In vitro preparation method for medial ganglionic eminence progenitor cells and GABA interneurons

Abstract

Provided are an in vitro preparation method for medial ganglionic eminence (MGE) progenitor cells and GABA interneurons, comprising the following steps: providing human-induced neural precursor cells, inoculating HiNPCs on a culture plate coated with poly-L-ornithine hydrobromide and laminin, and performing differentiation culture using an MGE differentiation culture medium until a cell cluster is formed; transferring the cell cluster and inoculating same into the MGE differentiation culture medium, removing dead cells from a surface of a cell sphere, tilting a culture device, sucking out a part of the culture medium, adding the MGE differentiation culture medium again, and performing culture for 14-15 days to obtain the medial ganglionic eminence progenitor cells; and collecting medial ganglionic eminence progenitor cell spheres and performing the differentiation culture on same again to obtain the GABA interneurons. According to the methods, an induced pluripotent stem cell stage is avoided from the source and the differentiation is confined to the neuroectoderm at the beginning, so that the differentiation direction is more accurate, and the medial ganglionic eminence progenitor cells can be obtained in a relatively short time.

Description

In vitro preparation of medial ganglionic eminence progenitor cells and GABA interneurons Technical Field

[0001] The present invention relates to the field of cell technology, and in particular to an in vitro preparation method of medial ganglionic eminence progenitor cells (MGE) and GABA interneurons. Background Art

[0002] Patent document CN101389290A discloses a method for transplanting neural cells. By implanting MGE cells into the brain, the transplanted cells can restore or enhance the function of inhibitory interneurons in vivo, potentially treating neurological disorders such as epilepsy. The patent also discloses that the MGE-derived cells possess the molecular and firing properties of interneurons.

[0003] Patent document CN109453203A discloses the use of inhibitory neural precursor cells for preparing drugs for treating Alzheimer's disease, and specifically discloses the use of inhibitory neural precursor cells derived from MGE obtained from mice.

[0004] However, the above patent documents do not disclose a method for preparing medial ganglionic eminence progenitor cells (MGE) in vitro.

[0005] Summary of the Invention

[0006] Based on this, it is necessary to provide a new method for forming human induced neural progenitor cells HiNPCs from peripheral blood mononuclear cells, then differentiating human induced neural progenitor cells HiNPCs into MGEs in vitro, and then differentiating MGEs into GABA (γ-aminobutyric acid) interneurons.

[0007] The present invention adopts the following technical solution: The present invention provides a method for preparing medial ganglionic eminence progenitor cells, comprising the following steps: providing human induced neural progenitor cells; inoculating the human induced neural progenitor cells on a culture plate coated with poly-L-ornithine hydrobromide (PLO) and laminin, and performing differentiation culture using MGE differentiation medium, wherein the MGE differentiation medium comprises: DMEM / F12, 1% N2 additive by volume, 1% NEAA by volume, and 1-2 μM purmorphamine (Pur). The differentiation culture process comprises: replacing the MGE differentiation medium with fresh one every other day until cell clusters are formed; transferring the cell clusters and inoculating them in fresh MGE differentiation medium, removing dead cells on the surface of the cell spheres, tilting the culture device, aspirating part of the medium, and adding fresh MGE differentiation medium again, and culturing for 14-15 days to obtain medial ganglionic eminence progenitor cells (MGE).

[0008] In some embodiments, the human induced neural progenitor cells are prepared by in vitro reprogramming of peripheral blood mononuclear cells.

[0009] In some embodiments, the method for obtaining human induced neural progenitor cells includes: extracting mononuclear cells (PBMNCs) from peripheral blood and amplifying and culturing them using PBMNC culture medium; transfecting the amplified PBMNCs with a Sendai virus vector carrying genes OCT3 / 4, SOX2, c-MYC, and KLF-4, separating the Sendai virus vector and the transfected cells, and resuspending the transfected cells in PBMNC culture medium and culturing overnight, with the transfection operation date being day 0; on day 1 after transfection, replacing the PBMNC culture medium with fresh one, and further removing the viral vector; on the second day after transfection, the medium was still replaced with fresh PBMNC medium to obtain passage cells; on the third day after transfection, the passage cells were transferred to a culture plate coated with matrigel and continued to be cultured; on the fourth day after transfection, the NPC differentiation medium was replaced, wherein the NPC differentiation medium consisted of: DMEM / F12 medium and Neurobasal medium in a volume ratio of 1:1, with the addition of 1% N2 additive, 2% B27, 1% NEAA, 1% GlutaMAX, 10 ng / mL rhLIF, 2-6 μM CHIR99021 and 2-4 μM SB431542, the medium was replaced every 2 days, and the culture was continued until human induced neural progenitor cell clones appeared.

[0010] In some embodiments, the method for extracting mononuclear cells (PBMNCs) from peripheral blood includes: obtaining venous peripheral blood, diluting it, adding a cell density gradient separation solution, centrifuging it, aspirating and discarding the yellow upper layer containing plasma, transferring the turbid white middle layer containing mononuclear cells (PBMNCs) to a new conical tube, resuspending it with PBS buffer, and centrifuging it to obtain a first cell pellet; mixing the first cell pellet with red blood cell lysis buffer, incubating it, diluting it with PBS buffer, and centrifuging it to obtain a second cell pellet; resuspending the second cell pellet with PBS buffer, centrifuging it, and further resuspending the pellet to obtain a PBMNC suspension; and adding the monocytes in the PBMNC suspension to a PBMNC culture medium for incubation and culture to obtain a peripheral blood mononuclear cell sample.

[0011] In some embodiments, the composition of the PBMNC culture medium includes: IMDM medium and Ham's F-12 medium in a volume ratio of 1:1, 1% ITS-X by volume, 1% chemically defined lipid concentrate by volume, 1% L-glutamine by mass volume, 30-80 μg / mL L-ascorbic acid, 5 mg / mL BSA, 200 μM monothioglycerol, 100 ng / mL recombinant human stem cell factor, 100 μg / mL holo-transferrin, 40 ng / mL IGF-1, 10 ng / mL IL-3, 2 U / mL EPO, and 1 μM dexamethasone.

[0012] The present invention also provides an in vitro differentiation method for medial ganglionic eminence progenitor cells, wherein the reprogrammed human induced neural precursor cells are placed in MGE differentiation medium for in vitro differentiation culture.

[0013] The present invention also provides an in vitro preparation method for GABA interneurons, comprising the following steps: preparing medial ganglionic eminence progenitor cells according to the in vitro preparation method for medial ganglionic eminence progenitor cells described above; collecting medial ganglionic eminence progenitor cell spheres, aspirating the supernatant, adding TrypLE to resuspend the medial ganglionic eminence progenitor cell spheres, incubating at 37°C until the spheres become loose, washing the medial ganglionic eminence progenitor cell spheres, aspirating the supernatant, adding GABA neuron differentiation medium, and transferring to a culture plate coated with poly-L-ornithine hydrobromide and laminin for culture for more than 15 days.

[0014] In some embodiments, the composition of the GABA neuron differentiation medium includes: 1-1.5 μM cAMP, 8-15 ng / mL IGF1, 10-20 ng / mL GDNF, and 8-12 ng / mL BDNF added to a medium containing Neurobasal medium, 1% N2 supplement by volume, and 1% NEAA by volume.

[0015] Compared to existing technologies, the present invention can generate safer HiNPCs from peripheral blood mononuclear cells, which can then differentiate in vitro into MGEs, which can then differentiate into GABAergic interneurons in vivo and in vitro. This method bypasses the induced pluripotent stem cell (iPS) stage and is initially restricted to the neuroectoderm, resulting in more precise differentiation and the ability to obtain medial ganglionic eminence (MGE) progenitors in a relatively short period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows changes in cell morphology during the reprogramming of healthy peripheral blood mononuclear cells (PBMNCs) into human induced neural progenitor cells (HiNPCs) in Example 1, with a scale bar of 100 μm; Figure A is a schematic diagram of the HiNPC reprogramming process, with bright field photographs showing changes in cell morphology during the reprogramming process; Figure B is an immunofluorescence labeling image of HiNPC cell clusters, in which neural stem cell markers are human paired nuclear gene 6 (PAX6), nestin (Nestin), and sex determining factor 2 (SOX2), and a pluripotent stem cell marker is octamer anchoring protein 4 (OCT4);

[0017] FIG2 is a graph showing the percentage of positive cells by fluorescence quantification of human induced neural progenitor cells (HiNPCs) obtained by reprogramming in Example 1;

[0018] FIG3 is a diagram showing the results of chromosome karyotype analysis of human induced neural progenitor cells (HiNPCs) obtained by reprogramming in Example 1;

[0019] FIG4 is a diagram showing changes in cell morphology during the differentiation of human neural progenitor cells into MGE and GABA in Examples 2 and 3;

[0020] Figure 5 shows the immunostaining results of cells at different culture days during the differentiation of human induced neural progenitor cells into MGE and GABA in Examples 2 and 3; wherein, Figure A shows the immunostaining results of NKX2.1 and FOXG1 on day 16 of differentiation, Figure B shows the immunostaining results of markers NKX2.1 and GABA on day 27 of differentiation, and Figure C shows the immunostaining results of Tuj 1 and synapsin-1 on day 37 of differentiation. Scale bar: 100 μm.

[0021] FIG6 is a diagram showing the quantitative results of NKX2.1 and FOXG1 during the 16th day of differentiation of human neural progenitor cells induced to differentiate into MGE and GABA in Examples 2 and 3;

[0022] FIG7 is a diagram showing the quantitative results of NKX2.1 and GABA on day 27 of differentiation during the process of inducing human neural progenitor cells to differentiate into MGE and GABA in Examples 2 and 3;

[0023] FIG8 is a diagram showing the quantitative results of Tuj 1 and synapsin-1 on day 37 of differentiation during the process of inducing human neural progenitor cells to differentiate into MGE and GABA in Examples 2 and 3. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0025] The following embodiments are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0026] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0027] In this application, the collected blood samples were obtained after signing the Medical Biological Sample Bank Sample Collection Consent Form.

[0028] Source or composition of raw materials such as reagents, antibodies, and culture media:

[0029] Blood collection tube containing sodium heparin: BD Vacutainer, cat.no.367871.

[0030] Cell density gradient separation medium: Ficoll-Paque Premium, P=1.077 (GE Healthcare, cat. no. 17-5442-02).

[0031] Erythrocyte Lysis Buffer: Add 4.145 g ammonium chloride, 0.5 g potassium bicarbonate, and 100 μl EDTA to 400 mL ultrapure water, adjust the pH to 7.2-7.4, and make up to 500 mL with ultrapure water. Filter-sterilize the buffer and store at 4°C until used.

[0032] Iscove's modified Dulbecco's medium (IMDM): purchased from Invitrogen, cat. no. 21056023.

[0033] Chemically defined lipid concentrate: Invitrogen, cat. no. 11905031.

[0034] Example 1

[0035] This embodiment provides a method for reprogramming peripheral blood mononuclear cells (PBMNCs) to form neural stem cells (HiNPCs), comprising the following steps:

[0036] S1, obtain peripheral blood mononuclear cells and culture them:

[0037] Obtain 15 mL of venous peripheral blood and collect it into an appropriate blood collection tube containing sodium heparin. Dilute to 35 mL with sterile PBS buffer, add 15 mL of cell density gradient separation medium (Ficoll-Paque Premium), and centrifuge at 750 g for 30 minutes at 25°C. Aspirate and discard the yellow upper layer containing plasma, and transfer the turbid white middle layer containing mononuclear cells (MNCs) to a new 50 mL conical tube.

[0038] Add 30 mL of sterile PBS buffer to the 50 mL conical tube containing the turbid white middle layer of mononuclear cells (PBMNC) and resuspend. Centrifuge at 350 g for 10 min at 4°C and discard the supernatant to obtain the first cell pellet.

[0039] The first cell pellet was mixed with 5 mL of red blood cell lysis buffer and resuspended in a 50 mL tube. The mixture was incubated at 25°C for 10 min. The red blood cell lysis buffer was then diluted with sterile PBS buffer. The mixture was centrifuged at 300 g for 10 min at 4°C. The supernatant was discarded to obtain the second cell pellet.

[0040] The second cell pellet was resuspended in 25 mL of sterile PBS buffer and centrifuged at 300 g for 10 min at 4°C. The supernatant was discarded and the cell pellet was resuspended in 25 mL of sterile PBS buffer and centrifuged to pellet the cells. The pellet was resuspended in 5 mL of sterile PBS buffer to obtain a PBMNC suspension.

[0041] Dilute the PBMNC suspension and count the number of viable cells (based on trypan blue exclusion). Aliquot the PBMNC suspension according to experimental needs, with approximately 5 million cells per ml of cryopreservative.

[0042] 5 to 10 million mononuclear cells (MNCs) were seeded in a six-well plate and 2 mL of PBMNC medium (containing IMDM medium and Ham's F-12 medium in a volume ratio of 1:1, 1% v / v insulin-transferrin-selenium supplement (ITS-X), 1% v / v chemically defined lipid concentrate, 1% m / v L-glutamine, 30-80 μg / mL L-ascorbic acid, 5 mg / mL bovine serum albumin (BSA), 200 μM 1-Thioglycerol, 100 ng / mL recombinant human stem cell factor, 100 μg / mL holo-transferrin, 40 ng / mL recombinant human insulin-like growth factor 1 (IGF-1), 10 ng / mL recombinant interleukin-3 (IL-3), 2 U / mL recombinant human erythropoietin (EPO), 1 μM dexamethasone], cultured in a 37°C, 5% CO2 incubator for two days, the cells and culture medium were removed with a sterile pipette and transferred to a sterile 15 mL centrifuge tube, centrifuged at 25°C, 200 g for 5 min, the supernatant discarded, and the precipitated cells resuspended in 1 mL of PBMNC culture medium.

[0043] Plate 0.5 to 1 million PBMNCs per ml of PBMNC culture medium in a six-well plate and incubate for 2 days. Repeat the cell counting and fresh medium replacement steps, but incubate the cells for an additional 2 days. Count the cells after 4 days of expansion. Observe that the initial cell population has approximately the same number of cells or more. Peripheral blood mononuclear cells (PBMNCs) are plated at a density of 5 × 10 5 Add cells and PBMNC culture medium into the middle part of a 24-well plate.

[0044] S2, reprogramming:

[0045] The day of transfection is designated as day 0. Refer to the instructions for the Sendai virus reprogramming kit (Cyto Tune™-Ips 2.0 Sendai Reprogramming Kit) and add Sendai virus containing OCT3 / 4, SOX2, KLF4, and c-MYC to the PBMNCs in a total volume of 1–1.5 mL. Securely place the cap on the tube. Centrifuge the cells and virus at 1000 g for 30 minutes at room temperature. After centrifugation, add 1 mL of PBMNC culture medium to the tube, resuspend the cells, and transfer them to one well of a 12-well plate (the volume should be between 2 and 2.5 mL). Incubate overnight at 37°C in a 5% CO2 incubator.

[0046] On day 1 after transfection, replace the old medium with fresh PBMNC medium to remove the Sendai reprogramming vector. On day 2, change the PBMNC medium. On day 3, passage the cells onto matrigel-coated 12-well plates. On day 4, switch to chemically defined NPC differentiation medium [1:1 volume ratio of DMEM / F12 medium and Neurobasal medium, supplemented with 1% N2 supplement (volume ratio), 2% B27, 1% NEAA (volume ratio), 1% GlutaMAX supplement (volume ratio), 10 ng / mL recombinant human leukemia inhibitory factor (rhLIF), 2-6 μM CHIR99021, and 2-4 μM SB431542]. Change the NPC differentiation medium every 2 days. Approximately 11 days after transfection, HiNPC colonies should emerge in NPC differentiation medium.

[0047] After observing the morphological changes of a small number of cultured cells for another 10 days, HiNPC clones were selected on poly-L-ornithine hydrobromide and laminin (PDL / laminin) (see Figure 1, Panel A) and immunostained for neural stem cell (NSC) and pluripotency markers.

[0048] HiNPCs expressed typical neural stem cell markers sex determining factor 2 (SOX2), nestin, and human paired nuclear gene 6 (PAX6) but did not express the pluripotent stem cell marker octamer anchoring protein 4 (OCT4) (see Figure 1B and Figure 2).

[0049] HiNPCs also had a normal karyotype ( FIG3 ).

[0050] Example 2

[0051] The present embodiment provides a method for differentiating the HiNPCs obtained in Example 1 into medial ganglionic eminence progenitor cells (MGE), comprising the following steps:

[0052] Neural stem cells (HiNPCs) were plated onto six-well plates coated with poly-L-ornithine hydrobromide / laminin (PLO / laminin) and maintained in MGE differentiation medium (DMEM / F12, 1% N2 supplement by volume, 1% NEAA by volume, 1-2 μM Pur), with the medium changed every other day. When the culture dish was confluent, the cell clusters were gently transferred to a 15 mL centrifuge tube and centrifuged at 50 g for 1 minute at room temperature. Alternatively, the cell clusters were allowed to rest for 2-5 minutes without centrifugation and then plated in fresh medium.

[0053] Gently pipette the MGE spheroids two or three times with a 10 mL pipette to remove dead cells from the surface of the spheroids. Tilt the culture dish at 45° and allow the spheroids to sink to one corner. Gently aspirate two-thirds of the culture medium without disturbing the spheroids. Refill with the same fresh culture medium and culture for 15 days to obtain MGEs.

[0054] HiNPCs were tested and differentiated into MGE progenitor cells in neural differentiation medium containing NEAA, N2 supplement, and Pur as a SHH agonist ( FIG4 ).

[0055] Example 3

[0056] This example provides a method for differentiating the MGE progenitor cells obtained in Example 2 into GABA interneurons, comprising the following steps:

[0057] Collect the prepared MGE progenitor cell spheroids into a 15 mL tube. Spin the tube at 50 g for 1 minute at room temperature. Aspirate the supernatant, add 1 mL of TrypLE to resuspend the MGE progenitor cell spheroids, and then incubate the mixture at 37°C for 2-4 minutes until the MGE progenitor cell spheroids become loose. Spin the tube at 50 g for 1 minute at room temperature. Aspirate the supernatant, then wash the spheroids. Aspirate the supernatant, add GABA neuron differentiation medium (Neurobasal medium, 1% N2 supplement by volume, 1% NEAA by volume, supplemented with 1-1.5 μM cAMP, 8-15 ng / mL IGF1, 10-20 ng / mL glial cell line-derived neurotrophic factor (GDNF), and 8-12 ng / mL brain-derived neurotrophic factor (BDNF), and culture on poly-L-ornithine hydrobromide / laminin (PLO / laminin)-coated 6-well plates.

[0058] Over the next 20 days, MGE progenitor cells gradually grew neurites and took on the shape of neurons (Figure 4); in particular, approximately 80% of the cells co-expressed the interneuron transcription factor NKX2.1 and the forkhead box gene G1 (see Figure 5A and Figure 6); approximately 70% of the cells co-expressed the interneuron transcription factor NKX2.1 and the γ-aminobutyric acid neurotransmitter marker (see Figure 5B and Figure 7).

[0059] On day 37, mature GABAergic neurons (β-tubulin Tuj1 positive; 95±10%, n=3) were accompanied by a large number of synapses (synapsin-1 positive; 94±7%, n=3) (see Figure 5C and Figure 8).

[0060] In addition, the inventor team found in a large number of exploratory experiments:

[0061] (1) During the reprogramming of PBMNCs into HiNPCs, a 1:1 volume ratio of DMEM / F12 medium and Neurobasal medium was used, with the addition of 1% volume N2 additive, 2% volume B27, 1% volume NEAA, 1% volume GlutaMAX, 10 ng / mL rhLIF, 2-6 μM CHIR99021, and 2-4 μM SB431542. The overall yield of HiNPCs was higher than that of other concentrations.

[0062] (2) During the differentiation of HiNPC into MGE, the differentiation efficiency was higher when using DMEM / F12, 1×N2 supplement, 1×NEAA, and 1-2 μM purmorphamine than other concentrations. The specific experimental comparison results are statistically shown in the following table:

[0063] As can be seen from the above table, during the differentiation of HiNPC into MGE, the highest differentiation efficiency is achieved at a concentration of 1-2 μM purmorphamine, which is at least 10% higher than that of other different culture media.

[0064] (3) During the in vitro differentiation of MGE into GABA neurons, the highest proportion of in vitro differentiation into GABA neurons was achieved when 1-1.5 μM cAMP, 8-15 ng / mL IGF1, 10-20 ng / mL GDNF, and 8-12 ng / mL BDNF were added to the Neurobasal medium, 1% N2 additive by volume, and 1% NEAA by volume culture medium.

[0065] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing medial ganglionic eminence progenitor cells, characterized in that: The steps include: Providing human induced neural progenitor cells; The human induced neural progenitor cells are inoculated on a culture plate coated with poly-L-ornithine hydrobromide and laminin, and are differentiated and cultured using an MGE differentiation medium, wherein the composition of the MGE differentiation medium includes: DMEM / F12, 1% N2 additive by volume, 1% NEAA by volume, and 1-2 μM purmorphamine. The differentiation and culture process includes: replacing the fresh MGE differentiation medium every other day until cell clusters are formed; transferring and inoculating the cell clusters in the fresh MGE differentiation medium, removing dead cells on the surface of the cell ball, tilting the culture device, sucking out part of the culture medium, adding fresh MGE differentiation medium again, and culturing for 14-15 days to obtain medial ganglionic eminence progenitor cells MGE.

2. The method for preparing medial ganglionic eminence progenitor cells according to claim 1, characterized in that: The human induced neural precursor cells are prepared by in vitro reprogramming of peripheral blood mononuclear cells.

3. The method for preparing medial ganglionic eminence progenitor cells according to claim 1, characterized in that: The method for obtaining human induced neural precursor cells comprises: Mononuclear cells (PBMNC) were extracted from peripheral blood and expanded and cultured using PBMNC medium; The amplified PBMNCs were transfected with Sendai virus vectors carrying genes OCT3 / 4, SOX2, c-MYC, and KLF-4, the Sendai virus vectors and transfected cells were separated, and the transfected cells were resuspended in PBMNC culture medium and cultured overnight. The transfection operation date was designated as day 0; On day 1 after transfection, the medium was replaced with fresh PBMNC culture medium to further remove the viral vector; On the second day after transfection, fresh PBMNC culture medium was used to replace the medium and culture to obtain passage cells; On the third day after transfection, the passaged cells were transferred to culture plates coated with Matrigel and continued to be cultured; On the 4th day after transfection, the NPC differentiation medium was replaced with a DMEM / F12 medium and a Neurobasal medium in a volume ratio of 1:1, and 1% of N2 supplement, 2% B27 by volume, 1% NEAA by volume, 1% GlutaMAX by volume, 10 ng / mL rhLIF, 2-6 μM CHIR99021 and 2-4 μM SB431542, the culture medium was replaced every 2 days, and the culture was continued until human induced neural progenitor cell clones appeared.

4. The method for preparing medial ganglionic eminence progenitor cells according to claim 1, characterized in that: The method for extracting mononuclear cells (PBMNC) from peripheral blood comprises: Obtain venous peripheral blood, dilute, add cell density gradient separation solution, centrifuge, aspirate and discard the yellow upper layer containing plasma, transfer the turbid white middle layer containing mononuclear cells PBMNC to a new conical tube, resuspend with PBS buffer, and centrifuge to obtain a first cell pellet; mix the first cell pellet with red blood cell lysis buffer, incubate, dilute with PBS buffer, and centrifuge to obtain a second cell pellet; resuspend the second cell pellet with PBS buffer, centrifuge, and continue to resuspend the pellet to obtain a PBMNC suspension; The mononuclear cells in the PBMNC suspension are added to the PBMNC culture medium for incubation to obtain a peripheral blood mononuclear cell sample.

5. The in vitro preparation method of medial ganglionic eminence progenitor cells according to claim 4, characterized in that: The composition of the PBMNC culture medium includes: IMDM culture medium and Ham's F-12 culture medium in a volume ratio of 1:1, ITS-X in a volume ratio of 1%, a lipid concentrate with a clear chemical composition in a volume ratio of 1%, L-glutamine in a mass volume ratio of 1%, 30-80 μg / mL L-ascorbic acid, 5 mg / mL BSA, 200 μM thioglycerol, 100 ng / mL recombinant human stem cell factor, 100 μg / mL holo-transferrin, 40 ng / mL IGF-1, 10 ng / mL IL-3, 2 U / mL EPO, and 1 μM dexamethasone.

6. An in vitro differentiation method for medial ganglionic eminence progenitor cells, characterized in that: The reprogrammed human induced neural progenitor cells were placed in MGE differentiation medium for in vitro differentiation culture.

7. A method for preparing GABA interneurons in vitro, characterized in that: The steps include: The medial ganglionic eminence progenitor cells are prepared according to the in vitro preparation method of the medial ganglionic eminence progenitor cells according to any one of claims 1 to 5; The medial ganglionic eminence progenitor cell spheres were collected, the supernatant was aspirated, TrypLE was added to resuspend the medial ganglionic eminence progenitor cell spheres, and the spheres were incubated at 37°C until the spheres became loose, the medial ganglionic eminence progenitor cell spheres were washed, the supernatant was aspirated, GABA neuron differentiation medium was added, and the spheres were transferred to poly-L-ornithine hydrobromide and laminin-coated culture plates for culture for more than 15 days.

8. The in vitro preparation method of GABA interneurons according to claim 7, characterized in that: The composition of the GABA neuron differentiation medium includes: Neurobasal medium, 1% N2 additive by volume, 1% NEAA by volume, 1-1.5 μM cAMP, 8-15 ng / mL IGF1, 10-20 ng / mL GDNF and 8-12 ng / mL BDNF added to the medium.

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