Induction preparation method of human astrocytes

Abstract

The invention discloses an induced preparation method of human-derived astrocytes, which comprises the following steps: taking venous peripheral blood, resuspending peripheral blood mononuclear cells by using a phosphate buffer solution, and centrifuging to obtain peripheral blood mononuclear cell suspension; the method comprises the following steps: transfecting peripheral blood mononuclear cells through a sendai virus vector to obtain amplified peripheral blood mononuclear cells, separating the sendai virus vector and transfected cells, and transferring subculture cells formed by transfection culture to a culture plate coated with matrigel for continuous culture after transfection culture, culturing the passage cells through a neural precursor cell differentiation medium to form human induced neural precursor cells; performing suspension culture through a glial progenitor cell induction medium to form glial progenitor cells; and carrying out adherent culture through the astrocyte induction culture medium to obtain astrocytes.

Description

Technical Field

[0001] The present application relates to the technical field of cell culture preparation, and specifically to a method for inducing preparation of human astrocytes. Background Art

[0002] Astrocytes play a vital role in the human body. They can regulate neurotransmitters, maintain ion balance, support the blood-brain barrier, provide energy, participate in circadian rhythm regulation, promote neuroprotection and repair, etc., which are essential for the health of the nervous system. In addition, astrocytes also play a dual role in neurodegenerative diseases: on the one hand, they may cause neuronal damage by releasing neurotoxic factors and participating in abnormal energy metabolism; on the other hand, they are also involved in neuroprotection. The activation state of these cells is closely related to the development of neuroinflammation and degeneration, making them a potential target for the treatment of these diseases. Related studies have shown that transplanting cultured astrocytes into the brains of white matter stroke patients can repair brain damage, induce healing of brain cells damaged by stroke, and stimulate damaged oligodendrocyte progenitor cells to restore myelin on newly grown axons. The traditional method of inducing the preparation of astrocytes has problems such as long induction time and poor safety risks. Summary of the invention

[0003] In order to overcome the above shortcomings, the purpose of the present application is to provide a method for inducing and preparing human astrocytes, thereby effectively solving the above technical problems.

[0004] In order to achieve the above objectives, this application adopts the following technical solutions:

[0005] The present application provides a method for inducing and preparing human astrocytes, comprising the following steps:

[0006] Taking venous peripheral blood, diluting the peripheral blood, adding a cell density gradient separation solution to the diluted peripheral blood, centrifuging the mixture of the peripheral blood and the cell density gradient separation solution to achieve a stratification effect, sucking out the yellow upper layer containing plasma in the stratified solution and transferring the white middle layer containing peripheral blood mononuclear cells, resuspending the peripheral blood mononuclear cells with phosphate buffer (PSB) and centrifuging to obtain a first cell precipitate, mixing the first cell precipitate with an erythrocyte lysis buffer and incubating, diluting the mixture of the first cell precipitate and the erythrocyte lysis buffer with a phosphate buffer and centrifuging to obtain a second cell precipitate, resuspending the second cell precipitate with a phosphate buffer and centrifuging, resuspending the precipitate after centrifugation to obtain a peripheral blood mononuclear cell suspension, adding a peripheral blood mononuclear cell culture medium to the peripheral blood mononuclear cells in the suspension for incubation and culture to obtain a peripheral blood mononuclear cell sample;

[0007] Taking a Sendai virus vector, the Sendai virus vector carries genes octamer binding transcription factor 3 / 4 (OCT3 / 4), sex determining region Y-box 2 (SOX2), myelocytoma oncogene (c-MYC), Krüppel-like factor 4 (KLF-4), transfecting peripheral blood mononuclear cells with the Sendai virus vector to obtain amplified peripheral blood mononuclear cells, separating the Sendai virus vector and the transfected cells, resuspending the transfected cells in a peripheral blood mononuclear cell culture medium for transfection culture, transferring the passage cells formed by the transfection culture to a culture plate coated with matrix gel for further culture after the transfection culture, taking a neural progenitor cell differentiation medium, and culturing the passage cells with the neural progenitor cell differentiation medium to form human induced neural progenitor cells;

[0008] Taking a glial progenitor cell induction medium, and suspending and culturing the human induced neural precursor cells with the glial progenitor cell induction medium to form glial progenitor cells;

[0009] An astrocyte induction medium is obtained, and the glial progenitor cells are subjected to adherent culture using the astrocyte induction medium to obtain astrocytes.

[0010] Furthermore, the peripheral blood mononuclear cell culture medium is formed by a volume ratio of 1:1 of Iskoff's modified Dulbecco's medium (IMDM culture medium) and Ham's F-12 nutrient mixture (Ham's F-12 culture medium), a volume ratio of 1% insulin-transferrin-selenium-ethanolamine culture medium supplement (ITS-X), a volume ratio of 1% chemically defined lipid concentrate, a mass volume ratio of 1% L-glutamine, 30-80 μg / ml L-ascorbic acid, 5 mg / ml bovine serum albumin, 200 μM thioglycerol, 100 ng / ml recombinant human stem cell factor, 100 μg / ml all-iron transferrin, 40 ng / ml insulin-like growth factor 1, 10 ng / ml interleukin 3, 2 U / ml erythropoietin, and 1 μM dexamethasone.

[0011] Furthermore, resuspending the transfected cells in a peripheral blood mononuclear cell culture medium for transfection culture comprises the following steps:

[0012] On the first day after transfection, replace the culture medium of peripheral blood mononuclear cells with new one to remove the viral vector.

[0013] On the second day after transfection, replace the new peripheral blood mononuclear cell culture medium to obtain passage cells.

[0014] On the third day after transfection, the passaged cells were transferred to the culture plate coated with matrix gel and continued to be cultured.

[0015] On the fourth day after transfection, the neural progenitor cell differentiation medium was replaced to obtain human induced neural progenitor cells.

[0016] Furthermore, after replacing the neural progenitor cell differentiation medium for culturing, the neural progenitor cell differentiation medium is replaced with a new one every two days, and the culturing is continued until human induced neural progenitor cells are obtained.

[0017] Furthermore, the neural precursor cell differentiation medium is formed by a 1:1 volume ratio of Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and a neurobasal medium (Neurobasal medium), with the addition of a 1% volume ratio of N2 additive, a 2% volume ratio of B27 additive, a 1% volume ratio of non-essential amino acids, a 1% volume ratio of glutamine supplement, 5-20ng / ml recombinant human leukemia inhibitory factor, 2-6μM 6-aminopurine derivatives and 2-4μM 4-[4-(1,3-benzodiazol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate (SB431542).

[0018] Furthermore, suspending and culturing the human induced neural precursor cells using the glial progenitor cell induction medium comprises:

[0019] Glial progenitor cell induction medium was used for suspension culture of human induced neural precursor cells (density was 5e5 / ml), and new glial progenitor cell induction medium was replaced every day until the suspension cell spheres were collected for adherent culture on the 14th day, and glial progenitor cell induction medium was used for culture again, and new glial progenitor cell induction medium was replaced every two days until the crawling glial progenitor cells were collected.

[0020] Furthermore, the glial progenitor cell induction medium is formed by adding 1% by volume of N2 additive, 2% by volume of B27 additive, 1% by volume of non-essential amino acids, 1% by volume of glutamine supplement, 40-80ng / ml triiodothyronine, 10-30ng / ml platelet-derived growth factor AA, 5-20ng / ml basic fibroblast growth factor, 80-200nM retinoic acid, and 0.5-2μM purmorphamine to a mixture of Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and neurobasal medium (Neurobasal medium) in a volume ratio of 1:1.

[0021] Furthermore, the adherent culture of the glial progenitor cells using the astrocyte induction medium comprises:

[0022] Astrocyte induction medium was used to culture the glial progenitor cells adherently, and new astrocyte induction medium was replaced every 2 days until the cells were passaged. After the cell passage, astrocyte induction medium was continued to be used for adherent culture, and new astrocyte induction medium was replaced every two days until astrocytes were obtained.

[0023] Furthermore, the astrocyte induction medium is formed by adding 1% by volume of N2 additive, 1% by volume of non-essential amino acids, 1% by volume of glutamine supplement, 10-30ng / ml epidermal growth factor, 10-30ng / ml basic fibroblast growth factor, 150-200ng / ml insulin-like growth factor 1, and 10-20ng / ml ciliary neurotrophic factor to a mixture of Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and neurobasal medium (Neurobasal medium) in a volume ratio of 1:1.

[0024] Beneficial Effects

[0025] The present application adopts a culture method of inducing the differentiation of human induced neural precursor cells in vitro to form astrocytes, first culturing them in suspension and then attaching them to balls, which greatly shortens the in vitro induced differentiation time of astrocytes and does not perform integrated gene editing operations on the cells, so it is relatively safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide an understanding of the technical solution of the present disclosure and constitute a part of the specification. Together with the embodiments of the present disclosure, they are used to explain the technical solution of the present disclosure and do not constitute a limitation on the technical solution of the present disclosure. The shapes and sizes of the components in the accompanying drawings do not reflect the actual proportions and are only intended to illustrate the content of the present application.

[0027] Figure 1 This is an embodiment of the present application providing changes in cell morphology during the process of reprogramming peripheral blood mononuclear cells into human induced neural progenitor cells.

[0028] Figure 2 This is the immunostaining result of human induced neural progenitor cells obtained by reprogramming provided in one embodiment of the present application.

[0029] Figure 3 This is the percentage of positive cells in the fluorescence quantification of human induced neural progenitor cells obtained by reprogramming provided in one embodiment of the present application.

[0030] Figure 4 This is a nuclear morphology diagram of human induced neural precursor cells obtained by reprogramming provided in one embodiment of the present application.

[0031] Figure 5This is an example of the present application providing changes in cell morphology during in vitro differentiation of human induced neural precursor cells HiNPC into astrocytes.

[0032] Figure 6 This is the immunostaining result of astrocytes on day 37 provided in one embodiment of the present application.

[0033] Figure 7 This is the percentage of positive cells in the fluorescence quantification of astrocytes on the 37th day provided in one embodiment of the present application. DETAILED DESCRIPTION

[0034] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present application and are not limited to the scope of the present application. The implementation conditions adopted in the examples can be further adjusted as the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0035] Unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should be understood by people with ordinary skills in the field to which the present application belongs. The "first", "second" and similar words used in the embodiments of the present disclosure do not indicate any order, quantity or importance, but are only used to distinguish different components. "Including" or "comprising" and similar words mean that the elements or objects appearing in front of the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connecting" or "connected" and similar words are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. In this article, "electrical connection" includes the situation where the constituent elements are connected together through an element with some electrical function. "Elements with some electrical function" are not particularly limited as long as they can transfer electrical signals between the connected constituent elements. "Elements with some electrical function" can be, for example, electrodes or wiring, or switching elements such as transistors, or other functional elements such as resistors, inductors or capacitors. "Up", "down", "left", "right", etc. are only used to indicate relative position relationships. When the absolute position of the object being described changes, the relative position relationship may also change accordingly.

[0036] In this application, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "middle", etc. are based on the directions or positional relationships shown in the drawings. These terms are mainly used to better describe the present application and its embodiments, and are not used to limit the indicated devices, elements or components to have a specific direction, or to be constructed and operated in a specific direction.

[0037] As mentioned in the background technology, the traditional method of inducing the preparation of astrocytes has problems such as long induction time and poor safety risk. Therefore, the inventor provides an induction preparation method for human astrocytes, which forms human induced neural precursor cells from peripheral blood mononuclear cells. This step is mentioned in the in vitro preparation method of medial ganglionic eminence progenitor cells and GABA interneurons in publication number CN117264892B, comprising: taking venous peripheral blood, diluting the peripheral blood, adding cell density gradient separation solution to the diluted peripheral blood, centrifuging the mixture of peripheral blood and cell density gradient separation solution to achieve a stratification effect, and aspirating the plasma in the stratified solution. After the yellow upper layer is removed, the white middle layer containing peripheral blood mononuclear cells is transferred out, the peripheral blood mononuclear cells are resuspended in phosphate buffer and centrifuged to obtain a first cell precipitate, the first cell precipitate is mixed with red blood cell lysis buffer and incubated, the mixture of the first cell precipitate and the red blood cell lysis buffer is diluted with phosphate buffer and centrifuged to obtain a second cell precipitate, the second cell precipitate is resuspended with phosphate buffer and centrifuged, the precipitate after centrifugation is resuspended to obtain a peripheral blood mononuclear cell suspension, and the peripheral blood mononuclear cells in the suspension are added to a peripheral blood mononuclear cell culture medium for incubation and culture to obtain a peripheral blood mononuclear cell sample;

[0038] Taking Sendai virus vector, the Sendai virus vector carries genes octamer binding transcription factor 3 / 4 (OCT3 / 4), sex determining region Y-box 2 (SOX2), myelocytoma oncogene (c-MYC), Krüppel-like factor 4 (KLF-4), transfecting peripheral blood mononuclear cells with the Sendai virus vector to obtain amplified peripheral blood mononuclear cells, separating the Sendai virus vector and the transfected cells, resuspending the transfected cells in a peripheral blood mononuclear cell culture medium for transfection culture, transferring the passage cells formed by the transfection culture to a culture plate coated with matrix gel for further culture, taking a neural progenitor cell differentiation medium, and culturing the passage cells with the neural progenitor cell differentiation medium to form human induced neural progenitor cells;

[0039] Taking a glial progenitor cell induction medium, suspending and culturing the human induced neural precursor cells with the glial progenitor cell induction medium to form glial progenitor cells;

[0040] An astrocyte induction medium is taken, and the glial progenitor cells are adherently cultured using the astrocyte induction medium to obtain astrocytes.

[0041] The peripheral blood mononuclear cell culture medium is formed by a volume ratio of 1:1 of Iskoff's modified Dulbecco's medium (IMDM medium) and Ham's F-12 nutrient mixture (Ham's F-12 medium), a volume ratio of 1% insulin-transferrin-selenium-ethanolamine medium supplement (ITS-X), a volume ratio of 1% chemically defined lipid concentrate, a mass volume ratio of 1% L-glutamine, 30-80 μg / ml L-ascorbic acid, 5 mg / ml bovine serum albumin, 200 μM thioglycerol, 100 ng / ml recombinant human stem cell factor, 100 μg / ml holo-transferrin, 40 ng / ml insulin-like growth factor 1, 10 ng / ml interleukin 3, 2 U / ml erythropoietin, and 1 μM dexamethasone.

[0042] The transfected cells are resuspended in a peripheral blood mononuclear cell culture medium for transfection culture, including: replacing the peripheral blood mononuclear cell culture medium with a new one on the first day after transfection to remove the viral vector, replacing the peripheral blood mononuclear cell culture medium with a new one on the second day after transfection to obtain passage cells, transferring the passage cells to a culture plate coated with matrix gel for further culture on the third day after transfection, and replacing the neural precursor cell differentiation culture medium on the fourth day after transfection to obtain human induced neural precursor cells.

[0043] After replacing the neural progenitor cell differentiation medium for culturing, the neural progenitor cell differentiation medium is replaced with a new one every two days, and the culture is continued until human induced neural progenitor cells are obtained.

[0044] The neural precursor cell differentiation medium is formed by a 1:1 volume ratio of Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and Neurobasal medium, with the addition of 1% by volume N2 supplement, 2% by volume B27 supplement, 1% by volume non-essential amino acids, 1% by volume glutamine supplement, 5-20 ng / ml recombinant human leukemia inhibitory factor, 2-6 μM 6-aminopurine derivatives and 2-4 μM 4-[4-(1,3-benzodiazol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate (SB431542).

[0045] The suspension culture of human induced neural progenitor cells using glial progenitor cell induction medium includes: taking glial progenitor cell induction medium with a density of 5e5 / ml to suspend and culture the human induced neural progenitor cells, replacing new glial progenitor cell induction medium every day until the 14th day, collecting the suspended cell ball adherence culture, culturing again using glial progenitor cell induction medium, replacing new glial progenitor cell induction medium every two days until the crawling glial progenitor cells are collected.

[0046] The glial progenitor cell induction culture is formed by adding 1% N2 additive by volume, 2% B27 additive by volume, 1% non-essential amino acids by volume, 1% glutamine supplement by volume, 40-80ng / ml triiodothyronine, 10-30ng / ml platelet-derived growth factor AA, 5-20ng / ml basic fibroblast growth factor, 80-200nM retinoic acid, and 0.5-2μM purmorphamine to the culture medium with a volume ratio of 1:1.

[0047] Adherent culture of glial progenitor cells using astrocyte induction medium includes using astrocyte induction medium to adhere to culture glial progenitor cells, replacing new astrocyte induction medium every 2 days until cell passage, and continuing to use astrocyte induction medium to adhere to culture after cell passage, replacing new astrocyte induction medium every two days until astrocytes are obtained.

[0048] The astrocyte induction medium is formed by mixing Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and neurobasal medium (Neurobasal medium) in a volume ratio of 1:1, adding 1% N2 additive by volume, 1% non-essential amino acids by volume, 1% glutamine supplement by volume, 10-30ng / ml epidermal growth factor, 10-30ng / ml basic fibroblast growth factor, 150-200ng / ml insulin-like growth factor 1, and 10-20ng / ml ciliary neurotrophic factor.

[0049] Next, combine Figure 1-7 The present application is described exemplarily.

[0050] This embodiment provides a method for inducing and preparing human astrocytes. Figure 1-7As shown, it includes: obtaining peripheral blood mononuclear cells and culturing them: obtaining 15 ml of venous peripheral blood, collecting it into an appropriate blood collection tube containing sodium heparin, adding sterile phosphate buffer to dilute it to 35 ml, adding 15 ml of cell density gradient separation solution (Ficoll-Paque Premium), putting it into a centrifuge, centrifuging it at 750 g for 30 minutes at 25°C, aspirating the yellow upper layer containing plasma and discarding it, transferring the turbid white middle layer containing mononuclear cells (MNC) to a new 50 ml conical tube, adding 30 ml of sterile phosphate buffer to the 50 ml conical tube containing the turbid white middle layer of peripheral blood mononuclear cells (PBMNC) to resuspend it, centrifuging it at 350 g for 10 minutes at 4°C, discarding the supernatant to obtain a first cell pellet, mixing the first cell pellet with 5 ml of red blood cell lysis buffer (red blood cell lysis buffer: 4.145 g of ammonium chloride, 0.5 g of potassium bicarbonate and 100 μl of EDTA were added to 400 ml of ultrapure water, and the pH was adjusted to 7.2-7.4. Ultrapure water was added to make up to 500 ml, and the buffer was filtered and sterilized, and stored at 4°C for later use) mixed and resuspended in a 50 ml tube, incubated at 25°C for 10 min, and then the red blood cell lysis buffer was diluted with sterile phosphate buffer, centrifuged at 300 g for 10 min at 4°C, and the supernatant was discarded to obtain a second cell pellet, and 25 ml of sterile phosphate buffer was added to the second cell pellet to resuspend it, and centrifuged at 300 g for 10 min at 4°C, and the supernatant was discarded. The cell pellet was repeatedly resuspended with 25 ml of sterile phosphate buffer and centrifuged to precipitate the cells, and the precipitated cells were resuspended with 5 ml of sterile phosphate buffer again to obtain a peripheral blood mononuclear cell (PBMNC) suspension.

[0051] Take the peripheral blood mononuclear cell (PBMNC) suspension and dilute it, calculate the number of live cells (based on trypan blue exclusion), and divide the peripheral blood mononuclear cell (PBMNC) suspension according to the experimental needs, and store about 5 million cells per ml of cell cryopreservation solution. Plant 5 million to 10 million mononuclear cells (MNC) in a six-well plate, add 2 ml of peripheral blood mononuclear cell (PBMNC) culture medium [containing 1:1 volume ratio of Iskoff's modified Dulbecco's medium (IMDM medium) and Ham's F-12 nutrient mixture (Ham's F-12 medium), 1% volume ratio of insulin-transferrin-selenium-ethanolamine medium supplement (ITS-X), 1% volume ratio of chemically defined lipid concentrate, 1% mass volume ratio of L-glutamine, 30-80μg / ml L-ascorbic acid, 5 mg / ml bovine serum albumin, 200 μM thioglycerol, 100 ng / ml recombinant human stem cell factor, 100 μg / ml all-iron transferrin, 40 ng / ml insulin-like growth factor 1, 10 ng / ml interleukin 3, 2 U / ml erythropoietin, 1 μM dexamethasone], cultured in a 37°C, 5% CO2 incubator for two days, removed the cells and culture medium with a sterile pipette, and transferred to a sterile 15 ml centrifuge tube, centrifuged at 25°C, 200 g for 5 min, discarded the supernatant, and resuspended the precipitated cells in 1 ml of peripheral blood mononuclear cell (PBMNC) culture medium.

[0052] Plate 500-1000000 PBMNCs per ml of PBMNC medium in six-well plates and incubate for 2 days. Repeat the steps of cell counting and fresh medium replacement, but incubate the cells for another 2 days. Count the cells after 4 days of expansion. Observe that approximately equal or greater number of cells in the initial cell population. Plate PBMNCs at 5×10 5 The cells and peripheral blood mononuclear cell (PBMNC) culture medium were added into the middle part of the 24-well plate.

[0053] Reprogramming: The day of transfection is recorded as day 0. Refer to the instructions of the Sendai virus reprogramming kit (Cyto Tune TM-Ips2.0 sendai reprogramming kit) and add Sendai virus transfection containing octamer binding transcription factor 3 / 4 (OCT3 / 4), sex determination region Y-box 2 (SOX2), KLF4 and myeloma oncogene (c-MYC) to peripheral blood mononuclear cells (PBMNC). The total volume should be between 1 and 1.5 ml. Put the lid tightly on the tube. Centrifuge the cells and virus at 1000g for 30 minutes at room temperature. After centrifugation, add 1 ml of peripheral blood mononuclear cell (PBMNC) culture medium to the test tube, resuspend the cells, and transfer them to 1 well of a 12-well plate (the volume should be between 2 and 2.5 ml) and incubate them overnight at 37°C and 5% CO2.

[0054] Day 1 after transfection: Replace the old culture medium with fresh PBMNC culture medium to remove the Sendai reprogramming vector. Day 2, replace the PBMNC culture medium. Day 3, passage the cells onto a 12-well plate coated with matrigel. On day 4, the cells were replaced with a chemically defined neural progenitor differentiation medium [1:1 volume ratio of Dulbecco's modified Eagle's medium / Ham's nutrient mixture F-12 (DMEM / F12 medium) and Neurobasal medium, with 1% N2 supplement, 2% B27 supplement, 1% non-essential amino acids, 1% glutamine supplement, 5-20 ng / ml recombinant human leukemia inhibitory factor (rhLEIF), 2-6 μM 6-aminopurine derivatives, and 2-4 μM 4-[4-(1,3-benzodiazol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate (SB431542)], and the neural progenitor differentiation medium was replaced every 2 days. About 11 days after transfection, neural progenitor clones appeared under the conditions of neural progenitor differentiation medium.

[0055] After observing the morphological changes of the cultured cells for another 10 days, neural progenitor cell clones were selected on poly-L-ornithine hydrobromide and laminin (PDL / laminin) (see Figure 1 ) and immunostained for neural stem cell (NSC) and pluripotency markers. Neural progenitor cells expressed typical neural stem cell markers SOX2, Nestin, and PAX6 and did not express octamer anchoring protein 4 (OCT4), a pluripotent stem cell marker (see Figure 2 , Figure 3Neural progenitor cells also have a normal karyotype ( Figure 4 ).

[0056] Method for inducing differentiation of human induced neural progenitor cells in vitro to form astrocytes: Inducing differentiation of human induced neural progenitor cells in vitro to form glial progenitor cells: The starting day is recorded as day 0, and the neural progenitor cells are suspended and cultured using glial progenitor cell induction medium (adding Y27632 with a final concentration of 10 μM), with a cell volume of 5e5 / ml, 4ml per well of a 6-well plate, and the well plate is placed on a horizontal shaker, and the horizontal shaker is placed in an incubator at 37°C and 5% CO2. Observe the cell spheres on day 1 ( Figure 5 ), the culture medium was aspirated and discarded, and 4 ml of glial progenitor cell induction medium was added to each well of the 6-well plate.

[0057] The medium was changed every day until the 14th day, and the cell spheres were collected; the cell spheres were seeded at a density of 2 to 3 cells / cm2 in a 6-well plate coated with poly-L-ornithine hydrobromide / laminin (PLO / laminin) using the glial progenitor cell induction medium (2 ml per well of a 6-well plate), and the 6-well plate was placed in an incubator at 37°C and 5% CO2. The glial progenitor cell induction medium was changed every 2 days until the 21st day ( Figure 5 ), 1 ml of TrypLE was added to each well, and then the well plate was placed in an incubator at 37°C and 5% CO2 for 5 minutes to digest the cells; the cell suspension was collected into a 15 ml centrifuge tube, settled at room temperature for 5 minutes, the supernatant was aspirated into a new 15 ml centrifuge tube, and centrifuged at 200 g for 5 minutes, and then the cell pellet was resuspended in astrocyte induction medium (2 ml per well of a 6-well plate, and Y27632 was added at a final concentration of 10 μM), and the cell pellet was seeded at a density of 2e4 / cm2 in a 6-well plate coated with poly-L-ornithine hydrobromide / laminin (PLO / laminin), and the 6-well plate was placed in an incubator at 37°C and 5% CO2.

[0058] [Glial progenitor cell induction medium: Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and Neurobasal medium in a volume ratio of 1:1, with the addition of 1% N2 supplement, 2% B27 supplement, 1% non-essential amino acids, 1% glutamine supplement, 40-80ng / ml triiodothyronine, 10-30ng / ml platelet-derived growth factor AA, 5-20ng / ml basic fibroblast growth factor, 80-200nM retinoic acid, and 0.5-2μM purmorphamine.

[0059] Astrocyte induction medium: Dulbecco's modified Eagle medium / Ham's F-12 nutrient mixture (DMEM / F12 medium) and Neurobasal medium in a volume ratio of 1:1, with 1% N2 supplement, 1% non-essential amino acids, 1% glutamine supplement, 10-30ng / ml epidermal growth factor, 10-30ng / ml basic fibroblast growth factor, 150-200ng / ml insulin-like growth factor 1, and 10-20ng / ml ciliary neurotrophic factor added.]

[0060] Induction of differentiation of glial progenitor cells into astrocytes in vitro: on the 22nd day, the culture medium was aspirated and 2 ml of astrocyte induction medium was added to each well of a 6-well plate; the astrocyte induction medium was replaced every 2 days until the 28th day, 1 ml of TrypLE was added to each well, and then the well plate was placed in an incubator at 37°C, 5% CO2 for 5 min to digest the cells; the cell suspension was collected into a 15 ml centrifuge tube, centrifuged at 200g for 5 min, and then the cell pellet was resuspended in astrocyte induction medium (2 ml per well of a 6-well plate, and Y27632 was added at a final concentration of 10 μM), and the cell pellet was seeded at a density of 2e4 / cm2 in a 6-well plate coated with poly-L-ornithine hydrobromide / laminin (PLO / laminin), and the 6-well plate was placed in an incubator at 37°C, 5% CO2. On the 29th day, the culture medium was discarded and 2 ml of astrocyte induction medium was added to each well of the 6-well plate; the astrocyte induction medium was replaced every 2 days until the 37th day, at which time high-purity astrocytes were obtained. Figure 5 shown.

[0061] The above embodiments are only for illustrating the technical concept and features of the present application, and their purpose is to enable people familiar with the technology to understand the content of the present application and implement it accordingly, and they cannot be used to limit the protection scope of the present application. Any equivalent transformation or modification made according to the spirit of the present application shall be included in the protection scope of the present application.

Claims

1. A method for inducing and preparing human astrocytes, characterized in that: The steps include: Taking venous peripheral blood, diluting the peripheral blood, adding a cell density gradient separation solution to the diluted peripheral blood, centrifuging the mixture of the peripheral blood and the cell density gradient separation solution to achieve a stratification effect, sucking out the yellow upper layer containing plasma in the stratified solution and transferring the white middle layer containing peripheral blood mononuclear cells, resuspending the peripheral blood mononuclear cells in a phosphate buffer and centrifuging to obtain a first cell precipitate, mixing the first cell precipitate with an erythrocyte lysis buffer and incubating, diluting the mixture of the first cell precipitate and the erythrocyte lysis buffer with a phosphate buffer and centrifuging to obtain a second cell precipitate, resuspending the second cell precipitate with a phosphate buffer and centrifuging, resuspending the precipitate after centrifugation to obtain a peripheral blood mononuclear cell suspension, adding a peripheral blood mononuclear cell culture medium to the peripheral blood mononuclear cells in the suspension for incubation and culture to obtain a peripheral blood mononuclear cell sample; Taking a Sendai virus vector, the Sendai virus vector carries a gene octamer binding transcription factor, a sex determining region, a myelocytoma oncogene, and a Krüppel-like factor, transfecting peripheral blood mononuclear cells with the Sendai virus vector to obtain amplified peripheral blood mononuclear cells, separating the Sendai virus vector and the transfected cells, resuspending the transfected cells in a peripheral blood mononuclear cell culture medium for transfection culture, transferring the passage cells formed by the transfection culture to a culture plate coated with matrix gel for further culture after the transfection culture, taking a neural progenitor cell differentiation medium, and culturing the passage cells with the neural progenitor cell differentiation medium to form human induced neural progenitor cells; Taking a glial progenitor cell induction medium, and suspending and culturing the human induced neural precursor cells with the glial progenitor cell induction medium to form glial progenitor cells; An astrocyte induction medium is obtained, and the glial progenitor cells are subjected to adherent culture using the astrocyte induction medium to obtain astrocytes.

2. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: The peripheral blood mononuclear cell culture medium is formed by Iskoff's modified Dulbecco's medium and Ham's F-12 nutrient mixture in a volume ratio of 1:1, 1% insulin-transferrin-selenium-ethanolamine culture medium supplement in a volume ratio, 1% lipid concentrate with a chemically defined composition in a volume ratio, 1% L-glutamine in a mass volume ratio, 30-80 μg / ml L-ascorbic acid, 5 mg / ml bovine serum albumin, 200 μM thioglycerol, 100 ng / ml recombinant human stem cell factor, 100 μg / ml all-iron transferrin, 40 ng / ml insulin-like growth factor 1, 10 ng / ml interleukin 3, 2 U / ml erythropoietin, and 1 μM dexamethasone.

3. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: Resuspending the transfected cells in a peripheral blood mononuclear cell culture medium for transfection culture comprises the following steps: On the first day after transfection, replace the culture medium of peripheral blood mononuclear cells with new one to remove the viral vector. On the second day after transfection, replace the new peripheral blood mononuclear cell culture medium to obtain passage cells. On the third day after transfection, the passaged cells were transferred to the culture plate coated with matrix gel and continued to be cultured. On the fourth day after transfection, the neural progenitor cell differentiation medium was replaced to obtain human induced neural progenitor cells.

4. The method for inducing and preparing human astrocytes according to claim 3, characterized in that: After replacing the neural progenitor cell differentiation medium for culturing, the neural progenitor cell differentiation medium is replaced with a new one every two days, and the culture is continued until human induced neural progenitor cells are obtained.

5. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: The neural precursor cell differentiation medium is formed by adding a volume ratio of 1:1 Dulbecco's modified Eagle medium or F-12 nutrient mixture and neural basal medium, a volume ratio of 1% N2 additive, a volume ratio of 2% B27 additive, a volume ratio of 1% non-essential amino acids, a volume ratio of 1% glutamine supplement, 5-20ng / ml recombinant human leukemia inhibitory factor, 2-6μM 6-aminopurine derivatives and 2-4μM 4-[4-(1,3-benzodiazol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate.

6. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: The method of suspending and culturing the human induced neural precursor cells by using the GPC induction medium comprises: Glial progenitor cell induction medium was used to perform suspension culture on human induced neural precursor cells. The density of the glial progenitor cell induction medium was 5e5 / ml. New glial progenitor cell induction medium was replaced every day until the suspension cell balls were collected for adherent culture on the 14th day. The glial progenitor cell induction medium was used again for culture. New glial progenitor cell induction medium was replaced every two days until the crawled glial progenitor cells were collected.

7. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: The glial progenitor cell induction medium is formed by adding 1% by volume of N2 additive, 2% by volume of B27 additive, 1% by volume of non-essential amino acids, 1% by volume of glutamine supplement, 40-80ng / ml triiodothyronine, 10-30ng / ml platelet-derived growth factor AA, 5-20ng / ml basic fibroblast growth factor, 80-200nM retinoic acid, and 0.5-2μM purmorphamine to Dulbecco's modified Eagle medium or F-12 nutrient mixture and neurobasal medium in a volume ratio of 1:

1.

8. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: The method of subjecting the glial progenitor cells to adherent culture using the astrocyte induction medium comprises: Astrocyte induction medium was used to culture the glial progenitor cells adherently, and new astrocyte induction medium was replaced every 2 days until the cells were passaged. After the cell passage, astrocyte induction medium was continued to be used for adherent culture, and new astrocyte induction medium was replaced every two days until astrocytes were obtained.

9. The method for inducing and preparing human astrocytes according to claim 1, characterized in that: The astrocyte induction medium is formed by adding 1% by volume of N2 additive, 1% by volume of non-essential amino acids, 1% by volume of glutamine supplement, 10-30ng / ml of epidermal growth factor, 10-30ng / ml of basic fibroblast growth factor, 150-200ng / ml of insulin-like growth factor 1, and 10-20ng / ml of ciliary neurotrophic factor to Dulbecco's modified Eagle medium or F-12 nutrient mixture and neural basal medium in a volume ratio of 1:1.

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