Umbilical cord mesenchymal stem cell heterogeneity analysis method based on single cell transcriptome sequencing

Abstract

The invention discloses an umbilical cord mesenchymal stem cell heterogeneity analysis method based on single-cell transcriptome sequencing, which comprises the following steps: (1) preparing a single-cell library, and performing single-cell transcriptome sequencing; (2) analyzing the obtained transcriptome data by using a bioinformatics tool; (3) identifying different subgroups of the UC-MSCs according to a clustering analysis result, and analyzing marker gene expression characteristics of each subgroup; (4) carrying out GO enrichment analysis to obtain enrichment items; and (5) verifying related functional genes. According to the invention, a single cell sequencing technology is utilized, subgroups of UC-MSCs of different donors are analyzed in a high-throughput and high-resolution manner, heterogeneity of the subgroups on a transcriptional level is disclosed, a scientific basis is provided for application of the UC-MSCs, more accurate cell characteristic data is provided for clinical application, and the application prospect is wide. Important theoretical basis and technical support are provided for stem cell treatment and regenerative medicine.

Description

Technical Field

[0001] The invention relates to an umbilical cord mesenchymal stem cell heterogeneity analysis method based on single-cell transcriptome sequencing, and belongs to the technical field of stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with self-renewal and multidirectional differentiation potentials, which can differentiate into various cell types such as osteocytes, chondrocytes, muscle cells, hepatocytes, and neurons. MSCs are widely present in various tissues and organs, such as bone marrow, fat, umbilical cord, and placenta. MSCs play an important role in regulating the inflammatory environment, supporting the development and maintenance of neurons, promoting angiogenesis, and wound healing, and have broad application prospects.

[0003] Umbilical cord mesenchymal stem cells (UC-MSCs) are a type of adult stem cells isolated from umbilical cord tissue. UC-MSCs have been used in regenerative medicine, tissue engineering, and cell therapy in recent years due to their easy access, low immunogenicity, and low ethical controversy. However, UC-MSCs from different donor sources have heterogeneity in biological characteristics and functions, which has a certain impact on their clinical application. Therefore, it is of great significance to establish an accurate and comprehensive detection method to evaluate the characteristics of UC-MSCs from different donor sources.

[0004] At present, the commonly used MSCs identification methods mainly include in vitro culture characteristics, surface marker expression profiles, differentiation potential, etc. However, these methods have certain limitations, such as complex operations and subjective results. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for analyzing the heterogeneity of umbilical cord mesenchymal stem cells based on single cell transcriptome sequencing (scRNA-seq). The present invention uses single cell sequencing technology that can quantitatively analyze gene expression at the single cell level, analyzes subpopulations of UC-MSCs from different donors in a high-throughput, high-resolution manner, and reveals their heterogeneity at the transcriptional level, thereby providing a scientific basis for the application of UC-MSCs, further providing more accurate cell characteristic data for clinical applications, and providing an important theoretical basis and technical support for stem cell therapy and regenerative medicine.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing, comprising the following steps:

[0008] (i) preparing a single-cell library of umbilical cord mesenchymal stem cells and performing single-cell transcriptome sequencing to obtain transcriptome data for each cell;

[0009] (ii) using bioinformatics tools to analyze the acquired transcriptome data;

[0010] (III) Based on the results of cluster analysis, different subpopulations of UC-MSCs were identified and the marker gene expression characteristics of each subpopulation were analyzed;

[0011] (IV) GO enrichment analysis was performed based on marker genes in different subgroups to obtain enriched entries;

[0012] (V) Verification of relevant functional genes of UC-MSCs.

[0013] Furthermore, the specific operation of step (1) is as follows:

[0014] (1) Using the C4_V2 library construction platform; adding the single cell suspension of umbilical cord mesenchymal stem cells, oil, and magnetic beads to the C4scRNA slide in sequence, preparing them into oil-in-water droplets using the DNBelab C-TaiM 4 device, and completing the process of cell lysis and magnetic bead capture of mRNA in the droplets;

[0015] (2) Reverse transcription to generate cDNA;

[0016] (3) adding a demulsifying agent to the cDNA, reacting at room temperature, centrifuging, aspirating the product, and distinguishing the oligo and cDNA products by magnetic bead screening;

[0017] (4) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, amplifying the cDNA, and purifying it;

[0018] (5) Physical examination of cDNA product to determine its concentration and fragment distribution;

[0019] (6) preparing the reaction system, reacting at an appropriate temperature for a certain period of time, amplifying the oligo product, adding index and purifying it, and waiting for cyclization;

[0020] (7) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, performing fragmentation, end repair and adding "A", and then purifying the product;

[0021] (8) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, connecting the linker, and then purifying the product;

[0022] (9) preparing a PCR reaction system, reacting at an appropriate temperature for a certain period of time, performing PCR amplification, and then purifying the product;

[0023] (10) After denaturing the cDNA product and the oligo product into single strands, respectively, preparing a cyclization reaction system and setting a reaction program to obtain a single-stranded circular product and digesting the uncyclized linear DNA molecules;

[0024] (11) The single-stranded circular DNA molecule replicates through rolling circles to form a DNA nanoball (DNB) containing multiple copies. The resulting DNA nanoball is added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using combined probe anchor polymerization (cPAS) technology.

[0025] Furthermore, the single cell suspension of umbilical cord mesenchymal stem cells is prepared by the following method:

[0026] (1) Rapidly transfer the cryopreserved human umbilical cord mesenchymal stem cells to a 37°C water bath (preheated for half an hour in advance), gently shake to thaw, open the lid and slowly add 1 mL of complete medium (90% DMEM + 10% FBS) preheated to 37°C, and gently pipette to mix;

[0027] (2) Transfer the cells to 9 mL of complete medium preheated to 37°C, gently invert to mix, centrifuge at 4°C, 300 g for 5 min, and discard the supernatant;

[0028] (3) Add 3 mL of complete medium to wash and resuspend, centrifuge at 4°C and 300 g for 5 min, and discard the supernatant;

[0029] (4) Resuspend the cells in an appropriate amount of culture medium and adjust the cell concentration to 1×10 6 cell / mL to prepare cell suspension.

[0030] Furthermore, in step (ii), the analysis includes data preprocessing, quality control, gene expression matrix construction and / or cluster analysis.

[0031] Furthermore, in steps (ii), (iii) and (iv), DNBelab_C4scRNA (v1.0.1) was used to process the original sequencing data of each sample to generate the original gene expression matrix, and then the R package Seurat (v3.2.0) was used for downstream analysis; the cells were quality controlled based on the number of genes detected and the proportion of mitochondrial reads.

[0032] Furthermore, the specific operations of steps (ii), (iii) and (iv) are as follows:

[0033] (1) Filter out cells with less than 200 genes or more than 90% of the maximum number of genes identified;

[0034] (2) Sort the cells in descending order according to the proportion of mitochondrial reads, and filter out the top 15% of cells;

[0035] (3) Doublet detection was used to identify and delete doublets, and cell cycle analysis was performed using Seurat (Cell Cycle Scoring function);

[0036] (4) The gene expression dataset was standardized, and then principal component analysis (PCA) was performed using the 2000 highly variable genes in the dataset;

[0037] (5) The data processing dimensionality reduction method UMAP (Uniform Manifold Approximation and Projection) was used for dimensionality reduction and clustering. For each cluster, the Find All Markers (logfc>0.25, minPct>0.1 and Padj≤0.05) function in the Seurat package was used to identify marker genes, and then the cell types were annotated using the SCSA method;

[0038] (6) Differential genes were identified using the Find Markers (logfc>0.25, minPct>0.1 and Padj≤0.05) function;

[0039] (7) Gene Ontology (GO) enrichment analysis was performed on genes using the phyper function in the R package. GO terms or pathways with FDR ≤ 0.05 were considered significantly enriched.

[0040] Furthermore, in the step (five), the relevant functional genes are any one, two or more, or all of the following nine genes: VCAM1, F3, MCAM, SDC2, PTPRN2, SERPINF1, ANPEP, NAV1, COL3A1, which are related to proliferation ability, angiogenesis ability, immune regulation, neurogenesis ability, etc.

[0041] Furthermore, in step (V), the specific operation of verifying the relevant functional genes is as follows:

[0042] (1) RNA extraction;

[0043] (2) RNA quality testing;

[0044] (3) Reverse transcription to obtain cDNA template;

[0045] (4) Perform relative quantitative analysis of genes to detect the expression levels of target genes and internal reference genes.

[0046] Furthermore, the method further comprises step (six): establishing a UC-MSCs heterogeneity database based on the above analysis results to provide a reference for stem cell research and application.

[0047] A UC-MSCs heterogeneity database is established using the above-mentioned umbilical cord mesenchymal stem cell heterogeneity analysis method based on single-cell transcriptome sequencing.

[0048] The application of the UC-MSCs heterogeneity database in the heterogeneity analysis of umbilical cord mesenchymal stem cells.

[0049] The single-cell transcriptome sequencing-based heterogeneity analysis method of umbilical cord mesenchymal stem cells of the present invention can comprehensively and deeply reveal the gene expression heterogeneity of UC-MSCs at the single-cell level, clarify the characteristics of each subgroup, and verify the expression of related functional genes, providing important theoretical basis and technical support for further research on its biological characteristics, differentiation mechanism and precision treatment in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : UMAP diagram of the clustering of human umbilical cord mesenchymal stem cells.

[0051] Figure 2 : Schematic diagram of the proportion of each subpopulation of human umbilical cord mesenchymal stem cells.

[0052] Figure 3 :Schematic diagram of the human umbilical cord mesenchymal stem cell Marker gene heat map results.

[0053] Figure 4 :Schematic diagram of the expression level of marker genes in human umbilical cord mesenchymal stem cells A.

[0054] Figure 5 :Schematic diagram of the expression level of marker genes in human umbilical cord mesenchymal stem cells B.

[0055] Figure 6 : Schematic diagram of the expression level of marker genes in human umbilical cord mesenchymal stem cells C.

[0056] Figure 7 :Schematic diagram of the expression level of marker genes in human umbilical cord mesenchymal stem cells D.

[0057] Figure 8: Schematic diagram of GO enrichment analysis of human umbilical cord mesenchymal stem cell marker genes A. From left to right and from top to bottom are the expression level results of marker genes of 6 subgroups, namely subgroup 0, subgroup 1, subgroup 2, subgroup 3, subgroup 4, and subgroup 5.

[0058] Fig. 9 : Schematic diagram of GO enrichment analysis of human umbilical cord mesenchymal stem cell marker genes B. From left to right are the expression level results of marker genes of three subgroups, namely subgroup 7, subgroup 8, and subgroup 9.

[0059] Fig.10 : Schematic diagram of RT-QPCR detection results of genes related to the functions of human umbilical cord mesenchymal stem cells, where from left to right and from top to bottom are the RT-QPCR detection results of 9 genes, including VCAM1, F3, MCAM, SDC2, PTPRN2, SERPINF1, ANPEP, NAV1, and COL3A1.

[0060] Note: The text or lines in the drawings of the present invention may be displayed too small or unclear. It is hereby declared that this situation has no effect on the content of the specification of the present invention, the content or meaning expressed in the drawings, and the understanding of the technical solution of the present invention. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0062] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0063] Analysis of heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing

[0064] 1 Experimental methods

[0065] 1.1 Isolation and culture of human umbilical cord mesenchymal stem cells

[0066] The umbilical cord used in this project was obtained from the First People's Hospital of Nanyang City. It was obtained from newborn umbilical cord samples after approval by the Ethics Committee and informed consent of the patient. The umbilical cord was cut into small pieces of about 2 cm in length using pointed straight scissors and washed with saline three times, and two arteries and one vein were stripped. The connective tissue between the umbilical artery and vein was taken, which is the Wharton's jelly. The Wharton's jelly was washed with saline three times and fully chopped into 1 mm pieces. 3 ~3mm 3 Size. Inoculate the umbilical cord tissue into the culture bottle according to the amount of processed tissue, and place it in a 37℃, 5% CO2 incubator for culture. After 7 days of culture, replace half of the medium and remove the floating tissue blocks. Replace half of the medium every 3 days. After dense clones appear in the cell culture bottle, aspirate the medium, rinse with saline, add 3mL of trypsin substitute for digestion, digest for 2 to 5 minutes, add 10mL of saline to flush the cell surface after the cells become round, collect the cells and centrifuge at 1000r for 10 minutes, and then follow 1×10 4 / cm 2 The cells of the fifth generation were taken for subsequent single-cell sequencing.

[0067] The present invention collected UC-MSCs from 2 umbilical cord tissue samples, which were named hUC-MSCs_1 and hUC-MSCs_2 respectively.

[0068] 1.2 Preparation of UC-MSCs single cell suspension

[0069] (1) Preheat a water bath to 37°C for half an hour and preheat 10 mL of complete culture medium (90% DMEM + 10% FBS) to 37°C.

[0070] (2) Quickly transfer the cryopreserved human umbilical cord mesenchymal stem cells to a 37°C water bath and gently shake to thaw until mung bean-sized ice cubes remain in the tube. Carefully open the lid and slowly add 1 mL of preheated complete culture medium. Gently pipette to mix.

[0071] (3) Transfer the cells to the remaining 9 mL of preheated complete medium, gently invert to mix, centrifuge at 4°C, 300 g for 5 min, and discard the supernatant.

[0072] (4) Add 3 mL of complete culture medium to wash and resuspend, centrifuge at 4°C and 300 g for 5 min, and discard the supernatant.

[0073] (5) Resuspend the cells in an appropriate amount of culture medium and adjust the cell concentration to 1×10 6 cell / mL to prepare cell suspension.

[0074] (6) The cell suspension was stained with 0.4% trypan blue and examined under a microscope to calculate the viability, clumping rate, background debris rate, and nuclear integrity. After passing the quality control, the library construction phase was initiated.

[0075] 1.3 Preparation of UC-MSCs single cell library

[0076] (1) Using the C4_V2 library construction platform, the prepared single-cell suspension, oil, and magnetic beads were added to the C4 scRNA slide in sequence, and prepared into oil-in-water droplets using the DNBelab C-TaiM 4 device, where the cell lysis and magnetic bead capture of mRNA were completed.

[0077] (2) Reverse transcription to generate cDNA.

[0078] (3) Add demulsification reagent to cDNA, react at room temperature, centrifuge, aspirate the product, and use magnetic bead screening to distinguish oligo and cDNA products.

[0079] (4) Prepare a reaction system, react at an appropriate temperature for a certain period of time, amplify the cDNA, and purify it.

[0080] (5) Analyze the concentration and fragment distribution of cDNA product.

[0081] (6) Prepare the reaction system, react at an appropriate temperature for a certain period of time, amplify the oligo product, add index (linker) and purify it, and wait for circularization.

[0082] (7) Prepare the reaction system, react at an appropriate temperature for a certain period of time, perform fragmentation, end repair and add "A", and then purify the product.

[0083] (8) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, connecting the linkers, and then purifying the product.

[0084] (9) preparing a PCR reaction system, reacting at an appropriate temperature for a certain period of time, performing PCR amplification, and then purifying the product.

[0085] (10) After the cDNA product and the oligo product are denatured into single strands, the cyclization reaction systems are prepared and the reaction procedures are set to obtain single-stranded circular products and digest the uncyclized linear DNA molecules.

[0086] (11) The single-stranded circular DNA molecule replicates through rolling circles to form a DNA nanoball (DNB) containing multiple copies. The resulting DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using combined probe anchor polymerization (cPAS) technology.

[0087] 1.4 Quality control of UC-MSCs single cell data

[0088] DNBelab_C4scRNA (v1.0.1) was used to process the original sequencing data of each sample to generate the original gene expression matrix, and then the R package Seurat (v3.2.0) was used for downstream analysis. The cells were quality controlled according to the number of genes detected and the proportion of mitochondrial reads. The specific operations are as follows:

[0089] (1) Cells with less than 200 genes or more than 90% of the maximum number of genes identified were filtered out.

[0090] (2) Sort the cells in descending order according to the proportion of mitochondrial reads, and filter out the top 15% of cells.

[0091] (3) Doublet detection was used to identify and delete doublets, and cell cycle analysis was performed using Seurat (Cell Cycle Scoring function).

[0092] (4) The gene expression dataset was normalized, and then PCA (n=15) analysis was performed using 2000 highly variable genes in the dataset.

[0093] (5) UMAP was used for further dimensionality reduction and clustering. For each cluster, the Find All Markers (logfc>0.25, minPct>0.1 and Padj≤0.05) function in the Seurat package was used to identify marker genes, and then the cell types were annotated using the SCSA method.

[0094] (6) Use the Find Markers (logfc>0.25, minPct>0.1 and Padj≤0.05) function to identify differentially expressed genes.

[0095] (7) GO enrichment analysis was performed on genes using the phyper function in the R package. GO terms or pathways with FDR ≤ 0.05 were considered significantly enriched.

[0096] The quality control is shown in Table 1 , and the proportions of different subgroups are shown in Table 2 .

[0097] Table 1 Quality control table of human umbilical cord mesenchymal stem cell single cell data

[0098] Data quality control hUC-MSCs_1 hUC-MSCs_2 The number of cells after filtration 9706 9224 The median UMI of each cell 15 370 8969 UMI’s Q30 data 88.24％ 86.89％ Median number of genes per cell 4459 3387 The average number of reads per cell 41 609 30 570

[0099] Table 2 Schematic diagram of the proportion of different subgroups

[0100] Subgroup Proportion 0 27.76％ 1 15.90％ 2 11.37％ 3 11.07％ 4 10.01％ 5 8.87％ 6 8.13％ 7 6.37％ 8 0.5％

[0101] 1.5 RT-qPCR detection of expression of related functional genes

[0102] (1) RNA extraction

[0103] ① Collect cells by centrifugation (3000g, 3min). 6 ~10×10 6 Add 1 mL of TRIzol to each animal cell and mix well by pipetting.

[0104] ② Place the sample at room temperature for 5 minutes to allow the nucleic acid-protein complex to completely separate.

[0105] ③ For every 1 mL of TRIzol used, add 0.2 mL of chloroform to the sample, cover the tube, shake vigorously for 15 seconds, and leave at room temperature for 3 to 5 minutes.

[0106] ④ Centrifuge at 4°C, 12000g for 10 min. The sample will separate into three layers, with RNA mainly in the upper aqueous phase. Transfer the aqueous phase to a new tube.

[0107] ⑤ Add isopropanol in an equal volume to the supernatant, mix well, and leave at room temperature for 10 to 20 minutes.

[0108] ⑥ Centrifuge at 4°C, 12000g for 10 min and discard the supernatant.

[0109] ⑦ Add 1 mL of 75% ethanol solution (prepared with DEPC water), invert and mix to wash the precipitate. For every 1 mL of TRIzoI used, add at least 1 mL of 75% ethanol solution.

[0110] ⑧ Centrifuge at 4℃, 7000g for 5min and discard the supernatant. Centrifuge briefly and quickly, and carefully discard the supernatant with a pipette, taking care not to absorb the precipitate. Open the lid and air-dry on the operating table for 5-10min, but the RNA precipitate cannot be completely dried to avoid reducing the solubility of RNA. Finally, dissolve the precipitate with an appropriate amount of DEPC water, and store it at -80℃ or for later use after it is completely dissolved.

[0111] (2) RNA quality testing

[0112] Turn on the ultra-micro nucleic acid analyzer, select the RNA detection program, use DEPC water as a blank, take a small amount (1.5 μL) of the RNA sample to be tested, and record the purity and concentration of the RNA (purity is OD 260 / OD 280 ).

[0113] (3) Reverse transcription

[0114] Prepare the PCR reaction solution, take a 0.2mL PCR tube and add the following reagents in sequence: 2.5μL primer Oligo dT, 5μgRNA, add DEPC water to 12.5μL. Adjust the reaction program, centrifuge the above mixture slightly, and immediately place it on the PCR instrument for amplification at 72℃ for 5min. When the temperature drops to 4℃, add the following reagents in sequence: 4.0μL 5xPrime ScriptBuffer, 2.0μL dNTP mix, 1.0μL Revert Aid Reverse, 0.5μL RNase Inhibitor. After the configuration is completed, 42℃, 60min, 72℃, 10min. After the end, the cDNA template is obtained.

[0115] (4) Quantitative system preparation

[0116] The configuration of the quantitative system: 5.0μL (1X) SYBR Master Mix (2X), 0.3μL R-Primer (10μM), 0.3μL F-Primer (10μM), 0.2μL cDNA, 0.42μL H2O. After the configuration is completed, set the PCR instrument to 95℃ for 3min, followed by 95℃ for 10s for 40 cycles, 59℃ for 20s for 40 cycles, and collect the fluorescence signal (Ct). –ΔΔCt Methods Relative quantitative analysis of genes was performed. The expression levels of target genes and reference genes in different samples were detected. The primer sequences are shown in Table 3.

[0117] Table 3 RT-qPCR gene primer sequences

[0118]

[0119]

[0120] 2 Results

[0121] 2.1 Heterogeneity of human umbilical cord mesenchymal stem cells

[0122] Single-cell transcriptome sequencing was performed on UC-MSCs from two different individuals, and the quality control results were as follows: Figure 1 As shown, the Q30 Bases in UMI were 88.24% and 86.89%, respectively, and the number of cells after filtration were 9706 and 9224, respectively. The data analysis results of the single-cell transcriptome of umbilical cord mesenchymal stem cells were reliable.

[0123] like Figure 2As shown in the figure, UC-MSCs from different individuals have limited heterogeneity. These cells can be clustered into 9 different cell subpopulations without bias after UAMP dimensionality reduction, including cluster0 (accounting for 27.76%), cluster1 (accounting for 15.90%), cluster2 (accounting for 11.37%), cluster3 (accounting for 11.07%), cluster4 (accounting for 10.01%), cluster5 (accounting for 8.87%), cluster6 (accounting for 8.13%), cluster7 (accounting for 6.37%), and cluster8 (accounting for 0.5%). Since cluster8 accounts for a relatively small proportion, it will not be included in the analysis in some subsequent results.

[0124] 2.2 Marker genes of human umbilical cord mesenchymal stem cells

[0125] The marker genes of each cluster of human umbilical cord mesenchymal stem cells are as follows Figure 3 As shown, they mainly include: TAGLN, PLAT, S100A4, TIMP1, TPM2, FTL, NNMT, MYL9, FTH1, S100A6, TUBA1A, PCNA, MCM3, MCM4, CDC6, FAM111B, FEN1, DUT, DEK, MCM7, HIST1H4C, CCNB1, CDC20, CKS2, UBE2S, CCNB2, CENPF, TPX2, PRC1, SMC4, TUBB4B, HIST1H4C, PCNA, TOP2A, HMGB2, CDK1, RRM2, UBE2T, SMC4, FEN1, CD KN1A, TOP2A, HMGB2, CKS2, UBE2C, ARL6IP1, CENPF, ASPM, CDK1, CENPE, KPNA2, EXT1, CDH13, BNC2, TRIO, MAML2, ASAP1, SAMD3, ADAMTS6, EYS, LRP 1B, PCDH15, CNTN5, PTPRD, CTNNA3, CNTNAP2, MACROD2, RBFOX1, DLG2, TPM2, ANXA2, TAGLN, CSRP1, MYL9, LINC01133, TPM1, CCND1, ACTA2, ACTG2.

[0126] The cluster classifications and some key marker genes are shown in Table 4. The expression level diagram of key marker genes is shown in Figures 4 to 7Taking the marker gene TAGLN as an example, the expression of the marker gene was analyzed: TAGLN was expressed in all clusters, but the expression level was highest in cluster1.

[0127] In summary, the expression of marker genes has a certain degree of heterogeneity.

[0128] Table 4 Key marker genes of each subgroup

[0129] Subgroup Marker genes of each subgroup 0 TAGLN, PLAT, S100A4, TIMP1, FTL 1 TUBA1A 2 PCNA, MCM3, MCM4, CDC6, FAM111B 3 CCNB1, CDC20, CKS2, UBE2S, CCNB2 4 HIST1H4C, PCNA, TOP2A, HMGB2, CDK1 5 TOP2A, HMGB2, CKS2, UBE2C, ARL6IP1 6 EXT1, CDH13, BNC2, TRIO, MAML2 7 CNTNAP2, LRP1B, PTPRD, DLG2, EYS 8 TAGLN, MYL9, CCND1, TPM2, ANXA2

[0130] 2.3 GO enrichment analysis of marker genes of human umbilical cord mesenchymal stem cells

[0131] According to the results of GO enrichment analysis, it can be seen that (such as Figure 8 , Fig. 9 As shown in the figure): cluster0 is enriched in the Relaxin signaling pathway; the cluster2 subgroup is basically in the S phase of the cell cycle and is involved in DNA replication; cluster3 is enriched in the P53 signaling pathway; cluster6 is enriched in the Wnt signaling pathway, PI3k-Akt signaling pathway, ErbB signaling pathway, and Rap1 signaling pathway; cluster7 is enriched in the ErbB signaling pathway, Hippo signaling pathway, MAPK signaling pathway, Ras and other signaling pathways.

[0132] 2.4 RT-QPCR detection results of genes related to human umbilical cord mesenchymal stem cells function

[0133] To validate this method, RT-QPCR was used to detect the expression levels of nine genes related to different functions of MSCs, including VCAM1, F3, MCAM, SDC2, PTPRN2, SERPINF1, ANPEP, NAV1, and COL3A1 (the results are shown in Fig.10 As shown), GAPHD was used as an internal reference. The above gene expression levels were found to be consistent with the results of the single-cell transcriptome sequencing analysis, and the expression levels of NAV1 and COL3A1 in hUC-MSCs-2 were the highest, indicating that hUC-MSCs-2 has stronger neurorepair and anti-aging abilities than hUC-MSCs-1. In addition, the expression levels of VCAM1, F3, MCAM, SDC2, PTPRN2, SERPINF1, and ANPEP in hUC-MSCs-1 were stronger than those in hUC-MSCs-2, indicating that hUC-MSCs - The angiogenesis-promoting and immunomodulatory abilities of hUC-MSCs-1 are stronger than those of hUC-MSCs-2, which provides strong data for the subsequent study of the heterogeneity of mesenchymal stem cells.

[0134] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for analyzing the heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing, characterized in that: The following steps are involved: (i) preparing a single-cell library of umbilical cord mesenchymal stem cells and performing single-cell transcriptome sequencing to obtain transcriptome data for each cell; (ii) using bioinformatics tools to analyze the acquired transcriptome data; (III) Based on the results of cluster analysis, different subpopulations of UC-MSCs were identified and the marker gene expression characteristics of each subpopulation were analyzed; (IV) GO enrichment analysis was performed based on marker genes in different subgroups to obtain enriched entries; (V) Verification of relevant functional genes of UC-MSCs.

2. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 1, characterized in that: The specific operation of step (1) is as follows: (1) Using the C4_V2 library construction platform; adding the single cell suspension of umbilical cord mesenchymal stem cells, oil, and magnetic beads to the C4scRNA slide in sequence, preparing them into oil-in-water droplets using the DNBelab C-TaiM 4 device, and completing the process of cell lysis and magnetic bead capture of mRNA in the droplets; (2) Reverse transcription to generate cDNA; (3) adding a demulsifying agent to the cDNA, reacting at room temperature, centrifuging, aspirating the product, and distinguishing the oligo and cDNA products by magnetic bead screening; (4) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, amplifying the cDNA, and purifying it; (5) Physical examination of cDNA product to determine its concentration and fragment distribution; (6) preparing the reaction system, reacting at an appropriate temperature for a certain period of time, amplifying the oligo product, adding index and purifying it, and waiting for cyclization; (7) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, performing fragmentation, end repair and adding "A", and then purifying the product; (8) preparing a reaction system, reacting at an appropriate temperature for a certain period of time, connecting the linker, and then purifying the product; (9) preparing a PCR reaction system, reacting at an appropriate temperature for a certain period of time, performing PCR amplification, and then purifying the product; (10) After denaturing the cDNA product and the oligo product into single strands, respectively, preparing a cyclization reaction system and setting a reaction program to obtain a single-stranded circular product and digesting the uncyclized linear DNA molecules; (11) The single-stranded circular DNA molecule replicates through rolling circles to form a DNA nanoball containing multiple copies; the obtained DNA nanoball is added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced using the combined probe anchor polymerization technology.

3. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 2, characterized in that: The single cell suspension of umbilical cord mesenchymal stem cells is prepared by the following method: (1) Rapidly transfer the cryopreserved tube of human umbilical cord mesenchymal stem cells to a 37°C water bath, gently shake to thaw, open the lid and slowly add complete culture medium preheated to 37°C, and gently pipette to mix; (2) Transfer the cells to complete medium preheated to 37°C, gently invert to mix, centrifuge, and discard the supernatant; (3) Add complete medium to wash and resuspend, centrifuge, and discard the supernatant; (4) Resuspend the cells in culture medium to prepare a cell suspension.

4. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 1, characterized in that: In steps (ii), (iii) and (iv), DNBelab_C4scRNA (v1.0.1) was used to process the original sequencing data of each sample to generate the original gene expression matrix, and then the R package Seurat (v3.2.0) was used for downstream analysis; the cells were quality controlled based on the number of genes detected and the proportion of mitochondrial reads.

5. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 4, characterized in that: The specific operations of steps (ii), (iii) and (iv) are as follows: (1) Filter out cells with less than 200 genes or more than 90% of the maximum number of genes identified; (2) Sort the cells in descending order according to the proportion of mitochondrial reads, and filter out the top 15% of cells; (3) Doublet detection was used to identify and remove doublets, and cell cycle analysis was performed using Seurat; (4) The gene expression dataset was normalized, and then PCA analysis was performed using the 2000 highly variable genes in the dataset; (5) Dimensionality reduction and clustering were performed using UMAP. For each cluster, the Find All Markers function in the Seurat package was used to identify marker genes, and then the cell types were annotated using the SCSA method. (6) Use the Find Markers function to identify differentially expressed genes; (7) GO enrichment analysis was performed on genes using the phyper function in the R package. GO terms or pathways with FDR ≤ 0.05 were considered significantly enriched.

6. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 1, characterized in that: In the step (V), the relevant functional genes are any one, two or more, or all of the following nine genes: VCAM1, F3, MCAM, SDC2, PTPRN2, SERPINF1, ANPEP, NAV1, and COL3A1.

7. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 1, characterized in that: In the step (V), the specific operation of verifying the relevant functional genes is as follows: (1) RNA extraction; (2) RNA quality testing; (3) Reverse transcription to obtain cDNA template; (4) Perform relative quantitative analysis of genes to detect the expression levels of target genes and internal reference genes.

8. The method for analyzing heterogeneity of umbilical cord mesenchymal stem cells based on single-cell transcriptome sequencing according to claim 1, characterized in that: The method further includes step (six): establishing a UC-MSCs heterogeneity database based on the above analysis results.

9. A UC-MSCs heterogeneity database, characterized by: The method is established by using the umbilical cord mesenchymal stem cell heterogeneity analysis method based on single-cell transcriptome sequencing according to any one of claims 1 to 8.

10. Use of the UC-MSCs heterogeneity database according to claim 9 in the heterogeneity analysis of umbilical cord mesenchymal stem cells.

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