CN111793608B

CN111793608B - HS5 conditioned medium for directionally inducing differentiation of hipscs into neural cell system

Info

Publication number: CN111793608B
Application number: CN202010752115.1A
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: 2022-05-17
Anticipated expiration: 2037-07-28
Also published as: CN107326013A; CN111793608A; ZA201802126B; CN111793607B; CN107326013B; CN111793607A; CN111849899A; WO2019019223A1; CN111849899B

Abstract

The invention discloses an HS5 conditioned medium for directionally inducing differentiation of hipscs into a neural cell system. 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 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

HS5 conditioned medium for directionally inducing differentiation of hipscs into neural cell system

The application is a divisional application of a patent application with the application date of 2017, 07, 28 and the application number of 201710632172.4 and the name of 'directionally inducing a neural cell system after hiPSC differentiation, an 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 advances in stem cell biology provide the basis for regenerative medicine, where directed differentiation of hiPSC cells can provide a variety of human neural cells for cell transplantation in treating patients with ischemic stroke (characterized by severe impairment of various neurons and glial cells). Efficient induction, purification and implantation of human neural cells are essential for the establishment of hiPSC cell-based neural cell therapies. Many protocols have been proposed for differentiating neural cells with high efficiency. However, these methods are insufficient to provide a mature neural cell lineage, resulting in promiscuous cells intermixed therein, leading to the formation of teratomas after intracerebral transplantation. In addition, some methods differentiate to produce a narrow spectrum of neural cell lines and do not satisfy the multiple cell types required for the treatment of ischemic stroke. More importantly, in order to apply the hiPSC cell-induced nerve cells to clinical application, it is necessary to separate and purify the induced nervous system cells to remove non-nervous system cells and naive cells. Thus, establishment of hiPSC cell-based nerve induction and purification systems has been expected for a long time (Rov, N.S.; Cleren, C.; Singh, S.K.; Yang, L.; Bell, M.F.; Goldman, S.A. functional expression of human ES cell-derived and chimeric genes with molecular gene-derived and chimeric gene-derived), Med.12: 1259- "1268; Yan, Y.P.; Yang, D.L.; Zarnowska, E.D.; Du Z.W.; Werbel, B.v.; Valliere, C.; Pear.A. Thomson, J.A. cell, E.D.; Du Z.W.; Werke cell, C.12423; U.S.S. J.S. Pat. No. D.D.; gradient 23; gradient cell, C.D.D.; moisture, C.D.D.D. D. D.D.D.; moisture, C.D.D.D.D. D. Pat. D. 7. blend, U.A. C. 7. blend, U.A. blend, moisture, U.A. Pat. C. Pat. 7. blend, U.A. 7. blend, U.A. C. Pat. 7. blend, U.A. 7. blend, U.A. 7. supplement, U.A. C. A. C. A. 7. blend No. 7. blend of U.A. 7. blend, U.A. C. blend, U.A. blend No. 7. blend, U.A. 7. blend No. 7. blend No. 7. blend of U.A. 7. blend, U.S. 7. blend, U.A. 7. blend No. 7. blend, U.S. 7. blend No. 7. blend, U.S. blend No. 7. blend, U.S. 7. blend, U.g. 7. blend No. 7. blend, U.S. 7. blend No. 7. blend, U.S. 7. blend No. blend, U.S. blend, U.S. 7. blend No. 7 et al, U.S. blend, U.S. 7. blend, U.S. blend No. blend, U.S. 7 et al, U.S. 7. blend No. 7. blend, U.S. blend No. 7.

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; preferably, the induction medium comprises the following components: 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 beta 3, 400-700 ng/ml of noggin and 2-3% of DMEM/F12 culture medium supplemented with B27, wherein the percentages are volume percentages. More preferably, in order to efficiently and simultaneously promote the proliferative activity of a cell differentiation product during differentiation of stem cells into a neural cell lineage, the induction medium further comprises 1-1.5% of a non-essential amino acid, 8-15ng/ml of a basic fibroblast growth factor, 0.08-0.15mM of β -mercaptoethanol, and 0.3-0.8mM of dibutyryl cyclic adenosine monophosphate.

Even more preferably, the culture medium comprises the following components: 20% serum replacement, 1% non-essential amino acids, 1mM glutamine0.1mM beta-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 beta 3, 0.5mM adenosine dibutyranyl cyclic phosphate, 500ng/ml noggin and 2% B27 cell culture additive DMEM/F12 medium. The serum substitute is preferably KnockOut^TMA serum replacement.

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

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 5X 10⁶～2×10⁷Inoculating the irradiated HS5 cells into 8-15ml of the induction 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; more preferably, the preparation method of the HS5 conditioned medium comprises the following steps: 1) will be 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 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 scheme for solving the problems is as follows: an induction medium for initially inducing differentiation of hipscs into neural cell systems, said medium comprising the following components: 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 beta 3, 400-700 ng/ml of noggin and 2-3% of DMEM/F12 culture medium with B27 additive, wherein the percentages are volume percentages; preferably, the culture medium further comprises 1-1.5% non-essential amino acids, 0.08-0.15mM beta-mercaptoethanol, 8-15ng/ml basic fibroblast growth factor, and 0.3-0.8mM bis-butyryl cyclic adenosine monophosphate. If the content of the above medium components is less than the lower limit of the above numerical range, the survival rate of hipscs during differentiation is low (the total survival rate of differentiation products is less than 40%); above the upper limit of the above numerical range, not only the survival rate of the differentiated product of hipscs is low (less than 45%), but also the differentiation efficiency of hipscs into neural lineage cells is significantly reduced (less than 24%).

In a preferred embodiment of the present invention, the culture medium comprises the following components: 20% serum replacement, 1% non-essential amino acids, 1mM glutamine, 0.1mM beta-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 beta 3, 0.5mM adenosine dibutyranyl cyclic phosphate, 500ng/ml noggin, and 2% B27 cell culture additive in DMEM/F12 medium; 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 the following preparation method: 1) will be 5X 10⁶～2×10⁷Inoculating the irradiated HS5 cells into 8-15ml of the induction medium; 2) collecting the supernatant of the cultured cells for 1-8 days continuously; 3) and mixing the supernatant and the induction culture medium in a ratio of 1: 1-8: 1. Wherein, in the step 1), if the cell number is less than 5 × 10⁶After cell inoculation and before irradiation, the survival rate of the cells is low (generally lower than 80 percent), and the concentration of the produced cell secretion is low; if the number of cells is higher than 2X 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 step 2), if the number of days for collecting the cell culture medium supernatant is less than 1 day, it is a waste of cell resourcesA fee; 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 the following preparation method: 1) will be 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). 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 (corrserving 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 results of reverse transcription PCR experiments. Shows that the expression of the genes Oct-4 and ALP related to the dry/undifferentiated state gradually disappears, and the expression completely disappears in the co-culture group at the end of the second phase; the expression of neural precursor cell markers Nestin and Musashi-1 is gradually enhanced; at the end of the third phase, more mature neuronal cell subtypes, including the glial cell marker GFAP, the late mitotic mature neuronal cell markers MAP-2, Nurr-1, showed strong expression status.

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

NT-3: neurotropin-3 neurotrophic factor-3

GFAP: 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

ALP: 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

The intracellular activation domain (NICD) of human Notch1 molecule was amplified by Polymerase Chain Reaction (PCR) for the 1759-2444 amino acid sequence in GenBank NM-017617 by pubmed search. Specific primers (upstream 5'-CGC GGA TCC ATG CGC AAG CGC CGG CGG CAG CAT-3'; downstream 5'-ACG TCT AGA CAC GTC TGC CTG GCT CGG-3'). The PCR product (2058bp) was digested with BamHI/XbaI cleavage sites and inserted into a mammalian expression vector pcDNA3.1(Invitrogen) to construct a pcDNA3.1/Notch1(pcNICD) expression vector. Cells were cultured at 2X 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 was completed, 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, and 20mM HEPES/NaOH; pH 7.4) for 15 minutes. Dopamine concentrations were determined by High Performance Liquid Chromatography (HPLC) (HTEC 500, Eicom corp., San Diego, CA, USA).

5. Immunoblot analysis

The total protein (20. mu.g) was subjected to 12% SDS-PAGE, and after membrane transfer (Millipore, Billerica, MA, USA), the antibody was added dropwise. Primary antibodies were rabbit anti-NICD (1: 1000; Cell signalling Technology, Beverly, MA, USA), rabbit anti-Hes 1(Hes 1; 1: 500; Chemicon, Temecula, Calif., USA), rabbit anti-Hes 5(1: 500; Chemicon) and murine anti-alpha-tubulin antibody (1: 2000; Chemicon), respectively. The secondary antibody was HRP (horseradish peroxidase) -labeled goat anti-rabbit or goat anti-mouse IgG or IgM (1: 2000; Millipore) and the band intensity was quantified using imaging software (Bio-Rad, Hercules, Calif., 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 g) 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 (2.5X 10) were injected into the corresponding area of the gerbil brain⁵Cells/5. mu.l of physiological saline, i.e., 2.5X 10⁵After suspending in 5. mu.l of physiological saline, the individual cells were transplanted to one side of the recipient brain; each animal brain received a total of 2 transplants on the left and right sides), (5X 10)⁵Individual/whole brain; the injection points are 0.5mm before bregma, 2mm outside midline and 4mm on ventral dura mater), and the same amount of physiological saline is injected into the corresponding part of the control gerbil. Human mesenchymal stem cells (hmscs) were used as cell control. The bone marrow aspiration collection procedure of healthy people is approved by regional ethics review committee and granted informed consent. Briefly, monocytes were collected at 1.6X 10 using 1073. mu.g/mL density gradient separation medium (GE Healthcare, Piscataway, NJ, USA)⁵/cm²The medium was a low sugar DMEM medium supplemented with 10% FBS, 1% NEAA and 1mM L-glutamine. After a period of culture, hMSC cell suspension was injected into gerbil brains in the same manner and at the same dose. Cyclosporine A (Novartis, Basel, Switzerland) was injected into gerbils 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 by adding 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 excised brain tissue was frozen in a thickness of 5 μm along the coronal plane. The number of donor cells was counted in the serial section as the number of immunoreactive positive cells in every fifth section and finally corrected by Abercrombic formula. In addition, tissues approximately 2-2.2mm between bregma and bregma were serially sectioned, stained with 0.2% thionin, photographed, and microscopically measured and counted in a 1mm by 0.25mm dimensional scale frame per frame image. Intact pyramidal neurons in the hippocampal CA1 region (large nuclei, clear nucleoli and clear cell membrane) were counted as described previously. Cells from the hippocampal CA1 region were graded according to the criteria described previously: level 0, the percentage of the fixed and condensed CA1 pyramidal neurons is less than 10% of the total number of cells in the frame; stage I, the number of the fixed and contracted CA1 pyramidal neurons accounts for 10% -40% of the total number of cells in the frame; stage II, 40-70% of solid contracted cells; grade III: the ratio of the solid-shrinkage cells is more than or equal to 70 percent.

11. Immunocytochemistry

Mouse anti-beta-tubulin III antibody (TuJ1,1: 500; Sigma), mouse anti-O₄Antibodies (1: 50; Chemicon), mouse anti-stage specific embryonic antigen-4 antibody (SSEA-4, 1: 100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-pax-6 antibody (1: 100; Santa Cruz Biotechnology); rabbit anti-Nestin antibody (1: 400; Chemicon), rabbit anti-collagen fibrin antibody (GFAP,1: 400; Chemicon), rabbit anti-synaptophysin I antibody (1: 500; Chemicon), rabbit anti-tyrosine hydroxylase antibody (TH,1: 100; Chemicon), rabbit anti-gamma-aminobutyric acid antibody (GABA,1: 200; Sigma), rabbit anti-choline acetyltransferase antibody (ChAT, 1: 200; Sigma), rabbit anti-5-hydroxytryptamine antibody (1: 100; Sigma) applied to cells; whereas murine anti-human mitochondrial antibodies (hmito, 1: 40; Sigma), murine anti-human nuclear antibodies (HuN,1: 100; Chemicon), murine anti-human neural cell adhesion molecule antibodies (hNCAM,1: 100; Santa Cruz Biotechnology) were applied to the tissue sections. CellsAnd the sections were further incubated with Fluorescein Isothiocyanate (FITC) or rhodamine tetramethyl isothiocyanate (TRITC) -labeled goat anti-mouse or anti-rabbit IgG or IgM (1: 100; Millipore) or with biotinylated goat anti-mouse IgG (1: 100; Vector, Burlingame, Calif., USA). Followed by addition of streptavidin-HRP (streptavidin-horseradish peroxidase) and visualization of the immunoreaction with diaminobenzidine. Cells or sections were washed and counterstained with 10. mu.g/mL propidium iodide (PI; Sigma) or 4', 6-diamidino-2-phenylindole (DAPI; Sigma).

12. Statistical analysis

Data are presented as mean ± sem. The SNK test and the t test are compared and analyzed pairwise, and the Nemenyi test is used for histological grading analysis of the hippocampal pyramidal cell layer. Using the SPSS17 software system (SPSS inc., Chicago, IL, USA), a P value < 0.05 was 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, CRL-11882) is utilized^TMAmerican Type Culture Collection (ATCC), Manassas, VA, USA) induces neural differentiation of hiPSC cells, and the composition of the induction medium is: 20% KSR, 1% NEAA, 1mM glutamine, 0.1mM beta-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 beta 3 (all from Invitrogen) and 500ng/ml Noggin (R)&D Systems, minneapolis, minnesota, usa). Wherein, the NEAA, the bFGF, the beta-mercaptoethanol and the dibutyryladenosine cyclophosphate are added to effectively and synchronously promote the proliferation activity of cell differentiation products in the process of differentiating stem cells to a neural cell lineage; b27 is 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 neurons and other central nervous system neurons. The B27 additive was a 50 Xconcentrated liquid that was diluted 1:50 at the time of use.

Establishment of direct Co-culture cell System in Co-culture with hiPSC cellsPreviously, HS5 was irradiated (gamma-irradiation) with an intensity of 80Gy at 1X 10⁶HS5 cells; the time is 30 minutes; the apparatus was Gamma acell 1000Elite 214, MDS Nordion, Ottawa, Ontario, Canada) followed by 2X 10⁵One/well, inoculated onto 6-well plates for one day of culture.

Establishment of non-direct contact Co-culture cell System HS5 cells were irradiated and cultured at 2X 10 before co-culturing with hiPSC cells⁵Seed/embedded wells, inoculated into tissue culture embedded wells (ThinCerts, friekenghasen, germany), and then nested in 6-well plates. HS5 conditioned Medium (HS5-CM), 1X 10, was prepared⁷Individual HS5 cells were irradiated and plated onto petri dishes containing 10ml of the induction medium described above. The culture supernatants were collected daily for 4 consecutive days. Before use, the collected HS5-CM was diluted at a ratio of 1:1 (equal volume of freshly prepared induction medium was mixed with HS5-CM at equal ratio), respectively. hiPSC cells at 2 × 10⁵One well, was inoculated in a 6-well plate precoated with Matrigel (the Matrigel was diluted in DMEM/F12 medium at a ratio of 1: 50). For experiments to inhibit the Notch pathway, 4. mu. M L-685458(Notch signaling pathway inhibitor (purchased from Calbiochem San Diego, Calif., USA)) was added on the first day of culture and incubated for 8 days.

Figure 1a shows that HS5 can secrete a variety of cytokines. After inducing differentiation for 8 days using the above two different methods, the cell survival rate and the Pax-6 positive rate of neural precursor cells were compared. The results showed that the cell survival rate and the positive rate for Pax-6 were 89.3. + -. 1.4% and 45.2. + -. 2.8%, respectively, after inducing differentiation for 8 days using the direct contact co-culture method, which was superior to the non-contact co-culture method (FIG. 1 b). The same results were obtained using another germline IMR90-1 cell of hiPSC cells for induced differentiation (fig. 1 c). The above results show that the product is based on HS5(human bone marrow stroral cell line, CRL-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

The first stage is as follows: the hiPSC cells were induced with HS5 cells in direct cocultureAfter 2 weeks of culture, the composition of the induction medium was the same as in example 1, and hiPSC cells were cultured in six-well plates at 2 × 10⁵One/well direct inoculation on 2X 10 pre-paved 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.

And a second stage: continuously culturing for 2 weeks by using HS5-CM medium diluted by 1: 1; preparation of HS5-CM medium: 1X 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).

And a third stage: after the second stage of culture, cells were subjected to enzymolysis at 2X 10⁵One well, inoculated into a 6-well plate previously coated with polyornithine and laminin, followed by stage 3 selection culture. Cultured for 2 weeks in neurobasal medium (Invitrogen) supplemented with 20ng/ml bFGF, 20ng/ml EGF, 2% B27, 1% N2 additive, 1% FBS (Invitrogen), 10. mu.M forskolin (forskolin, Calbiochem, San Diego, Calif., USA, also known as forskolin; line "adenylate cyclase activator") and 0.2mM ascorbic acid (Sigma, St. Louis, Mo., USA). Among them, the addition of N2 and FBS was performed in order to more effectively maintain the survival rate of mature neurons at the terminal stage of differentiation.

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

Since 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 corresponding receptors Notch1, Notch2 and Notch3 can be detected in hiPSC cells and derivatives thereof (fig. 2b), it is presumed that the Notch signaling pathway is a mediator of 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 may secrete a soluble molecule that has an activating effect on the Notch signaling pathway.

Expression levels of neuroectoderm-related genes such as Sox-1, Pax-6 and NFH were significantly increased in hiPSC-derived cells, while expression of endoderm-, mesoderm-and stem cell-related genes was decreased after 8 days of direct contact co-culture as compared to control non-direct contact co-culture (fig. 2). These data indicate that direct contact co-culture of HS5 can promote differentiation of hiPSC cells to neural cells while blocking their differentiation to non-neural cells. Subsequently, the present invention further tested whether Notch signaling was essential for HS 5-mediated neural induced differentiation. After 8 days of continuous culture by adding a Notch signaling pathway inhibitor L-685458, the expression level of the NICD, the Hes1 and the Hes5 in HS5 co-cultured cells is detected to be obviously reduced, wherein the reduced level of the NICD and the Hes5 has no obvious difference from the expression level of the control group using L-685458 (fig. 2c-d), which indicates that L-685458 can completely block the activation of a Notch pathway. However, L-685458 had a minor effect on the expression level of Hes1 (FIGS. 2c-d), which further confirms that expression of Hes1 is not dependent solely on the Notch signaling pathway. Furthermore, the direct contact co-culture of HS5 after addition of L-685458 promoted a diminished effect of Sox-1, Pax-6 and NFH expression, while in contrast, the expression of non-neural markers was increased by disinhibition (FIG. 2e), suggesting that the Notch signaling pathway plays a crucial role in HS5 mediated neural-induced differentiation of hipSC cells. L-685458 reduced the expression rate of Pax-6 positive cells in the experimental and control groups, but had no significant effect on cell viability (FIG. 2f, g). On the other hand, when iPS-1 cells transfected with pcNICD (construction method, see experimental method) were cultured for 8 days, Pax-6 positive cells increased significantly (fig. 2h) and tended to differentiate into neural cells, as compared with the control group transfected with only the vector (pcdna3.1). This suggests that NICD, an effector of the Notch signaling pathway, can push hiPSC cells to differentiate into neural cells. All data suggest that activation of Notch signaling may be, at least in part, a factor necessary for direct co-culture methods to promote differentiation of hiPSC cells into neural cell lineages.

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 (ALP) were not detected at the end of the second phase, while neural precursor cell markers Nestin and Musashi-1 expression were significantly increased. Subsequently, at the end of the third stage of culture, more mature (at the end of the third stage, a more mature neuronal cell subset including the glial cell marker GFAP, the mature neuronal marker MAP-2 in the post-mitotic phase, and the dopaminergic neuronal marker Nurr-1, showing a strongly expressed state; see FIG. 5) were found, including Tuj 1-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), indicating that the third stage of culture facilitated the normalized growth and maturation of the neuroprogenic precursor cells. Consistent with the above results, the transcription factors of the neural cell subtype-specific genes, including GFAP, a marker for astrocytes, MAP-2, a marker for postmitotic neurons, and Nurr-l, a dopaminergic neuron marker, were all detectable during stage 3 culture, while the expression of genes associated with mesoderm (c-kit, SOX-9, and PPAR γ) and endoderm development (AFP, glucose transporter-2, and amylase) was not detected, which further confirmed that the 3-stage culture method does not support the growth of non-neural cells.

Of particular note, some SSEA-4 positive hipscs were induced to gradually turn cylindrical, eventually forming tubular rosette structures 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 towards a mature phenotype, as evidenced by a significant increase in synaptonei expression in the large number of Tuj1 positive neurons at stage 3, suggesting synaptic formation. 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. At the same time, RT-PCR analysis showed that the mesodermal, endodermal and pluripotency markers gradually lost expression upon completion of the three-stage culture, while the controlThe c-kit, SOX-9 and AFP proteins in the group were expressed continuously. Compared with a control group, the 3-stage culture method has obvious advantages in inducing and generating Tuj1 positive neurons, GFAP positive astrocytes and O4 positive oligodendrocytes.

Markers for different nervous system cells were detected by corresponding antibodies (see Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H, Loonam K, Perrier AL, Brush J, Rubio ME, Topf N, Tabar V, Harrison NL, Beal MF, Moore MA, Stder L.neural subset specification of transformation and nuclear transfer complex cells and application in partial analysis. Nat Biotechnology. 2003; 21(10): 1200. quadrature. 1207; Fong SP, Tscan KS, Chan AB, Lu G, PoWS on, Li K, Baum LW, Ng HK. transport of simulation cell J2007: 2. expression J.1852. expression J.): mouse anti-pax-6 antibody (1: 100; Santa Cruz Biotechnology), rabbit anti-Nestin antibody (1: 400; Chemicon) detect markers for neural stem cells, pax-6, Nestin, broad spectrum neurons, dopaminergic neurons, GABAergic neurons, cholinergic neurons, 5-hydroxytryptophan neurons, astrocytes and oligodendrocytes, respectively, are beta-tubulin, tyrosine hydroxylase, gamma-aminobutyric acid, choline acetyltransferase, 5-hydroxytryptamine, glial fibrillary acidic protein and O4, respectively, and the corresponding antibodies are mouse anti-beta-tubulin III antibody (TuJ1,1: 500; Sigma), rabbit anti-tyrosine hydroxylase antibody (TH,1: 100; Chemicon), rabbit anti-gamma-aminobutyric acid antibody (GABA,1: 200; sigma), rabbit anti-choline acetyltransferase antibody (ChAT, 1: 200; sigma), rabbit anti-5-hydroxytryptamine antibody (1: 100; sigma), rabbit anti-glial fibrillary acidic protein antibody (GFAP,1: 400; chemicon) and mouse anti-O4 antibody (1: 50; chemicon), after the above antibody was applied to the cells (1-2 hours of antibody assignment), goat anti-mouse or anti-rabbit IgG or IgM (1: 100; millipore) for further reaction. Cells were washed and counterstained with 10. mu.g/mL propidium iodide (PI; Sigma) or 4', 6-diamidino-2-phenylindole (DAPI; Sigma). And (3) displaying a detection result: the induced nervous system cells comprise 59.3 plus or minus 1.9 percent of neural stem cells, 28.2 plus or minus 2.1 percent of various functional neurons (wherein 5.4 plus or minus 0.4 percent of dopaminergic neurons, 9.3 plus or minus 0.6 percent of acetylcholine neurons, 3.9 plus or minus 0.3 percent of gamma-aminobutyric acid neurons, 6.5 plus or minus 0.5 percent of 5-hydroxytryptamine neurons and 3.1 plus or minus 0.4 percent of juvenile neurons), 9.1 plus or minus 0.8 percent of astrocytes and 4.8 plus or minus 0.6 percent of oligodendrocytes in a fixed proportion (see the 'immunocytochemistry' method in an experimental method for details).

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 gerbil cerebral ischemia experimental operation, thionin staining showed extensive cell shrinkage and necrosis in the hippocampal CA1 region (sham operated control group compared with two groups 12 hours after 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, 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)²The number of intact pyramidal neurons in CA1 area was 39.2 ± 3.5 and 31.8 ± 3.2, respectively, with P ═ 0.43). 3 days after cerebral apoplexy operation, the 5 × 10 of the three-stage induction culture is obtained⁵Each (2.5 is multiplied by 10)⁵Lateral x 2 lateral) nerve cells were transplanted into the bilateral caudate nucleus of rats with ischemic stroke, when 82.3 ± 1.2% of the pyramidal neurons in the CA1 region of the hippocampus were extensively deleted. Gerbils which can reach the Morris water maze escape platform within 150 +/-4 s before stroke modeling operation are used for in-vivo experimental study. 92.3 percent after cerebral apoplexy operationThe gerbil can continue to survive and is accompanied by phenomena of lethargy, coma, lack of movement and the like. Six weeks after transplantation, no dead gerbils were found, and no abnormal behavior or progressive dyskinesia occurred, indicating that intracerebral transplantation of neural cells differentiated by hiPSC cells had no significant adverse effects on gerbils. Meanwhile, the 14-day behavioral assessment found that the time for which the animals subjected to intracerebral nerve cell transplantation stayed in the water maze was gradually reduced compared with the hMSC transplantation group and the saline injection group, confirming that the animals subjected to intracerebral nerve cell transplantation had better learning and memory abilities than the hMSC transplantation group and the saline injection 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 the time to reach the escape platform between the neural cell transplantation group (47.4 ± 3.1S) and the sham operation 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, whichIndicating that the donor cells can tolerate the internal environment of the organism of the animals with ischemic injury. The presence of donor cells was also found in the cortex, corpus callosum, hippocampus. Intracerebral hemorrhage, microglial infiltration or formation of teratoma were not found 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 in the original pyramidal cell layer in the hippocampus, and the cell morphology is similar to that of intact pyramidal neurons, which indicates that the neural cells differentiated by the hiPSC cells can directly participate in the structural reconstruction of the damaged hippocampus through further differentiation and migration. Similarly, HuN immunostaining (HuN, human nucleolus, also used for detecting human nerve cells in the brain of gerbils) showed the number of donor cells (10.4. + -. 0.7/0.25 mm) in the CA1 region of the hippocampal hippocampus 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 can stimulate the proliferation of endogenous progenitor cells, but also can promote the differentiation of the endogenous progenitor cells to the direction of nerve cells, the recruitment effect of the progenitor cells generated by donor cells is supposed to 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 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 is less than 0.0001) and the hMSC transplantation group (157.5 +/-9.4 pg/mg protein, P is 0.006), which indicates that the regulation process of endogenous regeneration of hippocampus is promoted by a paracrine mechanism after the basic fibroblast growth factor is secreted by donor cells. 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 combined culture medium for directionally inducing differentiation of hipscs into a nerve cell system is characterized in that the combined culture medium is a basal culture medium, an HS5 conditioned medium and an induction culture medium;

the HS5 conditioned medium 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, and the irradiation time is 28-35 minutes;

the induction culture medium comprises the following components: 20% KSR, 1% NEAA, 1mM glutamine, 0.1mM beta-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 beta 3 and 500ng/ml noggin in DMEM/F12;

the basic culture medium is neurobasal culture medium added with 20ng/mlbFGF, 20ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10 mu M forskolin and 0.2mM ascorbic acid, and the percentages are volume percentages;

the nerve cell system contains nerve system cells including neural stem cells, functional neurons, astrocytes and oligodendrocytes;

the functional neurons include dopaminergic neurons, acetylcholine neurons, gabaergic neurons, 5-hydroxytryptamine neurons, and naive neurons;

the HS5 conditioned medium is prepared by the following preparation method: 1) will be 5X 10⁶～2×10⁷Inoculating the irradiated HS5 cells into 8-15ml of induction culture medium; 2) collecting the supernatant of the cultured cells for 1-8 days continuously; 3) and mixing the supernatant and the induction culture medium in a ratio of 1: 1-8: 1.

2. HS5 conditioned medium according to claim 1, wherein the irradiation intensity is 80Gy and the irradiation time is 30 minutes.

3. The HS5 conditioned medium of claim 1, wherein the HS5 conditioned medium is prepared by the following method: 1) will be 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.