CN114958747A - Method for inducing pluripotent stem cells to generate excitatory and inhibitory neurons - Google Patents

Method for inducing pluripotent stem cells to generate excitatory and inhibitory neurons Download PDF

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CN114958747A
CN114958747A CN202210640116.6A CN202210640116A CN114958747A CN 114958747 A CN114958747 A CN 114958747A CN 202210640116 A CN202210640116 A CN 202210640116A CN 114958747 A CN114958747 A CN 114958747A
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刘长梅
刘佩佩
滕兆乾
徐雅洁
杜洪震
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Abstract

The invention discloses a method for generating excitatory and inhibitory neurons by inducing pluripotent stem cells. The invention discloses a method for inducing pluripotent stem cells to generate excitatory neurons, which comprises the steps of sequentially culturing human pluripotent stem cells in a culture medium II, an N2 culture medium, an N2+ lamin culture medium, an N2B27 culture medium and a neuron culture medium to obtain excitatory neurons; the method for inducing the pluripotent stem cells to generate the inhibitory neurons comprises the step of sequentially culturing the human embryonic stem cells in an hES culture medium, an NIM + pur culture medium, a VPA culture medium and an NDM culture medium to obtain the inhibitory neurons. The method is simple, can effectively reduce the induction time, can directionally differentiate into neurons in a single direction, has definite directivity, and has important significance for researching the development of the human cerebral cortex.

Description

Method for inducing pluripotent stem cells to generate excitatory and inhibitory neurons
Technical Field
The invention relates to a method for inducing and generating excitatory and inhibitory neurons by pluripotent stem cells in the field of stem cell biology.
Background
The study of the human cerebral cortex in terms of health and disease is limited by the model system. Establishing a differentiation method from human embryonic stem cells and induced pluripotent stem cells (collectively referred to as pluripotent stem cells, PSCs) has great potential for studying cortical development and constructing an in vitro model of cortical development diseases. Based on the knowledge of rodent cortical development, a robust, multi-step induction process was developed to study the cortical development of pluripotent stem cells, to direct induction of human embryonic stem cells, or to induce differentiation of pluripotent stem cells.
The cerebral cortex contains two types of neurons, 80% of which are excitatory neurons and glutamatergic neurons, which are produced by cortical stem/progenitor cells; inhibitory neurons (GABA) account for approximately 20%, being produced and migrated in cortical development. Once generated, different levels of cortical projection neurons form local microcircuits between the cortices, as well as longer intra-and extra-cortical connections, including corticospinal tracts, corticotalamic and corpus callosum projections.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare excitatory neurons and inhibitory neurons.
The invention provides, in a first aspect, a method of preparing an excitatory neuron, the method comprising:
1) culturing the human pluripotent stem cells in a culture medium II to obtain tissues cultured by the culture medium II;
the culture medium II is obtained by adding Y-27632 to a human pluripotent stem cell culture medium; wherein the content of Y-27632 in the culture medium II is 10 mu M;
2) culturing the tissue cultured by the culture medium II in an N2 culture medium to obtain a tissue cultured by an N2 culture medium;
the N2 culture medium is obtained by adding N2supplement (100X) and 100X NEAA in DMEM-F12 culture medium, the volume percentage content of N2supplement (100X) in the N2 culture medium is 1 percent, and the volume percentage content of 100X NEAA in the N2 culture medium is 1 percent;
3) culturing the tissue cultured by the N2 culture medium in an N2+ lamin culture medium to obtain a tissue cultured by an N2+ lamin culture medium;
the N2+ lamin culture medium is obtained by adding lamin to the N2 culture medium, and the concentration of lamin in the N2+ lamin culture medium is 1 mug/ml;
4) treating the tissue cultured by the N2+ lamin culture medium by using collagenase IV to obtain the tissue treated by the collagenase IV; culturing the tissue treated by the collagenase IV by using an N2B27 culture medium to obtain a tissue cultured by an N2B27 culture medium;
the N2B27 culture medium is obtained by adding N2supplement (100X), B27supplement (50X), RA and human fibroblast growth factor-2 into DMEM-F12 culture medium, wherein the volume percentage content of N2supplement (100X) in the N2B27 culture medium is 1%, the volume content of B27supplement (50X) in the N2B27 culture medium is 1/50, the content of RA in the N2B27 culture medium is 0.2 mu M, and the content of human fibroblast growth factor-2 in the N2B27 culture medium is 20 ng/ml;
5) digesting the tissue cultured by the N2B27 culture medium to obtain digested cells; culturing the digested cells in a neuron culture medium to obtain excitatory neurons;
the neuron culture medium is obtained by adding N2supplement (100X), B27supplement (50X), BDNF, GDNF, cAMP and ascorbic acid into a DMEM-F12 culture medium, wherein the volume percentage content of N2supplement (100X) in the neuron culture medium is 1%, the volume content of B27supplement (50X) in the neuron culture medium is 1/50, the content of BDNF in the neuron culture medium is 10ng/ml, the content of GDNF in the neuron culture medium is 10ng/ml, the content of cAMP in the neuron culture medium is 1 mu M, and the content of ascorbic acid in the neuron culture medium is 200 nM.
Wherein the excitatory neurons may be MAP2 and vgut 1 positive excitatory neurons.
The method of step 1) may further comprise a step of digesting the pluripotent stem cells before culturing the pluripotent stem cells.
The culturing in step 3) may be performed in a matrigel-coated culture plate.
The culturing in step 5) may be performed in ornithine and lamin coated plates.
The human pluripotent stem cell may be an induced pluripotent stem cell (hiPSC), an adult (adult) stem cell, a somatic (somatic) stem cell, a cancer stem cell, or any other pluripotent stem cell having differentiation capacity.
The culture in steps 1) -5) of the method can be carried out at 37 ℃ and 5% CO 2 Under the conditions of (1).
The time for culturing in step 1) of the above method may be 3 days.
The time for the culture in step 2) may be 4 days.
The time for the culture in step 3) may be 7 days.
The time for the culture in step 4) may be 6 days.
The time for the culture in step 5) may be 19 to 20 days.
The present invention also provides a method of making an inhibitory neuron, the method comprising:
1) culturing human embryonic stem cells in an hES culture medium to obtain a tissue cultured by the hES culture medium;
the hES culture medium is obtained by adding 100X NEAA, a serum substitute and beta-mercaptoethanol into a DMEM-F12 culture medium, wherein the volume percentage content of the 100X NEAA in the hES culture medium is 1%, the volume percentage concentration of the serum substitute in the hES culture medium is 20%, and the concentration of the beta-mercaptoethanol in the hES culture medium is 0.1 mM;
2) culturing the tissue cultured by the hES culture medium in an NIM culture medium to obtain a tissue 1 cultured by the NIM culture medium;
the NIM medium is obtained by adding N2supplement (100X), 100X NEAA and heparin into DMEM-F12 medium, wherein the volume percentage of N2supplement (100X) in the NIM medium is 1%, the volume percentage of 100X NEAA in the NIM medium is 1%, and the content of heparin in the NIM medium is 2 mu g/ml;
3) culturing the tissue 1 cultured by the NIM culture medium in a culture plate coated by matrigel by using the NIM culture medium to obtain a tissue 2 cultured by the NIM culture medium;
4) treating the tissue 2 cultured by the NIM culture medium obtained in the step 3) by using collagenase IV to obtain a tissue treated by collagenase IV; culturing the tissue treated by the collagenase IV by using an NIM + pur culture medium to obtain a tissue cultured by the NIM + pur culture medium;
the NIM + pur culture medium is obtained by adding Purmorphamine into the NIM culture medium, and the content of the Purmorphamine in the NIM + pur culture medium is 0.65 mu M;
5) digesting the tissue cultured by the NIM + pur culture medium to obtain digested cells; culturing the digested cells in a VPA culture medium to obtain cells cultured in the VPA culture medium;
the VPA culture medium is obtained by adding 100XGlutamax and valproic acid into a Neurobasal culture medium, wherein the volume percentage of 100XGlutamax in the VPA culture medium is 1%, and the content of valproic acid in the VPA culture medium is 10 mu M;
6) culturing the cells cultured by the VPA culture medium in an NDM culture medium to obtain inhibitory neurons;
the NDM culture medium is obtained by adding N2supplement (100X), B27supplement (50X), 100X Glutamax, BDNF, GDNF, cAMP and IGF1 into a Neurobasal culture medium, wherein the volume percentage content of N2supplement (100X) in the NDM culture medium is 1%, the volume percentage content of B27supplement (50X) in the NDM culture medium is 1/50, the volume percentage content of 100X Glutamax in the NDM culture medium is 1%, the volume percentage content of BDNF in the NDM culture medium is 10ng/ml, the volume percentage content of GDNF in the NDM culture medium is 10ng/ml, the volume percentage content of cAMP in the NDM culture medium is 1 muM, and the volume percentage content of IGF1 in the NDM culture medium is 10 ng/ml.
The method of step 1) may further comprise a step of digesting the pluripotent stem cells before culturing the pluripotent stem cells.
The culturing in step 5) may be performed in ornithine and lamin coated plates.
The culture in steps 1) -6) of the method can be carried out at 37 ℃ and 5% CO 2 Under the conditions of (1).
The time for culturing in step 1) of the above method may be 4 days.
The time for the culture in step 2) may be 3 days.
The time for the culture in step 3) may be 5 days.
The time for the culture in step 4) may be 14 days.
The time for the culture in step 5) may be 6 days.
The time for the culture in step 6) may be 15 days.
The inhibitory neuron may be a GABA inhibitory neuron.
The inhibitory neurons can be specifically NKX2.1 and FOXG1 positive inhibitory neurons.
The invention also provides a kit, which comprises the culture medium II, the N2 culture medium, the N2+ lamin culture medium, the N2B27 culture medium and the neuron culture medium, or comprises the hES culture medium, the NIM + pur culture medium, the VPA culture medium and the NDM culture medium.
The application of the kit in preparing excitatory neurons or inhibitory neurons or in preparing products inducing excitatory neurons or inhibitory neurons also belongs to the protection scope of the invention.
In the above, the culture medium for human pluripotent stem cells may be product # CA1007500 of Beijing seebeck Biotechnology Ltd.
Y-27632 may be seleck product, cat No. s 1049.
The DMEM-F12 medium can be Gibco-BRL product, cat.no. 11330.
lamin may be Invitrogen, cat.no. 23017-015.
Collagenase IV may be the product of GIBIO, cat.no. 17104019.
B27supplement (50X) can be Gibco-BRL product, cat.no. 17504044.
RA may be Sigma, cat.no. r 2625.
KOSR may be Gibco-BRL product, cat.no. 10828.
Purmorphamine may be CALBIOCHEM product, cat No. 540220.
The Neurobasal medium can be Gibco-BRL product, cat.no. 21103049.
N2supplement (100X) can be a Gibco-BRL product with a catalog number of 17502-.
100XNEAA, 100XMEM non-invasive amino acids solution (NEAA), product of Gibco-BRL, cat # 11140.
100X Glutamax is available from Gibco-BRL under the designation 35050.
The invention provides a method for generating excitatory neurons and inhibitory neurons, which is simple, can effectively reduce induction time, can directionally differentiate into neurons in a single direction, and has definite directivity. Contributes to the research of human cerebral cortex development.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
Drawings
FIG. 1 is a flow chart of the process of inducing the generation of mature excitatory neurons in human induced pluripotent stem cells.
FIG. 2 is a photograph of immunofluorescence staining at the stage of differentiation into neural stem cells. Among them, DAPI (blue) stained nuclei (first left panel), Nestin (green) stained neural stem cells (second left panel), and BrdU (red) stained proliferating cells (third left panel).
FIG. 3 is a graph of immunofluorescence staining during the differentiation to excitatory neuronal stage. Among them DAPI (blue) stained nuclei (first left panel), MAP2 (green) stained neurons (second left panel), vgut 1 (red) stained excitatory neurons (third left panel). The right-most panel shows statistics of the differentiation efficiency of excitatory neurons.
FIG. 4 is a flow chart of the induction of maturation inhibitory neurons by human induced pluripotent stem cells.
FIG. 5 is an immunofluorescence staining pattern of forebrain GABA interneuron precursor cells. Among these, DAPI (blue) stained nuclei (first left panel), FOXG1 (green) stained forebrain (second left panel), NKX2.1 (red) stained the protuberance of the Medial Ganglion (MGE) (third left panel).
FIG. 6 is a graph of immunofluorescence staining at the stage of differentiation into inhibitory neurons. Among them DAPI (blue) stained nuclei (first left panel), MAP2 (green) stained neurons (second left panel), GABA (red) stained inhibitory neurons (third left panel). The right-most panel is the statistics of inhibitory neuron differentiation efficiency.
Detailed Description
The experiments in the following examples, each set up three replicates.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products.
In the following examples, the humidity of the humidified incubator was 95%.
Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified.
1. Reagents used in the following examples:
(1) human pluripotent stem cell medium (PGM1 medium) (beijing seebeck biotechnology limited, # CA 1007500).
(2)1 XPhosphate buffer solution (PBS) (144 mg/L potassium dihydrogen phosphate, 9000mg/L sodium chloride, 795mg/L disodium hydrogen phosphate, pH7.4) (Invitrogen, # 10010023).
(3) ACCUTASE cell digest (BD, # 561527).
(4) Ascorbic Acid (Ascorbic Acid) (Sigma, cat. No. a 4403).
(5)B27 supplement(50X)(Gibco-BRL,cat.no.17504044)。
(6) Brain derived growth factor (BDNF) (PeproTech, cat. No. 450-02).
(7) Beta-mercaptoethanol (2-ME,14.3M, Sigma, cat. No. M7522).
(8) Cyclic adenosine monophosphate (cAMP) (Sigma, cat. No. d-0260).
(9) Dulbecco's modified eagle Medium/Nutrient mixture F-12(DMEM-F12) (1:1) (Gibco-BRL, cat. No. 11330).
(10) Human fibroblast growth factor-2 (FGF2) (PeproTech, cat. No.100-18 b).
(11) Glial cell line-derived neurotrophic factor (GDNF) (PeproTech, cat. No. 450-10).
(12) Heparin (Sigma, cat. No. h 3149).
(13) Insulin-like growth factor (IGF1) (PeproTech, cat. No. 100-11).
(14) Serum Replacement (KOSR) (Gibco-BRL, cat.no. 10828).
(15)100 XGlutamax (200mM L-alanyl-L-glutamine dipeptide, 0.85% NaCl) (Gibco-BRL, cat. No. 35050).
(16)100XMEM non-essential amino acids solution (NEAA) (glycine 10mM, L-alanine 10mM, L-asparaginase 10mM, L-aspartic acid 10mM, L-glutamic acid 10mM, L-proline 10mM, L-serine 10mM) (Gibco-BRL, cat. No. 11140).
(17) N2supplement (100X) (human transferrin (whole) 10000mg/L, recombinant insulin whole chain 500mg/L, progesterone 0.63mg/L, putrescine 1611mg/L, selenite 0.52mg/L) (Gibco-BRL, cat. No. 17502-048).
(18)Purmorphamine(CALBIOCHEM,cat.no.540220)。
(19)RA(Sigma,cat.no.R2625)。
(20) Neurobasal medium (Gibco-BRL, cat. No. 21103049).
(21)Y-27632(Selleck,cat.no.S1049)。
(22) 4% paraformaldehyde solution (cat. No. B1057-100, Beijing Runzukang Biotech Co., Ltd.).
(23) A rat anti-BrdU antibody (abcam, cat. No. ab6326).
(24) A murine anti-Nestin antibody (santa cruz, cat. No. sc-23927).
(25) A rabbit anti-FOXG 1 antibody (abcam, cat. No. ab18259).
(26) Murine anti-Nkx2.1 antibody (Millipore, cat. nomAB5460).
(27) Chicken anti-MAP 2 antibody (Bio-legend, cat. no 822501).
(28) Rabbit anti-Vgult 1 antibody (abcam, cat. noab 227805).
(29) Rabbit anti-GABA antibody (abcam, cat. noab 230136).
(30) Alexa Fluor 568 labeled goat anti-rat IgG (H + L) secondary antibody (Life, cat. No. a 11077).
(31) Alexa Fluor 488 labeled goat anti-mouse IgG (H + L) secondary antibody (Life, cat. No. a 11001).
(32) Alexa Fluor 568 labels a secondary goat anti-rabbit IgG (H + L) antibody (Life, cat. No. a 11011).
(33) Alexa Fluor 488 labeled goat anti-rabbit IgG (H + L) secondary antibody (Life, cat. No. a 11034).
(34) Alexa Fluor 568 labels a secondary goat anti-mouse IgG (H + L) antibody (Life, cat. No. a 11004).
(35) Alexa Fluor 488 labeled goat anti-chicken IgG (H + L) secondary antibody (Life, cat. No. a 11039).
(36) Ornithine (poly-l-ornithline hydrobromide) (Sigma-Aldrich, cat. No. p 3655).
(37)lamin 1mg/ml(Invitrogen,cat.no.23017-015)。
(38) Matrigel (Matrigel) (BD, cat. No. 356234).
(39)Tritonx-100(SIGMA-ALDRICH,#HPA014518)。
(40) Collagenase IV (GIBIO, cat. No. 17104019).
(41) Valproic acid (MCE, cat. No. HY-10585).
(42) Human embryonic stem cells (Beijing seebeck Biotechnology Co., Ltd. # CA 4001106).
2. The media and related reagent configurations used in the following examples:
culture medium I: human pluripotent stem cell medium (PGM1 medium).
And (3) a culture medium II: medium II was obtained by adding Y-27632 to PGM1 medium; wherein, the content of Y-27632 in the culture medium II is 10 MuM.
N2 medium: n2 medium was obtained by adding N2supplement (100X) and 100XNEAA to DMEM-F12 medium, N2supplement (100X) was diluted 100-fold in N2 medium, and 100XNEAA was diluted 100-fold in N2 medium. In the N2 culture medium, the volume percentage content of N2supplement (100X) is 1 percent; the concentrations of glycine, L-alanine, L-asparaginase, L-aspartic acid, L-glutamic acid, L-proline and L-serine in 100XNEAA were all 0.1 mM.
N2+ lamin medium: the N2+ lamin medium was obtained by adding lamin to N2 medium at a concentration of 1. mu.g/ml for lamin N2+ lamin medium.
N2B27 medium: the N2B27 medium was prepared by adding N2supplement (100X), B27supplement (50X), RA and human fibroblast growth factor-2 to DMEM-F12 medium, wherein N2supplement (100X) was diluted 100-fold in N2B27 medium, B27supplement (50X) was diluted 50-fold in N2B27 medium, RA content in N2B27 medium was 0.2. mu.M, and human fibroblast growth factor-2 content in N2B27 medium was 20 ng/ml. In the N2B27 medium, the volume percentage content of N2supplement (100X) is 1%, and the volume content of B27supplement (50X) is 1/50.
Neuron culture medium: the neuron culture medium is obtained by adding N2supplement (100X), B27supplement (50X), brain-derived growth factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP) and Ascorbic Acid (Ascorbic Acid) to DMEM-F12 culture medium, wherein N2supplement (100X) is diluted 100 times in the neuron culture medium, B27supplement (50X) is diluted 50 times in the neuron culture medium, the content of BDNF in the neuron culture medium is 10ng/ml, the content of GDNF in the neuron culture medium is 10ng/ml, the content of cAMP in the neuron culture medium is 1 muM, and the content of Ascorbic Acid in the neuron culture medium is 200 nM. In the neuron culture medium, the volume percentage content of N2supplement (100X) is 1%, and the volume content of B27supplement (50X) is 1/50.
hES medium: the hES medium was obtained by adding 100XNEAA, KOSR and β -mercaptoethanol to DMEM-F12 medium, 100XNEAA diluted 100-fold in hES medium, KOSR at 20% concentration by volume in hES medium and β -mercaptoethanol at 0.1mM concentration in hES medium. The concentrations of glycine, L-alanine, L-asparaginase, L-aspartic acid, L-glutamic acid, L-proline and L-serine in 100XNEAA were all 0.1mM in hES medium.
NIM medium: NIM medium was obtained by adding N2supplement (100X), 100XNEAA and heparin to DMEM-F12 medium, wherein N2supplement (100X) was diluted 100-fold in NIM medium, 100XNEAA was diluted 100-fold in NIM medium, and heparin was contained at 2. mu.g/ml in NIM medium. In NIM medium, the volume percentage of N2supplement (100X) is 1%; the concentrations of glycine, L-alanine, L-asparaginase, L-aspartic acid, L-glutamic acid, L-proline and L-serine in 100XNEAA were all 0.1 mM.
NIM + pur medium: NIM + pur medium was obtained by adding Purmorphamine to NIM medium, and the content of Purmorphamine in NIM + pur medium was 0.65. mu.M.
VPA medium: the VPA medium was obtained by adding 100XGlutamax and valproic acid to Neurobasal medium, wherein 100XGlutamax was diluted 100-fold in VPA medium and the amount of valproic acid in VPA medium was 10. mu.M. The concentration of L-alanyl-L-glutamine dipeptide 100XGlutamax was 2mM, 0.0085% NaCl in VPA medium.
NDM medium: the medium is obtained by adding N2supplement (100X), B27supplement (50X), 100X Glutamax, brain-derived growth factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP) and insulin-like growth factor (IGF1) to Neurobasal medium, wherein N2supplement (100X) is diluted 100 times in NDM medium, B27supplement (50X) is diluted 50 times in NDM medium, 100XGlutamax is diluted 100 times in NDM medium, BDNF is 10ng/ml, GDNF is 10ng/ml, cAMP is 1. mu.M, and IGF1 is 10ng/ml in NDM medium. In NDM medium, the volume percentage of N2supplement (100X) is 1%; the concentration of L-alanyl-L-glutamine dipeptide 100XGlutamax was 2mM, 0.0085% NaCl, and the volume content of B27supplement (50X) was 1/50.
Human fibroblast growth factor (FGF2) (100 μ g/ml): dissolve 1mgbFGF in 10ml hESC medium, split 500. mu.l × 20 tubes, take out one tube each time and add hESCM to 5ml, split 200. mu.L each tube.
Heparin (1 mg/ml): dissolving 10mg heparin in 10ml DMEM/F12, filtering with 0.22um filter membrane, subpackaging, storing in a refrigerator of-20 deg.C, and storing for more than 3 months.
Matrigel (Matrigel): 200 μ L per tube were dispensed on ice. For use, 49.8mL of cold DMEM-F12 was added, mixed well and plated onto a plate.
Purmorphamine (10 mM): 5mg of purmorphamine was dissolved in 480. mu.l of 100% ethanol and 480. mu.l of DMSO, and the mixture was stored at-20 ℃ for 8 weeks or more after being dispensed.
RA (100 mM): dissolve 50mg RA in 1.67ml DMSO. Subpackaging 50 μ l each tube, and keeping at-80 deg.C for more than six weeks in dark. Diluted with 4.95ml 100% ethanol and stored at working concentration. Can be stored at-20 deg.C for more than two weeks. Note that the working fluid does not exceed two weeks.
And (3) subpackaging Collagenase IV and Collagenase: 50ml of DMEM-F12 medium was added to 1000mg of collagenase per tube and dissolved thoroughly by gentle vortex shaking to prepare a 20mg/ml (i.e., 20X) stock. Then filtering and sterilizing by using a filter membrane with low protein binding property and 0.22 mu m, subpackaging into small parts, and freezing and storing at-20 ℃ in the dark. Before use, the product is thawed on ice to avoid repeated freezing and thawing. The usual concentrations for tissue and cell dispersion are: 0.5-2.5mg/ml, the optimum working concentration required is determined according to specific experimental conditions or by reference to corresponding literature data.
Ornithine was coated at 50 μ g/ml overnight and ornithine was soluble in water (50mg/ml) resulting in a clear solution.
The stock solution of Laminin was 500. mu.g/ml, diluted with DMEM/F12, 2.5ml of DMEM/F12 per 100. mu.l.
The preparation method of the sealing liquid comprises the following steps: weighing 40mL of 1 Xphosphate buffer solution, weighing 1g of bovine serum albumin, shaking and mixing uniformly until the bovine serum albumin is completely dissolved, adding 0.15mL of LTriton x-100, then adding 1 Xphosphate buffer solution to fix the volume to 50mL, mixing uniformly, filtering through a 0.22 μm filter, and storing at 4 ℃.
Example 1 Induction of differentiation of hESCs into excitatory neurons
This example induced hESCs to differentiate towards excitatory neurons, as schematically shown in FIG. 1, with the following steps:
induction of differentiation of hESCs into excitatory neurons
(1) Inoculation: culturing human embryonic stem cell, gently washing with PBS once when the cell grows to passage, adding 1ml ACCUTASE cell digestive juice at 37 deg.C and 5% CO 2 Digesting the cells in the humidifying culture box for 5min, taking out the digested cells after 2-3min, slightly blowing the digested cells if the digested cells do not fall off, adding half of PGM1 culture medium, centrifuging for 220g for 3min, and then removing the supernatant.
(2) Balling: adding culture medium II to resuspend cells into a new six-well plate, supplementing 1ml culture medium II, placing at 37 deg.C and 5% CO 2 The humidified incubator of (1) was left to stand for balling, and was cultured to day3 as day0 (i.e., day 0).
(3) Forming a pseudo-embryo body: day3 the original medium was aspirated, and N2 medium was added at 37 ℃ with 5% CO 2 The humidified incubator of (2) for cultivation. The medium was changed every other day to day7 with N2 medium to obtain embryoid bodies (EB balls). Note: standing for 1min under inclined condition, and directly sucking off the supernatant.
(4) Forming a rosette: day7 six well plates coated with matrigel were prepared, all EB spheres from the previous six well plates were resuspended in matrigel-coated plates, the supernatant was discarded after natural sedimentation, the supernatant was resuspended in N2+ lamin medium and the suspension continued at 37 ℃ with 5% CO 2 The rose rosette (similar to a poached egg) is formed every other day in the humidified incubator of (1), and every other day changing liquid is cultured by using N2+ lamin culture medium from day7 until day 14.
(5) Neural progenitor/stem cell formation: day14 discard part of the culture medium, add collagenase IV (collagenase IV concentration in the culture system is 20mg/mL) to the rest of the culture medium, and place at 37 ℃ with 5% CO 2 Digesting in a humidifying incubator for 20-30min (gently patting every 5min, and observing that the central thick ball-yolk begins to fall off after 10 min), after the ball at the center of each EB ball slowly falls off, suspending all the balls into a low-adsorption six-hole plate by using an N2B27 culture medium, and placing the six-hole plate into a chamber with the temperature of 37 ℃ and the content of 5% CO 2 The humidified incubator of (4) was cultured, and the medium was changed to day20 with N2B27 every other day. Digesting collagenase IV for 20min, beating every 5min, sucking into a centrifuge tube, settling for 3min, removing supernatant, and resuspending at 1:1 passage.
(6) Formation of neurons: day20, collecting all neurospheres in the six-well plate after the culture in step (5) is finished in an EP tube, sucking off the supernatant, digesting with preheated ACCUTASE cell digestive juice for 8min, sucking off ACCUTASE cell digestive juice, washing once with PBS, then blowing all the neurospheres 4-5 times with 1ml of N2B27 culture medium, inoculating into the six-well plate, culturing for 1 day in N2B27 culture medium, collecting small neurospheres after 24h, discarding the supernatant, digesting for 8min with preheated ACCUTASE cell digestive juice, sucking off ACCUTASE cell digestive juice, washing once with PBS, blowing all the neurospheres 4-5 times with 1ml of N2B27 culture medium, counting the blown single cells, and then re-suspending into the ornithine and laminin coated six-well plate (six-well plate is coated with ornithine and laminin for at least 4h in advance), and adding 2ml of neuron culture medium in advance. Fresh neuronal medium was then changed every other day.
(7) The day40 assay detects excitatory neurons and counts the differentiation efficiency (see FIG. 3).
Second, immunofluorescence staining is carried out on the neural stem cells
After the end of the culture in step one (5), the supernatant medium was aspirated and fixed with 500. mu.L of 4% paraformaldehyde solution for 20 minutes. Rinse three times with 500 μ L of 1 × phosphate buffer for 5 minutes each time and aspirate the supernatant. Add 500. mu.L of blocking solution and block for 1 hour at room temperature. The blocking solution was aspirated off, 200. mu.L of fresh blocking solution was added, and the volume ratio of 1: rat anti-BrdU antibody and murine anti-Nestin antibody (both antibodies were added simultaneously at the same fold dilution) were added at 1000 deg.C and incubated for 12 hours at 4 deg.C. The supernatant was aspirated off and rinsed three times with 500. mu.L of 1 XPhosphate buffer for 5 minutes each. The supernatant was aspirated off, 200 μ L of fresh blocking solution was added, and the volume ratio of 1: DAPI, Alexa Fluor 488-labeled secondary goat anti-mouse IgG (H + L) antibody and Alexa Fluor 568 secondary goat anti-rat IgG (H + L) antibody (both antibodies were added at the same dilution times) were added at the same time at a ratio of 1000, and after incubation for two hours in the dark, the supernatant was aspirated and discarded, and then rinsed three times in 500. mu.L of 1 XPhosphate buffer in the dark for 5 minutes each time. After the slides were picked up and mounted with the anti-quenching mounting plate, the marker genes for neural progenitors/stem cells (i.e., BrdU and Nestin genes, see FIG. 2) were successfully stained by fluorescence microscopy, indicating that the differentiation of BrdU and Nestin-positive neural progenitors/stem cells was successful.
Performing immunofluorescence staining on mature excitatory neurons
After the culture in the first step (7) is finished, the obtained culture product is subjected to immunofluorescence staining according to the method in the second step, the rat anti-BrdU antibody and the mouse anti-Nestin antibody are replaced by the chicken anti-MAP 2 antibody and the rabbit anti-Vglut 1 antibody, the Alexa Fluor 488 labeled secondary goat anti-mouse IgG (H + L) antibody and the Alexa Fluor 568 secondary goat anti-rat IgG (H + L) antibody are replaced by the Alexa Fluor 568 labeled secondary goat anti-rabbit IgG (H + L) antibody and the Alexa Fluor 488 labeled secondary goat anti-chicken IgG (H + L) antibody. After mounting, the staining was observed by fluorescence microscopy, and the marker genes for excitatory neurons (i.e., MAP2 and Vglut1 genes, see FIG. 3) were successfully stained. This example successfully obtained MAP2 and vgut 1 positive excitatory neurons, accounting for more than 80% of the total cells.
Example 2 Induction of differentiation of hESCs into inhibitory neurons
This example induced hESCs to differentiate towards inhibitory neurons, the schematic diagram is shown in fig. 4, and the specific steps are as follows:
induction of hESCs differentiation into inhibitory neurons
(1) Inoculation: culturing human embryonic stem cell, gently washing with PBS once when the cell grows to passage, adding 1ml ACCUTASE cell digestive juice at 37 deg.C and 5% CO 2 The cells are digested in the humidifying incubator for 5min, the cells are taken out when the cells are digested for 2-3min, if the cells do not fall off, the cells are lightly beaten, half of the PGM1 culture medium is added, the cells are centrifuged for 3min at 220g, and then the supernatant is discarded.
(2) Forming a pseudo-embryo body: adding hES culture medium to resuspend cells into a new six-well plate, supplementing 1ml of hES culture medium, placing at 37 ℃ and 5% CO 2 The humidified incubator of (1) was left to stand as a ball (i.e., EB ball), and was recorded as day0 when the day (i.e., day 0). day4 the original medium was aspirated, NIM medium was added at 37 ℃ with 5% CO 2 The humidified incubator of (2) for cultivation. Change the medium every other day with NIM medium until day 7. Note: standing for 1min under inclined condition, and directly sucking off the supernatant.
(3) Rosette formation: day7 six well plates coated with matrigel were prepared and all EB spheres from the previous six well plates were resuspended in matrigel-coatedIn the plate, the supernatant was discarded after natural sedimentation, suspended with NIM medium and continued at 37 ℃ with 5% CO 2 The rose rosettes (similar to poached eggs) are formed every other day in the humidified incubator of (1), and every other day change is performed by using NIM medium from day7 until day 12.
(4) GABA interneural precursor cell formation: day12, discarding part of the culture medium, adding collagenase IV (collagenase IV concentration in the culture system is 20mg/mL) to the rest of the culture medium, placing at 37 deg.C and 5% CO 2 Digesting in a humidifying incubator for 20-30min (gently patting every 5min, and observing that the central thick ball-yolk begins to fall off after 10 min), after the ball (i.e. neurosphere) at the center of each EB ball slowly falls off, re-suspending all the balls into a low-adsorption six-hole plate by using an NIM + pur culture medium, placing the six-hole plate into a chamber with the temperature of 37 ℃ and 5% CO 2 The humidified incubator of (4) was cultured, and the medium was changed to day26 using NIM + pur medium every other day.
(5) GABA interneuron precursor formation: day 26A pre-warmed ACCUTASE cell digest was prepared and the six-well plate was previously coated with 50. mu.g/ml ornithine and laminin, both of which had to be coated for more than 4 hr. Collecting all neurospheres in the six-well plate after the culture in the step (4) is finished in an EP tube, sucking away supernatant, digesting with preheated ACCUTASE cell digestive juice for 8min, sucking away the ACCUTASE cell digestive juice, washing with PBS once, blowing all the neurospheres under 4-5 by using 1ml of VPA culture medium, counting the blown single cells, then re-suspending the cells in the plate coated with ornithine and laminin to culture to day32, and adding 2ml of VPA culture medium in the plate in advance. Then, the culture medium was changed every other day, and cultured for one week.
(6) GABA inhibitory neuron formation: culture was continued with day32 changed to NDM medium, and day47 was used to examine inhibitory neurons, and the differentiation efficiency was counted (see FIG. 6).
Forebrain GABA interneuron precursor cell immunofluorescence staining
After the end of the culture in step one (5), the supernatant medium was aspirated and fixed with 500. mu.L of 4% paraformaldehyde solution for 20 minutes. Rinse three times with 500 μ L of 1 × phosphate buffer for 5 minutes each time and aspirate the supernatant. Add 500. mu.L of blocking solution and block for 1 hour at room temperature. The blocking solution was aspirated off, 200. mu.L of fresh blocking solution was added, and the volume ratio of 1: the murine anti-NKX 2.1 antibody and the rabbit anti-FOXG 1 antibody (both antibodies diluted in the same fold) were added simultaneously at a ratio of 1000 and incubated at 4 ℃ for 12 hours. The supernatant was aspirated off and rinsed three times with 500. mu.L of 1 XPhosphate buffer for 5 minutes each. The supernatant was aspirated off, 200. mu.L of fresh blocking solution was added, and the reaction mixture was mixed at 1: DAPI was added simultaneously at a ratio of 1000, and a secondary Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) antibody and a secondary Alexa Fluor 568 goat anti-mouse IgG (H + L) antibody (diluted by the same fold) were added, and after incubation for two hours in the dark, the supernatant was aspirated and rinsed three times with 500 μ L1 × phosphate buffer in the dark for 5 minutes each. After the slide glass is picked up and the piece is sealed by the anti-quenching sealing tablet, the marker genes of GABA interneuron precursor cells (namely NKX2.1 and FOXG1 genes, see figure 5) can be successfully stained by observing the staining condition by using a fluorescence microscope, namely, the GABA interneuron precursor cells positive to NKX2.1 and FOXG1 can be successfully differentiated.
Performing immunofluorescence staining on GABA inhibitory neurons
After the culture in the first step (6) is finished, performing immunofluorescence staining on the obtained culture product according to the second step, replacing the mouse anti-NKX 2.1 antibody and the rabbit anti-FOXG 1 antibody with a chicken anti-MAP 2 antibody and a rabbit anti-GABA antibody, replacing the Alexa Fluor 488 labeled secondary goat anti-rabbit IgG (H + L) antibody and the Alexa Fluor 568 secondary goat anti-mouse IgG (H + L) antibody with an Alexa Fluor 568 labeled secondary goat anti-rabbit IgG (H + L) antibody and an Alexa Fluor 488 labeled secondary goat anti-chicken IgG (H + L) antibody. After mounting, the staining was observed by fluorescence microscopy, and the marker gene of GABA inhibitory neurons (i.e., MAP2 and GABA gene, see FIG. 6) was successfully stained. As an illustration, this example succeeded in obtaining MAP2 and GABA-positive GABA-inhibitory neurons, and statistics of the differentiation efficiency showed that the differentiation efficiency of GABA-inhibitory neurons was more than 85% (see FIG. 6).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Claims (10)

1. A method of preparing an excitatory neuron, comprising:
1) culturing the human pluripotent stem cells in a culture medium II to obtain tissues cultured by the culture medium II;
the culture medium II is obtained by adding Y-27632 to a human pluripotent stem cell culture medium; wherein the content of Y-27632 in the culture medium II is 10 mu M;
2) culturing the tissue cultured by the culture medium II in an N2 culture medium to obtain a tissue cultured by an N2 culture medium;
the N2 culture medium is obtained by adding N2supplement (100X) and 100X NEAA in DMEM-F12 culture medium, the volume percentage content of N2supplement (100X) in the N2 culture medium is 1 percent, and the volume percentage content of 100X NEAA in the N2 culture medium is 1 percent;
3) culturing the tissue cultured by the N2 culture medium in an N2+ lamin culture medium to obtain a tissue cultured by an N2+ lamin culture medium;
the N2+ lamin culture medium is obtained by adding lamin to the N2 culture medium, and the concentration of lamin in the N2+ lamin culture medium is 1 mug/ml;
4) treating the tissue cultured by the N2+ lamin culture medium by using collagenase IV to obtain the tissue treated by the collagenase IV; culturing the tissue treated by the collagenase IV by using an N2B27 culture medium to obtain a tissue cultured by an N2B27 culture medium;
the N2B27 culture medium is obtained by adding N2supplement (100X), B27supplement (50X), RA and human fibroblast growth factor-2 into DMEM-F12 culture medium, wherein the volume percentage content of N2supplement (100X) in the N2B27 culture medium is 1%, the volume content of B27supplement (50X) in the N2B27 culture medium is 1/50, the content of RA in the N2B27 culture medium is 0.2 mu M, and the content of human fibroblast growth factor-2 in the N2B27 culture medium is 20 ng/ml;
5) digesting the tissue cultured by the N2B27 culture medium to obtain digested cells; culturing the digested cells in a neuron culture medium to obtain excitatory neurons;
the neuron culture medium is obtained by adding N2supplement (100X), B27supplement (50X), BDNF, GDNF, cAMP and ascorbic acid into a DMEM-F12 culture medium, wherein the volume percentage content of N2supplement (100X) in the neuron culture medium is 1%, the volume content of B27supplement (50X) in the neuron culture medium is 1/50, the content of BDNF in the neuron culture medium is 10ng/ml, the content of GDNF in the neuron culture medium is 10ng/ml, the content of cAMP in the neuron culture medium is 1 mu M, and the content of ascorbic acid in the neuron culture medium is 200 nM.
2. The method of claim 1, wherein: in the step 1), before the culture of the pluripotent stem cells, the method further comprises the step of digesting the stem cells;
and/or, the culturing in step 3) is carried out in a matrigel coated culture plate;
and/or, the culturing in step 5) is performed in an ornithine and laminin coated culture plate;
and/or, the human pluripotent stem cell is an induced pluripotent stem cell, an adult stem cell, an somatic stem cell, a cancer stem cell, or any other pluripotent stem cell that has the ability to differentiate.
3. The method according to claim 1 or 2, characterized in that: the culture in steps 1) -5) was carried out at 37 ℃ and 5% CO 2 Under the conditions of (1).
4. A method according to any one of claims 1-3, characterized in that: the culture time in the step 1) is 3 days;
and/or, the culturing time in the step 2) is 4 days;
and/or, the culturing time in the step 3) is 7 days;
and/or, the culture time in the step 4) is 6 days;
and/or, the culture time in the step 5) is 19 to 20 days.
5. A method of making an inhibitory neuron, comprising:
1) culturing human embryonic stem cells in an hES culture medium to obtain a tissue cultured by the hES culture medium;
the hES culture medium is obtained by adding 100X NEAA, a serum substitute and beta-mercaptoethanol into a DMEM-F12 culture medium, wherein the volume percentage content of the 100X NEAA in the hES culture medium is 1%, the volume percentage concentration of the serum substitute in the hES culture medium is 20%, and the concentration of the beta-mercaptoethanol in the hES culture medium is 0.1 mM;
2) culturing the tissue cultured by the hES culture medium in an NIM culture medium to obtain a tissue 1 cultured by the NIM culture medium;
the NIM medium is obtained by adding N2supplement (100X), 100X NEAA and heparin into DMEM-F12 medium, wherein the volume percentage of N2supplement (100X) in the NIM medium is 1%, the volume percentage of 100X NEAA in the NIM medium is 1%, and the content of heparin in the NIM medium is 2 mu g/ml;
3) culturing the tissue 1 cultured by the NIM culture medium in a culture plate coated by matrigel by using the NIM culture medium to obtain a tissue 2 cultured by the NIM culture medium;
4) treating the tissue 2 cultured by the NIM culture medium obtained in the step 3) by using collagenase IV to obtain a tissue treated by collagenase IV; culturing the tissue treated by the collagenase IV by using an NIM + pur culture medium to obtain a tissue cultured by the NIM + pur culture medium;
the NIM + pur culture medium is obtained by adding Purmorphamine into the NIM culture medium, and the content of the Purmorphamine in the NIM + pur culture medium is 0.65 mu M;
5) digesting the tissue cultured by the NIM + pur culture medium to obtain digested cells; culturing the digested cells in a VPA culture medium to obtain cells cultured in the VPA culture medium;
the VPA culture medium is obtained by adding 100XGlutamax and valproic acid into a Neurobasal culture medium, wherein the volume percentage of 100XGlutamax in the VPA culture medium is 1%, and the content of valproic acid in the VPA culture medium is 10 mu M;
6) culturing the cells cultured by the VPA culture medium in an NDM culture medium to obtain inhibitory neurons;
the NDM culture medium is obtained by adding N2supplement (100X), B27supplement (50X), 100X Glutamax, BDNF, GDNF, cAMP and IGF1 into a Neurobasal culture medium, wherein the volume percentage content of N2supplement (100X) in the NDM culture medium is 1%, the volume percentage content of B27supplement (50X) in the NDM culture medium is 1/50, the volume percentage content of 100X Glutamax in the NDM culture medium is 1%, the volume percentage content of BDNF in the NDM culture medium is 10ng/ml, the volume percentage content of GDNF in the NDM culture medium is 10ng/ml, the volume percentage content of cAMP in the NDM culture medium is 1 muM, and the volume percentage content of IGF1 in the NDM culture medium is 10 ng/ml.
6. The method of claim 5, wherein: in the step 1), before the culture of the pluripotent stem cells, the method further comprises the step of digesting the stem cells;
and/or, the culturing in step 5) is performed in an ornithine and lamin coated culture plate.
7. The method according to claim 5 or 6, characterized in that: the culture in steps 1) -6) is carried out at 37 ℃ and 5% CO 2 Under the conditions of (1).
8. The method according to any one of claims 5-7, wherein: the culture time in the step 1) is 4 days;
and/or, the culturing time in the step 2) is 3 days;
and/or, the culturing time in the step 3) is 5 days;
and/or, the culture time in the step 4) is 14 days;
and/or, the time of the culture in the step 5) is 6 days;
and/or, the time of the culture in the step 6) is 15 days;
and/or, the inhibitory neuron is a GABA inhibitory neuron.
9. A kit comprising the medium II, the N2 medium, the N2+ lamin medium, the N2B27 medium, and the neuron medium of claim 1, or the hES medium, the NIM + pur medium, the VPA medium, and the NDM medium of claim 5.
10. Use of the kit of claim 9 in the preparation of excitatory or inhibitory neurons, or in the preparation of products for inducing excitatory or inhibitory neurons.
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100317103A1 (en) * 2006-06-07 2010-12-16 Je Il Pharmaceutical Co., Ltd. Efficient generation of neural progenitors, neurons, and dopaminergic neurons from human embryonic stem cells
US20120244567A1 (en) * 2009-09-04 2012-09-27 Buck Institute For Research On Aging Human embryonic stem cells for high throughout drug screening
US20130065834A1 (en) * 2011-07-25 2013-03-14 Salk Institute For Biological Studies Modulators of proteasome activity
CN103266082A (en) * 2013-02-06 2013-08-28 赛业（广州）生物科技有限公司 Method of embryonic stem cell differentiation into neurons by in vitro induction
CN105331634A (en) * 2014-08-08 2016-02-17 中国科学院动物研究所 Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts
US20160053226A1 (en) * 2013-04-15 2016-02-25 INSERM (Institut National de la Santé et de la Recherche Médicale Method and Medium for Amplifying Neural Precursor Cells
CN108048400A (en) * 2017-12-26 2018-05-18 南开大学 A kind of preparation method of monkey monoploid neural stem cell
CN110205295A (en) * 2019-06-25 2019-09-06 中国科学院动物研究所 A kind of multipotential stem cell induction generates the method and kit of microglia
CN111344302A (en) * 2017-08-31 2020-06-26 胡默波斯生物医学公司 Multispecific antibodies that specifically bind to epitopes of Zika virus and uses thereof
CN112840018A (en) * 2019-09-25 2021-05-25 S生物医药公司 Method for inducing differentiation of dopamine neural precursor cells from stem cells
CN113604434A (en) * 2020-11-30 2021-11-05 浙江霍德生物工程有限公司 Generation of neural precursor cells from embryonic stem cells or induced pluripotent stem cells

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100317103A1 (en) * 2006-06-07 2010-12-16 Je Il Pharmaceutical Co., Ltd. Efficient generation of neural progenitors, neurons, and dopaminergic neurons from human embryonic stem cells
US20120244567A1 (en) * 2009-09-04 2012-09-27 Buck Institute For Research On Aging Human embryonic stem cells for high throughout drug screening
US20130065834A1 (en) * 2011-07-25 2013-03-14 Salk Institute For Biological Studies Modulators of proteasome activity
CN103266082A (en) * 2013-02-06 2013-08-28 赛业（广州）生物科技有限公司 Method of embryonic stem cell differentiation into neurons by in vitro induction
US20160053226A1 (en) * 2013-04-15 2016-02-25 INSERM (Institut National de la Santé et de la Recherche Médicale Method and Medium for Amplifying Neural Precursor Cells
CN105331634A (en) * 2014-08-08 2016-02-17 中国科学院动物研究所 Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts
CN111344302A (en) * 2017-08-31 2020-06-26 胡默波斯生物医学公司 Multispecific antibodies that specifically bind to epitopes of Zika virus and uses thereof
CN108048400A (en) * 2017-12-26 2018-05-18 南开大学 A kind of preparation method of monkey monoploid neural stem cell
CN110205295A (en) * 2019-06-25 2019-09-06 中国科学院动物研究所 A kind of multipotential stem cell induction generates the method and kit of microglia
CN112840018A (en) * 2019-09-25 2021-05-25 S生物医药公司 Method for inducing differentiation of dopamine neural precursor cells from stem cells
CN113604434A (en) * 2020-11-30 2021-11-05 浙江霍德生物工程有限公司 Generation of neural precursor cells from embryonic stem cells or induced pluripotent stem cells
WO2022110654A1 (en) * 2020-11-30 2022-06-02 Zhejiang Huode Bioengineering Company Generation of neural progenitor cells from embryonic stem cells or induced pluripotent stem cells

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