CN107326013B

CN107326013B - Neural cell system after directional induction of hiPSC differentiation, induction method and application

Info

Publication number: CN107326013B
Application number: CN201710632172.4A
Authority: CN
Inventors: 杨涛; 隋昳
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-07-28
Filing date: 2017-07-28
Publication date: 2020-08-07
Anticipated expiration: 2037-07-28
Also published as: WO2019019223A1; CN111793607A; CN111849899A; CN111849899B; CN111793608A; CN107326013A; CN111793608B; ZA201802126B; CN111793607B

Abstract

The invention discloses a neural cell system after directional induction of hiPSC differentiation, an induction method and application. The inventive method comprises culturing the hipscs in stages to induce neural differentiation thereof, said stages comprising: stage a. co-culturing the hipscs with bone marrow stromal cells HS5 in an induction medium; stage b. continuously culturing the hipscs in HS5 conditioned medium; continuing to culture the hipscs in a basal medium that cultures the neuronal cells. The method of the present invention induces the directed differentiation of human hipscs into neural cells, while inhibiting the generation of non-neural cells, thereby obtaining a mature, broad-spectrum neural cell population. The nerve cell group is verified to be mature neuron with electric impulse distribution in vitro, and the nerve cell group is also verified in vivo experiments of mice, and has the effect of effectively treating nervous system diseases (such as cerebral apoplexy and cerebral injury).

Description

Neural cell system after directional induction of hiPSC differentiation, induction method and application

Technical Field

The invention belongs to the field of neurobiology, and particularly relates to a neural cell system after directional induction of hiPSC differentiation, an induction method and application.

Background

Human induced pluripotent stem (hiPSC) derived neural cells have a significant effect on the treatment of ischemic stroke (characterized by severe depletion of multiple different lineages of neural cells) by cell transplantation. However, the conventional induced hiPSC cells have low efficiency of differentiation into neural cells and poor stability.

Recent research in dry cell biology provides the basis for regenerative medicine, wherein directed differentiation of hiPSC Cells can provide a variety of human nerve Cells for patients with ischemic stroke (characterized by severe defects of various neurons and glial Cells). many protocols on efficiently differentiating nerve Cells have been proposed, however, these methods are not sufficient to provide a mature nerve cell lineage, resulting in intermixing of immature Cells therein, leading to formation of teratomas after intracerebral transplantation, furthermore, some methods can only differentiate to produce a narrow spectrum of nerve cell lines, failing to satisfy the variety of cell types required for treating ischemic stroke.more importantly, separation and purification of the nerve Cells induced by hiPSC Cells, removal of non-nerve system Cells and immature Cells are essential, thus establishment of a neural induction and purification system based on hiPSC Cells is expected to be applied to Cells of clinical, induced nerve system Cells, cell, scientific, No. 23, No. 7, a.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defect that the immature neural cell lineage provided by the prior art is immature so that the immature cells are mixed in the immature neural cell lineage to cause teratoma formation after intracerebral transplantation, and provide a method for directionally inducing the differentiation of hipscs into the neural cell lineage, a prepared neural cell lineage and application thereof. The method of the present invention induces the directed differentiation of human hipscs into neural cells, while inhibiting the generation of non-neural cells, thereby obtaining a mature, broad-spectrum neural cell population. The nerve cell group is verified to be mature neuron with electric impulse distribution in vitro, and the nerve cell group is also verified in vivo experiments of mice, and has the effect of effectively treating nervous system diseases (such as cerebral apoplexy and cerebral injury).

One of the technical solutions of the present invention for solving the above problems is: a method of directionally inducing differentiation of hipscs into neural cell systems, comprising culturing said hipscs in stages to induce neural differentiation thereof, said stages comprising:

stage a. co-culturing the hipscs with bone marrow stromal cells HS5 in an induction medium;

stage b. continuously culturing the hipscs with HS5 conditioned medium, i.e. induction medium containing the secretion of HS 5;

continuing to culture the hipscs in a basal medium that cultures the neuronal cells.

Wherein the induction medium in the stage a is an induction medium conventional in the art, and preferably comprises 20-25% serum substitute, 0.5-1.5mM glutamine, 8-20ng/ml epidermal growth factor, 8-12ng/ml brain-derived neurotrophic factor, 8-15ng/ml neurotrophic factor-3, 0.5-1.5ng/ml transforming growth factor β 3, 400-700 ng/ml noggin and 2-3% DMEM/F12 medium supplemented with B27, wherein the percentages are percentages by volume, more preferably, the induction medium further comprises 1-1.5% non-essential amino acid, 8-15ng/ml basic fibroblast growth factor, 0.08-0.15mM β -mercaptoethanol and 0.3-0.8mM bis-butyryl cyclic adenosine monophosphate in order to effectively and synchronously promote the proliferation activity of cell differentiation products during the differentiation of stem cells into a neural cell lineage.

Even more preferably, the culture medium comprises 20% serum replacement, 1% nonessential amino acids, 1mM glutamine, 0.1mM β -mercaptoethanol, 10ng/ml basic fibroblast growth factor, 10ng/ml epidermal growth factor, 10ng/ml brain-derived neurotrophic factor, 10ng/ml neurotrophic factor-3, 1ng/ml transforming growth factor β 3, 0.5mM adenosine dibutyryl cyclic phosphate, 500ng/ml noggin and 2% B27 cell culture additive DMEM/F12 culture medium^TMA serum replacement.

The co-cultivation in stage a may be a direct contact co-cultivation or an indirect contact co-cultivation, preferably a direct contact co-cultivation.

The bone marrow stromal cell HS5 in the stage a is HS5 inhibiting division; preferably, the method of inhibiting cleavage is irradiation; more preferably, the irradiation conditions are: the gamma ray irradiation intensity is 75-85Gy, the irradiation time is 28-35 minutes, the irradiation intensity is preferably 80Gy, and the irradiation time is preferably 30 minutes.

The period of co-cultivation described in stage a is conventional in the art, preferably 10-18 days, more preferably 2 weeks.

The duration of the continuous culture described in phase b is conventional in the art, and is preferably 8 to 18 days, more preferably 2 weeks.

The period of continued cultivation as described in stage c is conventional in the art, preferably 10-18 days, more preferably 2 weeks.

The HS5 conditioned medium described in stage b was prepared by 1) mixing 5 × 10⁶～2×10⁷Inoculating irradiated HS5 cells into 8-15ml of induction culture medium, 2) continuously collecting supernatant of the cultured cells for 1-8 days, 3) mixing the supernatant and the induction culture medium according to the proportion of 1: 1-8: 1, and preferably, the preparation method of the HS5 conditioned medium comprises the steps of 1) inoculating 1 × 10 to the induction culture medium⁷Inoculating 10ml of the induction medium with the irradiated HS5 cells; 2) collecting the supernatant of the cultured cells for 4 consecutive days; 3) mixing the supernatant and the induction culture medium in a ratio of 1: 1.

The basic culture medium in the stage c is added with: neurobasal medium of 15-30ng/ml bFGF, 15-30ng/ml EGF, 1-3% B27 additive, 8-12 μ M forskolin and 0.1-0.3mM ascorbic acid; preferably, the basal medium further comprises 0.5-1.5% of N2 additive and 0.5-1.5% of fetal bovine serum in order to effectively maintain the survival rate of mature neurons at the terminal stage of differentiation; more preferably, the basic medium in stage c is supplemented with: 20ng/ml bFGF, 20ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10. mu.M forskolin, and 0.2mM ascorbic acid in neurobasal medium, said percentages being by volume.

The second technical scheme for solving the problems is as follows: a nerve cell system obtained by the method. Preferably, the neural cell system comprises: 59.3 +/-1.9 percent of neural stem cells, 28.2 +/-2.1 percent of various functional neurons, 9.1 +/-0.8 percent of astrocytes and 4.8 +/-0.6 percent of oligodendrocytes, wherein the percentage is the percentage of the number of the whole neural cell system. Wherein the multifunctional neuronal cell comprises: dopaminergic neuron 5.4 + -0.4%, acetylcholine neuron 9.3 + -0.6%, GABAergic neuron 3.9 + -0.3%, 5-hydroxytryptamine neuron 6.5 + -0.5%, and juvenile neuron 3.1 + -0.4%; the percentage is the number percentage of the whole nerve cell system.

The third technical scheme for solving the problems is as follows: an application of the nerve cell system in preparing a brain tissue cell repair preparation; preferably, the preparation is used for treating cerebral injury caused by ischemic stroke, cerebral hemorrhage or trauma.

The fourth technical solution of the present invention is to provide an induction medium for initially inducing differentiation of hipscs into neural cell systems, wherein the medium is a DMEM/F12 medium comprising 20-25% serum replacement, 0.5-1.5mM glutamine, 8-20ng/ml epidermal growth factor, 8-12ng/ml brain-derived neurotrophic factor, 8-15ng/ml neurotrophic factor-3, 0.5-1.5ng/ml transforming growth factor β 3, 400-700 ng/ml noggin and 2-3% B27 additive, wherein the percentages are percentages by volume, preferably, the medium further comprises 1-1.5% non-essential amino acids, 0.08-0.15mM β -mercaptoethanol, 8-15ng/ml basic fibroblast growth factor and 0.3-0.8mM dibutyryladenosine cyclic adenosine phosphate, wherein the contents of the components are less than the lower limit of the above-mentioned medium, the survival rate of the hipscs is significantly lower than the upper limit of differentiation survival rate of the above-psc (the above mentioned medium is lower than the above-mentioned differentiation medium), and the survival rate of the above mentioned hipscs is significantly lower than the above mentioned differentiation medium (the above mentioned differentiation medium is not only if the above mentioned psc) is lower than the above mentioned differentiation medium, and the above mentioned differentiation medium is also lower than the above mentioned differentiation medium, the above mentioned 40% differentiation medium.

In a preferred embodiment of the invention, the medium is a DMEM/F12 medium comprising 20% serum replacement, 1% nonessential amino acids, 1mM glutamine, 0.1mM β -mercaptoethanol, 10ng/ml basic fibroblast growth factor, 10ng/ml epidermal growth factor, 10ng/ml brain derived neurotrophic factor, 10ng/ml neurotrophic factor-3, 1ng/ml transforming growth factor β 3, 0.5mM adenosine dibutyryl cyclic phosphate, 500ng/ml noggin, and 2% B27 cell culture additive, more preferably the serum replacement is KnockOut^TMA serum replacement.

The fifth technical scheme for solving the problems is as follows: an HS5 conditioned medium for directionally inducing the differentiation of hipscs into a nerve cell system, which is an induction medium containing a secretion of bone marrow stromal cells HS 5; the HS5 is irradiated HS 5; the irradiation conditions were: the gamma ray irradiation intensity is 75-85Gy, the irradiation time is 28-35 minutes, the irradiation intensity is preferably 80Gy, and the irradiation time is preferably 30 minutes;

preferably, the HS5 conditioned medium is prepared by 1) preparing 5 × 10⁶～2×10⁷Inoculating irradiated HS5 cells into 8-15ml of induction culture medium, 2) continuously collecting supernatant of the cultured cells for 1-8 days, and 3) mixing the supernatant and the induction culture medium in a ratio of 1: 1-8: 1, wherein in the step 1), if the number of the cells is less than 5 × 10⁶The survival rate of the cells after inoculation and before irradiation is low (generally lower than 80 percent), the concentration of the produced cell secretion is low, and if the cell number is higher than 2 × 10⁷If the number of the cells is too large, the cells are too crowded, and the death rate after irradiation is higher (higher than 50%). In the step 2), if the number of days for collecting the cell culture medium supernatant is less than 1 day, cell resources are wasted; when the number of days is more than 8 days, HS5 cells gradually die after irradiation, and thus, many inflammatory factors and apoptosis factors are produced, which is not favorable for induction culture of hipscs. In the step 3), the mixing ratio of the collected culture medium supernatant to the freshly prepared induction culture medium is lower than 1:1, so that the concentration of HS5 cell secretion contained in the mixture is too low to induce neural differentiation of the hipscs; if the mixing ratio is higher than 8:1, the ratio of the culture supernatant is too high, and the culture supernatant contains not only effective inducing components but also high concentrations of metabolic wastes and pro-apoptotic factors of HS5, which is more beneficial for the induction of hiPSC.

More preferably, the HS5 conditioned medium is prepared by 1) preparing 1 × 10⁷Inoculating 10ml of the induction medium with the irradiated HS5 cells; 2) collecting the supernatant of the cultured cells for 4 consecutive days; 3) mixing the supernatant and the induction culture medium in a ratio of 1: 1.

The sixth technical scheme for solving the problems is as follows: a basal medium for directionally inducing differentiation of hipscs into neuronal cells in a neuronal cell system, the basal medium comprising: 15-30ng/ml bFGF, 15-30ng/ml EGF, 1-3% B27 additive, 8-12 μ M forskolin and 0.1-0.3mM ascorbic acid in neurobasal medium. If the content of the medium component is less than the lower limit of the above numerical range, the survival rate of mature neurons differentiated by hiPSC is low (which may be less than 60%); above the upper limit of the above range, apoptosis of the mature neurons may be induced, resulting in low survival rate (which may be less than 50%).

Preferably, the basic culture medium further comprises 0.5-1.5% of N2 additive and 0.5-1.5% of fetal bovine serum. More preferably, the basic medium is neurobasal medium comprising 20ng/ml bFGF, 20ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10. mu.M forskolin and 0.2mM ascorbic acid, said percentages being by volume.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention co-cultures Bone Marrow Stromal Cells (BMSC) HS5 and hipSC, can induce human hipSC cells to directionally differentiate into nervous system cells, and simultaneously inhibit the generation of non-nervous system cells, thereby obtaining mature and broad-spectrum nerve cell populations. The nerve cell group is verified to be mature neuron with electric impulse distribution in vitro, and the nerve cell group is also verified in vivo experiments of mice, and has the effect of effectively treating nervous system diseases (such as cerebral apoplexy and cerebral injury). The stepwise culture method and a series of additives used in the invention can form a standardized and commercialized in vitro culture process, thereby meeting the clinical and scientific research requirements for iPS induction and transplantation therapy in China and abroad.

Drawings

FIG. 1 shows that HS5 can secrete multiple cytokines (a). In terms of overall cell viability (b; c) and Pax-6⁺(early development marker of nervous system cells) cell ratio (b; c) was used as an evaluation index, and the direct contact induction culture of HS-5 was most effective for neural differentiation of hipSC cells (b, c against two cell lines of hipSC, namely ips-1 and IMR90-1, respectively).

FIG. 2 is a contact induced culture of HS5 directed induction of hipSC cells to differentiate into nervous system cells [. sup.p ] by activating Notch receptors of hipSC cells<0.05vs. control;^▲p<0.05vs. control or corresponding group (corresponding group) + L-685458;^#p<0.05vs. control + L-685458]。

FIG. 3 shows the differentiation of hPSC cells into Pax-6 cells at different time points⁺(early differentiation marker of nervous system cells) dynamic change of cell proportion, and hiPSC cells in undifferentiated state (SSEA-4)⁺) Dynamic change of the ratio.

Fig. 4 shows that the hiPSC-derived neural cells (donor cells) exhibited extensive migration ability within the brain parenchyma of the host 8 weeks after transplantation into the gerbil model. (a) At 8 weeks post-transplantation, donor cells diffused along the injection needle track, migrating into the surrounding striatal parenchyma, indicating that the donor cells can tolerate the intracorporeal environment of ischemic stroke animals. The gray lines have generally marked the needle track extent of the donor cells. (b) Donor cells spread and migrate to the peripheral brain parenchyma 8 weeks after transplantation. The antibody used for fluorescent staining was a mouse anti-human nuclear antibody (HuN,1: 100; Chemicon). The immunofluorescent-stained positive (green) cell nucleus is the donor cell nucleus. Similarly, donor cells were also found in the cortex, corpus callosum, hippocampus of recipient animals. (c) Hippocampus of recipient animals (gerbils). (d-f) the presence of donor cells in the hippocampus of the recipient animal. The antibody used for fluorescent staining was a mouse anti-human nuclear antibody (HuN,1: 100; Chemicon). The immunofluorescent-stained positive (green) cell nucleus is the donor cell nucleus. Shown in the control group (physiological saline injection only; d), there were no donor cells in the hippocampus; in the human bone marrow stromal cell (hMSC; e) injection group, the number of positive cells in the host hippocampus is small; whereas in the hiPSC-derived neural cell injection group (f), donor cells were more in the hippocampus of the host.

FIG. 5 shows the results of reverse transcription-PCR experiments, showing that the expression of genes Oct-4 and A L P related to the sternness/undifferentiated state gradually fades away and completely disappears in the co-cultured group at the end of the second phase, while the expression of the neural precursor cell markers Nestin and Musashi-1 gradually increases, and at the end of the third phase, the more mature neural cell subtypes including the marker GFAP of glial cells, the mature neural cell markers MAP-2 and Nurr-1 at the later stage of mitosis show the strong expression state.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Letter abbreviations:

hiPSC: human Induced Pluripotent Stem cells from Human Induced Pluripotent Stem cells

MEF: mouse embryo fibroblasts

KSR: knock-out serum replacement

bFGF: basic fibroblast growth factor

EGF: epidermal growth factor

NEAA: non-essential amino acids of non-essential amino acids

BDNF: brain-derived neurotrophic factor of brain-derived neurotrophic factor

NT-3: neurotropin-3 neurotrophic factor-3

GFAP: glial fibrilliary acid protein glial fibrillary acidic protein

MAP-2: microtubuli-associated protein 2

Nurr-1: nuclear receptor related protein-1 nuclear receptor associated protein 1

Oct-4: octamer binding transcription factor 4

A L P alkaline phosphatase

In addition, the percentage (%) not particularly specified in the present invention is generally a volume percentage.

The experimental method comprises the following steps:

1. construction of expression vectors

Human Notch1 molecule intracellular activation domain (NICD), pubmed, searched, for 1759-2444 amino acid sequence in GenBank NM-017617, amplified by Polymerase Chain Reaction (PCR). specificity primer (upstream 5'-CGC GGA TCC ATG CGCAAG CGC CGG CGG CAG CAT-3'; downstream 5'-ACG TCT AGA CAC GTC TGC CTG GCT CGG-3'). PCR product (2058bp) was digested by BamHI/XbaI cleavage site, inserted into mammalian expression vector pcDNA3.1(Invitrogen), constructed pcDNA3.1/Notch1(pcNICD) expression vector, cells with 2 × 10⁴/cm²Inoculated in 6-well plates (0.5ml medium/cm)²) And after 24 hours of culture, transfection was carried out. Transfected cells were selected with G418 at a dose of 250. mu.g/ml for approximately 2-4 weeks until single clones appeared.

2. Reverse transcription PCR

The first strand of cDNA obtained after reverse transcription is subjected to amplification of a specific gene sequence. Fluorescent quantitative PCR amplification reactions were performed using the SYBR Green method (Applied Biosystems, Foster City, Calif., USA). The control group and the experimental group showed significant fold difference using comparative CT, as shown in fig. 2.

3. Electrophysiological analysis

Cell voltage recordings were performed at room temperature using an Axopatch-200B amplifier (Axon Instruments Inc., Foster City, Calif., USA). The patch clamp was attached to a pipette containing 120mM potassium gluconate, 20mM KCl, 10mM NaCl, 10mM EGTA, 1mM CaCl2, 2mM Mg-ATP, 0.3mM Na-GTP and 10mM HEPES/KOH (pH 7.2, 280mOsmol/kg) with a resistance of about 3-5 Ω. The current was guaranteed to be 0pA before recording the membrane potential.

4. Dopamine release test

After the 3-stage culture, the cells were washed with a low-concentration KCl solution (4.7nM) and then with 2ml of a high-concentration KCl solution (60mM KCl,85mM NaCl,2.5mM CaCl)₂,1.2mM MgSO₄,1.2mM KH₂PO₄11mM D-glucose, and20mM HEPES/NaOH; pH 7.4) for 15 minutes the dopamine concentration was determined by high performance liquid chromatography (HP L C) (HTEC 500, Eicom Corp., San Diego, Calif., USA).

5. Immunoblot analysis

Total protein (20. mu.g) was electrophoresed on 12% SDS-PAGE and subjected to membrane transfer (Millipore, Billerica, MA, USA) and antibodies were added drop-wise to primary antibodies, rabbit anti-NICD (1: 1000; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Hes 1(Hes 1; 1: 500; Chemicon, Temecula, CA, USA), rabbit anti-Hes 5(1: 500; Chemicon) and mouse anti- α -tubulin antibodies (1: 2000; Chemicon) secondary antibodies were HRP (horseradish peroxidase) -labeled goat anti-rabbit or goat anti-mouse IgG or IgM (1: 2000; Millipore), and the band intensities were quantified using image software (Bio-Rad, Hercules, CA, USA).

6. Brain surgery

Animal experiments were in compliance with relevant regulations of the national institutes of health, and were approved by local governments. Adult male Mongolian gerbils (age 12-13 weeks, body weight 60-80 grams) were anesthetized with 2% halothane and released 5 minutes after occlusion with an aneurysm clip (Ridenour TR et al brain Res.1991; 565(1): 116-. Control gerbils were subjected to a sham procedure without bilateral arterial occlusion.

7. Cell transplantation

3 days after brain surgery, nerve cells (2.5 × 10) were injected into the corresponding areas of the gerbil brain⁵Cells/5. mu.l of physiological saline, i.e., 2.5 × 10⁵The cells were suspended in 5. mu.l of physiological saline and transplanted into one side of the recipient brain, each animal brain received 2 transplants in total for the left and right sides), (5 × 10⁵The injection points are 0.5mm before bregma, 2mm outside midline and 4mm ventral dura mater), and the corresponding parts of the control group gerbil are injected with physiological saline in equal amount, human mesenchymal stem cells (hMSC) are taken as cell control, the puncture collection operation of healthy human bone marrow is approved by regional ethics examination committee and granted informed consent, the brief operation is that 1073 mug/m L density gradient separation liquid (GEHealthcare, Piscataway, NJ, USA) is used for collecting singleNuclear cells, 1.6 × 10⁵/cm²The culture medium was a low sugar DMEM medium supplemented with 10% FBS, 1% NEAA and 1mM L-glutamine, after a period of culture, the hMSC cell suspension was injected into the gerbil brain in the same manner and at the same dose, cyclosporine A (Novartis, Basel, Switzerland) was injected into gerbil by intraperitoneal injection at a dose of 10 mg/kg/day.

8. Behavioral analysis

The Morris water maze experiment (Harvard Apparatus, Holliston, MA, USA) was used to evaluate the spatial cognitive ability of model animals. Six weeks after transplantation, animals were trained for 13 days, 4 times daily, and formal behavioral assessments were performed on day 14, along with space exploration experiments. Briefly, on day 14, the pre-set animal rest platform below the water surface was removed and the animals were allowed to remain in the water maze for 60 seconds. And recording the times of the gerbil crossing the original platform position within 60 seconds, and simultaneously recording the time of the gerbil staying in the quadrant region of the original platform.

9. Enzyme immunoassay

Hippocampus was isolated from isolated brain tissue, homogenized with a buffer (20mM Tris-HCl,137mM NaCl,1mM DTT, 0.5% Triton X-100and 0.5mM PMSF; pH 8.0) and centrifuged at 14,000g for 30 minutes at 4 ℃. The basic fibroblast growth factor was quantitatively determined using a detection kit (Quantikine HS; R & D Systems) as indicated.

10. Histological analysis

The number of donor cells is measured by the number of positive cells in each fifth slice during continuous slicing, and finally corrected by Abercrombic for mula formula, in addition, tissues with the length of about 2-2.2mm between bregma and bregma are continuously sliced and stained with 0.2% of thionin, each frame of images under a microscope is measured and counted by a scale frame with the dimension of 1mm × 0.25.25 mm, the whole pyramidal neurons (large nucleus, obvious nucleolus and clear cell membrane) in a hippocampal CA1 area are counted according to the method described previously, the cells in the hippocampal CA1 area are graded according to the previously described standard, wherein 0 grade of the compacted CA1 pyramidal neurons accounts for less than 10% of the total number of cells in the frame, and I grade 1 pyramidal neurons account for 10% -40% of the total number of cells in the frame, 40% -70% of the compacted cells, and 70% of the compacted cells.

11. Immunocytochemistry

Mouse anti- β -tubulin III antibody (TuJ1,1: 500; Sigma), mouse anti-O₄Antibodies (1: 50; Chemicon), mouse anti-stage-specific embryonic antigen-4 antibodies (SSEA-4, 1: 100; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), mouse anti-pax-6 antibodies (1: 100; Santa Cruz Biotechnology), rabbit anti-Nestin antibodies (1: 400; Chemicon), rabbit anti-collagen fibrin antibodies (GFAP,1: 400; Chemicon), rabbit anti-synaptophI antibodies (1: 500; Chemicon), rabbit anti-tyrosine hydroxylase antibodies (TH,1: 100; Chemicon), rabbit anti-gamma-aminobutyric acid antibodies (GABA,1: 200; Sigma), rabbit anti-choline acetyltransferase antibodies (ChAT, 1: 200; Sigma), rabbit anti-5-hydroxytryptamine antibodies (1: 100; Sigma) were applied to cells, while mouse anti-human mitochondrial antibodies (hmito, 1: 40; mouse anti-human milch antibodies (Sigma, 1: 200; Sigma), rabbit anti-5-hydroxytryptamine antibodies (1: 100; Sigma) were applied to cells, while mouse anti-human mitochondrial antibodies (hmiton, goat anti-peroxidase, goat anti-rabbit anti-bovine serum albumin (1: 100; goat anti-rabbit anti-rat anti-rabbit anti-peroxidase) antibodies (goat anti-rat antibody (goat anti-rat), goat anti-rat anti-rabbit anti-rat-rabbit anti-rat-rabbit anti-rat-rabbit anti-tyrosine hydroxylase antibody (goat anti-rat-rabbit anti-rat-rabbit antibody (goat anti-rabbit antibody (1: 100; goat anti-rabbit), goat anti-rat-rabbit anti-rat-rabbit anti.

12. Statistical analysis

Data are presented as mean ± standard error, two-by-two comparisons of SNK and t tests, histological grading of hippocampal pyramidal cell layers by Nemenyi test SPSS17 software system (SPSS inc., Chicago, I L) was used, P values < 0.05 were considered statistically significant.

Example 1 establishment of Co-culture System of bone marrow stromal cell line HS5 and hipSC cells

Human bone marrow stromal cell line HS5(human bone marrow stromal cell line, CR L-11882)^TMAmerican Type Culture Collection (ATCC), Manassas, VA, USA) induces neural differentiation of hipSC cells, and the induction medium comprises 20% KSR, 1% NEAA, 1mM glutamine, 0.1mM β -mercaptoethanol, 10ng/ml bFGF, 10ng/ml EGF, 10ng/ml BDNF, 10ng/ml NT-3, 2% B27, 0.5mM adenosine dibutyranyl cyclic phosphate, 1ng/ml transforming growth factor β 3 (all of which are available from Invitrogen), and 500ng/ml Noggin (R) (Noggin)&DSystems, minneapolis, minnesota, usa, where NEAA, bFGF, β -mercaptoethanol and dibutyryladenosine cyclophosphate were added to effectively synchronize the proliferation activity of cell differentiation products during differentiation of stem cells into the neural lineage, B27 was a serum-free additive (mainly containing vitamin a, various antioxidants and insulin) for growth and maintenance of short-term or long-term activity of hippocampal and other central nervous system neurons, B27 was a 50 × concentrated liquid that was used at 1:50 dilution.

Establishment of a direct Co-culture cell System HS5 was irradiated (gamma-irradiation) with an intensity of 80Gy at 1 × 10 before co-culture with hipSC cells⁶HS5 cells for 30 minutes in a Gamma cell1000Elite 214, MDS Nordion, Ottawa, Ontario, Canada) at 2 × 10⁵One/well, inoculated onto 6-well plates for one day of culture.

Establishment of non-direct contact Co-cultured cell System HS5 cells were irradiated and cultured at 2 × 10 before co-culturing with hiPSC cells⁵(ii) a seed/embedding tank, inoculated into a tissue culture embedding tank (ThinCerts, Flikenhausen, Germany), followed by nesting in a 6-well plate HS5 conditioned medium (HS5-CM), 1 × 10⁷HS5 cells were irradiated and plated on a petri dish containing 10ml of the induction medium described above, culture supernatant was collected daily and collected for 4 consecutive days, prior to use, collected HS5-CM was diluted at a ratio of 1:1 (equal volume of freshly prepared induction medium mixed with HS5-CM at equal ratio), respectively, hipSC cells were diluted at 2 × 10⁵One well, inoculated in a Matrigel precoated (DMEM/F12 culture)Matrigel) in 6-well plates were diluted at a ratio of 1: 50. for experiments inhibiting the Notch pathway, 4. mu.M L-685458 (Notch signaling pathway inhibitor (from Calbiochem San Diego, Calif., USA) was added on the first day of culture and incubated for 8 days.

FIG. 1a shows that HS5 can secrete various cytokines, and the cell survival rate and the Pax-6 positive rate of neural precursor cells are compared 8 days after induced differentiation by the two different methods, and the results show that the cell survival rate and the Pax-6 positive rate are 89.3 +/-1.4% and 45.2 +/-2.8% respectively after induced differentiation by the direct contact co-culture method for 8 days, which are superior to those of the non-contact co-culture method (FIG. 1b) and induced differentiation by another germ line IMR90-1 cell of hipSC cells, and the results are the same (FIG. 1c), and the results show that the neural stem cell line based on HS5(human bone marrow stromal cell line, CR L-11882)^TMATCC, Manassas, VA, USA), the direct co-culture method is more advantageous in promoting the proliferation and differentiation of neural precursor cells than other methods.

Example 2 three-stage culture of neural differentiation of hiPSC cells

First stage, hiPSC cells and HS5 cells were induced for 2 weeks in direct cocultivation, the composition of the induction medium was the same as in example 1, and the hiPSC cells were cultured in six-well plates at 2 × 10⁵One/well was inoculated directly to pre-plated 2 × 10 cells 1 day ago⁵One/well HS5 cell layer; for the parallel control group, hiPSC cells were not co-cultured with HS5 cells, but were directly seeded in Matrigel-coated 6-well plates, followed by

phase

2 and 3 culture in the same manner. The liquid is changed every other day.

The second stage is continuous culture with HS5-CM diluted 1:1 for 2 weeks, and HS5-CM medium preparation 1 × 10⁷After being irradiated, the HS5 cells are inoculated into a culture dish containing 10ml of hipSC induction culture medium, waste liquid is collected every day, and HS5-CM culture medium is obtained after continuous collection for 4 days. Before use, the collected HS5-CM was diluted at a ratio of 1:1 for application (freshly prepared hipSC induction medium was mixed with HS5-CM at equal volume ratio).

The third stage is that after the second stage culture is finished, the cells are enzymolyzed and then treated with 2 × 10⁵Seed/well, inoculationIn 6-well plates previously coated with polyornithine and laminin, 3 rd stage selection culture was performed, with Neurobasal medium (Invitrogen) supplemented with 20ng/ml bFGF, 20ng/ml EGF, 2% B27, 1% N2 additive, 1% FBS (Invitrogen), 10 μ M forskolin (Forskolin, Calbiochem, San Diego, CA, USA, also known as forskolin; the line "adenylate cyclase activator"), and 0.2mM ascorbic acid (Sigma, St. L ouis, MO, USA) for 2 weeks, wherein the addition of N2 additive and FBS was done to more effectively maintain survival of mature neurons in the terminal stage of differentiation.

Example 3 direct contact Co-culture activated Notch signaling pathway in hiPSC cells

Since the Notch ligands Delta1, Delta3, Jagged1 and Jagged2 can be easily detected in HS5 cells by using the method of reverse transcription PCR in the experimental method (FIG. 2a), and the corresponding receptors Notch1, Notch2 and Notch3 can be detected in hipSC cells and derivatives thereof (FIG. 2b), the Notch signaling pathway is presumed to be a medium for the interaction between HS5 and hipSC cells. The NICD protein is dissociated from Notch receptors by gamma-secretase and is targeted to downstream molecules Hes1 and Hes 5. After inducing differentiation for 8 days, the expression level of NICD protein in each group of hiPSC-derived cells was examined using the "immunoblot analysis" method among experimental methods. The results show that the direct contact co-culture group was significantly higher than the non-contact co-culture group and the control group (fig. 2c-d), indicating that the direct contact co-culture method was able to significantly activate Notch signaling pathway in hiPSC cell derivatives. It is noteworthy that non-contact co-culture can also increase the expression levels of NICD, Hes1, Hes5, albeit to a very small extent. This suggests that HS5 might secrete a soluble molecule that has an activating effect on the Notch signaling pathway.

After 8 days of direct contact coculture, the expression levels of neuroectodermal related genes such as Sox-1, Pax-6 and NFH in hiPSC-derived cells are obviously increased compared with those of a control group of indirect contact coculture, while the expression levels of endodermal, mesodermal and stem cell related genes are reduced (FIG. 2). these data show that HS5 direct contact coculture can promote the differentiation of hiPSC cells to nerve cells and simultaneously block the differentiation of the hiPSC cells to non-nerve cells. subsequently, the invention further tests whether Notch signals are necessary for HS 5-mediated nerve-induced differentiation. after 8 days of continuous culture, the test shows that the expression levels of NICD, Hes1 and Hes5 in HS5 coculture cells are obviously reduced, wherein the levels after the reduction of NICD and Hes5 have no obvious difference from those of L-685458 in the control group of cells using L-685458 (FIG. 2c-d), which shows that the activation and activation of the activity of the nock pathways of the hepsc 6-685458 can be completely blocked but the cells are not only slightly influenced by the neural differentiation of the neural cells after the neural cells cultured by the neural cells (see the contact of the nock-transfected cells), but the control group of the neural cells, the neural cells which is not only the neural differentiation of the neural cells which is obviously reduced by the influence the neural differentiation of the neural cells which is induced by the neural differentiation of the nock-transfected cells which is shown by the nock-transfected cells, and the nock-transfected cells which is not only the nock signal of the nock 466, the nock-transfected cells which is shown by the nock-transfected cells which is increased (FIG. 7-466-transfected cells), and the nock-466, the nock-transfected cells which is not only by the nock-transfected cells which is obviously (FIG. 7-transfected cells which is not only by the factor of the nock-transfected cells which is shown by the factor of the nock, the nock 466, the induction of the nock-transfected cells which is not only by the nock-transfected cells which is increased, the nock 466-transfected cells which is not only by the nock-transfected cells which is shown by the nock-27, the nock-transfected.

Example 4 identification of the composition of the hiPSC cell-derived neural cell system

After the induction culture of HS5, the neural precursor cells with positive Pax-6 expression rapidly increase in the continuous culture of the second stage, even the positive rate reaches 87.2 +/-1.9% at the end of the culture, meanwhile, the number of undifferentiated cells with positive SSEA-4 is gradually reduced to zero (figure 3), the RT-PCR reverse transcription is carried out for cDNA synthesis, the reaction conditions are 50min at 42 ℃ and 5min at 95 ℃, and then the cells are stored in the environment with 4 ℃. Then, the target gene in the cDNA is amplified by PCR. The basic reaction conditions of PCR are as follows: (1) pre-denaturation of cDNA: 5min at 94 ℃; (2) and (3) PCR amplification: 30 cycles of 94 ℃ for 30sec, 60 ℃ for 30sec, and 72 ℃ for 45 sec; (3) and (3) final extension stage: 7min at 72 ℃. See table 1 for primer sequences.

TABLE 1 primer sequences for reverse transcription PCR and target Gene Length

The analysis further confirmed (FIG. 5) that sternness gene transcription factor Oct-4 and alkaline phosphatase (A L P) were not detected at the end of the second phase, while the expression of the neural precursor cell markers Nestin and Musashi-1 was significantly increased, immediately after which a more mature (at the end of the third phase, a more mature subset of nerve cells, including the glial cell marker GFAP, the mature neuronal marker MAP-2 at the end of mitosis, and the dopaminergic neuronal marker Nurr-1, showing a strong expression status; see FIG. 5) was found at the end of the third phase of culture, including Tuj1 positive neurons (28.2 + -2.1%), GFAP positive astrocytes (9.1 + -0.8%) and O4 positive oligodendrocytes (4.8 + -0.6%) (FIG. 3), while the number of Pax-6 positive neuroprogenic precursor cells gradually decreased to 59.3 + -1.9% (FIG. 3), which indicates that the growth of both the sternness gene transcription factor Oct-4 and alkaline phosphatase (A L P) were detectable in the third phase of neural precursor cells in culture, and the neural stem cells, and the stem cells were able to be detected by the neural markers GFX-2, and stem cells were found to be non-stem cells.

Of particular note, some SSEA-4 positive hipSC cells were induced to gradually turn cylindrical, eventually forming a tubular rosette structure composed of Pax-6 positive neural precursor cells and Tuj1 positive neurons, and spontaneously dispersed into small masses at stage 2, followed by further differentiation toward a mature phenotype, as synaptonectin in Tuj1 positive neurons that emerged abundantly at stage 3The expression of I is obviously increased, which indicates the formation of synapses. At the same time, dopamine subtypes (TH) were also detected in these cell populations⁺)、GABA(GABA⁺) Choline (ChAT)⁺) And 5-hydroxytryptamine (serotonin)⁺) The positive expression of neurons, corresponding to the cell ratio (percentage of the total cell product) of 5.4 + -0.4%, 3.9 + -0.3%, 9.3 + -0.6% and 6.5 + -0.5%, respectively. Furthermore, the total number of hiPSC-derived cells at each culture stage was significantly higher than the parallel control group, indicating that the HS 5-based method of inducing differentiation had the effect of promoting cell growth, which is consistent with the results of the detection of multiple growth factors in HS5 cells. Finally, methods using "electrophysiological analysis" and "dopamine release test" were used: by high concentration of K⁺Depolarizing stimulation, detecting action potential of neuron, detecting every 10 by high performance liquid chromatography⁶The cells released 8.6. + -. 0.6pmol acetylcholine and 72.5. + -. 6.2pmol dopamine (N. RTM.12), indicating that the neural cells produced by the 3-stage culture method were functional. Meanwhile, RT-PCR analysis showed that the mesodermal, endodermal and pluripotency markers gradually lost expression upon completion of the three-stage culture, while c-kit, SOX-9 and AFP proteins continued to be expressed in the control group. Compared with the control group, the 3-stage culture method has obvious advantages in inducing and generating Tuj1 positive neurons, GFAP positive astrocytes and O4 positive oligodendrocytes.

The antibody for detecting different nervous system cells (see the following documents: Barberi T, Klivenyi P, Calingasan NY, & ltl TtT translation =. L "& gTt L & ltl/T & gTt ee H, Kawamata H, L onam K, Perrier A L, Brusses J, RubioME, Topf N, Tabar V, Harrison N L, Beal MF, Moore MA, Studer L. Neural subtype and section transfer cells [ T5 ] A.7: 10: 7; broad spectrum of transfer and nuclear transfer neuron and tissue transfer in tissue protein tissue protein.Nat Biotechnology.2003; 21: 10: Tt 1: T5. mu. T5. T10: T-rat neuron 5. T5. 10: T5. mu. T-10. alpha. 1. alpha. 10. D.5. D.7. A. No. 5. A. No. 5. T.5. A. D.7. A. No. 5. No. 2. A. No. 2. A. No. 2. A. No. 2. A. No. 2. No. A. No. 2. A. No. 2. A. No. A. No. 2. A. No. 2. No. A. No. 2. A. No. 2. A. No. A.

In conclusion, the three-stage culture method can induce the generation of a multi-layered neural cell lineage while limiting the generation of non-neural cell derivatives.

Effect example 1 Induction of intracerebral transplantation of nerve cells can improve cognitive function in stroke animals

The hippocampus is one of the more vulnerable sites after cerebral ischemia reperfusion. After 12 hours of cerebral ischemia experiment operation, thionin staining was shown to be present in the hippocampal CA1 regionTwo groups after 12h in the extensive cell contraction and necrosis (sham operated control group compared with stroke group, every 0.25mm²The complete pyramidal neuron counts in CA1 region were 218.6 + -9.5 and 121.3 + -8.9, respectively, P < 0.0001). Over time, the number of morphologically intact pyramidal neurons (large nuclei, clear nucleoli and intact cell borders) in CA1 zone continued to decrease within 3 days after brain surgery (every 0.25mm on

days

1 and 3 after stroke surgery)²The number of intact neurons in the CA1 region was 77.5 + -5.1 and 39.2 + -3.5, respectively, P<00.001). In the following time window (5 days after the surgical operation of gerbil stroke), the number of pyramidal neurons was no longer significantly reduced (every 0.25mm on days 3 and 5)²Number of intact pyramidal neurons in CA1 region 39.2 + -3.5 and 31.8 + -3.2, respectively, P0.43. after 3 days of cerebral apoplexy, three-stage induction culture yielded 5 × 10⁵2.5 × 10⁵Lateral × 2 side) nerve cells were transplanted into bilateral caudate putamen of rats with ischemic stroke, in which 82.3 + -1.2% of pyramidal neurons in the CA1 region of hippocampus were widely lost, rats that could reach the escape platform of Morris water maze within 150 + -4 s before the operation of stroke molding were used for in vivo experimental study, after the operation of stroke, 92.3% of rats could continue to survive with phenomena of lethargy, coma, lack of movement, etc. six weeks after transplantation, no dead rats were found, and no abnormal behavior or progressive dyskinesia was found, which indicates that the nerve cells differentiated by hiPSC cells were not significantly adversely affected to rats in intracerebral transplantation, and at the same time, 14 days of behavioral assessment revealed that the time of retention of the animals transplanted by intracerebral nerve cells in the water maze was gradually reduced compared to those of hMSC transplanted group and physiological saline injected group, confirming that the animals transplanted by intracerebral nerve cells had better learning and memory abilities than those of hMSC transplanted group and physiological saline injected group.

Behavioral assessment experiments (i.e., 56 days after transplantation, see "behavioral analysis" in experimental methods for details) on day 14 showed that gerbils in the nerve cell transplantation group reached the escape plateau (47.4 ± 3.1s) significantly less than in the saline control group (165.8 ± 7.4s, P < 0.0001) and the hMSC transplantation group (96.7 ± 6.6s, P < 0.0001). There was no significant difference in the index of time to reach the escape plateau between the neural cell transplantation group (47.4 ± 3.1S) and the sham-operated control group (39.2 ± 2.8S, P ═ 0.282). A150-centimeter straight-line path experiment shows that the swimming speeds of all groups of gerbils are similar, so that the time spent by the gerbils to find an escape platform in a water maze is shortened, and the cognitive abilities of learning, memory and the like play a critical role. In addition, in the space exploration test, the number of times that the gerbil in the nerve cell transplantation group passes through the position of the original escape platform is obviously more than that of the normal saline control group and the hMSC transplantation group. The same result is also shown when the retention time of the gerbil in the target quadrant of the original escape platform is measured (namely, the retention time of the gerbil in the target quadrant of the original escape platform in the nerve cell transplantation group is the longest). In conclusion, the implantation of neural cells differentiated from hiPSC cells is helpful for improving the spatial learning and memory ability of animals with ischemic stroke, and the effect is obviously better than that of hMSC transplantation.

Effect example 2 intracerebral transplantation of cells to promote recovery of damaged hippocampus

Brain transplantation implanted cells within the gerbil brain were traced after 8 weeks, and the results are shown in fig. 4: a large number of donor cells were found in the tail shell core and migrated to the surrounding striatal tissue, suggesting that donor cells may be resistant to the intracorporeal environment of the ischemia damaged animal. The presence of donor cells was also found in the cortex, corpus callosum, hippocampus. Intracerebral hemorrhage, microglial infiltration or teratoma formation were not observed in the experiment. The number of intact pyramidal neurons in CA1 region of hippocampus of gerbil in cerebral ischemia nerve cell transplantation group (190.6 + -11.1/0.25 mm)²CA1 area) was significantly higher than that of the non-NSC transplant group (saline control group: 68.8 +/-4.1/0.25 mm²CA1 region, P < 0.0001; hMSC transplant group: 143.2 + -6.5/0.25 mm²CA1 region, P ═ 0.0002). Nerve cell transplantation group (190.6 + -11.1/0.25 mm)²Area CA 1) and sham operation group (212.3 + -10.3/0.25 mm)²CA1 area, P ═ 0.075) density of pyramidal neurons in the hippocampal CA1 area was similar. Meanwhile, hNCAM was found in hippocampus of gerbil transplanted with nerve cells in cerebral ischemia⁺Cells (human neural cell addition molecules, used to detect human nerve cells in the brains of gerbils) indicate that the donor cells have active migratory homing activity. These hNCAMs⁺The cells are located inThe original layer of pyramidal cells in the hippocampus, whose cell morphology resembles intact pyramidal neurons, suggests that neural cells differentiated from hiPSC cells can directly participate in the structural reconstruction of the damaged hippocampus by further differentiation and migration. Similarly, HuN immunostaining (HuN, humannuclear, also used to detect human nerve cells in the brains of gerbils) showed the number of donor cells (10.4. + -. 0.7/0.25 mm) in the hippocampal CA1 region of the nerve cell transplant group²CA1 area) is obviously higher than hMSC transplantation group (3.2 +/-0.4/0.25 mm)²CA1 region, P < 0.0001), which means that intracerebral transplantation of neural cells differentiated from hiPSC cells is more advantageous in promoting the structural reconstruction of the damaged hippocampus and the recovery of the number of pyramidal neurons than hMSC transplantation. A small amount of endogenous regeneration was also detected in the hippocampal region of the brain ischemia saline control group (the number of neurons in CA1 region ranged from 39.2. + -. 3.5/0.25mm 8 weeks ago²Increase to 68.8 +/-4.1/0.25 mm²P < 0.01), indicating that spontaneous endogenous regeneration promoted a slight increase in CA1 area neurons, although the loss of neurons in CA1 area of hippocampus of brain ischemia saline control (66.3 ± 2.3%) remained significant compared to the normal hippocampus of sham operated groups.

On the other hand, neurons in the CA1 region of the neural cell transplantation group were increased by about 121.9. + -. 8.9/0.25mm after 8 weeks of transplantation, as compared with the brain ischemia physiological saline control group². However, histological analysis showed only about 10.4. + -. 0.7/0.25mm in the hippocampal CA1 region²Individual neurons HuN stained positive, indicating that exogenous donor cells promoted the reconstruction of cellular structures in the hippocampal CA1 region primarily by promoting endogenous regenerative mechanisms. Since the intracerebroventricular basic fibroblast growth factor not only stimulates the proliferation of endogenous progenitor cells, but also promotes the differentiation of the endogenous progenitor cells towards nerve cells, it is speculated that the progenitor cell recruitment effect produced by donor cells may be related to the paracrine of the basic fibroblast growth factor. To test this guess, the present invention measured the level of basic fibroblast growth factor in the hippocampal tissues of gerbils 8 weeks after transplantation. The results show that the level of the basic fibroblast growth factor (195.8 +/-11.8 pg/mg protein) in the nerve cell transplantation group is obviously higher than that in the normal saline control group (133.2 +/-8.9 pg/mg protein, P < 0.0001) and the hMSC transplantationGroup (157.5 ± 9.4pg/mg protein, P ═ 0.006), which indicates that basic fibroblast growth factor is secreted from donor cells and promotes the regulation process of endogenous regeneration of hippocampus by paracrine mechanism. Similarly, histological grading analysis shows that the cell structure of the hippocampal pyramidal layer of the nerve cell transplantation group is less damaged and recovers faster compared with the brain ischemia normal saline control group and the hMSC transplantation group. In summary, the hiPSC cell-derived neural cells are integrated into different ischemia-damaged brain regions through migration and homing behaviors after transplantation; on the other hand, the reconstruction of the damaged hippocampus of the cerebral ischemic animal is promoted by promoting an endogenous regeneration mechanism, so that the cognitive function of the cerebral ischemic animal is improved.

Claims

1. A method for directionally inducing differentiation of hipscs into neural cell systems, comprising culturing said hipscs in stages to induce neural differentiation thereof, said stages comprising:

continuously culturing the hipscs with HS5 conditioned medium, the HS5 conditioned medium being an induction medium containing the secretion of HS 5;

continuing to culture the hipscs in a basal medium that cultures neuronal cells;

the induction culture medium in the stage a is a DMEM/F12 culture medium which comprises 20-25% of serum substitute, 0.5-1.5mM of glutamine, 8-20ng/ml of epidermal growth factor, 8-12ng/ml of brain-derived neurotrophic factor, 8-15ng/ml of neurotrophic factor-3, 0.5-1.5ng/ml of transforming growth factor β 3, 400-700 ng/ml of noggin and 2-3% of B27 additive;

the HS5 conditioned medium described in stage b was prepared by 1) mixing 5 × 10⁶~ 2×10⁷Inoculating the irradiated HS5 cells into 8-15ml of the induction culture medium; 2) collecting the supernatant of the cultured cells for 1-8 days continuously; 3) mixing the supernatant and the induction culture medium in a ratio of 1: 1-8: 1 to obtain the compound;

the basic culture medium in the stage c is added with: a neurobasal medium of 15-30ng/ml bFGF, 15-30ng/ml EGF, 1-3% B27 additive, 8-12 mu M forskolin and 0.1-0.3mM ascorbic acid;

the percentages are volume percentages.

2. The method of claim 1, wherein said induction medium further comprises 1-1.5% non-essential amino acids, 8-15ng/ml basic fibroblast growth factor, 0.08-0.15mM β -mercaptoethanol, and 0.3-0.8mM bis-butyryl cyclic adenosine monophosphate;

the percentages are volume percentages.

3. The method of claim 1, wherein said induction medium is a DMEM/F12 medium comprising 20% serum replacement, 1% non-essential amino acids, 1mM glutamine, 0.1mM β -mercaptoethanol, 10ng/ml basic fibroblast growth factor, 10ng/ml epidermal growth factor, 10ng/ml brain derived neurotrophic factor, 10ng/ml neurotrophic factor-3, 1ng/ml transforming growth factor β 3, 0.5mM dibutyryladenosine cyclophosphate, 500ng/ml noggin, and 2% B27 cell culture additive;

the percentages are volume percentages.

4. The method of any one of claims 1 to 3, wherein the serum replacement is KnockOut^TMA serum replacement; the percentages are volume percentages.

5. The method of any one of claims 1 to 3, wherein the co-cultivation in stage a is direct contact co-cultivation;

and/or the bone marrow stromal cells HS5 are irradiated HS 5;

and/or the co-culture time is 10-18 days.

6. The method of claim 5, wherein the conditions of the irradiation are: the gamma ray irradiation intensity is 75-85Gy, and the irradiation time is 28-35 minutes.

7. The method of claim 6, wherein the irradiation intensity is 80Gy and the irradiation time is 30 minutes.

8. The method of claim 5, wherein the co-cultivation is for a period of 2 weeks.

9. The method according to any one of claims 1 to 3, wherein the continuous cultivation in phase b is carried out for a period of 8 to 18 days;

and/or, the period of continued culturing in phase c is 10-18 days.

10. The method of claim 9, wherein the period of continuous culture in stage b is 2 weeks.

11. The method of claim 9, wherein the HS5 conditioned medium is prepared by 1) mixing 1 × 10⁷Inoculating 10ml of the induction medium with the irradiated HS5 cells; 2) collecting the supernatant of the cultured cells for 4 consecutive days; 3) mixing the supernatant and the induction culture medium in a ratio of 1: 1.

12. The method of claim 9, wherein the period of continued culturing in phase c is 2 weeks.

13. The method of claim 9, wherein the medium in stage c further comprises 0.5-1.5% N2 additive and 0.5-1.5% fetal bovine serum;

the percentages are volume percentages.

14. The method of claim 9, wherein the basal medium of stage c is supplemented with: 20ng/ml bFGF, 20ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10 μ M forskolin, and 0.2mM ascorbic acid in neurobasal medium, the percentages being volume percentages.

15. A neural cell system induced by the method of any one of claims 1 to 14, comprising: 59.3 +/-1.9 percent of neural stem cells, 28.2 +/-2.1 percent of multifunctional neurons, 9.1 +/-0.8 percent of astrocytes and 4.8 +/-0.6 percent of oligodendrocytes, wherein the percentage is the percentage of the number of the whole neural cell system.

16. A neural cell system as claimed in claim 15, wherein the multifunctional neuronal cell comprises: dopaminergic neuron 5.4 + -0.4%, acetylcholine neuron 9.3 + -0.6%, GABAergic neuron 3.9 + -0.3%, 5-hydroxytryptamine neuron 6.5 + -0.5%, and juvenile neuron 3.1 + -0.4%; the percentage is the number percentage of the whole nerve cell system.

17. Use of a neural cell system as claimed in claim 15 or claim 16 in the preparation of a brain tissue cell repair preparation.

18. The use of claim 17, wherein the agent is an agent for treating cerebral injury resulting from ischemic stroke, cerebral hemorrhage or trauma.