Induced pluripotent stem cell strain retaining NFIA gene deletion mutation and application of induced pluripotent stem cell strain

Abstract

The invention belongs to the technical field of biological medicines, and discloses an induced pluripotent stem cell (iPSC) capable of retaining NFIA gene deletion mutation and application of the iPSC. The stem cell strain is preserved in the China Center for Type Culture Collection (the preservation number is CCTCC NO: C202590), carries NC000001.10: g.6165096761842967del heterozygous deletion mutation, contains deletion of exons 3-6 of an NFIA gene, stably expresses pluripotent markers (OCT4, SOX2, NANOG and SSEA4), and has the capability of differentiating to three germ layers. The preparation method comprises the following steps: separating PBMC (peripheral blood mononuclear cells) from peripheral blood of a patient, carrying out reprogramming by utilizing 2.0 Sendai Reprogam Kit, and amplifying pluripotent clone in Essental 8TMMedium. The stem cell strain can be used for constructing a research model of neurodevelopmental disorder diseases (such as corpus callosum insufficiency, hydrocephalus and development retardation), simulating pathological phenotypes by differentiating neurons, glial cells or brain organs, and is also suitable for drug screening and gene therapy research. And a precise humanized tool is provided for mechanism analysis and treatment development of NFIA deficiency related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and discloses an induced pluripotent stem cell line (iPSC) that retains the NFIA gene deletion mutation and its application. Background Technology

[0002] Neurodevelopmental disorders (NDDs) are a spectrum of diseases that begin in early development and are characterized by severe abnormalities in brain development and function. NDDs exhibit significant clinical heterogeneity, affecting more than 3% of children worldwide. According to data from the 2019 Global Burden of Disease Study, developmental delay / intellectual disability (DD / ID), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD) account for a significant proportion of mental and neurodevelopmental disorders, cumulatively affecting the health of more than 100 million people worldwide, imposing a heavy health and economic burden on patients, families, and society.

[0003] The NFIA gene is a member of the nuclear factor I (NFI) transcription factor family, which also includes proteins such as NFIB, NFIC, and NFIX. These proteins bind to DNA in a dimer form, exhibiting similar or identical DNA-binding specificity. NFIA plays a crucial role in the development of the central nervous system and multiple other organs, regulating cell proliferation and differentiation. Studies have shown that in the mouse embryonic brain, NFIA is specifically expressed in the developing cerebral cortex. NFIA gene knockout mouse models exhibit perinatal lethality, hydrocephalus, and agenesis of the corpus callosum, the mechanism of which is closely related to impaired self-renewal and differentiation of neural progenitor cells. In human diseases, mutations or deletions of the NFIA gene lead to a neurodevelopmental disorder characterized by agenesis or absence of the corpus callosum, hydrocephalus or ventricular enlargement, and developmental delay. Furthermore, patients may exhibit macrocephaly, seizures, cognitive impairment, nonspecific malformations, ventricular dilatation, and hypotonia, which may further exacerbate motor developmental delays and feeding difficulties in infancy. Currently, research on NFIA-related diseases mainly relies on animal models and in vitro overexpression systems. However, animal models cannot fully simulate the human-specific gene regulatory network, while overexpression systems cannot accurately reflect the pathogenic mechanisms within the patient's genomic context. Furthermore, the limited proliferation capacity and difficulty in directing the differentiation of primary cells from patients (such as peripheral blood mononuclear cells, PBMCs) into target cells for the nervous or urinary systems present technical bottlenecks, severely hindering the elucidation of disease mechanisms and the progress of drug development.

[0004] In recent years, patient-derived induced pluripotent stem cell (iPSC) technology has shown great potential in disease modeling and drug screening for rare neurological genetic diseases. iPSCs not only solve the problem of the limited availability and quantity of primary human neurons, but also preserve the patient's genomic characteristics, thus reproducing the neurophysiological and anatomical pathological phenotypes at the cellular and molecular levels. Taking amyotrophic lateral sclerosis (ALS) as an example, iPSCs have been successfully used to reconstruct the heterogeneity and complex phenotypes of the disease, providing an important model for studying disease mechanisms. Furthermore, significant progress has been made in brain organoid culture technology based on human iPSCs, which can better simulate the cellular structure of the brain during development, providing unique advantages for studying the molecular and cellular mechanisms of embryonic neural development and modeling neurodevelopmental disorders. However, to date, there are no reports on research on iPSCs or organoids derived from patients with NFIA mutations or deletions, a gap that urgently needs to be filled. Summary of the Invention

[0005] To address the aforementioned issues, this invention marks the first successful reprogramming of an induced pluripotent stem cell (iPSC) line with complete patient genomic characteristics from peripheral blood mononuclear cells (PBMCs) of patients with NFIA deficiency. This cell line not only stably retains the patient-specific NFIA gene deletion mutation (NC_000001.10:g.61650967_61842967del), but also exhibits typical pluripotency marker expression (such as OCT4, SOX2, and NANOG) and trigerm layer differentiation capabilities. Compared to existing technologies, this invention is the first to construct a humanized research model capable of unlimited proliferation and differentiation into disease-related functional cells (such as neurons, glial cells, and brain organoids), filling the gap in the field of precise disease models and providing an irreplaceable research tool for elucidating the molecular mechanisms of neurodevelopmental disorders caused by NFIA deficiency.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An induced pluripotent stem cell line retaining the NFIA gene deletion mutation is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202590.

[0008] Furthermore, the aforementioned induced pluripotent stem cell line retaining the NFIA gene deletion mutation possesses any one of the characteristics in (a)-(c):

[0009] (a) The stem cell line carries the NC_000001.10:g.61650967_61842967del heterozygous deletion mutation, which includes the deletion of exons 3-6 of the NFIA gene;

[0010] (b) The stem cell line expressed the pluripotency markers OCT4, SOX2, NANOG and SSEA4;

[0011] (c) The stem cell line has the ability to differentiate into the three germ layers and expresses the ectoderm marker PAX6, the mesodermal markers HAND1 and TBXT, and the endoderm marker FOXA2 after differentiation.

[0012] Furthermore, the aforementioned induced pluripotent stem cell line retaining the NFIA gene deletion mutation has a normal male karyotype (46, XY).

[0013] This invention also discloses a method for preparing the above-mentioned induced pluripotent stem cell line, comprising the following steps:

[0014] (1) Isolate peripheral blood mononuclear cells (PBMCs) from the peripheral blood of patients carrying the NFIA gene deletion mutation;

[0015] (2) Use The -iPS 2.0 Sendai Reprogramming Kit reprograms PBMCs and contains Sendai virus vectors encoding SOX2, KLF4 and c-MYC.

[0016] (3) In Essential 8 TM Reprogrammed cells were cultured in Medium medium, and clones expressing pluripotency markers were screened and amplified.

[0017] Furthermore, in the above preparation method, in step (1), PBMCs are separated using Ficoll-Paque gradient centrifugation and the cells are cryopreserved using fetal bovine serum containing 10% DMSO.

[0018] Furthermore, in the above preparation method, in step (3), the culture conditions are 37°C, 5% CO2, and the culture medium base is a Matrigel-coated culture dish.

[0019] This invention also discloses the application of the above-mentioned induced pluripotent stem cell line in the preparation of research models for neurodevelopmental disorders, characterized in that: the stem cell line is differentiated into neurons, glial cells or brain organoids to simulate the pathological phenotype of neurodevelopmental disorders caused by NFIA deficiency.

[0020] Furthermore, in the above applications, the neurodevelopmental disorders include agenesis of the corpus callosum, hydrocephalus, developmental delay, or intellectual disability.

[0021] This invention also discloses the application of the above-mentioned induced pluripotent stem cell lines in drug screening, characterized in that: candidate compounds with therapeutic effects on NFIA-related neurodevelopmental disorders are screened through the stem cell lines or their differentiation products.

[0022] This invention also discloses the application of the above-mentioned induced pluripotent stem cell line in the preparation of gene therapy drugs, characterized in that: the NFIA gene deletion mutation of the stem cell line is repaired by gene editing technology, and its effect on reversing neurodevelopmental disorder phenotypes is evaluated.

[0023] Compared with the prior art, the present invention has the following outstanding advantages:

[0024] 1. Originality and Precision: For the first time, an iPSC strain was successfully constructed from PBMCs of patients with NFIA deletion, retaining the patient-specific heterozygous deletion mutation (NC_000001.10:g.61650967_61842967del), filling the gap in the lack of humanized disease models for this gene mutation. Compared with animal models and overexpression systems, this model truly reflects the pathogenic mechanism in the context of the patient's genome, significantly improving the accuracy of the research.

[0025] 2. Stable pluripotency and differentiation capacity: The stem cell line continuously expresses core pluripotency markers such as OCT4 and SOX2 (flow cytometry positive rate >94%), and can differentiate into cells from all three germ layers (ectoderm marker PAX6, mesoderm HAND1 / TBXT, and endoderm FOXA2), meeting the functional cell requirements for disease modeling. The chromosome karyotype (46, XY) is normal, and there is no mycoplasma contamination, ensuring experimental reliability.

[0026] 3. High-efficiency reprogramming technology: Employs a non-integrated Sendai virus vector ( The iPS 2.0 Kit avoids the risk of foreign gene insertion, has high reprogramming efficiency (clones are formed in 15-21 days), and PBMCs are readily available and can be cryopreserved for a long time, providing technical support for large-scale preparation.

[0027] 4. Wide range of applications:

[0028] Disease mechanism research: By differentiating neurons, glial cells and brain organoids, we simulate the phenotypes such as agenesis of the corpus callosum and hydrocephalus caused by NFIA deficiency, and reveal the molecular pathways of neurodevelopmental disorders.

[0029] Drug screening platform: Utilizes patient-specific cell models to screen small molecule compounds or biologics, accelerating the development of therapeutic drugs for diseases related to NFIA deficiency.

[0030] Gene therapy evaluation: Combining gene editing technologies such as CRISPR / Cas9 to repair NFIA deletion mutations, verifying phenotypic reversal effects, and providing an experimental basis for personalized gene therapy.

[0031] Clinical translational potential: This model overcomes the limitations of primary cell proliferation restriction and species differences in animal models, providing an irreplaceable tool for precision medicine research on rare neurodevelopmental disorders, and has significant scientific research and industrialization value.

[0032] The preservation information for the strain is as follows:

[0033] Name of depositary institution: China Center for Type Culture Collection (CCTCC);

[0034] Address of the depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0035] Preservation date:

[0036] Accession number CCTCC NO.C202590

[0037] Taxonomic nomenclature: Attached Figure Description

[0038] Figure 1 CNV-seq analysis of .DPNJMUi003-A iPSCs showed a heterozygous deletion of the NFIA gene at NC_000001.10:g.61650967_61842967, containing exons 3-6 of the NFIA gene;

[0039] Figure 2 The cell line was passaged to the 15th generation and cultured on day 3 (P15 Day 3). Under a phase-contrast microscope, the cells exhibited typical clonal growth with clear boundaries. Figure 2 A, objective lens 4X), the cells within the cell cluster are tightly packed and have a high nucleocytic ratio (A). Figure 2 B, objective lens 10X);

[0040] Figure 3 The in vitro differentiation RT-qPCR results of the cell lines showed that the mRNA expression levels of lineage-specific markers (red) in the three germ layers (A. ectoderm: PAX6; B. mesoderm: HAND1 and TBXT; C. endoderm: FOXA2) were increased compared to undifferentiated iPSCs (blue), and the mRNA expression levels of undifferentiated markers (SOX2, NANOG, OCT4) were downregulated; sample size N=3; ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, which are considered to be significant differences;

[0041] Figure 4 The cell line immunofluorescence staining results showed that the undifferentiated markers Oct4, SSEA4 (red) and Nanog, Sox2 (green) were expressed. The cell nuclei were stained with DAPI (blue). The results showed that Oct4, SSEA4, Nanog, and Sox2 were expressed in DPNJMUi003-A iPSCs.

[0042] Figure 5 The flow cytometry analysis results of the cell lines, including the simultaneous expression of SSEA4 and Oct3 / 4 in DPNJMUi003-A iPSCs; Figure 5 A showed that 99.0% of iPSCs expressed Oct3 / 4. Figure 5 B showed that 94.6% of the cells were SSEA4 positive, indicating that DPNJMUi003-A iPSCs had high expression of undifferentiated markers and were a highly pure induced pluripotent stem cell line.

[0043] Figure 6 The mycoplasma contamination detection results for the cell lines are as follows: Lane 1: DNA marker: DL2000; Lane 2: The cell line shows only a 150bp band, indicating no mycoplasma contamination; Lane 3: The positive control shows two bands, 150bp and 250bp; Lane 4: The negative control shows only one band, indicating no mycoplasma infection.

[0044] Figure 7 The karyotype analysis results of the cell line showed a normal male karyotype (46, XY);

[0045] Figure 8 Analysis of 28 STR loci in the cell line and peripheral blood of the patient; A represents the STR analysis of the cell line, and B represents the STR analysis of the patient's peripheral blood; all 28 STR loci showed high agreement, indicating that the cell line and the patient's peripheral blood are from the same biological source;

[0046] Figure 9 RT-qPCR results showed that, compared with normal human iPSCs (blue), the expression of the NFIA gene in DPNJMUi003-A iPSCs (red) was significantly reduced; GAPDH was used as the internal control; **p<0.01 was considered to be significant. Detailed Implementation

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] Example

[0049] 1. Patient recruitment and ethical approval.

[0050] The donor was a 3-year-old Chinese boy suffering from a neurodevelopmental disorder caused by a deletion of the NFIA gene. He first presented to our hospital in April 2020 with global developmental delay. A detailed medical history, physical examination, laboratory tests, and genetic testing were conducted. Figure 1 As shown, the patient carries a deletion mutation in the NFIA gene. The Children's Hospital Affiliated to Nanjing Medical University approved the gene analysis and clinical characterization study (Approval No.: 202211214-1).

[0051] 2. PBMCs do not require integrated reprogramming.

[0052] 2.1 Extraction of PBMCs from Human Peripheral Blood

[0053] (1) Take two 15ml centrifuge tubes and add 7ml of Ficoll solution. Transfer 5ml of whole blood into the centrifuge tubes containing Ficoll, being careful to avoid mixing the two solutions.

[0054] (2) Set the centrifuge speed to 3, the deceleration speed to 0, the centrifugal force to 1200g, and the temperature to 20℃. After centrifugation for 15 minutes, the cells will separate into layers. The cell layer containing PBMCs is white. Transfer this cell layer to another clean 15ml centrifuge tube. Add 10ml of RPMI 1640 medium, set the centrifuge speed to 5, the deceleration speed to 5, and the centrifugal force to 750g. After centrifugation for 5 minutes, remove the supernatant.

[0055] (3) Prepare cell culture medium in advance: RPMI 1640 + 10% FBS + 1% P / S; add 10ml of culture medium to resuspend cells for subsequent counting culture or plate plating.

[0056] 2.2 Reprogramming PBMCs to iPSCs

[0057] Using Cytotune TM The iPS2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) reprograms PBMCs using a Sendai virus-based vector that encodes Yamanaka reprogramming factors (SOX2, KLF4, and CMYC).

[0058] (1) Inoculation and culture of PBMCs: Transfer the PBMCs from step 2.1 to a 15 ml centrifuge tube. Slowly add 5–10 mL of pre-warmed whole PBMC culture medium and perform viable cell counting (PBMC culture medium consists of whole PBMCs). The cell suspension was centrifuged at 200g for 10 min in a medium containing suitable cytokines (composition of -34). The supernatant was discarded, and the cells were resuspended in PBMC complete medium. The cells were cultured in a 37°C incubator for 4 days, with the medium changed daily.

[0059] (2) Cell counting and virus transfection: The volume of each virus required to reach the target was calculated using the MOI (Mean Intent of Immunity) based on viable cell count and titer information on the CoA. 2.5 × 10⁶ cells were inoculated. 5 –5×10 5 Cells are transferred to round-bottom centrifuge tubes for transfection. 2.0 Sendai reagent was thawed from -80°C and immediately placed on ice. The three viruses were added to 1 mL of PBMC medium according to the calculated volume, preheated to 37°C, and gently shaken to mix thoroughly. The prepared reprogrammed virus mixture was added to a round-bottom centrifuge tube containing PBMC, with a total volume between 1 and 1.5 mL. The centrifuge tube was tightly capped and sealed with a sealing film. The cells and virus were centrifuged at 1000g for 30 minutes, then 1 mL of PBMC medium was added to the tube to resuspend the cells. The cells were then transferred to a 12-well plate and incubated overnight at 37°C in a 5% CO2 incubator.

[0060] (3) Change the culture medium and maintain the culture: The next day, transfer the cells and culture medium to 15 mL centrifuge tubes. Wash the wells with 1 mL of culture medium. Centrifuge at 200 g for 10 min, discard the supernatant, resuspend the cells in 0.5 mL of PBMCs culture, and seed them into each well of a 24-well plate. Incubate at 37 °C and 5% CO2 for 2 days.

[0061] (4) Seeding iPSCs: Coat 6-well plates with Geltrex for 1 hour, count cells, and seed 1 x 10⁴–1 x 10⁵ live PBMCs per well with 2 mL of StemPro-34 medium. Use 1 mL of fresh... -34°C cytokine-free medium, change half the medium every other day for two days. Afterwards, change the medium halfway through with Essential 8. TM After 24 hours, replace all the culture medium with E8 medium and continue culturing for the specified number of days.

[0062] (5) Clones are selected: on days 15-21 after transfection, clones are selected and cultured continuously.

[0063] 3. Cultivation and maintenance of iPSCs.

[0064] iPSCs were cultured in Geltrex-coated petri dishes at 37°C and 5% CO2 using Essential 8. TM Culture medium maintenance. When cell confluence reaches 70%-80%, discard the old culture medium, wash once with DPBS, add an appropriate amount of 0.5 mM EDTA to cover the cell surface, and observe under a microscope until the cells are fully dissociated. Discard the EDTA, add DPBS to wash away any remaining EDTA, and then add an appropriate amount of Essential 8. TM The culture medium was passaged at a ratio of 1:8 to 1:10. For example... Figure 2 The image shows the cell cloning results on day 3 (P15 Day 3) of the 15th generation of the cell line. Under a phase-contrast microscope, the cells exhibit typical clonal growth with clear boundaries. Figure 2 A, objective lens 4X), the cells within the cell cluster are tightly packed and have a high nucleocytic ratio (A). Figure 2 B, objective lens 10X).

[0065] 4. Trigerm layer differentiation experiment of iPSCs.

[0066] In the 20th generation, STEMdiff was used. TM Trigerm layer differentiation kit (STEMCELL Technologies, 05230) was used for trigerm layer differentiation experiments.

[0067] (1) Coat 24-well plates with Matrigel and incubate at 37°C for 1 hour. Preheat mTeSR containing 10 μM Y-27632. TM 1. DMEM / F-12 and cell dissociation reagent TyPLE were heated to room temperature for passage.

[0068] (2) Wash the wells to be passaged with 1 mL DPBS, remove the washing solution, add 1 mL of cell dissociation reagent TyPLE to each well, incubate at 37°C for 3-5 minutes, and then use a pipette with a 1 mL tip to blow up and down 1-3 times to ensure that all remaining cell clusters are broken into single cells.

[0069] (3) Transfer the cells to a test tube containing 1 mL of DMEM / F-12. Wash each well once with 1 mL of DMEM / F-12, collect the remaining cells, and transfer them to a test tube. Centrifuge at 300 g for 5 minutes.

[0070] (4) Remove the supernatant. Resuspend the cells in mTeSR. TM In culture medium. Live cells were counted using trypan blue and a hemocytometer.

[0071] (5) Add 0.5 mL of mTeSR to each well first. TM1. Culture medium. Use mTeSR according to the cell number requirements of different germ layers. TM 1. Resuspend cells in culture medium and seed them into 24-well plates. (Ectodermal: 400,000 cells / well, Mesodermal: 100,000 cells / well, Endoderm: 400,000 cells / well)

[0072] (7) Place the 24-well plate in a 37°C incubator. Move the culture dish quickly and briefly back and forth and left and right several times to ensure even distribution of cells. Do not disturb the culture dish for 24 hours.

[0073] (8) Differentiate the monolayer culture into three germ layer lineages: The culture medium was removed from the cell culture the following day. Add 1 ml of the corresponding STEMdiff solution to each well. TM Trilayer culture medium. Incubate at 37°C for 24 hours. Repeat this process until day 5 (mesoderm and endoderm lineage) or day 7 (ectoderm lineage). Collect RNA at fixed time points.

[0074] (9) Collect cells at specified time points and use... Total RNA was extracted using reagents (Life Technologies). 5×PrimeScript was used. TM cDNA was synthesized using RT Master Mix (Takara). Real-time quantitative PCR (RT-qPCR) was performed using AceQ qPCR SYBR GreenMaster Mix (Vazyme). The PCR reaction system was prepared according to the table below, with a total volume of 10 μL: 5 μL of 2×qPCR SYBR Green Master Mix, 2.2 μL of RNase-free H2O, 0.4 μL of Forward Primer, 0.4 μL of Reverse Primer, and 2 μL of cDNA. The prepared liquid was mixed and incubated briefly. According to the planned sample loading order, the prepared mixture and cDNA were added to the 96-well plate. After incubation, the 96-well plate was placed in a Roche LightCycler container. Amplification was performed using a 96 real-time quantitative PCR instrument with the following program: 94℃, 10 min; 95℃, 15 s; 60℃, 1 min, 40 cycles; 95℃, 15 s; 60℃, 15 s. The mRNA expression levels of ectoderm marker PAX6, mesodermal markers HAND1 and TBXT, endoderm marker FOXA2, and undifferentiated markers OCT4, NANOG, and SOX2 were detected using this method. Primer sequences are shown in Table 1. mRNA expression levels were standardized using GAPDH as an internal control, and relative expression levels were calculated using the formula: relative expression level = 2^ ﹣ΔΔCTData are presented as mean ± standard deviation (SD), with N = 3 biological replicates. P-values ​​were calculated using a two-tailed unpaired t-test. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Graphs were generated using GraphPadPrism (v.9.5).

[0075] (10) such as Figure 3 As shown in the in vitro differentiation RT-qPCR results of the cell lines, the mRNA expression levels of lineage-specific markers (red) of the three germ layers (A. ectoderm: PAX6; B. mesoderm: HAND1 and TBXT; C. endoderm: FOXA2) were all increased compared to undifferentiated iPSCs (blue), and the mRNA expression levels of undifferentiated markers (SOX2, NANOG, OCT4) were downregulated.

[0076] 6. Immunofluorescence detection of undifferentiated markers in iPSCs.

[0077] (1) iPSCs were seeded onto cell slides coated with Geltrex in advance, and the cells were harvested after the confluence was above 50%.

[0078] (2) Wash with PBS 3 times, 5 min each time, and fix with 4% paraformaldehyde for 15 min.

[0079] (3) Wash with PBS 3 times, 5 min each time, and permeate with PBS containing 0.3% Triton X-100 for 10 min.

[0080] (4) Wash with PBS 3 times, 5 min each time, and block with PBS containing 3% fetal bovine serum at 37°C for 1 hour.

[0081] (5) Dilute the primary antibodies for iPSC undifferentiated markers (Oct3 / 4, Sox2, Nanog and SSEA4) with PBS containing 1% BSA. The dilution ratios are shown in Table 1. Incubate the primary antibodies overnight at 4°C.

[0082] (6) Half an hour after warming up the next day, the primary antibody was recovered and washed with PBS three times for 5 minutes each time. The secondary antibody was diluted with PBS containing 1% BSA. The dilution ratio is shown in Table 1. The secondary antibody was incubated at room temperature for 1 hour.

[0083] (7) The cell nuclei were stained with DAPI for 10 min, washed with PBS 3 times for 5 min each time, discarded the PBS and air-dried, and then mounted with anti-quenching glycerol.

[0084] (8) Images were taken using an OLYMPUS IX73 microscope. For example... Figure 4As shown, DPNJMUi003-AiPSCs expressed Oct4, SSEA4, Nanog, and Sox2. Oct3 / 4, SSEA4 (red), Nanog, and Sox2 (green). Cell nuclei were stained with DAPI (blue).

[0085] 7. Flow cytometry detection of undifferentiated markers in iPSCs.

[0086] (1) Preparation of single-cell suspension: Wash each well with 1 mL DPBS, remove the washing solution, add 1 mL of cell dissociation reagent TyPLE to each well, incubate at 37°C for 3-5 minutes, then use a pipette with a 1 mL tip to blow up and down 1-3 times to ensure that all remaining cell clusters are broken into single cells, add 4 times the volume of DPBS to stop digestion, centrifuge at 300g for 5 minutes and discard the supernatant, then resuspend the cells in DPBS.

[0087] (2) Cell fixation and permeabilization: Fix with 4% PFA at room temperature for 10-15 minutes, and wash 3 times with DPBS. Permeabilization is required when staining Oct3 / 4, treat with 0.1% Triton X-100 for 10-15 minutes, and wash 3 times with DPBS.

[0088] (3) Blocking: Block with PBS containing 1% BSA at room temperature for 15-30 minutes.

[0089] (4) Primary antibody incubation: Add fluorescently labeled primary antibody Oct3 / 4 or SSEA4 and isotype control, incubate in the dark for 30 min, wash 3 times with DPBS by centrifugation, and resuspend in DPBS containing 1% fetal bovine serum.

[0090] (5) Instrumentation and Data Analysis: Calibrate the DxFLEX flow cytometer (Beckman Coulter), set thresholds to exclude debris, and collect 10,000-50,000 cell events. Select the target cell population, exclude debris and cell clumps, set an isotype control gate, and determine the positive signal threshold. Analyze the data using the flow cytometry software FlowJo.

[0091] (5) Figure 5 A showed that 99.0% of iPSCs were Oct3 / 4 positive, such as Figure 5 B showed that 94.6% of the cells were SSEA4 positive, indicating that DPNJMUi003-A iPSCs had high expression of undifferentiated markers and were a highly pure induced pluripotent stem cell line.

[0092] 8. Mycoplasma detection

[0093] The test was performed using a mycoplasma detection kit (ExCell Bio, MB000-1591).

[0094] (1) A few days before the experiment, allow iPSCs to grow to more than 80% in a 24-well culture plate. Discard the culture medium, wash twice with PBS, add 100 μL of lysis buffer, and incubate at room temperature for 5 minutes.

[0095] (2) Collect the cell lysate and place it in a centrifuge tube. Incubate at 95°C for 5 minutes. Centrifuge at 13000 rpm for 5 minutes and transfer the supernatant to a new centrifuge tube. Use 2 μL of the supernatant as a template for PCR reaction.

[0096] (3) After briefly centrifuging the 8-tube pack containing the pre-added reaction primers and Loading Dyed components provided in the kit, carefully open the pack and add the following reagents in sequence: first, add ddH2O to hydrate the dry powder in the tube, then add the sample, namely the supernatant, positive and negative samples, and finally add the enzyme-containing Mix. Mix the PCR tube thoroughly and centrifuge briefly. Start the reaction under the following conditions: 50℃ for 2 min, 95℃ for 10 min, then 95℃ for 30 sec, 56℃ for 30 sec, 72℃ for 30 sec, for a final cycle of 72℃ for 5 min; take 5 μL of the amplification product and perform direct electrophoresis on a 2% agarose gel. Figure 6 As shown, the cell line is free of mycoplasma contamination.

[0097] 9. Chromosome karyotype analysis and STR analysis.

[0098] In the 20th generation, such as Figure 7 As shown, chromosome karyotype analysis of hiPSCs was performed using the G-banding method, analyzing at least 20 metaphase cells at a resolution of 300-400 bands. The Nonaplex I PCR amplification kit (Biotype) performs individual STR analysis at 28 independent genomic loci using 2 ng of DNA. Figure 8 As shown, all 28 STR loci are highly congruent, indicating that the cell line and the peripheral blood of the child are of the same biological origin.

[0099] The obtained stem cell line was deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: C202590. The stem cell line carries the heterozygous deletion mutation NC_000001.10:g.61650967_61842967del, which includes the deletion of exons 3-6 of the NFIA gene. The stem cell line expresses the pluripotency markers OCT4, SOX2, NANOG, and SSEA4. The stem cell line has the ability to differentiate into three germ layers and, after differentiation, expresses the ectoderm marker PAX6, the mesodermal markers HAND1 and TBXT, and the endoderm marker FOXA2.

[0100] 10. NFIA gene expression level detection

[0101] The cell line was passaged to the 20th generation, and the cells were collected and used. Total RNA was extracted using reagents (Life Technologies). RT-qPCR reagents, methods, and statistical analysis were the same as in Experiment 4 above to detect the mRNA expression level of the NFIA gene. Primer sequences are shown in Table 1. Figure 9 As shown, the expression level of the NFIA gene in the cell line was significantly lower than that in the control cells.

[0102] Table 1 Antibody information obtained from immunofluorescence and flow cytometry analysis.

[0103]

[0104]

[0105] The primer sequences in Table 1 are numbered from top to bottom as SEQ ID No. 1-18.

[0106] From the above embodiments, the technical solutions of the embodiments have the following significant advantages:

[0107] 1. Safe and efficient non-integration reprogramming technology

[0108] Cytotune, based on Sendai virus (non-integrating vector), was used. TM The iPS2.0 system avoids the risk of exogenous gene insertion into the host genome, significantly improving the safety of iPSCs in clinical applications. By optimizing transfection conditions (such as centrifugation to enhance infection efficiency), efficient reprogramming of PBMCs has been successfully achieved, shortening the clone formation cycle (15-21 days).

[0109] 2. Establishment and verification of high-quality iPSCs

[0110] Pluripotency verification: The ability of iPSCs to differentiate into three germ layers was confirmed by the three germ layer differentiation experiment (ectoderm marker PAX6, mesoderm HAND1 / TBXT, endoderm FOXA2);

[0111] Undifferentiated state detection: Immunofluorescence and flow cytometry showed high levels of expression of pluripotency markers (OCT4, NANOG, SOX2, SSEA4), and RT-qPCR showed significant expression of undifferentiated genes (p<0.01–0.0001), ensuring the undifferentiated characteristics of cells;

[0112] Genetic stability: Karyotype analysis of the 20th generation cells (G-banding method) showed the normal number and structure of chromosomes (20 metaphase cells, resolution 300-400 bands), and STR analysis confirmed the uniqueness of cell origin and ruled out cross-contamination.

[0113] 3. A strict cell quality control system

[0114] Contamination-free assurance: The mycoplasma detection kit, combined with PCR amplification, verifies that the cells are free of mycoplasma contamination;

[0115] Standardized culture process: using Essential 8 TM The culture medium and Geltrex substrate, combined with the mild EDTA subculture method, maintained the stable amplification of iPSCs to more than 20 generations, showing good long-term culture stability.

[0116] 4. Clinical translation potential and ethical compliance

[0117] Minimal invasive cell source: Peripheral blood PBMCs are used as the starting material, which minimizes patient trauma and makes samples easy to obtain;

[0118] Ethical Standardization: The study passed the ethical review of the Children's Hospital Affiliated to Nanjing Medical University (approval number 202211214-1), meeting the ethical standards for clinical research and laying the foundation for subsequent research on disease mechanisms or personalized treatment.

[0119] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An induced pluripotent stem cell line retaining the NFIA gene deletion mutation, characterized in that, The stem cell line is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: C202590.

2. The induced pluripotent stem cell line according to claim 1, characterized in that, It has any of the characteristics in (a)-(c): (a) The stem cell line carries the NC_000001.10:g.61650967_61842967del heterozygous deletion mutation, which includes the deletion of exons 3-6 of the NFIA gene; (b) The stem cell line expressed the pluripotency markers OCT4, SOX2, NANOG and SSEA4; (c) The stem cell line has the ability to differentiate into the three germ layers and expresses the ectoderm marker PAX6, the mesodermal markers HAND1 and TBXT, and the endoderm marker FOXA2 after differentiation.

3. The induced pluripotent stem cell line according to claim 1, characterized in that: The karyotype of the stem cell line is a normal male karyotype (46,XY).

4. A method for preparing an induced pluripotent stem cell line retaining the NFIA gene deletion mutation, characterized in that, Includes the following steps: (1) Isolate peripheral blood mononuclear cells (PBMCs) from the peripheral blood of patients carrying the NFIA gene deletion mutation; (2) Use The iPS2.0 Sendai Reprogramming Kit reprograms PBMCs and contains Sendai virus vectors encoding SOX2, KLF4 and c-MYC. (3) In Essential 8 TM Reprogrammed cells were cultured in Medium medium, and clones expressing pluripotency markers were screened and amplified.

5. The preparation method according to claim 4, characterized in that: In step (1), PBMCs were separated using Ficoll-Paque gradient centrifugation and the cells were cryopreserved using fetal bovine serum containing 10% DMSO.

6. The preparation method according to claim 4, characterized in that: In step (3), the culture conditions are 37°C, 5% CO2, and the culture medium is a Matrigel-coated culture dish.

7. The application of the induced pluripotent stem cell line as described in any one of claims 1-3 in the preparation of research models for neurodevelopmental disorders, characterized in that: The stem cell lines are differentiated into neurons, glial cells, or brain organoids to mimic the pathological phenotype of neurodevelopmental disorders caused by NFIA deficiency.

8. The application according to claim 7, characterized in that: The neurodevelopmental disorders include agenesis of the corpus callosum, hydrocephalus, developmental delay, or intellectual disability.

9. The application of the induced pluripotent stem cell line as described in any one of claims 1-3 in drug screening, characterized in that: Candidate compounds with therapeutic effects on NFIA-related neurodevelopmental disorders are screened using the stem cell lines or their differentiation products.

10. The use of the induced pluripotent stem cell line according to any one of claims 1-3 in the preparation of gene therapy drugs, characterized in that: The gene editing technology was used to repair the NFIA gene deletion mutation in the stem cell line, and its effect on reversing the neurodevelopmental disorder phenotype was evaluated.

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