CN113832110A

CN113832110A - Induced pluripotent stem cell carrying GCH1 gene mutation and preparation method for differentiating induced pluripotent stem cell into neural precursor cell

Info

Publication number: CN113832110A
Application number: CN202111275360.9A
Authority: CN
Inventors: 黄国滨; 周红艳; 李爱群; 鲁浩; 龚春丽; 陈传云; 葛圆圆
Original assignee: Zhuhai Leviregen Medical Technology Co ltd
Current assignee: Zhuhai Leviregen Medical Technology Co ltd
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2021-12-24

Abstract

The invention discloses an induced pluripotent stem cell carrying GCH1 gene mutation and a preparation method for differentiating the induced pluripotent stem cell into a neural precursor cell, and relates to the technical field of induced pluripotent stem cell differentiation. The invention provides a method for preparing induced pluripotent stem cells with GCH1 gene mutation, which can invert peripheral blood mononuclear cells into induced pluripotent stem cells, thereby obtaining the induced pluripotent stem cells with GCH1 gene mutation. Under specific conditions, the induced pluripotent stem cells can be induced to differentiate into neural progenitor cells, and the neural progenitor cells can be further induced to differentiate into corresponding neuronal cells, such as NGN2 neurons. The induced pluripotent stem cells are induced to differentiate, so that various neurons such as mesencephalon dopamine neurons can be directly prepared.

Description

Induced pluripotent stem cell carrying GCH1 gene mutation and preparation method for differentiating induced pluripotent stem cell into neural precursor cell

Technical Field

The invention relates to the technical field of induced pluripotent stem cell differentiation, in particular to an induced pluripotent stem cell carrying GCH1 gene mutation and a preparation method for differentiating the induced pluripotent stem cell into a neural precursor cell.

Background

Induced Pluripotent Stem Cells (ipscs) were introduced into somatic Cells (skin fibroblasts, blood Cells, etc.) after four transcription factors were first combined by a viral vector by enchanting japan scientists mountain, and Cells in an embryonic-like state were obtained. The iPSC has the capability of unlimited self-renewal and proliferation, and can be differentiated into specific functional cells, even tissues, organs and the like. The differentiation of the iPSC into cells of specific tissues avoids the immune rejection phenomenon caused by individual differences, and more importantly, compared with embryonic stem cells, the iPSC also avoids the debate in ethical aspects, so that the iPSC has wide application prospect in the biomedical field, is an important life resource and medical resource in the biomedical industry, plays an irreplaceable role in the fields of personal storage and application, disease models, drug research and development, cell therapy, precise medical treatment, health management and the like, and has great market demand.

Stem cells and gene editing techniques are well known as new directions for treating diseases and delaying human aging. Brain aging and brain diseases, especially Alzheimer's disease, Parkinson's disease and other nervous system diseases increasingly threaten the economic development of China.

In the human mendelian genetics database, it is described that the GCH1(GTP cyclohydrophase 1) gene is a causative gene for dopa-responsive dystonia with or without hyperphenylalaninemia and tetrahydrobiopterin-deficient hyperphenylalaninemia type B. Among them, dopamine-responsive dystonia (DRD) is a rare disorder of dystonia in nervous system metabolism. The disease is typically characterized by lower limb dystonia, often referred to as plantar flexion inversion, resulting in gait disorders such as falls or falls in the patient. The symptoms fluctuate day and night, worsen at night, improve during the day after sleep, and aggravate by physical exercise. The disease usually develops into a general dystonia, and some patients, especially those in adolescence or adulthood, also suffer from Parkinson's disease.

GCH1 plays a role as a rate-limiting enzyme in the synthesis of tetrahydrobiopterin, while BH4 is an essential cofactor in the biosynthesis of catecholamines, and thus in the nigrostriatal system, the absence or mutation of GCH1 in dopamine neurons leads to a decrease in tyrosine hydroxylase synthesis and thus to a decrease in dopamine levels. The c.626C > G of GCH1 gene is homozygous variation (C at position 626 is replaced by G) at the p.T209R site, the variation at the site is missense mutation, the variation site is positioned at the last base in the No. 5 exon, and the variation can influence the shearing of mRNA. Furthermore, since the mutation is located in GTP cyclohydrolase I region, it is likely to affect its protein function. The variation at this site was shown in the GnomAD database to be 3.98e-6 in the total population and 5.44e-5 in the east Asian population. Furthermore, different variations of this amino acid, such as p.T209I or p.T209P, have been reported in patients with dopa-responsive dystonia or Parkinson's disease, but homozygous variation of the C.626C > G: p.T209R locus of the GCH1 gene is not clear for the development of the disease.

In view of this, the invention is proposed to provide a highly efficient and accurate human cell model for the study of nervous system diseases.

Disclosure of Invention

The present invention aims at providing induced pluripotent stem cells carrying GCH1 gene mutation and a preparation method for differentiating the induced pluripotent stem cells into neural precursor cells so as to solve the technical problems.

The invention is realized by the following steps:

the invention provides a preparation method of induced pluripotent stem cells carrying GCH1 gene mutation, which comprises the following steps: carrying out cell reprogramming on isolated human peripheral blood mononuclear cells carrying GCH1 gene mutation so as to obtain pluripotent stem cells carrying GCH1 gene mutation; then carrying out mixed culture on the pluripotent stem cells carrying the GCH1 gene mutation, viruses carrying a regulation expression cassette and viruses carrying a reaction expression cassette to obtain induced pluripotent stem cells carrying the GCH1 gene mutation;

the regulation expression box comprises a specific promoter and an antisense tetracycline activator, and the reaction expression box comprises a TRE, a tetracycline inducible promoter, a transcription factor coding gene and a screening gene;

cell reprogramming includes: infecting isolated human peripheral blood mononuclear cells carrying GCH1 gene mutation with Sendai virus carrying c-Myc gene, SOX2 gene, KLF4 gene and OCT4 gene, and culturing until obtaining GCH1 gene mutated pluripotent stem cells; the GCH1 gene is mutated into T209R of GCH1 gene.

The inventors provide a method for preparing induced pluripotent stem cells having a GCH1 gene mutation, which can reverse peripheral blood mononuclear cells to induced pluripotent stem cells, thereby obtaining induced pluripotent stem cells having a GCH1 gene mutation. Under specific conditions, the induced pluripotent stem cells can be induced to differentiate into neural progenitor cells, and the neural progenitor cells can be further induced to differentiate into corresponding neuronal cells, such as NGN2 neurons. The induced pluripotent stem cells are induced to differentiate, so that various neurons such as mesencephalon dopamine neurons can be directly prepared.

Based on the establishment of the GCH1 gene mutation iPS cell strain and the success of the preparation of related neuron cells, people can better understand the pathogenic mechanism of GCH1 gene mutation, and can better understand the protection mechanism of neural stem cells based on the pathogenic mechanism. The method is also helpful for developing new drug targets and screening new small molecule compounds. Thereby providing convenience for drug development, disease models, poison screening and clinical cell transplantation treatment for the repair and regeneration of nerve injury. Therefore, the disease condition is prevented from continuously developing, the symptoms are relieved, the mood of the patient is soothed, and a new hope is brought to the patient.

The peripheral blood mononuclear cells homozygous and mutated at the c.626C > G and p.T209R sites of the GCH1 gene are reprogrammed in vitro, so that somatic cells are reversed into pluripotent stem cells.

In a preferred embodiment of the present invention, the method further comprises subjecting the human peripheral blood mononuclear cells to amplification culture in an amplification medium before mixing the isolated human peripheral blood mononuclear cells carrying the GCH1 gene mutation with Sendai virus; the amplification medium was StemPro-34 complete medium containing 20-30ng/ml IL3, 20-30ng/ml IL6, 100-150ng/ml SCF and 100-150ng/ml FLT 3.

The amplification medium is obtained by screening through a large number of experiments for a long time by the inventor, and the 4 components synergistically promote the proliferation of precursor B cells by adding specific concentrations of IL3, IL6, SCF (stem cell growth factor) and FLT3(Fms-like tyrosine kinase 3). The number of pluripotent stem cells can be maintained by the synergistic effect of SCF and other cytokines.

In an alternative embodiment, the cultured cells are plated after 4-5 days of expansion culture and Matrigel is added for subsequent viral infection. Matrigel is a cell-based mixture containing laminin and various growth factors. Available from commercial purchase.

In a preferred embodiment of the invention, the transcription factor encoding gene comprises the hNGN2 gene; the screening gene is selected from at least one of eGFP gene, YFP, puromycin resistance gene, hygromycin resistance gene and neomycin gene (Neo); in other embodiments, the type of selection gene to be inserted into the expression cassette may be selected as desired, as long as the selection of cells is satisfactory.

The specific promoter is selected from UBC promoter; the tetracycline-inducible promoter was chosen for the CMV promoter.

In other embodiments, the specific promoter may be selected from the group consisting of an albumin promoter, an apolipoprotein E promoter, a phosphoenolpyruvate carboxykinase promoter, an alpha-I-antitrypsin promoter, a thyroid hormone binding globulin promoter, an alpha-fetoprotein promoter, an alcohol dehydrogenase promoter, an IGF-II promoter, a factor VIII promoter, an HBV basic core protein promoter, an HBV pre-s 2 protein promoter, a thyroxine-binding globulin promoter, a hybrid promoter of HCR-Ap0CII, an HCR-hAAT hybrid promoter, an AAT promoter associated with an enhancer element of the mouse albumin gene, a low density lipoprotein promoter, a pyruvate kinase promoter, a lecithin-cholesterol acyltransferase promoter, an apolipoprotein H promoter, an iron transfer protein promoter, an alpha-fetoprotein promoter, an alcohol dehydrogenase promoter, an IGF-II promoter, a factor VIII promoter, an HBV basic core protein promoter, an HBV pre-s 2 protein promoter, a thyroxine-binding globulin promoter, a hybrid promoter, an HCR-hAAT promoter, an hAAT promoter associated with an enhancer element of a mouse albumin gene, an apolipoprotein H promoter, an apolipoprotein E promoter, an alpha-hAAT promoter, an alpha-fetoprotein gene, an alpha-fetoprotein promoter, an alpha-, A transthyretin promoter, promoters of alpha-fibrinogen and beta-fibrinogen, an alpha-I-antichymotrypsin promoter, an alpha-2-HS glycoprotein promoter, a haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a complement protein promoter, a complement C3 activator promoter, a hemopexin promoter and an alpha-I-acidic glycoprotein promoter.

In a preferred embodiment of the present invention, the titer of Sendai virus MOI is KOS: c-Myc: KLF4 was 5:5: 2.5-3. The KOS reagent was Sendai virus containing the SOX2 gene and the OCT4 gene. Through the screening of the inventor, the efficient reprogramming of the cells can be met under the condition of the mixing ratio. It should be noted that, with the above mixing ratio provided by the inventors, only a trace volume (80-100 ul of virus + cell sap) is required to complete reprogramming of cells.

In a preferred embodiment of the invention, the virus carrying the regulatory expression cassette and the virus carrying the response expression cassette are mixed in a ratio of 1:1 to 1.1; and adding polybrene with final concentration of 8-10 μ g/ml for culture. Under the mixing ratio of the pluripotent stem cells and the viruses, virus infection of iPSC can be efficiently realized, so that induced pluripotent stem cells are obtained.

In an alternative embodiment, after the pluripotent stem cells are infected by the virus carrying the regulatory expression cassette and the virus carrying the response expression cassette for 40-48h, the cells are cultured for 7-10 days by replacing the StemFlex complete medium with 400-420. mu.g/ml G418, and the culture is continued by replacing the StemFlex complete medium with 200-220. mu.g/ml G418 to obtain the induced pluripotent stem cells carrying the GCH1 gene mutation.

The method for differentiating the induced pluripotent stem cell carrying the GCH1 gene mutation into the neural precursor cell, which is prepared by the preparation method, comprises the following steps: and inducing the induced pluripotent stem cells in a neural precursor cell induction culture medium to enable the induced pluripotent stem cells to differentiate towards the neural precursor cells.

In a preferred embodiment of the present invention, the neural precursor cell induction medium is N2 basal medium containing Thx and B27 medium; the culture time is 13-15 days.

Thx is N-benzyl-2- (pyrimidin-4-ylamino) thiazole-4-carboxamide.

The method for differentiating the neural precursor cell prepared by the above preparation method into the NGN2 neuron, comprising: the neural precursor cells were seeded on the Matrigel-treated plate, cultured in NPC medium containing Thx at a final concentration of 2 to 2.2 μ M and Dox at 1 to 1.2ug/ml, then replaced with NPC medium containing Dox, replaced with neural medium containing Dox (inducer), cultured for 9 to 10 days while changing the medium, then replaced with neural medium, and the culture was continued for 7 to 10 days.

In a preferred embodiment of the invention, the neural medium is an Ara-C medium containing 50-60 nM.

The method for differentiating the induced pluripotent stem cell carrying the GCH1 gene mutation into the midbrain dopamine neuron, which is prepared by the preparation method, comprises the following steps:

and inoculating the induced pluripotent stem cells into a midbrain dopamine neuron differentiation culture medium for culture.

In an alternative embodiment, induced pluripotent stem cells are seeded into a Matrigel-treated plate, cultured in a stemFlex complete medium containing THX, and then the stemFlex complete medium is replaced on the day of differentiation with a KSR basal medium containing an ALK inhibitor at a final concentration of 10-12. mu.M and a 100-plus 110nM BMP I receptor kinase inhibitor, and then replaced with a KSR basal medium containing an ALK inhibitor at a final concentration of 10-12. mu.M, a 100-plus 110nM BMP I receptor kinase inhibitor, 200-plus 210ng/ml SHH, 2-2.2. mu.M purine morpholine and 100-plus 110ng/ml heparin-binding growth factor for 2-3 days, and then replaced with a KSR basal medium containing an ALK inhibitor at a final concentration of 10-12. mu.M, a 100-plus 110nM BMP I kinase inhibitor, and a KSR basal medium containing 10-12. mu.M, 200-210ng/ml SHH, 2-2.2. mu.M purine morpholine, 100-110ng/ml heparin binding growth factor and 2-3. mu.M GSK-3 inhibitor for 2-3 days;

then, the medium was replaced with KSR basal medium containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, 2-2.2. mu.M purine morpholine, 100-110ng/ml heparin binding growth factor and 2-3. mu.M GSK-3 inhibitor at the final concentration, and N2 basal medium containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, 2-2.2. mu.M purine morpholine, 100-110ng/ml heparin binding growth factor and 2-3. mu.M GSK-3 inhibitor, and the culture was continued for 2-3 days with the mixing volume ratio of KSR basal medium and N2 basal medium being 70-75%: 25 to 30 percent;

then, the KSR basal medium containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, and 2-3. mu.M GSK-3 inhibitor at the final concentration and the N2 basal medium containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, and 2-3. mu.M GSK-3 inhibitor were replaced for further culturing for 2-3 days, and the mixing volume ratio of the KSR basal medium and the N2 basal medium was 1: 1.2;

then, the KSR basal medium containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, and 2-3. mu.M GSK-3 inhibitor at the final concentration and the N2 basal medium containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, and 2-3. mu.M GSK-3 inhibitor were replaced for further culturing for 2-3 days, and the mixing volume ratio of the KSR basal medium to the N2 basal medium was 25-30%: 70-75%;

then replacing the culture medium with a culture medium which is added with BDNF with the final concentration of 20-25ng/ml, GDNF with the final concentration of 10-12ng/ml, ascorbic acid with the final concentration of 0.2-0.3mM, TGF beta 3 with the final concentration of 1-1.2ng/ml, cAMP with the final concentration of 0.5-0.7mM, DAPT with the final concentration of 10-12 mu M and GSK-3 inhibitor with the final concentration of 2-3 mu M on the basis of a nerve substrate culture medium, glutamine, an N2 basic culture medium and a B27 basic culture medium; continuously culturing for 2-3 days;

then replacing the culture medium with a culture medium which is added with BDNF with the final concentration of 20-25ng/ml, GDNF with the final concentration of 10-12ng/ml, ascorbic acid with the final concentration of 0.2-0.3mM, TGF beta 3 with the final concentration of 1-1.2ng/ml, cAMP with the final concentration of 0.5-0.7mM and DAPT with the final concentration of 10-12 mu M on the basis of a nerve base culture medium, glutamine, an N2 basic culture medium and a B27 basic culture medium; the culture is continued for 7-20 days.

In an alternative embodiment, the GSK-3 inhibitor is CHIR-99021, the heparin binding growth factor is FGF-8b, the ALK inhibitor is SB431542, and the BMP I receptor kinase inhibitor is selected from LDN 193289.

The SHH is fully called as follows: recombinant Human Sonic Hedgehog N-Terminanus.

The invention has the following beneficial effects:

the invention provides a method for preparing induced pluripotent stem cells with GCH1 gene mutation, which can invert peripheral blood mononuclear cells into induced pluripotent stem cells, thereby obtaining the induced pluripotent stem cells with GCH1 gene mutation. Under specific conditions, the induced pluripotent stem cells can be induced to differentiate into neural progenitor cells, and the neural progenitor cells can be further induced to differentiate into corresponding neuronal cells, such as NGN2 neurons. The induced pluripotent stem cells are induced to differentiate, so that various neurons such as mesencephalon dopamine neurons can be directly prepared.

Based on the establishment of the GCH1 gene mutation iPS cell strain and the success of the preparation of related neuron cells, people can better understand the pathogenic mechanism of GCH1 gene mutation, and can better understand the protection mechanism of neural stem cells based on the pathogenic mechanism. The method is not only beneficial to developing new drug targets, but also beneficial to screening out new small molecule compounds. Thereby providing convenience for drug development, disease model construction, poison screening and clinical cell transplantation treatment for repairing and regenerating nerve injury. And the symptoms are relieved by preventing the disease from continuing to develop, the mood of the patient is further soothed, and a new hope is brought to the patient.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the immunofluorescence results of pluripotent stem cell iPSC;

FIG. 2 shows immunofluorescence results of neural precursor cells;

FIG. 3 is the immunofluorescence results of NGN2 neurons;

FIG. 4 is the immunofluorescence results for mesencephalic dopamine neurons.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The partial reagents and culture media used in the invention are as follows:

KSR basal medium: knockout DMEM, 15% Knockout serum replacement, 1 Xglutamine, 10. mu.M beta-mercaptoethanol.

NB/N2/B27 basal medium: nerve base, 1 Xglutamine, 1 XN 2, 1 XB 27.

N2 basal medium: DMEM/F12, 1 XMEM non-essential amino acids, 1 Xglutamine, 0.16% D-glucose, 1 XN 2, 200. mu.M ascorbic acid.

NPC medium: DMEM/F12, 1 Xglutamine, 1 XN 2, 1 XB 27, 20ng/ml FGF2 and 1mg/ml Lamin.

N2/B27 Medium: DMEM/F12, 1 Xglutamine, 1 XN 2, 1 XB 27, 0.1uM LDN193189, 10uM SB-431542.

Example 1

This example provides a method for preparing pluripotent stem cells carrying a mutation in the GCH1 gene.

1. Separation of peripheral blood mononuclear cells:

(1) 10ml of peripheral blood (blood from a nurse collected patient) was collected using a heparin sodium-containing blood collection tube, and separation of peripheral blood mononuclear cells was completed within 2 hours.

(2) Peripheral blood from the blood collection tube was transferred to a 50ml centrifuge tube, another sterile 50ml centrifuge tube was prepared, and the separation medium was added to the centrifuge tube.

(3) Use the pipette to suck peripheral blood of mixing, slowly add peripheral blood in the centrifuging tube that contains the parting liquid, both proportions are, whole blood: separation liquid 4: 3.

(4) the mixture was centrifuged horizontally at 2000rpm at 25 ℃ for 18 min. The centrifuge is lifted up at 3 and lowered down at 1, and slowly lifted up and lowered down.

(5) And (3) gently taking out the centrifuge tube from the centrifuge, and after centrifugation, showing that the tube is divided into 4 layers, wherein the first layer is plasma, the second layer is a cell layer, the third layer is separation liquid, and the fourth layer is a red blood cell layer.

(6) The upper plasma layer was transferred to a new 15ml centrifuge tube, and the cell layer was carefully transferred to a new 50ml centrifuge tube and supplemented with PBS to 50 ml.

(7) Centrifuging at 1500rpm for 10min at room temperature, and discarding the supernatant when cell precipitation is observed at the bottom of the tube.

(8) Media was added to resuspend the cells and cell counts were performed.

(9) After cell counting, a part of cells are taken for high-throughput sequencing, and the detection content comprises 6259 monogenic genetic diseases. The sequencing result shows that a mutation site exists in the GCH1 gene in the detected sample, and the mutation site c.626C > G is homozygous mutation.

(10) The remaining cells were subjected to cryopreservation and subsequent cell reprogramming.

2. Reprogramming the peripheral blood mononuclear cells in the step 1 into ipscs:

(1) the separated PBMCs are counted at 1X10⁶Cell transfer toCentrifuge in 15ml centrifuge tubes at 1200rpm for 10min at room temperature.

(2) Cells were resuspended in 200. mu.l expansion medium (StemPro-34 complete medium containing 20ng/ml IL3, 20ng/ml IL6, 100ng/ml SCF, and 100ng/ml FLT 3) and transferred to 96-well low adsorption plates and cultured overnight in a cell culture incubator.

(3) The culture was continued for the next 4 days, with fresh amplification medium being replaced each day.

(4) Spreading 96-well plate one day before infecting cells, diluting Matrigel at a ratio of 1:100, adding into 96-well plate, sealing the plate with sealing film, and standing in 4 deg.C refrigerator overnight.

(5) Reprogramming was performed using Sendai virus (CytotUne iPS2.0 Sendai replication Kit from ThermoFisher) and the procedure for infecting cells was as follows:

1) resuspend the cells in a 96-well plate, remove 5X 104 cells to a 1.5ml EP tube after cell counting.

2) To the EP tube cell suspension was added the calculated amount of virus (Sendai virus titer MOI 5:5:3 (KOS: c-Myc: KLF4)) to bring the cell suspension to a final volume of 100 ul. Sendai virus carries OCT4 gene, SOX2 gene, KLF4 gene and c-Myc gene.

3) The cell suspension was transferred to a 96-well plate, centrifuged at 2250rpm for 30min at room temperature, and then placed in an incubator overnight after centrifugation.

(6) The next day of virus infection of cells, fluid change was performed and fresh complete medium was changed to remove Sendai virus.

(7) The culture medium with the volume ratio of StemPro-34 to StemFlex being 1:1 was replaced on the fourth day after virus infection of the cells, and the culture was continued overnight in the incubator.

(8) The fifth day after virus infection of cells, the StemFlex medium was changed every other day.

(9) The formation of clones was seen around the fourteenth day, after which the fresh StemFlex medium was changed daily.

(10) When the colonies grew sufficiently, the colonies were picked under a microscope and transferred to a 24-well plate for culture.

(11) The monoclonals grow in 24-well plates to a density of about 90%, and the cells are digested.

(12) And analyzing, identifying and storing the purified monoclonal to obtain the iPSC cell strain.

Example 2

This example provides a method for preparing induced pluripotent stem cells carrying a mutation in the GCH1 gene.

1. Viruses carrying regulatory expression cassettes and viruses carrying response expression cassettes were prepared separately. Specifically, packaging and concentration of rtTA lentivirus and NGN2 lentivirus, respectively, was performed.

(1) The inoculation amount was 1X10⁷The HEK293 cells were plated onto 10cm diameter cell culture dishes and incubated overnight in an incubator.

(2) Two centrifuge tubes, designated as Plasmid (pLV-TetO-hNGN2-eGFP-Neo, CMV-rtTA) and PEI, were labeled and Opti MEM medium was added to both centrifuge tubes.

(3) Add plasmid and PEI to the designated centrifuge tube, mix by gentle pipetting, and let stand at room temperature for 5 min.

(4) After the end of the quiescence, the medium in the centrifuge tube named PEI was transferred to a Plasmid tube, mixed well with a pipette and allowed to stand at room temperature for 15 min.

(5) After the standing is finished, the mixed solution in the centrifugal tube is added into HEK293 cells drop by drop, and the HEK293 cells are placed in an incubator for 6 hours and then replaced by fresh culture medium.

(6) The first virus supernatant was collected 48h after cell transfection, and the second virus supernatant was collected 72h after cell transfection, and the collected supernatants were filtered through 0.45 μm filters.

(7) And (3) carrying out centrifugal concentration on the collected virus supernatant at 4 ℃ and 19400rpm, removing the supernatant after centrifugation, adding a basic culture medium to resuspend the virus, and subpackaging and storing in a negative 80 refrigerator. Lentiviruses carrying pLV-TetO-hNGN2-eGFP-Neo and CMV-rtTA were obtained.

2. Screening of NGN2/rtTA lentivirus infected iPSC and stable cell strain

(1) When the cell density reached ninety percent, iPS cells were digested from the culture wells, lentiviruses carrying pLV-TetO-hNGN2-eGFP-Neo and lentiviruses of CMV-rtTA were added to the cell suspension, and polybrene was added to a final concentration of 10. mu.g/ml and incubated overnight in an incubator.

(2) The complete medium was replaced with fresh StemFlex and incubated overnight in the incubator.

(3) 48 hours after virus infection of cells, the complete StemFlex medium containing 400. mu.g/ml G418 was replaced and incubated overnight in an incubator.

(4) After culturing for 7 days using the complete StemFlex medium containing 400. mu.g/ml G418, the culture was continued by replacing the complete StemFlex medium containing 400. mu.g/ml G418.

(5) An iPS cell line with Dox-induced expression of NGN2 was obtained.

Example 3

This example provides a method for differentiating the induced pluripotent stem cells prepared in example 2 into Neural Precursor Cells (NPCs).

(1) The iPSC after G418 screening is inoculated into a 6-well plate processed by Matrigel for culture.

(2) The cell density reached around 90%, the cells were digested with 0.5mM EDTA for 3 minutes, the EDTA was discarded after the digestion was completed, and 3ml of N2/B27 medium containing Thx was added to resuspend the cells.

(3) The cell suspension was gently transferred to a low adsorption well plate and incubated overnight in an incubator to form embryoid bodies.

(4) Cell change was performed, and the cell pellet in the well plate was gently transferred to a 15ml centrifuge tube containing 5ml of DMEM basal medium and left to stand for 15 min.

(5) After standing, cell pellets were visible to settle at the bottom of the centrifuge tube, and the centrifuge tube supernatant medium was carefully aspirated away.

(6) The cell pellet was gently resuspended by adding 3ml of N2/B27 medium to the centrifuge tube and the cell suspension was transferred back to the well plate and incubated overnight in an incubator.

(7) Repeating the steps (4) to (6) every day, and continuously culturing for 7 days.

(8) The cell pellet formed in the well plate was carefully transferred to a Matrigel-treated 24-well plate, 500. mu.l of N2/B27 medium containing Thx was added to the plate, and cultured overnight in an incubator.

(9) Cell change was performed and culture was continued by adding 500. mu. l N2/B27 medium.

(10) On the next 6 days, cell changes were made daily and culture continued by adding fresh N2/B27 medium.

(11) Cell digestion was performed using Accutase digest, cells were resuspended in DMEM basal medium and transferred to centrifuge tubes.

(12) Centrifuge at 1200rpm for 3min at room temperature.

(13) After centrifugation, the supernatant was aspirated off, the cells were resuspended in 2ml NPC medium containing Thx, and plated onto Matrigel-treated well plates overnight.

(14) The culture was continued the next day by replacing fresh NPC medium.

(15) And (5) carrying out cell passage and cryopreservation when the cell density reaches about 90%.

Example 4

This example provides a method of differentiating the Neural Precursor Cells (NPCs) prepared in example 3 into NGN2 neurons.

(1) NPCs were inoculated into Matrigel-treated 24-well plates and cultured overnight using NPC medium containing Thx and 1ug/ml Dox.

(2) The next day, the culture was continued overnight by replacing fresh Dox-containing NPC medium.

(3) On the third day, the medium was changed to Dox-containing nerve medium, and thereafter, cell culture was performed every 2 days until day 13.

(4) On days 13 to 21, the culture was continued by replacing the medium with a neural medium containing 50nM Ara-C to obtain NGN2 neurons.

Example 5

This example provides a method for differentiating the induced pluripotent stem cells prepared in example 2 into mesencephalic dopamine neurons.

(1) Induced pluripotent stem cells prepared in example 2 were seeded into Matrigel-treated 24-well plates and cultured overnight using StemFlex complete medium containing Thx.

(2) The next day, fresh StemFlex complete medium was changed and culture was continued until the cell density reached about 90% and differentiation began. Relevant basal media for differentiation were as follows:

1) on the day of differentiation, fresh medium was changed by adding SB431542 at a final concentration of 10. mu.M and LDN193289 at 100nM to KSR basal medium.

2) The day after differentiation, the fresh medium was changed by adding SB431542 at a final concentration of 10. mu.M, LDN193289 at 100nM, SHH at 200ng/ml, purine morpholine at 2. mu.M and FGF-8b at 100ng/ml to KSR basal medium.

3) On day 4 of differentiation fresh medium was changed by addition of 10. mu.M SB431542, 100nM LDN193289, 200ng/ml SHH, 2. mu.M purine morpholine, 100ng/ml FGF-8b and 3. mu.M CHIR-99021 on the basis of KSR basal medium.

4) On day 6 of differentiation, fresh medium was replaced by 75% KSR basal medium and 25% N2 basal medium, to which LDN193289, SHH at 200ng/ml, purine morpholine at 2. mu.M, FGF-8b at 100ng/ml and CHIR-99021 at 3. mu.M were added to a final concentration of 100 nM.

5) On day 8 of differentiation fresh medium was changed, based on 50% KSR basal medium and 50% N2 basal medium, LDN193289 at a final concentration of 100nM, SHH at 200ng/ml and CHIR-99021 at 3. mu.M.

6) Ten days of differentiation fresh medium was changed, based on 25% KSR basal medium and 75% N2 basal medium, with LDN193289 at a final concentration of 100nM, SHH at 200ng/ml and CHIR-99021 at 3. mu.M.

7) On day 12 of differentiation, fresh medium was replaced by adding BDNF, GDNF, 0.2mM Ascorbic acid, 1ng/ml TGF β 3, 0.5mM cAMP, 10. mu.M DAPT and 3. mu.M CHIR-99021 to NB/N2/B27 basal medium to a final concentration.

8) On day 14 of differentiation, fresh medium was replaced by adding BDNF, GDNF, 0.2mM Ascorbic acid, 1ng/ml TGF β 3, 0.5mM cAMP and 10. mu.M DAPT to NB/N2/B27 basal medium at a final concentration of 20 ng/ml.

9) Fresh medium was changed every 2 days after the 14 th day of differentiation.

10) On day 21 of differentiation, more and more dopaminergic neurons/TH positive cells appeared.

11) Differentiation was performed by immunofluorescence analysis until day 30.

Experimental example 1

Immunofluorescence experiments were performed on the pluripotent stem cell ipscs prepared in example 1, the neural precursor cells obtained by differentiation in example 3, the NGN2 neurons obtained by differentiation in example 4, and the mesencephalon dopamine neurons obtained by differentiation in example 5, respectively, and the experimental procedures were as follows:

(1) and (4) carrying out immunofluorescence analysis when the cell density in the pore plate reaches about 70%.

(2) The medium in the well plate was aspirated off and the cells were rinsed twice with PBS.

(3) PBS was aspirated off, and 4% paraformaldehyde was added to the well plate and fixed for 10min at room temperature.

(4) Paraformaldehyde was aspirated, cells were rinsed twice with PBS and incubated in a blocking chamber for 1 hour.

(5) The blocking solution was aspirated and primary antibody was added and incubated overnight in a refrigerator at 4 ℃.

(6) After the incubation was completed, the primary antibody was recovered, PBST was added to rinse the cells three times, and a secondary antibody was added to incubate for 1 hour at room temperature.

(7) After incubation, PBST was added to rinse the cells three times and recorded by photographing under a fluorescence microscope.

FIG. 1 shows the immunofluorescence result of pluripotent stem cell iPSC, and it can be seen from the immunofluorescence result that the cells express iPSC specific marker proteins SOX2 and OCT4, and the cell morphology can be preliminarily determined, and the reprogramming of PBMC into iPS cells can be successfully realized.

FIG. 2 shows the immunofluorescence results of neural precursor cells, and from the immunofluorescence results, it can be seen that the cells express NPC specific marker proteins Nestin and SOX1, and from the cell morphology, the cells show obvious "rosette ring" shape, and it can be preliminarily determined that iPSC successfully achieves differentiation into NPC.

FIG. 3 shows the immunofluorescence results of NGN2 neurons, and it can be seen from the immunofluorescence results that an inducer Dox is added into a culture medium, NGN2 can be seen under a fluorescence microscope to realize expression, obvious NGN2 neuron generation can be seen from cell morphology, and the successful differentiation of iPSC into NGN2 neurons can be preliminarily determined. Fig. 3 is a micrograph from left to right in the same field of view, with magnification increasing from left to right.

Fig. 4 is an immunofluorescence result of a midbrain dopamine neuron, and it can be seen from the immunofluorescence result that the midbrain dopamine neuron specific marker proteins TH and Tuj1 are expressed by cells, obvious midbrain dopamine neuron generation is seen from the cell morphology, and the successful differentiation of ipscs into midbrain dopamine neurons can be preliminarily determined.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing induced pluripotent stem cells carrying GCH1 gene mutation, which comprises the following steps: carrying out cell reprogramming on isolated human peripheral blood mononuclear cells carrying GCH1 gene mutation so as to obtain pluripotent stem cells carrying GCH1 gene mutation; then carrying out mixed culture on the pluripotent stem cells carrying the GCH1 gene mutation, viruses carrying a regulation expression cassette and viruses carrying a reaction expression cassette to obtain induced pluripotent stem cells carrying the GCH1 gene mutation;

the reprogramming of the cell comprises: infecting the isolated human peripheral blood mononuclear cells carrying the GCH1 gene mutation with Sendai virus carrying the c-Myc gene, the SOX2 gene, the KLF4 gene and the OCT4 gene, and culturing until obtaining the GCH1 gene mutated pluripotent stem cells; the GCH1 gene is mutated into T209R of GCH1 gene.

2. The method according to claim 1, further comprising subjecting the isolated human peripheral blood mononuclear cells carrying the GCH1 gene mutation to amplification culture in an amplification medium before mixing the isolated human peripheral blood mononuclear cells with Sendai virus; the amplification medium is StemPro-34 complete medium containing 20-30ng/ml IL3, 20-30ng/ml IL6, 100-150ng/ml SCF and 100-150ng/ml FLT 3;

preferably, the cultured cells are plated after 4-5 days of expansion culture and Matrigel is added for subsequent viral infection.

3. The method of claim 1 or 2, wherein the transcription factor-encoding gene comprises the hNGN2 gene; the screening gene is selected from at least one of eGFP gene, YFP, puromycin resistance gene, hygromycin resistance gene and neomycin gene;

the specific promoter is selected from UBC promoters; the tetracycline-inducible promoter is selected from the CMV promoter.

4. The method according to claim 1, wherein the titer MOI of Sendai virus is KOS: c-Myc: KLF4 was 5:5: 2.5-3.

5. The method according to claim 1, wherein the virus carrying the regulatory expression cassette and the virus carrying the response expression cassette are mixed at a ratio of 1:1 to 1.1; adding polybrene with final concentration of 8-10 μ g/ml for culturing;

preferably, after the pluripotent stem cells are infected by the virus carrying the regulatory expression cassette and the virus carrying the response expression cassette for 40-48h, the cells are cultured for 7-10 days by replacing the complete culture medium of StemFlex with 400-420 mu G/ml G418, and the cells are further cultured by replacing the complete culture medium of StemFlex with 200-220 mu G/ml G418 to obtain the induced pluripotent stem cells carrying the GCH1 gene mutation.

6. The method for differentiating the induced pluripotent stem cell carrying the GCH1 gene mutation into a neural precursor cell according to any one of claims 1 to 5, comprising: and inducing the induced pluripotent stem cells in a neural precursor cell induction culture medium to enable the induced pluripotent stem cells to differentiate towards the neural precursor cells.

7. The method of claim 6, wherein the neural precursor cell induction medium is N2 basal medium containing Thx and B27 medium; the culture time is 13-15 days.

8. A method for differentiating neural precursor cells obtained by the production method according to any one of claims 6 to 7 into NGN2 neurons, comprising: the neural precursor cells were inoculated into the Matrigel-treated plate, cultured in NPC medium containing Thx at a final concentration of 2-2.2 μ M and Dox at 1-1.2ug/ml, then replaced with NPC medium containing Dox, and then replaced with neural medium containing Dox, during which culture was carried out for 9-10 days, during which the medium was replaced, then replaced with neural medium, and culture was continued for 7-10 days.

9. The method of claim 8, wherein the neural medium is Ara-C medium containing 50-60 nM.

10. The method for differentiating the induced pluripotent stem cell carrying the GCH1 gene mutation into a mesencephalon dopamine neuron according to any one of claims 1 to 5, which comprises:

inoculating the induced pluripotent stem cells into a midbrain dopamine neuron differentiation culture medium for culture;

preferably, the induced pluripotent stem cells are inoculated into the Matrigel-treated plate, cultured in a stemFlex complete medium containing THX, and then the stemFlex complete medium is replaced with a KSR basal medium containing an ALK inhibitor at a final concentration of 10-12. mu.M and a 100-plus 110nM BMP I receptor kinase inhibitor on the day of differentiation, and then the culture is continued for 2-3 days in a KSR basal medium containing an ALK inhibitor at a final concentration of 10-12. mu.M, a 100-plus 110nM BMP I receptor kinase inhibitor, 200-plus 210ng/ml SHH, 2-2.2. mu.M purine morpholine and 100-plus 110ng/ml heparin-binding growth factor, and then replaced with a KSR basal medium containing an ALK inhibitor at a final concentration of 10-12. mu.M, a 100-plus 110nM BMP I kinase inhibitor, 200-plus 210ng/ml SHH, 100-plus 110nM BMP I kinase inhibitor, and 100-plus 110ng/ml SHR, 2-2.2. mu.M purine morpholine, 100-110ng/ml heparin binding growth factor and 2-3. mu.M GSK-3 inhibitor for 2-3 days;

then replacing the basic medium with KSR containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, 2-2.2 muM purine morpholine, 100-110ng/ml heparin binding growth factor and 2-3 muM GSK-3 inhibitor at the final concentration, and the basic medium with N2 containing 100-110nM BMP I receptor kinase inhibitor, 200-210ng/ml SHH, 2-2.2 muM purine morpholine, 100-110ng/ml heparin binding growth factor and 2-3 muM GSK-3 inhibitor, and continuing culturing for 2-3 days, wherein the mixing volume ratio of the KSR basic medium and the N2 basic medium is 70-75%: 25 to 30 percent;