A simple and efficient method for differentiating neural stem cells and astrocytes from EPSC

Abstract

The present invention discloses a method for simply and efficiently differentiating EPSCs into neural stem cells and astrocytes. The EPSC-derived neural stem cells prepared by the method provided by the present invention have extremely strong potential for differentiating into astrocytes and neurons, and are significantly superior to the neural stem cells induced and differentiated by the methods reported in the prior art. It is applicable to the large-scale production of EPSC-derived neural stem cells and astrocytes, and has the advantages of simple operation, low cost, high differentiation efficiency, short differentiation cycle, and large number of differentiated cells, etc.

Description

Technical Field

[0001] The present invention belongs to the field of stem cell biotechnology. Specifically, the present invention relates to a method for simply and efficiently differentiating neural stem cells and astrocytes from EPSCs. Background Art

[0002] The spinal cord, as a part of the central nervous system (CNS), plays a crucial role in the transmission of motor and sensory information. Spinal cord injury (SCI) results in impaired spinal cord function, which in turn leads to the loss of sensory, motor, and visceral functions and often ends in paralysis. Neural stem cell (NSC) transplantation is considered a promising therapeutic strategy for promoting functional recovery after spinal cord injury. In recent years, NSC transplantation has been widely used in the treatment of neurological diseases such as stroke, amyotrophic lateral sclerosis, and spinal cord injury, as well as in anti-cancer gene therapy based on NSCs. There are various protocols for manufacturing NSCs in vitro, such as NSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), and NSCs reprogrammed from somatic stem cells. These NSCs have the potential to be used in NSC transplantation therapy.

[0003] Astrocytes are the most important type of glial cells in the central nervous system and are involved in various physiological functions such as the formation of the blood-brain barrier, neural synapse pruning, neuronal nutrient supply and homeostasis maintenance, maintaining the normal operation of the glymphatic system, and clearing microglial debris. Astrocytes have the ability to uptake glutamate in the brain tissue through glutamate transporters (EAATs) to prevent neurotoxicity caused by the accumulation of glutamate. Abnormal astrocyte function is associated with central nervous system aging and dysfunction as well as various neurodegenerative diseases. Vieira R et al. found that intrathecal reinfusion therapy using healthy astrocytes can replace the originally functionally impaired astrocytes in the brain and play a therapeutic role in some central nervous system diseases (Literature 1: Vieira R, Mariani J N, Huynh N P T, et al. Young glial progenitor cells competitively replace aged and diseased human glia in the adult chimeric mouse brain [J]. Nature Biotechnology, 2023: 1-12.).

[0004] Swistowski A et al. first formed embryoid bodies (EBs) from embryonic stem cells (ESCs), and then continued to culture them in suspension in neural induction medium (DMEM / F12 basal medium, 1% Glutamax, 1% NEAA, 0.5% N2 and 20 ng / mL FGF2) for 2-3 days. The cells were then transferred to a culture plate pre-coated with CellstartTM for adherent culture. After 2-3 days, neural rosette structures appeared. The differentiated NSCs needed to be picked up with a glass pipette and transferred to a new well to improve the purity (Reference 2: Swistowski A, Peng J, Han Y, et al. Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them [J]. PloS one, 2009, 4 (7): e6233). However, the pluripotent stem cells used in this scheme are hESCs, which poses ethical issues. The obtained NSCs have a decent ability to differentiate into dopaminergic neurons, but the glial cell differentiation potential, especially astrocyte (ATC) differentiation potential, is low.

[0005] Liu BC et al. used human embryonic stem cells (hEMS) or induced pluripotent stem cells (iPSC) as precursor cells for differentiation. During the culture process, new CL medium was replaced every day until neural rosettes appeared. The cells had double positive characteristics of NESTIN and N-cadherin. After digestion, the CL medium was continued to be half-changed for culture. Neurospheres were formed after 6-7 days (Reference 3: Liu BC, Liu FY, Gao XY, et al. Global transcriptional analyses of the Wnt-induced development of neural stem cells from human pluripotent stem cells [J]. International Journal of Molecular Sciences, 2021, 22 (14): 7473). The hESC used in this method also has ethical issues. In addition, the stemness and neural differentiation potential of iPSC are not as good as EPSC. The composition of CL medium is complex, the NSC sphere culture steps are long (14 days), and the neurons and astrocytes differentiated from NSC obtained by this scheme are not typical neurons and astrocytes in morphology.

[0006] Chen Y et al. used hESCs or iPSCs, first cultured in E8 medium containing Y-27632. During the neural induction culture stage, through screening, it was found that when using E6 as the basal medium and adding DN-193189 + 2 μM A83-01 (LA), the differentiation efficiency was the highest, and the positive cell rate of PAX6 reached 90% on the 6th day (Literature 4: Chen Y, Tristan C A, Mallanna S K, et al. Chemically Defined Neural Conversion of Human Pluripotent Stem Cells [J]. Neural Stem Cells: Methods and Protocols, 2019: 59-72). However, the ability of the NSCs formed by this method to differentiate into astrocytes is weak, and a large number of small molecule inhibitors need to be used, resulting in problems such as long differentiation time, high cost, and poor safety.

[0007] It can be seen that most current neural stem cell differentiation protocols are cumbersome and require various activators or inhibitors to change the fate of stem cells, resulting in problems such as long differentiation time and high cost. Some existing protocols use embryonic stem cells as the source of iNSCs, which have ethical issues in research and clinical applications. Other existing protocols use iPSCs as the source of iNSCs, and the maintenance of the stemness of iPSCs and their potential to differentiate into the neuroectoderm are not good, specifically manifested as the weak potential of the obtained iNSCs to differentiate into glial cells. Extended potential stem cells or expanded pluripotent stem cells (EPSCs) are a new type of more stable cell line with gene expression more similar to that of the four-cell or eight-cell stage, having extended developmental potential, capable of forming embryonic and extra-embryonic (ExEm) lineages, with a high single-cell cloning rate, high efficient chimeric ability, and high efficient extra-embryonic tissue development ability, and are proven to be stem cells with better developmental potential than iPSCs.

[0008] In view of this, in order to overcome the above technical problems existing in the current field, the present invention provides a method for differentiating human extended pluripotent stem cells into neural stem cells, which is simple and efficient, low-cost and short in differentiation time, and the obtained neural stem cells can be efficiently induced to differentiate into astrocytes and neurons. Summary of the Invention

[0009] The object of the present invention is to overcome the above technical problems existing in the current field and provide a method for differentiating human extended pluripotent stem cells into neural stem cells for the field.

[0010] The above object of the present invention is achieved by the following technical solutions:

[0011] The first aspect of the present invention provides a method for inducing the differentiation of expanded pluripotent stem cells into neural stem cells.

[0012] Furthermore, the method comprises the following steps: culturing expanded pluripotent stem cells in a neural stem cell induction medium to obtain neural stem cells derived from expanded pluripotent stem cells;

[0013] The neural stem cell induction medium comprises the following components: basal medium DMEM / F12, basal medium Neurobasal Medium, N2 supplementary, B27 supplementary, Glutmax, NEAA, 2-mercaptoethanol, Penicillin-Streptomycin Solution.

[0014] Furthermore, the contents of the components in the neural stem cell induction medium are as follows: basal medium DMEM / F12:Neurobasal Medium 1:(0.5 - 1.5), N2 supplementary (0.1 - 1)%, B27 supplementary (0.5 - 1.5)%, Glutmax (0.5 - 1.5)%, NEAA (0.5 - 1.5)%, 2-mercaptoethanol (0.05 - 0.5)%, Penicillin-Streptomycin Solution (0.5 - 1.5)%;

[0015] Preferably, the contents of the components in the neural stem cell induction medium are as follows: basal medium DMEM / F12:Neurobasal Medium 1:1, N2 supplementary 0.5%, B27 supplementary 1%, Glutmax 1%, NEAA 1%, 2-mercaptoethanol 0.1%, Penicillin-Streptomycin Solution 1%.

[0016] Furthermore, the method comprises the following steps:

[0017] (1) On Day 0, culture expanded pluripotent stem cells in the neural stem cell induction medium;

[0018] (3) On Day 6 - 8, obtain neural stem cells derived from expanded pluripotent stem cells;

[0019] Preferably, the neural stem cell induction medium is replaced every 24 hours.

[0020] In some embodiments, there are no particular limitations on the expanded pluripotent stem cells (EPSCs), which can be derived from substantially any suitable source. Suitable sources of expanded pluripotent stem cells can be expanded pluripotent stem cells derived from various mammalian somatic cell sources, or expanded pluripotent stem cells from various suitable stem cell sources.

[0021] In some embodiments, the somatic cells include, but are not limited to: various somatic cells from mammalian sources (such as from rodents, cats, dogs, pigs, goats, sheep, horses, cows, or primates, such as humans, preferably humans), such as muscle cells, chondrocytes, endothelial cells, epithelial cells, etc. Alternatively, the expanded pluripotent stem cells are obtained from a public cell resource bank or are a commercial product purchased.

[0022] In some embodiments, the suitable stem cells include, but are not limited to: embryonic stem cells (ESCs), haploid embryonic stem cells (haESCs), induced pluripotent stem cells (iPSCs), adult stem cells (somatic stem cells, such as stem cells extracted from bone marrow, fat, nerve, or skin).

[0023] In a specific embodiment, the expanded pluripotent stem cells of the present invention are prepared by the following method: Dilute matrigel with DMEM / F12 medium to a final concentration of 0.5%, coat the cell culture plate, and place it in a CO2 incubator for more than 2 hours. Coating volume: For a 6-well plate, add 2 mL of the diluted matrigel to each well; for a T25 flask, add 5 mL of the diluted matrigel, and place it in a CO2 incubator for more than 2 hours. Pre-warm the E8 medium at room temperature, resuscitate the iPSCs in E8 complete medium containing Y-27632 (ROCKi), and prepare the medium at a rate of 5 million cells / 10 - 15 mL. After 24 hours, change the medium to EPSC medium, and change the medium every day for culture. When the confluence reaches 70% - 80%, passage at a ratio of 1:4, digest the cells with 0.5 mM EDTA, and transfer the cells into a new well or T25 flask. After 5 - 7 passages, the EPSCs are obtained.

[0024] In a specific embodiment, the EPSC medium comprises the following components: Advanced DMEM / F-12, Neurobasal Medium, KSR, Glutamax, NEAA, ITSX, N2 supplementary, B27 supplementary, L-AA-pi, Y-27632 (ROCKi), LIF, Activin A, SDM, CHIR99021, Minocycline HCl, XAV939, GSK126, Trolox.

[0025] In a specific embodiment, the contents of the components in the EPSC medium are as follows: Advanced DMEM / F-12 46%, Neurobasal Medium 46%, KSR 3%, Glutamax 1%, NEAA 1%, ITSX 1%, N2 supplementary 1%, B27 supplementary 0.5%, L-AA-pi 100 μg / mL, Y-27632 (ROCKi) 1.6 μg / mL, LIF 10 ng / mL, Activin A 40 ng / mL, SDM 0.9 μg / mL, CHIR99021 0.5 μg / mL, Minocycline HCl 0.85 μg / mL, XAV939 0.65 μg / mL, GSK126 0.53 μg / mL, Trolox 2.6 μg / mL.

[0026] In a specific embodiment, the method further comprises the following steps: On Day-1, the expandable pluripotent stem cells prepared in the examples of the present invention are cultured in the EPSC medium as described above until the confluence reaches approximately 90%. TryplE digestive enzyme is diluted with DPBS at a ratio of 1:1. The EPSC is washed once with DPBS and then digested with the TryplE dilution solution. Under the microscope, when the cells are shiny around and there are gaps between cells, they are collected and dispersed into single cells, and plated into a 6-well plate at a density of 60-80%. The 6-well plate needs to be pre-coated with Matrigel diluted 1:300 for 1 hour. After 24 hours (Day0), the EPSC medium is replaced with the neural stem cell induction medium in the method as described above for the culture process as described above.

[0027] In a specific embodiment, it is possible to determine whether the extended pluripotent stem cells have been successfully induced to differentiate into neural stem cells by observing the morphological changes of cells at different stages and detecting the expression of neural stem cell markers NESTIN / SOX2. Among them, detecting the expression of the marker refers to measuring the relative level of the RNA transcript of the gene (marker) or its expression product. The methods for detecting gene expression / transcription, namely gene expression / transcription profiling, include methods based on polynucleotide hybridization analysis, methods based on polynucleotide sequencing, immunohistochemical methods, and proteomics-based methods. These methods generally detect the expression / transcription products (such as mRNA) of the genes of interest. In some embodiments, PCR-based methods such as reverse transcription PCR (RT-PCR) and array-based methods such as microarrays can be used.

[0028] The neural stem cells derived from extended pluripotent stem cells prepared by the method of the present invention have extremely strong differentiation potential, including the ability to differentiate into functional astrocytes and the ability to differentiate into functional neurons, and are significantly superior to the neural stem cells induced and differentiated by the neural stem cell induction media reported in the prior art. The neural stem cells prepared by the method of the present invention can be directly applied to various medical and scientific research purposes, or can be induced to differentiate into different target cells and then applied to various medical and scientific research purposes.

[0029] The second aspect of the present invention provides a neural stem cell induction medium for inducing the differentiation of extended pluripotent stem cells into neural stem cells.

[0030] Furthermore, the neural stem cell induction medium comprises the following components: basal medium DMEM / F12, basal medium Neurobasal Medium, N2 supplementary, B27 supplementary, Glutmax, NEAA, 2-mercaptoethanol, Penicillin-Streptomycin Solution;

[0031] Preferably, the content of each component in the neural stem cell induction medium is as follows: basal medium DMEM / F12:Neurobasal Medium 1:(0.5 - 1.5), N2 supplementary (0.1 - 1)%, B27 supplementary (0.5 - 1.5)%, Glutmax (0.5 - 1.5)%, NEAA (0.5 - 1.5)%, 2-mercaptoethanol (0.05 - 0.5)%, Penicillin-Streptomycin Solution (0.5 - 1.5)%;

[0032] More preferably, the contents of the components in the neural stem cell induction medium are as follows: basal medium DMEM / F12: Neurobasal Medium 1:1, N2 supplementary 0.5%, B27 supplementary 1%, Glutmax 1%, NEAA 1%, 2-mercaptoethanol 0.1%, Penicillin-Streptomycin Solution 1%.

[0033] In some embodiments, the components of the present invention refer to any compound or other material that can be used in a cell culture medium to maintain and / or promote cell growth and / or differentiation, regardless of whether its source is chemical or biological. The terms "component" and "constituent" can be used interchangeably. Conventional components for cell culture media can include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other components for promoting and / or maintaining ex vivo or in vitro cell cultures can be selected by those of ordinary skill in the art according to the needs of the desired effect.

[0034] In some embodiments, the present invention does not particularly limit the specific contents of the components in the neural stem cell induction medium, as long as the neural stem cell induction medium composed of the components with specific contents can exert or basically exert the expected effect (for example: can effectively or basically effectively induce the differentiation of extended pluripotent stem cells into neural stem cells), it is within the protection scope of the present invention.

[0035] In a specific embodiment, the present invention verified through comparative experiments that, compared with the neural stem cells induced and differentiated by using the conventional neural stem cell induction media reported in the prior art (for example: CL medium and N-induce medium), the neural stem cells induced and differentiated by using the neural stem cell induction medium of the present invention have a stronger potential to differentiate into astrocytes and neurons.

[0036] The third aspect of the present invention provides a method for inducing the differentiation of extended pluripotent stem cells into astrocytes.

[0037] Furthermore, the method includes the following steps: inducing and differentiating extended pluripotent stem cells into neural stem cells by using the method of the first aspect of the present invention, culturing the obtained neural stem cells in an astrocyte medium to obtain astrocytes;

[0038] Preferably, astrocytes are obtained after culturing in the astrocyte medium for 28 days;

[0039] Preferably, the astrocyte medium is changed every 48h.

[0040] Furthermore, the astrocyte medium contains the following components: Advanced DMEM / F12, Neurobasal Medium, N2 supplementary, B27 supplementary, Glutamax, NEAA, 2-mercaptoethanol, Penicillin-Streptomycin Solution, CNTF;

[0041] Preferably, the contents of the components in the astrocyte medium are as follows: Advanced DMEM / F12 (20-60)%, Neurobasal Medium (20-60)%, N2 supplementary (0.1-1)%, B27 supplementary (0.5-1.5)%, Glutamax (0.5-1.5)%, NEAA (0.5-1.5)%, 2-mercaptoethanol (0.05-0.5)%, Penicillin-Streptomycin Solution (0.5-1.5)%, CNTF (5-15) ng / mL;

[0042] More preferably, the contents of the components in the astrocyte medium are as follows: Advanced DMEM / F12 48%, Neurobasal Medium 48%, N2 supplementary 0.5%, B27 supplementary 1%, Glutamax 1%, NEAA 1%, 2-mercaptoethanol 0.1%, Penicillin-Streptomycin Solution 1%, CNTF 10 ng / mL.

[0043] In a specific embodiment, the successful induction of differentiation of expanded pluripotent stem cell-derived neural stem cells into neural stem cells can be determined by detecting the expression of astrocyte markers such as S100B, AQP4, NFIB, OCT4, EAAT1, etc.

[0044] The fourth aspect of the present invention provides a method for inducing the differentiation of expanded pluripotent stem cells into neurons.

[0045] Furthermore, the method includes the following steps: inducing the differentiation of expanded pluripotent stem cells into neural stem cells by using the method described in the first aspect of the present invention, and culturing the obtained neural stem cells in a neural expansion medium to obtain neurons;

[0046] Preferably, neurons are obtained after culturing in a neural expansion medium for 5 days;

[0047] Preferably, the neural expansion medium is replaced every 24 hours.

[0048] Furthermore, the neural expansion medium comprises the following components: Neurobasal Medium, B27 supplementary, Glutamax, Penicillin-Streptomycin Solution, bFGF;

[0049] Preferably, the contents of the components in the neural expansion medium are as follows: Neurobasal Medium (93 - 97)%, B27 supplementary (1 - 3)%, Glutamax (0.5 - 2)%, Penicillin-Streptomycin Solution (0.5 - 2)%, bFGF (10 - 30) ng / mL;

[0050] More preferably, the contents of the components in the neural expansion medium are as follows: Neurobasal Medium 96%, B27 supplementary 2%, Glutamax 1%, Penicillin-Streptomycin Solution 1%, bFGF 20 ng / mL.

[0051] In a specific embodiment, the successful induction of differentiation of neural stem cells derived from expandable pluripotent stem cells into neurons can be determined by detecting the expression of neural cell markers such as MAP2.

[0052] The fifth aspect of the present invention provides an astrocyte medium for inducing the differentiation of neural stem cells into astrocytes and a neural expansion medium for inducing the differentiation of neural stem cells into neurons.

[0053] Furthermore, the astrocyte medium is the astrocyte medium described in the third aspect of the present invention, and the neural expansion medium is the neural expansion medium described in the fourth aspect of the present invention.

[0054] The sixth aspect of the present invention provides an application of any of the following aspects, and the application includes:

[0055] (1) The application of the neural stem cell induction medium described in the second aspect of the present invention in inducing the differentiation of expandable pluripotent stem cells into neural stem cells;

[0056] (2) Use of the neural stem cell induction medium described in the second aspect of the present invention and / or the astrocyte medium described in the fifth aspect of the present invention in inducing the differentiation of expandable pluripotent stem cells into astrocytes;

[0057] (3) Use of the neural stem cell induction medium described in the second aspect of the present invention, the astrocyte medium described in the fifth aspect of the present invention, and / or the neural expansion medium described in the fifth aspect of the present invention in inducing the differentiation of expandable pluripotent stem cells into neurons;

[0058] (4) Use of the neural stem cells prepared by the method described in the first aspect of the present invention, the astrocytes prepared by the method described in the third aspect of the present invention, and / or the neurons prepared by the method described in the fourth aspect of the present invention in the construction of cell models related to neurological diseases, drug screening related to neurological diseases, and in vitro experimental studies related to neurological diseases.

[0059] In some embodiments, the neurological diseases include but are not limited to: stroke, amyotrophic lateral sclerosis, spinal cord injury, motor neuron disease, brain atrophy, multiple sclerosis, demyelinating disease, Huntington's chorea, Alzheimer's disease, Parkinson's disease, cerebral palsy, brain trauma, brain tumor, congenital brain hypoplasia, brain nerve tissue damage caused by chemical drug poisoning, acute infectious polyneuritis, poliomyelitis. Any brain diseases such as cerebral palsy and mental retardation caused by genetics or congenital dysplasia, any sequelae of acute brain injury caused by trauma, any sequelae of stroke caused by cerebrovascular diseases, and any chronic degenerative diseases caused by damage to central nerve fibers and subsequent senescence and degeneration of nerve cells are within the scope of the neurological diseases described in the present invention.

[0060] Advantages and beneficial effects of the present invention compared with the prior art:

[0061] (1) Compared with the current conventional methods for inducing the differentiation of iPSC / ESC into NSC, the method provided by the present invention uses EPSC instead of iPSC and ESC, without the ethical risk of ESC, and the differentiation potential towards the neuroectoderm is significantly better than that of iPSC. In addition, the differentiation process of the method provided by the present invention is simple. After plating, the medium is changed daily until the required number of days, without the need for steps such as forming embryoid bodies and monoclonal neurospheres; the differentiation time is short, a large number of NESTIN-positive cells can appear on D3 - D4, and the cells have strong proliferation ability and a large harvest.

[0062] (2) The culture medium components used in the method provided by the present invention are simple and do not contain one or more of small molecule drugs such as CHIR99032, XAV929, LDN193189, SB431542, LIF, and putrescine. The present invention uses the readily available antioxidant 2-mercaptoethanol and is coated with the readily available matrigel, with lower differentiation costs. Moreover, the differentiated neural stem cells have strong potential to differentiate into astrocytes and neurons. The method provided by the present invention can achieve large-scale production of neural stem cells and astrocytes derived from EPSCs, and has the advantages of simple operation, low cost, high differentiation efficiency, short differentiation cycle, and large number of differentiated cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Bright field images of neural stem cells differentiated from EPSCs on the 4th day (D4) and the 7th day (D7);

[0064] Figure 2 Results of immunofluorescence staining identification of iNSCs derived from EPSCs;

[0065] Figure 3 Results of detection of the expression levels of NESTIN, PAX6, and the neuronal marker TUBB3, which are related markers of iNSCs derived from EPSCs;

[0066] Figure 4 Results of GFAP staining of neurons derived from iNSCs;

[0067] Figure 5 Results of GFAP staining of astrocytes derived from iNSCs;

[0068] Figure 6 Results of detection of the expression levels of S100B, AQP4, NFIB, OCT4, and EAAT1, which are characteristic proteins related to astrocytes differentiated from neural stem cells derived from EPSCs;

[0069] Figure 7 Results of co-culture of neurons and astrocytes derived from iNSCs to form synapse-end foot structures;

[0070] Figure 8 Results of comparison of the potential of iNSCs differentiated from different neural stem cell induction media to differentiate into astrocytes; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0071] Specific information on the main instruments, consumables, and experimental reagents used in the embodiments of the present invention is shown in Table 1 and Table 2 below.

[0072] Table 1 Instruments and Consumables Used in the Present Invention

[0073] Name of Instrument / Consumable Specification or Model Manufacturer Laser Confocal Microscope 02-06-0026 Nikon Single-channel Manual Adjustable Pipette 100 - 1000 μL Dalong Single-channel Manual Adjustable Pipette 20 - 200 μL Dalong Single-channel Manual Adjustable Pipette 0.5 - 10 μL Dalong Medical Centrifuge TDZ5 - WS Hunan Xiangyi Micro Centrifuge Mini - 6K Hangzhou Ausheng 1.5 mL EP Tube 509 - GRD - Q QSP 1000 μL Low - adsorption Pipette Tip 112NXL - Q QSP 200 μL Low - adsorption Pipette Tip T090 - Q QSP 10 μL Low - adsorption Pipette Tip 104-Q QSP 12 - well Cell Culture Plate 150628 Thermo SPL 10 mL Pipette SPL91010 SPL SPL 5 mL Pipette SPL91005 SPL 50 mL Centrifuge Tube 339652 QSP

[0074] Table 2 Reagent information used in the present invention

[0075] Name of Reagent Catalog Number Manufacturer Neurobasal 21103049 Gibco Advanced DMEM / F - 12 12634-010 Thermo N2 17502048 Thermo B27 12587010 Thermo Glutamax 35050061 Gibco NEAA 11140050 Gibco 2 - mercaptoethanol 21985023 Gibco Penicillin - streptomycin 15140122 Gibco bFGF PHG0367 Thermo CNTF 107-08 sciencell BMP4 ab87063 Abcam Accutase A6964 Sigma DPBS SH30028.02 Cytiva LDN193189 S7507 SELLECK XAV939 ab120897 Abcam SB431542 ab120163 Abcam CHIR99021 72054 Stemcell KSR 10828-028 Gibco ITSX 51500-056 Gibco L - AA - pi A8960 Sigma Y - 27632(ROCKi) ab120129 Abcam LIF GMP - TL633 Tongli Haiyuan Activin A 120-14 peprotech SDM B6734 APEXBIO Minocycline HCl A4452 APEXBIO

[0076] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: without departing from the principles and purposes of the present invention, various changes, modifications, substitutions, and variations can be made to these embodiments, and the scope of the present invention is defined by the claims and their equivalents. For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0077] Example 1 Preparation of neural stem cells from human extended pluripotent stem cells (EPSCs)

[0078] 1. Experimental method

[0079] (1) Induction and subculture of human extended pluripotent stem cells (EPSCs)

[0080] EPSCs were prepared by our company itself, and the preparation method is as follows: Dilute matrigel with DMEM / F12 medium to a final concentration of 0.5%, coat the cell culture plate, and place it in a CO2 constant temperature incubator for more than 2 h. Coating volume: For a 6-well plate, add 2 mL of the diluted matrigel to each well; for a T25 flask, add 5 mL of the diluted matrigel and place it in a CO2 constant temperature incubator for more than 2 h. The E8 medium was pre-warmed at room temperature, and iPSCs were resuscitated in the E8 complete medium containing Y-27632 (ROCKi), and the medium was prepared according to the amount of 5 million cells / 10 - 15 mL. After 24 h, the medium was changed to the EPSC medium (the components of the EPSC medium are shown in Table 3 below), and the cells were cultured with medium change every day. When the confluence reached 70% - 80%, subculture was carried out at a ratio of 1:4, and the cells were digested with 0.5 mM EDTA and transferred into new wells or T25 flasks. After 5 - 7 subcultures, EPSCs were obtained. The expression of pluripotent genes Oct4, nanog, and sox2 in EPSCs was up-regulated, and they were able to be induced to differentiate into trophoblast stem cells (TSS).

[0081] Table 3 Composition table of EPSC medium

[0082]

[0083]

[0084] (2) Inductive Differentiation Culture of Human Expansible Pluripotent Stem Cells (EPSCs) into Neural Stem Cells (NSCs)

[0085] Culture the EPSCs in the EPSC medium (the components are shown in Table 3) until the confluence reaches approximately 90%. Dilute TryplE digestive enzyme and DPBS at a ratio of 1:1. Wash the EPSCs once with DPBS and then digest them with the TryplE dilution solution. Observe under the microscope until the cells are shiny around and there are gaps between cells. Collect and disperse them into single cells, and plate them in a 6-well plate at a density of 60 - 80%. The 6-well plate needs to be pre-coated with Matrigel diluted 1:300 for 1 hour in advance. At this time, the differentiation time is recorded as D - 1.

[0086] After 24 hours, change the EPSC medium to the neural stem cell induction medium (N - M medium). At this time, the differentiation day is D0. Subsequently, change the NSC induction medium every 24 hours and culture for 6 - 8 days to obtain neural stem cells. Observe the morphological changes of the cells during the inductive differentiation process, and detect the expression of the neural stem cell markers NESTIN, PAX6 and the neuron - specific marker TUBB3.

[0087] Components of the neural stem cell induction medium (N - M medium) (see Table 4): Basal medium DMEM / F12:Neurobasal Medium 1:1, N2 supplementary 0.5%, B27 supplementary 1%, Glutmax 1%, NEAA 1%, 2 - mercaptoethanol 0.1%, Penicillin - Streptomycin Solution 1%.

[0088] Table 4 N - M Medium

[0089]

[0090]

[0091] 2. Experimental Results

[0092] (1) Morphological Changes of Cells during the Differentiation of EPSCs into iNSCs

[0093] The results are as Figure 1 shown. The results show that under the condition of the neural stem cell induction medium, EPSCs proliferate rapidly, and neural rosette structures can appear on the fourth day, and a large number of neural rosette structures can appear on the seventh day.

[0094] (2) Expression of Neural Stem Cell Markers during the Differentiation of EPSCs into iNSCs

[0095] To identify the characteristics of neural stem cells at day 7 of differentiation (D7), the differentiated cells were stained with the neural stem cell marker NESTIN, and a radial flower ring structure positive for NESTIN could be seen ( Figure 2 left). After digesting them into single cells and replating, NESTIN / SOX2 staining was performed, and it could be seen that NESTIN was localized to the cytoskeleton and SOX2 was localized to the nucleus, which was identified as neural stem cells ( Figure 2 right).

[0096] The expression levels of the neural stem cell markers NESTIN and PAX6, as well as the expression changes of the neuron-specific marker TUBB3, were detected at days 0, 2, 3, 4, 5, and 7 of differentiation. The results were as Figure 3 shown. The results showed that from day 0 to day 7 of differentiation, the expression levels of the neural stem cell markers NESTIN and PAX6 gradually increased, while the neuron-specific marker TUBB3 remained basically unchanged after day 3 of differentiation. This indicated that during the differentiation of neural stem cells, the cells could be maintained at the neural stem cell stage without differentiating into neurons.

[0097] Example 2 Functional detection of neural stem cells differentiated from EPSC

[0098] 1. Experimental method

[0099] (1) Inducing the differentiation of induced neural stem cells (iNSC) into astrocytes and neurons

[0100] When the confluence of iNSC reached 60%, it was passaged at a ratio of 1:2 to a 12-well plate, and the astrocyte medium (ATC medium, the components are shown in Table 5) was replaced to induce its differentiation into astrocytes. When the cell confluence reached 90%, it was passaged at a ratio of 1:2 (reference: Shaltouki A, Peng J, Liu Q, et al. Efficient generation of astrocytes from human pluripotent stem cells in defined conditions [J]. Stem cells, 2013, 31(5): 941 - 952).

[0101] Table 5 Composition table of ATC medium

[0102] Component Concentration Advanced DMEM / F - 12 48％ Neurobasal 48％ N2 0.5％ B27 1％ Glutamax 1％ NEAA 1％ 2 - mercaptoethanol 0.1％ PS 1％ CNTF 10 ng / mL

[0103] After culturing EPSC-derived iNSCs in neural stem cell induction medium (N-M medium) for 14 days, they were digested with accutase, passaged at low density, and cultured in neural expansion medium (N-expanding medium, the composition is shown in Table 6) for 5 days to be induced into MAP2-positive neuron-like cells.

[0104] Differentiation of EPSC-derived iNSCs into astrocytes: EPSC-derived iNSCs were cultured in astrocyte induction medium (ATC medium). Astrocytes were obtained after 28 days of culture. The medium was changed every 48 h during this period. When reaching 90%, they were passaged at a ratio of 1:2.

[0105] Differentiation of EPSC-derived iNSCs into neurons: EPSC-derived iNSCs were cultured in neural expansion medium (N-expanding medium). Neurons were obtained after 5 days of culture. The medium was changed every 24 h during this period (the neurons obtained by this method were used for co-culture experiments with astrocytes).

[0106] Table 6 N-expanding medium

[0107] Component Concentration Neurobasal 96％ B27 2％ Glutamax 1％ PS 1％ bFGF 20 ng / mL

[0108] (2) Detection of astrocyte and neuron markers derived from induced neural stem cells (iNSCs)

[0109] Seven days after the differentiation of EPSC-derived neural stem cells into neurons, immunofluorescence was used to detect the neural cell marker MAP2. Four weeks after the induction of EPSC-derived iNSCs into astrocytes, immunofluorescence was used to detect the astrocyte-specific marker GFAP; The 6th generation cells of EPSC-derived iNSCs induced to differentiate into astrocytes were used for RT-qPCR to detect astrocyte markers S100B, AQP4, NFIB, EAAT1, and the pluripotent stem cell marker Oct4.

[0110] 2. Experimental results

[0111] A large number of neural synapses appeared seven days after the differentiation of EPSC-derived neural stem cells into neurons. Immunofluorescence staining showed that the neural cell marker MAP2 was positive (see Figure 4 ). It indicates that the EPSC-derived neural stem cells have the ability of neuron differentiation.

[0112] Four weeks after the induction of EPSC-derived iNSCs into astrocytes, specific marker GFAP staining was performed. The results showed that the cells were GFAP positive and presented the cell morphology of astrocytes (see Figure 5), indicating that the neural stem cells derived from EPSCs have the ability to differentiate into astrocytes.

[0113] The iNSCs derived from EPSCs were induced into astrocytes, and qPCR detection of relevant markers was performed after 6 passages. The results showed that the expressions of astrocyte-related characteristic proteins S100B, AQP4, NFIB, and EAAT1 were up-regulated, and the expression of the pluripotent gene Oct4 was down-regulated. The cells were GFAP-positive and showed the cell morphology of astrocytes (see Figure 6 ), indicating that the neural stem cells derived from EPSCs have the ability to differentiate into astrocytes.

[0114] Astrocytes are involved in the remodeling of neural synapses in vivo, and morphologically, the end-feet of astrocytes can bind to neural synapses. The neurons derived from iNSCs differentiated from EPSCs were co-cultured with astrocytes, and the results showed the co-localization of the end-feet of astrocytes and neural synapses (see Figure 7 ), indicating that the obtained iNSCs have the ability to differentiate into functional astrocytes.

[0115] Comparative example Comparison of the effects of the neural stem cell induction medium (N-M medium) used in the present invention with the neural stem cell induction media reported in the prior art

[0116] 1. Experimental method

[0117] Under the condition that other steps and methods remain unchanged, the N-M medium in step (2) of Example 1 was replaced with the CL medium used in Document 3 cited in the background art and the N-induce medium used in Document 4 (the components are shown in Tables 7 and 8 respectively) as controls. The potential of the neural stem cells obtained by using the N-M medium of the present invention, the CL medium used in the existing literature, and the N-induce medium to differentiate into astrocytes was compared.

[0118] Table 7 Composition table of CL medium

[0119] Component Concentration N - M Medium (used in this invention) 100％ CHIR99021 1.4 μg / mL LIF 10 ng / mL

[0120] Table 8 Composition table of N-induce medium

[0121] Component Concentration Advanced DMEM / F - 12 49％ Neurobasal 49％ N2 0.5％ B27 1％ Glutamax 1％ 2 - mercaptoethanol 0.1％ LDN193189 3.84 μg / mL XAV939 0.65 μg / mL SB431542 40 ng / mL

[0122] 2. Experimental results

[0123] Compared with the C-L medium (C-L) and N-induce medium (N-in) used in the existing literature, the iNSCs differentiated using the N-M medium of the present invention showed a significant increase in the expression of astrocyte marker proteins GFAP, AQP4, and S100B after being induced in the astrocyte induction medium (ATC medium) for 2 weeks (see Figure 8 ), indicating that the iNSCs differentiated using the neural stem cell induction medium N-M medium of the present invention have a stronger potential to differentiate into astrocytes.

[0124] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for inducing the differentiation of extended pluripotent stem cells into neural stem cells, characterized in that, The method cultures expandable pluripotent stem cells in a neural stem cell induction medium to obtain neural stem cells derived from expandable pluripotent stem cells; The neural stem cell induction medium consists of the following components : basal medium DMEM / F12, basal medium Neurobasal Medium, N2 supplementary, B27 supplementary, Glutmax, NEAA, 2-mercaptoethanol, Penicillin-Streptomycin Solution; The contents of the components in the neural stem cell induction medium are as follows: the ratio of basal medium DMEM / F12 to Neurobasal Medium is 1:0.5 - 1:1.5, N2 supplementary is 0.1% - 1%, B27 supplementary is 0.5% - 1.5%, Glutmax is 0.5% - 1.5%, NEAA is 0.5% - 1.5%, 2-mercaptoethanol is 0.05% - 0.5%, and Penicillin-Streptomycin Solution is 0.5% - 1.5%.

2. The method according to claim 1, wherein The contents of the components in the neural stem cell induction medium are as follows: the ratio of basal medium DMEM / F12 to Neurobasal Medium is 1:1, N2 supplementary is 0.5%, B27 supplementary is 1%, Glutmax is 1%, NEAA is 1%, 2-mercaptoethanol is 0.1%, and Penicillin-Streptomycin Solution is 1%.

3. The method according to claim 1, characterized in that The method is as follows: (1) On Day 0, culture expandable pluripotent stem cells in the neural stem cell induction medium; (2) On Day 6 - 8, obtain neural stem cells derived from expandable pluripotent stem cells.

4. The method according to claim 3, wherein Replace the neural stem cell induction medium every 24 h.

5. A method for inducing the differentiation of expanded pluripotent stem cells into astrocytes, characterized in that, The method includes the following steps: inducing and differentiating expandable pluripotent stem cells into neural stem cells by using the method described in any one of claims 1 - 4, and culturing the obtained neural stem cells in an astrocyte medium to obtain astrocytes; The astrocyte medium consists of the following components: Advanced DMEM / F12, Neurobasal Medium, N2 supplementary, B27 supplementary, Glutamax, NEAA, 2-mercaptoethanol, Penicillin-Streptomycin Solution, CNTF; The contents of the components in the astrocyte medium are as follows: Advanced DMEM / F12 is 20%-60%, Neurobasal Medium is 20%-60%, N2 supplementary is 0.1%-1%, B27 supplementary is 0.5%-1.5%, Glutamax is 0.5%-1.5%, NEAA is 0.5%-1.5%, 2-mercaptoethanol is 0.05%-0.5%, Penicillin-Streptomycin Solution is 0.5%-1.5%, and CNTF is 5 ng / mL-15 ng / mL.

6. The method according to claim 5, characterized in that, Astrocytes are obtained after culturing in the astrocyte medium for 28 days.

7. The method according to claim 5, wherein The astrocyte medium is changed every 48 h.

8. The method according to claim 5, characterized in that, The contents of the components in the astrocyte medium are as follows: Advanced DMEM / F12 is 48%, Neurobasal Medium is 48%, N2 supplementary is 0.5%, B27 supplementary is 1%, Glutamax is 1%, NEAA is 1%, 2-mercaptoethanol is 0.1%, Penicillin-Streptomycin Solution is 1%, and CNTF is 10 ng / mL.

9. A method for inducing the differentiation of expanded pluripotent stem cells into neurons, characterized in that, The method comprises the following steps: inducing and differentiating expandable pluripotent stem cells into neural stem cells by using the method according to any one of claims 1-4, and culturing the obtained neural stem cells in a neural amplification medium to obtain neurons; The neural amplification medium consists of the following components : Neurobasal Medium, B27 supplementary, Glutamax, Penicillin-Streptomycin Solution, bFGF; The contents of the components in the neural amplification medium are as follows: Neurobasal Medium is 93%-97%, B27 supplementary is 1%-3%, Glutamax is 0.5%-2%, Penicillin-Streptomycin Solution is 0.5%-2%, and bFGF is 10 ng / mL-30 ng / mL.

10. The method according to claim 9, characterized in that, Neurons are obtained after culturing in the neural amplification medium for 5 days.

11. The method according to claim 9, wherein The neural amplification medium is changed every 24 h.

12. The method according to claim 9, characterized in that The contents of the components in the neural amplification medium are as follows: Neurobasal Medium is 96%, B27 supplementary is 2%, Glutamax is 1%, Penicillin-Streptomycin Solution is 1%, and bFGF is 20 ng / mL.

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