CN117264891B - Method for obtaining high-purity nerve cells - Google Patents
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Abstract
The invention relates to a method for obtaining high-purity nerve cells, which relates to the field of biology, and is used for knocking out HBO1 genes of human pluripotent stem cells to obtain human pluripotent stem cell strains knocking out the HBO1 genes; and differentiating the human pluripotent stem cell strain with the HBO1 gene knocked out to nerve ectoderm to prepare the nerve cell. The cell of the HBO1 gene of the knocked-out human pluripotent stem cell can be withdrawn from the multipotency, and can be spontaneously differentiated into a neuron-like cell without induction, so that the high-purity nerve cell can be obtained.
Description
Technical Field
The invention relates to the field of biology, in particular to a method for obtaining high-purity nerve cells.
Background
Studies have now demonstrated that implantation of dopaminergic neurons into the brain of patients suffering from parkinson or huntington, neurons not only survive but also alleviate disease symptoms, bringing promise for treatment of the disease. But the neural cells required for transplantation are a major problem. It is extremely difficult to isolate large numbers of purified cells of the neural lineage in vivo, so in vitro differentiation of cells producing the human neural lineage is the most viable approach for cell therapy.
In order to obtain cells of the neural lineage in vitro, scientists have previously studied to overexpress neural-related transcription factors in somatic cells, successfully converting the somatic cells into cells of the neural lineage. However, this mode of transdifferentiation is inefficient and does not result in a large number of cells of the neural lineage available for transplantation, so its application prospect is limited.
Human embryonic stem cells (human Embryonic STEM CELLS, HESCS) have the potential of unlimited proliferation and can differentiate into all types of somatic cells, so that the human embryonic stem cells have good application prospect in the field of regenerative medicine. There are two common methods currently used to induce hESCs into cells of the neural lineage. The first is to use hESCs to have the potential of multidirectional differentiation, induce the hESCs to form EB balls, and separate the EB balls from the EB balls to obtain neural stem cells; the second is to induce differentiation into neural stem cells using a chemically defined medium based on developmental biology knowledge. The classical differentiation method utilizes N2 and B27 culture mediums and adds small molecules SB431542 and Dorsomophin to inhibit TGF beta-SMAD signal paths, so that the efficiency of hESCs differentiation into neural stem cells is improved. However, both of these methods have a significant disadvantage in that they do not efficiently direct the specific differentiation of hESCs into the neural lineage, and cells of other lineages appear during induction.
In addition, the neural stem cells spontaneously differentiate into neurons and glial cells when cultured in vitro, and even if proliferation of the neural stem cells is promoted by factors such as FGF2 and EGF, the spontaneous differentiation thereof is not prevented, which causes difficulty in amplifying the neural stem cells in vitro in large quantities. In view of this, the present invention is a method for obtaining high purity neural cells.
Disclosure of Invention
The invention aims to solve the technical problem of how to solve the phenomenon of insufficient purity when a great deal of neural stem cells are amplified in vitro. The object is to provide a method for obtaining high purity nerve cells to obtain high purity nerve cells.
The technical scheme for solving the technical problems is as follows:
in a first aspect, the present invention provides a method of obtaining high purity neural cells, comprising the steps of:
(1) Knocking out the HBO1 gene of the human pluripotent stem cell to obtain a human pluripotent stem cell strain knocking out the HBO1 gene;
(2) And differentiating the human pluripotent stem cell strain with the HBO1 gene knocked out to nerve ectoderm to prepare the nerve cell.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the HBO1 gene of the human pluripotent stem cell is knocked out, and the nucleotide of 182bp from 34144 to 34325 is deleted in a ninth exon, and the sequence is shown as SEQ ID NO:1, and inserting a stop codon at the deleted position, wherein the sequence of the stop codon is shown as SEQ ID NO: 2.
Further, the human pluripotent stem cells include human embryonic stem cells and human induced pluripotent stem cells, and the human embryonic stem cells are human embryonic stem cell H1 lines and the like.
In addition, it is known from the prior experimental results and related literature reports that the same phenotype should be found in primed embryonic stem cells.
Further, the step (1) specifically comprises: and (3) knocking out the HBO1 gene of the human pluripotent stem cell by using a CRISPR/Cas9 technology to obtain the human pluripotent stem cell strain with the HBO1 gene knocked out.
Further, the method for knocking out the HBO1 gene of the human pluripotent stem cells by using the CRISPR/Cas9 technology comprises the following specific steps:
(1-1) designing sgRNA of a targeted HBO1 gene, cloning the sgRNA into a CRISPR/Cas9 plasmid, constructing a plasmid of the targeted HBO1 gene knockout, and constructing a donor plasmid containing a homology arm;
(1-2) transfecting the targeting HBO1 gene knockout plasmid and the donor plasmid containing the homology arm in the step (1-1) into the human pluripotent stem cells, and screening to obtain the human pluripotent stem cell strain with the HBO1 gene knocked out.
Constructing a donor plasmid containing a homology arm, and repairing a core structural domain of the accurate knockout HBO1 by using cell DNA homologous recombination; the homology arm has two segments, the left homology arm is called the left arm, and the right homology arm is called the right arm. In the genome, the sequences in the middle of the left arm and the right arm are the regions of the invention which are planned to be knocked out; in addition, a stop codon is added in the reverse direction of the left arm, so that the reliability of gene knockout is ensured; left arm upstream primer sequence AGAGGCAGGGTTTGCTGGCTAC; left arm downstream primer sequence CTCCTATTGCCAAGAAATCAAAAGTCA; right arm upstream primer sequence GCACATGGTGAGTTGTCTTGGGTT; the right arm downstream primer sequence CTGGAGAACCTGAGTCAAAGCAACA.
Further, the nucleotide sequence of the sgRNA in step (1-1) is shown as SEQ ID NO. 3.
Further, in step (1-1), the plasmid targeting the HBO1 gene knockout is a pX330 plasmid containing Cas9, and the donor plasmid containing the homology arm is a pUC57 plasmid containing the donor.
Further, the step (2) of differentiating the human pluripotent stem cell strain from which the HBO1 gene is knocked out into neuroectoderm specifically comprises the following steps: and culturing the human pluripotent stem cell strain with the HBO1 gene knocked out in a culture medium (such as mTESR1 culture medium), and carrying out passage differentiation to obtain the nerve cell.
In a second aspect, there is provided the use of a neural cell obtained by the method in a medicament for the treatment of parkinson's or huntington.
The invention discovers that the acetyl transferase HBO1 plays an important role in hESCs self-updating and early fate decision process in the research process, and the human pluripotent stem cells lacking HBO1 exit from pluripotency, lose the potential of differentiation to mesoderm and endoderm and have the tendency of differentiation to neuroectoderm. Thus, human pluripotent stem cells that can induce knockout of HBO1 were constructed as seed cells for obtaining high-purity human neural lineage cells. And constructed and verified by the following experiments:
(1) Knocking out HBO1 in a human embryonic stem cell line H1 by combining a CRISPR/Cas9 technology and a homologous recombination mode, finding that H1-HBO1 -/- hESCs exit pluripotency and specifically differentiate towards neuroectoderm, and finally forming a neuron-like cell;
(2) By constructing H1 hESCs (H1-OE-HBO 1 -/- hESCs) capable of inducting and knocking out HBO1, the expression of the HBO1 is induced to maintain self-renewal under the condition of adding doxycycline (Doxycycline, dox), and the HBO1 is deleted under the condition of not adding Dox, the multipotent and specific differentiation to neuroectoderm can be exited, and finally, neuron-like cells are formed;
(3) By teratoma experiments and BMP 4-induced gastrulation models, it was found that HBO 1-deficient H1 hESCs lost the potential to differentiate towards the mesoderm, more prone to differentiation towards the neuroectoderm.
Compared with the prior art, the invention has the beneficial effects that: the cell which knocks out the HBO1 gene of the human pluripotent stem cell can be withdrawn from the pluripotent cell and can be spontaneously differentiated into a neuron-like cell without induction. For example, H1-OE-HBO1 -/- hESCs maintain self-renewal and proliferate in large amounts upon addition of Dox, and neuronal-like cells can be produced after 15 days without inducing spontaneous differentiation to the neural lineage upon removal of Dox. H1-OE-HBO1 -/- hESCs lost the potential to differentiate towards the mesoderm and endoderm, and tended to differentiate towards the neuroectoderm.
Drawings
FIG. 1 is a graph of experimental results provided in example 1 of the present invention for the knockout of HBO1 (H1-HBO 1 -/- hESCs) in H1 hESCs resulting in its ability to exit multipotency and spontaneously differentiate into neuronal cells; wherein a is the ninth exon for HBO 1; b is a PCR glue pattern; c is a histogram of transcription levels of wild-type H1 and HBO1 from the knockout cell line analyzed by RT-qPCR; d is a cell morphology graph of HBO1 knockout cell line after 30 days of culture, and the scale is 200 μm; e is expression of multipotent genes OCT4, SOX2, NANOG and neuroectodermal genes PAX6, SOX1, MAP2 and NGN2 in wild type H1 and HBO1 knockout cell lines by RT-qPCR analysis, and wild type H1 is a control; f is the expression of SOX2, TUJ1, MAP2, NEUN (all red) in immunofluorescence assay wild type H1 and HBO1 knockout cell lines, DAPI (blue) indicates nuclei, scale 50 μm;
FIG. 2 is a schematic and experimental result diagram of the construction of inducible knockout HBO1 cell lines (H1-OE-HBO 1 -/- hESCs) in H1 hESCs provided in example 2 of the present invention; wherein A is through a Tet-on overexpression system; b is verified by western blotting; c is RT-qPCR, wherein DOX can regulate and control the expression of HBO1, DOX can induce the expression of HBO1, the expression of HBO1 is recovered to a normal level after DOX is removed, and wild type H1 is used as a control; d is a schematic diagram for constructing inducible knockout HBO 1; e is a PCR glue pattern; f is that the RT-qPCR proves that DOX can regulate and control the expression of HBO1, DOX can induce the expression of HBO1, and after DOX is removed, HBO1 is not expressed;
FIG. 3 is a graph of the experimental results provided in example 2 of the present invention for the addition of Dox to H1-OE-HBO1 -/- hESCs to maintain self-renewal, and for the removal of Dox from spontaneous specification into cells of the neural lineage; wherein A is an inducible knockout cell line, 5 days, 10 days, 15 days, 20 days after addition of DOX (+DOX) and withdrawal of DOX (-DOX); b is the expression of multipotent genes OCT4, NANOG, SOX2, mesendoderm genes T, RUNX, SOX17, neuroectoderm PAX6, SOX1, MAP2 of the inducible knockout cell line in the presence of +DOX and D5, D10, D15, D20 of-DOX by RT-qPCR analysis; c is an immunofluorescence photograph showing the expression of TUJ1 (green)/SOX 2 (red), MAP2 (red)/TUJ 1 (green), TH (green)/GABA (red) neuro lineage marker genes in D35 cells of +dox and-DOX, DAPI (blue) indicating the location of the nucleus, scale 50 μm; d is a western blot experiment photograph showing the enrichment level of the pluripotency genes OCT4, SOX2, NANOG of the wild type H1 and the inducible knockout cell line in cells of +DOX and-DOX D5, D10, D15, D20;
FIG. 4 is a graph of experimental results of the loss of potential for differentiation into the mesendoderm by H1 hESCs lacking HBO1 provided in example 3 of the present invention, wherein A is a western blot photograph and GAPDH is a control; b is a schematic representation of induction of gastrulation by BMP 4; c is a cell morphology map of prointestinal embryos formed by BMP 4-induced wild H1 and inducible knockout cell line-DOX for 3 days; d is immunofluorescence to detect expression of wild type H1, inducible knockout cell line SOX2 (red)/NES (green), GATA3 (green)/T (red), GATA3 (green)/SOX 17 (red), OCT4 (green)/GATA 6 (red) three days after BMP4 induction, DAPI (blue) indicates the location of the nucleus with a scale of 50 μm; e is an H & E staining pattern of teratomas formed under conditions of wild-type H1 and DOX depletion of the inducible knockout cell line.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
Knockout of HBO1 in human embryonic stem cells results in its withdrawal from pluripotency and specific transformation into cells of the neural lineage. Results as shown in FIG. 1, FIG. 1 is a graph of experimental results provided in example 1 of the present invention in which knockout of HBO1 (H1-HBO 1 -/- hESCs) in H1 hESCs resulted in their ability to exit pluripotency and spontaneously differentiate into neuronal cells; wherein a is the ninth exon for HBO 1; b is a PCR glue graph, the genotype of the HBO1 knockout cell line is identified, wild seeds are knocked out, and sgRNA and homology arm schematic diagrams are designed. The H1 of the genotype was the negative control. C is a histogram of transcription levels of wild-type H1 and HBO1 from the knockout cell line analyzed by RT-qPCR; wild type H1 is a control; d is a cell morphology graph of HBO1 knockout cell line after 30 days of culture, and the scale is 200 μm; e is the expression of multipotent genes OCT4, SOX2, NANOG and neuroectodermal genes PAX6, SOX1, MAP2 and NGN2 in wild type H1 and HBO1 knockout cell lines by RT-qPCR analysis; wild type H1 is a control; f is the expression of SOX2, TUJ1, MAP2, NEUN (all red) in immunofluorescence assay wild type H1 and HBO1 knockout cell lines, DAPI (blue) indicates nuclei, scale 50 μm.
The specific process is as follows:
1. Construction of embryonic Stem cell lines for obtaining neural cells
A method of constructing an embryonic stem cell line for obtaining neural cells, comprising the steps of: the HBO1 gene of human embryonic stem cells (purchased at WICELL RESEARCH Institute, 5/4/2018) was knocked out using CRISPR/Cas9 technology to obtain embryonic stem cell lines for obtaining neural cells.
Specifically, the method comprises the following specific steps: the invention designs a targeted knockout aiming at MYST structural domain of HBO1, deletes 182bp (gene sequence) in a ninth exon, and adds a stop codon to ensure the effectiveness of gene knockout by early termination of translation (figure 1A). After constructing the plasmid required for knockout, gene knockout experiments were performed by electrotransformation in wild type H1.
(1) Construction of knockout plasmid
Double strand breaks can be generated in a targeted genome by using CRISPR/Cas9 technology, and the double strand breaks of DNA activate DNA repair functions of cells, and can be divided into homologous recombination and non-homologous end repair according to whether the repair needs a template or not. Compared with random uncontrollable repair of non-homologous ends, homologous recombination provides that homologous arm DNA can be recombined by taking a homologous arm as a template, and the DNA sequence after repair can be consistent with the homologous arm, so that the CRISPR/Cas9 combined homologous recombination technology is selected to design knockout of a target gene core structural domain.
1) SgRNA design principle: to achieve the goal of gene knockout, key domains of related genes were found at NCBI (https:// www.ncbi.nlm.nih.gov /). HBO1 is an acetyltransferase and we knocked out the MYST domain which has acetyltransferase activity.
2) The sgRNA design method comprises the following steps: the mRNA sequence encoding the key domain is uploaded to a website (https:// cctop. Cos. Uni-heidelberg. De /) where the sgRNA can be automatically designed, and the sgRNA with higher efficiency and fewer off-target sites is sought. sequence ACCAGGTATCAAGCTCATAG of sgRNA (SEQ ID NO: 3).
3) Annealing and renaturation: the related primer synthesis was completed in the department of Optimago, and after primer synthesis, the primers were diluted to 10. Mu.M with ddH2O, and the two complementary primers were annealed and renatured. After the system is prepared, annealing renaturation is finished by using a PCR instrument, and an annealed product can be stored at 4 ℃ for a long time.
4) And (3) enzyme digestion of plasmids: the annealed product was ligated to the pX330 support. The pX330 vector was digested (fast-cutting enzyme Bpi I) and incubated at 37℃for 1 hour before ligation. The products after cleavage were subjected to DNA agarose gel electrophoresis to identify whether the plasmid was cleaved (the plasmid after cleavage was linear and the migration rate was slower than that of the original plasmid). The enzyme digestion product is purified and recovered by using a gel recovery kit (Nuo-vozan), and the purified and recovered enzyme digestion plasmid can be stored for a long time at-20 ℃.
5) And (3) connection: and (3) connecting the annealed product with the digested pX330 plasmid, and connecting the annealed product with the digested pX330 plasmid for 1 hour in a metal bath at the temperature of 16 ℃, wherein the connected product can be directly subjected to connection transformation.
6) Conversion: mu.L of ligation product was mixed with 50. Mu.L of E.coli DH 5. Alpha. Competence in a 1.5mL EP tube and allowed to stand in ice for 30 minutes. Then put into a water bath kettle with the temperature of 42 ℃ for 90 seconds, and then put into ice for 2 minutes. 120 mu L of LB liquid medium was added to the EP tube in an ultra clean bench, and the mixture was uniformly spread on a solid plate of LB and incubated at 37℃for 16 hours.
7) Sequencing and identification: after colony was grown, single colonies were selected and cultured in a LB liquid medium of 700. Mu.L for several hours at 37℃in a shaker. The bacterial liquid is turbid and then sent to a company for sequencing, and related sequencing is completed in Guangzhou Ai Ji company and the Optimago company. Selecting bacterial liquid of sequencing pair, extracting plasmid with endotoxin removal kit
(2) Construction of knockout cell lines
1) Cell culture: when the H1 cell density reaches 80%, the cells can be used for electrotransformation. Electrotransformation refers to the transfer of a previously constructed sgRNA together with a donor plasmid into H1 cells. The target gene is knocked out by using CRISPR/Cas9 and homologous recombination repair technology.
2) Cell electrotransformation: after cells grow to the proper density, the medium is aspirated and the cells are washed with DMEM/F12. Cells were digested by adding 700. Mu.L of Actutase, and the mixture was placed in a cell incubator, and digested for about 5 minutes, whereby the cells became single cells. The digestion was stopped by dilution of Accutase with an equal volume of DMEM/F12, gently swirled to resuspend cells, and collected in 1.5mL EP tubes and centrifuged at 200g for 5 min. The supernatant was removed, and the electrotransfer reagent (82. Mu.L solution+18. Mu.L support) and the previously constructed electrotransfer plasmid (4. Mu.g pUC57-LPR and 4. Mu.g pX 330-sgRNA) were added to the EP tube, and the resuspended cells were gently blown, and the cell suspension transferred to an electrotransfer cup. And placing the electric rotating cup into an electric rotating instrument, and selecting an electric rotating program B-016 to perform electric rotation. Immediately after that, 800. Mu.L of mTESR1 was added to the electric rotating cup, the cells were resuspended, and the cells were aliquoted into 3 wells of a 6-well plate at a ratio of 1:3, fresh medium was added to the wells to 2mL, thiazovin with a final concentration of 0.5. Mu.M was added, and after shaking, they were placed into a cell incubator for culturing. Then culturing according to the normal cell culture mode.
3) Cell screening: if the cell is successfully knocked out, either the single allele or the double allele is knocked out, the cell has puromycin resistance. Whereas cells that were not knocked out were very sensitive to puromycin, they generally died after one day of puromycin addition to a final concentration of 1. Mu.g/mL. Therefore, when the cells after the electric transformation are grown into clones from single cells (clone size: about 100 μm), 1. Mu.g/mL puromycin is added for screening for 5 to 7 days. When the length of the selected cell clone is about 500 mu m, the cell clone can be singly selected and placed into a 24-well plate for continuous culture, at the moment, puromycin can be removed, and only the fresh culture medium mTESR1 is replaced for normal culture. Culturing for about 7-10 days, adding 0.5mM EDTA to digest when the cell clone grows to about 1000 μm, taking half of the cells for genome identification, and transferring the rest cells into a new 24-well plate for continuous culture.
4) Genome identification: the cells removed were centrifuged at 12000g for 2 minutes, and the supernatant was removed. Extraction of DNA was performed using an animal genomic DNA rapid extraction kit (specific steps are described in the specification) as a template for PCR identification. The primers for knocking out and identifying are designed on the homology arm, the sequence of the primers is shown in the appendix I, and then PCR identification is carried out. The PCR system is shown in tables 3-5 (total volume 10. Mu.L) and the PCR procedure is shown in tables 3-6. After PCR amplification products were obtained, agarose gel electrophoresis was performed to identify that the band of the Wild Type (WT) was about 700bp, and that of the knocked-out DNA was about 1500 bp. Depending on the number and location of the bands we can determine whether the cell is single-knocked (both bands), double-knocked (only one 1500bp band), or wild-type (only one 700bp band). After the identification, we transferred the double knocked cells to 6-well plates for further culture for subsequent mRNA identification and protein identification. After the related identification is completed, the primary task is completed to construct a knockout cell line, and the method can be used for subsequent experiments.
The invention designs a specific knockout verification primer upstream primer CCTCTGCCTCTTGGGCGATTC; the downstream primer GAAATGAAGAGCAGTAGGCAGCAAG is used for carrying out PCR identification on the obtained cell clone, the band size of the wild type H1 is 737bp, the band size of the HBO1 double-allele knockout cell is 1947bp, and the PCR results of two HBO1 double-allele knockout strains are shown in the invention (figure 1B). The invention then proceeds to detect the level of transcription of both strains of cells. The qPCR experiment shows that the transcription level of HBO1 is obviously reduced (figure 1C), and the knockout of the HBO1 is proved to be successful, so that the human embryonic stem cells (H1-HBO 1 -/- hESCs) with the HBO1 gene knocked out are obtained.
2. H1-HBO1 -/- hESCs exit pluripotency and differentiate towards the neural lineage
Surprisingly, the present invention uses mTESR1 medium (purchased from STEMCELL Technologies company) to culture the HBO1 knockout cell line 1-HBO1 -/- hESCs, which has a tendency to spontaneously differentiate during normal subculture, accompanied by the appearance of neuronal-like cells (FIG. 1D).
RT-qPCR analysis was performed on HBO1 knockout cells 1-HBO1 -/- hESCs after 30 days of culture, and it was found that the expression of the pluripotency-related genes OCT4 and NANOG was significantly decreased in the HBO1 knockout cell line of the present invention as compared with wild-type H1, but that the expression of SOX2 was significantly increased (FIG. 1E), and that the expression of the nerve-related genes PAX6, SOX1, MAP2 and NGN2 was significantly up-regulated (FIG. 1E). Immunofluorescence experiments were then performed to examine the expression of the neurolineage markers, and it was found that the neuronal markers TUJ1, MAP2, NEUN of the HBO1 knockout cell line were all expressed as compared to wild type H1 (FIG. 1F).
In summary, the present invention has found that acetyl transferase HBO1 is necessary for the maintenance of human embryonic stem cell stem properties, and that knockout of HBO1 gene results in human embryonic stem cells that differentiate toward neural directions; this suggests that HBO1 is important not only for the maintenance of human embryonic stem cell stem properties, but also plays an important and unique role in lineage differentiation.
Example 2
The invention constructs H1 hESCs (H1-OE-HBO 1 -/- hESCs) capable of inducting and knocking out HBO1, and the addition of Dox can maintain self-renewal, remove the withdrawal pluripotency of Dox and differentiate towards neuroectoderm.
HBO1 knockout is performed on the basis of a cell line (H1-HBO 1-OE hESCs) capable of inducing expression of HBO1 by constructing the cell line. The results are shown in fig. 2 and 3:
FIG. 2 is a schematic and experimental result diagram of the construction of inducible knockout HBO1 cell lines (H1-OE-HBO 1 -/- hESCs) in H1 hESCs provided in example 2 of the present invention; wherein A is a schematic diagram for constructing inducible expression HBO1 through a Tet-on overexpression system; b is that the Western blot verification proves that DOX can regulate and control the expression of HBO1, DOX can induce the expression of HBO1, and after DOX is removed, HBO1 is not expressed; c is RT-qPCR, wherein DOX can regulate and control the expression of HBO1, DOX can induce the expression of HBO1, the expression of HBO1 is recovered to a normal level after DOX is removed, and wild type H1 is used as a control; d is a schematic diagram for constructing inducible knockout HBO 1; e is a PCR glue pattern, and the genotype of an inducible knockout HBO1 cell line is identified, and the wild type H1 is a negative control; f is that the RT-qPCR proves that DOX can regulate and control the expression of HBO1, DOX can induce the expression of HBO1, and after DOX is removed, HBO1 is not expressed;
FIG. 3 is a graph of the experimental results provided in example 2 of the present invention for the addition of Dox to H1-OE-HBO1 -/- hESCs to maintain self-renewal, and for the removal of Dox from spontaneous specification into cells of the neural lineage; wherein A is an inducible knockout cell line, 5 days, 10 days, 15 days, 20 days after addition of DOX (+DOX) and withdrawal of DOX (-DOX); b is the expression of multipotent genes OCT4, NANOG, SOX2, mesendoderm genes T, RUNX, SOX17, neuroectoderm PAX6, SOX1, MAP2 of the inducible knockout cell line in the presence of +DOX and D5, D10, D15, D20 of-DOX by RT-qPCR analysis; c is an immunofluorescence photograph showing the expression of TUJ1 (green)/SOX 2 (red), MAP2 (red)/TUJ 1 (green), TH (green)/GABA (red) neuro lineage marker genes in D35 cells of +dox and-DOX, DAPI (blue) indicating the location of the nucleus, scale 50 μm; d is a western blot photograph showing the levels of enrichment of the multipotent genes OCT4, SOX2, NANOG in cells of +dox and-DOX D5, D10, D15, D20 for wild type H1 and inducible knockout cell lines.
The specific process is as follows:
1. Construction of H1 hESCs (H1-OE-HBO 1 -/- hESCs) that can induce knockout of HBO1
Constructing H1 hESCs capable of inducing to knock out HBO1, comprising the following specific steps:
(1) H1 hESCs construction inducible expression of HBO 1:
The invention uses a Tet-on system to realize the inducible expression of genes. The gene is expressed when Dox is present and not expressed when Dox is not present. FUW-HBO1 (purchased from addgene) (a plasmid that induces expression of HBO 1) and rtTA were packaged separately into lentiviruses by a second generation lentivirus packaging system. Cell H1hESCs were co-infected with packaged FUW and rtTA lentiviruses, and inducible expression cell lines were constructed by screening (FIG. 2A). The invention carries out western blotting and RT-qPCR detection on a cell line capable of inducing the over-expression of HBO1, and the result shows that the expression of HBO1 can be induced by adding Dox; after Doxycycline days of withdrawal, HBO1 expression was restored to levels consistent with wild-type H1 (fig. 2B, 2C).
(2) Construction of inducible knockout HBO1 cell lines:
Subsequently, on the basis of the H1-HBO1-OE hESCs cell line constructed as described above, a knockout experiment of HBO1 was performed, and after removing Dox for two days, electrotransfer knockout was performed (the specific operation is the same as that described in example 1), and after passing through a drug sieve, the expression of HBO1 was induced by adding Dox, and after the cell clone was grown up, it was identified (FIG. 2D). By PCR identification, the invention obtains a cell line (H1-OE-HBO 1 -/- hESCs) with double-allele knockout of HBO1 in cells capable of inducing expression of HBO1, and shows the PCR identification results of two strains of cells (FIG. 2E). Through RT-qPCR verification, compared with wild type H1, the removal of Dox leads to the significant reduction of the expression of the HBO1 gene of the inducible knockout HBO1 cell line; and HBO1 gene expression was restored after Dox addition (fig. 2F). It can be seen that the invention successfully constructs a human embryonic stem cell line capable of inducting and knocking out HBO 1.
2. Differentiation verification of H1-OE-HBO1 -/- hESCs
The present invention found that H1-OE-HBO1 -/- hESCs maintained normal ES morphology in Dox-containing medium, however, upon removal of Dox, the induced knockout of HBO1 cell line exhibited spontaneous differentiation and neuronal-like cells (FIG. 3A) with cell morphology similar to that of the present invention in wild-type H1 direct knockout of HBO1 (FIG. 1D).
The invention takes the day of removing Dox as the 0 th day, and sequentially collects cells on the 5 th, 10 th, 15 th and 20 th days for detection. Through RT-qPCR analysis, the expression of the pluripotency genes OCT4 and NANOG is gradually reduced, the expression of SOX2 is in an ascending trend, the mesoderm marker T, RUNX and the endoderm marker T, RUNX and the SOX17 are not significantly changed, and the ectoderm marker genes PAX6, SOX1 and MAP2 are significantly up-regulated in expression (figure 3B). Immunofluorescence experiments also prove that the specific markers TUJ1 and MAP2 of the neurons have obvious expression, and in addition to TH (dopaminergic neurons) and GABA (inhibitory neurons) have certain expression (figure 3C). Immunoblotting experiments showed that the protein level of HBO1 was significantly reduced after removal of Dox compared to wild-type H1 and cells with Dox-cultured H1-OE-HBO1 -/- hESCs, the expression level of the pluripotency markers OCT4, NANOG was gradually reduced, while the expression level of SOX2 was gradually increased (FIG. 3D). SOX2 is not only a marker for pluripotency, but also a marker gene for neural stem cells.
In summary, the inducible knockout HBO1 cell line constructed in the present invention is consistent with the phenotype of direct knockout HBO1 in wild-type H1 hESCs in that withdrawal of Dox results in spontaneous differentiation into the neural direction. That is, H1-OE-HBO1 -/- hESCs maintain self-renewal upon addition of Dox, exit pluripotency after removal of Dox and differentiate towards the neural lineage. It is shown that HBO1 does play a critical regulatory role in early fate decisions of H1 hESCs, which spontaneously differentiate towards the neural lineage after HBO1 depletion.
Example 3
In vivo and in vitro experiments demonstrated that HBO 1-deficient hESCs lost mesodermal differentiation. The results are shown in FIG. 4; FIG. 4 is a graph of experimental results of the loss of potential for mesoendodermal differentiation of H1 hESCs lacking HBO1 provided in example 3 of the present invention, wherein A is a western blot experimental photograph showing expression levels of HBO1 and OCT4 on days D1, D2, D3 of inducible knockout cell lines +DOX and-DOX, GAPDH is a control; b is a schematic representation of induction of gastrulation by BMP 4; c is a cell morphology map of prointestinal embryos formed by BMP 4-induced wild H1 and inducible knockout cell line-DOX for 3 days; d is immunofluorescence to detect expression of wild type H1, inducible knockout cell line SOX2 (red)/NES (green), GATA3 (green)/T (red), GATA3 (green)/SOX 17 (red), OCT4 (green)/GATA 6 (red) three days after BMP4 induction, DAPI (blue) indicates the location of the nucleus with a scale of 50 μm; e is an H & E staining pattern of teratomas formed under conditions of wild-type H1 and DOX depletion of the inducible knockout cell line.
The specific process is as follows:
1. In vitro validation
Under BMP4 ((Bone Morphogenetic Protein 4)) induction, the absence of HBO1 results in loss of mesodermal and endodermal differentiation capacity of hESCs.
After DOX was removed in FIG. 3, H1-OE-HBO1 -/- hESCs lost multipotency to begin spontaneous differentiation, but expression of mesoderm and endoderm genes was not detected in the present invention, so it was hypothesized that HBO 1-deleted hESCs lost the differentiation ability of mesoderm and endoderm.
To further verify the hypothesis of the present invention, first the present invention examined the expression level of HBO1 at day 3 when Dox was removed by western blot experiments (fig. 4A), and the results showed that the protein level of HBO1 at day 3 when Dox was removed was not substantially detected, and the expression of the multipotent core transcription factor OCT4 was not substantially changed at this time, so the present invention selects cells at this time point to examine the ability of the tricdermal differentiation thereof.
The present invention uses high concentrations of BMP4 (final concentration 50 ng/mL) to induce hESCs differentiation to form a gastrulation model (fig. 4B). Three days after BMP4 induction, both wild-type H1 hESCs and H1-OE-HBO1 -/- hESCs cells appeared in the circular structure of the gastrulation (fig. 4C). Immunofluorescence was performed on BMP4 induced differentiation D3 day cells, and the present invention found that wild type H1 hESCs formed a complete gastrula structure with the outermost layer being GATA3 positive trophoblast, followed by T positive and SOX17 positive mesoderm, endoderm and GATA6 positive primitive endoderm, with SOX2 positive neuroectoderm and undifferentiated OCT4 positive pluripotent stem cells. However, the gastrulation model formed by H1-OE-HBO1 -/- hESCs was significantly different, and although the outermost layer was still GATA3 positive trophectoderm, T positive and SOX17 positive mesoderm, endoderm and GATA6 positive primitive endoderm were absent, and the intermediate SOX2 positive neuroectoderm was significantly increased, OCT4 positive pluripotent stem cells were significantly decreased (FIG. 4D).
In summary, the present invention found that during BMP4 induced differentiation, the absence of HBO1 resulted in the loss of the ability of hESCs to differentiate into mesoderm and endoderm, which is more prone to differentiation into neuroectoderm.
2. In vivo verification
Teratomas formed by hESCs lacking HBO1 are more prone to differentiation towards neuroectoderm.
To verify whether HBO1 knockdown hESCs lost the potential for mesodermal and endodermal differentiation, they tended to differentiate towards neuroectodermal. 200 ten thousand H1 hESCs and H1-OE-HBO1 -/- hESCs cells are respectively collected, injected into the groin of an immunodeficiency mouse (purchased from Ji-Cui-kang) to form teratomas, the mouse is killed in a cervical mode after 6-8 weeks of teratomas grow to a proper size, the teratomas are taken out, and the teratomas are placed in 4% PFA for one day at 4 ℃. Teratomas were embedded in paraffin and subsequently sectioned, and visualized by photographing with hematoxylin-eosin (H & E) staining.
Compared with teratomas formed by wild type H1 hESCs, the teratomas formed by the H1-OE-HBO1 -/- hESCs have smaller and more irregular tissue structures. Wherein the neural tissue significance increased, accounting for approximately 70% of the total tissue structure, while the tissue significance of the mesoderm and endoderm decreased (fig. 4E). This result demonstrates that HBO 1-deficient hESCs are more prone to forming neuroectodermal structures.
In conclusion, the invention discovers that the acetyl transferase HBO1 plays an important role in the hESCs self-updating and early fate decision process, the hESCs lacking the HBO1 exit pluripotency, lose the potential of differentiation to the mesoderm and the endoderm and have the tendency of differentiation to the neuroectoderm; hESCs, which are inducible to knock out HBO1, are used as seed cells to obtain high purity human neural lineage cells.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (8)
1. A method for obtaining high purity neural cells, comprising the steps of:
(1) Knocking out the HBO1 gene of the human embryonic stem cell H1 line to obtain the human embryonic stem cell H1 line knocking out the HBO1 gene;
(2) And differentiating the human embryonic stem cell H1 strain from which the HBO1 gene is knocked out into neuroectoderm to prepare the nerve cell.
2. The method for obtaining high purity neural cells according to claim 1, wherein the knockout of HBO1 gene of human embryonic stem cell line H1 is deletion of nucleotide of 182bp from 34144 to 34325 in the ninth exon, and the sequence is as shown in SEQ ID NO:1, and inserting a stop codon at the deleted position, wherein the sequence of the stop codon is shown as SEQ ID NO: 2.
3. The method of obtaining high purity nerve cells according to any one of claims 1 to 2, wherein step (1) is specifically: the CRISPR/Cas9 technology is utilized to knock out the HBO1 gene of the human embryonic stem cell H1 line, and the human embryonic stem cell H1 line with the HBO1 gene knocked out is obtained.
4. A method of obtaining high purity nerve cells according to claim 3 wherein:
The method for knocking out the HBO1 gene of the H1 line of the human embryonic stem cells by using the CRISPR/Cas9 technology comprises the following specific steps:
(1-1) designing sgRNA of a targeted HBO1 gene, cloning the sgRNA into a CRISPR/Cas9 plasmid, constructing a plasmid of the targeted HBO1 gene knockout, and constructing a donor plasmid containing a homology arm;
(1-2) transfecting the targeting HBO1 gene knockout plasmid and the donor plasmid containing the homology arm in the step (1-1) into the human embryonic stem cell H1 line, and screening to obtain the human embryonic stem cell H1 line from which the HBO1 gene is knocked out.
5. The method of obtaining high purity nerve cells according to claim 4, wherein the nucleotide sequence of the sgRNA in step (1-1) is shown in SEQ ID NO. 3.
6. The method according to claim 4, wherein in the step (1-1), the plasmid targeting the knockout of HBO1 gene is a pX330 plasmid containing Cas9, and the plasmid containing the homology arm donor is a pUC57 plasmid containing donor.
7. The method for obtaining high purity neural cells according to claim 1, wherein the differentiation of the human embryonic stem cells H1 line, from which the HBO1 gene is knocked out, into neuroectoderm in step (2) is specifically as follows: and culturing the human embryonic stem cell H1 line from which the HBO1 gene is knocked out in a culture medium, and carrying out passage differentiation to obtain the nerve cell.
8. Use of a neural cell obtained by the method of claims 1 to 7 for the preparation of a medicament for the treatment of parkinson's or huntington.
Priority Applications (1)
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