WO2019019223A1 - Neural cell system following directed induction of hipsc differentiation, induction method and application - Google Patents

Abstract

A neural cell system following the directed induction of human induced pluripotent stem cells (hiPSC) differentiation, an induction method and an application, the method comprising culturing hiPSC in various stages to induce the neural differentiation thereof, the stages comprising: stage a. co-culturing the hiPSC and bone marrow stromal cells (HS5) in an induction culture medium; stage b. continuously culturing the hiPSC by using an HS5 conditioned culture medium; stage c. continuously culturing the hiPSC by using a basic culture medium for culturing neuron cells. The method induces human hiPSC cells to be directionally differentiated into neural system cells, while inhibiting the production of non-neural system cells, thereby obtaining a mature, broad-spectrum neural cell population. The neural cell population has been verified in vitro as mature neurons which release electrical impulses, and has also has been confirmed in mice in vivo to be an effective treatment for nervous system diseases (such as stroke and brain damage).

Description

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

This application claims priority from Chinese patent application CN201710632172.4, filed on July 28, 2017. This application cites the entire text of the above-mentioned Chinese patent application.

Technical field

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

Background technique

Human induced pluripotent stem cells (hiPSC)-derived neural cells have a significant effect on cell transplantation for ischemic stroke (characterized by severe neuronal loss in many different lineages). However, the existing induction of differentiation of hiPSC cells into neural cells is inefficient and poor in stability.

Recent advances in stem cell biology provide a basis for regenerative medicine, in which directed differentiation of hiPSC cells can provide for cell transplantation for patients with ischemic stroke (characterized by severe neuronal and glial defects). A variety of human nerve cells. Effective induction, purification, and implantation of human neural cells are necessary to establish neural cell therapy based on hiPSC cells. A number of draft methods for the efficient differentiation of nerve cells have been proposed. However, these methods are not sufficient to provide a mature neuronal lineage, resulting in the mixing of naive cells, leading to the formation of teratomas after transplantation in the brain. In addition, some methods can only differentiate to produce a narrow-spectrum neuronal cell line that does not meet the many cell types required to treat ischemic stroke. More importantly, in order to apply hiPSC-induced neural cells to the clinic, it is necessary to separate and purify the inducible nervous system cells to eliminate the nervous system cells and naive cells. Therefore, the establishment of a neural induction and purification system based on hiPSC cells has been expected for a long time (this application refers to the full text of the following documents Rov, NS; Cleren, C.; Singh, SK; Yang, L.; Beal, MF; Goldman) SAFunctional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat. Med. 12: 1259–1268; 2006; Yan, YP; Yang, DL; Zarnowska, ED; Du, ZW; Werbel, B.; Valliere, C.; Pearce, RA; Thomson, JA; Zhang, SCDirected differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23:781–790;2005; Gerrard, L.; Rodgers, L.;Cui,W.Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells 23:1234–1241;2005;Keirstead,HS;Nistor,G.;Bernal,G.; Totoiu, M.; Cloutier, F.; Sharp, K.; Steward, O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25:4694–4705;2005.).

Summary of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the neuronal lineage provided by the prior art, causing the immature cells to be mixed therein to cause teratoma formation after intracerebral transplantation, and to provide a directed induction of differentiation of hiPSC into neural cells. Systematic methods, resulting neuronal cell systems and their applications. The method of the present invention induces the differentiation of human hiPSC cells into neural system cells while inhibiting the production of non-neural cells, thereby obtaining a mature, broad-spectrum neural cell population. The neural cell population has not only been verified in vitro as a mature neuron with electrical impulses, but also has been confirmed in mice in vivo, and has an effective treatment for neurological diseases (such as stroke and brain damage). effect.

One of the technical solutions to solve the above problems is: a method for inducing differentiation of hiPSC into a neural cell system, comprising culturing the hiPSC in stages to induce neural differentiation thereof, the stage comprising:

Stage a. co-cultivating the hiPSC and bone marrow stromal cells HS5 in an induction medium;

Stage b. continuously culturing the hiPSC with HS5 conditioned medium, ie, an induction medium containing the secretion of the HS5;

Stage c. Continue to culture the hiPSC with a basal medium that cultures the neuronal cells.

Wherein the induction medium described in stage a is a conventional induction medium in the art; preferably, the induction medium comprises the following components: 20-25% serum substitute, 0.5-1.5 mM 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 β3, 400-700ng /ml Noggin and 2-3% B27 additive in DMEM/F12 medium, the percentage being volume percent. More preferably, in order to effectively promote the proliferative activity of the cell differentiation product in the process of differentiation of the stem cells into the neural cell lineage, the induction medium further comprises 1-1.5% non-essential amino acids, 8-15 ng/ml basic fibrils. Cell growth factor, 0.08-0.15 mM β-mercaptoethanol, and 0.3-0.8 mM dibutyryl cyclic adenosine monophosphate.

Further preferably, the medium comprises the following components: 20% serum substitute, 1% non-essential amino acid, 1 mM glutamine, 0.1 mM β-mercaptoethanol, 10 ng/ml basic fibroblast growth factor, 10 ng/ml epidermal growth factor, 10 ng/ml brain-derived neurotrophic factor, 10 ng/ml neurotrophic factor-3, 1 ng/ml transforming growth factor β3, 0.5 mM dibutyryl cyclic adenosine monophosphate, 500 ng/ml noggin and 2% B27 cell culture additive DMEM/F12 medium. The serum replacement is preferably KnockOut ^TM serum replacement.

The co-cultivation described in stage a can be direct contact co-cultivation or indirect contact co-cultivation, preferably direct contact co-cultivation.

The bone marrow stromal cell HS5 described in the stage a is HS5 which inhibits division; preferably, the method for inhibiting division is irradiation; more preferably, the irradiation condition is: γ-ray irradiation intensity 75-85 Gy, irradiation The time is 28-35 minutes, the irradiation intensity is preferably 80 Gy, and the irradiation time is preferably 30 minutes.

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

The time of continuous culture as described in stage b is conventional in the art, preferably 8-18 days, more preferably 2 weeks.

The time of continued culture as described in stage c is conventional in the art, preferably from 10 to 18 days, more preferably 2 weeks.

The preparation method of the HS5 conditioned medium described in the stage b is 1) inoculation of 5×10 ⁶ to 2×10 ⁷ irradiated HS5 cells into 8-15 ml of the induction medium; 2) continuous 1~ The supernatant of the cultured cells is collected for 8 days; 3) the supernatant is mixed with the induction medium at a ratio of 1:1 to 8:1; more preferably, the preparation method of the HS5 conditioned medium is: 1) 1×10 ⁷ irradiated HS5 cells were inoculated into 10 ml of the induction medium; 2) the supernatant of the cultured cells was collected for 4 consecutive days; 3) the supernatant and the induction medium were at a ratio of 1:1 Mix and get.

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

The second technical solution of the present invention to solve the above problems is: a neural cell system induced by the above method. Preferably, the neural cell system comprises: neural stem cells 59.3±1.9%, multiple functional neurons 28.2±2.1%, astrocytes 9.1±0.8%, and oligodendrocytes 4.8±0.6%. The percentage is the percentage of the entire neural cell system. Wherein, the multifunctional neuronal cells include: 5.4±0.4% of dopaminergic neurons, 9.3±0.6% of acetylcholinergic neurons, 3.9±0.3% of γ-aminobutyric acid neurons, and serotonergic neurons 6.5 ± 0.5% and 3.1 ± 0.4% of immature neurons; the percentage is the percentage of the entire neural cell system.

The third technical solution of the present invention for solving the above problems is: the application of the above-mentioned nerve cell system in preparing a brain tissue repair preparation; preferably, the preparation is for treating ischemic stroke, cerebral hemorrhage or trauma Preparation of brain damage.

The fourth technical solution of the present invention to solve the above problems is: an induction medium for initially inducing differentiation of hiPSC into a neural cell system, the medium comprising the following components: 20-25% serum substitute, 0.5-1.5 mM Glutamine, 8-20 ng/ml epidermal growth factor, 8-12 ng/ml brain-derived neurotrophic factor, 8-15 ng/ml neurotrophic factor a sub--3, 0.5-1.5 ng/ml transforming growth factor β3, 400-700 ng/ml noggin, and 2-3% B27 additive in DMEM/F12 medium, the percentage being a volume percentage; preferably, the culture The group also includes 1-1.5% non-essential amino acids, 0.08-0.15 mM β-mercaptoethanol, 8-15 ng/ml basic fibroblast growth factor, and 0.3-0.8 mM dibutyryl cyclic adenosine monophosphate. If the content of the above medium component is lower than the lower limit of the above numerical range, the survival rate of hiPSC during differentiation is low (the total survival rate of the differentiation product is less than 40%); if it is higher than the upper limit of the above numerical range, The differentiation product of hiPSC not only has a low survival rate (less than 45%), but also the differentiation efficiency of hiPSC to neural lineage cells is significantly reduced (less than 24%).

In a preferred embodiment of the invention, the medium comprises the following components: 20% serum substitute, 1% non-essential amino acid, 1 mM glutamine, 0.1 mM β-mercaptoethanol, 10 ng/ml alkaline Fibroblast growth factor, 10 ng/ml epidermal growth factor, 10 ng/ml brain-derived neurotrophic factor, 10 ng/ml neurotrophic factor-3, 1 ng/ml transforming growth factor β3, 0.5 mM dibutyryl cyclic adenosine monophosphate, 500 ng/ ml noggin and 2% B27 cell culture additives DMEM / F12 medium; more preferably, the serum replacement is KnockOut ^TM serum replacement.

The fifth technical solution of the present invention for solving the above problems is: an HS5 conditioned medium for inducing differentiation of hiPSC into a neural cell system, which is an induction medium containing a secretion of bone marrow stromal cells HS5; the HS5 is irradiated HS5; the irradiation conditions are: gamma ray irradiation intensity 75-85 Gy, irradiation time 28-35 minutes, the irradiation intensity is preferably 80 Gy, and the irradiation time is preferably 30 minutes.

Preferably, the HS5 conditioned medium is prepared by the following preparation method: 1) inoculating 5×10 ⁶ to 2×10 ⁷ irradiated HS5 cells into 8-15 ml of the induction medium; The supernatant of the cultured cells is collected for 1 to 8 consecutive days; 3) the supernatant is mixed with the induction medium at a ratio of 1:1 to 8:1. Wherein, in step 1), if the number of cells is less than 5×10 ⁶ , the survival rate after cell inoculation and before irradiation is low (generally lower than 80%), and the concentration of cell secretion is low; When the number of cells is higher than 2 × 10 ⁷ , the cell inoculation is overcrowded, and the mortality after irradiation is high (may be higher than 50%). In step 2), if the number of days in which the supernatant of the cell culture medium is collected is less than one day, it is a waste of cell resources; if the number of days is higher than 8 days, the HS5 cells are more likely to die due to gradual death after irradiation. Inflammatory factors and apoptotic factors are not conducive to the induction culture of hiPSC. In step 3), the mixed ratio of the collected culture supernatant to the freshly prepared induction medium is less than 1:1, and the concentration of the secretion of HS5 cells contained after mixing is too low to induce neural differentiation of hiPSC; When the ratio is higher than 8:1, the proportion of the supernatant of the medium is too high, and it contains not only effective inducing components, but also a higher concentration of HS5 metabolic waste and pro-apoptotic factors, which is disadvantageous for the induction of hiPSC. More than good.

More preferably, the HS5 conditioned medium is prepared by the following preparation method: 1) inoculation of 1×10 ⁷ irradiated HS5 cells into 10 ml of the induction medium; 2) collection and culture for 4 consecutive days The supernatant of the cells; 3) The supernatant is mixed with the induction medium in a 1:1 ratio.

The sixth technical solution of the present invention to solve the above problems is: a basal medium for inducing differentiation of hiPSC into neuronal cells in a neuronal cell system, the basal medium comprising: 15-30 ng/ml bFGF, 15- 30 ng/ml EGF, 1-3% B27 additive, 8-12 μM forskolin and 0.1-0.3 mM ascorbic acid in neurobasal medium. If the content of the above-mentioned medium component is lower than the lower limit of the above numerical range, the survival rate of mature neurons which can be differentiated by hiPSC is low (less than 60%); if it is higher than the upper limit of the above numerical range, It can cause apoptosis of mature neurons and cause low survival rate (less than 50%).

Preferably, the basal medium further comprises 0.5-1.5% N2 additive and 0.5-1.5% fetal calf serum. More preferably, the basal medium is a neurobasal culture comprising 20 ng/ml bFGF, 20 ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10 μM forskolin and 0.2 mM ascorbic acid. Base, the percentage is a volume percentage.

Based on the common knowledge in the art, the above various preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

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

The positive effects of the present invention are:

The invention co-cultures human bone marrow stromal cells (BMSC) HS5 and hiPSC, can induce human hiPSC cells to differentiate into nervous system cells, and inhibit the formation of non-neural cells, thereby obtaining mature, broad-spectrum nerves. Cell population. The neural cell population has not only been verified in vitro as a mature neuron with electrical impulses, but also has been confirmed in mice in vivo, and has an effective treatment for neurological diseases (such as stroke and brain damage). effect. By using the stepwise culture method and a series of additives used in the invention, a standardized and commercial in vitro culture process can be formed, thereby catering to the clinical and scientific needs of "hiPSC induction and transplantation therapy" both domestically and internationally.

DRAWINGS

Figure 1 shows that HS5 secretes a variety of cytokines (a). The total cell viability (b; c) and Pax-6 ⁺ (early developmental markers of nervous system cells) cell proportion (b; c) were used as evaluation indicators, and HS-5 was directly contacted to induce culture of hiPSC cells ( b and c are the most effective for the differentiation of the two cell lines of hiPSC, namely ips-1 and IMR90-1.

Figure 2 shows that contact-induced culture of HS5 induces differentiation of hiPSC cells into nervous system cells by activating the Notch receptor of hiPSC cells [*p<0.05 vs. control; ^▲ p<0.05 vs. control or corresponding group (corresponding group) ) +L-685458; ^# p<0.05vs.Control +L-685458].

Figure 3 shows the dynamic changes of the proportion of Pax-6 ⁺ (the early differentiation markers of nervous system cells) differentiated by hiPSC cells at different time points, and the proportion of hiPSC cells (SSEA-4 ⁺ ) in undifferentiated state. Variety.

Figure 4 shows the broad migration ability exhibited in the host brain parenchyma 8 weeks after transplantation of hiPSC-derived nerve cells (donor cells) into the gerbil model. (a) 8 weeks after transplantation, donor cells spread along the injection needle and migrated into the surrounding striatum, indicating that donor cells can tolerate the in vivo environment of ischemic stroke animals. The gray line has roughly labeled the range of needle channels for donor cells. (b) The case where the donor cells diffuse and migrate to the surrounding brain parenchyma 8 weeks after transplantation. The antibody used for fluorescent staining was mouse anti-human nuclear antibody (HuN, 1:100; Chemicon). Immunofluorescence staining positive (green) nuclei are the nuclei of donor cells. Similarly, the presence of donor cells was also found in the cortex, corpus callosum, and hippocampus of recipient animals. (c) The hippocampus of the recipient animal (gerbil). (d-f) There are donor cells in the hippocampus of recipient animals. The antibody used for fluorescent staining was mouse anti-human nuclear antibody (HuN, 1:100; Chemicon). Immunofluorescence staining positive (green) nuclei are the nuclei of donor cells. Shown in the control group (only saline injection; d), there is no donor cell 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; and in the hippocampus-derived nerve In the cell injection group (f), there were more donor cells in the host hippocampus.

Figure 5 shows the results of reverse transcription PCR experiments. The expression of Oct-4 and ALP in the dry/undifferentiated state gradually disappeared, and the expression disappeared completely in the co-culture group at the end of the second stage; while the expression of neural precursor cell markers Nestin and Musashi-1 gradually increased; At the end of the third stage, the more mature subtypes of nerve cells, including the glial cell marker GFAP, the mature neuronal cell markers MAP-2 and Nurr-1 in the late mitosis, showed strong expression status.

Detailed ways

The invention is further described by way of examples, without the invention being limited to the scope of the embodiments. The experimental methods in the following examples which do not specify the specific conditions are selected according to conventional methods and conditions, or according to the product specifications.

Alphabetic abbreviation:

hiPSC: Human Induced Pluripotent Stem Cell Human induced pluripotent stem cells

MEF: mouse embryonic fibroblasts mouse embryonic fibroblasts

KSR: knockout serum replacement serum replacement

bFGF: basic fibroblast growth factor basic fibroblast growth factor

EGF: epidermal growth factor

NEAA: non-essential amino acid non-essential amino acid

BDNF: brain-derived neurotrophic factor

NT-3: neurotrophin-3 neurotrophic factor-3

GFAP: glial fibrillary acidic protein glial fibrillary acidic protein

MAP-2: microtubule-associated protein 2 microtubule-associated protein 2

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

Oct-4: octamer binding transcription factor 4 octamer binding transcription factor 4

ALP: alkaline phosphatase alkaline phosphatase

Further, the percentage (%) not specifically described in the present invention is generally a volume percentage.

experimental method:

1. Construction of expression vector

The Notch intracellular domain (NICD) of human Notch1 molecule, which was searched by pubmed, is the amino acid sequence of 1759-2444 in GenBank NM_017617, and amplified by polymerase chain reaction (PCR). . 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 (2058 bp) was digested with the BamHI/XbaI restriction site, and inserted into the mammalian expression vector pcDNA3.1 (Invitrogen) to construct a pcDNA3.1/Notch1 (pcNICD) expression vector. The cells were seeded at 2 × 10 ⁴ /cm ² in a 6-well plate (0.5 ml medium/cm ² ), and cultured for 24 hours and then transfected. Transfected cells were screened with G418 at a dose of 250 μg/ml for approximately 2–4 weeks until a single clone appeared.

2. Reverse transcription PCR

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

3. Electrophysiological analysis

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

4. Dopamine release test

After the completion of the 3-stage culture, the cells were first washed with a low concentration of KCl solution (4.7 nM), and then 2 ml of a high concentration KCl solution (60 mM KCl, 85 mM NaCl, 2.5 mM CaCl ₂ , 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , Incubate for 15 minutes with 11 mM D-glucose, and 20 mM HEPES/NaOH; pH 7.4). Dopamine concentration was determined by high performance liquid chromatography (HPLC) (HTEC 500, Eicom Corp., San Diego, CA, USA).

5, immunoblot analysis

Total protein (20 μg) was added to the antibody by 12% SDS-PAGE electrophoresis, and after transfection (Millipore, Billerica, MA, USA). Primary antibodies were rabbit anti-NICD (1:1000; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Hes1 (Hes1; 1:500; Chemicon, Temecula, CA, USA), rabbit anti-Hes5 (1:500) ; Chemicon) and murine anti-α-tubulin antibody (1:2000; Chemicon). The secondary antibody was HRP (horseradish peroxidase)-labeled goat anti-rabbit or goat anti-mouse IgG or IgM (1:2000; Millipore), and the banding intensity was quantified using image software (Bio-Rad, Hercules, CA, USA). ).

6, brain surgery

Animal experiments are in compliance with the relevant regulations of the National Institutes of Health and are approved by the local government. Adult male Mongolian gerbils (age 12-13 weeks, body weight 60-80 g) were exposed to 2% halothane and exposed to bilateral common carotid arteries. They were released after 5 minutes of occlusion with an aneurysm clip (Ridenour TR et al. Brain Res. 1991;565(1):116-122). The gerbils of the control group underwent sham operation without bilateral arterial occlusion.

7, cell transplantation

Three days after brain surgery, nerve cells (2.5 × 10 ⁵ cells / 5 μl saline) were injected into the corresponding areas of the gerbil brain, that is, 2.5 × 10 ⁵ cells were suspended in 5 μl of physiological saline, and transplanted into the recipient brain. Side; each animal brain received a total of 2 left and right unilateral transplantation), 5 × 10 ⁵ / whole brain; injection point is 0.5mm before the anterior iliac crest, 2mm outside the midline and 4mm ventral side of the dura mater, control group The corresponding parts of the mouse were injected with the same amount of physiological saline. Human mesenchymal stromal cells (hMSCs) were used as cell controls. The healthy person's bone marrow aspiration collection operation was approved by the regional ethics review committee and informed consent was obtained. Briefly, mononuclear cells were collected with a 1073 μg/mL density gradient separation solution (GE Healthcare, Piscataway, NJ, USA) and plated at a density of 1.6×10 ⁵ /cm ² in a culture dish with 10% added. FBS, 1% NEAA and 1 mM L-glutamine in low glucose DMEM medium. After culturing for a period of time, the hMSC cell suspension was injected into the gerbil brain in the same manner and at the same dose. Cyclosporin A (Novartis, Basel, Switzerland) was injected into gerbils by intraperitoneal injection at a dose of 10 mg/kg/day.

8, behavior analysis

The Morris water maze test (Harvard Apparatus, Holliston, MA, USA) was used to assess the spatial cognition capabilities of model animals. Six weeks after the transplant, the animals were trained for 13 days, 4 times a day, and a formal behavioral assessment was performed on the 14th day, while a space exploration experiment was conducted. In short, on the 14th day, the animal rest platform preset under the water was removed and the animals stayed in the water maze for 60 seconds. Record the number of times the gerbils pass through the original platform position within 60 seconds, and record the time when the gerbils stay in the quadrant area where the original platform is located.

9, enzyme immunoassay

The hippocampus was isolated from excised brain tissue, homogenized by adding buffer (20 mM Tris-HCl, 137 mM NaCl, 1 mM DTT, 0.5% Triton X-100 and 0.5 mM PMSF; pH 8.0), and centrifuged at 14,000 g for 30 minutes at 4 °C. Press Basic fibroblast growth factor was quantified using a detection kit (Quantikine HS; R&D Systems).

10. Histological analysis

Isolated brain tissue was made into 5 μm thick frozen sections along the coronal plane. The number of donor cells was measured by the number of immunoreactive cells per fifth slice during serial sectioning and finally corrected by the Abercrombie formula. In addition, about 2-2.2 mm of tissue between the anterior and posterior iliac crest was serially sliced and stained with 0.2% sulphur, and each frame of the microscope was measured and counted in a scale frame of 1 mm × 0.25 mm dimension. Intact pyramidal neurons in the hippocampal CA1 region (large nuclei, distinct nucleoli and clear cell membranes) were counted according to the methods described previously. The cells in the hippocampal CA1 region were graded according to the previously described criteria: grade 0, condensed CA1 pyramidal neurons accounted for 10% of the total number of cells in the framework; grade I, condensed CA1 pyramidal neurons accounted for cells within the framework The total number is 10%–40%; Grade II, condensed cells account for 40%–70%; Grade III: condensed cells account for ≥70%.

11. Immunocytochemistry

Mouse anti-β-tubulin III antibody (TuJ1, 1:500; Sigma), mouse anti-O ₄ antibody (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-colloid Fibrillary acidic protein antibody (GFAP, 1:400; Chemicon), rabbit anti-synaptophysin I antibody (1:500; Chemicon), rabbit anti-tyrosine hydroxylase antibody (TH, 1:100; Chemicon), rabbit anti- Γ-aminobutyric acid antibody (GABA, 1:200; Sigma), rabbit anti-choline acetyltransferase antibody (ChAT, 1:200; Sigma), rabbit anti-serotonin antibody (1:100; Sigma) applied to cells Mouse anti-human mitochondrial antibody (hmito, 1:40; Sigma), mouse anti-human nuclear antibody (HuN, 1:100; Chemicon), mouse anti-human neural cell adhesion molecule antibody (hNCAM, 1:100; Santa Cruz) Biotechnology) is applied to tissue sections. Cells and sections were either fluorescein isothiocyanate (FITC) or rhodamine tetramethyl isothiocyanate (TRITC)-labeled goat anti-mouse or anti-rabbit IgG or IgM (1:100; Millipore) or biotinylated Goat anti-mouse IgG (1:100; Vector, Burlingame, CA, USA;) for further incubation. Subsequently, streptavidin-HRP (streptavidin-horseradish peroxidase) was added and visualized immunological reaction was carried out with diaminobenzidine. The cells or sections were washed, 10 μg/mL propidium iodide (PI; Sigma) or 4',6-diamidino-2-phenylindole (DAPI; Sigma) for counterstaining.

12. Statistical analysis

Data are expressed as mean ± standard error. The SNK test and the t-test were used for comparative analysis of the two groups. The Nemenyi test was performed to analyze the histological grade 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 a co-culture system of bone marrow stromal cell line HS5 and hiPSC cells

Using human bone marrow stromal cell line HS5 (human bone marrow stromal cell line , CRL-11882 TM, American Type Culture Collection (ATCC), Manassas, VA, USA) hiPSC cells induced neural differentiation induction medium was composed of: 20% KSR 1% NEAA, 1 mM glutamine, 0.1 mM β-mercaptoethanol, 10 ng/ml bFGF, 10 ng/ml EGF, 10 ng/ml BDNF, 10 ng/ml NT-3, 2% B27, 0.5 mM dibutyryl cyclic adenosine monophosphate 1 ng/ml transforming growth factor β3 (all of the above components were purchased from Invitrogen) and 500 ng/ml Noggin (R&D Systems, Minneapolis, Minnesota, USA). Among them, NEAA, bFGF, β-mercaptoethanol and dibutyryl cyclic adenosine are added to effectively promote the proliferative activity of cell differentiation products in the process of stem cell differentiation into the neural cell lineage; B27 is a serum-free additive ( It mainly contains vitamin A, various antioxidants and insulin) for the growth and maintenance of short-term or long-term activity of hippocampal neurons and other central nervous system neurons. The B27 additive is a 50x concentrated liquid that requires a 1:50 dilution when used.

Direct co-cultured cell system was established. Before co-culture with hiPSC cells, HS5 was irradiated (γ-irradiation intensity 80 Gy on 1×10 ⁶ HS5 cells; time 30 min; device was Gammacell 1000 Elite 214, MDS Nordion, Ottawa, Ontario, Canada) was inoculated to a 6-well plate at 2 x 10 ⁵ cells/well for one day.

The establishment of a non-direct contact co-cultured cell system, before co-culture with hiPSC cells, HS5 cells were inoculated into tissue culture-embedded cells at 2 × 10 ⁵ cells/embedded cells after irradiation (ThinCerts, Frickenhausen, Germany), then nested in a 6-well plate. HS5 conditioned medium (HS5-CM) was prepared, and 1 × 10 ⁷ HS5 cells were irradiated and inoculated into a petri dish containing 10 ml of the above induction medium. The culture supernatant was collected daily for 4 days. Prior to use, the collected HS5-CM was diluted in a 1:1 ratio (equal volume freshly prepared induction medium mixed with HS5-CM in equal proportions). The hiPSC cells were seeded at 2 x 10 ⁵ cells/well in 6-well plates pre-coated with Matrigel (DMEM/F12 medium diluted Matrigel at a ratio of 1:50). For the inhibition of the Notch pathway, 4 μM L-685458 (Notch signaling pathway inhibitor; purchased from Calbiochem San Diego, CA, USA) was added on the first day of culture and incubated for 8 days.

Figure 1a shows that HS5 secretes a variety of cytokines. After 8 days of differentiation induced by the above two different methods, the cell survival rate and the Pax-6 positive rate of the neural precursor cells were compared. The results showed that the cell viability and Pax-6 positive rate were 89.3±1.4% and 45.2±2.8%, respectively, after 8 days of differentiation induced by direct contact co-culture, which was better than the non-contact co-culture method (Fig. 1b). Another germline IMR90-1 cell of hiPSC cells was used for induction of differentiation with the same results (Fig. 1c). The above results indicate that in the neural induction differentiation method based on HS5 (human bone marrow stromal cell line, CRL-11882 ^TM , ATCC, Manassas, VA, USA), the direct co-culture method is more favorable for promoting the nerve front than other methods. Somatic cell proliferation and differentiation.

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

The first stage: the hiPSC cells and HS5 cells were induced to culture in direct co-culture for 2 weeks, and the composition of the induction medium was the same as in Example 1. In the six-well plate, hiPSC cells were directly inoculated at 2×10 ⁵ cells/well. On the 2×10 ⁵ /well HS5 cell layer pre-plated before the day; for the parallel control group, the hiPSC cells were not co-cultured with HS5 cells, but were directly inoculated into Matrigel-coated 6-well plates, followed by the same Methods The second and third stages of culture were carried out. Change the fluid once every other day.

The second stage: continuous culture with 1:1 diluted HS5-CM medium for 2 weeks; preparation of HS5-CM medium: 1 × 10 ⁷ HS5 cells were irradiated and inoculated into a Petri dish containing 10 ml of hiPSC induction medium. The waste liquid was collected every day, and the HS5-CM medium was collected continuously for 4 days. Prior to use, the collected HS5-CM was diluted in a 1:1 ratio (freshly prepared hiPSC induction medium mixed with HS5-CM in equal volume ratio).

The third stage: after the second stage of culture, the cells were inoculated into 2 well plates coated with polyornithine and laminin at 2 × 10 ⁵ /well after enzymatic hydrolysis, followed by the third stage selection. to cultivate. With 20ng/ml bFGF, 20ng/ml EGF, 2% B27, 1% N2 additive, 1% FBS (Invitrogen), 10μM forskolin (forskolin, Calbiochem, San Diego, CA, USA, also known as: hair The laryngin; "Adenylate cyclase activator") and 0.2 mM ascorbic acid (Sigma, St. Louis, MO, USA) in neurobasal medium (Invitrogen) were cultured for 2 weeks. Among them, the addition of N2 additive and FBS is to more effectively maintain the survival rate of mature neurons at the end stage of differentiation.

Example 3 Direct contact co-culture activates Notch signaling pathway in hiPSC cells

In view of the fact that the Notch ligands Delta1, delta3, Jagged1 and Jagged2 can be easily detected in HS5 cells by the method of "reverse transcription PCR" in the experimental method (Fig. 2a), Notch can be detected in hiPSC cells and their derivatives. The corresponding receptors Notch1, Notch2 and Notch3 (Fig. 2b) speculate that the Notch signaling pathway is a mediator of HS5 and hiPSC cell interactions. After being acted upon by γ-secretase, the NICD protein dissociates from the Notch receptor and targets the downstream molecules Hes1 and Hes5. Eight days after induction of differentiation, the expression level of NICD protein in each group of hiPSC-derived cells was determined by the "immunoblot analysis" method in the experimental method. The results showed 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 can significantly activate the Notch signaling pathway in the hiPSC cell derivative. It is worth noting 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 activates the Notch signaling pathway.

Compared with the non-direct contact co-culture of the control group, the expression levels of neuroectodermal-related genes such as Sox-1, Pax-6 and NFH were significantly increased in hiPSC-derived cells after 8 days of direct contact co-culture, while endoderm, mesoderm and Stem cell-related gene expression is reduced (Figure 2). These data indicate that HS5 direct contact co-culture can promote the differentiation of hiPSC cells into neural cells and simultaneously block their differentiation into non-neuronal cells. Subsequently, the present invention further tests Notch Whether the signal is essential for HS5-mediated neural-induced differentiation. After 8 days of continuous incubation with Notch signaling pathway inhibitor L-685458, the expression levels of NICD, Hes1 and Hes5 in HS5 co-cultured cells were significantly decreased, and the levels of NICD and Hes5 decreased and the expression of L-685458 was used in the control group. There was no significant difference in levels (Fig. 2c-d), indicating that L-685458 completely blocked the activation and activation of the Notch pathway. However, L-685458 had a small effect on Hes1 expression levels (Fig. 2c-d), which further confirmed that Hes1 expression is not solely dependent on the Notch signaling pathway. In addition, the addition of L-685458, HS5 direct contact co-culture promoted the expression of Sox-1, Pax-6 and NFH decreased, on the contrary, the expression of non-neuron markers increased due to de-inhibition (Fig. 2e), indicating Notch signal The pathway plays a pivotal role in HS5-mediated neural 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, compared with the control group which only transfected the vector (pcDNA3.1), iPS-1 cells transfected with pcNICD (construction method see experimental method) showed a significant increase in Pax-6 positive cells after 8 days of culture (Fig. 2h), has a tendency to differentiate into nerve cells. This suggests that NICD, as an effector of the Notch signaling pathway, can promote the differentiation of hiPSC cells into neural cells. All data suggest that activation of the Notch signal may be, at least in part, a factor necessary for direct co-culture methods to promote differentiation of hiPSC cells into the neuronal lineage.

Example 4 Composition Identification of a HiPSC Cell-derived Neuronal Cell System

After induction of HS5, the neural precursor cells positive for Pax-6 increased rapidly in the second stage of continuous culture, even at the end of culture, the positive rate reached 87.2±1.9%, and at the same time, SSEA-4 positive undifferentiated cells. The number was gradually reduced until it reached zero (Fig. 3), and RT-PCR reverse transcription was performed for cDNA synthesis under the reaction conditions of 42 ° C for 50 min, 95 ° C for 5 min, and then stored at 4 ° C environment. The target gene in the cDNA is then amplified by PCR. The basic reaction conditions of PCR were: (1) pre-denaturation of cDNA: 94 ° C for 5 min; (2) PCR amplification: 94 ° C for 30 sec, 60 ° C for 30 sec, 72 ° C for 45 sec, for 30 cycles; (3) final extension phase: 72 ° C 7min. See Table 1 for primer sequences.

Table 1 Primer sequences and gene lengths of interest for reverse transcription PCR

The analysis further confirmed (Fig. 5) that the dry gene transcription factors Oct-4 and alkaline phosphatase (ALP) were not detected at the end of the second phase, while the expression of neural precursor cell markers Nestin and Musashi-1 was significantly increased. Subsequently, more mature was found at the end of the third stage of culture (at the end of the third stage, the more mature neuronal subtypes, including the glial cell marker GFAP, the mature neuronal marker MAP-2 in the late mitosis stage) And the dopaminergic neuron marker Nurr-1, showing a strong expression state; see Figure 5) of the neuronal subtypes, 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 neural precursor cells gradually decreased to 59.3 ± 1.9% (Fig. 3). It is shown that the third stage of culture is conducive to the standardized growth and maturation of neural precursor cells. Consistent with the above results, transcription factors of neuronal cell subtype-specific genes, including astrocyte marker GFAP, mitotic neuronal marker MAP-2 and dopaminergic neuron marker Nurr-1, Can be detected in the third stage culture period, and the expression of genes related to mesoderm (c-kit, SOX-9 and PPARγ) and endoderm development (AFP, glucose transporter-2 and amylase) were not detected. This further confirms that the 3-stage culture method does not support the growth of non-neuronal cells.

It is particularly noted that some SSEA-4 positive hiPSC cells gradually change to a cylindrical shape after induction, and finally form a tubular garland structure composed of Pax-6-positive neural precursor cells and Tuj1-positive neurons, and in the second The stage spontaneously disperses into small clumps, and then further differentiates into mature phenotypes, just as the expression of synaptophysin I is significantly increased in Tuj1-positive neurons that emerged in the third stage, which indicates the formation of synapses. At the same time, in these cell populations, neurons positively expressed by dopamine subtype (TH ⁺ ), GABA (GABA ⁺ ), choline (ChAT ⁺ ) and serotonin ⁺ (serotonin ⁺ ) were also detected, and the corresponding cell proportion (% of total cell product total) were 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 was significantly higher in each culture stage than in the parallel control group, indicating that the method based on HS5-induced differentiation has an effect of promoting cell growth, which is consistent with the detection results of various growth factors in HS5 cells. Finally, using the "electrophysiological analysis" and "dopamine release test" method: the action potential of neurons can be detected by high concentration K ⁺ depolarization stimulation, and every 10 ⁶ cells can be detected by high performance liquid chromatography. 8.6 ± 0.6 pmol acetylcholine and 72.5 ± 6.2 pmol dopamine (N = 12) were released, indicating that the neural cells produced by the three-stage culture method are functional. At the same time, RT-PCR analysis showed that the mesoderm, endoderm and pluripotency markers gradually lost expression after completing the three-stage culture, while the c-kit, SOX-9 and AFP proteins were continuously expressed in the control group. Compared with the control group, the 3-stage culture method has obvious advantages in inducing Tuj1-positive neurons, GFAP-positive astrocytes and O4-positive oligodendrocytes.

Markers of different nervous system cells are detected by the corresponding antibodies (see Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H, Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N, Tabar V, Harrison) NL, Beal MF, Moore MA, Studer L. Neural subtype specification of Fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol. 2003;21(10):1200-1207; Fong SP, Tsang KS, Chan AB, Lu G, Poon WS, Li K, Baum LW, Ng HK .Trophism of neural progenitor cells to embryonic stem cells: neural induction and transplantation in a mouse ischemic stroke model. J Neurosci Res. 2007;85(9):1851-1862.): mouse anti-pax-6 antibody (1: 100; Santa Cruz Biotechnology), rabbit anti-Nestin antibody (1:400; Chemicon) detected neural stem cell markers pax-6, Nestin, broad-spectrum neurons, dopaminergic neurons, gamma-aminobutyric acid nerves Metabolic, cholinergic neurons, serotonergic neurons, astrocytes, and oligodendrocyte markers are β-tubulin, tyrosine hydroxylase, γ-aminobutyric acid, Choline acetyltransferase, serotonin, glial fibrillary acidic protein and O4, the corresponding antibodies are mouse anti-β-tubulin III antibody (TuJ1, 1:500; Sigma), rabbit anti-tyramine Acid hydroxylase antibody (TH, 1:100; Ch Emicon), rabbit anti-gamma-aminobutyric acid antibody (GABA, 1:200; Sigma), rabbit anti-choline acetyltransferase antibody (ChAT, 1:200; Sigma), rabbit anti-5-serotonin 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 is applied to cells (antibody-administered 1~) After 2 hours), further with fluorescein isothiocyanate (FITC) or rhodamine tetramethyl isothiocyanate (TRITC)-labeled goat anti-mouse or anti-rabbit IgG or IgM (1:100; Millipore) Reaction. The cells were washed and counterstained with 10 μg/mL propidium iodide (PI; Sigma) or 4',6-diamidino-2-phenylindole (DAPI; Sigma). The results showed that the induced nervous system cells included a fixed proportion of neural stem cells of 59.3±1.9% and multiple functional neurons of 28.2±2.1% (including 5.4±0.4% of dopaminergic neurons and 9.3±0.6% of acetylcholinergic neurons). Γ-aminobutyric acid neuron 3.9±0.3%, serotonergic neurons 6.5±0.5% and immature neurons 3.1±0.4%), astrocytes 9.1±0.8%, and oligodendrocytes 4.8 ±0.6% (see the "Immunocytochemistry" method in the experimental method).

In summary, the three-stage culture method can induce the formation of a multi-level neuronal lineage while limiting the production of non-neuronal cell derivatives.

Effect Example 1 Induced intracerebral transplantation of nerve cells can improve cognitive function in stroke animals

Hippocampus is one of the more vulnerable sites after cerebral ischemia and reperfusion. After 12 hours of gerbil cerebral ischemia test, sulphur staining showed extensive cell pyknosis and necrosis in the hippocampal CA1 region (sinus surgery control group and stroke group 12 h after comparison, each 0.25 mm ² CA1 region intact The number of pyramidal neurons was 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 edges) in the CA1 region continued to decrease within 3 days after brain surgery (every 0.25 on day 1 and day 3 after stroke surgery) The number of intact neurons in the mm ² CA1 region were 77.5 ± 5.1 and 39.2 ± 3.5, respectively, P < 0.001. In the subsequent time window (5 days after the operation of the gerbil stroke), the number of pyramidal neurons was no longer significantly reduced (Days 3 and 5 per 0.25 mm ^{2 of the} number of intact pyramidal neurons in the CA1 region) They were 39.2 ± 3.5 and 31.8 ± 3.2, respectively, P = 0.43). Three days after stroke surgery, 5×10 ⁵ (2.5×10 ⁵ / side×2 side) nerve cells produced by three-stage induction culture were transplanted into the bilateral caudate putamen of ischemic stroke gerbils. At this time, hippocampal CA1 area 82.3 ± 1.2% of pyramidal neurons were extensively absent. The gerbils who were able to reach the Morris water maze escape platform within 150 ± 4 s before stroke surgery were used for in vivo experimental studies. After stroke surgery, 92.3% of gerbils were able to survive, accompanied by drowsiness, coma, and lack of exercise. Six weeks after transplantation, no dead gerbils were found, and no abnormal behavior or progressive dyskinesia occurred. This indicates that intracerebral transplantation of neurons differentiated by hiPSC cells has no obvious adverse reactions in gerbils. At the same time, the 14-day behavioral evaluation found that the time of staying in the water maze by the animals transplanted with nerve cells in the brain was gradually reduced compared with the hMSC transplantation group and the saline injection group, confirming the learning and learning of the animals transplanted through the brain cells. The memory ability was stronger than that of the hMSC transplantation group and the saline injection group.

On the 14th day behavioral evaluation experiment (ie, 56 days after transplantation, see “Behavioral Analysis” in the experimental method), the time of arrival of the gerbils in the neuron transplantation group (47.4±3.1 s) was significantly less than that of normal saline. The control group (165.8 ± 7.4 s, P < 0.0001) and the hMSC transplantation group (96.7 ± 6.6 s, P < 0.0001). There was no significant difference between the nerve cell transplantation group (47.4±3.1 s) and the sham control group (39.2±2.8S, P=0.282) on the time to reach the escape platform. The 150 cm straight path experiment showed that the swimming speeds of the gerbils in each group were similar, which indicated the length of time it took for the gerbils to find the escape platform in the water maze, and the cognitive ability such as learning and memory played a key role. In addition, in the space exploration experiment, it was also found that the number of gerbils passing through the original escape platform in the nerve cell transplantation group was significantly higher than that in the saline control group and the hMSC transplantation group. The same result was obtained when measuring the length of stay of the gerbils in the target quadrant of the original escape platform (ie, the gerbils in the neuron transplantation group had the longest stay in the target quadrant of the original escape platform). In summary, the implantation of neural cells differentiated from hiPSC cells helps to improve the spatial learning and memory ability of ischemic stroke animals, and the effect is significantly better than hMSC transplantation.

Effect Example 2 Cellular intracerebral transplantation promotes recovery of damaged hippocampus

After 8 weeks of brain transplantation, the implanted cells in the brain of gerbils were followed. The results are shown in Figure 4: a large number of donor cells were found in the caudate putamen and spread to the surrounding striatum, indicating donor The cells can tolerate the in vivo environment of ischemic injured animals. The presence of donor cells was also found in the cortex, corpus callosum, and hippocampus. No intracerebral hemorrhage, microglia infiltration or teratoma formation was found in the experiment. The number of intact pyramidal neurons in the hippocampal CA1 area of the cerebral ischemic nerve cell transplantation group (190.6±11.1/0.25mm ² CA1 area) was significantly higher than that of the non-NSC transplantation group (normal saline control group: 68.8±4.1/0.25mm ^{2 )} CA1 area, P <0.0001; hMSC transplantation group: 143.2 ± 6.5 / 0.25 mm ² CA1 area, P = 0.0002). The density of pyramidal neurons in the hippocampal CA1 region was similar in the neuronal cell transplantation group (190.6±11.1/0.25 mm ² CA1 region) and the sham operation group (212.3±10.3/0.25 mm ² CA1 region, P=0.075). At the same time, hNCAM ⁺ cells (human neural cell adhesion molecule) were detected in the hippocampus of gerbils transplanted by cerebral ischemia. The donor cells have active migration. Homing activity. These hNCAM ⁺ cells are located in the original pyramidal cell layer in the hippocampus, and their cell morphology resembles intact pyramidal neurons, indicating that the neural cells differentiated by hiPSC cells can directly participate in the structural reconstruction of the injured hippocampus by further differentiation and migration. Similarly, HuN immunostaining (HuN, human nucleus, also used to detect human nerve cells in the brain of gerbils) showed the number of donor cells in the hippocampal CA1 region of the neuronal transplantation group (10.4±0.7/0.25 mm ² CA1 region). It was significantly higher than the hMSC transplantation group (3.2±0.4/0.25mm ² CA1 area, P<0.0001), which means that the brain transplantation of neurons differentiated from hiPSC cells is more conducive to the structural reconstruction of damaged hippocampus than hMSC transplantation. Recovery with the number of pyramidal neurons. A small amount of endogenous regeneration was also detected in the hippocampus of the cerebral ischemia saline control group (the number of neurons in the CA1 region increased from 39.2±3.5/0.25 mm ² 8 weeks ago to 68.8±4.1/0.25 mm ² , P<0.01), indicating that spontaneous endogenous regeneration promoted a slight increase in neurons in the CA1 region. However, compared with the normal hippocampus in the sham-operated group, the neuronal loss in the hippocampal CA1 region of the cerebral ischemic saline control group was observed. (66.3 ± 2.3%) is still significant.

On the other hand, compared with the cerebral ischemic saline control group, after 8 weeks of transplantation, the number of neurons in the CA1 region of the nerve cell transplantation group was about 121.9±8.9/0.25 mm ² . However, histological analysis showed that only about hippocampal CA1 area 10.4 ± 0.7 / 0.25mm ² HuN neurons stained positive, indicating that an exogenous donor cells promote cell CA1 pyramidal structure mainly by stimulating the endogenous regenerative mechanism reconstruction. In view of the fact that intraventricular basal fibroblast growth factor can not only stimulate the proliferation of endogenous progenitor cells, but also promote its differentiation into nerve cells, it is speculated that the recruitment of progenitor cells by donor cells may be related to basic fibroblasts. Paracrine associated with growth factors. To test this hypothesis, the present invention measures the level of basic fibroblast growth factor in the hippocampus of gerbils after 8 weeks of transplantation. The results showed that the level of basic fibroblast growth factor (195.8±11.8pg/mg protein) in the nerve cell transplantation group was significantly higher than that in the saline control group (133.2±8.9pg/mg protein, P<0.0001) and hMSC transplantation group (157.5). ±9.4 pg/mg protein, P=0.006), indicating that basic fibroblast growth factor is secreted by donor cells and promotes the regulation of endogenous regeneration in hippocampus by a paracrine mechanism. Similarly, histological grading analysis showed that compared with the cerebral ischemic saline control group and the hMSC transplantation group, the pyramidal cell structure in the hippocampus of the nerve cell transplantation group was less damaged and recovered faster. In summary, after transplantation, the neurons derived from hiPSC cells are integrated into different brain regions of ischemic damage through migration and homing behavior; on the other hand, they promote cerebral ischemia by promoting endogenous regeneration mechanisms. Destruction of the hippocampus, thereby improving its cognitive function.

While the invention has been described with respect to the preferred embodiments of the embodiments of the embodiments of the invention modify. Accordingly, the scope of the invention is defined by the appended claims.

Claims (10)

1. A method for directionally inducing differentiation of hiPSC into a neural cell system, characterized in that it comprises culturing the hiPSC in stages to induce neural differentiation thereof, the stage comprising:

   Stage a. co-cultivating the hiPSC and bone marrow stromal cells HS5 in an induction medium;

   Stage b. continuously culturing the hiPSC with HS5 conditioned medium, the HS5 conditioned medium being an induction medium containing the secretion of the HS5;

   Stage c. Continue to culture the hiPSC with a basal medium that cultures the neuronal cells.
2. The method of claim 1 wherein said induction medium in stage a comprises the following components: 20-25% serum replacement, 0.5-1.5 mM glutamine, 8-20 ng/ml Epidermal growth factor, 8-12 ng/ml brain-derived neurotrophic factor, 8-15 ng/ml neurotrophic factor-3, 0.5-1.5 ng/ml transforming growth factor β3, 400-700 ng/ml noggin and 2-3% DMEM/F12 medium of B27 additive; preferably, the medium further comprises 1-1.5% non-essential amino acids, 8-15 ng/ml basic fibroblast growth factor, 0.08-0.15 mM β-mercaptoethanol, and 0.3- 0.8 mM dibutyryl cyclic adenosine monophosphate; more preferably, the medium comprises the following components: 20% serum substitute, 1% non-essential amino acid, 1 mM glutamine, 0.1 mM β-mercaptoethanol, 10 ng/ml Basic fibroblast growth factor, 10 ng/ml epidermal growth factor, 10 ng/ml brain-derived neurotrophic factor, 10 ng/ml neurotrophic factor-3, 1 ng/ml transforming growth factor β3, 0.5 mM dibutyryl cyclic adenosine monophosphate, 500 ng/ml noggin and 2% B27 cell culture additive in DMEM/F12 medium; further preferably, the serum is replaced Product is KnockOut ^TM serum substitute; the percentages are percentages by volume.
3. The method according to at least one of claims 1 and 2, wherein the co-cultivation described in stage a is direct contact co-cultivation;

   And/or, the bone marrow stromal cells HS5 are HS5 after irradiation; preferably, the irradiation conditions are: γ-ray irradiation intensity 75-85 Gy, irradiation time 28-35 minutes, the irradiation intensity Preferably, it is 80 Gy, and the irradiation time is preferably 30 minutes;

   And/or, the co-cultivation time is 10-18 days, preferably 2 weeks.
4. The method according to at least one of claims 1 to 3, wherein the continuous culture time in the stage b is 8-18 days, preferably 2 weeks;

   And/or the preparation method of the HS5 conditioned medium described in the stage b is: 1) inoculating 5×10 ⁶ to 2×10 ⁷ irradiated HS5 cells into 8-15 ml of the induction medium; 2) collecting the supernatant of the cultured cells for 1 to 8 consecutive days; 3) mixing the supernatant with the induction medium at a ratio of 1:1 to 8:1; more preferably, the HS5 conditioned medium The preparation method comprises the following steps: 1) inoculating 1×10 ⁷ irradiated HS5 cells into 10 ml of the induction medium; 2) collecting the supernatant of the cultured cells for 4 consecutive days; 3) separating the supernatant and the supernatant The induction medium is mixed in a 1:1 ratio;

   And/or, the time of continuing cultivation as described in stage c is 10-18 days, preferably 2 weeks;

   And/or, the basal medium described in stage c is: 15-30 ng/ml bFGF, 15-30 ng/ml EGF, 1-3% B27 additive, 8-12 μM forskolin and 0.1-0.3 mM Ascorbic acid neurobasal medium; preferably, the medium further comprises 0.5-1.5% N2 additive and 0.5-1.5% fetal calf serum; more preferably, the basal medium described in stage c is added: 20 ng/ml bFGF, 20 ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10 μM forskolin and 0.2 mM ascorbic acid in neurobasal medium, the percentage being volume percent.
5. A neural cell system induced by the method according to at least one of claims 1 to 4.
6. The neural cell system according to claim 5, which comprises: neural stem cells 59.3 ± 1.9%, multiple functional neurons 28.2 ± 2.1%, astrocytes 9.1 ± 0.8%, and oligodendrocytes. The cells are 4.8±0.6%; preferably, the multifunctional neuronal cells include: 5.4±0.4% of dopaminergic neurons, 9.3±0.6% of acetylcholinergic neurons, and 3.9±0.3% of γ-aminobutyric acid neurons. Serotonergic neurons 6.5 ± 0.5% and immature neurons 3.1 ± 0.4%; the percentage is the percentage of the entire neural cell system.
7. Use of a nerve cell system according to at least any of claims 5 and 6 for preparing a brain tissue repair preparation; preferably, the preparation is for treating brain damage caused by ischemic stroke, cerebral hemorrhage or trauma Preparation.
8. An induction medium for inducing differentiation of hiPSC into a neural cell system, characterized in that the induction medium comprises the following components: 20-25% serum substitute, 0.5-1.5 mM 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 β3, 400-700ng/ml head protein and 2 -3% B27 additive in DMEM/F12 medium, the percentage being a volume percentage; preferably, the medium further comprises 1-1.5% non-essential amino acids, 8-15 ng/ml basic fibroblast growth factor, 0.08-0.15 mM β-mercaptoethanol and 0.3-0.8 mM dibutyryl cyclic adenosine; more preferably, the induction medium comprises the following components: 20% serum substitute, 1% non-essential amino acid, 1 mM glutamine Amide, 0.1 mM β-mercaptoethanol, 10 ng/ml basic fibroblast growth factor, 10 ng/ml epidermal growth factor, 10 ng/ml brain-derived neurotrophic factor, 10 ng/ml neurotrophic factor-3, 1 ng/ml transformation growth Factor β3, 0.5 mM dibutyryl cyclic adenosine monophosphate, 500 ng/ml noggin and 2% B27 cell culture The additive DMEM / F12 medium; Further more preferably, the serum replacement is KnockOut ^TM serum replacement.
9. An HS5 conditioned medium for inducing differentiation of hiPSC into a neural cell system, characterized in that it is an induction medium according to claim 8 containing a secretion of bone marrow stromal cells HS5; the HS5 is an irradiated HS5 The irradiation conditions are: γ-ray irradiation intensity of 75-85 Gy, irradiation time of 28-35 minutes, The irradiation intensity is preferably 80 Gy, and the irradiation time is preferably 30 minutes;

   Preferably, the HS5 conditioned medium is prepared by the following preparation method: 1) inoculating 5×10 ⁶ to 2×10 ⁷ irradiated HS5 cells to 8-15 ml of the induced culture according to claim 8. 2) collecting the supernatant of the cultured cells for 1 to 8 consecutive days; 3) mixing the supernatant with the induction medium at a ratio of 1:1 to 8:1; more preferably, the HS5 The conditioned medium was prepared by the following preparation method: 1) inoculation of 1×10 ⁷ irradiated HS5 cells into 10 ml of the induction medium; 2) collecting the supernatant of the cultured cells for 4 consecutive days; 3) The supernatant is obtained by mixing the induction medium in a 1:1 ratio.
10. A basal medium for inducing differentiation of hiPSC into neuronal cells in a neural cell system, characterized in that the basal medium is added with: 15-30 ng/ml bFGF, 15-30 ng/ml EGF, 1- 3% B27 additive, 8-12 μM forskolin and 0.1-0.3 mM ascorbic acid neurobasal medium; preferably, the basal medium further comprises 0.5-1.5% N2 additive and 0.5-1.5% fetal bovine serum; Preferably, the basal medium is a neurobasal culture supplemented with 20 ng/ml bFGF, 20 ng/ml EGF, 2% B27 additive, 1% N2 additive, 1% fetal bovine serum, 10 μM forskolin and 0.2 mM ascorbic acid. Base, the percentage is a volume percentage.

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