CN107760649A - It is a kind of to be used for the nourishing additive agent of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation - Google Patents
It is a kind of to be used for the nourishing additive agent of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation Download PDFInfo
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Abstract
The present invention discloses a kind of human's nervous system cell culture and people's multipotent stem cells of being used for the nourishing additive agent of neural system cell differentiation, and the nourishing additive agent constituent and final concentration are respectively：The 20mg/L of actrapid monotard 0.1, the 200mg/L of vitamin C 10, the 100mg/L of glutathione 10, the 5mg/L of leukotrienes 0.05,5 500 μM of 0.2 20mg/L, N acetylcysteine of carnitine, the 10mg/L of monoethanolamine 0.01, the 5mg/L of linoleic acid 0.05.The invention also discloses above-mentioned nourishing additive agent in culture human's nervous system cell and to induce people's multipotent stem cells to the application on neural system cell differentiation.Nourishing additive agent of the present invention formula is simple, and cost is cheap, specific chemical components, and without animal derived materials, without protein extract and hydrolysate, stable homogeneous between batch is safer.
Description
Technical field
The present invention relates to technical field of cell culture.More particularly, to one kind be used for human's nervous system cell culture and
Nourishing additive agent of people's multipotent stem cells to neural system cell differentiation.
Background technology
The incidence of disease of various neurotrosises and retrograde the nervous system disease is in ascendant trend year by year, traditional medical science side
Method is difficult thoroughly to cure this kind of disease, and cell replacement treatment is thoroughly to cure this kind of disease potentially maximally effective means.
People's multipotent stem cells have unlimited multiplication capacity and are divided into the potential of all kinds body cell in human body, are
The preferable seed resource cell of cell replacement treatment.Under certain inductive condition, people's versatility can be divided into nerve
Stem cell, various types of neural precursors, neuron, star spongiocyte and few coign cell plastid, are nervous system
The treatment of the cell replacement of damage class disease and degenerative disease continuously provides cell.
At present, during people's multipotent stem cells are divided into nervous system cell, in NSC, neural precursor
In the incubation of cell and neuron etc., researcher, which is substantially all, employs serum free medium, i.e. basal medium
Ensure Liquid additive, basal medium are responsible for providing necessary inorganic salts of cell growth, vitamin, amino acid, glucose, organic
The materials such as thing, nourishing additive agent are responsible for providing the compositions such as the necessary albumen of cell growth, hormone, trace element.At present, use
Most nourishing additive agents is N2 and B27 commercially.Wherein N2 compositions are simple, can only meet that people's multipotent stem cells are divided into god
Process through stem cell.People's multipotent stem cells are divided into various types of neural precursors and neuron, NSC,
The culture of neural precursor and neuron etc. needs to use the more rich B27 of nutritional ingredient.But B27 complicated components, contain
Animal extracts proteins Bovine Serum Albumin, not only results in unstability between batch, also introduces potential animal sources venereal disease
The risk of poison, potential potential safety hazard is brought for cell replacement treatment.
In order that free serum culture based additive is safer, and it is more stable between batch, carried in removal animal or people
In terms of taking albumen, scientist has done substantial amounts of effort, and achieves some achievements (such as patent CN107119010A and CN
106282114A).But while seralbumin is removed, in order to reach identical culture effect, CN107119010A adds
The indefinite banana tegument extract of chemical composition is added, the A of CN 106282114 have used the indefinite ginseng extraction of chemical composition
Thing ginsenoside, not only makes the composition of additive become relative complex, and be difficult to control there is also stability between batch
Problem.
Accordingly, it is desirable to provide a kind of new human's nervous system cell culture and people's multipotent stem cells of being used for are to nerveous system
The nourishing additive agent for cell differentiation of uniting.
The content of the invention
It is an object of the present invention to provide a kind of new to do for human's nervous system cell culture and people's versatility
Cell is to the nourishing additive agent of neural system cell differentiation, and nourishing additive agent formula is simple, and cost is cheap, and chemical composition is bright
Really, without animal derived materials, without protein extract and hydrolysate.
It is another object of the present invention to provide a kind of above-mentioned nourishing additive agent in culture human's nervous system cell and to lure
People's multipotent stem cells are led to the application on neural system cell differentiation.
To reach above-mentioned purpose, the present invention uses following technical proposals：
The invention provides one kind to be used for human's nervous system cell culture and people's multipotent stem cells to nervous system cell
The nourishing additive agent of differentiation, the nourishing additive agent include consisting of composition：Actrapid monotard, vitamin C, glutathione, Asia
Numb acid, carnitine, N-acetylcystein, monoethanolamine and linoleic acid；
Wherein, the final concentration of each constituent is respectively：Actrapid monotard 0.1-20mg/L, vitamin C 10-
200mg/L, glutathione 10-100mg/L, leukotrienes 0.05-5mg/L, carnitine 0.2-20mg/L, N-acetylcystein 5-
500 μM, monoethanolamine 0.01-10mg/L, linoleic acid 0.05-5mg/L.
Preferably, the final concentration of each constituent of the nourishing additive agent is respectively：Actrapid monotard 0.5-
15mg/L, vitamin C 20-100mg/L, glutathione 20-90mg/L, leukotrienes 0.1-4mg/L, carnitine 0.5-15mg/L,
10-400 μM of N-acetylcystein, monoethanolamine 0.05-8mg/L, linoleic acid 0.1-4mg/L.
Most preferably, the final concentration of each constituent of the nourishing additive agent is respectively：Actrapid monotard 1-
10mg/L, vitamin C 30-80mg/L, glutathione 30-80mg/L, leukotrienes 1-3mg/L, carnitine 1-10mg/L, N- second
50-100 μM of acyl cysteine, monoethanolamine 0.1-5mg/L, linoleic acid 1-3mg/L.
The human's nervous system cell can be NSC, neuron, neural precursor, spongiocyte etc..
The preparation of nourishing additive agent of the present invention is prepared according to conventional method of the present invention；Of the invention specific real
Apply in mode, the final concentration that each constituent in nourishing additive agent is prepared in hundred grades of gnotobasis concentrates 100 times
Dense liquid storage, the dense liquid storage of 100 × nourishing additive agent is added in basal medium in proportion during use.
Done invention further provides above-mentioned nourishing additive agent in culture human's nervous system cell and induction people's versatility
Cell is to the application on neural system cell differentiation.
Further, the application is that the nourishing additive agent coordinates basal medium to use.
Further, the basal medium is one or two kinds of in DMEM/F12 medium, Neurobasal medium；
Preferably, volume ratio 1:1 DMEM/F12 medium and Neurobasal medium.
Further, the nourishing additive agent can also coordinate promote human's nervous system cell growth growth factor and/or
Small molecule and promotion people's multipotent stem cells use to the growth factor and/or small molecule of neural system cell differentiation.
Wherein, the growth factor of the promotion human's nervous system cell growth is basic fibroblast growth factor
(bFGF), epithelial cell growth factor (EGF), colloid derived neurotrophic factor (GDNF), BDNF
(BDNF), one or more of combinations in Sonic hedgehog (SHH)；
The small molecule for promoting human's nervous system cell growth activates related small molecule and retinoic acid for SHH signal paths
Signal path activates related small molecule.
It is described to promote people's multipotent stem cells to activate phase to the growth factor of neural system cell differentiation for Wnt signal paths
Close combination one or two kinds of in growth factor and SHH signal paths activation relevant growth factors；
It is described to promote people's multipotent stem cells to suppress phase to the small molecule of neural system cell differentiation for TGF signal betas path
Molecule is turned down, Wnt signal paths activate related small molecule, and SHH signal paths activate related small molecule, and retinoic acid signal path swashs
One or more of combinations in related small molecule living.
Specifically,
The nourishing additive agent can coordinate basic fibroblast growth factor and epithelial cell growth factor to use, and use
In culture human nerve stem cell；The human nerve stem cell source can be separately cultured acquisition in vivo, induce people's versatility
It is that stem cell differentiation obtains, the acquisition of body cell transdifferentiation etc..
The nourishing additive agent can coordinate colloid derived neurotrophic factor and BDNF to use, and be used for
Cultivate human neure；The source of the human neure can be separately cultured acquisition in vivo, induction people multipotent stem cells point
Change obtaining, the acquisition of body cell transdifferentiation etc..
The nourishing additive agent can coordinate SHH signal paths activator (such as small molecule SAG, purmorphamine or life
Long factor Sonic hedgehog) and retinoic acid signal path activator (such as retinoic acid) use, it is thin for all kinds people's neural precursor
The culture of born of the same parents；The source of people's neural precursor can be separately cultured acquisition in vivo, induce people's multipotent stem cells
What differentiation obtains, body cell transdifferentiation obtained etc..
The nourishing additive agent can coordinate TGF signal betas path to suppress related small molecule such as SB431542, LDN193189
Related small molecule such as CHIR99021,6-BIO etc. are activated Deng, Wnt signal paths, SHH signal paths activate related small molecule such as
SAG, purmorphamine etc., it is related that retinoic acid signal path activates such as retinoic acid, Wnt the signal paths activation of related small molecule
Growth factor such as WNT3a and SHH signal paths activation relevant growth factors such as SHH etc. is used, for inducing people's versatility dry thin
Born of the same parents are to human's nervous system cell differentiation.
The present invention experiments prove that, nourishing additive agent of the present invention no matter in terms of backer's nervous system cell growth also
It is in backer's multipotent stem cells in terms of neural system cell differentiation, it is relative that effect is better than the chemical composition reported at present
Nourishing additive agent complicated, containing protein extracts such as human transferrin, human serum albumins.
In addition, unless otherwise specified, reagent, component used in the present invention can be by commercially available commercially available, if do not had
Have a special instruction, any scope described in the present invention include any numerical value between end value and end value and with end value or
Any subrange that any number between end value is formed.
Beneficial effects of the present invention are as follows：
The present invention is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive is by screening and optimizing acquisition on a large scale, and it is formulated simply, and cost is cheap, specific chemical components, without dynamic
Thing derived component, without protein extract and hydrolysate, stable homogeneous between batch is safer.In addition, the nourishing additive agent
There is no potential introducing animal derived and the risk of humanized's pathogenic microorganism, it is safer, it can apply in clinical research and face
In the culture of cell needed for bed experiment.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 shows NSC aspect graph (experimental group, control group).
Fig. 2 shows NSC marker gene Nestin streamings figure (experimental group, control group).
Fig. 3 shows NSC marker gene Nestin immunofluorescences figure (experimental group).
It is (real that Fig. 4 shows that people's multipotent stem cells are divided into the spinal motor nerve precursor cell differentiation aspect graph of the 8th day
Test group, control group).
Fig. 5 shows that people's multipotent stem cells are divided into the 8th day spinal motor nerve of spinal motor nerve precursor cell differentiation
Precursor marker gene Olig2 streamings figure (experimental group, control group).
Fig. 6 shows that people's multipotent stem cells are divided into the 8th day spinal motor nerve of spinal motor nerve precursor cell differentiation
Precursor marker gene Olig2 immunofluorescences figure (experimental group).
Embodiment
In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings
It is bright.Similar part is indicated with identical reference in accompanying drawing.It will be appreciated by those skilled in the art that institute is specific below
The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.
Basal medium DMEM/F12 medium and Neurobasal medium in the present invention used in embodiment,
B27 is purchased from Life Technologies companies；Growth factor and small molecule are public purchased from Sigma, Stemgent, Enzo, Tocris
Department, other materials is material commonly used in the art.
Embodiment 1 is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive
It is a kind of to add for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Add agent, the constituent of the nourishing additive agent and final concentration are respectively：Actrapid monotard 0.1mg/L, vitamin C
10mg/L, glutathione 10mg/L, leukotrienes 0.05mg/L, carnitine 0.2mg/L, 5 μM of N-acetylcystein, monoethanolamine
0.01mg/L, linoleic acid 0.05mg/L.
Embodiment 2 is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive
It is a kind of to add for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Add agent, the constituent of the nourishing additive agent and final concentration are respectively：Actrapid monotard 20mg/L, vitamin C
200mg/L, glutathione 100mg/L, leukotrienes 5mg/L, carnitine 20mg/L, 500 μM of N-acetylcystein, monoethanolamine
10mg/L, linoleic acid 5mg/L.
Embodiment 3 is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive
It is a kind of to add for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Add agent, the constituent of the nourishing additive agent and final concentration are respectively：Actrapid monotard 0.5mg/L, vitamin C
20mg/L, glutathione 20mg/L, leukotrienes 0.1mg/L, carnitine 0.5mg/L, 10 μM of N-acetylcystein, monoethanolamine
0.05mg/L, linoleic acid 0.1mg/L.
Embodiment 4 is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive
It is a kind of to add for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Add agent, the constituent of the nourishing additive agent and final concentration are respectively：Actrapid monotard 15mg/L, vitamin C
100mg/L, glutathione 90mg/L, leukotrienes 4mg/L, carnitine 15mg/L, 400 μM of N-acetylcystein, monoethanolamine
8mg/L, linoleic acid 4mg/L.
Embodiment 5 is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive
It is a kind of to add for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Add agent, the constituent of the nourishing additive agent and final concentration are respectively：Actrapid monotard 1mg/L, vitamin C 30mg/
L, glutathione 30mg/L, leukotrienes 1mg/L, carnitine 1mg/L, 50 μM of N-acetylcystein, monoethanolamine 0.1mg/L, Asia
Oleic acid 1mg/L.
Embodiment 6 is used for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Additive
It is a kind of to add for the nutrition of human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation
Add agent, the constituent of the nourishing additive agent and final concentration are respectively：Actrapid monotard 10mg/L, vitamin C
80mg/L, glutathione 80mg/L, leukotrienes 3mg/L, carnitine 10mg/L, 100 μM of N-acetylcystein, monoethanolamine 5mg/
L, linoleic acid 3mg/L.
The contrast experiment of the Culture of neural stem cells of embodiment 7
1st, the formula of the nutrient solution for cultivating human nerve stem cell is as follows：
Basal medium (volume ratio)：50%DMEM/F12medium and 50%Neurobasal medium
Nourishing additive agent
BFGF (final concentration)：20ng/mL
EGF (final concentration)：20ng/mL
Wherein,
The constituent of the nourishing additive agent of experimental group and final concentration are the same as embodiment 6；
The nourishing additive agent of control group is the nourishing additive agent that current human nerve stem cell culture generally uses, i.e. B27.
2nd, the culture of human nerve stem cell
The culture of human nerve stem cell includes adhere-wall culture and the culture two ways that suspends：
The adhere-wall culture method of 2.1 NSCs
According to 105Cells/mL density is inoculated into be coated with the culture dish of matrix in advance, 37 DEG C, 5%CO2Saturation is wet
Incubator culture is spent, changes liquid once within two days, cell confluency degree carries out Secondary Culture when reaching more than 90%.
The method for coating of culture dish matrix is as follows：Poly-ornithine (Poly-L-ornithine, PLO) is diluted with PBS
To 15 μ g/mL, it is added in culture dish, the amount of addition is incubated 2h or 4 DEG C overnight for 1,37 DEG C see table, pays attention to during being incubated
The bottom of culture dish is not allowed to be killed.PLO is discarded, with twice of PBS rinses, DMEM/F12 rinses one time.By Laminin lens
(Laminin) 5 μ g/mL are diluted to DMEM/F12, are added to the culture dish that PLO was coated with, the amount of addition is see 1,37 DEG C of table
It is incubated 2h or 4 DEG C overnight, pays attention to not allowing the bottom of culture dish to be killed during being incubated.
Table 1 is coated with the amount of the PLO and Laminin needed for culture dish
| Culture dish specification | Grow area (cm2) | The amount (mL) of addition |
| 6 orifice plates | 10cm2/well | 1mL/well |
| 12 orifice plates | 4cm2/well | 0.4mL/well |
| 24 orifice plates | 2cm2/well | 0.2mL/well |
| 35mm culture dishes | 10cm2 | 1mL |
| 60mm culture dishes | 20cm2 | 2mL |
| 100mm culture dishes | 60cm2 | 6mL |
2.2 NSC suspension culture methods
According to 105Cells/mL density is inoculated into low absorption culture dish, 37 DEG C, 5%CO2Saturated humidity incubator is trained
Support, change liquid once within two days, neural ball and nutrient solution are transferred in 50mL centrifuge tubes when changing liquid, natural subsidence, carefully discard training
Nutrient solution, add fresh medium and neural ball is resuspended, be transferred in culture dish.Enter when most of neural bulb diameter reaches 250 μm
Row Secondary Culture.
As Figure 1-3, shown in Fig. 1 is human nerve stem cell in experimental group culture medium, control group culture to cultivation results
In base growth five instead of after aspect graph, it can be seen that NSC in experimental group growth conditions than in control group culture
Growth conditions in base will get well；Shown in Fig. 2 is that human nerve stem cell grows five in experimental group culture medium, control group culture medium
The expression of NSC Nestin genes after instead of, it can be seen that NSC is in experimental group culture medium
Nestin positive ratio will be significantly higher than control group；Shown in Fig. 3 is that human nerve stem cell is cultivated in experimental group culture medium
Nestin immunofluorescence picture after five generations.
It can thus be seen that in Culture of neural stem cells, the effect ratio of nourishing additive agent of the invention contain animal or
People extract albumen, complicated component, it is good using more extensive nourishing additive agent B27 effects.
Embodiment 8 induces people's cell multipotent stem cells to be divided into the contrast experiment 1 of spinal motor nerve precursor, use
The differentiation basic culture solution formula that spinal motor nerve precursor is divided into induction people's cell multipotent stem cells is as follows：
Basal medium (volume ratio)：50%DMEM/F12medium and 50%Neurobasal medium
Nourishing additive agent
Wherein,
The constituent of the nourishing additive agent of experimental group and final concentration are the same as embodiment 6；
The nourishing additive agent of control group is current induction people's multipotent stem cells to general during neural system cell differentiation
All over the nourishing additive agent used, i.e. B27.
2nd, the method that induction people's cell multipotent stem cells are divided into spinal motor nerve precursor
D0 by the good human embryo stem cell of growth conditions be digested to it is unicellular after according to 105Cells/mL density inoculation
Into culture dish, nutrient solution for differentiation basic culture solution containing 40 μM of SB431542,100nM LDN193189,3 μM
CHIR99021.D2, by nutrient solution be changed to differentiation basic culture solution containing 40 μM of SB431542,100nM LDN193189,3 μM
CHIR99021,500nM SAG, 100nM retinoic acids.After D4, by nutrient solution be changed to differentiation basic culture solution SAG containing 500nM,
100nM retinoic acids, change liquid once within every two days.D8 starts, it is possible to obtains the positive spinal motor nerve precursors of Olig2.
Differentiated result as Figure 4-Figure 6, shown in Fig. 4 respectively in the differentiation basic culture solution described in experimental group, control group,
People's multipotent stem cells are induced differentiation into the aspect graph of spinal motor nerve precursor induction differentiation the 8th day.Can be with by figure
Find out, induce the growth conditions of cell in atomization does not have difference in experimental group and control group.Tested respectively shown in Fig. 5
In differentiation basic culture solution described in group and control group, it is thin that people's multipotent stem cells are induced differentiation into spinal motor nerve precursor
Born of the same parents induce the 8th day people's spinal motor nerve precursor marker gene Olig2 of differentiation expression, it can be seen that people's multipotency
Property stem cell, which is divided into differentiation efficiency of the spinal motor nerve precursor in experimental group, to be significantly higher than in control group
Differentiation efficiency.Shown in Fig. 6 is in experimental group culture medium, and induction people's multipotent stem cells are divided into spinal motor nerve precursor
Cell, breaking up the 8th day spinal motor nerve precursor marker gene Olig2 immunofluorescence figure.
It can thus be seen that during induction people's multipotent stem cells are divided into spinal motor nerve precursor,
The effect of the nourishing additive agent of the present invention than containing animal or people extract albumen, complicated component, use relatively more extensive battalion
It is good to support factor additive B27 effects.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair
The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, all embodiments can not be exhaustive here, it is every to belong to this hair
Row of the obvious changes or variations that bright technical scheme is extended out still in protection scope of the present invention.
Claims (10)
- A kind of 1. nutrition addition for human's nervous system cell culture and people's multipotent stem cells to neural system cell differentiation Agent, it is characterised in that the nourishing additive agent includes consisting of composition：Actrapid monotard, vitamin C, glutathione, flax Acid, carnitine, N-acetylcystein, monoethanolamine and linoleic acid；Wherein, the final concentration of each constituent is respectively：Actrapid monotard 0.1-20mg/L, vitamin C 10-200mg/ L, glutathione 10-100mg/L, leukotrienes 0.05-5mg/L, carnitine 0.2-20mg/L, 5-500 μM of N-acetylcystein, Monoethanolamine 0.01-10mg/L, linoleic acid 0.05-5mg/L.
- 2. nourishing additive agent according to claim 1, it is characterised in that each constituent of the nourishing additive agent is most Whole concentration is respectively：Actrapid monotard 0.5-15mg/L, vitamin C 20-100mg/L, glutathione 20-90mg/L, flax Sour 0.1-4mg/L, carnitine 0.5-15mg/L, 10-400 μM of N-acetylcystein, monoethanolamine 0.05-8mg/L, linoleic acid 0.1-4mg/L。
- 3. nourishing additive agent according to claim 2, it is characterised in that each constituent of the nourishing additive agent is most Whole concentration is respectively：Actrapid monotard 1-10mg/L, vitamin C 30-80mg/L, glutathione 30-80mg/L, leukotrienes 1-3mg/L, carnitine 1-10mg/L, 50-100 μM of N-acetylcystein, monoethanolamine 0.1-5mg/L, linoleic acid 1-3mg/L.
- A kind of 4. application of nourishing additive agent as described in claim 1-3 is any on culture human's nervous system cell.
- 5. a kind of nourishing additive agent as described in claim 1-3 is any is inducing people's multipotent stem cells to nervous system cell Application in differentiation.
- 6. the application according to claim 4 or 5, it is characterised in that the application is that the nourishing additive agent coordinates basis Culture medium uses.
- 7. application according to claim 6, it is characterised in that the basal medium be DMEM/F12medium, It is one or two kinds of in Neurobasal medium；Preferably, volume ratio 1:1 DMEM/F12medium and Neurobasal medium。
- 8. application according to claim 6, it is characterised in that the nourishing additive agent, which can also coordinate, promotes people's nerveous system The growth factor and/or small molecule and promotion small molecule of people's multipotent stem cells to neural system cell differentiation for cell growth of uniting And/or growth factor uses.
- 9. application according to claim 8, it is characterised in that the growth factor for promoting human's nervous system cell growth For basic fibroblast growth factor, epithelial cell growth factor, colloid derived neurotrophic factor, brain-derived neurotrophy because One or more of combinations in son, Sonic hedgehog；The small molecule for promoting human's nervous system cell growth is led to for SHH signals Activate related small molecule and the related small molecule of retinoic acid signal path activation in road.
- 10. application according to claim 8, it is characterised in that promotion people's multipotent stem cells are thin to nervous system The growth factor of born of the same parents' differentiation activates relevant growth factors for Wnt signal paths and SHH signal paths are activated in relevant growth factors One or two kinds of combinations；It is described to promote people's multipotent stem cells to lead to the small molecule of neural system cell differentiation for TGF signal betas Road suppresses related small molecule, Wnt signal paths activate related small molecule, SHH signal paths activate related small molecule, retinoic acid letter One or more of combination in number Pathway Activation correlation small molecule.
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