Induced pluripotent stem cell carrying angel syndrome specific genetic information and application thereof

Abstract

The invention discloses an induced pluripotent stem cell carrying angel syndrome specific genetic information and application of the induced pluripotent stem cell, and relates to the technical field of stem cells. The induced pluripotent stem cell carries angel syndrome specific genetic information and can be further induced into excitatory cortical neuronal cells, and a good cell model is provided for exploration of pathogenesis of neurodevelopment disorder diseases, drug screening and the like.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, and more specifically, to an induced pluripotent stem cell carrying Angelman syndrome-specific genetic information and its applications. Background Technology

[0002] Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of function of the UBE3A gene in the q11-q13 region of chromosome 15, originating from the mother. Clinically, it is characterized by developmental delay, language impairment, motor balance disorder, seizures, and characteristic pleasurable behaviors (such as frequent smiling and clapping). Patients often exhibit distinctive facial expressions (open mouth and protruding tongue, prominent jaw) and gait (rigidity, ataxia), clinically presenting as an "angelic face," hence the name. The global incidence is approximately 1 in 12,000–1 in 24,000.

[0003] Due to the limited availability of patient-derived in vitro neural cells and the short survival time of primary neurons in vitro, animal models are currently the primary means of reproducing the phenotypes of AS patients for research. AS mouse models are constructed by knocking out the maternal UBE3A gene to reproduce core phenotypes such as motor incoordination, susceptibility to epilepsy, and abnormal synaptic plasticity. However, animal models cannot fully mimic the complete phenotype of human AS; for example, only 30%–50% of mice develop epilepsy. Furthermore, the severity of human AS is correlated with different genetic molecular subtypes (e.g., the UBE3A deletion type has the most severe symptoms), but mouse models are highly homogeneous, making it difficult to simulate the phenotypic differences between patients with different molecular subtypes. Additionally, mouse models cannot simulate the complexity of human epigenetic imprinting. These limitations in technology and phenotype mean that using animal models to study the pathogenesis of AS and to screen drugs will result in discrepancies compared to humans.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an induced pluripotent stem cell carrying Angelman syndrome-specific genetic information and its application.

[0006] There are four genetic types of AS, all of which lead to the loss of maternal UBE3A function. Among them, 70–75% of patients have a deletion of the 15q11-13 region of the maternal chromosome (including UBE3A), 10–20% of patients have point mutations / small fragment deletions of the UBE3A gene, 3–5% of patients have methylation errors in the imprinting center (IC) leading to silencing of maternal UBE3A, and 2–5% of patients have both chromosomes 15 inherited from the father, resulting in paternal uniparental diploidity and causing the loss of maternal UBE3A.

[0007] Induced pluripotent stem cells (iPS cells) are pluripotent stem cells reprogrammed from terminally differentiated somatic cells by introducing specific transcription factors. iPSC technology does not raise ethical concerns in its application and can prepare stem cells from patient-derived cells, significantly reducing the likelihood of immune rejection. Furthermore, because patient-derived iPSCs possess the patient's own genetic background, they can better reflect the cellular characteristics of human diseases when simulating disease pathogenesis. These characteristics bring enormous potential to the application of iPSC technology in disease mechanism research and drug screening, and also present new opportunities for the treatment of ankylosing spondylitis (AS).

[0008] This invention isolates PBMCs from blood samples of AS carriers, then reprograms the PBMCs and cultures them into iPSCs, obtaining iPSCs that can cover the four existing AS genetic subtypes. These iPSCs can be induced into excitatory cortical neurons, providing a good cell model for exploring the pathogenesis of neurodevelopmental disorders and drug screening.

[0009] Based on this, on the one hand, the present invention provides an induced pluripotent stem cell carrying Angelman syndrome-specific genetic information, with accession numbers CCTCC NO: C2025177, CCTCC NO: C2025178, CCTCC NO: C2025179, or CCTCC NO: C2025180.

[0010] The four induced pluripotent stem cells provided by this invention were deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on September 3, 2025, with accession numbers CCTCC NO: C2025177, CCTCC NO: C2025178, CCTCC NO: C2025179 and CCTCC NO: C2025180, respectively; their taxonomic name is *Homo sapiens*.

[0011] The four induced pluripotent stem cells provided by this invention cover the four existing AS genetic subtypes and have differentiation potential. They can be further induced into excitatory cortical neurons, providing a good cell model for exploring the pathogenesis of neurodevelopmental disorders and drug screening.

[0012] The establishment of iPSCs for four AS disease subtypes solves the problems of difficulty in obtaining patient neural cells in vitro and short survival time of primary in vitro cultures. It provides a new approach to obtaining samples from rare disease patients and avoids ethical controversies.

[0013] The iPSCs of this invention use patients with four subtypes: large heterozygous deletion of UBE3A region, UBE3A region imprinting defect, paternally uniparental diploid of UBE3A region, and UBE3A gene point mutation. This preserves the patient's original genetic background, allowing for a faster, more direct, and objective evaluation of the patient's unique neuronal characteristics, morphology, and function. This, in turn, helps explain the principles of the patient's neurodevelopmental disorders and provides important support and evidence for the study of Angelman syndrome and other related neurodevelopmental disorders.

[0014] Optionally, in some embodiments of the present invention, the UBE3A gene function of the induced pluripotent stem cells is lost.

[0015] Optionally, in some embodiments of the present invention, the UBE3A gene of the induced pluripotent stem cells has at least one of the following defects: large heterozygous deletion of a segment in the UBE3A gene region, imprinting defect in the UBE3A gene region, paternally uniparental diploid type of the UBE3A gene, and point mutation in the UBE3A gene.

[0016] The UBE3A gene is located at chr15:25,578,875-25,684,203 (hg19) in the genome.

[0017] Optionally, in some embodiments of the present invention, the UBE3A gene of induced pluripotent stem cells with accession number CCTCC NO: C2025177 has a large segment of heterozygous deletion.

[0018] The UBE3A gene of iPSCs (AS-IPSC 4-1 derived from patients with Angelman syndrome) with accession number CCTCC NO: C2025177 has a large heterozygous deletion, specifically: chr15:23,609,469-28,544,684 (hg19).

[0019] Optionally, in some embodiments of the present invention, the UBE3A gene of the induced pluripotent stem cells (AS-IPSC 4-2 derived from patients with Angelman syndrome) with accession number CCTCC NO: C2025178 has an imprinting defect.

[0020] Optionally, in some embodiments of the present invention, the UBE3A gene of the induced pluripotent stem cells (AS-IPSC 4-3 derived from patients with Angelman syndrome) with accession number CCTCC NO: C2025179 is paternally uniparental diploid.

[0021] Optionally, in some embodiments of the present invention, the UBE3A gene of the induced pluripotent stem cells (AS-IPSC 4-4 derived from patients with Angelman syndrome) with accession number CCTCC NO: C2025180 has a point mutation.

[0022] The UBE3A gene of iPSCs with accession number CCTCC NO: C2025180 has a point mutation, specifically: GRCh37 / hg19: chr15-25615817, NM_130838.1, c.1444C>T, p.R482*.

[0023] On the other hand, the present invention provides the application of induced pluripotent stem cells as described above in constructing Angelman syndrome disease models.

[0024] Optionally, in some embodiments of the present invention, the disease model is a cell model or a tissue model.

[0025] Optionally, in some embodiments of the present invention, the application includes: differentiating the induced pluripotent stem cells into excitatory cortical neurons.

[0026] These excitatory cortical neurons can serve as a disease model for Angelman syndrome and can be used for screening therapeutic drugs or studying the mechanisms of Angelman syndrome.

[0027] It should be noted that the specific method for inducing differentiation of human cortical neurons can be referred to the method described in Embodiment 2 of the present invention. For those skilled in the art, the specific differentiation and culture method is conventional. Based on the induced pluripotent stem cells provided by the present invention, excitatory cortical neurons can be obtained by using conventional differentiation methods in the art.

[0028] On the other hand, the present invention provides the use of the induced pluripotent stem cells and their differentiation products as described above in the preparation or screening of drugs for the treatment of Angelman syndrome.

[0029] The induced pluripotent stem cells (iPSCs) provided by this invention, originating from human patients, possess the patient's own genetic background. Therefore, in simulating disease pathogenesis, they can better reflect the cellular characteristics of human diseases at the cellular level. These characteristics bring enormous potential to the application of iPSC technology in disease mechanism research and drug screening, and also bring new opportunities for the treatment of ankylosing spondylitis (AS).

[0030] An exemplary method for screening drugs could be to contact the candidate drug with the aforementioned induced pluripotent stem cells or their differentiation products, such as excitatory cortical neurons; if the induced pluripotent stem cells or their differentiation products, such as excitatory cortical neurons, exhibit upregulated expression of the UBE3A gene, then the candidate drug is indicated to have therapeutic potential.

[0031] On the other hand, the present invention provides a method for preparing a disease model of Angelman syndrome, comprising: culturing induced pluripotent stem cells as described in any of the preceding claims under suitable conditions to differentiate into excitatory cortical neurons.

[0032] Optionally, in some embodiments of the present invention, the disease model is a cell model.

[0033] On the other hand, the present invention provides a method for screening drugs for treating Angelman syndrome, wherein the candidate drug is contacted with induced pluripotent stem cells or their differentiation products as described in any of the preceding claims.

[0034] On the other hand, the present invention provides a cell culture comprising induced pluripotent stem cells as described in any of the preceding claims and a culture medium.

[0035] For those skilled in the art, the types and formulations of iPSC culture media for culturing pluripotent stem cells are readily available (e.g., Sendai virus CytoTune™ 2.0), and the culture and passage of induced pluripotent stem cells can be achieved using media conventional in the art. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1a Bright-field plot of iPSC clone C2025177;

[0038] Figure 1b Bright-field plot of iPSC clone C2025178;

[0039] Figure 1c Bright-field plot of iPSC clone C2025179;

[0040] Figure 1d Bright-field plot of the C2025180 iPSC clone;

[0041] Figure 2a Marker coloring diagram for C2025177 iPSC;

[0042] Figure 2b Marker coloring diagram for C2025178 iPSC;

[0043] Figure 2cMarker coloring diagram for C2025179 iPSC;

[0044] Figure 2d Marker coloring diagram for C2025180 iPSC;

[0045] Figure 3a AP staining map of C2025177 iPSC;

[0046] Figure 3b AP staining map of C2025178 iPSC;

[0047] Figure 3c AP staining map of C2025179 iPSC;

[0048] Figure 3d AP staining map of C2025180 iPSC;

[0049] Figure 4a Electrophoresis image for mycoplasma identification of iPSC C2025177;

[0050] Figure 4b Electrophoresis image for mycoplasma identification of iPSC C2025178;

[0051] Figure 4c Electrophoresis image for mycoplasma identification of iPSC C2025179;

[0052] Figure 4d Electrophoresis image for mycoplasma identification of iPSC C2025180;

[0053] Figure 5a Karyotype analysis diagram of iPSC C2025177;

[0054] Figure 5b Karyotype analysis diagram of iPSC C2025178;

[0055] Figure 5c Karyotype analysis diagram of iPSC C2025179;

[0056] Figure 5d The karyotype analysis diagram of the C2025180 iPSC;

[0057] Figure 6a The STR test results for iPSC C2025177;

[0058] Figure 6b The STR test results for iPSC C2025178;

[0059] Figure 6cThe STR test results for iPSC C2025179;

[0060] Figure 6d The STR test results for iPSC C2025180;

[0061] Figure 7a The results of viral residual qPCR for iPSC C2025177;

[0062] Figure 7b The results of viral residual qPCR for iPSC C2025178;

[0063] Figure 7c The results of viral residual qPCR for iPSC C2025179;

[0064] Figure 7d The results of viral residual qPCR for iPSC C2025180;

[0065] Figure 8a qPCR results for the three germ layer differentiation marker of C2025177iPSC;

[0066] Figure 8b qPCR results for the three germ layer differentiation marker of iPSC C2025178;

[0067] Figure 8c qPCR results for the trigerm layer differentiation marker of iPSC C2025179;

[0068] Figure 8d qPCR results for the three germ layer differentiation marker of C2025180 iPSC;

[0069] Figure 9 a is a bright-field plot of cortical excitatory neurons differentiated from C2025177 iPSCs;

[0070] Figure 9 b is a bright-field plot of cortical excitatory neurons differentiated from C2025178 iPSCs;

[0071] Figure 9 c is a bright-field plot of cortical excitatory neurons differentiated from C2025179 iPSCs;

[0072] Figure 9 d is a bright-field plot of cortical excitatory neurons differentiated from C2025180 iPSCs;

[0073] Figure 10aMarker staining diagrams of cortical excitatory neurons differentiated from iPSCs (a is C2025177, b is C2025178, c is C2025179, d is C2025180).

[0074] Figure 10b Marker staining map of cortical excitatory neurons differentiated from C2025178 iPSCs;

[0075] Figure 10c Marker staining map of cortical excitatory neurons differentiated from C2025179 iPSCs;

[0076] Figure 10d Marker staining map of cortical excitatory neurons differentiated from C2025180 iPSCs;

[0077] Figure 11 Detection of UBE3A gene expression levels in cortical excitatory neurons differentiated from iPSCs;

[0078] Among them, C2025177, C2025178, C2025179, and C2025180 represent iPSCs with accession numbers CCTCC NO: C2025177, CCTCC NO: C2025178, CCTCC NO: C2025179, and CCTCC NO: C2025180. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0080] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0081] Explanation of abbreviations in this embodiment:

[0082] iPSC: Induced pluripotent stem cells;

[0083] Ficoll: Lymphocyte separation medium;

[0084] PBMCs: Peripheral blood mononuclear cells;

[0085] MOI: multiplicity of infection, refers to the ratio of the number of viruses that can infect cells to the total number of cells in a system;

[0086] Metrigel: A natural hydrogel rich in various proteins and growth factors, which can be used for cell culture;

[0087] mTeSR Plus: A commercially available iPSC culture medium;

[0088] hES: Human embryonic stem cells, a type of pluripotent stem cell;

[0089] DPBS: Duchenne phosphate buffer;

[0090] qPCR: Real-time quantitative PCR;

[0091] ATCC: American Type Culture Collection;

[0092] P / S: Penicillin / Streptomycin; Puro: Puromycin; Dox: Doxycycline;

[0093] Example 1

[0094] Preparation of Angelman syndrome induced pluripotent stem cells

[0095] The method for preparing Angelman syndrome-induced pluripotent stem cells includes the following steps:

[0096] 1) The cell donors were four Angelman syndrome patients who visited the Children's Hospital of Fudan University. Genetic diagnosis had been completed, identifying four subtypes (large heterozygous deletion in the UBE3A region; UBE3A region imprinting defect; paternally uniparental diploid type in the UBE3A region; and UBE3A gene point mutation type). Blood was collected from each patient for subsequent procedures.

[0097] 2) Isolation of mononuclear cells from peripheral blood

[0098] a) Draw 3 ml of Ficoll into a 15 ml centrifuge tube using a syringe, and then slowly add 4 ml of peripheral blood along the tube wall to the surface of the Ficoll solution, being careful not to break the surface.

[0099] b) Gently transfer the centrifuge tube to the centrifuge and centrifuge at 18°C, 400g, for 40 minutes. Clear stratification will occur after centrifugation.

[0100] c) Aspirate the upper plasma layer down to about 0.5 ml from the white membrane layer, and carefully transfer the white membrane layer cells to a new 15 mL centrifuge tube. Add 3 times the volume of physiological saline, gently shake to mix, and centrifuge at 18°C, 500 g, for 10 min.

[0101] d) Discard the supernatant, add 6 ml of physiological saline to resuspend the precipitate, and mix well. Centrifuge at 18℃, 100g, for 15 min.

[0102] e) Discard the supernatant, resuspend the cells in an appropriate amount of PBMC medium (StemSpan™ SFEM II), transfer to 12-well plates (2 ml PBMC medium per well), and place in an incubator. Change the medium every two days and culture for 4-15 days until the cells enter the logarithmic growth phase. Count the cells; once the desired cell number is reached, reprogramming can proceed.

[0103] 3) Construction of induced pluripotent stem cells,

[0104] A) Day -4: Inoculation with PBMCs. Four days prior to viral infection, collect at least 2 × 10⁶ PBMCs, resuspend them, centrifuge at 200 × g for 10 minutes, and discard the supernatant. Resuspend the cells in complete PBMC medium and adjust the cell concentration to 5 × 10⁶. 5 1 cell / mL. Add 1 mL of cell suspension to the center of each well of the 24-well plate (to reduce excessive evaporation of the medium during incubation). Use at least 4 wells to ensure a sufficient number of cells on day 0. Incubate the cells in a humidified incubator at 37°C and 5% CO2.

[0105] B) Day -3 to Day -1: Observe the cells and add fresh culture medium. Change the medium daily: Gently remove 0.5 mL of culture medium from each well and replace it with 0.5 mL of fresh, complete PBMC medium, avoiding disturbing the cells as much as possible. If the 0.5 mL removed from the well contains cells, centrifuge the cell suspension at 200×g for 10 minutes, discard the supernatant, resuspend the cells in 0.5 mL of fresh PBMC medium, and then add them back to the well plate.

[0106] C) Day 0: Cell count and transduction. For the Sendai virus CytoTune™ 2.0 reprogrammed vector, initial transduction used MOIs of 5, 5, and 3 (i.e., KOS MOI=5, hc-Myc MOI=5, hKlf4 MOI=3). Before transduction, 3 × 10⁻⁶ cells were... 5Pipe the PBMC cells into a round-bottom tube. Remove the CytoTune™ 2.0 Sendai virus tubes from -80°C storage. Thaw one tube at a time, first immersing the bottom of the tube in a 37°C water bath for 5–10 seconds, then removing it from the water bath and allowing it to thaw at room temperature. After thawing, briefly centrifuge the tubes and immediately place them on ice. Add the corresponding volumes of the three CytoTune™ 2.0 Sendai virus tubes (i.e., KOS MOI=15 μl, hc-Myc MOI=15 μl, hKlf4 MOI=10 μl) to 1 mL of PBMC medium preheated to 37°C. Ensure the solution is thoroughly mixed by gently pipetting the mixture up and down. Within 5 minutes, add this reprogrammed virus mixture to the round-bottom tube containing the PBMCs. The total volume should be between 1–1.5 mL. Tightly cap the tubes and seal them with sealing film. Centrifuge the cells and virus at 1000×g for 30 minutes at room temperature. After centrifugation, add an additional 1 mL of PBMC medium to the tube to resuspend the cells, and transfer it to one well of a 12-well plate (the total volume should now be between 2 and 2.5 mL). Incubate the plate overnight in a humidified incubator at 37°C and 5% CO2.

[0107] D) Day 1: Change the medium and culture the cells. Remove the cells and medium from the culture plate and transfer them to 15 mL centrifuge tubes. Gently rinse the well walls with 1 mL of medium to ensure most cells are collected. Remove CytoTune™ 2.0 Sendai virus by centrifuging the cell suspension at 200×g for 10 minutes, discarding the supernatant, and resuspending the cells in 0.5 mL of complete PBMC medium per well of a 24-well plate. Observe cell morphology changes under a microscope during the first 48 hours to verify transduction; large, aggregated cells are expected. Culture the cells in a humidified incubator at 37°C and 5% CO2 for 2 days. No medium change is required during this period.

[0108] E) Day 3: Plate the cells onto Metarigel-coated culture dishes. Add 1×10⁶ cells... 5 One live cell / well was seeded into a 6-well Metrigel-coated culture plate, and 2 mL of cytokine-free PBMC medium was added to each well. The cells were incubated in a humidified incubator at 37°C and 5% CO2.

[0109] F) Days 4–6: Replace waste medium. Every other day, gently remove 1 mL (half) of waste medium from the cells and replace it with 1 mL of fresh, cytokine-free PBMC medium, avoiding disturbing the cells.

[0110] G) Day 7: Begin transitioning cells to mTeSR Plus medium (Stemcell). Prepare mTeSR Plus medium. Remove 1 mL of medium (half) from the cells and replace it with 1 mL of mTeSR Plus medium to begin acclimating the cells to the new medium. Incubate the cells overnight in a 37°C, 5% CO2 incubator.

[0111] H) Days 8 to 28: Medium Change and Cell Monitoring. After 24 hours (day 8), replace all the medium with mTeSR Plus medium, and change the waste medium daily thereafter. Starting from day 8, observe the culture plate under a microscope every other day, looking for cell clumps indicating the presence of reprogrammed cells. Manually pick clones and transfer them to prepared metrigel-coated 6-well culture plates.

[0112] 4) Quality control of induced pluripotent stem cells

[0113] A) Morphology. iPSC characteristics: Cells grow in colonies, clustering into clonal shapes. Cells are relatively small, with large nuclei, a high nucleoplasm-to-cytoplasm ratio, and smooth, even clonal surfaces with regular edges and no free cells. Figures 1a-1d .

[0114] B) Surface marker identification.

[0115] 1. iPSCs possess the ability to self-renew in vitro, thus allowing for the identification of induced cells at the cellular level through fluorescent staining of the cell surface, such as Oct4, Nanog, Sox2, SSEA1, and SSEA4. Generally, hiPSCs (human iPSCs) do not express SSEA1 but do express hES cell-specific surface antigens, such as SSEA3, SSEA4, and Nanog.

[0116] 2. The iPSC surface marker staining steps are as follows:

[0117] 3. Wash the iPSCs cultured in the 24-well plate with 1 x PBS (catalog number: 10010072, Gibco) and fix them with 4% PFA (catalog number: P0099-500mL, Beyotime);

[0118] 4. Soak in membrane rupture buffer at room temperature until permeabilized;

[0119] 5. Add blocking buffer at room temperature, block with 10% Goat Serum (catalog number: 16210072, Thermo), wash twice with DPBS, and incubate at room temperature with primary antibody OCT4 (mouse-derived, catalog number sc-5279, Santa Cruz Biotechnology) and TRA-1-60 (mouse-derived, catalog number: MAB4360, Millipore) antibody dilution buffer.

[0120] Wash twice with 6.1x PBS, then add secondary antibodies Goat anti-Mouse IgG (Alexa Fluor 488) (labeled green, catalog number: A-11029, Invitrogen) and Goat anti-Mouse IgG (Alexa Fluor 546) (labeled red, catalog number: A-11003, Invitrogen).

[0121] 7. Wash twice with 1xPBS, then counterstain with Hoechst nuclear staining buffer (catalog number: H3570, Invitrogen) diluted 1:5000, wash twice with 1xPBS, photograph, and see results below. Figures 2a-2d .

[0122] C) Stem Cell Detection (AP Identification). Alkaline phosphatase (AP) is a type of hydrolase that removes the phosphate group from substrate molecules by hydrolyzing phosphate monoesters, generating phosphate ions and free hydroxyl groups. Its dephosphorylation substrates include nucleotides, proteins, and alkaloids, and it is most effective under alkaline conditions. BCIP / NBT is a commonly used substrate for AP. Under the catalysis of AP, BCIP is hydrolyzed to produce a highly reactive substrate, which reacts with NBT to form insoluble dark blue to blue-violet NBT-formazan. iPSCs can express tissue-nonspecific phosphatases; therefore, we used a pluripotent stem cell alkaline phosphatase colorimetric kit (catalog number: C3250S) to identify induced pluripotent stem cells (iPSCs).

[0123] 1. Cell preparation: Usually cultured in 24-well plates, prepare 2-4 wells for each cell line to facilitate repeated experiments and sample backup.

[0124] 2. Coat culture dishes with Matrigel, incubate at 37 degrees Celsius for 30 minutes, seed cells at an appropriate ratio, and allow them to grow to a density of about 40%-50% in 2-3 days;

[0125] 3. Preparation of staining reagents: Prepare staining solution according to the sample volume, and mix solution A and solution B in a 1:1 ratio;

[0126] 4. Fix the sample: Gently aspirate the culture medium, wash once with 1X PBS, add 250 μl of fixative (4% PFA) to each well of a 24-well plate, and fix at room temperature for 10 min (do not fix for more than 20 min, otherwise AP activity will be inactivated);

[0127] 5. Staining: Wash twice with 1xPBS, add freshly prepared AP staining solution, incubate at room temperature for 15-20 min, discard the staining solution, add 1XPBS to stop the reaction, and wash once.

[0128] 6. Photography: Images were taken and analyzed using an inverted microscope. Under normal conditions, iPSCs should mostly stain dark blue. Results are shown below. Figures 3a-3d .

[0129] D) Mycoplasma detection.

[0130] 1. Take 100 μL of the cell culture medium to be tested (changed more than 12 hours ago) and transfer it to a new sterile 1.5 ml EP tube.

[0131] 2. Treat with a metal bath at 95℃ for 8 minutes.

[0132] 3. Centrifuge at 12000 rpm for 5 min, transfer the supernatant to a sterile new 1.5 ml EP tube, and use 2 μL of the supernatant as a template for PCR amplification.

[0133] 4. Take 8 μL of PCR amplification product and perform direct electrophoresis. The result was negative, indicating no mycoplasma contamination. The result is shown in Figure 4a-. Figure 4d .

[0134] E) Karyotype analysis. Karyotype analysis is the process of pairing, numbering, and grouping the chromosomes of the cells to be tested according to the inherent morphological characteristics and rules of human cells, and then performing morphological analysis. Karyotype analysis can be used to check whether the chromosome number is normal, detect large chromosomal structural aberrations, and determine sex. The analysis showed that the iPSC chromosome number was normal, with no loss or increase, and the structure was normal. The karyotype analysis results of the four samples are shown in Figure 5a- Figure 5d .

[0135] F) STR typing and identification. DNA was extracted from appropriate amounts of PBMCs and iPSCs using the Microread Genomic DNA Kit. Sixteen STR loci and sex identification loci were amplified using the Microreader™ 21 ID System. PCR products were detected using an ABI 3730xl genetic analyzer. The results were analyzed using GeneMapper ID-X software (Applied Biosystems) and compared with the ATCC and DSMZ databases. This confirmed that the iPSCs originated from their corresponding PBMCs. The results are shown in Figure 6a-. Figure 6d .

[0136] G) Sendai virus residue detection. iPSCs were passaged to the 11th generation, and Sendai virus residue (SeV, KOS, and Klf4 factor) was detected by qPCR. The results showed no Sendai virus residue, as shown in Figure 7a-. Figure 7d .

[0137] H) Differentiation potential assay. iPSCs were subjected to trilaminar differentiation using different differentiation media. RNA samples were collected from each of the three germ layers, and qPCR was used to detect the expression of specific biomarkers for each germ layer: endoderm SOX17, mesoderm T, and ectoderm SOX1. Upregulated expression of these biomarkers demonstrated the ability to differentiate into all three germ layers; the results are shown in Figure 8a-. Figure 8d .

[0138] The obtained iPSCs include four cell lines, corresponding to the four subtypes of Angelman syndrome: 1) a large heterozygous deletion of the UBE3A region, named AS-IPSC 4-1; 2) a UBE3A region imprinting defect, named AS-IPSC 4-2; 3) a paternally uniparental diploid type of the UBE3A region, named AS-IPSC 4-3; and 4) a UBE3A gene point mutation type, named AS-IPSC 4-4. The four cell lines were deposited on September 3, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession numbers CCTCC NO: C2025177, CCTCC NO: C2025178, CCTCC NO: C2025179, and CCTCC NO: C2025180, respectively.

[0139] Among them, the iPSCs with accession number CCTCC NO: C2025177 had a large heterozygous deletion in the UBE3A gene, specifically: chr15:23,609,469-28,544,684(hg19). The induced pluripotent stem cells with accession number CCTCC NO: C2025178 had an imprinted defect in the UBE3A gene. The induced pluripotent stem cells with accession number CCTCC NO: C2025179 had a paternally uniparental diploid UBE3A gene. The iPSCs with accession number CCTCC NO: C2025180 had a point mutation in the UBE3A gene, specifically: GRCh37 / hg19:chr15-25615817,NM_130838.1,c.1444C>T,p.R482*.

[0140] Example 2

[0141] Application of Angelman syndrome-induced pluripotent stem cells in the preparation of cortical excitatory neuron cell models.

[0142] Directed differentiation of cortical excitatory neurons. Differentiation methods: Differentiation can be divided into two stages: lentiviral transfection of iPSCs and amplification, and neuronal differentiation and maturation stages.

[0143] 2.1 Transfection and Propagation Stage

[0144] D0: iPSC at 1E5 / cm 2 The samples were seeded at a density in 24-well plates and cultured with mTeSR Plus + ROCKi (10 μM).

[0145] D1: Perform virus transfection: Replace with fresh culture medium, add virus, and set up a GFP control group to observe transfection efficiency.

[0146] D2: Replace with fresh mTeSR Plus.

[0147] D3: Seed cells in 6cm culture dishes.

[0148] D4-7: Change the medium daily until the cells are 80%-90% confluent. You can then choose to continue the culture or freeze the cells.

[0149] 2.2 Differentiation Stage

[0150] D0: Transfected iPSCs requiring differentiation at a rate of 2E5 / cm³ 2 Density plated and cultured using mTeSR Plus + ROCKi (10 μM).

[0151] D1, D2, D3: Discard the culture medium, rinse with PBS, and replace with fresh differentiation medium daily.

[0152] D4: Options include cryopreservation or subculturing. The digestion solution is Accutase. The subculturing density is 1.5E5 / cm², and the culture medium is a stable medium supplemented with ROCKi.

[0153] D5: Complete medium replacement using stable culture medium.

[0154] D8: Discard the culture medium, rinse twice with PBS, and replace with fresh maintenance culture medium. Thereafter, perform a half-change of medium every 2-3 days.

[0155] D15: Can be used to observe the state and purity of excitatory neurons in the cortex.

[0156] 2.3 Quality Inspection of Cortical Excitatory Neurons

[0157] A) Morphology

[0158] Bright-field images of excitatory neurons in the cortex of each group were taken at D15. Full neuronal cell bodies and healthy neural networks were observed. Results are shown in [Figure number missing]. Figure 9 a- Figure 9 d.

[0159] B) Immunofluorescence detection

[0160] Neurons from D15 were fixed with 4% PFA and blocked with immunofluorescence blocking solution. Anti-TUJ1 and anti-VGLUT1 markers were selected. After incubation with primary and secondary antibodies, DAPI staining was performed. For statistical analysis, three fields of view were randomly selected to determine the proportion of VGLUT1-positive cells to DAPI-positive cells. The purity of excitatory cortical neurons differentiated using this method reached over 80%, as shown in Figure 10a-. Figure 10d .

[0161] C) Detection of target gene expression levels

[0162] RNA was extracted from D15 cortical excitatory neurons using the Trizol method, reverse transcribed, and then subjected to qPCR to detect the difference in UBE3A expression levels in cortical excitatory neurons of healthy individuals and patients. The results are as follows: Figure 11 As shown, compared to healthy individuals, patients exhibited significantly downregulated UBE3A expression levels.

[0163] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An induced pluripotent stem cell carrying Angelman syndrome-specific genetic information, characterized in that, Its accession numbers are CCTCC NO: C2025177, CCTCC NO: C2025178, CCTCC NO: C2025179, or CCTCC NO: C2025180.

2. The induced pluripotent stem cells according to claim 1, characterized in that, The induced pluripotent stem cells lost the function of the UBE3A gene.

3. The induced pluripotent stem cells according to claim 2, characterized in that, The induced pluripotent stem cells have at least one of the following defects in their UBE3A gene: large heterozygous deletion of a segment in the UBE3A gene region, hypomethylation in the UBE3A gene region, paternally uniparental diploidity of the UBE3A gene, and point mutation in the UBE3A gene.

4. The induced pluripotent stem cells according to claim 3, characterized in that, The UBE3A gene of induced pluripotent stem cells with accession number CCTCC NO: C2025177 has a large heterozygous deletion.

5. The induced pluripotent stem cells according to claim 3, characterized in that, The UBE3A gene of induced pluripotent stem cells with accession number CCTCC NO: C2025178 is hypomethylated.

6. The induced pluripotent stem cells according to claim 3, characterized in that, The UBE3A gene of the induced pluripotent stem cells with accession number CCTCC NO: C2025179 is paternally uniparental diploid.

7. The induced pluripotent stem cells according to claim 3, characterized in that, The UBE3A gene of induced pluripotent stem cells with accession number CCTCC NO: C2025180 has a point mutation.

8. The use of the induced pluripotent stem cells according to any one of claims 1-7 in constructing a disease model of Angelman syndrome.

9. The application according to claim 8, characterized in that, The application includes differentiating the induced pluripotent stem cells into excitatory cortical neurons.

10. The use of the induced pluripotent stem cells and their differentiation products as described in any one of claims 1-7 in the preparation or screening of drugs for the treatment of Angelman syndrome.

11. A method for preparing a disease model of Angelman syndrome, characterized in that, It includes: Under suitable conditions, the induced pluripotent stem cells according to any one of claims 1-7 are differentiated and cultured to obtain excitatory cortical neurons.

12. A method for screening drugs to treat Angelman syndrome, characterized in that, Contact the candidate drug with the induced pluripotent stem cells or their differentiation products as described in any one of claims 1-7.

13. A cell culture, characterized in that, It includes the induced pluripotent stem cells as described in any one of claims 1-7 and the culture medium.

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