CN110172442B - Human induced pluripotent stem cell, construction method and application thereof - Google Patents

Abstract

The invention relates to pluripotent stem cells (iPSCs) capable of being directionally differentiated into endothelial progenitor cells. The invention also relates to a construction method of the pluripotent stem cell and application thereof in pharmacy. The basic technical scheme claimed by the invention is as follows: a human-induced pluripotent stem cell characterized in thatF8The coding sequence of the B region of the gene is deleted in-frame near the mutation site, or the entire B region coding sequence is targeted for deletion. The invention overcomes the disadvantages ofF8A method for the aberrant expression of a gene comprising providing a modified human induced pluripotent stem cell in which the gene is abnormally expressedF8The sequence near the mutation site in the coding sequence of the B region of the gene is deleted in frame or the entire B region coding sequence is targeted for deletion. TheIn the method for obtaining the human induced pluripotent stem cells, a donor vector does not need to be constructed, only the ssODN needs to be directly synthesized, the technical realization is relatively easy, no screening gene is added, and the efficiency is higher.

Description

Human induced pluripotent stem cell, construction method and application thereof

Technical Field

The invention relates to pluripotent stem cells (iPSCs) capable of being directionally differentiated into endothelial progenitor cells. The invention also relates to a construction method of the pluripotent stem cell and application thereof in pharmacy.

Background

Hemophilia A (HA) is an X-linked hereditary hemorrhagic disease caused by a lack of functional FVIII, with an incidence of about 1/5000 in men^[1,2]. It is mainly manifested as coagulation disorders of different degrees, and clinically classified into mild, intermediate and severe types according to the coagulation activity of plasma FVIII. Light patients (FVIII clotting activity 5% -30%) typically experience bleeding after trauma or major surgery, accounting for about 40% of HA patients; intermediate patients (FVIII clotting activity 1% -5%) occasionally bleed spontaneously, with severe post-operative bleeding accounting for about 10% of HA patients; heavy patients (FVIII clotting activity)<1%) accounts for about 50% of HA patients, joint repeated bleeding and disability often occur, and even intracranial bleeding can occur to endanger life.

Currently, there is no radical cure method for HA, and clinically, replacement therapy is mainly adopted for the disease, i.e., bleeding is prevented by infusing FVIII separated from plasma or recombinant FVIII protein, or bleeding is stopped by timely infusion after bleeding. FVIII, however, has a very short half-life and is often administered by repeated lifelong infusions, which also risk potential viral infections and, more importantly, long term repeated infusions that produce neutralizing antibodies and thus render the treatment ineffective^[3,4]。

HA is caused by the fact that FVIII molecular structure defects or FVIII content reduction caused by F8 gene mutation cannot play a normal blood coagulation function. F8 as one of the largest cloned genes with a full length of 186kb^[5]Contains 26 exons, of which exon No.14 is the largest exon (3.1 kb). Process for preparation of F8The types of mutations are many, and 3231 types of mutations recorded in The Human Mutation Database (HGMD) comprise missense Mutation, nonsense Mutation, small fragment deletion and insertion and The like, which cause The function reduction or loss of FVIII^[6]。

F8 encodes a precursor polypeptide containing 2351 amino acids, and is modified by a series of processes to form FVIII mature protein containing 2332 amino acids. Based on sequence similarity, the FVIII structure can be divided into several different functional regions, including 3A-regions, 1B-region and 2C-regions, and the structure composition is N-A1-A2-B-A3-C1-C2-C (as shown in FIG. 1)^[7,8]. Wherein the binding site of calcium ion is present in region A and plays a role in the endogenous coagulation pathway.

During secretion of FVIII, the single-chain form of the protein is cleaved by furin hydrolase to produce a heterodimer consisting of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2) linked by cuprous ions. After thrombin hydrolysis, the B region is cleaved to form A1, A2, A3-C1-C2 linked by cuprous ions, which becomes active FVIII, i.e., FVIIIa, thereby exerting coagulation activity (as shown in FIG. 2)^[7,9]。

In addition, the B region is highly glycosylated, and researches show that the B region is not greatly related to the FVIII activity, and the deletion of most of the B region does not influence the FVIII activity^[10]. Although the B domain is not present in FVIII which ultimately exerts clotting activity at the protein functional level, of all mutations in the F8 gene that have been included, nearly 500 mutations occur in the B domain coding sequence, of which more than 90% cause premature translation termination of the FVIII protein, loss of FVIII clotting function, and HA^[6]。

HA HAs been considered one of the most likely monogenic genetic diseases to achieve gene therapy. One of the desirable ways is in situ repair of the mutated gene or functional correction by appropriate in situ gene editing, i.e. precise gene repair by homologous recombination at the point of the defect gene in situ or elimination of the pathogenic effects of the mutation. The method not only restores the function of the gene, but also retains the in-situ regulatory element of the gene, and transfers the mutant gene to the maximumThe physiological state of the human body is changed to be consistent with that of a normal human body. At present, HA gene in-situ repair strategies aiming at inverted mutation of intron 22 and intron 1 of F8 gene^[11,12]. Another gene therapy method is gene replacement, which is to integrate a therapeutic gene with a promoter into defective cells at random or at a fixed point, thereby achieving the purpose of treatment. Due to technical limitations, most of the gene therapy studies conducted today use gene replacement strategies, such as random integration strategies mediated by viruses, and gene addition strategies not mediated by viral vectors.

The random integration strategy mediated by virus is to transfect cells with F8 deficiency by using a plasmid containing the coding sequence of F8 and a promoter sequence, and the expression frame of F8 is integrated into the genome with a certain probability, so that the expression of FVIII protein plays a role. The disadvantages are: (1) f8 is very large, the coding sequence is about 8kb, even the B region deletion version is larger than 4kb, and the promoter sequence is added, the plasmid is very large, generally, the larger the plasmid is, the more troublesome the construction is, and the integration efficiency is very low; (2) if the gene is randomly integrated into the genome, the expression of the gene may be affected by the position effect, and the expression efficiency may be low or even impossible; (3) if the plasmid is integrated into another endogenous gene site, it may destroy the endogenous gene or activate a harmful gene that was not expressed, resulting in undesirable consequences.

The non-viral vector-mediated gene addition strategy has the following defects: (1) because F8 is very large, the coding sequence is about 8kb, even the B region deletion version is larger than 4kb, and the promoter sequence is very large, generally speaking, the larger the plasmid is, the more troublesome the construction is, the integration efficiency is very low, and exogenous screening genes are generally introduced, and the long-term influence of the residue of exogenous gene fragments on cells cannot be determined temporarily; (2) exogenous genes are integrated into a safe site in the genome, which is regulated by exogenous promoters and regulatory elements, potentially affecting expression efficiency.

Earlier studies show that an exogenous gene targeting a B region deletion type F8(B domain deletion F8, BDD-F8) can effectively express functional BDD-FVIII^[13-15]And adoptThe recombinant BDD-FVIII also has certain clinical treatment effect^[10,16]. Therefore, aiming at the pathogenic mutation of the B region coding sequence of the F8 gene, the invention provides a brand-new treatment strategy based on in-situ gene editing, namely, the pathogenic mutation is converted into the micro in-frame deletion of the B region coding sequence in situ by a targeted gene editing technology, so that the translation of FVIII protein is recovered, the pathogenic effect of FVIII translation early termination caused by the mutation is eliminated, and the coagulation activity of endogenous FVIII is recovered. On the basis, the in-frame deletion is further expanded to the deletion of the whole B region coding sequence, and the novel HA in-situ gene therapy strategy is applied to all B region pathogenic mutation patients.

Disclosure of Invention

One of the objects of the present invention is to provide human Induced Pluripotent Stem Cells (iPSCs) capable of committed differentiation into endothelial progenitor cells and normally expressing the F8 gene in human body.

Another purpose of the invention is to provide a method for obtaining the iPSCs.

It is a further object of the present invention to provide a use thereof for HA therapy, in accordance with the iPSCs obtained.

The basic technical scheme claimed by the invention is as follows: a human induced pluripotent stem cell wherein the F8 gene is deleted in-frame near the mutation site in the B region coding sequence or the entire B region coding sequence is deleted in a targeted manner.

The applicant obtained accession numbers: two cell lines of C201968 and C201990, which are only one specific example of the technical solution to be protected by the present invention. One of the cells is a cell of which the iPSCs of the patient lack 54 bases and is deposited with a deposit number of C201968, and the other cell is a cell of which the iPSCs of the patient lack the whole B area and is deposited with a deposit number of C201990.

Due to different methods of deleting mutation sites in the B region coding sequence of the F8 gene in frame or deleting the whole B region coding sequence in a targeted manner, specific sequence details of related genes in the obtained human induced pluripotent stem cells have certain differences, but the differences do not influence the expression of the related genes, so that the method belongs to the technical scheme of the invention.

According to an embodiment of the invention, the mutation site to which it is directed is a c.3167delctga variation.

In order to efficiently obtain iPSCs with in-frame deletions of in-situ B region coding sequences, with reference to the prior art, possible methods include, but are not limited to: artificial nucleases can be used. At present, artificial nucleases mainly comprise clustered regularly spaced short palindromic repeats (CRISPRs) gene editing systems, Zinc-finger enzymes (ZFNs) and TALE nucleases (TALENs)^[17,18]. These gene editing tools are used to cleave target DNA by recognition, causing double-strand breaks in DNA (DSBs)^[19]. DSB is repaired mainly by the imprecise non-homologous end joining (NHEJ) pathway^[20]Efficient gene knockout can be achieved^[21,22]. Simultaneously, DSB can activate Homologous Recombination (HR) activity near the fracture site, and can obviously improve gene targeting efficiency in the presence of a homologous repair template^[23]And the addition, the replacement, the deletion of precise gene segments or point mutation of genes are realized.

The invention needs to particularly propose the following method: utilizing the advantages of ssODN (Single-stranded oligodeoxyribonucleotide) mediated homologous recombination^[24]And precisely deleting the gene fragment by using the ssODN as a homologous repair template. Specifically, HA-iPSCs derived from patients are subjected to nuclear transfer through CRISPR/Cas9 and ssODN to obtain iPSCs with in-situ B region coding sequence micro-in-frame deletion or whole B region coding sequence targeted deletion.

If only the position near the mutation sequence is deleted, the sgRNA of the nearest CRISPR/Cas9 (the design of the Cas9 is limited by the PAM sequence) is designed according to the sequence near the mutation site, and the multiple bases of the total 3 including the base of the mutation site are deleted, namely, the in-frame deletion is realized. On the other hand, there are hundreds of types of mutations in the B region that cause HA, and we designed a strategy for deletion of the entire B region that can be directed to any variation in the B region, and we also performed experiments on deletion of the entire B region in normal human iPSCs in order to exclude the influence of the genetic background of possible patients, see example 2 (7). This experiment demonstrated that deletion of the entire B region did not affect the expression of the F8 gene.

The SQ type ligation with 14 amino acids on both sides is more common in the specific embodiment of the present invention. If only multiple bases of a part of 3 nearby a specific mutation site are deleted, different ssODNs need to be designed according to the purpose of constructing in-frame deletion nearby the specific mutation site, specifically, about 40 bases are respectively taken at the upstream and downstream of the in-frame deletion base to synthesize a ssODN about 80nt to mediate deletion of precise gene segments^[25]. Each specific case is not the same; if treatment of all pathogenic mutations in the B region is achieved directly by deletion of the entire B region coding sequence, it is most common to obtain this type of B region deletion in SQ junction (fusion of Ser743 at the N-terminus of the B region with Gln1638 at the C-terminus), so that the sequence F8-BDD-ssODN, SEQ ID NO.20, is used for the ssODN. The ssODN can be applied to all mutations in the B-region, and if other types of linked B-region deletion forms of F8 are to be constructed, such as RH linkage (fusion of the N-terminal Arg747 to the C-terminal His1646 in the B-region), the sequence of the ssODN is adjusted accordingly.

According to an embodiment of the present invention, HA-iPSCs are cells of urine collected from a patient and induced into HA patient-specific iPSCs (HA-iPSCs). Induction method references used^[11]。

After iPSCs are obtained, the iPSCs are directionally induced and differentiated into endothelial progenitor cells for cell transplantation to play a therapeutic role. Reference is made to how to direct induced differentiation into endothelial progenitor cells^[26]。

For practical clinical application, the somatic cells of a patient are collected and induced into HA-iPSCs, then the mutation sites in the B region coding sequence in the F8 gene are subjected to micro-in-frame deletion or targeted deletion of the whole B region coding sequence by adopting the method provided by the invention, so as to obtain genetically corrected human induced pluripotent stem cells, and the stem cells are transplanted after being directionally differentiated into endothelial progenitor cells, so that rejection reaction can be reduced.

The invention provides a method for overcoming F8 gene expression abnormality, and provides a modified human induced pluripotent stem cell, wherein a sequence near a mutation site in a B region coding sequence of an abnormally expressed F8 gene is deleted in frame or the whole B region coding sequence is deleted in a targeted mode. In the method for obtaining the human induced pluripotent stem cells, a donor vector does not need to be constructed, only the ssODN needs to be directly synthesized, the technical realization is relatively easy, no screening gene is added, and the efficiency is higher. The method for obtaining the iPSCs can be called as in-situ gene correction, so that the normal expression of the genes can be realized under the original promoters and regulatory elements of the genes, and the method is relatively safe.

Drawings

FIG. 1 is a diagram of the FVIII structural pattern;

FIG. 2 is a diagram of the structural change pattern of the FVIII molecular structure and the activation process; wherein the red arrow indicates the cleavage site by furin hydrolase, the yellow arrow indicates the cleavage site by thrombin, and the purple M indicates the metal ion linking the heavy and light chains. Full length FVIII means Full length FVIII, FVIIIa means activated FVIII;

FIG. 3 is a schematic diagram of a process of the method provided by the present invention;

FIG. 4 is a drawing showing the induction and identification of mutant iPSCs in the B region coding sequence of the F8 gene; a, collecting and culturing urine cells to express beta-catenin, KRT7, ZO-1 and the like, and indicating the source of renal tubular epithelial cells; B. reprogramming the obtained iPSC clone morphology; C. the obtained iPSCs have no abnormal karyotype detection; D. the obtained iPSCs still retain the deletion mutation of the B region; E. immunofluorescence shows that iPSCs express multiple stem cell marker proteins; F. in vivo teratoma experiments confirm that the iPSCs can be differentiated in vivo to form tissues of three germ layers sources.

FIG. 5 shows the result of the small in-frame deletion of the B region coding sequence of F8 gene mediated by CRISPR gene editing system; schematic diagram of in-frame knockout of F8 gene B region mediated by CRISPR gene editing system; B. the efficiencies of the constructed sgrnas were measured at 41.3% and 18.92%, respectively, green bases were sgRNA sequences, blue bases were PAM, dotted lines indicated deletion, black arrows indicated insertion, red bases were inserted base sequences, + indicated insertion, Δ indicated deletion, and x indicated frequency. C. After gene targeting experiments, the clones obtained were preliminarily identified by PCR. D, sequencing and verifying a PCR positive strip;

FIG. 6 shows the result of the PCR and sequencing identification of the N-iPSCs with the accurate deletion of 54 bp; wherein, the picture A is an electrophoretogram of N-del 54-15-iPSCs and N-del 54-42-iPSCs which are subjected to PCR amplification by F8-E14-F/R, and the N-iPSCs are control cells; FIG. B shows the sequencing results of the PCR products of N-del 54-15-iPSCs and N-del 54-42-iPSCs, in which the black vertical line represents the deleted fragment, and the N-del 54-15-iPSCs and N-del 54-42-iPSCs are directly connected after 54bp of basic groups are deleted;

FIG. 7 is the identification of stem cell surface markers after gene targeting of HA-iPSCs; wherein Nanog, Oct4 is marked as green fluorescence, SSEA-1, SSEA-4 is marked as red fluorescence; DAPI was used to stain nuclei;

FIG. 8 is the identification of stem cell surface markers after gene targeting of N-iPSCs; wherein Nanog, Oct4 is marked as green fluorescence, SSEA-1, SSEA-4 is marked as red fluorescence; DAPI was used to stain nuclei;

FIG. 9 is detection of HA-iPSCs in-frame deletion of 54bp clone karyotype; wherein, the picture A is 2-6-iPSCs karyotype, the picture B is 2-46-iPSCs karyotype, and the karyotype analysis shows 46, XY, no abnormality is found;

FIG. 10 shows the detection of the 54bp clone karyotype of the N-iPSCs in-frame deletion; wherein, the picture A is the N-del 54-42-iPSCs karyotype, the picture B is the N-del 54-15-iPSCs karyotype, and the karyotype analysis shows 46 XY which is not abnormal.

FIG. 11 shows the transcription of iPSCs F8 after 54bp of HA-iPSCs frame internal deletion is detected by RT-PCR; wherein the iPSCs stage detects F8 expression through RT-PCR transcription level, H₂O is blank control, HA-iPSCs is patient group, 2-6-iPSCs and 2-46-iPSCs are frame deletion group, N-iPSCs is normal control group, GAPDH is internal reference, F8(E14) is precise gene deletion region amplification, and F8(E23-26) is 23-26 exon primer amplification;

FIG. 12 shows the transcription of iPSCs F8 after 54bp of in-frame deletion of N-iPSCs in RT-PCR detection; wherein the expression of F8 is detected by RT-PCR transcription level in the iPSCs stage, H2O is blank control, N-iPSCs is normal control group, N-del 54-42-iPSCs and N-del 54-42-iPSCs are accurate deletion 54bp clone group, GAPDH is internal reference, F8(E14) is accurate gene deletion region amplification, and F8(E23-26) is trans-23-26 exon primer amplification;

FIG. 13 is an iPSCs stage FVIII secretion assay; wherein ELISA is used for detecting FVIII expression in cell lysate and cell culture supernatant at the iPSCs stage;

FIG. 14 shows expression detection of LMAN1 in iPSCs stage; wherein the expression of LMAN1 in cells in an iPSCs stage is detected by Western blot, and beta-actin is used as an internal reference.

FIG. 15 shows flow assays of day 5 cells of endothelial progenitor cell differentiation; wherein the upper row of drawings comprises the following components in sequence from left to right: 1. performing subsequent analysis on a cell scatter diagram to be analyzed by taking a group of cells concentrated in an in-gate comparison as a target group of cells to be analyzed; analysis results of HA-iEPCs components, wherein the upper right quadrant shows the positive proportion of CD31/CD34, and the positive proportion is 23.24%; 3.2-6-iEPCs component analysis result, the ratio of double positive in the upper right quadrant is 20.85%; 4.2-46-iEPCs component analysis result, the ratio of double positive in the upper right quadrant is 11.27%; the lower row of drawings comprises from left to right: the analysis result of the N-iEPCs component shows that the ratio of double positive in the upper right quadrant is 10.09%; 2, the analysis result of the N-del 54-42-iEPCs component shows that the double positive proportion of the upper right quadrant is 27.37 percent; 3, analyzing the N-del 54-15-iEPCs component, wherein the double positive proportion of the upper right quadrant is 24.54%;

FIG. 16 is a flow assay after sorting of endothelial progenitor cells; wherein the upper row of drawings comprises the following components in sequence from left to right: 1. the sorted endothelial progenitor cells have the morphology and high proliferation speed and are epithelial-like cells; 2. after sorting, detecting a scatter diagram by using a cell flow method, wherein the intra-portal cell population is a target cell population to be analyzed; 3. the single-labeled CD34-PE group, wherein the upper left quadrant is a positive cell population; the lower row of drawings comprises from left to right: 1. the single-label CD31-FITC group, the right lower quadrant is positive cell group; 2.2-6 component cell selection analysis results, the proportion of double positive cells is 93.24%; the analysis result of the sorting cells of the N-iPSCs component shows that the proportion of double positive cells is 92.36 percent.

FIG. 17 is an immunofluorescence assay of endothelial cells following sorting; wherein the CD34 label is red fluorescent; CD31 labeled red fluorescence; CD144 label as green fluorescence, DAPI was used to stain nuclei;

FIG. 18 shows immunofluorescence identification of mature endothelial cells; wherein the CD31 label is red fluorescence, the vWF label is green fluorescence, and DAPI is used to stain the nucleus;

FIG. 19 is an immunofluorescence assay for mature endothelial cell N-terminal FVIII; wherein the FVIII-N marker is red fluorescence, the vWF marker is green fluorescence, and DAPI stains a nucleus;

FIG. 20 is a mature endothelial cell C-terminal FVIII immunofluorescence assay; wherein the FVIII-C label is red fluorescence, the vWF label is green fluorescence, and DAPI stains a nucleus;

FIG. 21 shows the mature endothelial cell stage FVIII ELISA assay;

FIG. 22 is a graph of endothelial cell stage LMAN1 expression assay; detecting the expression of LMAN1 in cells at an endothelial cell stage by Western blot, wherein beta-actin is used as an internal reference;

FIG. 23 is a measurement of FVIII clotting activity of endothelial progenitor cells following in vivo transplantation in mice;

FIG. 24 is a mouse survival curve after tail-biting experiments; wherein HA mic (n 9); HA-iEPCs (n ═ 9); 2-6-iEPCs (n-12); 2-46-iEPCs (n ═ 10); N-iEPCs (N ═ 9); n-del 54-42-iEPCs (N ═ 9). Ns, no statistical difference compared to HA mic; p <0.001, p <0.01, compared to HA-iEPCs (log-rank test);

FIG. 25 shows the mean survival time of mice in tail-cutoff experiments; in which tail-biting experiments were performed, mice surviving after the time of experimental recording (48 hours) were not counted. ns, no statistical difference compared to HA mic. P <0.001, p <0.01, p <0.05, compared to the HA-iEPCs group;

FIG. 26 shows the detection of human cells in mouse liver; wherein the CD31 label is red fluorescence, the vWF label is green fluorescence, and DAPI stains the nucleus;

FIG. 27 shows the detection of human cells in the major organs of mice; wherein the CD31 label is red fluorescence, the vWF label is green fluorescence, and DAPI stains the nucleus;

FIG. 28 is a schematic illustration of targeting a deletion of the B region coding sequence; wherein black bases are internal sequences of a B region of an F8 gene, green represents a designed recognition sequence of CRISPR/Cas9, blue is a PAM sequence, a red small arrow is a cutting position, a yellow shaded part is an upstream homologous sequence of the ssoDN, a gray shaded part is a downstream homologous sequence of the ssoDN, orange yellow bases are two positions of synonymous mutation, and a B region Reframed F8(BDD F8) is a structural schematic diagram of F8 after in-situ deletion of the internal sequences of the B region;

FIG. 29 is a CRISPR/Cas9 cleavage efficiency assay for targeted deletions of the B region coding sequence; wherein, the picture A is the efficiency of identifying F8-BDU-sg1 by using a T vector connection PCR product resequencing method, and the indels generation ratio is 13.33%; FIG. B shows the efficiency of F8-BDD-sg4 identified by the T vector ligation PCR product resequencing method, with an indels production rate of 35.71%. WT shows the original sequence, the blue labeled part is PAM sequence, the dotted line shows the deleted part, the red labeled base is mutation and insertion base, delta is the number of base of deletion mutation, + is the number of base of insertion mutation;

FIG. 30 shows sequencing to identify the clone of HA-iPSCs lacking B region; the graph A shows the electrophoresis result of BD21-iPSCs and BD25-iPSCs after PCR amplification through F8-E14-F/R, wherein the Marker is DL 2000, HA-iPSCs are untargeted cells and amplify 498bp bands, BD21-iPSCs and BD25-iPSCs do not amplify bands, and N-iPSCs are normal controls and amplify 502bp bands. And the graph B shows the electrophoresis result of BD21-iPSCs and BD25-iPSCs after PCR amplification through F8-BUF/BDR, wherein Marker is DL 2000, HA-iPSCs are untargeted cells and amplify 3019bp bands, BD21-iPSCs and BD25-iPSCs amplify 341bp bands, and N-iPSCs are normal controls and amplify 3023bp bands. FIG. C shows the result of Sanger sequencing of PCR products amplified by F8-BUF/BDR of BD21-iPSCs and BD25-iPSCs, Predicted is the theoretical sequence of HA-iPSCs lacking B region (keeping SQ sequence), black vertical line in the figure represents the missing fragment, peak diagram is that BD21-iPSCs and BD25-iPSCs lack base in B region and then two side sequences are directly connected, and blue arrow points are synonymous mutation introduced in SQ sequence;

FIG. 31 shows the results of PCR and sequencing identification of N-iPSCs deletion B region; FIG. A shows the electrophoresis results of N-BD9-iPSCs and N-BD14-iPSCs after PCR amplification by F8-E14-F/R, wherein the Marker is DL 2000, the N-iPSCs are normal controls, a 502bp band is amplified, and the N-BD9-iPSCs and N-BD14-iPSCs are not amplified. And the graph B shows the electrophoresis result of N-BD9-iPSCs and N-BD14-iPSCs after PCR amplification by F8-BUF/BDR, wherein Marker is DL 2000, N-iPSCs is a normal control, a 3023bp strip is amplified, and N-BD9-iPSCs and N-BD14-iPSCs are amplified to form a 341bp strip. FIG. C shows the result of Sanger sequencing of PCR products amplified by F8-BUF/BDR of N-BD9-iPSCs and N-BD14-iPSCs, Predicted is the theoretical sequence of N-iPSCs lacking B region (keeping SQ sequence), black vertical line in the figure represents deletion fragment segment, peak diagram is that BD21-iPSCs and BD25-iPSCs lack base in B region and then two side sequences are directly connected, and blue arrow points are synonymous mutation introduced in SQ sequence.

FIG. 32 is the identification of the surface marker of the cloned stem cell deleted from the B region of HA-iPSCs; nanog, Oct4 labeled green fluorescence, SSEA-1, SSEA-4 labeled red fluorescence; DAPI was used to stain nuclei.

FIG. 33 is the identification of the surface marker of the cloned stem cell lacking in the B region of N-iPSCs; nanog, Oct4 labeled green fluorescence, SSEA-1, SSEA-4 labeled red fluorescence; DAPI was used to stain nuclei.

FIG. 34 shows the detection of the deletion clone karyotype of HA-iPSCs B region; the karyotype analysis of BD21-iPSCs in panel A and BD25-iPSCs in panel B all showed 46, XY, no abnormality.

FIG. 35 shows the detection of N-iPSCSSB region deletion clone karyotype; the karyotype analysis of N-BD9-iPSCs in panel A and N-BD14-iPSCs in panel B showed 46 XY, no abnormality.

FIG. 36 is RT-PCR detection of iPSCs stage F8 transcription; f8 expression detection through RT-PCR transcription level in iPSCs stage, H₂O is blank control, HA-iPSCs is patient group, BD21-iPSCs and BD25-iPSCs are B region deletion group, N-iPSCs is normal control group, GAPDH is internal reference, F8(BD) is amplified by crossing 14 exon primer, and F8(E23-26) is amplified by crossing 23-26 exon primer.

FIG. 37 is RT-PCR detection of iPSCs stage F8 transcription; detection of F8 expression, H, by RT-PCR transcript level at iPSCs stage₂O is blank control, N-iPSCs are normal control groups, N-BD9-iPSCs and N-BD14-iPSCs are B region deletion clone groups, GAPDH is internal reference, F8(BD) is amplified by a primer spanning No.14 exon, and F8(E23-26) is amplified by a primer spanning No. 23-26 exon.

FIG. 38 is an iPSCs stage FVIII secretion assay; and (3) detecting FVIII expression of the cell lysate and the cell culture supernatant in the iPSCs stage by ELISA.

FIG. 39 shows expression detection of LMAN1 in iPSCs stage; the expression of LMAN1 in cells at the iPSCs stage is detected by Western blot, and beta-actin is used as an internal reference.

FIG. 40 flow assays of day 5 differentiated cells; from left to right are: 1. performing subsequent analysis on a cell scatter diagram to be analyzed by taking a group of cells concentrated in an in-gate comparison as a target group of cells to be analyzed; the analysis result of the BD21-iEPCs component shows that the upper right quadrant shows the positive proportion of the CD31/CD34 double-label, and the positive proportion is 20.59 percent; analysis results of the BD25-iEPCs component show that the ratio of double positive in the upper right quadrant is 16.84%; the analysis result of the N-BD9-iEPCs component shows that the ratio of the right upper quadrant double positive is 19.55%; analysis of the N-BD14-iEPCs showed a 21.23% bipolarity ratio in the upper right quadrant.

FIG. 41 shows immunofluorescence identification of endothelial progenitor cells after sorting; CD31 labeled red fluorescence; the CD144 marker was green fluorescent and DAPI was used to stain the nuclei.

FIG. 42 is an immunofluorescence assay for mature endothelial cells; CD31 was labeled red fluorescence, vWF was labeled green fluorescence, and DAPI was used to stain the nucleus.

FIG. 43 detection of iECs stage F8 transcription by RT-PCR; detection of F8 expression, H, by RT-PCR transcript levels during the iECs stage₂O is blank control, HA-iECs is patient group, BD21-iECs and BD25-iECs are B region deletion group, N-iECs is normal control group, GAPDH is internal reference, F8(BD) is amplified by crossing 14 exon primer, and F8(E23-26) is amplified by crossing 23-26 exon primer.

FIG. 44 is RT-PCR detection of iECs stage F8 transcription; detection of F8 expression, H, by RT-PCR transcript levels during the iECs stage₂O is blank control, N-iECs is normal control group, N-BD9-iECs and N-BD14-iECs are B region deletion clone group, GAPDH is internal reference, F8(BD) is amplified by crossing No.14 exon primer, and F8(E23-26) is amplified by crossing No. 23-26 exon primer.

FIG. 45 is a mature endothelial cell N-terminal FVIII immunofluorescence assay; FVIII-N is labeled as red fluorescence, vWF is labeled as green fluorescence, and DAPI stains nuclei.

FIG. 46 is a mature endothelial cell C-terminal FVIII immunofluorescence assay; FVIII-C is labeled red fluorescence, vWF is labeled green fluorescence, and DAPI stains nuclei.

FIG. 47 shows mature endothelial cell stage FVIII ELISA assay;

FIG. 48 is a graph of endothelial cell stage LMAN1 expression assays; expression of LMAN1 in cells at an endothelial cell stage is detected by Western blot, and beta-actin is used as an internal reference.

FIG. 49 shows FVIII clotting activity assays of endothelial progenitor cells following in vivo transplantation in mice; p <0.001, n ═ 6 compared to the HA-iEPCs group.

FIG. 50 is a graph of mouse survival after tail-off experiments; ns, no statistical difference compared to HA mic; p <0.001, compared to HA-iEPCs (log-rank test), (n ═ 9).

FIG. 51 is the mean survival time of mice from tail-biting experiments; mice surviving the tail-biting experiment and survived after the time of the experimental recording (48 hours) without statistics. ns, no statistical difference compared to HA mic. P <0.001, p <0.01, compared to the HA-iEPCs group.

FIG. 52 is a diagram showing PCR amplification thermal cycling conditions of F8-E14-sg1, F8-E14-sg2, F8-BDU-sg1, and F8-BDD-sg4 groups.

Fig. 53 is a diagram of thermal cycling conditions of a CRISPR/Cas9 targeted site-directed deletion detection PCR system in example step 4.2.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The names and sequences of the primers involved in the examples of the present invention are shown in Table 1 below.

TABLE 1 primer names, sequences and corresponding uses

The experimental method and the steps implemented by the invention are as follows:

CRISPR/Cas9 expression plasmid construction

Suitable short-chain guide RNAs (sgRNAs) for CRISPR/Cas9 (http:// CRISPR. mit. edu /) [27] were designed using the manipulated CRISPR Design provided by FengZhang Lab. Ordering Cas9 codon optimized CRISPR/Cas9 backbone pX330 provided by Feng Zhang Lab.

(1) Designing sgRNA (about 20 nt) according to the experimental purpose, wherein the sequences are shown as sequences SEQ ID NO.1, SEQ ID number 2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 in a table 1, adding a recognition sequence CACC of Bbs I at the 5 'end of the sgRNA sequence, and adding AAAC at the 5' end of a sgRNA complementary sequence;

(2) the pX330 backbone was cleaved with BbsI as follows:

and (3) carrying out enzyme digestion at 37 ℃ for 3 hours, carrying out agarose gel electrophoresis on the enzyme digestion product, and identifying the enzyme digestion completion through electrophoresis, namely carrying out gel recovery on the product, adding 12 mu L of ddH2O lysate after recovery for connection.

(3) Respectively annealing oligonucleotides synthesized by Shanghai Biotechnology Limited according to four different systems to form double chains containing sticky ends;

the annealing system of F8-E14-sg1 is as follows:

after 5 minutes at 95 ℃, naturally cooling to room temperature for connection;

the annealing system of F8-E14-sg2 is as follows:

after 5 minutes at 95 ℃, naturally cooling to room temperature for connection;

the annealing system of F8-BDU-sg1 is as follows:

after 5 minutes at 95 ℃, naturally cooling to room temperature for connection;

the annealing system of F8-BDD-sg4 is as follows:

after 5 minutes at 95 ℃, naturally cooling to room temperature for connection;

(4) the system is as follows:

ligation was performed overnight at 16 ℃;

(5) transformation and monoclonal colony picking

Taking out DH5 alpha competence from a-75 ℃ ultralow temperature refrigerator, adding 10 mu L of the ligation product into 50 mu L of competence, and lightly blowing and uniformly mixing by using a middle gun head;

standing on ice for 30 minutes, and simultaneously preheating a circulating water bath tank to 42 ℃;

placing a 1.5mL centrifuge tube after the ice bath is finished in a circulating water bath at 42 ℃ for heat shock for 45 seconds, and immediately standing on ice for 2 minutes;

operating in a clean bench, adding 500 μ L of liquid LB culture medium without resistance;

shaking by an air shaking table at the temperature of 37 ℃ for 45 minutes at the speed of 180 r/min;

taking out the bacterial liquid from the shaking table, centrifuging for 5 minutes at 2500g, discarding part of supernatant in a super clean workbench, and reserving about 100 mu L of bacterial liquid to be mixed uniformly;

adding the bacterial liquid into a pre-poured LB solid plate with ampicillin resistance, and uniformly coating the bacterial liquid on a solid LB culture dish by using a coating rod; and (4) inversely placing the mixture in a constant-temperature incubator at 37 ℃ for culturing for 12-16 hours.

And (3) selecting a single colony to 500 mu L of LB liquid culture medium containing benzyl resistance, carrying out rotation growth on an air shaking table 220 at 37 ℃ for 4-6 hours, and then sending a bacterium solution to carry out sequencing, wherein the sequencing primer is a U6 universal primer.

And (3) performing amplification culture again on the bacterial colony with the sequencing result consistent with the theoretical result, inoculating the bacterial liquid into about 60mL of liquid LB culture medium added with the ampicillin, and growing for 14-16 hours at the temperature of 37 ℃ on an air shaking bed at 280 revolutions. The successful CRISPR/Cas9 expression vector can be obtained by extracting the plasmid by using an OMEGA Endofree plasmid Midi Kit and operating according to the instruction.

2. Hemophilia A patient specific iPSCs induction (HA-iPSCs)

One example of heavy HA patient urine with a mutant type of F8 gene c.3167delCTGA leading to p.D1055fsX5 is collected, urine cells of the heavy HA patient urine are separated and cultured, and the urine cells of the heavy HA patient urine are reprogrammed into iPSC-like clones by a classical four-factor method and identified.

2.1 Collection of urine specimens

Under the premise that a patient signs an informed consent, the method for collecting urine of the patient in a non-invasive manner comprises the following specific steps:

(1) disinfecting the urethral orifice, dipping a clean cotton swab in iodophor, lightly wiping the urethral orifice and the periphery, replacing a new cotton swab and repeating for 1 time, and dipping in 70% alcohol and wiping for two times;

(2) the urine can be collected after the alcohol is dried for a few seconds. And opening the cover of the collecting bottle, discharging the initial 20mL of urine into the urinal, collecting the middle-section urine into the collecting bottle, discharging the last part into the urinal when the urine is discharged quickly, and carefully covering the cover of the collecting bottle. Typically 100 and 200mL of urine are collected.

2.2 culture of urine cells

(1) Subpackaging urine in a collection bottle into 50mL centrifuge tubes in a sterile room, centrifuging at 400g room temperature for 10 minutes, carefully removing supernatant, reserving less than 1mL of residual liquid, merging all residual liquid after centrifuging urine subpackaged into 50mL centrifuge tubes into one 50mL centrifuge tube, adding 10mL of DPBS, centrifuging at 200g room temperature for 10 minutes, carefully removing supernatant, reserving 200 mu L of residual liquid, adding 1mL of initial culture medium to resuspend cells, and inoculating the cells into one hole of a 12-hole cell culture plate coated with 0.1% gelatin;

(2) the next day 500. mu.L of fresh initial medium was added directly;

(3) on day three, 500 μ L of fresh initial medium was added directly;

(4) and starting on the fourth day, half-replacing the liquid with the REBM complete culture medium every day, wherein a few cell colonies can be observed about 5 days after inoculation in general, the REBM complete culture medium can be replaced in full after the cell colonies are observed, the culture is continued until the confluency of the cells reaches about 50%, the cells are subcultured and recorded as P1, and the REBM complete culture medium is used for culture.

(5) Cells from the P1-P3 generation were counted and plated into six well plates and two six well plates were plated at 6 ten thousand cells/well. The remaining cells were cryopreserved.

2.3 Induction of iPS cells

Two retroviral infections were required during induction, and the time for the first viral infection was defined as Day 0.

(1) HEK293T cells were counted by Day-3 at 6 pm in six well plates and seeded at 80 ten thousand cells/well;

(2) day-2 packaged virus, 4 pm, yesterday inoculated HEK293T cells were replaced with 1.5mL of complete medium, and transfection was started after 2 hours, and the cell density at transfection was about 80%.

Preparing a calcium phosphate transfection solution:

firstly adding Milli-Q water into an EP tube, then adding plasmids, mixing uniformly, then adding 2M CaCl2, mixing uniformly, and finally adding 2 xHBS to blow and beat forcibly to generate bubbles. The transfection solution is added dropwise to the cells, and the transfection solution can cover all the areas of the holes as much as possible;

(3) day-1 morning transfected HEK293T was carefully replaced with 2mL fresh DMEM complete medium (12 hours post-transfection); in the afternoon, 6 million cells to be induced were seeded per well in six well cell culture plates, two wells. Day 0, cells to be induced in six well plates were replaced with 2mL REGM fresh medium. The cell culture supernatant of HEK293T containing retrovirus (about 36 hours after transfection) was collected, filtered with 0.45 μm needle filter and added to the wells of the cells to be induced. Four virus supernatants of Oct4, Sox, Klf4 and c-Myc were added to the wells to be induced, only the virus supernatant containing GFP was added to the control wells, and polybrene was added to the system at a final concentration of 8. mu.g/mL to increase the infection efficiency. HEK293T cells 2mL fresh DMEM complete medium was added per well;

(4) day 1, virus supernatants were changed to REGM after overnight infection to promote cell recovery. In the afternoon, the virus supernatant was collected again and infected again, and the HEK293T cells were not cultured again after use as before. Day 2 was changed to REGM medium in the morning. Day 3 changes REGM medium and can judge virus packaging and infection efficiency by observing GFP efficiency in control wells, which should be close to 100% normally. The method is characterized in that the REGM culture medium is replaced by Day 4, so that the morphological change of partial cells can be observed, the cells become small, the morphology becomes polygonal and flat, the nucleus-to-cytoplasm ratio increases, the agglomeration and aggregation growth trend is generated at the same time, the proliferation speed becomes high, the morphological change is gradually enhanced along with the continuous culture, and the density and the morphology of the cells are strongly compared with those of GFP control cells;

(5) MEFs are typically prepared when Day 5 can be transferred to MEFs, and it is anticipated that cells will approach confluence the next Day, which is followed by digestion and counting with Accutase. Inoculating cells into 10 cm cell culture dishes, inoculating 10 ten thousand or 20 ten thousand cells into each dish, culturing by using an ES/DFBS 1:1 culture medium, and changing the culture solution every day;

(6) VPA was added every other day to a final concentration of 1mM for 7 consecutive days. From Day 6, a large number of small clones appeared in the dish, which showed clear smooth borders, high refractive index and high nuclear mass ratio. But these miniclones started to differentiate gradually or died as culture continued from Day 9;

(7) from Day 16, it can be seen that some ES-morphology clones grew gradually, some formed individually, and some grew out of the middle of the previously differentiated clones, and when the clones grew up to a 10-fold field of view of the objective lens, they were picked up by Pasteur tubes for amplification and identification.

2.4 iPSCs Resuscitation

(1) Culturing a cell pore plate coated with Matrigel one day before cell recovery;

(2) opening a water bath tank, setting the temperature to 37 ℃, taking out the cryopreserved cells from a liquid nitrogen tank when the temperature of the water bath tank is stabilized at 37 ℃, unscrewing a cryopreserved pipe cover to volatilize residual liquid nitrogen, quickly screwing the cryopreserved pipe cover, quickly placing the cryopreserved pipe cover in a water bath tank at 37 ℃, taking out the cryopreserved pipe cover when ice blocks in the pipe are melted to the size of only one mung bean, and quickly entering a sterile room for resuscitation;

(3) paving an operation table in an operating room of a sterile room, taking a clean 15mL centrifuge tube, adding 9 times of volume of mTeSR1 pre-warmed in advance into the liquid in the frozen tube, sucking the cells in the frozen tube into the centrifuge tube added with the culture medium, and centrifuging at the room temperature for 5 minutes at 170 g;

(4) gently sucking out the supernatant, discarding, adding an appropriate amount of mTeSR1 culture medium to resuspend the cell precipitate, adding Y27632 to a final concentration of 10 μ M, mixing, inoculating to a pore plate pre-coated with Matrigel, shaking uniformly, and culturing in a 37 ℃ cell culture box.

2.5 iPSCs culture, passage and cryopreservation

(1) After iPSCs are recovered, the solution is changed every day for maintaining culture, and the culture is carried out by mTeSR1 culture medium;

(2) when the cell confluence reaches 70%, the Matrigel is coated, and a proper pore plate is coated according to the experimental requirement. Passage the next day after Matrigel coating, at passage digested with 0.5mM EDTA: firstly sucking out the culture medium, removing the culture medium, adding 0.5mM EDTA which can submerge the bottom surface of the culture hole, rinsing twice, sucking up residual liquid, adding a little more 0.5mM EDTA, standing and digesting for 3 minutes at room temperature, sucking up liquid, adding a proper amount of mTeSR1, blowing out the required amount of cells, inoculating the cells into a pore plate coated with Matrigel, shaking up in a cross manner, and culturing in a 37 ℃ cell culture box;

(3) when the cell confluence reaches 80-90%, the cells can be frozen and stored according to the needs, the digestion process is the same as passage, the frozen solution (90% mTeSR1+ 10% DMSO, precooling at 4 ℃ in advance) is directly used for resuspending the cells in the last step, the cells are directly transferred to a freezing tube, the freezing tube is placed in a freezing box for overnight at the ultralow temperature of-80 ℃, and then the freezing tube is transferred to a liquid nitrogen tank for recording.

2.6 identification of Stem cell surface markers

(1) Subculturing HA-iPSCs cultured on the Matrigel into a 24-well plate coated with a Matrigel cell slide, and performing immunofluorescence identification when continuously culturing for 2-3 days;

(2) discarding the culture medium, rinsing the DPBS for 2 times, completely sucking residual liquid, adding 4% paraformaldehyde, and fixing at room temperature for 15 minutes;

(3) residual liquid is absorbed completely, PBST (DPBS +1/1000Triton-100) is added for permeabilization for 15 minutes;

(4) PBST is discarded, DPBS is rinsed for 2 to 3 times, and 5% BSA is blocked for 30 minutes;

(5) diluting Oct4/Nanog/SSEA-1/SSEA-4 primary antibody with a blocking solution at a ratio of 1:200, and incubating the primary antibody at room temperature for 1 hour;

(6) discarding the primary antibody, adding DPBS to rinse for 2-3 times, and adding DPBS to rinse for 3 times, each time for 10 minutes; blocking with 5% BSA for 30 min;

(7) diluting the secondary antibody with a confining liquid at a ratio of 1:300, selecting a proper secondary antibody corresponding to the primary antibody species, and incubating for 1 hour at room temperature in a dark place;

(8) discarding the liquid, rinsing the DPBS for 2-3 times, adding the DPBS to wash for 5 times, and each time for 5 minutes;

(9) adding 1 mu g/mL DAPI, incubating at room temperature in dark place for 6min, rinsing with DPBS for 2-3 times, adding DPBS, and washing for 5 times, each for 5 min;

(10) rinsing with ultrapure water once, adding 90% glycerol, sealing with nail polish, and taking pictures under a laser confocal microscope for storage.

2.7 karyotype detection

(1) When the confluence degree of HA-iPSCs in the six-hole plate is up to 80%, adding colchicine into the culture medium to a final concentration of 40ng/mL, and culturing for 5 hours in a cell culture box at 37 ℃;

(2) sucking the culture medium, adding normal saline to rinse twice, digesting for 3 minutes at 37 ℃ by 0.05 percent EDTA-pancreatin, and adding the culture medium to stop digestion;

(3) collecting cells into a 15mL centrifuge tube, and centrifuging at 1200g for 5 minutes;

(4) discarding the supernatant, adding 8mL of 0.07% KCl hypotonic solution, and incubating in a water bath at 37 ℃ for 10 minutes for hypotonic;

(5) after hypotonic reaction, 1.5mL of 37 ℃ preheated fixing solution (glacial acetic acid and methanol are mixed according to the proportion of 3: 1) is added and lightly blown and uniformly mixed, and the mixture is placed in a 37 ℃ water bath box for 5 minutes for pre-fixing;

(6) centrifugation at 1200g for 10 minutes at room temperature after the pre-fixation is complete;

(7) discarding the supernatant, adding 8mL of 37 ℃ preheated stationary liquid, and uniformly blowing and beating;

(8) centrifuging at 1200g for 10 minutes at room temperature;

(9) discarding the supernatant, repeating the fixation once, slightly sucking the supernatant, and adding a proper amount of fresh fixing solution to resuspend the cells (until the crude glass sample is turbid);

(10) dropping the slices in a drop-slice dispersion instrument, numbering, baking the slices at 75 ℃ for 3 hours, dyeing Giemsa, observing under a mirror, and collecting images.

2.8 in vivo three germ layer differentiation

(1) Because of the existence of cellular immunogenicity, immunological rejection can occur in the xenograft, and therefore, the human iPSC is injected into a nude mouse subcutaneously to carry out an in-vivo three-germ layer differentiation experiment. Preparing HA-iPSCs with the confluence degree of more than 80% in 26 cm dis;

(2) 0.05% EDTA-pancreatin digests cells for about 5 minutes at 37 ℃, after termination of digestion in mTeSR1 medium, centrifuge at 1200g for 5 minutes, aspirate the supernatant, resuspend the cells in 140. mu.L of mTeSR1 medium, add 70. mu.L of Matrigel, mix well and place on ice;

(3) fixing a nude mouse with age of 6-8 weeks, locally sterilizing the inguinal position of the lower limb of the nude mouse by iodophor, slowly and subcutaneously injecting cell suspension to the inguinal position of the nude mouse at uniform speed by using a 1mL injector, and locally pressing after injection;

(4) cutting the naked mouse toe, and recording;

(5) local small protrusions were visible at the injection site for about two weeks, and after 6 weeks, a clear tumor mass was visible. The teratoma can be harvested by operation when the teratoma grows to about 1cm in diameter after about 8 weeks;

(6) taking out the cells together with teratoma envelope completely, measuring tumor diameter and taking a picture, fixing 4% paraformaldehyde overnight, and then making paraffin tissue section and parallel HE staining. After staining was complete, the specimens were observed under an optical microscope and photographed.

CRISPR/Cas9 cleavage activity assay

In order to detect the CRISPR/Cas9 cleavage activity, a CRISPR/Cas9 expression plasmid is transferred into an HA-iPSCs cell in a nuclear transfection mode to express CRISPR/Cas9, when the CRISPR/Cas9 expression plasmid plays a cleavage role to generate DSB, the cell is repaired through a mechanism of NHEJ to generate indels, and the cleavage activity of the sgRNA can be reflected by detecting the proportion of target genes containing the indels.

3.1 Nuclear transfection

Inoculating HA-iPSCs into a six-hole plate coated with Matrigel, culturing by using mTeSR1, and targeting when the confluence degree of the six-hole plate is about 90%;

2.5 hours before gene targeting, changing the liquid, adding fresh culture medium with the final concentration of 10 mu M Y27632 to continuously culture in a 37 ℃ incubator;

③ Human Stem Cell, a nuclear transfer reagent according to the company Lonza

The Kit 2 instruction is operated, 82 mu L Solution II and 18 mu L Supplement I in the Kit are taken and mixed, and then the mixture is placed at the room temperature and the temperature is balanced; standing at room temperature for 15 minutes, respectively adding a CRISPR/Cas9 expression plasmid for expressing corresponding sgRNA into each tube, and standing at room temperature for 5 minutes;

(iv) digesting the cells with TrypLE SELECT: washing 3-4 times with DPBS, adding trypLE SELECT, digesting in 37 deg.C incubator for 3-5 min, observing cell clone edge curl under microscope, adding ES culture medium to stop digestion, sucking off cells, transferring to centrifuge tube, counting with hemocyte counting plate, taking out 170g cell suspension containing 100 ten thousand cells, and centrifuging at room temperature for 5 min;

fifthly, abandoning the supernatant after centrifugation, completely absorbing residual liquid of the culture medium, adding the liquid added with the plasmids in the third step into a centrifuge tube, resuspending the cells, transferring the cells into an electrode cup, carrying out nuclear transfer by utilizing a LONZA nuclear transfer instrument B-016 program, immediately adding 400 mu L mTeSR1 into the electrode cup, and standing for 5 minutes at room temperature;

sixthly, adding 1.5mL of mTeSR1 into a six-hole plate with matrix laid in advance, transferring cells in an electrode cup to the six-hole plate with the mTeSR1, adding Y27632, shaking uniformly, and culturing in an incubator at 37 ℃;

seventhly, changing core for 12 hours, changing fresh culture medium, adding Y27632, culturing for 3 days, and extracting cell DNA.

3.2 gDNA extraction

(1) Rinsing cells to be subjected to DNA extraction by using DPBS for 3 times, carrying out pancreatin digestion at 37 ℃ for 5 minutes, adding an ES culture medium to terminate digestion, collecting the cells into a 1.5mL centrifuge tube, and centrifuging the cells at the room temperature of 5000g for 5 minutes;

(2) centrifuging, removing supernatant, adding 500 μ L cell nucleus lysate, adding 1/200 proteinase K and 1/1000RNase, blowing, mixing, adding 70 μ L10% SDS, mixing, placing in a shaking table, standing at 37 deg.C, and performing lysis at 100 r overnight;

(3) taking out overnight cracked cells, adding equal volume of Tris-phenol, repeatedly reversing and uniformly mixing until fine and uniform tiny oily particles appear, centrifuging at 17000g room temperature for 10 minutes;

(4) after the centrifugation is finished, the liquid in the tube is layered, the upper layer liquid is sucked out to a new 1.5mL centrifuge tube, the 1:1 phenol/chloroform mixed solution with the same volume is added, the mixture is repeatedly inverted and mixed evenly until uniform chylomicron particles are formed, the mixture is centrifuged at 17000g room temperature for 10 minutes;

(5) after the centrifugation is finished, liquid in the tube is layered, upper layer liquid is absorbed into a new 1.5mL centrifuge tube, 2 times volume of precooled absolute ethyl alcohol is added, the mixture is repeatedly inverted and mixed evenly, when white filamentous DNA appears, a low-temperature centrifuge is used for centrifugation, and 17000g of the mixture is centrifuged for 10 minutes at 4 ℃;

(6) after centrifugation, the supernatant was decanted, 500. mu.L of 75% absolute ethanol pre-chilled in advance was added, the mixture was inverted 10 times, and centrifuged at 17000g for 10 minutes at 4 ℃;

(7) and pouring off the supernatant, carrying out spot separation, completely sucking residual liquid, airing in a clean bench, and adding an appropriate amount of ddH20 for dissolution.

The obtained gDNA was used as a template to PCR amplify sequences near the cleavage site as follows: (groups F8-E14-sg1 and F8-E14-sg2 were amplified using F8-E14-F/R; group F8-BDU-sg1 was amplified using F8-BU-F/R; group F8-BDD-sg4 was amplified using F8-BD-F/R)

The thermal cycling conditions are shown in figure 52.

3.3 detection by sequencing method

Performing electrophoresis on the PCR product, recovering target fragments by glue, and performing T-vector connection on the recovered product, wherein the connection system is as follows:

connecting in a water bath at 16 ℃ overnight, then taking 10 mu L for transformation, carrying out blue-white screening, growing the transformed bacteria for about 14 hours, picking white bacterial colonies into liquid LB added with ampicillin, shaking the bacteria for about 3 hours, carrying out sequencing identification on the bacterial liquid, carrying out sequencing by using a universal primer T7, carrying out sequence comparison by using DNA star sequence comparison software, counting the person with indels near a cleavage site as a cutter, and calculating the corresponding CRISPR/Cas9 cleavage efficiency.

CRISPR/Cas9 combined ssODN nuclear transfer HA-iPSCs and N-iPSCs (same as 3.1)

For deletion of the mini-in-frame deletion in the B region, the transfected plasmids were divided into two groups, one of which was 4. mu. g F8-E14-sg1 and 4. mu. g F8-E14-sg2, and the other was 4. mu. g F8-E14-sg1, 4. mu. g F8-E14-sg2 and 50pmol F8-E14-del54-ssODN, and left to stand at room temperature for 5 minutes;

for deletion of the entire block B, the transfection plasmid was set to 1 group of 4. mu. g F8-BDU-sg1, 4. mu. g F8-BDD-sg4 and 50pmol F8-BDD-ssODN, and left to stand at room temperature for 5 minutes;

culturing for 3-5 days after nuclear transfer, counting 10000 cells when the cell amount is more, inoculating the cells on an MEF feeder layer to obtain monoclone, and freezing the rest mixed cells for subsequent application;

after the single cells are inoculated on MEF, the ES culture medium needs to be replaced every day for about 12 days, when the clone grows to 10 times of the visual field 1/2-2/3, the single clone is picked into a 48-hole plate paved with Matrigel for clonal amplification; extracting DNA and carrying out PCR identification to obtain positive clones.

4.1 g of DNA extract (as in 3.2)

4.2 PCR preliminary detection of site-directed deletions

Preliminary identification of deletion clone in minute frame in B region by using F8-E14-F/R primer

The CRISPR/Cas9 targeted site-specific deletion detection PCR system is as follows:

the thermal cycling conditions are shown in fig. 52.

Carrying out electrophoresis on the PCR product through 1% agarose gel, and amplifying 498bp bands of untargeted HA-iPSCs or untargeted unintegrated cells; the PCR product of the positive site-directed deletion clone is a band of 448bp in size. And (3) sequencing the PCR product with the amplified band of about 448bp, performing sequence comparison by using F8-E14-F as a sequencing primer and SeqMan software, amplifying and freezing the completely and accurately deleted clone, and performing the next experiment. For N-iPSCs, 502bp bands can be amplified by non-targeted persons or non-integrators, and the PCR product of positive site-directed deletion clones is a 448bp band.

Preliminary identification of whole B region targeted deletion clone by using F8-BU-F/F8-BD-R primer

The CRISPR/Cas9 targeted site-specific deletion detection PCR system is as follows:

the thermal cycling conditions are shown in fig. 53.

Carrying out electrophoresis on the PCR product through 1% agarose gel, and amplifying 3019bp bands of untargeted HA-iPSCs or targeted unintegrated cells; the PCR product of the positive site-directed deletion clone is a 341bp band. And (3) sequencing the PCR product with amplified band of about 341bp, carrying out sequence comparison by using F8-BU-F as a sequencing primer and SeqMan software, amplifying and freezing the clone which is completely and accurately deleted, and simultaneously carrying out the next step of experiment.

4.3 identifying the cell surface marker of the cloned stem cell by site-directed precise deletion, and the steps are the same as 2.6.

RT-PCR detection of F8 expression

Culturing HA-iPSCs and gene targeted iPSCs and N-iPSCs in a twelve-well plate, and extracting total RNA of cells by a Trizol method when the confluence degree reaches more than 80%; culturing a clone N-del 54(N-del 54-15, N-del 54-42) with 54bp deletion of N-iPSCs in a 12-well plate, and extracting cell total RNA by a Trizol method; simultaneously culturing clones (BD21, BD25) with the whole B region missing in a twelve-well plate, and extracting the total RNA of the cells by a Trizol method when the confluence reaches more than 80%; culturing a clone N-BD (N-BD9, N-BD14) of the N-iPSCs with the deletion of the whole B region in a 12-well plate, and extracting the total RNA of the cells by a Trizol method;

secondly, DNase I of Thermo scientific company is utilized to digest and remove possible DNA;

③ 1. mu.g of total RNA was transferred to a PCR tube, treated at 70 ℃ for 10 minutes in a PCR apparatus, placed on ice for 5 minutes, and a Reverse Transcription System was prepared using the Reverse Transcription System of Promega corporation:

42 ℃, 3Omin → 95 ℃, 5min → 5min on ice

Reverse transcription was performed on a PCR instrument, reaction program:

fourthly, the cDNA reverse transcribed in the previous step is taken as a template, and a PCR system is prepared as follows:

primers for the internal reference gene GAPDH were GAPDH-F and GAPDH-R (theoretical size: 453 bp). For deletion 54bp clones, the F8 transcription assay was performed by two pairs of RT primers: F8-E14-F/R, F8-RT-E23F and F8-RT-E26R were subjected to PCR. For clones lacking the entire B region, the F8 transcription assay was performed by two pairs of RT primers: F8-RT-BDF/R, F8-RT-E23F and F8-RT-E26R were subjected to PCR. The thermal cycling conditions are shown in fig. 53.

5 μ L of the product was analyzed by agarose gel electrophoresis.

ELISA detection of FVIII expression

Respectively collecting culture supernatants of HA-iPSCs cultured in a 12-well plate, identifying fixed point precise deletion clone 2-6 and 2-46 and normal control human iPSCs (N-iPSCs) cells for 24 hours, centrifuging the collected culture supernatants for 5 minutes at 5000g, taking the supernatants, adding 1/100 protease inhibitor, and storing at-80 ℃ for later use. Simultaneously collecting the supernatant of a clone N-del 54(N-del 54-15, N-del 54-42) of which the N-iPSCs lack 54bp, and correspondingly counting cells in a culture well plate after collecting the supernatant; for clones lacking the entire B region, the cell supernatants were also collected and counted;

② detection was carried out by using Paired Antibodies for ELISA-Factor VIII of CEDARLANE.

Differentiation of iPSCs into committed endothelial cells

In order to detect whether the gene repair strategy adopted by the research is effective, the evaluation of in vivo treatment effect is required, and endothelial cells are the main cell types for synthesizing and secreting FVIII, so that iPSCs are directionally differentiated into endothelial progenitor cells with proliferation capacity, and favorable conditions are provided for subsequent preclinical research and clinical application.

Referring to the existing literature report [26], the directional differentiation of iPSCs into endothelial progenitor cells is realized by a small molecule inhibitor, and 6 μ M CHIR99021 is adopted in the experiment for differentiation, and the specific differentiation steps are as follows:

(1) rinsing iPSCs cultured in a Matrigel-coated six-hole plate for 3 times by using DPBS, adding 500 mu L of Accutase, standing and digesting for 5 minutes in a 37 ℃ incubator, adding mTeSR1 culture medium to terminate digestion, collecting cells into a sterile 15mL centrifuge tube, counting by using a blood cell counting plate, centrifuging for 5 minutes at the room temperature of 170g, sucking supernatant, adding an appropriate amount of culture medium to suspend the cells, inoculating the cells into the Matrigel-coated culture hole plate at the speed of 5 ten thousand cells/cm 2, adding Y27632 to the final concentration of 10 mu M, culturing by using mTeSR1, shaking uniformly, and culturing in a 37 ℃ incubator, wherein the concentration is marked as Day-3;

(2) day-2 and Day-1 only need to change the medium mTeSR1 every Day;

(3) changing the culture medium LaSR basal medium in Day 0, adding 6 μ M CHIR99021 (since CHIR99021 is prepared by DMSO, adding LaSR basal medium to be used in fresh state, mixing, and changing liquid);

(4) the culture medium of the Day 1 is replaced like the culture medium of the Day 0, and dead cells are increased when the culture medium is observed under a microscope;

(5) beginning with Day 2, replacing the LaSR basic culture medium every Day until Day 5;

(6) flow cytometry at Day 5 for endothelial progenitor cell surface markers CD31 and CD34 to examine differentiation efficiency;

meanwhile, the Day 5 can also carry out CD31 magnetic bead sorting to obtain endothelial progenitor cells with higher purity;

7.1 endothelial progenitor cell sorting

Cells that differentiated Day 5 were ready for CD31 magnetic bead sorting:

(1) discarding the differentiation culture medium, adding DPBS (double DPBS) to rinse for 3-4 times, and removing residual liquid by suction;

(2) adding Accutase, digesting for 5 minutes at 37 ℃, and stopping digestion by using a LaSR basic culture medium;

(3) collecting cells into a clean sterile 50mL centrifuge tube, filtering the cell pellet with a cell sieve, and centrifuging at 170g for 5 minutes at room temperature;

(4) discarding the supernatant, adding 10mL of precooled MACS buffer solution, resuspending the cells, and centrifuging at 170g for 5 minutes at room temperature;

(5) slightly sucking and discarding the supernatant, and when a small amount of residual liquid is left, dotting and separating to suck the residual liquid completely;

(6) adding 60 mu L of MACS buffer solution, blowing, beating and mixing uniformly, then adding 20 mu L of FcR Blocking Reagent, mixing uniformly, adding 20 mu L of CD31MicroBeads, blowing, beating and mixing uniformly;

(7) placing the mixture in a refrigerator for incubation for 15 minutes at 4 ℃;

(8) after incubation, 1mL of pre-cooled MACS buffer was added and centrifuged at 170g for 5min at room temperature;

(9) the supernatant was aspirated off, and 1mL of pre-cooled MACS buffer was added to resuspend the cells;

(10) preparing a sorting column, and fixing an LS sorting column on a MACS sorting adapter rack;

(11) firstly, balancing a sorting column by using 3mL of precooled MACS buffer solution, namely adding the MACS buffer solution into an LS sorting column, and preparing a 50mL centrifuge tube to collect filtrate after the MACS buffer solution naturally flows through the LS sorting column;

(12) adding the cell suspension into the balanced LS sorting column, adding 3mL of precooled MACS buffer solution to wash the column for 3 times after the cell suspension flows freely, and avoiding the generation of bubbles in the operation process;

(13) after the liquid completely flows out, taking down the LS separation column to separate from the magnetic field, and preparing a sterile 15mL centrifuge tube;

(14) 5mL of pre-cooled MACS buffer solution is added into the LS sorting column, the buffer solution is rapidly pushed into the LS sorting column at one time by using a sterile piston in the LS sorting column kit, and the buffer solution is collected into a 15mL centrifuge tube;

(15) the cell suspension was counted and centrifuged at 170g for 5 minutes at room temperature, inoculated into a well-coated Collagen IV well in a well plate, and cultured in EGM-2.

7.2 flow cytometry for detecting differentiation efficiency of endothelial progenitor cells

(1) Differentiating the endothelial progenitor cells by Day 5, carrying out flow detection on the differentiation efficiency of the endothelial progenitor cells, rinsing the differentiated cells by DPBS (double stranded buffer solution) for 3 times, adding prewarmed Accutase, digesting for 5 minutes at 37 ℃, stopping digestion of a LaSR (LaSR) basic culture medium, collecting the cells into a sterile and clean 15mL centrifuge tube, and centrifuging for 5 minutes at the room temperature of 170 g;

(2) after the centrifugation is finished, the supernatant is discarded, 5mL of DPBS is added for re-suspending the cells, and the cells are centrifuged again at the room temperature of 170g for 5 minutes;

(3) the supernatant was aspirated and added with 400. mu.L of DPBS resuspended cells and split into 4 clean sterilized 1.5mL centrifuge tubes, 100. mu.L of each 1.5mL centrifuge tube;

(4) one tube is used as a blank control, 20 mu L of CD34-PE Antibody is added into one tube, the mixture is lightly blown and uniformly mixed, the mixture is placed on ice and incubated for 30 minutes in the dark, 20 mu L of CD31-FITC Antibody is added into the other tube, the mixture is lightly blown and uniformly mixed, the mixture is placed on ice and incubated for 30 minutes in the dark, and 20 mu L of CD34-PE Antibody and 20 mu L of CD31-FITC Antibody are added into the other tube, the mixture is lightly blown and uniformly mixed, and then the mixture is placed on ice and incubated for 30 minutes in the dark;

(5) after the antibody incubation was completed, 1mL of DPBS was added to each tube, and after gently inverting and mixing, centrifugation was performed at 5000g for 5 minutes at room temperature;

(6) after the centrifugation is finished, the supernatant is slightly sucked and discarded, 300 mu L of DPBS (double stranded phosphate) is added for resuspending cells, the sample is placed on ice and is away from the sun to a Flow cell chamber in Hematology of Hunan ya hospital for detection, CellQuest Pro software in a BD FACSCaliburTM Flow Cytometer instrument is used for detection, and data are counted and analyzed.

7.3 flow cytometry detection sorting efficiency (same procedure as 7.2)

After sorting the endothelial progenitor cell differentiation Day 5 by CD31 magnetic bead labeled antibody, the sorting efficiency of the endothelial progenitor cells is detected by flow.

7.4 immunofluorescence identification of surface markers by endothelial cells

(1) Inoculating the sorted endothelial progenitor cells cultured on the Collagen IV into a 24-well plate coated with Matrigel cell slide at room temperature, and performing immunofluorescence identification respectively when the cells are cultured for 1 day and 7 days, wherein the detected marker is CD31/CD144/CD34 primary antibody diluted at the ratio of 1:100 in the same step as 2.6 when the cells are cultured for 1 day; the markers detected at 7 days in culture were vWF and N-terminal and C-terminal FVIII antibodies at 1:100 dilution.

8. Detection of endothelial stage F8 expression

8.1 RT-PCR detection of F8 expression

(1) Culturing the differentiated endothelial cells in a twelve-well plate, and extracting total RNA by a Trizol method when the confluence reaches more than 80%; the rest steps are the same as the step 5.

8.2 ELISA detection of FVIII expression

Culture supernatants of endothelial cells differentiated from iPSCs cultured in 12-well plates for 24 hours were collected, respectively, and the collected culture supernatants were centrifuged at 5000g for 5 minutes, followed by collecting the supernatants, adding 1/100 protease inhibitor, and storing at-80 ℃ for future use. Collecting the cells in the wells after the supernatant and correspondingly counting; detection was carried out using Paired Antibodies for ELISA-Factor VIII from CEDARLANE.

Western blot detection of intracellular LMAN1 expression

9.1 extraction of Total cellular protein

(1) Sucking clean the cell culture medium in a 12-hole plate, adding DPBS (double priming solution) and rinsing for 2-3 times;

(2) adding 200 μ L RIPA lysate (added with 1/100 protease inhibitor), standing and digesting for 5 minutes on ice, and scraping digested cell lysate into a 1.5mL centrifuge tube by using a middle gun head;

(3) performing ultrasonic interruption on the cell lysate by using an ultrasonic instrument, inserting an ultrasonic probe below the liquid level, performing ultrasonic continuously for 5 seconds, standing for 5 seconds, and repeating for 5-6 times;

(4) heating at 95 deg.C for 10 min for denaturation;

(5) centrifuge at 14000g for 10 minutes at 4 deg.C, aspirate the supernatant into a clean 1.5mL centrifuge tube, label, and store at-80 deg.C.

9.2 BCA protein quantification

(1) Adding RIPA lysate into 9 mu L of extracted protein according to a ratio of 1:4 to dilute the protein;

(2) meanwhile, taking a BSA standard substance (protein concentration is 2mg/mL) to dilute according to an equal ratio, and diluting with RIPA lysate in 6 gradients of 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0 mg/mL;

(3) and (3) setting two multiple wells for all sample and standard detection, calculating the amount of the required BCA detection solution according to the number of the wells to be detected, and adding 200 mu L of the BCA detection solution into each well. The BCA detection solution is divided into solution A and solution B, the working solution is the mixture of the solution A and the solution B according to the ratio of 1:50, after the mixture is inverted and mixed evenly, 200 mu L of BCA detection solution is added into each hole of the ELISA plate, and 20 mu L of a sample to be detected or a standard substance is added. Care should be taken to avoid air bubbles during sample application;

(4) placing the ELISA plate added with the sample to be detected and the BCA detection solution in a water-proof constant temperature incubator at 37 ℃ for incubation for 30 minutes;

(5) after the incubation is finished, detecting the absorbance corresponding to the sample under the absorption wavelength of 570nm in a microplate reader, drawing a standard curve through the standard substance, and converting the concentration of the sample by combining the dilution. R2 of the standard curve fitting curve is required to be more than 0.99, so that the detection is accurate;

(6) different samples were diluted uniformly to the same concentration using RIPA lysate and then mixed in a 5 × loading buffer as 4: 1, uniformly mixing the protein sample with the protein;

(7) boiling at 100 deg.C for 10 min to obtain protein, and performing electrophoresis.

9.3 Western blot

(1) Preparing polyacrylamide gel (10% separation gel): cleaning glass rubber plates, fixing the glass rubber plates on a Bio-Rad rubber frame, preparing separation rubber according to the formula in the following table, mixing the separation rubber uniformly, adding the separation rubber between the rubber plates in a soft manner to about 7mL, and adding deionized water to the upper layer for sealing;

(2) preparing upper layer concentrated glue (5%) after the separation glue is solidified: preparing concentrated glue according to the formula in the following table, uniformly mixing, pouring the deionized water used for sealing glue in the step (1), adding the concentrated glue, and inserting the concentrated glue into the comb teeth;

(3) electrophoresis: adding 1X electrophoresis solution into a vertical electrophoresis tank, putting the prepared glue and a glue plate into the electrophoresis tank, enabling a short glass plate to face the inner side, pulling out comb teeth, adding 20 mu g of protein added with a loading buffer into a sample application hole, adding 5 mu L of pre-stabilized protein ladder into one sample application hole, carrying out electrophoresis at 80V for about 30 minutes, and adjusting the voltage to 120V when a protein sample is electrophoresed into separation glue, wherein the electrophoresis time is determined according to the size of a strip and the concentration of the glue;

(4) film transfer: preparing 1 Xmembrane transferring liquid about 20 minutes before electrophoresis is finished, and precooling at-20 ℃. After electrophoresis is finished, taking out the gel, selecting a target area to cut off the surrounding gel, cutting a PVDF membrane with the same size as the PAGE gel of the membrane to be transferred according to the size of the PAGE gel, soaking the PVDF membrane into methanol for activation for 5 minutes, then soaking the gel and the PVDF membrane into precooled 1 multiplied membrane transferring liquid, placing a black clamping part of the membrane transferring liquid below, sequentially placing a sponge pad, double-layer filter paper, the gel, the PVDF membrane, double-layer filter paper and the sponge pad, covering and clamping the white clamping part of the membrane transferring liquid, placing the clamping part in a membrane transferring groove, placing a black clamping part of the membrane transferring liquid corresponding to the black surface in the groove, placing a-20 ℃ precooled Bioice box of the Bioice in the membrane transferring groove, inserting an electrode, and transferring the membrane for 90 minutes by using a 252mA constant current;

(5) primary antibody incubation: after the membrane is transferred, the membrane is placed into a membrane washing box with the right side facing upwards, the membrane is rinsed once by 0.1 percent TBST, 5 percent skimmed milk is sealed on a decolouring shaker for 1 hour at room temperature, and after the membrane is sealed, a primary antibody prepared by 5 percent skimmed milk is added on the decolouring shaker for gentle shaking at 4 ℃ overnight (beta-actin is diluted by 1:10000, LMAN1 is diluted by 1: 1000);

(6) and (3) secondary antibody incubation: recovering primary antibody, washing with 0.1% TBST on decolorizing shaker for 10 min for 3 times, adding secondary antibody prepared with 5% skimmed milk, and incubating on decolorizing shaker for 1 hr (anti-mouse secondary antibody diluted at 1:10000 and anti-rabbit secondary antibody diluted at 1: 10000);

(7) and (3) developing: after the secondary antibody incubation is finished, shaking and washing for 3 times with 0.1% TBST on a decoloring shaker for 10 minutes each time, preparing ECL developing solution, mixing solution A and solution B at a ratio of 1:1, dripping the mixture on a membrane, incubating for 3-5 minutes at room temperature in a dark place, placing the membrane into a Biorad developing instrument for developing, adjusting exposure time according to strip brightness, and storing pictures.

10 in vivo transplantation of HA model mice by EPCs

HA model mice were a F8tm1Kaz strain purchased from Jackson laboratories^[28]. The homozygous mice of the strain can bleed spontaneously at joints or soft tissues, but pregnant mice generally do not bleed during pregnancy and have no production difficulty. The strain HA model mouse shows the key characteristics of HA, and provides a good model for HA research and gene therapy strategy exploration.

Taking half of male mice of female mice of 6-8 weeks old, correspondingly dividing the mice required by each experimental group into cages before the experiment, distributing the mice of the same sex into one cage, and carrying out cell transplantation experiment after the mice are adapted for 2-3 days.

Cell transplantation experiments: washing endothelial progenitor cells cultured for 1-3 days after sorting CD31 magnetic bead antibodies by DPBS (platelet-derived polysubtility model) for 3 times, adding Accutase, digesting for 5 minutes in an incubator at 37 ℃, adding 5% FBS/DPBS to stop digestion, collecting the cells into a sterile 15mL centrifuge tube, counting by using a blood cell counting plate, centrifuging for 5 minutes at 170g room temperature, sucking and removing supernatant, adding 5mL of DPBS (platelet-derived polysubstrate serum), centrifuging for 5 minutes at 170g room temperature, sucking and removing supernatant, dotting, sucking supernatant liquid, adding 100 mu L of DPBS (platelet-derived polysubstrate per 200 million cells, dividing 200 million into one tube, and transplanting HA mouse cells.

Mice of hemophilia a model were intraperitoneally injected with 10 μ L avermectin (25mg/mL)/g (mouse body weight) for anesthesia, about 5 minutes after injection, after the mice were anesthetized and stunned, 100 μ L of cell suspension, i.e., 200 ten thousand endothelial progenitor cells, were aspirated with an insulin needle, and carefully purged of air bubbles, injected into the mice via retro-orbital veins, the mice were returned to their cages, housed after they were awakened, on IVC racks, 24 hours later, each mouse was intraperitoneally injected with 40 μ L FK506 for immunosuppression, and then injected with FK506 once every other day.

The partial deletion type gene correction of the B region in the experiment is divided into 8 groups:

HA model mouse group, no cells injected, FK506 only injected;

in the DPBS group, 100 μ L of DPBS was injected retroorbitally into HA model mice, and the other treatments were the same;

the HA-iEPCs group, i.e., the group injected with 200 ten thousand HA-iEPCs;

2-6-iEPCs group, i.e. the group of endothelial progenitor cells differentiated by injecting 200 ten thousand 2-6-iPSCs;

2-46-iEPCs group, i.e. the group of endothelial progenitor cells differentiated by injecting 200 ten thousand 2-46-iPSCs;

the N-iEPCs group is an endothelial progenitor cell group differentiated by injecting 200 ten thousand normal control N-iPSCs;

the group of N-del 54-42-iEPCs, i.e., the group of endothelial progenitor cells differentiated by injecting 200 ten thousand of N-del 54-42-iPSCs;

the C57BL/6 group, i.e., the wild type mouse group, was injected with FK506 alone without cells.

The total deletion type gene correction of the B region in the experiment is divided into 8 groups:

HA model mouse group, no cells injected, FK506 only injected;

in the DPBS group, 100 μ L of DPBS was injected retroorbitally into HA model mice, and the other treatments were the same;

the HA-iEPCs group, i.e., the group injected with 200 ten thousand HA-iEPCs;

BD21-iEPCs group, which is a group of endothelial progenitor cells differentiated by injecting 200 ten thousand 2-6-iPSCs;

BD25-iEPCs group, which is a group of endothelial progenitor cells differentiated by injecting 200 ten thousand cells through 2-46-iPSCs;

the N-iEPCs group is an endothelial progenitor cell group differentiated by injecting 200 ten thousand normal control N-iPSCs;

the group of N-BD14-iEPCs, i.e., the group of endothelial progenitor cells differentiated by injecting 200 ten thousand N-del 54-42-iPSCs;

the C57BL/6 group, i.e., the wild type mouse group, was injected with FK506 alone without cells.

11. Detection of blood coagulation activity after cell transplantation and tail-end breakage experiment

After cells are transplanted for 14 days, after the mice are anesthetized by intraperitoneal injection of avermectin, blood is taken through eyeballs, sodium citrate is anticoagulated, plasma is separated, and FVIII coagulation activity in the plasma is detected. Meanwhile, a mouse with transplanted cells for 14 days is taken, after Abutilin is anesthetized in an abdominal cavity, four limbs are fixed by using a pin, 75% alcohol is used for disinfecting the abdomen, the skin and the peritoneum are cut off, the auricle is cut off, about 25mL of DPBS is quickly injected from the apex of the heart, when the liquid flowing out is basically transparent, the DPBS is changed into 4% paraformaldehyde, the DPBS is injected into the mouse from the apex of the heart, the perfusion is stopped when the mouse shows protein denaturation such as tail tilting, limb spasm and the like, the main organs such as the liver, the heart, the spleen, the lung, the kidney and the like of the mouse are taken out, the mouse is placed in the 4% paraformaldehyde for fixation, after 24 hours, the mouse is changed into 15% sucrose for dehydration for 24 hours, then the mouse is changed into 30% sucrose for dehydration for 24 hours, the thickness is cut into 15-20 microns, and after the cutting, the chip is baked for 30 minutes at 55 ℃, and the chip can be used for immunofluorescence staining.

Tail breaking experiment: after the mice after 14 days of cell transplantation are subjected to Abametin abdominal cavity anesthesia, the tail of the mice is cut off at the position which is about 1.5mm away from the tail of the mice at the tail end of the mice, the mice are allowed to vertically bleed for 5 minutes, then the proximal part of the cut end is pressed for hemostasis for 1 minute, and then the mice are placed back into a cage for observation and the survival time of the mice is recorded.

12. Immunofluorescence staining of mouse tissue frozen section

(1) Taking out the frozen tissue section subjected to the baking sheet for immunofluorescence staining to verify that human cells enter a mouse liver, wherein the injected cells are endothelial progenitor cells, the main surface marker is CD31/CD144/vWF, and the vWF antibody can be identified through preliminary experiments to distinguish human sources from mouse sources, so that the immunofluorescence is used for staining the anti-human vWF antibody in the research;

(2) because the tissue has been fixed by paraformaldehyde, the permeabilization can be directly carried out, PBST (DPBS containing 1/1000Triton-100) is added at the position of the slide with the tissue block for permeabilization for 17 minutes; the rest of the subsequent steps are the same as 2.6.

Examples

1. A 54 base fragment of the four bases containing the deletion was deleted near the HA patient mutation site for the c.3167delctga variant, thereby restoring the frameshift mutation to normal reading:

(1) identification of specific iPSCs for hemophilia A patients (HA-iPSCs)

One sample of urine from a patient containing HA with a frameshift mutation in the B region (c.3167delCTGA results in p.D1055fsX5) was collected at the early stage, and the epithelial cells (expressing ZO-1, KRT7, β -catenin) were cultured (FIG. 4A), and then reprogrammed to iPSCs in the form of clonal growth with regular edges and high nuclear-to-mass ratio (FIG. 4B); the karyotype detection showed no chromosomal level variation (fig. 4C), and sequencing of the induced iPSCs confirmed that it still retained patient-specific c.3167delctga variation (fig. 4D), which is a good disease cell model. The stem cell surface marker is identified through immunofluorescence, NANOG, OCT4 and SSEA-4 can be expressed, SSEA-1 is not expressed, and the expression characteristics of the stem cell surface marker are met (figure 4E); induced iPSCs were found to form teratomas in nude mice after in vivo three-germ layer differentiation, and HE staining revealed the presence of gut epithelial cells of endoderm, mesodermal cartilage tissue and neuroepithelial-like tissue of ectoderm within teratomas (fig. 4F).

(2) Construction and efficiency detection of CRISPR/Cas9 expression plasmid for accurate deletion of 54bp of mutation site and acquisition of fixed-point accurate deletion clone by nuclear transfer HA-iPSCs

The applicant introduces a CRISPR/Cas9 gene editing system from a Zhang Feng laboratory, designs sgRNA aiming at the specific mutation site of the patient, and combines ssODN to perform in-situ gene therapy in the B region mutant HA-iPSCs (as shown in figure 5A); performing cutting efficiency detection on the constructed CRISPR/Cas9 by using a T vector connection sequencing mode (FIG. 5B); performing gene targeting on HA-iPSCs by the CRISPR/Cas9 and the ssODN which are constructed in a combined manner, performing primary identification on targeted clones by PCR, randomly selecting two 2-6 and 2-46 genes for subsequent experiments, and obtaining a PCR identification result as shown in figure 5C; the PCR products were Sanger sequenced which confirmed that the two clones had exactly 54 bases deleted including 4 bases of the patient variation (fig. 5D).

The HA-iPSCs are subjected to gene targeting by combining the CRISPR/Cas9 and the ssODN, meanwhile, the HA-iPSCs are subjected to nuclear transfer by independently using the CRISPR/Cas9, after single cells are inoculated to an MEF feeding layer, single clones are picked, DNA is extracted after amplification, and the number of the obtained positive clones is counted through PCR and sequencing identification, wherein as shown in the following table (table 2), the targeting efficiency of a combination ssODN group is 14.71%, and the targeting efficiency of a person independently using the CRISPR/Cas9 is 2.08%. This indicates that the combination of CRISPR/Cas9 and ssODN mediated deletion of the precise gene fragment is more efficient.

TABLE 2 CRISPR/Cas9 in combination with ssoDN compared to CRISPR/Cas9 alone for nuclear transfer of HA-iPSCs efficiency

(3) Accurate deletion of 54bp by combining CRISPR/Cas9 and ssODN nuclear transfer N-iPSCs

In order to avoid the genetic background influence of HA-iPSCs, the 54bp fragment of normal control N-iPSCs is accurately deleted. Due to the fact that the efficiency of combining CRISPR/Cas9 and ssoDN to mediate deletion of accurate gene fragments is higher, accurate deletion of 54bp of N-iPSCs is carried out in a mode of combining CRISPR/Cas9 and ssoDN. After nuclear transfer, inoculating single cells of mixed cells to MEF, when the cell clone grows to about ten times of visual field 1/2-2/3, picking out the single clone, amplifying, extracting DNA, carrying out PCR identification, carrying out PCR amplification to obtain a 448bp strip, sending the strip to be sequenced, identifying the strip to be positive as the clone with the deletion of an accurate gene segment, and randomly selecting N-del 54-15-iPSCs and N-del 54-42-iPSCs for subsequent experiments (figure 6).

(4) Positive clone immunofluorescence identification obtained by targeting

Stem cell surface marker immunofluorescence identification is carried out on the obtained positive clone, the positive clone obtained after targeting can express surface markers consistent with embryonic stem cells, Oct4, Nanog and SSEA-4 are positive in expression, SSEA-1 is negative in expression (figures 7 and 8), and the fact that the targeting process has no influence on the characteristics of iPSCs is verified.

(5) Detection of positive clone karyotypes by targeting

Karyotyping was performed after 10 passages of the gene repair clones, consistent with that before targeting, with a karyotype of 46, XY, (fig. 9 and 10) showing no significant variation at the chromosomal level.

(6) iPSCs stage F8 expression detection

In the iPSCs stage, the F8 transcript level detection is carried out on the positive clone which is obtained by target deletion and 54bp through RT-PCR (figure 11 and figure 12), and the exon 14 position of the accurate fragment deletion clone transcript level is found to be an accurate deletion type in the iPSCs stage, and the patient, the repair clone and the normal human exon 23 position are found to be out of 26 positionsThe transcription between the exons has bands with the same size, which indicates that the patient does not have nonsense-mediated RNA degradation, and the results are consistent with the reports in the literature^[29]。

FVIII was expressed in the cytoplasm and secreted in the culture supernatant by ELISA, and it was found that FVIII was secreted in a low amount in the iPSCs phase (fig. 13). It has been reported in the literature that [30] the secretion of FVIII requires the involvement of an endoplasmic reticulum-Golgi intermediate, which is a complex formed by LMAN1 and MCFD 2. Clinically, homozygous mutation of any one protein can cause combined deficiency of FV and FVIII, the expression of LMAN1 is detected by Western blot (figure 14), and the phenomenon that LMAN1 protein expression is not found in cells in iPSCs stage can be explained, so that the phenomenon that the FVIII secretion amount is low in the iPSCs stage can be explained.

(7) iPSCs directional endothelial progenitor cell differentiation and F8 expression detection of endothelial cell stage

Cells from day 5 of differentiation were flow assayed using CD31-FITC/CD34-PE to determine endothelial progenitor differentiation efficiency. As can be seen from FIG. 15, the differentiation efficiency of different strains of cells is different, the proportion of CD31/CD34 double positive cells is about 10% -30%, and the differentiated endothelial progenitor cells have the proliferation capacity, which provides the possibility for obtaining a large amount of endothelial progenitor cells for transplantation.

After sorting and purifying endothelial progenitor cells by using CD31 magnetic beads, performing flow detection by using CD31-FITC/CD34-PE to determine the sorting effect of the sorting system, as shown in FIG. 16, morphologically observed sorted cells are typical forms of epithelial-like cells, uniform cells and high proliferation speed, the proportion of CD31/CD34 double-positive cells is more than 90%, which indicates that the sorting system used by people is feasible, and the purity of the endothelial progenitor cells obtained after sorting is good, thereby being beneficial to subsequent experiments.

The sorted endothelial progenitor cells were subjected to immunofluorescence detection of relevant surface markers, as shown in FIG. 17, and all CD31/CD144/CD34 were expressed positively, further confirming that the sorted cells were endothelial progenitor cells.

When the sorted endothelial progenitor cells were cultured in EGM-2 medium for 6-7 days, the proliferation rate of endothelial cells was reduced, and the expression of mature endothelial cell marker vWF was detected (fig. 18).

The endothelial cells can synthesize FVIII and vWF, and the vWF can be synthesized and stored in Weibel-Palade corpuscles, when the blood vessels are damaged, the vWF and the FVIII are jointly released and combined to form stable vWF/FVIII, the FVIII is prevented from being rapidly degraded, and therefore, the vWF and the FVIII can participate in the hemostasis process more effectively. We therefore examined FVIII expression and localization in cells by staining vWF with N-terminal and C-terminal FVIII antibodies. As shown in fig. 19, since FVIII expression by patient cells was terminated early, and thus an N-terminal FVIII positive signal could be detected in endothelial cells induced by HA-iPSCs, both gene repair clones and normal human-derived mature endothelial cells co-expressed FVIII and vWF. However, no obvious positive signal was observed in C-terminal FVIII staining of mature endothelial cells differentiated by iPSCs induced in patients (fig. 20), and the endothelial cells differentiated by gene repair clones and normal iPSCs could detect not only N-terminal FVIII but also C-terminal FVIII, which confirmed the effectiveness of the repair strategy used in this study from the protein level.

FVIII is detected in cytoplasm expression amount of mature endothelial cells and secretion amount of culture supernatant by ELISA, as shown in figure 21, FVIII secretion amount is increased in mature endothelial cell stage compared with iPSCs stage, LMAN1 protein is detected in endothelial cells, and LMAN1 protein is found to be expressed in endothelial cells (figure 22), thus FVIII secretion amount in endothelial cell stage is higher than that in iPSCs stage.

(8) Endothelial progenitor cell HA model mouse in vivo transplantation

Transplanting HA-iEPCs, EPCs (2-6-iEPCs and 2-46-iEPCs) formed by C-iPSCs, EPCs (N-iEPCs) formed by normal control N-iPSCs differentiation, N-iPSCs (N-iEPCs) formed by deletion of 54bp clone of N-iPSCs, N-del 54-42-iEPCs formed by differentiation of N-del 54-42-iPSCs are transplanted in mice in vivo, and five groups of cell transplantation groups are obtained, and one group is injected with DPBS as a control group, and when the group is transplanted for 14 days, the plasma of each group of mice is taken to detect FVIII coagulation activity, and the coagulation activity of each group after transplantation is shown in proportion of the actual coagulation activity of each group of mice to the coagulation activity of wild type mice. As shown in FIG. 23, the blood coagulation activity of the transplanted groups of 2-6-iEPCs and 2-46-iEPCs is significantly higher than that of the transplanted groups of DPBS and HA-iEPCs, and HAs no significant difference with the transplanted groups of N-iEPCs and N-del 54-42-iEPCs, and the in vivo experiments prove that the repair strategy used by the inventor is effective.

The therapeutic effect of this strategy was judged by tail-off experiments, and for the survival time of mice after tail-off, as can be seen from FIGS. 24 and 25, the survival time of mice in the groups of 2-6-iEPCs, 2-46-iEPCs, N-iEPCs and N-del 54-42-iEPCs was significantly longer than that of the group of HA-iEPCs, and it is noted that the survival curves show that at the end of the tail-off experiments (48 hours), mice in the groups of 2-6-iEPCs, 2-46-iEPCs and N-del 54-42-iEPCs survived the tail-off experiments, which is very significant for the treatment of diseases. 4 of 12 mice in the group of 2-6-iEPCs that underwent the tail-breaking test survived, 4 of 10 mice in the group of 2-46-iEPCs that underwent the tail-breaking test survived, and 1 of 9 mice in the group of N-del 54-42-iEPCs that underwent the tail-breaking test survived. This further confirms the effectiveness of the gene repair strategy.

Detection of transplanted human cells in vivo in mice

After the endothelial progenitor cells are injected into a mouse body through the retroorbital vein, the human cells need to enter and be implanted in the mouse body, and the lasting effect can be exerted. After perfusion, the liver of the mouse with the transplanted cell group was taken out, frozen section was performed, endothelial cells were labeled with CD31, immunofluorescence staining was performed with anti-human vWF antibody, and it was observed that vWF staining-positive cells could be observed in the liver section of the mouse into which the cells were injected, and no positive signal was observed in the DPBS group, as compared with the DPBS negative control group (fig. 26). Meanwhile, other main organs of the mouse, such as heart, spleen, lung, kidney and brain tissues, are taken, and are subjected to frozen section and immunofluorescence staining, so that human cells are found in the lung of the mouse (figure 27), and an experimental basis is provided for the long-term effectiveness of the treatment strategy.

2. The iPSCs of HA patients for the c.3167delctga variant were targeted to delete the entire B region coding sequence, thereby restoring the frameshift mutation to normal reading:

(1) construction and efficiency detection of CRISPR/Cas9 expression plasmid with targeted deletion of B region coding sequence

Synthesizing an sgRNA primer sequence as shown in a targeting schematic diagram (figure 28), annealing and connecting the sgRNA primer sequence to a pX330 framework to construct two CRISPR/Cas 9-sgRNAs, precisely deleting a B structure domain by combining the two CRISPR/Cas 9-sgRNAs and one ssODN, and simultaneously reserving an SQ sequence, wherein the SQ sequence comprises amino acids 741 and 743, amino acids 1638 and amino acids 1639 are glutamine, and the amino acids 1639 and asparagine, due to the limitation of a PAM sequence, the 744 and 745 amino acids are reserved in the research, the gene sequence is CAGAAT, the two amino acids are consistent with the amino acids coded by the CAAAAC at 1638 and 1639, and are glutamine and asparagine, so two synonymous mutations are equivalently introduced. For HA patient specific iPSCs, a 2678bp fragment is precisely deleted, so that the frame shift mutation is corrected, and the reading frame is recovered to be normal. For normal control iPSCs, a 2682bp fragment is precisely deleted, and iPSCs with in-situ B region deletion are constructed.

CRISPR/Cas9 efficiency detection

The T-vector is used for connecting PCR products and then sequencing, so that the NHEJ ratio generated by the CRISPR/Cas9 is detected, and the efficiency of the CRISPR/Cas9 is suggested. The cleavage efficiency of F8-BDU-sg1 was 13.33% (FIG. 29A) and that of F8-BDD-sg4 was 35.71% (FIG. 29B).

(2) Gene targeting of iPSCs by combining CRISPR/Cas9 and ssODN

And performing HA-iPSCs nuclear transfer by combining CRISPR/Cas9 and ssODN to obtain cells with the deletion of the accurate gene fragment in the B region. Performing nuclear transfer on HA-iPSCs by using a Lonza nuclear transfer instrument, after the nuclear transfer, counting cells, inoculating the cells by using single cells, picking and amplifying the single cells, extracting cell genome DNA, performing PCR identification, simultaneously identifying by using two pairs of primers, amplifying by using F8-E14-F/R, amplifying by using the clone with the deletion of a B region, wherein the band cannot be amplified by using the clone with the deletion of the B region, and amplifying by using F8-BUF/BDR, wherein the band with the size of 341bp can be amplified by using the clone with the deletion of the B region. Sequencing the PCR product when the 341bp band is amplified by PCR, performing Sanger sequencing by taking F8-BD-R as a primer, and identifying the cell which is positive by sequencing as the cell with the accurate deletion B area. Two clones identified as precise gene fragment deletions, BD21-iPSCs and BD25-iPSCs (FIG. 30), were randomly picked for further experiments.

In order to avoid the genetic background influence of HA-iPSCs, B region fragment deletion is carried out on normal control N-iPSCs simultaneously in the experiment. And simultaneously, two pairs of primers are used for identification, a band cannot be amplified by using F8-E14-F/R, a band cannot be amplified by using the clone with the deletion of the B region, the band with the size of 341bp can be amplified by using F8-BUF/BDR for amplification, and the band can be amplified by using the clone with the deletion of the B region. Sequencing the PCR product when the 341bp band is amplified by PCR, performing Sanger sequencing by taking F8-BD-R as a primer, and identifying the cell which is positive by sequencing as the cell with the accurate deletion B area. N-BD9-iPSCs and N-BD14-iPSCs were randomly picked for subsequent experiments (FIG. 31).

(3) B-region deletion clone immunofluorescence identification and karyotype detection obtained by targeting

And performing immunofluorescence identification on stem cell surface markers of the obtained positive clones deleted from the B region, wherein the positive clones obtained after targeting can express surface markers consistent with embryonic stem cells, and the expression of Oct4, Nanog and SSEA-4 is positive, the expression of SSEA-1 is negative (figure 32 and figure 33), and the targeting process is proved to have no influence on the characteristics of iPSCs.

Karyotyping of the B-region deletion clones after passage of 10 passages was 46, consistent with that before targeting, and XY (FIGS. 34 and 35) showed no significant variation at the chromosomal level.

(4) iPSCs stage F8 expression detection

In the iPSCs stage, the F8 transcription level detection is carried out on the B region deleted positive clone obtained by the target shooting through RT-PCR (figure 36 and figure 37), and the B region deleted clone transcription level in the iPSCs stage is found to generate a 552bp band by crossing with a 14 # exon primer, while the band is not generated by the patient and the normal control; the patient and the B region deletion clone and the transcription between the normal control No. 23 exon and No. 26 exon have bands with the same size, which indicates that the patient does not have nonsense-mediated RNA degradation, the document reports that the frameshift mutation in the B region leads to the early termination of protein translation but does not have nonsense-mediated RNA degradation, and the research and the document report have the same results^[29]。

Detection of FV by ELISAIII expressed in the cytoplasm and secreted in the culture supernatant, FVIII was found to be secreted in a lower amount in the iPSCs phase (fig. 38). The secretion of FVIII requires the involvement of an endoplasmic reticulum-Golgi intermediate, which is a complex formed by LMAN1 and MCFD2^[30]. Expression of LMAN1 was detected by Western blot (fig. 39), and LMAN1 protein expression was not detected in iPSCs stage cells, and thus, it is likely that the iPSCs stage FVIII secretion was low because the relevant proteins required for iPSCs stage FVIII secretion were not expressed.

(5) Directed differentiation of iPSCs into endothelial progenitor cells

Method for adding small molecule inhibitor to iPSCs with B region deleted^[26]Directionally differentiating into endothelial progenitor cells, and detecting the differentiation efficiency of the endothelial progenitor cells by flow type on the fifth day of differentiation. As shown in FIG. 40, the differentiation efficiency of different strains of cells is slightly different, and the proportion of CD31/CD34 double positive cells is about 15% -30%. Because of their proliferative capacity, sufficient endothelial progenitor cells can be obtained by culture expansion.

The differentiated endothelial progenitor cells are sorted by using CD31 magnetic beads, and the sorted endothelial progenitor cells detect cell surface markers through immunofluorescence, as shown in FIG. 41, CD31/CD144 are all positive in expression.

When the sorted endothelial progenitor cells were cultured in EGM-2 medium for 6-7 days, the proliferation rate of endothelial cells was reduced, and the expression of mature endothelial cell marker vWF was detected (fig. 42).

(6) Endothelial cell stage F8 expression assay

In the endothelial cell stage, the F8 transcript level detection (FIGS. 43 and 44) was performed on the B region deleted positive clone obtained by targeting through RT-PCR, and it could be observed that in the endothelial cell stage, the transcript level of the B region deleted clone amplified a 552bp band across the 14 # exon primer, while the band was not amplified in the patients and normal controls; the patient and B-domain deletion clones and the normal control transcript between exon 23 to exon 26 were bands of identical size.

Endothelial cells are not only the main cell type for the synthesis of FVIII^[31,32]And can synthesize vWF for storageIn Weibel-Palade corpuscles, vWF and FVIII are released together when blood vessels are injured, so that rapid degradation of FVIII can be avoided, and the blood stanching process can be effectively participated. In this subject, expression and localization of FVIII in cells was examined by staining vWF with N-terminal and C-terminal FVIII antibodies. Because of the frameshift mutation in patient F8 gene, resulting in premature translation termination, an N-terminal FVIII positive signal could be detected in HA-iECs but no C-terminal FVIII positive signal could be detected as indicated in the first chapter of testing. Both positive signals were detected for N-terminal FVIII (FIG. 45) and C-terminal FVIII (FIG. 46), whether from patient-derived endothelial cells differentiated from the B-domain deleted clone or from normal human mature endothelial cells differentiated from the B-domain deleted clone. This also confirms the effectiveness of the B-domain deletion repair strategy employed in this study from the protein level.

FVIII secretion in mature endothelial cell culture supernatant was measured by ELISA, as shown in FIG. 47, FVIII secretion increased during the mature endothelial cell phase compared to the iPSCs phase, LMAN1 protein expression was measured in endothelial cells, and LMAN1 protein expression was found in endothelial cells (FIG. 48), which may be a cause of increased FVIII secretion during the endothelial cell phase.

(7) Endothelial progenitor cell HA model mouse in vivo transplantation

Transplanting HA-iEPCs, EPCs (BD21-iEPCs and BD25-iEPCs) differentiated from iPSCs with deleted B region, EPCs (N-iEPCs) differentiated from normal control N-iPSCs, cloning obtained by deleting B region from N-iPSCs, and transplanting the N-BD14-iPSCs differentiated from N-BD14-iEPCs into mice in vivo, wherein five groups of cell transplantation groups are formed, and one group is injected with DPBS as a control group. At the time of 14 days of transplantation, plasma of each group of mice was taken and the coagulation activity of FVIII was measured, and the coagulation activity of each group after transplantation was expressed as the ratio of the actual coagulation activity of each group of mice to the coagulation activity of wild type mice. As shown in FIG. 49, the clotting activities of the transplanted group of BD21-iEPCs and BD25-iEPCs were significantly higher than those of the transplanted group of DPBS and HA-iEPCs, and were not significantly different from those of the transplanted group of N-iEPCs and N-BD14-iEPCs, and in vivo experiments confirmed that the in situ B deletion strategy used by us was effective for hemophilia A therapy.

The therapeutic effect of the in situ B-domain deletion strategy was judged by tail-breaking experiments, and the survival time of mice was recorded after tail-breaking, as can be seen from FIGS. 50 and 51, the survival time of mice in groups BD21-iEPCs, BD25-iEPCs, N-iEPCs and N-BD14-iEPCs was significantly longer than that of HA-iEPCs, and it is noted that the survival curves show that at the end of the tail-breaking experimental observation (48 hours), mice in groups BD21-iEPCs, BD25-iEPCs, N-iEPCs and N-BD14-iEPCs survived after tail-breaking experiments, which is very significant for the treatment of diseases. 1 of 9 mice in the BD21-iEPCs group that underwent tail-off, 2 of 9 mice in the BD25-iEPCs group that underwent tail-off, 1 of 9 mice in the N-iEPCs group that underwent tail-off, and 2 of 9 mice in the N-BD14-iEPCs group that underwent tail-off. This further confirms the effectiveness of the deletion strategy of zone B in situ for hemophilia a treatment.

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The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.

SEQUENCE LISTING

<110> university of south-middle school

<120> human induced pluripotent stem cell, construction method and use thereof

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Claims (4)

1. A human induced pluripotent stem cell with accession number C201968.

2. A method for constructing human induced pluripotent stem cells is characterized in that iPSCs derived from hemophilia A patients are subjected to nuclear transfer through CRISPR/Cas9 and ssODN to obtain iPSCs containing in-frame deletion mutation sites of in-situ B region coding sequences, wherein the iPSCs derived from hemophilia A patients are obtained by inducing urine cells of the patients, the ssODN sequence is shown as SEQ ID NO 9, and the sequence of sgRNA adopted during CRISPR/Cas9 editing is shown as SEQ ID number 1 or SEQ ID NO 3.

3. Use of the human induced pluripotent stem cell according to claim 1 or the human induced pluripotent stem cell constructed according to the method of claim 2 for the manufacture of a medicament for the treatment of hemophilia a disease.

4. A medicament for treating hemophilia a comprising the human induced pluripotent stem cell according to claim 1 or the human induced pluripotent stem cell constructed by the method according to claim 2.

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