WO2019192504A1 - 功能大脑皮层细胞的诱导分化方法 - Google Patents

功能大脑皮层细胞的诱导分化方法 Download PDF

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WO2019192504A1
WO2019192504A1 PCT/CN2019/081156 CN2019081156W WO2019192504A1 WO 2019192504 A1 WO2019192504 A1 WO 2019192504A1 CN 2019081156 W CN2019081156 W CN 2019081156W WO 2019192504 A1 WO2019192504 A1 WO 2019192504A1
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differentiation
cells
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French (fr)
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范靖
王安欣
邹潭
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浙江霍德生物工程有限公司
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Priority to US17/044,916 priority Critical patent/US20210163885A1/en
Priority to EP19781651.5A priority patent/EP3778878A4/en
Priority to JP2020553533A priority patent/JP7223448B2/ja
Publication of WO2019192504A1 publication Critical patent/WO2019192504A1/zh
Priority to JP2022197509A priority patent/JP2023027243A/ja

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    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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Definitions

  • the invention belongs to the technical field of induced differentiation of functional cerebral cortical cells, and particularly relates to a method for rapidly inducing differentiation of human neural stem cells into functional cerebral cortical cells.
  • human neural cells have been induced to differentiate from human induced pluripotent stem cells (hiPSC) or embryonic stem cells (hESC) in vitro, and are used for neurological disease research, drug screening, drug neurotoxicity. Tests, transplant trials, etc. have been the focus of research and drug development, and related articles have continued to grow. Among them, although the methods of inducing differentiation are endless, it is still a big problem to obtain a composition and proportion close to the brain and a mature function of the nerve cell population.
  • hiPSC human induced pluripotent stem cells
  • hESC embryonic stem cells
  • an object of the present invention is to provide a method for inducing differentiation of functional cerebral cortical cells, and the method provided by the present invention can obtain a nerve cell having a main function and being stable and healthy in a short period of time.
  • the invention provides a method for inducing differentiation of functional cerebral cortical cells, comprising the following steps:
  • the neural stem cells or neural precursor cells were digested and inoculated onto the cell culture plate, and the culture was started on the first day using the neural differentiation medium A; on the seventh day, the culture was continued using the neural differentiation medium B;
  • the neural differentiation medium A includes one or more of retinoic acid, BDNF, GDNF, ascorbic acid, nutritional supplement, Neurobasal medium, and B27 additive without vitamin A;
  • the neural differentiation medium B includes one or more of BDNF, GDNF, ascorbic acid, nutritional supplement, Neurobasal medium, and vitamin A-free B27 additive;
  • the nutritional additive is selected from one or more of SU 5402, BIBF 1120, IBM X, and glucose.
  • the present invention adds a specific factor, such as an inhibitor of FGF, a VEGF signaling pathway, an activator of cAMP, etc., to accelerate the differentiation and maturation of nerve cells at a specific time when neural stem cells or neural precursor cells are induced to differentiate into various nerve cells. It can induce the differentiation from human neural precursor cells for about 7 to 14 days to obtain the main function and stable and healthy nerve cells, including excitatory and inhibitory neurons, so that the human neural cells cultured in vitro can be truly applied to drugs. In areas such as screening, reducing manufacturing costs and shortening production cycles.
  • a specific factor such as an inhibitor of FGF, a VEGF signaling pathway, an activator of cAMP, etc.
  • the neural stem cells or neural precursor cells are first digested and inoculated onto the cell culture plate. Specifically, the neural stem cells or neural precursor cells are digested with accutase and inoculated at 5 ⁇ 10 5 /cm 2 to poly-D. On the cell culture plate coated with -lysine and laminin, the culture was started using the neural differentiation medium A on the first day; the culture was continued on the seventh day using the neural differentiation medium B.
  • the neural differentiation medium A includes retinoic acid, BDNF (brain-derived neurotrophic factor), GDNF (glial cell-derived neurotrophic factor), ascorbic acid, nutritional additive One or more of Neurobasal medium and B27 additive without vitamin A;
  • the neural differentiation medium B includes BDNF, GDNF, ascorbic acid, nutritional supplement, Neurobasal medium, and vitamin A-free One or more of the B27 additives.
  • the invention adds nutrient additives to the nerve cell culture medium, and can accelerate the differentiation and maturation of nerve cells.
  • the nutritional additive is selected from one or more of SU5402, BIBF1120, IBMX, and glucose.
  • SU5402 and BIBF1120 are used to inhibit FGF and VEGF receptor signaling pathways, inhibit cell aggregation, promote migration of immature neurons, and allow nerve cells to have sufficient distance gaps to facilitate the growth of neurites and further mature into functional true neurons. . Because the aggregation of cells leads to the high density of neurons, which leads to the loss of the opportunity for cells to extend the neurites, which is not conducive to cell maturation. Existing differentiation and neuronal culture methods generally affect cell health, maturation and homogeneity due to the accumulation of nerve cells.
  • nerve cells cultured with the addition of SU5402 and BIBF1120 medium can be maintained for up to 3 years, and nerve cells obtained by differentiating human neural stem cells by other methods are generally cultured in vitro for only 1 to 2 months and will die in large numbers. .
  • IBMX is a non-specific inhibitor of intracellular cAMP degrading enzyme-phosphodiesterase (PDE), which can increase the level of intracellular cAMP by inhibiting the degradation of cAMP.
  • PDE intracellular cAMP degrading enzyme-phosphodiesterase
  • the role of IBMX requires the participation of glucose to exert its effects.
  • the differentiation and maturation of neurons requires a large amount of cAMP to provide energy, usually using a large amount of cAMP, which is relatively expensive; and IBMX can indirectly increase the concentration of intracellular cAMP by inhibiting the activity of cAMP degrading enzyme-phosphodiesterase.
  • the present invention also provides a medium for functional differentiation of cerebral cortex cells, including a neural cell culture medium and a nutritional supplement.
  • the nutritional additive comprises one or more of SU5402, BIBF1120, IBMX, and glucose.
  • the nutritional additive comprises SU5402.
  • the nutritional additive comprises one or more of 100 nM to 100 ⁇ M of SU 5402, 1 to 500 ng/ml of BIBF 1120, 1 to 100 ⁇ M of IBMX, and 1 to 10 mM of glucose.
  • the nutritional supplement comprises 100 nM to 100 ⁇ M of SU 5402, 1 to 500 ng/ml of BIBF 1120, 1 to 100 ⁇ M of IBMX, and 1 to 10 mM of glucose.
  • the nutritional supplement comprises 80-120 nM SU 5402, 150-250 ng/ml BIBF 1120, 5-15 ⁇ M IBMX, and 3-8 mM glucose.
  • the nutritional supplement comprises 100 nM SU 5402, 200 ng/ml BIBF 1120, 10 ⁇ M IBMX, and 5 mM glucose.
  • the neural cell culture medium is selected from a cerebral neural cell culture medium, and a different combination of factors may also be added.
  • the neural cell culture medium comprises retinoic acid, BDNF, GDNF, ascorbic acid, Neurobasal medium, and B27 additive without vimentine A; in one embodiment, the The neural cell culture medium includes BDNF, GDNF, ascorbic acid, Neurobasal medium, and B27 additive without vitamin A.
  • the culture was started using the neural differentiation medium A on the first day; the culture was continued on the seventh day using the neural differentiation medium B.
  • the neural differentiation medium A includes a nerve cell culture medium A and a nutritional additive.
  • the neural cell culture medium A includes retinoic acid, BDNF, GDNF, ascorbic acid, Neurobasal medium, and B27 additive (vitamamine A-free).
  • the neural differentiation medium A comprises 2 ⁇ M retinoic acid, 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 100 nM SU 5402, 200 ng/ml BIBF 1120, 10 ⁇ M IBMX 5mM glucose and supplemental Neurobasal medium and B27 additive without vitamin A, wherein the ratio of the amount of Neurobasal medium and B27 additive is 50:1 (except for special instructions, the ratio of the amount used in the present invention is mass ratio) ).
  • the neural differentiation medium B includes a nerve cell culture medium B and a nutritional additive.
  • the neural cell culture medium B includes BDNF, GDNF, ascorbic acid, Neurobasal medium, and B27 additive without vimentine A.
  • the neural differentiation medium B comprises 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 100 nM SU 5402, 200 ng/ml BIBF 1120, 10 ⁇ M IBMX, 5 mM glucose, and complemented Neurobasal Medium and B27 additive without vimentine A, wherein the ratio of Neurobasal medium to B27 additive without vimentin A is 50:1.
  • the nerve differentiation medium A was used to culture in a 37 ° C, 5% CO 2 cell incubator, and the medium was changed every 3 to 5 days; on the 7th day, the nerve differentiation medium B was used to continue.
  • the medium is cultured in a 5% CO 2 cell incubator at 37 ° C, and the medium is changed every 3 to 5 days. From the 7th day, the maturity of the nerve cells can be detected by electrophysiological and immunofluorescence staining, and when the target function index is reached. It can be used later.
  • the present invention adds a specific factor, such as an inhibitor of FGF, a VEGF signaling pathway, an activator of cAMP, etc., to accelerate the differentiation and maturation of nerve cells at a specific time when neural stem cells or neural precursor cells are induced to differentiate into various nerve cells. It can induce the differentiation from human neural precursor cells for about 7 to 14 days to obtain the main function and stable and healthy nerve cells, including excitatory and inhibitory neurons, so that the human neural cells cultured in vitro can be truly applied to drugs. In areas such as screening, reducing manufacturing costs and shortening production cycles.
  • the method provided by the method of the present invention uses a serum-free and animal-free factor, and is therefore suitable for use in clinical transplantation tests.
  • FIG. 1 is a schematic flow chart of a method for inducing differentiation of functional cerebral cortical cells provided by the present invention
  • Figure 2 is a bright field photograph of human nerve cells on days 1, 7, 14, and 84 of differentiation induced in Example 1;
  • Figure 3 is an example of action potentials of single cell patch clamp recording human nerve cells on day 14 of differentiation induced by Example 1, examples of miniIPSC (inhibitory neuronal firing) and miniEPSC (excitatory neuronal firing);
  • Figure 4 is a graph showing the magnitude and frequency of excitatory and inhibitory neuronal firing of cells recorded on day 14 of the induction of differentiation in Example 1;
  • Figure 5 is an exemplary recording of sEPSC (spontaneous excitatory nerve release) of single-cell patch clamp recording human nerve cells on day 14 of differentiation induction;
  • sEPSC spontaneous excitatory nerve release
  • Figure 6 is an exemplary record of spontaneous neuronal release of human neural cells on day 14 of differentiation induction of Example 1.
  • Figure 7 is the result of MAP2 fluorescent immunostaining on day 14 of differentiation
  • Figure 8 is a histogram of MAP2 fluorescent immunostaining results on day 14 of differentiation
  • Figure 9 is the result of Synapsin fluorescent immunostaining on day 14 of differentiation
  • Figure 10 is a histogram of the results of Synapsin fluorescence immunostaining on day 14 of differentiation;
  • Figure 11 shows the results of vGlut fluorescent immunostaining on day 21 of differentiation
  • Figure 12 is a bar graph showing the results of vGlut fluorescence immunostaining on day 21 of differentiation.
  • Fig. 13 is a bright-field photograph of human nerve cells at the 21st day of differentiation of Example 2 and Comparative Example 1.
  • Fig. 14 is a bar graph showing statistical results of the degree of dispersion of human nerve cells at the 21st day of differentiation in Example 2 and Comparative Example 1.
  • Fig. 15 is a bar graph showing the results of Synapsin fluorescence immunostaining on the 21st day of differentiation of Example 2 and Comparative Example 1.
  • Fig. 16 is a bar graph showing the results of fluorescent immunostaining of FOXG1 on the 21st day of differentiation of Example 2 and Comparative Example 1.
  • Fig. 17 is a graph showing the spontaneous nerve release record of human nerve cells at the 7th day of differentiation of Example 2 and Comparative Example 1.
  • Step 1 The neural progenitor cells (hNPC) obtained by differentiating the human induced pluripotent stem cell line DYR0100 were digested with accutase, and then inoculated into a poly-D-lysine and laminin coated cell culture at 5 ⁇ 10 5 /cm 2 . Board
  • Step 2 starting from day 1 using neural differentiation medium A, cultured in a 37 ° C, 5% CO 2 cell incubator for 7 days, half a day for every 3 days; the neural differentiation medium A contains a final concentration of 2 ⁇ M retinoic Acid, 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/ml BIBF1120, 10 ⁇ M IBMX and 5 mM glucose in Neurobasal medium and B27 additive without vitamin A, among which Neurobasal medium and B27 The dosage ratio of the additive is 50:1;
  • Step 3 From the 7th day, use the neural differentiation medium B to culture in a 37 ° C, 5% CO 2 cell incubator, and change the amount every 3 days; the neural differentiation medium B contains a final concentration of 20 ng / ml BDNF, 20 ng /ml GDNF, 0.2mM ascorbic acid, 100nM SU5402, 200ng/ml BIBF1120, 10 ⁇ M IBMX and 5mM glucose Neurobasal medium and B27 additive without vitamin A, wherein the ratio of Neurobasal medium and B27 additive is 50:1 From the 7th day, the maturity of nerve cells can be detected by electrophysiological and immunofluorescence staining, and can be used when the target function index is reached.
  • FIG. 1 is a schematic flow chart of a method for inducing differentiation of functional cerebral cortex cells according to the present invention. After digesting hNSC or hNPC, differentiation is induced, and nerve differentiation medium A is used on the first day of induction differentiation, day 7 Beginning to use the neural differentiation medium B, basic neuronal function and maturation markers can be detected from the 14th day, and the human brain nerve cells can be obtained by continuing to mature.
  • the neural differentiation medium A contains Neurosual and B27 (excluding 2 ⁇ M retinoic acid, 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/ml BIBF1120, 10 ⁇ M IBMX and 5 mM glucose). Vitamine A) medium; neural differentiation medium B contains Neurobasal and B27 at a final concentration of 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/ml BIBF 1120, 10 ⁇ M IBMX and 5 mM glucose (excluding Vitamine A) medium.
  • Neurosual and B27 excluding 2 ⁇ M retinoic acid, 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/ml BIBF1120, 10 ⁇ M
  • FIG. 2 is a bright-field photograph of human nerve cells on days 1, 7, 14, and 84 of differentiation induced by Example 1
  • FIG. 3 is a single-cell membrane on the 14th day of differentiation induced in Example 1.
  • Figure 4 shows excitatory and inhibitory neuronal firing of cells recorded on day 14 of induction of differentiation in Example 1.
  • Statistics of amplitude and frequency As can be seen from Fig. 2, Fig. 3 and Fig. 4, the nerve cells having excitatory and inhibitory neurons can be obtained by the method of the present invention for 14 days, have electrophysiological functions, and can be cultured for a long period of time.
  • Example 1 The difference from Example 1 is that the common neural cell culture medium (Neurobasal media + B27) is used instead of the neural differentiation medium A and the neural differentiation medium B;
  • the common neural cell culture medium Neurorobasal media + B27
  • Example 1 The difference from Example 1 was that the neural differentiation medium A and the neural differentiation medium B were replaced with a medium (BrainPhys) which promotes the electrophysiological maturity of nerve cells by the famous stem cell reagent company StemCell technologies (CompA).
  • BrainPhys a medium which promotes the electrophysiological maturity of nerve cells by the famous stem cell reagent company StemCell technologies (CompA).
  • Example 1 The difference from Example 1 is that the neural differentiation medium A and the neural differentiation medium B are replaced by a medium supplement promoting nerve cell maturity of BrainXell (CompB), a company of the well-known professor of stem cell neural differentiation in the United States.
  • CompB BrainXell
  • FIG. 5 is an exemplary recording of sEPSC (spontaneous excitatory nerve release) of single-cell patch clamp recording human nerve cells on the 14th day of differentiation induction of Example 1 and Comparative Examples 1 to 3, from top to bottom. Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1 were used;
  • Figure 6 is an exemplary recording of spontaneous neuronal firing of human nerve cells on Day 14 of differentiation induction of Example 1 (Multi-Elode Array by Axion Bioscience) , multi-electrode array electrophysiological recorder) recorded, you can see through the analysis of the software, find out the correlation of cluster-like synergistic action action potential between multiple electrodes.
  • sEPSC spontaneous excitatory nerve release
  • FIG. 7 shows the results of MAP2 fluorescence immunostaining on the 14th day of differentiation of Example 1 and Comparative Examples 1 to 3, wherein from left to right, Comparative Example 1, Comparative Example 2, Comparative Example 3, and
  • the bright field photograph, the DAPI staining result, and the MAP2 staining result are sequentially from top to bottom
  • FIG. 8 is a histogram of the MAP2 fluorescent immunostaining result on the 14th day of differentiation of Example 1 and Comparative Examples 1-3, from left to right.
  • the right side is Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1, respectively.
  • FIG. 9 is a result of Synapsin fluorescent immunostaining on the 14th day of differentiation of Example 1 and Comparative Examples 1 to 3, wherein Comparative Example 1, Comparative Example 3, and Example 1 are sequentially from left to right. From top to bottom, Merge results, MAP2 staining results, and Synapsin staining results;
  • Figure 10 is a histogram of Synapsin fluorescence immunostaining results on the 14th day of differentiation between Example 1 and Comparative Examples 1-3, from left to right for comparative examples. 1. Comparative Example 3 and Example 1.
  • FIG. 11 is a result of vGlut fluorescence immunostaining on the 21st day of differentiation of Example 1 and Comparative Examples 1 to 3, wherein from left to right, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1 is a Merge staining map, a DAPI staining map, and a vGlut staining map from top to bottom;
  • FIG. 12 is a histogram of vGlut fluorescent immunostaining results on the 21st day of differentiation of Example 1 and Comparative Examples 1 to 3, from left to The right side is Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1, respectively.
  • the method provided by the present invention can rapidly obtain nerve cells having excitatory and inhibitory neurons.
  • Step 1 The neural progenitor cells (hNPC) obtained by differentiating the human induced pluripotent stem cell line DYR0100 were digested with accutase, and then inoculated into a poly-D-lysine and laminin coated cell culture at 5 ⁇ 10 5 /cm 2 . Board
  • Step 2 starting from day 1 using neural differentiation medium A, cultured in a 37 ° C, 5% CO 2 cell incubator for 7 days, half a day for every 3 days; the neural differentiation medium A contains a final concentration of 2 ⁇ M retinoic Acid, 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 10 ⁇ M SU5402 Neurobasal medium and B27 additive without vitamin A, wherein the ratio of Neurobasal medium and B27 additive is 50:1;
  • Step 3 From the 7th day, use the neural differentiation medium B to culture in a 37 ° C, 5% CO 2 cell incubator, and change the amount every 3 days; the neural differentiation medium B contains a final concentration of 20 ng / ml BDNF, 20 ng /ml GDNF, 0.2mM ascorbic acid, 10 ⁇ M SU5402 Neurobasal medium and B27 additive (excluding vitamin A), wherein the ratio of Neurobasal medium and B27 additive is 50:1; from the 7th day, electrophysiology can be passed And immunofluorescence staining and other methods to detect the maturity of nerve cells, can be used when the target function indicators are reached.
  • FIG. 13 is a bright-field photograph of human neural cells on the 21st day of differentiation induced in Example 2 and Comparative Example 1
  • FIG. 14 is a dispersion degree of cells on the 21st day of differentiation induced in Example 2 and Comparative Example 1. Statistical data.
  • FIG. 15 is a histogram of the results of Synapsin fluorescent immunostaining on the 21st day of differentiation induced in Example 2 and Comparative Example 1
  • FIG. 16 is a fluorescent immunoassay of FOXG1 on the 21st day of differentiation induced in Example 2 and Comparative Example 1. Dyeing results histogram;
  • Fig. 17 is a statistical result of a record of spontaneous nerve release of human nerve cells (obtained by MEA of Axion Bioscience) on the 7th day of differentiation induction in Example 2 and Comparative Example 1. Cells in which Neural medium was used did not detect electrophysiological signals on day 7.
  • the method provided by the present invention can inhibit cell aggregation, promote neuronal maturation, and make nerve cells have sufficient distance and space, which is favorable for the growth of neurites and further mature into functional true neurons.
  • Step 1 The neural progenitor cells (hNPC) obtained by differentiating the human induced pluripotent stem cell line DYR0100 were digested with accutase, and then inoculated into a poly-D-lysine and laminin coated cell culture at 5 ⁇ 10 5 /cm 2 . Board
  • Step 2 starting from day 1 using neural differentiation medium A, cultured in a 37 ° C, 5% CO 2 cell incubator for 7 days, half a day for every 3 days; the neural differentiation medium A contains a final concentration of 2 ⁇ M retinoic Acid, 20ng/ml BDNF, 20ng/ml GDNF, 0.2mM ascorbic acid, 5 ⁇ M SU5402 and 50 ⁇ M IBMX Neurobasal medium and B27 additive without vitamin A, wherein the ratio of Neurobasal medium and B27 additive is 50:1 ;
  • Step 3 From the 7th day, use the neural differentiation medium B to culture in a 37 ° C, 5% CO 2 cell incubator, and change the amount every 3 days; the neural differentiation medium B contains a final concentration of 20 ng / ml BDNF, 20 ng /ml GDNF, 0.2 mM ascorbic acid, 5 ⁇ M SU5402 and 50 ⁇ M IBMX Neurobasal medium and B27 additive (without vitamin A), wherein the ratio of Neurobasal medium and B27 additive is 50:1.
  • the results showed that the method provided in Example 3 was able to effectively inhibit cell aggregation as compared with Comparative Example 1, Comparative Example 2, and Comparative Example 3.
  • Step 1 The neural progenitor cells (hNPC) obtained by differentiating the human induced pluripotent stem cell line DYR0100 were digested with accutase, and then inoculated into a poly-D-lysine and laminin coated cell culture at 5 ⁇ 10 5 /cm 2 . Board
  • Step 2 starting from day 1 using neural differentiation medium A, cultured in a 37 ° C, 5% CO 2 cell incubator for 7 days, half a day for every 3 days; the neural differentiation medium A contains a final concentration of 2 ⁇ M retinoic Acid, 20ng/ml BDNF, 20ng/ml GDNF, 0.2mM ascorbic acid, 5ng/ml BIBF1120 and 50 ⁇ M IBMX Neurobasal medium and B27 additive without vitamin A, wherein the ratio of Neurobasal medium and B27 additive is 50 :1;
  • Step 3 From the 7th day, use the neural differentiation medium B to culture in a 37 ° C, 5% CO 2 cell incubator, and change the amount every 3 days; the neural differentiation medium B contains a final concentration of 20 ng / ml BDNF, 20 ng /ml GDNF, 0.2 mM ascorbic acid, 5 ng/ml BIBF1120 and 50 ⁇ M IBMX Neurobasal medium and B27 additive (vitamamine A free), wherein the ratio of Neurobasal medium and B27 additive is 50:1.
  • IBMX Neurobasal medium and B27 additive vitamin A free

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Abstract

提供了一种功能大脑皮层细胞诱导分化用培养基以及诱导分化方法，所述培养基包括神经细胞培养基和营养添加剂，其中营养添加剂选自SU5402、BIBF1120、IBMX和葡萄糖中的一种或多种。所述方法由神经干细胞或神经前体细胞向多种神经细胞诱导分化的特定时间添加特定的因子，例如FGF、VEGF信号通路的抑制剂，cAMP的激活剂等，加速神经细胞的分化和成熟，可以在从人神经前体细胞开始诱导分化7～14天左右得到具有主要功能且稳定健康的神经细胞，从而降低制造成本，缩短生产周期。

Description

功能大脑皮层细胞的诱导分化方法 技术领域
本发明属于功能大脑皮层细胞的诱导分化技术领域，尤其涉及一种人神经干细胞快速诱导分化为功能大脑皮层细胞的方法。
背景技术
自从2008年(Dimos et al.2008)开始，从人诱导多能干细胞(hiPSC)或胚胎干细胞(hESC)体外诱导分化得到人的神经细胞，并用于神经系统疾病研究，药物筛选，药物的神经毒性测试，移植试验等就一直是科研和药物研发的热点，相关的文章一直在持续增长。其中，诱导分化的方法虽然层出不穷，但是能得到接近大脑的组成和比例，以及成熟功能的神经细胞群体依然是一大难题。
人神经前体细胞被诱导分化为成熟有功能的大脑皮层细胞一般需要30～60天，且很难具有真正成熟的神经结构和功能，如神经递质受体在突触的分布，自发、较强且频繁的兴奋性和抑制性电生理信号等。而且，在高通量药物筛选的多孔板中，神经细胞无法稳定健康的长时间培养，且不适宜再次换液，所以需要较长培养时间和不够成熟这两点极大地限制了这些细胞在疾病研究，药物筛选及毒性测试方面的大规模应用，同时也影响到所获得数据的代表性和可靠性。
发明内容
有鉴于此，本发明的目的在于提供一种功能大脑皮层细胞的诱导分化方法，本发明提供的方法用时较短即可获得具有主要功能且稳定健康的神经细胞。
本发明提供了一种功能大脑皮层细胞的诱导分化方法，包括以下步骤：
将神经干细胞或神经前体细胞消化后接种至细胞培养板上，第1天开始使 用神经分化培养基A进行培养；第7天开始使用神经分化培养基B继续培养；
所述神经分化培养基A包括维甲酸(retinoic acid)、BDNF、GDNF、抗坏血酸(ascorbic acid)、营养添加剂、Neurobasal培养基和不含维生素A的B27添加剂中的一种或多种；
所述神经分化培养基B包括BDNF、GDNF、抗坏血酸(ascorbic acid)、营养添加剂、Neurobasal培养基和不含维生素A的B27添加剂中的一种或多种；
所述营养添加剂选自SU5402、BIBF1120、IBMX和葡萄糖(glucose)中的一种或多种。
本发明在由神经干细胞或神经前体细胞向多种神经细胞诱导分化的特定时间添加特定的因子，例如FGF、VEGF信号通路的抑制剂，cAMP的激活剂等，加速神经细胞的分化和成熟，可以在从人神经前体细胞开始诱导分化7～14天左右得到具有主要功能且稳定健康的神经细胞，其中包含兴奋性及抑制性神经元，从而使得体外培养的人神经细胞可以真正应用于药物筛选等领域，降低制造成本，缩短生产周期。
本发明首先将神经干细胞或神经前体细胞消化后接种至细胞培养板上，具体而言，将神经干细胞或神经前体细胞用accutase消化后按5×10 5/cm 2接种至用poly-D-lysine和laminin包被的细胞培养板上，然后第1天开始使用神经分化培养基A进行培养；第7天开始使用神经分化培养基B继续培养。
在本发明中，所述神经分化培养基A包括维甲酸(retinoic acid)、BDNF(脑源性神经营养因子)、GDNF(胶质细胞源性神经营养因子)、抗坏血酸(ascorbic acid)、营养添加剂、Neurobasal培养基和不含维生素A的B27添加剂中的一种或多种；所述神经分化培养基B包括BDNF、GDNF、抗坏血酸(ascorbic acid)、营养添加剂、Neurobasal培养基和不含维生素A的B27添加剂中的一种或多种。
本发明在神经细胞培养基中添加营养添加剂，能够加速神经细胞的分化和成熟。在本发明中，所述营养添加剂选自SU5402、BIBF1120、IBMX和葡萄糖(glucose)中的一种或多种。
SU5402和BIBF1120用于抑制FGF和VEGF受体信号通路，抑制细胞聚集，促进未成熟神经元迁移，使得神经细胞具有足够的距离空隙，有利于神经突起的生长，进一步成熟为功能性的真神经元。因为细胞的聚集会导致神 经元的高密度，导致细胞失去延伸神经突起的机会，不利于细胞的成熟，现有分化和神经元培养方法普遍都有因神经细胞聚集而影响细胞健康，成熟和均一性，甚至在成熟前大量死亡的问题，添加SU5402和BIBF1120可以通过减少细胞聚集而有效的防止这些情况的出现，使得神经细胞可以较早成熟，同时保持健康及均一的密度，降低死亡率，从而可以满足科研及产业界大批量和功能上的需求，可用于高通量筛选等。而且，使用添加SU5402和BIBF1120培养基所培养的神经细胞可维持长达3年以上，而通过其它方法分化人神经干细胞得到的神经细胞一般在体外只能培养至1～2个月就会大量死亡。
IBMX是胞内cAMP降解酶-磷酸二酯酶(PDE)的非特异性抑制剂,可通过抑制cAMP的降解而提高胞内cAMP的水平，IBMX的作用需要glucose的参与才能发挥效果。神经元的分化成熟需要大量的cAMP提供能量，通常都使用大量的cAMP,较高成本；而IBMX通过抑制cAMP降解酶-磷酸二酯酶的活性，可以间接提高细胞内cAMP的浓度。使用IBMX和glucose替代大量的cAMP，一方面使得神经细胞可以更早成熟，另一方面可以降低细胞培养的成本，从而可以满足科研及产业界大批量和功能上的需求，适合用于高通量筛选。
本发明还提供了一种功能大脑皮层细胞诱导分化用培养基，包括神经细胞培养基和营养添加剂。
在一个实施例中，所述营养添加剂包括SU5402、BIBF1120、IBMX和葡萄糖中的一种或多种。
在一个实施例中，所述营养添加剂包括SU5402。
在一个实施例中，所述营养添加剂包括100nM～100μM的SU5402、1～500ng/ml BIBF1120、1～100μM IBMX和1～10mM的葡萄糖中的一种或多种。
在一个实施例中，所述营养添加剂包括100nM～100μM的SU5402、1～500ng/ml BIBF1120、1～100μM IBMX和1～10mM的葡萄糖。
在一个实施例中，所述营养添加剂包括80～120nM SU5402、150～250ng/ml BIBF1120、5～15μM IBMX和3～8mM的葡萄糖(glucose)。
在一个实施例中，所述营养添加剂包括100nM SU5402、200ng/ml BIBF1120、10μM IBMX和5mM的葡萄糖(glucose)。
在本发明中，神经细胞培养基选自大脑神经细胞培养基，也可添加不同的因子组合。在一个实施例中，所述神经细胞培养基包括维甲酸(retinoic acid)、BDNF、GDNF、抗坏血酸(ascorbic acid)、Neurobasal培养基和不含vitamine A的B27添加剂；在一个实施例中，所述神经细胞培养基包括BDNF、GDNF、抗坏血酸(ascorbic acid)、Neurobasal培养基和不含vitamine A的B27添加剂。
在诱导分化神经干细胞或神经前体细胞时，第1天开始使用神经分化培养基A进行培养；第7天开始使用神经分化培养基B继续培养。
具体而言，所述神经分化培养基A包括神经细胞培养基A和营养添加剂。所述神经细胞培养基A包括维甲酸(retinoic acid)、BDNF、GDNF、抗坏血酸(ascorbic acid)、Neurobasal培养基和B27添加剂(不含vitamine A)。在一个实施例中，所述神经分化培养基A包括2μM维甲酸(retinoic acid)、20ng/ml BDNF、20ng/ml GDNF、0.2mM抗坏血酸(ascorbic acid)、100nM SU5402、200ng/ml BIBF1120、10μM IBMX、5mM的葡萄糖和补足的Neurobasal培养基和不含vitamine A的B27添加剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1(除特殊说明外，本发明所述用量比例均为质量比)。
具体而言，所述神经分化培养基B包括神经细胞培养基B和营养添加剂。所述神经细胞培养基B包括BDNF、GDNF、抗坏血酸(ascorbic acid)、Neurobasal培养基和不含vitamine A的B27添加剂。在一个实施例中，所述神经分化培养基B包括20ng/ml BDNF、20ng/ml GDNF、0.2mM抗坏血酸(ascorbic acid)、100nM SU5402、200ng/ml BIBF1120、10μM IBMX、5mM的葡萄糖和补足的Neurobasal培养基和不含vitamine A的B27添加剂，其中，Neurobasal培养基和不含vitamine A的B27添加剂的用量比例是50:1。
在本发明中，第1天开始使用神经分化培养基A在37℃，5％CO 2细胞培养箱中进行培养，每3～5天半量换液；第7天开始使用神经分化培养基B继续在37℃，5％CO 2细胞培养箱中培养，每3～5天半量换液，从第7天开始可以通过电生理及免疫荧光染色等方法检测神经细胞的成熟度，当达到目的功能指标后即可使用。
本发明在由神经干细胞或神经前体细胞向多种神经细胞诱导分化的特定 时间添加特定的因子，例如FGF、VEGF信号通路的抑制剂，cAMP的激活剂等，加速神经细胞的分化和成熟，可以在从人神经前体细胞开始诱导分化7～14天左右得到具有主要功能且稳定健康的神经细胞，其中包含兴奋性及抑制性神经元，从而使得体外培养的人神经细胞可以真正应用于药物筛选等领域，降低制造成本，缩短生产周期。另外，本发明提供的方法采用的培养基的成分都是无血清无动物源的因子，因此也适合用于临床移植试验。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本发明提供的功能大脑皮层细胞的诱导分化方法的流程示意图；
图2是实施例1诱导分化第1、7、14及84天的人神经细胞的明场照片；
图3是实施例1诱导分化第14天时单细胞膜片钳记录人神经细胞的动作电位示例，miniIPSC(抑制性神经发放)和miniEPSC(兴奋性神经发放)的示例；
图4是实施例1诱导分化第14天所记录的细胞的兴奋及抑制性神经发放的幅度及频率的统计数据；
图5是分化诱导第14天时单细胞膜片钳记录人神经细胞的sEPSC(自发性兴奋神经发放)的示例记录；
图6是实施例1分化诱导第14天时人神经细胞的自发性神经发放的示例记录；
图7是分化第14天时的MAP2荧光免疫染色结果；
图8是分化第14天时的MAP2荧光免疫染色结果柱状图；
图9是分化第14天时的Synapsin荧光免疫染色结果；
图10是分化第14天时的Synapsin荧光免疫染色结果柱状图；
图11是分化第21天时的vGlut荧光免疫染色结果；
图12是分化第21天时的vGlut荧光免疫染色结果柱状图。
图13是实施例2及比较例1分化第21天时的人神经细胞的明场照片。
图14是实施例2及比较例1分化第21天时的人神经细胞的分散程度统计结果柱状图。
图15是实施例2及比较例1分化第21天时的Synapsin荧光免疫染色结果柱状图。
图16是实施例2及比较例1分化第21天时的FOXG1荧光免疫染色结果柱状图。
图17是实施例2及比较例1分化第7天时的人神经细胞的自发性神经发放记录统计图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
步骤1、将人诱导多能干细胞系DYR0100分化所得到的神经前体细胞(hNPC)用accutase消化后，按5×10 5/cm 2接种至poly-D-lysine和laminin包被好的细胞培养板上；
步骤2、从第1天开始使用神经分化培养基A在37℃，5％CO 2细胞培养箱中培养7天，每3天半量换液；所述神经分化培养基A含有终浓度为2μM retinoic acid、20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、100nM SU5402、200ng/ml BIBF1120、10μM IBMX和5mM glucose的Neurobasal培养基及不含vitamine A的B27添加剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；
步骤3、从第7天开始使用神经分化培养基B在37℃，5％CO 2细胞培养箱中进行培养，每3天半量换液；神经分化培养基B含有终浓度20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、100nM SU5402、200ng/ml BIBF1120、10μM IBMX和5mM glucose的Neurobasal培养基及不含vitamine A的B27添加 剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；从第7天开始可以通过电生理及免疫荧光染色等方法检测神经细胞的成熟度，当达到目的功能指标后即可使用。
参见图1，图1为本发明提供的功能大脑皮层细胞的诱导分化方法的流程示意图，将hNSC或hNPC消化后进行诱导分化，在诱导分化第1天开始使用神经分化培养基A，第7天开始使用神经分化培养基B，第14天起即可检测到基本的神经细胞功能及成熟标志物，继续成熟即可获得人大脑神经细胞。其中，神经分化培养基A含有终浓度为2μM retinoic acid、20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、100nM SU5402、200ng/ml BIBF1120、10μM IBMX和5mM glucose的Neurobasal及B27(不含vitamine A)培养基；神经分化培养基B含有终浓度为20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、100nM SU5402、200ng/ml BIBF1120、10μM IBMX和5mM glucose的Neurobasal及B27(不含vitamine A)培养基。
参见图2、图3和图4，图2是实施例1诱导分化第1、7、14及84天的人神经细胞的明场照片；图3是实施例1诱导分化第14天时单细胞膜片钳记录人神经细胞的动作电位示例，miniIPSC(抑制性神经发放)和miniEPSC(兴奋性神经发放)的示例；图4是实施例1诱导分化第14天所记录的细胞的兴奋及抑制性神经发放的幅度及频率的统计数据。由图2、图3和图4可知，采用本发明提供的方法14天即可获得具有兴奋性及抑制性神经元的神经细胞，具有电生理功能，且可以长期培养。
比较例1
与实施例1的差别在于，采用普通神经细胞培养基(Neurobasal media+B27)代替神经分化培养基A和神经分化培养基B；
比较例2
与实施例1的差别在于，采用加拿大著名干细胞试剂公司StemCell technologies(CompA)的促进神经细胞电生理成熟度的培养基(BrainPhys)代替神经分化培养基A和神经分化培养基B。
比较例3
与实施例1的差别在于，采用美国著名的干细胞神经分化的教授所经营公司BrainXell(CompB)的促进神经细胞成熟度的培养基添加物代替神经分化 培养基A和神经分化培养基B。
参见图5和图6，图5是实施例1及比较例1～3分化诱导第14天时单细胞膜片钳记录人神经细胞的sEPSC(自发性兴奋神经发放)的示例记录，从上到下依次是用比较例1、比较例2、比较例3和实施例1；图6是实施例1分化诱导第14天时人神经细胞的自发性神经发放的示例记录(由Axion Bioscience的MEA(Multi Electrode Array，多电极阵列电生理记录仪)记录得到)，可以看到经过软件的分析，找出了多个电极间簇状协同发放动作电位的关联。这代表着在第14天，实施例1提供的方法分化得到的神经细胞已经形成了一定的网状连接(更成熟的功能表现)，而在使用其他方法分化获得的细胞上均没有观测到这一现象(第14天)，并且根据已知的文献，其它分化方法及培养基培养得到人神经细胞甚至在分化2个月时都很难观测到这样的现象。
参见图7和图8，图7是实施例1及比较例1～3分化第14天时的MAP2荧光免疫染色结果，其中，由左到右依次为比较例1、比较例2、比较例3和实施例1，由上到下依次为明场照片、DAPI染色结果和MAP2染色结果，图8是实施例1及比较例1～3分化第14天时的MAP2荧光免疫染色结果柱状图，由左至右分别为比较例1、比较例2、比较例3和实施例1。
参见图9和图10，图9是实施例1及比较例1～3分化第14天时的Synapsin荧光免疫染色结果，其中，由左到右依次为比较例1、比较例3和实施例1，由上到下依次为Merge结果、MAP2染色结果、Synapsin染色结果；图10是实施例1及比较例1～3分化第14天时的Synapsin荧光免疫染色结果柱状图，由左至右分别为比较例1、比较例3和实施例1。
参见图11和图12，图11是实施例1及比较例1～3分化第21天时的vGlut荧光免疫染色结果，其中，由左到右依次为比较例1、比较例2、比较例3和实施例1，由上到下依次为Merge染色图、DAPI染色图、vGlut染色图；图12是实施例1及比较例1～3分化第21天时的vGlut荧光免疫染色结果柱状图，由左至右分别为比较例1、比较例2、比较例3和实施例1。
由图7～图12可知，本发明提供的方法能够快速获得具有兴奋性及抑制性神经元的神经细胞。
实施例2
步骤1、将人诱导多能干细胞系DYR0100分化所得到的神经前体细胞 (hNPC)用accutase消化后，按5×10 5/cm 2接种至poly-D-lysine和laminin包被好的细胞培养板上；
步骤2、从第1天开始使用神经分化培养基A在37℃，5％CO 2细胞培养箱中培养7天，每3天半量换液；所述神经分化培养基A含有终浓度为2μM retinoic acid、20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、10μM SU5402的Neurobasal培养基及不含vitamine A的B27添加剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；
步骤3、从第7天开始使用神经分化培养基B在37℃，5％CO 2细胞培养箱中进行培养，每3天半量换液；神经分化培养基B含有终浓度20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、10μM SU5402的Neurobasal培养基及B27添加剂(不含vitamine A)，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；从第7天开始可以通过电生理及免疫荧光染色等方法检测神经细胞的成熟度，当达到目的功能指标后即可使用。
参见图13和图14，图13是实施例2及比较例1诱导分化第21天的人神经细胞的明场照片；图14是实施例2及比较例1诱导分化第21天时细胞的分散程度统计数据。
参见图15和图16，图15是实施例2及比较例1诱导分化第21天时的Synapsin荧光免疫染色结果柱状图；图16是实施例2及比较例1诱导分化第21天时的FOXG1荧光免疫染色结果柱状图；
参见图17,图17是实施例2及比较例1诱导分化第7天时人神经细胞的自发性神经发放的记录(由Axion Bioscience的MEA记录得到)统计结果。其中使用Neural培养基的细胞在第7天没有检测到电生理信号。
由图13～图17可知，本发明提供的方法能够抑制细胞聚集，促进神经元成熟，使得神经细胞具有足够的距离空隙，有利于神经突起的生长，进一步成熟为功能性的真神经元。
实施例3
步骤1、将人诱导多能干细胞系DYR0100分化所得到的神经前体细胞(hNPC)用accutase消化后，按5×10 5/cm 2接种至poly-D-lysine和laminin包被好的细胞培养板上；
步骤2、从第1天开始使用神经分化培养基A在37℃，5％CO 2细胞培养箱中 培养7天，每3天半量换液；所述神经分化培养基A含有终浓度为2μM retinoic acid、20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、5μM SU5402和50μM IBMX的Neurobasal培养基及不含vitamine A的B27添加剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；
步骤3、从第7天开始使用神经分化培养基B在37℃，5％CO 2细胞培养箱中进行培养，每3天半量换液；神经分化培养基B含有终浓度20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、5μM SU5402和50μM IBMX的Neurobasal培养基及B27添加剂(不含vitamine A)，其中，Neurobasal培养基和B27添加剂的用量比例是50:1。结果显示与比较例1、比较例2和比较例3相比，实施例3提供的方法能够有效抑制细胞聚集。
实施例4
步骤1、将人诱导多能干细胞系DYR0100分化所得到的神经前体细胞(hNPC)用accutase消化后，按5×10 5/cm 2接种至poly-D-lysine和laminin包被好的细胞培养板上；
步骤2、从第1天开始使用神经分化培养基A在37℃，5％CO 2细胞培养箱中培养7天，每3天半量换液；所述神经分化培养基A含有终浓度为2μM retinoic acid、20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、5ng/ml BIBF1120和50μM IBMX的Neurobasal培养基及不含vitamine A的B27添加剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；
步骤3、从第7天开始使用神经分化培养基B在37℃，5％CO 2细胞培养箱中进行培养，每3天半量换液；神经分化培养基B含有终浓度20ng/ml BDNF、20ng/ml GDNF、0.2mM ascorbic acid、5ng/ml BIBF1120和50μM IBMX的Neurobasal培养基及B27添加剂(不含vitamine A)，其中，Neurobasal培养基和B27添加剂的用量比例是50:1。结果显示与比较例1、比较例2和比较例3相比，实施例3提供的方法能够有效抑制细胞聚集。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种功能大脑皮层细胞诱导分化用培养基，包括神经细胞培养基和营养添加剂，所述营养添加剂选自SU5402、BIBF1120、IBMX和葡萄糖中的一种或多种。
  2. 根据权利要求1所述的培养基，其特征在于，所述营养添加剂包括100nM～100μM的SU5402、1～500ng/ml BIBF1120、1～100μM IBMX和1～10mM的葡萄糖中的一种或多种。
  3. 根据权利要求2所述的培养基，其特征在于，所述营养添加剂包括100nM～100μM的SU5402、1～500ng/ml BIBF1120、1～100μM IBMX和1～10mM的葡萄糖。
  4. 一种功能大脑皮层细胞的诱导分化方法，其特征在于，包括：
    将神经干细胞或神经前体细胞消化后接种至细胞培养板上，第1天开始使用神经分化培养基A进行培养；第7天开始使用神经分化培养基B继续培养；
    所述神经分化培养基B包括BDNF、GDNF、抗坏血酸、营养添加剂、Neurobasal培养基和不含维生素A的B27添加剂中的一种或多种；
    所述营养添加剂选自SU5402、BIBF1120、IBMX和葡萄糖中的一种或多种。
  5. 根据权利要求4所述的诱导分化方法，其特征在于，所述营养添加剂包括100nM～100μM的SU5402、1～500ng/ml BIBF1120、1～100μM IBMX和1～10mM的葡萄糖中的一种或多种。
  6. 根据权利要求5所述的诱导分化方法，其特征在于，所述营养添加剂包括80～120nM SU5402、150～250ng/ml BIBF1120、5～15μM IBMX和3～8mM的葡萄糖中的一种或多种。
  7. 根据权利要求6所述的诱导分化方法，其特征在于，所述营养添加剂包括100nM SU5402、200ng/ml BIBF1120、10μM IBMX和5mM的葡萄糖。
  8. 根据权利要求4～7任意一项所述的诱导分化方法，其特征在于，所述神经分化培养基A包括2μM维甲酸、20ng/ml BDNF、20ng/ml GDNF、0.2mM抗坏血酸、100nM SU5402、200ng/ml BIBF1120、10μM IBMX、5mM的葡萄 糖和补足的Neurobasal培养基和不含vitamine A的B27添加剂，其中，Neurobasal培养基和B27添加剂的用量比例是50:1；
    所述神经分化培养基B包括20ng/ml BDNF、20ng/ml GDNF、0.2mM抗坏血酸、100nM SU5402、200ng/ml BIBF1120、10μM IBMX、5mM的葡萄糖和补足的Neurobasal培养基和不含vitamine A的B27添加剂，其中，Neurobasal培养基和不含vitamine A的B27添加剂的用量比例是50:1。
  9. 根据权利要求8所述的诱导分化方法，其特征在于，第1天开始使用神经分化培养基A进行培养，每3～5天半量换液；第7天开始使用神经分化培养基B继续培养，每3～5天半量换液。
  10. 根据权利要求9所述的诱导分化方法，其特征在于，将神经干细胞或神经前体细胞用accutase消化后按5×10 5/cm 2接种至用poly-D-lysine和laminin包被的细胞培养板上。
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