Down's syndrome induced pluripotent stem cell as well as construction method and application thereof

Abstract

The invention relates to the field of molecular biology, in particular to Down's syndrome induced pluripotent stem cells as well as a construction method and application thereof. Four transcription factor reprogramming methods of KLF4, OCT4, c-MYC and SOX2 are utilized to construct Down's syndrome induced pluripotent stem cells, an expression profile of non-coding RNA in derived exosomes of the Down's syndrome induced pluripotent stem cells is systematically analyzed, differentially expressed non-coding RNA is screened, an RT-ddPCR technology is further adopted to analyze the expression condition of the differentially expressed non-coding RNA in maternal peripheral blood, and the DDown's syndrome induced pluripotent stem cells are obtained. And new information is provided for screening of novel markers for antenatal diagnosis of Down's syndrome and establishment of a detection method.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to induced pluripotent stem cells for Down syndrome, their construction methods, and applications. Background Technology

[0002] Down syndrome (DS), also known as trisomy 21, is a serious chromosomal aneuploidy caused by the nondisjunction of chromosome 21 during meiosis. Clinical manifestations of DS include developmental delay, congenital heart disease, hearing impairment, and autoimmune deficiencies. Currently, prenatal screening methods for DS mainly include serological testing during pregnancy and fetal ultrasound screening. Serological testing involves detecting serum markers such as alpha-fetoprotein (AFP), β-human chorionic gonadotropin (β-hCG), unconjugated estriol (uE3), and inhibin A in the peripheral blood of pregnant women during early and mid-pregnancy. Fetal ultrasound screening is used to identify imaging features such as increased nuchal translucency in fetuses with DS. However, these methods have certain limitations in terms of sensitivity and specificity: serological tests are easily affected by the individual characteristics of pregnant women (body mass index, race, accuracy of gestational age calculation), while the accuracy of ultrasound screening depends on the operator's experience and the quality of the equipment, which may lead to a high false positive or false negative rate.

[0003] Disease-specific models are effective tools in the screening and discovery of biomarkers; however, existing animal models of DS (such as Dp(10)1Yey / +, Dp(16)1Yey / +, Dp(17)1Yey / +, and Ts65Dn mice) all have significant limitations. For example, the Hsa21 gene is distributed on multiple chromosomes in mice, making it difficult to completely reproduce the phenotypic and genotypic characteristics of human DS. Furthermore, due to differences in genetic background between species, phenotypic expressions also vary. Given the limitations of current invasive and non-invasive prenatal diagnostic methods, developing novel, reliable, and cost-effective biomarkers for prenatal screening and diagnosis of DS remains of significant research value. In recent years, induced pluripotent stem cell (iPSC) technology has become a powerful tool for studying genetic diseases. However, due to the relatively complex process of constructing iPSCs and the relatively low success rate, there are currently few reports on DS-specific iPSCs. Moreover, related studies mainly focus on the developmental abnormalities of the nervous system in DS, and there is still a lack of reports on their potential value in prenatal diagnostic marker screening. In addition, studies have shown that non-coding RNAs (ncRNAs) carried by exosomes have significant potential value in the field of disease diagnosis due to their unique biological characteristics [Zhou L, Wang W, Wang F, et al. Plasma-derived exosomalmiR-15a-5p as a promising diagnostic biomarker for early detection of endometrial carcinoma[J]. Molecular Cancer, 2021, 20(1): 57.]. Because ncRNAs exhibit higher stability and tissue specificity compared to traditional mRNA biomarkers, they have gradually become hot targets for molecular diagnostic biomarker screening [Matulić M, Gršković P, Petrović A, et al. miRNA in Molecular Diagnostics[J]. Bioengineering, 2022, 9(9): 459.]. Previous studies have shown that ncRNAs, such as microRNAs (miRNAs), pre-miRNAs, and small nucleolar RNAs (snoRNAs), play a crucial role in gene expression regulation and have become important research subjects in the field of molecular diagnostics. However, systematic studies on the expression profiles of ncRNAs in DS-iPSC-derived exosomes have not yet been reported, and their potential application value in prenatal diagnosis remains unclear.In view of this, the present invention provides induced pluripotent stem cells for Down syndrome, methods for constructing them, and their applications. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide induced pluripotent stem cells for Down syndrome, their construction methods, and applications. The aim is to construct induced pluripotent stem cells for Down syndrome (DS-iPSCs), systematically analyze the expression profile of ncRNAs (non-coding RNAs) in their derived exosomes, screen for differentially expressed ncRNAs, and further analyze the expression of differentially expressed ncRNAs in maternal peripheral blood using RT-ddPCR technology. This is intended to provide new information for the establishment of novel biomarkers and testing methods for prenatal diagnosis of Down syndrome.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first aspect concerns the method for constructing induced pluripotent stem cells for Down syndrome, which includes the following steps: Somatic cells carrying triploid chromosome 21 were transduced with Sendai virus vectors carrying genes OCT4, SOX2, c-MYC, and KLF-4 and then cultured to obtain Down syndrome induced pluripotent stem cells. The specific culture method followed the instructions on the ReproeasyhiPSC reprogramming kit (Beijing Saibei Biotechnology Co., Ltd.).

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, the somatic cells carrying the triploid chromosome 21 include at least one of amniotic fluid cells and peripheral blood cells.

[0008] Secondly, a Down syndrome induced pluripotent stem cell, wherein the Down syndrome induced pluripotent stem cell is obtained by the aforementioned construction method.

[0009] Thirdly, an application of Down syndrome induced pluripotent stem cells, wherein the aforementioned Down syndrome induced pluripotent stem cells are used to screen for biomarkers of Down syndrome.

[0010] Fourthly, a biomarker for Down syndrome, said biomarker for Down syndrome is obtained by a Down syndrome-induced pluripotent stem cell.

[0011] Furthermore, the biomarkers for Down syndrome include at least one of hsa-mir-4327, hsa-let-7c, and SNORA32.

[0012] Furthermore, the hsa-let-7c nucleotide sequence is shown in SEQ ID NO:1; The nucleotide sequence of hsa-mir-4327 is shown in SEQ ID NO:2; The nucleotide sequence of SNORA32 is shown in SEQ ID NO:4.

[0013] Fifthly, the application of a biomarker for Down syndrome, wherein the biomarker for Down syndrome is used in the preparation of products for prenatal screening of Down syndrome.

[0014] Furthermore, the products used for prenatal screening include any one of the following: testing reagents and testing kits.

[0015] Furthermore, the detection reagent includes primers, the nucleotide sequences of which are shown in SEQ ID NO:5-13.

[0016] The present invention has conducted research in the following specific aspects: (1) Collect and culture cell samples remaining after clinical chromosome karyotype analysis, and construct Down syndrome-specific induced pluripotent stem cells (DS-iPSCs) using four transcription factor reprogramming methods: KLF4, OCT4, c-MYC and SOX2.

[0017] (2) The expression of pluripotency markers (TRA-1-81, SSEA4) was detected by flow cytometry, and the karyotype of chromosomes was detected by G-banding technology to analyze their biological characteristics. The results of G-banding karyotype analysis showed that both cell clones carried an extra chromosome 21 (karyotype: 47, XY, +21); flow cytometry showed that they both expressed pluripotency markers TRA-1-81 and SSEA4 normally, and two DS-iPSCs were successfully established.

[0018] (3) Collect the culture medium from DS-iPSCs and control induced pluripotent stem cells during the culture process and isolate exosomes.

[0019] (4) Microarray chip technology was used to analyze the expression profile of differentially expressed ncRNAs in exosomes, and bioinformatics methods were combined to predict the potential functions of ncRNAs. Microarray chip technology was used to analyze DS-iPSCs-derived exosomes and found a total of 595 differentially expressed ncRNAs, including 210 upregulated pre-miRNAs, 175 downregulated pre-miRNAs, 123 upregulated snoRNAs and 87 downregulated snoRNAs. Biological function enrichment analysis showed that these differentially expressed ncRNAs are mainly involved in key biological processes such as gene silencing, gene expression regulation and polypeptide biosynthesis.

[0020] (5) The differentially expressed ncRNAs initially screened were validated using reverse transcription droplet digital polymerase chain reaction (RT-ddPCR), and their expression in peripheral blood samples from pregnant women carrying fetuses with DS was detected to screen for biomarkers with potential prenatal diagnostic value. RT-ddPCR analysis revealed that the average relative expression levels of hsa-mir-4327, hsa-let-7c, and SNORA32 in DS-iPSC-derived exosomes were 7.04±2.19, 9.71±3.05, and 19.66±10.67, respectively. This result was consistent with the microarray analysis results, and the expression was upregulated in peripheral blood samples from pregnant women carrying fetuses with DS, with statistically significant differences. P <0.05).

[0021] The beneficial effects of this invention are: This invention successfully constructed disease-specific iPSCs using DS-derived cells, which carry triploid chromosome 21. This invention also established a method for screening disease biomarkers based on disease-specific iPSCs and screened a series of biomarkers that may have potential diagnostic value for DS. This method provides a new approach for the screening and functional study of disease diagnostic biomarkers, which is quite challenging. Attached Figure Description

[0022] Figure 1 These are images of amniotic fluid cells cultured in vitro under an inverted microscope; (A) shows the morphology of cells with relatively active growth in vitro; (BD) shows the morphology of cells with slow growth and poor activity in vitro; (AD) scale bar = 200 μm. Figure 2 The images show the morphological characteristics of cells after reprogramming in vitro. Among them, (AD) shows the morphological characteristics of failed reprogrammed cells where cell adhesion gradually decreased and no stem cell-like cell clones appeared; (E, F) show the morphological characteristics of cells after reprogramming where stem cell-like cell clones appeared; (A, C) 5X, (B, D) 10X, (E, F) scale bar = 500μm. Figure 3 This is a morphological diagram of cell clones formed by in vitro culture of DS-iPSCs of the present invention; Figure 4 The diagram shows the karyotype analysis of DS-iPSCs and NC-iPSCs of the present invention; wherein, (AD) is the karyotype of the two DS-iPSCs; and (EF) is the karyotype of the two NC-iPSCs. Figure 5The graph shows the flow cytometry results of DS-iPSCs and NC-iPSCs of the present invention; where flow cytometry shows that both DS-iPSCs and both NC-iPSCs clearly express the pluripotent markers TRA-1-81 and SSEA4. (A, B) DS-iPSCs, (C, D) NC-iPSCs, (AD) X-axis is the fluorescence intensity of TRA-1-81-APC, and Y-axis is the fluorescence intensity of SSEA4-PE; Figure 6 The images show the morphology of exosomes from iPSCs under TEM; (A, C) exosomes from DS-iPSCs (scale bar = 100 nm); (B, D) exosomes from NC-iPSCs (scale bar = 500 nm). Figure 7 Scatter plots showing the distribution of ncRNAs in the DS and NC groups; (A) scatter plot of differentially expressed pre-miRNAs, with red representing 210 upregulated pre-miRNAs and blue representing 175 downregulated pre-miRNAs; (B) scatter plot of differentially expressed snoRNAs, with red representing 123 upregulated snoRNAs and blue representing 87 downregulated snoRNAs. Figure 8 Figure 1 shows the GO enrichment analysis results of differentially expressed pre-miRNAs in microarray analysis; Figure 9 Figure 1 shows the GO enrichment analysis results of differentially expressed snoRNAs in microarray analysis; Figure 10 One-dimensional plots of iPSC exosomes SNORA32, SNORD74B, hsa-let-7c, and hsa-mir-4327 for RT-ddPCR analysis; where (A) SNORA32 (blue droplets); (B) SNORD74B (green droplets); (C) hsa-let-7c (pink droplets); (D) hsa-mir-4327 (green droplets); (E) U6 internal control (orange droplets), and gray droplets are negative droplets; Figure 11 The diagram shows the relative expression levels of ncRNAs in the exosomes of iPSCs from the DS and NC groups. Figure 12 One-dimensional plot of hsa-mir-4327 and hsa-let-7c in maternal peripheral blood for RT-ddPCR analysis; where (A) hsa-mir-4327 (green droplets); (B) hsa-let-7c (pink droplets); (C) U6 internal control (orange droplets); gray indicates negative droplets; Figure 13One-dimensional plot of SNORA32 and SNORD74B in maternal peripheral blood for RT-ddPCR analysis; where (A) SNORA32 (blue droplets); (B) SNORD74B (green droplets); (C) U6 internal control (orange droplets); gray indicates negative droplets; Figure 14 To compare the differential expression of hsa-let-7c, hsa-mir-4327, SNORA32, and SNORD74B in the peripheral blood of pregnant women in the DS and NC groups using a t-test; p <0.01;*, p <0.05; among them, (A) hsa-mir-4327; (B) hsa-let-7c; (C) SNORA32; (D) SNORD74B. Detailed Implementation

[0023] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] 1. Experimental methods.

[0025] 1.1 Experimental Samples.

[0026] We reviewed the karyotype analysis data of samples from the Genetics and Precision Medicine Center of the Second Affiliated Hospital of Guilin Medical University from January 2023 to December 2024. We collected amniotic fluid (AF) cell samples from 4 cases of patients diagnosed with DS after karyotype analysis and 1 peripheral blood cell sample to construct DS-iPSCs. We used two previously constructed non-DS-derived iPSCs (karyotype: 46, XY) as control samples (NC-iPSCs, normal control induced pluripotent stem cells) [Ou M, Li C, Tang D, et al. Genotyping, generation and proteomic profilingof the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells[J]. Stem Cell Research & Therapy, 2019, 10(1): 251.Li D,Ou M, Dai G, et al. Genotypic Characterization of a Chinese Family with Osteogenesis Imperfecta and Generation of Disease-Specific [Induced Pluripotent Stem Cells[J]. Frontiers in Bioscience (Landmark Edition), 2023,28(12): 336.]. Simultaneously, six peripheral blood samples remaining from prenatal screening of pregnant women diagnosed with DS fetuses were randomly selected as the experimental group (DS group), and six peripheral blood samples remaining from prenatal screening of matched healthy pregnant women without DS fetuses were selected as the control group (NC group). The age of the DS group was 38±5 years, and the gestational age was 18±3 weeks; the age of the NC group was 37±3 years, and the gestational age was 17±5 weeks. These samples were used for subsequent differentially expressed ncRNAs analysis experiments. All subjects signed informed consent forms, and this invention was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Guilin Medical University (NO. GLMC202303039).

[0027] 1.2 Cell culture and activation.

[0028] Amniotic fluid cells remaining from chromosome karyotype analysis were seeded into culture medium and cultured as primary adherent cells in a 37°C, 5% CO2 incubator. Cell adhesion was assessed using an inverted phase-contrast microscope (×10). The medium was changed when the number of cell clones reached 5 or more (regardless of size) and cell activity was deemed "active," until sufficient cells were available for subsequent iPSC construction. For peripheral blood cells, mononuclear cells were isolated using density gradient centrifugation and resuspended in PBMCeasy® blood cell activation medium (Beijing Saibei Biotechnology Co., Ltd.) in 24-well plates. After 5 days of activation culture, cells were directly reprogrammed for subsequent iPSC construction.

[0029] 1.3 Construction of DS-iPSCs.

[0030] The cell reprogramming method used non-integrating Sendai virus (SeV) as the vector. This vector-mediated transfection contained four transcription factors, namely OCT4, SOX2, KLF4, and c-MYC, and was performed according to the instructions of the Reproeasy hiPSC reprogramming kit (Beijing Saibei Biotechnology Co., Ltd.); details are as follows: (1) After digestion, the cells are seeded in a culture plate (three density gradients are set according to the cell growth, and three replicates are set for each gradient, with about 5,000 to 15,000 cells per well), and then cultured in a 37°C, 5% CO2 constant temperature and humidity incubator until the cell confluence reaches 70%-90%, and then cell reprogramming is performed.

[0031] (2) Day 0: Observe the confluence and state of cells under a microscope, and select one replicate well of different gradients for digestion and counting. Select wells with 10,000-20,000 cells for reprogramming.

[0032] First, centrifuge the reprogramming additive II. Then, add 100 µL of reprogramming medium A to the reprogramming additive II and mix well to prepare reprogramming medium B. Add the reprogramming medium B to a selected 96-well plate that meets the conditions and place the plate back into a 37°C, 5% CO2 constant temperature and humidity incubator, and record it as day 0.

[0033] (3) Days 1-2: Observe cell morphology under a microscope and take photos. When the cell morphology changes significantly, remove the reprogramming medium B and continue culturing with the reprogramming medium A; if the cell changes are not significant, there is no need to change the medium.

[0034] (4) Day 3: Under the microscope, the cell morphology has undergone obvious changes and the growth rate is relatively fast, reaching a state of full confluence. Then, trypsin digestion and passage are performed. If the cell quantity is insufficient under the microscope at this time, passage is postponed for one to two days. Then, according to the cell state and cell quantity, the cells are passaged to 2-6 wells of a 6-well plate (the 6-well plate needs to be coated with PSCeasy human pluripotent stem cell seeding solution one day in advance) and reprogramming medium C is added to try to form single-cell adhesion.

[0035] (5) Day 4: Observe the cell adhesion under a microscope. If most cells adhere well, remove the reprogrammed culture medium C and replace it with fresh AF cell culture medium.

[0036] (6) Day 5: Observe under a microscope. When small clusters of clones with more than 4 cells are formed, replace the AF cell culture medium with Reproeasy human cell reprogramming medium. If small clusters of clones have not yet formed, continue to observe for 1-2 days before replacing with Reproeasy human cell reprogramming medium.

[0037] Days 6-8: Under a microscope, small cell clusters will enlarge, with each individual clonal clump containing more than 10 cells. At this point, replace the Reproeasy human cell reprogramming medium with PGM1 human pluripotent stem cell culture medium. If a large number of dead cells are observed under the microscope before changing the medium, wash the cells with PBS equilibrated at room temperature before changing the medium.

[0038] (7) Days 9-20: Observe under a microscope daily and take pictures to record changes in cell morphology. At the same time, replace the culture medium with fresh PGM1 human pluripotent stem cells after equilibration at room temperature.

[0039] (8) Day 21: When a single cell clone fills the entire 10x field of view, cut the clone with a 1ml syringe needle and transfer it to a 48-well plate pre-coated with PSCeasy human pluripotent stem cell substrate. If the clone is in good condition, the cells are thick and growing fast, then directly pick it into a 24-well plate for culture.

[0040] (9) After the clones are picked out, they are seeded with PSCeasy human pluripotent stem cell resuscitation medium. After the cells adhere, they are replaced with PGM1 human pluripotent stem cell medium and cultured until the cell quantity is sufficient for subsequent experiments and preservation.

[0041] 1.4 Analysis of the biological characteristics of iPSCs.

[0042] (1) iPSC karyotype analysis: ① After adding an appropriate amount of colchicine (Guangdong Comsen Biotechnology Co., Ltd.), 0.025% trypsin was added to detach the cells from the cell wall. ② Hypotonic treatment: 0.05 mol / L KCl hypotonic solution was used. ③ Pre-fixation; ④ Fixation: Fixation with fixative; ⑤ Slide preparation and baking; ⑥ Banding; ⑦ Staining: The digested slides were placed in a staining jar for staining. ⑧ Karyotype analysis strictly followed the ISCN (2020) international standard and was performed using the CytoVision chromosome automated analysis system.

[0043] (2) Flow cytometry analysis of iPSCs: ① Cell collection and pretreatment; ② Cell resuspension and antibody labeling: To label cell surface-specific antigens, validated fluorescently labeled antibodies, namely anti-Tra-1-81 and anti-SSEA4 (Thermo Fisher Scientific, Inc.), were added to each cell suspension; ③ Washing; ④ Flow cytometry detection; ⑤ Data processing and statistical analysis: After data acquisition, the flow cytometry data were analyzed in detail using FlowJo software.

[0044] 1.5 Isolation and identification of iPSCs exosomes.

[0045] (1) Isolation of iPSCs exosomes: ① Collection and preservation of iPSC cell supernatant: During iPSC culture, when the cell confluence is observed to be about 70% under an inverted microscope and the cells are in good condition, the culture medium is aspirated into a 50 ml pre-cooled centrifuge tube using a sterile Pasteur pipette and stored at -80℃.

[0046] ② Ultracentrifugation to separate iPSCs exosomes: First, low-speed centrifugation is used to remove cell debris and large particulate impurities, and then ultracentrifugation is used to precipitate the exosomes.

[0047] (2) Identification of iPSCs exosomes: ① Mesh pretreatment; ② Preparation of staining solution: Prepare 2% uranium acetate staining solution (USA Spectroscopy); ③ Sample loading and adsorption; ④ Washing and staining: Add 5 μl of 2% uranium acetate staining solution and stain at room temperature for 1 minute; ⑤ TEM observation.

[0048] (3) Extraction of exosomal RNA from iPSCs: ① Mixed extraction: The phenol-guanidine isothiocyanate complex in the reagent disrupts the exosome membrane structure, releasing RNA and inhibiting RNase activity. ② Two-phase separation; ③ RNA precipitation; ④ RNA washing; ⑤ Redissolving the RNA precipitate; and determining the RNA concentration and purity using a NanoDrop ND-1000 micro spectrophotometer.

[0049] 1.6 Microarray chip hybridization and data analysis.

[0050] Exosomal RNA extracted from iPSCs was used for microarray hybridization of ncRNAs. GeneSpring GX software was used for quantitative normalization and subsequent data processing, ensuring that at least one probe signal in each sample had a "Present (P)" or "Marginal (M)" marker. Multiple probe signals from the same ncRNA were merged into one ncRNA level. Differentially expressed ncRNAs were screened using FC and P values ​​(default threshold |FC|>1.5). P <0.05). FC refers to the ratio of ncRNA expression levels in the DS group to those in the NC group. The smaller the P-value, the more statistically significant the difference in ncRNA expression.

[0051] 1.7 Analysis of ncRNAs derived from chromosome 21.

[0052] Sequence alignments were performed using the UCSC Genome Explorer, based on the December 2013 human genome assembly (GRCh37 / hg19). RNA secondary structures were predicted using RNA Structure software version 6.5. Sequence conservation was analyzed using the Clustal Omega tool, accessible at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0053] 1.8 RT-ddPCR detection (reverse transcription droplet digital polymerase chain reaction).

[0054] The extracted RNA samples were subjected to quality testing and then used for absolute quantification analysis by RT-ddPCR.

[0055] (1) The reaction system and procedure are shown in Table 1, and the primer sequences are shown in Table 2: Table 1 Reaction Mixture Table 2 Primer sequences Note: hsa-mir-4327, hsa-1et-7c, SNORA32, SNORD74B, and the internal reference U6snRNA primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. All components were prepared according to the manufacturer's recommended concentrations to ensure the sensitivity and specificity of the reaction system.

[0056] Amplification procedure: ① Reverse transcription step: Incubate at 55℃ for 30 minutes to convert RNA into cDNA; ② Pre-denaturation: Incubate at 95℃ for 10 minutes to activate the enzyme system; ③ Cyclic amplification: A total of 45 cycles, each cycle including: denaturation at 95℃ for 30 seconds; annealing at 56℃ for 1 minute; extension at 98℃ for 10 minutes; and finally, incubate at 16℃ to end the reaction; ④ Data acquisition and analysis: After amplification, the fluorescence signal in each droplet was quantitatively detected using a MicroDrop-400B microdrop analyzer; ⑤ Using Quant Drop analysis software, the absolute abundance of target RNA in each sample was calculated based on the Poisson distribution principle, providing data support for subsequent statistical analysis and results discussion.

[0057] (2) RT-ddPCR detection of iPSC exosomes of differentially expressed ncRNAs derived from chromosome 21: The obtained iPSC exosomal RNA was validated by RT-ddPCR. The exosomal RNA was extracted using the guanidine isothiocyanate-phenol-chloroform single-phase lysis method as described above, and the quality of the RNA was detected using a NanoDrop ND-1000 spectrophotometer, followed by RT-ddPCR detection.

[0058] (3) RT-ddPCR detection to verify differentially expressed ncRNAs in peripheral blood serum samples from pregnant women: To detect the expression of differentially expressed ncRNAs in the peripheral blood of pregnant women, this invention uses the EasBead magnetic bead extraction kit (Guangdong Yixin Biotechnology Co., Ltd.) to extract and purify RNA from the peripheral blood serum of pregnant women carrying DS fetuses and normal pregnant women. The entire procedure was performed under RNase-free conditions, and all consumables underwent rigorous pretreatment.

[0059] (4) Statistical analysis: Data obtained from RT-ddPCR were analyzed using GraphPad Prism software (version 10.1.2). Statistical significance between the two groups was assessed using a t-test. P A value < 0.05 indicates statistical significance.

[0060] 2. Experimental results.

[0061] 2.1 In vitro cell expansion culture and reprogramming: For the remaining amniotic fluid cell samples diagnosed with DS after chromosome karyotype analysis, during in vitro culture, the amniotic fluid cells from one case showed relatively active growth. After approximately 7 days of in vitro culture, numerous adherent cell clones were observed under a microscope, forming cell clones covering one or more complete 10× fields of view. Figure 1(Case A); while the other three cases showed slower growth of amniotic fluid cells, poor cell adhesion, slow growth, poor activity, and the absence or scarcity of round cells. Figure 1 (BD).

[0062] In subsequent cell reprogramming using a non-integrated SeV vector containing four transcription factors, two cell culture plates showed the emergence of some stem cell-like cell clones within one week, and individual clones filling the entire 10x field of view were observed under a microscope within two to three weeks. However, in the other three slower-growing amniotic fluid cells, adherent cells gradually decreased on days 2 and 6 of reprogramming, and no stem cell-like cell clones were observed. Figure 2 (AD). Next, the present invention selected cell clones for culture and successfully obtained two cases of DS-specific stem cell-like cells (AD). Figure 2 (E, F). The results in this section suggest that the growth activity of cells before reprogramming may have a significant impact on the construction of iPSCs.

[0063] 2.2 Analysis of the biological characteristics of iPSCs.

[0064] To observe the culture characteristics of reprogrammed cells, this invention cultured two DS-specific stem cell-like cells in vitro. From the cell culture morphology, both cell lines grew in a dense cell cluster or clonal form, exhibiting a dome-shaped or hemispherical raised structure with clear edges. The cell morphology showed a high degree of similarity to embryonic stem cells. Figure 3 ).

[0065] To verify the genomic stability of the reprogrammed cells, this invention used G-banding technology to analyze the karyotypes of the newly constructed iPSCs. It was found that the karyotypes of both DS-iPSCs were (47, XY, +21), and the karyotypes of both NC-iPSCs were (46, XY). Figure 4 (AF). The karyotype diagrams show that both DS-iPSCs have abnormal chromosome 21 numbers, but normal autosome and sex chromosome structures. This indicates that no abnormal chromosome numbers (such as polyploidy or aneuploidy) or structural aberrations (such as translocation, deletion, or inversion) outside of trisomy 21 occurred during cell reprogramming and passage, which is consistent with the typical cytogenetic characteristics of iPSCs.

[0066] Furthermore, to analyze the expression of pluripotent markers in the two successfully reprogrammed cell lines, flow cytometry was used to detect the expression of typical markers of iPSCs (including SSEA4 and TRA-1-81). The results showed that 97.74% and 98.15% of the two DS-iPSC lines co-expressed these two markers, respectively. In contrast, the control cells showed 99.27% ​​and 99.31%, respectively. Figure 5 (Chinese AD).

[0067] The results of morphology, chromosome karyotype, and expression of pluripotent markers in in vitro cell culture clones indicate that DS-iPSCs possess the typical biological characteristics of iPSCs.

[0068] 2.3 Isolation and identification of iPSCs exosomes.

[0069] To analyze potential biomarkers in DS-iPSC cell exosomes, this invention utilizes TEM for morphological evaluation of exosomes, such as... Figure 6 As shown in Figure AD, the isolated exosomes exhibit a typical cup-shaped or disc-shaped morphology with a flat, circular structure, and their diameter ranges from 30 nm to 150 nm. This indicates that the present invention has isolated typical exosomes.

[0070] 2.4 Expression profile analysis of ncRNAs in iPSC-derived exosomes.

[0071] 2.4.1 Microarray Chip Analysis: To further analyze ncRNAs in iPSC exosomes that may serve as biomarkers for prenatal diagnosis of DS, microarray analysis was performed; the microarray analysis results showed that 210 pre-miRNAs and 123 snoRNAs were upregulated ( P <0.05, FC>1.5), 175 pre-miRNAs and 87 snoRNAs were downregulated ( P <0.05, FC<1 / 1.5). This invention lists the top 10 significantly upregulated and downregulated pre-miRNAs and snoRNAs in microarray analysis.

[0072] The above 595 differentially expressed ncRNAs ( P Volcano plot, scatter plot, and cluster plot analyses were performed using values ​​<0.05 and |FC|>1.5. The results are shown below: Scatter plot analysis: Figure 7The scatter plot in AB is based on the differentially expressed ncRNAs obtained above. The green dashed line is the break line (the default FC cutoff value is 1.5). The ncRNAs above or below the green break line show a change of more than 1.5-fold between the two comparison samples. The scatter plot can intuitively show the differential expression of various ncRNAs between the NC group and the DS group.

[0073] It is noteworthy that some of these pre-miRNAs and snoRNAs are encoded by chromosome 21, including two pre-miRNAs (hsa-mir-4327 and hsa-let-7c) and two snoRNAs (SNORD74B and SNORA32) (Table 3), which may be related to DS. Given that previous studies have shown a close interaction between pre-miRNAs and snoRNAs, the analysis in this invention focuses primarily on these two classes of ncRNAs.

[0074] Table 3 lists the ncRNAs encoded by chromosome 21. 2.4.2 Biological function analysis of differentially expressed ncRNAs.

[0075] To further explore the potential biological functions of differentially expressed ncRNAs discovered by microarrays and their possible roles in the pathogenesis of disseminated syndromes (DS), this invention performed gene ontology (GO) annotation analysis on the host genes encoding pre-miRNAs and snoRNAs. Figure 8 , Figure 9The analysis covered three subdomains: cellular components, biological processes, and molecular functions. The top ten enriched pre-miRNAs and snoRNAs associated with DS were found in these subdomains. GO enrichment analysis showed that differentially expressed snoRNAs were mainly enriched in biological processes such as cytoplasmic translation, polypeptide biosynthesis, and amide biosynthesis. Cellular components were mainly enriched in cytoplasmic ribosomes, large cytoplasmic ribosome subunits, and the eukaryotic translation initiation factor 4F complex. Significant enrichment was also observed in molecular functions such as ribosome structural components, mRNA 5'-UTR binding, RNA binding, 5S rRNA binding, and insulin receptor substrate binding. Differentially expressed pre-miRNAs are mainly involved in miRNA-mediated gene silencing, post-transcriptional gene silencing, and post-transcriptional gene expression regulation. Cellular components were mainly located in the RISC complex, RNAi effector complex, ribonucleoprotein complex, extracellular space, extracellular regions, and extracellular vesicles. Significant enrichment was observed in molecular functions related to mRNA base pairing-mediated post-transcriptional repression and translational regulation. These results not only reveal the multiple roles of differentially expressed ncRNAs in intracellular regulatory networks, but also provide new perspectives and theoretical basis for the molecular pathogenesis of DS.

[0076] 2.5 RT-ddPCR detection.

[0077] 2.5.1 RT-ddPCR analysis of the expression of ncRNAs in DS-iPSC exosomal cells: This invention utilizes RT-ddPCR to analyze four exosomal ncRNAs derived from chromosome 21. Figure 10 (AD). Using the U6 copy number detected by RT-ddPCR as an internal control, and the copy numbers of hsa-let-7c, hsa-mir-4327, SNORA32, and SNORD74B as observation indicators, the relative expression levels of each ncRNA were calculated based on (copy number of DS group ncRNAs / U6 internal control copy number) / (copy number of NC group ncRNAs / U6 internal control copy number) (with 1 representing a copy number of zero). It was found that the trends of the RT-ddPCR detection results for three of them (hsa-let-7c, hsa-mir-4327, and SNORA32) were consistent with the microarray chip analysis results. Figure 11 This laid the foundation for subsequent functional verification and mechanism research.

[0078] 2.5.2 RT-ddPCR analysis of differentially expressed ncRNAs in maternal peripheral blood: To further verify whether differentially expressed ncRNAs related to chromosome 21 derived from iPSCs have the potential to serve as prenatal diagnostic biomarkers, this invention performed RT-ddPCR analysis on peripheral blood samples from 6 pregnant women with DS fetuses and 6 pregnant women with normal fetuses. Based on previous screening results, this invention mainly analyzed the relative expression of hsa-mir-4327, hsa-let-7c, SNORA32, and SNORD74B derived from chromosome 21. The main criteria for selecting these ncRNAs for analysis were: 1) the coding genes of these indicators are located on chromosome 21; 2) existing literature reports that they are closely related to neurodevelopmental abnormalities. In the actual RT-ddPCR detection process, normalization was performed based on the target ncRNA copy number / internal reference copy number. Considering the limited amount of remaining samples from prenatal screening, peripheral blood samples from 3 pregnant women with DS fetuses and 3 pregnant women with normal fetuses were used for the analysis of hsa-mir-4327 and hsa-let-7c. Figure 12 AC (Chinese)

[0079] In addition, peripheral blood samples from 3 pregnant women carrying DS fetuses and 3 pregnant women carrying normal fetuses were used for the analysis of SNORA32 and SNORD74B. Figure 13 (AC), the results showed that the expression levels of hsa-mir-4327, hsa-let-7c, and SNORA32 in the peripheral blood of pregnant women carrying fetuses with DS were significantly higher than those in the control group, and the differences were statistically significant. P <0.05)( Figure 14 The relative expression levels of SNORD74B did not differ statistically between the two groups. P >0.05), this result is inconsistent with the chip analysis results.

[0080] In summary, this invention utilizes somatic cells from patients to establish DS-iPSCs cell lines with the same genomic and chromosomal genetic background. The constructed DS-iPSCs not only retain the typical trisomy 21 karyotype but also express relevant biomarkers of pluripotent stem cell potential (SSE4, TRA-1-81). This provides an ideal cell model for further screening of DS-specific biomarkers and exploring the pathogenesis of DS.

[0081] This invention successfully isolated exosomes from DS-iPSCs. Their morphology, after ultracentrifugation and TEM observation, exhibited typical characteristics such as cup-shaped, butterfly-shaped, and flattened round shapes. To investigate the effect of DS chromosome dosage changes on ncRNA expression, this invention performed microarray analysis on the ncRNAs in the isolated exosomes. The results showed that hundreds of ncRNAs exhibited significant differential expression. GO enrichment analysis of pre-miRNAs and snoRNAs indicated that these ncRNAs are mainly involved in gene silencing, gene expression regulation, and snRNA modification in their host genes, and their functions are closely related to increased chromosome 21 dosage, suggesting that they may play a regulatory role in the pathological process of DS. RT-ddPCR technology was used for verification, and the results showed that the relative expression levels of hsa-let-7c, hsa-mir-4327, and SNORA32 located on chromosome 21 were increased in DS-iPSCs, which is consistent with the microarray detection results of this invention. Furthermore, to investigate the differential expression of these ncRNAs in maternal peripheral blood, this invention further employed RT-ddPCR to detect ncRNAs derived from chromosome 21 in the peripheral blood of pregnant women with DS. The results showed that the expression levels of hsa-mir-4327, hsa-let-7c, and SNORA32 in the peripheral blood of mothers with DS fetuses were significantly higher than those in the control group, and the differences were statistically significant. This suggests that they may serve as diagnostic biomarkers for DS screening.

[0082] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing induced pluripotent stem cells for Down syndrome, characterized in that, Includes the following steps: Sendai virus vectors carrying genes OCT4, SOX2, c-MYC, and KLF-4 were transduced into somatic cells carrying triploid chromosome 21 and cultured to obtain Down syndrome induced pluripotent stem cells.

2. The method for constructing induced pluripotent stem cells for Down syndrome according to claim 1, characterized in that, The somatic cells carrying triploid chromosome 21 include at least one of amniotic fluid cells and peripheral blood cells.

3. A type of induced pluripotent stem cell for Down syndrome, characterized in that, The Down syndrome induced pluripotent stem cells are obtained by the construction method according to any one of claims 1 to 2.

4. An application of induced pluripotent stem cells for Down syndrome, characterized in that, The Down syndrome induced pluripotent stem cell of claim 3 is used to screen for biomarkers of Down syndrome.

5. A biomarker for Down syndrome, characterized in that, The biomarkers for Down syndrome are obtained using a Down syndrome induced pluripotent stem cell as described in claim 3.

6. A biomarker for Down syndrome according to claim 5, characterized in that, The biomarkers for Down syndrome include at least one of hsa-mir-4327, hsa-let-7c, and SNORA32.

7. A biomarker for Down syndrome according to claim 6, characterized in that, The hsa-let-7c nucleotide sequence is shown in SEQ ID NO:1; The nucleotide sequence of hsa-mir-4327 is shown in SEQ ID NO:2; The nucleotide sequence of SNORA32 is shown in SEQ ID NO:

4.

8. The application of a biomarker for Down syndrome, characterized in that, The biomarker of Down syndrome as described in any one of claims 5 to 7 may be used in the preparation of a product for prenatal screening of Down syndrome.

9. The application of a biomarker for Down syndrome according to claim 8, characterized in that, The products mentioned in the prenatal screening include any one of the following: testing reagents and testing kits.

10. The application of a biomarker for Down syndrome according to claim 9, characterized in that, The detection reagent includes primers, the nucleotide sequences of which are shown in SEQ ID NO:5-13.

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