Disclosure of Invention
The invention aims to solve the technical problem of providing a traditional Chinese medicine composition for promoting differentiation of pluripotent stem cells into neural stem cells, which has reliable components, definite structure and no toxic or side effect and is safer.
The invention also solves the technical problem of providing a culture medium for promoting differentiation of pluripotent stem cells into neural stem cells, which is serum-free, animal-derived component-free, feeder layer cell-free, and the obtained neural stem cells have no toxic or side effect and are safer.
The invention also solves the technical problem of providing a method for differentiating the pluripotent stem cells into the neural stem cells, which has wide applicability and can efficiently and rapidly induce and differentiate the monolayer human pluripotent stem cells into the neural stem cells.
In order to solve the technical problems, a traditional Chinese medicine composition for promoting differentiation of pluripotent stem cells into neural stem cells comprises icariin, osthole and resveratrol;
wherein, the addition concentration of the icariin is 1-20 mug/mL; the addition concentration of the osthole is 0.2-10 mug/mL; the adding concentration of the resveratrol is 0.1-5 mug/mL.
In one embodiment, the icariin is added at a concentration of 5-15 μg/mL; the addition concentration of the osthole is 2-8 mug/mL; the adding concentration of the resveratrol is 1-3 mug/mL;
the icariin is an epimedium extract; the osthole is osthole extract; the resveratrol is natural polyphenols in peanut, grape, giant knotweed or mulberry extract.
Preferably, the addition concentration of icariin is 10 mug/mL; the addition concentration of the osthole is 5 mug/mL; the adding concentration of the resveratrol is 2 mug/mL;
correspondingly, the invention provides application of the traditional Chinese medicine composition for promoting differentiation of the pluripotent stem cells into the neural stem cells in preparation of a culture medium for promoting differentiation of the pluripotent stem cells into the neural stem cells.
In order to solve the problems, the invention provides a culture medium for promoting differentiation of pluripotent stem cells into neural stem cells, which comprises the traditional Chinese medicine composition and a basal culture medium, wherein the basal culture medium comprises DMEM/F12, vitamin A, ascorbic acid, riboflavin, DL-alpha-tocopherol, transferrin, D galactose, L-glutamine, lipoic acid, putrescine and sodium selenite.
In one embodiment, the basal medium comprises DMEM/F12, 0.1-0.5. Mu.M vitamin A, 0.05-0.15 mM ascorbic acid, 175-190. Mu.M riboflavin, 1-3. Mu.M DL-alpha-tocopherol, 0.01-0.15. Mu.M transferrin, 10-20. Mu.g/ml D galactose, 1-3 mM L-glutamine, 5-6 mM lipoic acid, 175-190. Mu.M putrescine, and 0.05-1.5. Mu.M sodium selenite;
in the traditional Chinese medicine composition, the addition concentration of icariin is 1-20 mug/mL; the addition concentration of the osthole is 0.2-10 mug/mL; the adding concentration of the resveratrol is 0.1-5 mug/mL.
Preferably, the basal medium comprises DMEM/F12, 0.3. Mu.M vitamin A, 0.1mM ascorbic acid, 183. Mu.M riboflavin, 2. Mu.M DL-alpha-tocopherol, 0.06. Mu.M transferrin, 15. Mu.g/ml D galactose, 2mM L-glutamine, 5.5mM lipoic acid, 183. Mu.M putrescine and 0.08. Mu.M sodium selenite.
In the traditional Chinese medicine composition, the addition concentration of icariin is 5-15 mug/mL; the addition concentration of the osthole is 2-8 mug/mL; the adding concentration of the resveratrol is 1-3 mug/mL.
More preferably, in the traditional Chinese medicine composition, the addition concentration of the icariin is 10 mug/mL; the addition concentration of the osthole is 5 mug/mL; the adding concentration of the resveratrol is 2 mug/mL.
In order to solve the above problems, the present invention provides a method for differentiating pluripotent stem cells into neural stem cells, comprising the steps of:
s1, culturing pluripotent stem cells by using a cell culture medium, and performing cell digestion when the cell fusion degree reaches a preset value to obtain single cells, and inoculating the single cells into a cell culture plate for culture after the single cells are resuspended;
s2, replacing the cell culture medium in the cell culture plate with the induction culture medium, and continuously culturing for a plurality of days under preset induction culture conditions to obtain the differentiated neural stem cells.
In one embodiment, in step S1, the pluripotent stem cells are cultured by using a cell culture medium, the pluripotent stem cells with good state are selected when the cell fusion degree is 65-75%, and the cells are digested for 3-5 min at 36.5-37.5 ℃ by using Ackutase, so that the cell clones are dissociated into single cells;
the single cells were washed and resuspended in cell culture medium containing 8-12. Mu.M ROCK inhibitor, followed by a 2X 10 protocol4 ～3×104 /cm2 Is inoculated in Matrigel coated cell culture plates for 24h.
Preferably, the pluripotent stem cells are cultured by using a cell culture medium, the pluripotent stem cells with good states are selected when the cell fusion degree is 70%, and the cells are digested for 4min at 37 ℃ by using Ackutase, so that the cell clones are dissociated into single cells;
the single cells were washed and resuspended in cell culture medium containing 10. Mu.M ROCK inhibitor, followed by a 2.5X10-fold protocol4 /cm2 Is inoculated in Matrigel coated cell culture plates for 24h.
In one embodiment, the cell culture medium is one of mTeSRTM1, tesrt-E8, and mTeSRTM Plus; preferably, the cell culture medium is mTeSRTM Plus.
The pluripotent stem cells are one of human embryonic stem cells and human induced pluripotent stem cells;
the ROCK inhibitor is Y-27632 or Thiazovivin.
In one embodiment, in step S2, the mixture is heated at 36.5 to 37.5℃and 4 to 6% CO2 Under the condition of induction culture, changing the cell culture medium in the cell culture plate into the induction culture medium every day, and continuously culturing for 6-8 days to obtain the differentiated neural stem cells.
Preferably, in step S2, at 37℃and 5% CO2 Under the condition of induction culture, new induction culture medium is changed every day, and differentiated neural stem cells are obtained after continuous culture for 7 days.
The implementation of the invention has the following beneficial effects:
1. the traditional Chinese medicine composition for promoting differentiation of the pluripotent stem cells into the neural stem cells provided by the invention has the advantages of reliable components, definite structure, capability of effectively promoting differentiation of the human pluripotent stem cells into the neural stem cells under the synergistic effect of icariin, osthole and resveratrol, no toxic or side effect and safer obtained neural stem cells.
2. The culture medium for promoting the differentiation of the pluripotent stem cells into the neural stem cells provided by the invention has no serum, no animal source component and no feeder layer cells, and the traditional animal source growth factors, chemical synthesis small molecules, hormones and the like are replaced by the traditional Chinese medicine composition of icariin, osthole and resveratrol, so that the problems of the animal source culture method and the toxic and side effects of the chemical molecules on the physiological functions of the cells, which restrict the clinical research and application of the neural stem cells, are solved.
3. The method for differentiating the pluripotent stem cells into the neural stem cells has wide applicability, and can efficiently and rapidly induce and differentiate the single-layer human pluripotent stem cells into the neural stem cells with high purity, synchronous differentiation and differentiation potential. Meanwhile, the induction method without feeder cells avoids the pollution of other cells, so that the obtained neural stem cells are safer, and an effective way is provided for treating neurodegenerative diseases and nerve regeneration by cell transplantation.
Detailed Description
The present invention will be described in further detail with reference to the drawings and the detailed description, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
In order to solve the technical problems, a traditional Chinese medicine composition for promoting differentiation of pluripotent stem cells into neural stem cells comprises icariin, osthole and resveratrol;
wherein, the addition concentration of the icariin is 1-20 mug/mL; the addition concentration of the osthole is 0.2-10 mug/mL; the adding concentration of the resveratrol is 0.1-5 mug/mL.
The traditional Chinese medicine composition for promoting differentiation of the pluripotent stem cells into the neural stem cells provided by the invention has the advantages of reliable components, definite structure, capability of effectively promoting differentiation of the human pluripotent stem cells into the neural stem cells under the synergistic effect of icariin, osthole and resveratrol, no toxic or side effect and safer obtained neural stem cells.
Specifically, the icariin is a kidney-tonifying traditional Chinese medicine icariin extract, and is flavonol glycoside. Icariin can affect proliferation of various cells including neural stem cells, mesenchymal stem cells, smooth muscle cells, etc., but has dual effects of promoting and inhibiting regulation of cell proliferation. In fate conversion of pluripotent stem cells and neural stem cells, icariin can mediate Notch or GFs signaling pathway respectively to control, the Notch signaling pathway is a key regulation and control link in a network system for stem cell proliferation and differentiation, and the Notch pathway plays different regulation and control roles in different stages of stem cell differentiation. Further, the addition concentration of icariin affects the differentiation effect, and the addition concentration is in the range of 1-20 mug/mL, so that the icariin has good cell growth and has the effects of promoting cell survival and differentiation to neural stem cells. When the concentration is lower than 1 mug/mL, the differentiation efficiency of the pluripotent stem cells to the neural stem cells is not obviously improved compared with that of a control group without addition; at concentrations above 20. Mu.g/mL, cytotoxic effects are exhibited, i.e., the cells shed and do not proliferate during differentiation. Preferably, the addition concentration of the icariin is 5-15 mug/mL; more preferably, the icariin is added at a concentration of 10 μg/mL.
The osthole is an extract of fructus Cnidii, is a potential inhibitor of histamine H1 receptor, and has various pharmacological effects such as antiinflammatory, analgesic, antibacterial, antipruritic, antioxidant and neuroprotection. Further, the concentration of osthole is 0.2-10 mug/mL. When the addition concentration of osthole is more than 10. Mu.g/mL, the cell differentiation efficiency is lowered, and an increase in concentration shows a toxic effect. Preferably, the addition concentration of the osthole is 2-8 mug/mL; more preferably, the osthole is added at a concentration of 5 μg/mL.
The resveratrol is natural polyphenols in peanut, grape, giant knotweed or mulberry extract, and the source of the resveratrol is not limited to the above list. Resveratrol has antioxidant, antiinflammatory, heart protecting and anticancer effects. Resveratrol inhibits the growth of cells in a dose-dependent manner, and the comprehensive effect of cell differentiation induction is best under the condition that the adding concentration of the resveratrol is 0.1-5 mug/mL. Preferably, the adding concentration of the resveratrol is 1-3 mug/mL; more preferably, the resveratrol is added at a concentration of 2 mug/mL, and under the condition, the comprehensive effect of cell viability and differentiation efficiency is optimal.
In summary, the traditional Chinese medicine composition provided by the invention, wherein icariin with specific addition concentration can play a role in regulating cells by regulating the expression of key proteins or genes Notch1, NICD and Hes in a Notch signal channel; the osthole with specific concentration and resveratrol can play the roles of anti-inflammatory, antioxidant and cell protection. The icariin, the osthole and the resveratrol are cooperatively matched, so that the effect of cell repair can be achieved, and the differentiation of the pluripotent stem cells into the neural stem cells can be promoted through the influence on cell passages on the premise of not generating toxicity and inhibition on the cells, so that the induced differentiation efficiency and the proliferation and differentiation capacity of the neural stem cells are greatly improved.
Correspondingly, the invention provides application of the traditional Chinese medicine composition for promoting differentiation of the pluripotent stem cells into the neural stem cells in preparation of a culture medium for promoting differentiation of the pluripotent stem cells into the neural stem cells.
The culture medium for promoting differentiation of the pluripotent stem cells into the neural stem cells comprises the traditional Chinese medicine composition and a basal culture medium, wherein the basal culture medium comprises DMEM/F12, vitamin A, ascorbic acid, riboflavin, DL-alpha-tocopherol, transferrin, D galactose, L-glutamine, lipoic acid, putrescine and sodium selenite.
The culture medium for promoting the differentiation of the pluripotent stem cells into the neural stem cells provided by the invention has no serum, no animal source component and no feeder layer cells, and the traditional animal source growth factors, chemical synthesis small molecules, hormones and the like are replaced by the traditional Chinese medicine composition of icariin, osthole and resveratrol, so that the problems of the animal source culture method and the toxic and side effects of the chemical molecules on the physiological functions of the cells, which restrict the clinical research and application of the neural stem cells, are solved.
In particular, wherein the DMEM/F12 medium provides essential nutrients for cell growth; transferrin and lipoic acid can improve the efficiency of cell uptake, utilization and conversion of nutrient components, thereby promoting the growth and metabolism of cells; l-glutamine is an important source of capability for cells while promoting the entry of various amino acids into the cell membrane.
In one embodiment, the basal medium comprises DMEM/F12, 0.1-0.5. Mu.M vitamin A, 0.05-0.15 mM ascorbic acid, 175-190. Mu.M riboflavin, 1-3. Mu.M DL-alpha-tocopherol, 0.01-0.15. Mu.M transferrin, 10-20. Mu.g/ml D galactose, 1-3 mM L-glutamine, 5-6 mM lipoic acid, 175-190. Mu.M putrescine, and 0.05-1.5. Mu.M sodium selenite;
in the traditional Chinese medicine composition, the addition concentration of icariin is 1-20 mug/mL; the addition concentration of the osthole is 0.2-10 mug/mL; the adding concentration of the resveratrol is 0.1-5 mug/mL.
Preferably, the basal medium comprises DMEM/F12, 0.3. Mu.M vitamin A, 0.1mM ascorbic acid, 183. Mu.M riboflavin, 2. Mu.M DL-alpha-tocopherol, 0.06. Mu.M transferrin, 15. Mu.g/ml D galactose, 2mM L-glutamine, 5.5mM lipoic acid, 183. Mu.M putrescine and 0.08. Mu.M sodium selenite.
In the traditional Chinese medicine composition, the addition concentration of icariin is 5-15 mug/mL; the addition concentration of the osthole is 2-8 mug/mL; the adding concentration of the resveratrol is 1-3 mug/mL.
More preferably, in the traditional Chinese medicine composition, the addition concentration of the icariin is 10 mug/mL; the addition concentration of the osthole is 5 mug/mL; the adding concentration of the resveratrol is 2 mug/mL.
Correspondingly, the invention also provides a method for differentiating the pluripotent stem cells into the neural stem cells, which comprises the following steps:
s1, culturing pluripotent stem cells by using a cell culture medium, and performing cell digestion when the cell fusion degree reaches a preset value to obtain single cells, and inoculating the single cells into a cell culture plate for culture after the single cells are resuspended;
in one embodiment, in step S1, the pluripotent stem cells are cultured by using a cell culture medium, the pluripotent stem cells with good state are selected when the cell fusion degree is 65-75%, and the cells are digested for 3-5 min at 36.5-37.5 ℃ by using Ackutase, so that the cell clones are dissociated into single cells;
the single cells were washed and resuspended in cell culture medium containing 8-12. Mu.M ROCK inhibitor, followed by a 2X 10 protocol4 ～3×104 /cm2 Is inoculated in Matrigel coated cell culture plates for 24h.
It should be noted that, the cell lysis medium density has a great influence on the differentiation efficiency, when the cell inoculation density is too low, the cells have the phenomena of no adherence, low survival rate, no proliferation and the like, when the cell inoculation density is too high, the cells are easy to be in clonal growth before differentiation, and clone medium cells are stacked to be apoptotic or not differentiated in the induced differentiation process.
Preferably, the pluripotent stem cells are cultured by using a cell culture medium, the pluripotent stem cells with good states are selected when the cell fusion degree is 70%, and the cells are digested for 4min at 37 ℃ by using Ackutase, so that the cell clones are dissociated into single cells;
the single cells were washed and resuspended in the presence of 10. Mu.M ROCIn cell culture medium with K inhibitor, followed by 2.5X104 /cm2 Is inoculated in Matrigel coated cell culture plates for 24h.
In one embodiment, the cell culture medium is one of mTeSRTM1, tesrt-E8, and mTeSRTM Plus; preferably, the cell culture medium is mTeSRTM Plus.
The pluripotent stem cells are one of human embryonic stem cells and human induced pluripotent stem cells;
the ROCK inhibitor is Y-27632 or Thiazovivin.
S2, replacing the cell culture medium in the cell culture plate with the induction culture medium, and continuously culturing for a plurality of days under preset induction culture conditions to obtain the differentiated neural stem cells.
In one embodiment, in step S2, the mixture is heated at 36.5 to 37.5℃and 4 to 6% CO2 Under the condition of induction culture, changing the cell culture medium in the cell culture plate into the induction culture medium every day, and continuously culturing for 6-8 days to obtain the differentiated neural stem cells.
Preferably, in step S2, at 37℃and 5% CO2 Under the condition of induction culture, new induction culture medium is changed every day, and differentiated neural stem cells are obtained after continuous culture for 7 days.
The method for differentiating the pluripotent stem cells into the neural stem cells has wide applicability, and can efficiently and rapidly induce and differentiate the single-layer human pluripotent stem cells into the neural stem cells with high purity, synchronous differentiation and differentiation potential. The neural stem cells may be further induced to differentiate into neurons, astrocytes and oligodendrocytes.
Meanwhile, the induction method without feeder cells avoids the pollution of other cells, so that the obtained neural stem cells are safer, and an effective way is provided for treating neurodegenerative diseases and nerve regeneration by cell transplantation.
The invention is illustrated below by means of specific examples.
Example 1
Method for transforming human pluripotent stem cells into neural stem cells
S1, culturing pluripotent stem cells by using a cell culture medium, and performing cell digestion when the cell fusion degree reaches a preset value to obtain single cells, and inoculating the single cells into a cell culture plate for culture after the single cells are resuspended;
the method specifically comprises the following steps: culturing pluripotent stem cells with mTESR-plus medium, and culturing at a cell fusion rate of about 70% at 1ml/10cm2 Adding Ackuas, and incubating for 4min at 37 ℃ to digest the cells into single cells;
after digestion, the Ackutase is discarded, the single cells are washed by DMEM/F12 and then resuspended in mTESR-plus medium containing 8-12 mu M ROCK inhibitor, and the supernatant is discarded after centrifugation at 1000rpm for 5 min;
then according to 2.5X104 /cm2 Is inoculated in Matrigel coated cell culture plate, 5% CO2 Culturing at 37deg.C for 24 hr.
Wherein, figure 1 is a morphological diagram (magnification is 100 times) of human embryonic stem cells H9 during culture; FIG. 2 shows an immunofluorescence staining pattern of Tra-1-60 protein of cells before induced differentiation (magnification 400); FIG. 3 is a graph showing the cell seeding density (magnification 100 times) induced from day 0.
S2, replacing a cell culture medium in the cell culture plate with an induction culture medium, and continuously culturing for a plurality of days under preset induction culture conditions to obtain differentiated neural stem cells.
Specifically, the induction medium comprises a traditional Chinese medicine composition and a basal medium, wherein the basal medium comprises DMEM/F12, vitamin A with the final concentration of 0.3 mu M, ascorbic acid with the final concentration of 0.1mM, riboflavin with the final concentration of 183 mu M, DL-alpha-tocopherol with the final concentration of 2 mu M, transferrin with the final concentration of 0.06 mu M, D galactose with the final concentration of 15 mu g/ml, L-glutamine with the final concentration of 2mM, lipoic acid with the final concentration of 5.5mM, putrescine with the final concentration of 183 mu M and sodium selenite with the final concentration of 0.08 mu M. The traditional Chinese medicine composition comprises icariin, osthole and resveratrol, wherein the final concentration of the icariin in an induction culture medium is 10 mug/mL, the final concentration of the osthole in the induction culture medium is 5 mug/mL, and the final concentration of the resveratrol in the induction culture medium is 2 mug/mL.
The mTESR-plus medium in S1 was pipetted off, replaced with the induction medium, at 37℃and 5% CO2 Under the condition of induction culture, the new induction culture medium is changed every day, and even distribution of the garland structure of the neural stem cells can be observed after 7 days of culture. FIGS. 4 to 10 are cell morphology diagrams showing the induced differentiation of human pluripotent stem cells into neural stem cells according to example 1.
Example 2
Method for transforming human pluripotent stem cells into neural stem cells
S1, culturing pluripotent stem cells by using a cell culture medium, and performing cell digestion when the cell fusion degree reaches a preset value to obtain single cells, and inoculating the single cells into a cell culture plate for culture after the single cells are resuspended;
the method specifically comprises the following steps: culturing pluripotent stem cells with mTESR-plus medium, and culturing at a cell fusion rate of about 70% at 1ml/10cm2 Adding Ackuas, and incubating for 4min at 37 ℃ to digest the cells into single cells;
after digestion, the Ackutase is discarded, the single cells are washed by DMEM/F12 and then resuspended in mTESR-plus medium containing 8-12 mu M ROCK inhibitor, and the supernatant is discarded after centrifugation at 1000rpm for 5 min;
then according to 2.5X104 /cm2 Is inoculated in Matrigel coated cell culture plate, 5% CO2 Culturing at 37deg.C for 24 hr.
S2, replacing a cell culture medium in the cell culture plate with an induction culture medium, and continuously culturing for a plurality of days under preset induction culture conditions to obtain differentiated neural stem cells.
Specifically, the induction medium comprises a traditional Chinese medicine composition and a basal medium, wherein the basal medium comprises DMEM/F12, vitamin A with the final concentration of 0.1 mu M, ascorbic acid with the final concentration of 0.15mM, riboflavin with the final concentration of 190 mu M, DL-alpha-tocopherol with the final concentration of 3 mu M, transferrin with the final concentration of 0.01 mu M, D galactose with the final concentration of 10 mu g/ml, L-glutamine with the final concentration of 3mM, lipoic acid with the final concentration of 5mM, putrescine with the final concentration of 175 mu M and sodium selenite with the final concentration of 0.05 mu M. The traditional Chinese medicine composition comprises icariin, osthole and resveratrol, wherein the final concentration of the icariin in an induction culture medium is 5 mug/mL, the final concentration of the osthole in the induction culture medium is 2 mug/mL, and the final concentration of the resveratrol in the induction culture medium is 1 mug/mL.
The mTESR-plus medium in S1 was pipetted off, replaced with the induction medium, at 37℃and 5% CO2 Under the condition of induction culture, the new induction culture medium is changed every day, and even distribution of the garland structure of the neural stem cells can be observed after 7 days of culture.
Example 3
Method for transforming human pluripotent stem cells into neural stem cells
S1, culturing pluripotent stem cells by using a cell culture medium, and performing cell digestion when the cell fusion degree reaches a preset value to obtain single cells, and inoculating the single cells into a cell culture plate for culture after the single cells are resuspended;
the method specifically comprises the following steps: culturing pluripotent stem cells with mTESR-plus medium, and culturing at a cell fusion rate of about 70% at 1ml/10cm2 Adding Ackuas, and incubating for 4min at 37 ℃ to digest the cells into single cells;
after digestion, the Ackutase is discarded, the single cells are washed by DMEM/F12 and then resuspended in mTESR-plus medium containing 8-12 mu M ROCK inhibitor, and the supernatant is discarded after centrifugation at 1000rpm for 5 min;
then according to 2.5X104 /cm2 Is inoculated in Matrigel coated cell culture plate, 5% CO2 Culturing at 37deg.C for 24 hr.
S2, replacing a cell culture medium in the cell culture plate with an induction culture medium, and continuously culturing for a plurality of days under preset induction culture conditions to obtain differentiated neural stem cells.
Specifically, the induction medium comprises a traditional Chinese medicine composition and a basal medium, wherein the basal medium comprises DMEM/F12, vitamin A with the final concentration of 0.5 mu M, ascorbic acid with the final concentration of 0.05mM, riboflavin with the final concentration of 175 mu M, DL-alpha-tocopherol with the final concentration of 1 mu M, transferrin with the final concentration of 0.15 mu M, D galactose with the final concentration of 20 mu g/ml, L-glutamine with the final concentration of 1mM, lipoic acid with the final concentration of 6mM, putrescine with the final concentration of 190 mu M and sodium selenite with the final concentration of 0.15 mu M. The traditional Chinese medicine composition comprises icariin, osthole and resveratrol, wherein the final concentration of the icariin in an induction culture medium is 15 mug/mL, the final concentration of the osthole in the induction culture medium is 8 mug/mL, and the final concentration of the resveratrol in the induction culture medium is 3 mug/mL.
The mTESR-plus medium in S1 was pipetted off, replaced with the induction medium, at 37℃and 5% CO2 Under the condition of induction culture, the new induction culture medium is changed every day, and even distribution of the garland structure of the neural stem cells can be observed after 7 days of culture.
Identification of neural stem cells obtained in example 1:
1. sox1 protein immunofluorescence staining identification of neural stem cells
The cells induced and cultured to day 7 in example 1 were collected, and the neural stem cell marker Sox1 protein was detected by immunofluorescent staining, specifically by the following steps: sucking and discarding neural stem cell induction medium, digesting cells by Ackutase at 37 ℃ for 2min, discarding digestive juice, using human neural stem cell expansion medium Stempro NSC SFM suspension cells, inoculating cells into new Matrigel coated culture plate, 5% CO2 Culturing at 37 ℃ for 24 hours. Cells were fixed with 4% paraformaldehyde and PBS 0.2% triton X-100 permeabilized and blocked, using 1: a100-proportion of rabbit monoclonal anti-human Sox1 antibody and Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L) were incubated and observed under a fluorescence microscope after counterstaining with DAPI. The observation results are shown in FIG. 11.
2. Nestin protein flow assay for neural stem cells
The culture medium induced to the 7 th day cells in example 1 was aspirated, the Ackutase digested the cells at 37℃for 2min, the digested solution was discarded, the cells were resuspended to post-count using Cell Stain Buffer after washing, and the diluted cells were 5X 105 Transferring to flow detection tube at volume of 100 μl, sealing cells, adding FITC anti-human nestin moloclonal antibody, incubating, and Cell standAnd (5) performing machine-on detection after Buffer washing and re-suspension. The detection results are shown in FIG. 12.
3. In vitro induction of neural stem cells into motor neurons
The cells induced to culture on day 7 in example 1 were collected as follows: sucking and discarding neural stem cell induction medium, digesting cells by Ackutase at 37 ℃ for 2min, discarding digestive juice, using human neural stem cell expansion medium Stempro NSC SFM suspension cells, inoculating cells into new Matrigel coated culture plate, 5% CO2 Culturing at 37 ℃ for 24 hours. And changing the culture medium to a motor neuron induction culture medium for continuous culture for 6 days to finish differentiation, thus obtaining the cell expressing the Tubulin protein. The formula of the motor neuron induction medium is as follows: neurobasal medium 1% N is added2 And 10ng/ml BDNF, GDNF and IGF1.
Comparative example 1
Comparative example 1 is different from example 1 in that the traditional Chinese medicine composition of the induction medium does not contain osthole and resveratrol, and the rest is the same as example 1.
Comparative example 2
Comparative example 1 is different from example 1 in that icariin and resveratrol are not contained in the traditional Chinese medicine composition of the induction medium, and the rest is the same as example 1.
Comparative example 3
Comparative example 1 is different from example 1 in that icariin and osthole are not contained in the Chinese medicinal composition of the induction medium, and the rest is the same as example 1.
The neural stem cell differentiation efficiency of example 1 and comparative examples 1 to 3 was evaluated as shown in table 1:
table 1 shows the results of differentiation efficiency of human neural stem cells of example 1 and comparative examples 1 to 3
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Nestin protein positive cell fraction | 97.8％ | 86.5％ | 92.7％ | 72％ | 59.6％ | 27.5％ |
Further, the cells induced and cultured in examples 1 to 3 until day 7 were collected, and the cells were cultured in Stempro NSC SFM medium, and the metabolic activity of the cells was examined by CCK8 colorimetry. Cells were seeded in 96-well cell culture plates and 1 group of cells were taken every 24h for detection, and detection was continued for 5 days. CCK8 contains WST-8 (chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazolium monosodium salt), and is reduced into yellow formazan products with high water solubility by dehydrogenase in cells under the action of electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate, the quantity of formazan products generated is proportional to the quantity of living cells, and the OD value can reflect the metabolic activity of the cells by detecting on an enzyme-labeled instrument. As is clear from FIG. 13, the number of living cells increases with the increase of the culture time, the proliferation of cells is fast, the activity is good, and the activity of differentiated cells is optimal in example 1.
The result shows that the method for differentiating the pluripotent stem cells into the neural stem cells adopts the induction culture medium provided by the invention for induction differentiation, the induction culture medium has no serum, no animal source component and no feeder layer cells, the traditional Chinese medicine composition has reliable components and definite structure, and the method can effectively promote the differentiation of the human pluripotent stem cells into the neural stem cells under the synergistic effect of icariin, osthole and resveratrol, and the obtained neural stem cells have no toxic or side effect and are safer. In addition, the induction method provided by the invention can efficiently and rapidly induce and differentiate the monolayer human pluripotent stem cells into the neural stem cells with high purity, synchronous differentiation and differentiation potential.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.