inhibitor comprises 12.5uM LY2157299, 5uM JW 55;
the other components comprise 12.5ug/ml D (+) -galactose, 6.3ng/ml progesterone, 23ug/ml putrescine and 20mg/L insulin;
the serum substitute is one or more of serum albumin, transferrin and fatty acid, and the dosage is 100 ng/ml.
7. A method of inducing differentiation of pluripotent stem cells into cortical neuronal cells in a culture medium according to claim 4, comprising the steps of:
s1, inducing and differentiating the pluripotent stem cells into neural stem cells: performing single-layer adherent culture on the pluripotent stem cells in a serum-free basal culture medium, and inducing the culture medium to perform adherent culture to obtain neural stem cells which form a neural rosette and are uniformly arranged;
s2, inducing and differentiating cortical neurons by neural stem cells: and (3) inducing and differentiating the neural stem cells obtained from the S1 by using a cortical neuron induction culture medium to obtain cortical neurons.
8. The method of claim 7, wherein said adherent culture comprises a basement membrane preparation selected from the group consisting of one or more of basement membrane glue, laminin and vitronectin.
9. The method of claim 7, wherein the step S1 further comprises identifying neural stem cell markers Nestin, ZO-1, SOX2, PAX6 and Nestin to verify the induction result of the neural stem cells.
10. The method of claim 7, wherein the step S2 further comprises identifying cortical neuron expression genes GAD1, GRID1, GRIN1, VGLUT1 and VGLUT2 to verify the differentiation inducing result of the cortical neuron.
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