A method and kit for evaluating the differentiation state of stem cells

Abstract

The present invention belongs to the technical field of gene detection, and particularly relates to a method and a kit for evaluating the differentiation state of stem cells. The method first screens out genes related to the differentiation of stem cells to be evaluated as target genes, designs primers for the screened target genes, performs multiplex PCR amplification, and performs high-throughput sequencing on the PCR products, so as to quickly and efficiently obtain the expression of the target genes, thereby helping to quickly and accurately evaluate the differentiation state of stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a method and a kit for rapidly evaluating the differentiation state of somatic stem cells by detecting the expression of related genes. Background Art

[0002] Currently, the commonly used methods for detecting gene expression after stem cell differentiation treatment in the literature are fluorescence RT-PCR technology or single-cell RNA sequencing technology. Fluorescence RT-PCR technology uses fluorescent dyes or specific probes labeled with fluorescence to label and track PCR products, monitors the reaction process in real-time online, and can analyze the products by combining corresponding software to detect the expression of target genes. Single-cell RNA sequencing (scRNA-seq) is a high-throughput experimental technology that quantitatively analyzes the gene expression profiles of specific cell populations at the single-cell level using RNA sequencing.

[0003] The advantages of using fluorescence RT-PCR to detect gene expression are short time, low cost, and high sensitivity. However, due to limited fluorescence channels, it is impossible to simultaneously detect the expression of multiple (more than a dozen) genes in the same reaction.

[0004] Although single-cell RNA sequencing can perform heterogeneity analysis of gene expression in each cell, its process is complex, time-consuming, and costly.

[0005] Therefore, it is necessary to develop a detection method that can cover more genes and can rapidly evaluate the differentiation state of stem cells by detecting the expression of related genes after stem cell differentiation treatment. Summary of the Invention

[0006] The object of the present invention is to solve the problems of fewer genes covered by existing detection technologies or complex detection processes, long time consumption, and high cost, and provides a method and a kit for evaluating the differentiation state of somatic stem cells, which can improve the accuracy and speed of detection and help rapidly evaluate the differentiation state of stem cells.

[0007] The present invention designs primers near the target gene region, performs multiplex PCR amplification, and performs high-throughput sequencing on the PCR products, so as to rapidly and efficiently obtain the expression of target genes.

[0008] The technical solution adopted by the present invention is as follows:

[0009] A method for evaluating the differentiation state of stem cells, comprising the following steps:

[0010] S1: Extract RNA of stem cells to be detected;

[0011] S2: Synthesize cDNA;

[0012] S3: Add primers for detecting target genes to perform the first-round multiplex PCR amplification. The target genes are the FEV gene, ONECUT2 gene, zkscan1 gene, stat1 gene, ARID3A gene, RFX6 gene, pax4 gene, neurod1 gene, TPH1 gene, LMX1A gene, INS gene, MAFA gene, IAPP gene, FOSB gene, HOPX gene, NR4A1 gene, SAMD11 gene, SIX3 gene, ATF3 gene, FOS gene, MEIS2 gene, NROB1 gene, ATF4 gene, CEBPD gene, DDIT3 gene, JUNB gene, EGR1 gene, SIX2 gene, TSC22D1 gene, ARNTL2 gene, HSF4 gene, ELF3 gene, HSF2 gene, IRF1 gene, UCN3 gene, ngn3 gene, MNX1 gene, and ZNF358 gene;

[0013] S4: Add adapter sequences to perform the second-round PCR reaction;

[0014] S5: Magnetic bead purification;

[0015] S6: Sequencing analysis, and evaluate the stem cell differentiation status according to the sequencing results.

[0016] Preferably, the primer sequences for detecting target genes are as follows:

[0017] The primer sequence of the FEV gene is shown as SEQ ID NO: 1-2; the primer sequence of the ONECUT2 gene is shown as SEQ ID NO: 3-4

[0018] ; the primer sequence of the zkscan1 gene is shown as SEQ ID NO: 5-6; the primer sequence of the stat1 gene is shown as SEQ ID NO: 7-8

[0019] ; the primer sequence of the ARID3A gene is shown as SEQ ID NO: 9-10; the primer sequence of the RFX6 gene is shown as SEQ ID

[0020] Shown in NO:11 - 12; the primer sequences of the pax4 gene are shown in SEQ ID NO:13 - 14; the primer sequences of the neurod1 gene are shown in SEQ ID NO:15 - 16; the primer sequences of the TPH1 gene are shown in SEQ ID NO:17 - 18; the primer sequences of the LMX1A gene are shown in SEQ ID NO:19 - 20; the primer sequences of the INS gene are shown in SEQ ID NO:21 - 22; the primer sequences of the MAFA gene are shown in SEQ ID NO:23 - 24; the primer sequences of the IAPP gene are shown in SEQ ID NO:25 - 26; the primer sequences of the FOSB gene are shown in SEQ ID NO:27 - 28; the primer sequences of the HOPX gene are shown in SEQ ID NO:29 - 30; the primer sequences of the NR4A1 gene are shown in SEQ ID NO:31 - 32; the primer sequences of the SAMD11 gene are shown in SEQ ID NO:33 - 34; the primer sequences of the SIX3 gene are shown in SEQ ID NO:35 - 36; the primer sequences of the ATF3 gene are shown in SEQ ID NO:37 - 38; the primer sequences of the FOS gene are shown in SEQ ID NO:39 - 40; the primer sequences of the MEIS2 gene are shown in SEQ ID NO:41 - 42; the primer sequences of the NROB1 gene are shown in SEQ ID NO:43 - 44; the primer sequences of the ATF4 gene are shown in SEQ ID NO:45 - 46; the primer sequences of the CEBPD gene are shown in SEQ ID NO:47 - 48; the primer sequences of the DDIT3 gene are shown in SEQ ID NO:49 - 50; the primer sequences of the JUNB gene are shown in SEQ ID NO:51 - 52; the primer sequences of the EGR1 gene are shown in SEQ IDNO:53 - 54; the primer sequences of the SIX2 gene are shown in SEQ ID NO:55 - 56; the primer sequences of the TSC22D1 gene are shown in SEQID

[0021] Shown in NO:57 - 58; the primer sequences of the ARNTL2 gene are shown in SEQ ID NO:59 - 60; the primer sequences of the HSF4 gene are shown in SEQ ID NO:61 - 62; the primer sequences of the ELF3 gene are shown in SEQ ID NO:63 - 64; the primer sequences of the HSF2 gene are shown in SEQ ID NO:65 - 66; the primer sequences of the IRF1 gene are shown in SEQ ID NO:67 - 68; the primer sequences of the UCN3 gene are shown in SEQ ID NO:69 - 70; the primer sequences of the ngn3 gene are shown in SEQ ID NO:71 - 72; the primer sequences of the MNX1 gene are shown in SEQ ID NO:73 - 74; the primer sequences of the ZNF358 gene are shown in SEQ ID NO:75 - 76.

[0022] Preferably, while adding primers for detecting the target gene in step S2, primers for detecting reference genes are also added, and the reference genes are GAPDH gene and ACTB gene.

[0023] Preferably, the primer sequences of the reference genes are as follows:

[0024] The primer sequences of the GAPDH gene are shown as SEQ ID NO:77-78; the primer sequences of the ACTB gene are shown as SEQ ID

[0025] NO:79-80.

[0026] Preferably, the primers for detecting the target gene are configured into a primer pool solution according to the following ratio:

[0027] Gene Name Primer Concentration Added Volume Gene Name Primer Concentration Added Volume NR4A1 10uM 4ul IAPP 10uM 2ul CEBPD 10uM 4ul UCN3 10uM 2ul MAFA 10uM 4ul DDIT3 10uM 2ul SIX2 10uM 4ul ELF3 10uM 2ul HSF4 10uM 4ul zkscan1 10uM 2ul EGR1 10uM 1ul SIX3 10uM 2ul TSC22D1 10uM 4ul MEIS2 10uM 2ul INS 10uM 1ul HOPX 10uM 2ul ARNTL 10uM 4ul ngn3 20uM 2ul FOS 10uM 2ul ZNF358 10uM 2ul JUNB 10uM 2ul MNX1 10uM 2ul FOSB 30uM 2ul ONECUT2 10uM 2ul SAMD11 10uM 2ul RFX6 10uM 2ul IRF1 20uM 2ul neurod1 10uM 2ul ATF4 10uM 2ul ARID3A 10uM 2ul ATF3 20uM 2ul FEV 10uM 2ul HSF2 60uM 2ul stat1 10uM 2ul ACTIN 10uM 2ul pax4 10uM 2ul GAPDH 10uM 2ul TPH1 10uM 2ul NROB1 20uM 2ul LMX1A 10uM 2ul

[0028] In step S3, 2 μL of the primer pool solution is added to every 20 μL of the cDNA solution.

[0029] Preferably, the stem cells are pancreatic islet stem cells.

[0030] The present invention also provides a kit for evaluating the differentiation state of stem cells. The kit contains primers for detecting target genes, and the target genes are FEV gene, ONECUT2 gene, zkscan1 gene, stat1 gene, ARID3A gene, RFX6 gene, pax4 gene, neurod1 gene, TPH1 gene, LMX1A gene, INS gene, MAFA gene, IAPP gene, FOSB gene, HOPX gene, NR4A1 gene, SAMD11 gene, SIX3 gene, ATF3 gene, FOS gene, MEIS2 gene, NROB1 gene, ATF4 gene, CEBPD gene, DDIT3 gene, JUNB gene, EGR1 gene, SIX2 gene, TSC22D1 gene, ARNTL2 gene, HSF4 gene, ELF3 gene, HSF2 gene, IRF1 gene, UCN3 gene, ngn3 gene, MNX1 gene and ZNF358 gene.

[0031] Preferably, the primer sequences for detecting target genes are as follows: The primer sequences for the FEV gene are shown in SEQ ID NO: 1-2; the primer sequences for the ONECUT2 gene are shown in SEQ ID NO: 3-4; the primer sequences for the zkscan1 gene are shown in SEQ ID NO: 5-6; the primer sequences for the stat1 gene are shown in SEQ ID NO: 7-8; the primer sequences for the ARID3A gene are shown in SEQ ID NO: 9-10; the primer sequences for the RFX6 gene are shown in SEQ ID NO: 11-12; the primer sequences for the pax4 gene are shown in SEQ ID NO: 13-14; the primer sequences for the neurod1 gene are shown in SEQ ID NO: 15-16; the primer sequences for the TPH1 gene are shown in SEQ ID

[0032] Shown in NO:17 - 18; The primer sequences of the LMX1A gene are shown in SEQ ID NO:19 - 20; The primer sequences of the INS gene are shown in SEQ ID NO:21 - 22; The primer sequences of the MAFA gene are shown in SEQ ID NO:23 - 24; The primer sequences of the IAPP gene are shown in SEQ ID NO:25 - 26; The primer sequences of the FOSB gene are shown in SEQ ID NO:27 - 28; The primer sequences of the HOPX gene are shown in SEQ ID NO:29 - 30; The primer sequences of the NR4A1 gene are shown in SEQ ID NO:31 - 32; The primer sequences of the SAMD11 gene are shown in SEQ ID NO:33 - 34; The primer sequences of the SIX3 gene are shown in SEQ ID NO:35 - 36; The primer sequences of the ATF3 gene are shown in SEQ ID NO:37 - 38; The primer sequences of the FOS gene are shown in SEQ ID NO:39 - 40; The primer sequences of the MEIS2 gene are shown in SEQ ID NO:41 - 42; The primer sequences of the NROB1 gene are shown in SEQ ID NO:43 - 44; The primer sequences of the ATF4 gene are shown in SEQ ID NO:45 - 46; The primer sequences of the CEBPD gene are shown in SEQ ID NO:47 - 48; The primer sequences of the DDIT3 gene are shown in SEQ ID NO:49 - 50; The primer sequences of the JUNB gene are shown in SEQ ID NO:51 - 52; The primer sequences of the EGR1 gene are shown in SEQ ID NO:53 - 54; The primer sequences of the SIX2 gene are shown in SEQ ID NO:55 - 56; The primer sequences of the TSC22D1 gene are shown in SEQ ID NO:57 - 58; The primer sequences of the ARNTL2 gene are shown in SEQ ID NO:59 - 60; The primer sequences of the HSF4 gene are shown in SEQ ID NO:61 - 62; The primer sequences of the ELF3 gene are shown in SEQ ID NO:63 - 64; The primer sequences of the HSF2 gene are shown in SEQ ID NO:65 - 66; The primer sequences of the IRF1 gene are shown in SEQ ID NO:67 - 68; The primer sequences of the UCN3 gene are shown in SEQ ID NO:69 - 70; The primer sequences of the ngn3 gene are shown in SEQ ID NO:71 - 72; The primer sequences of the MNX1 gene are shown in SEQ ID NO:73 - 74; The primer sequences of the ZNF358 gene are shown in SEQ ID NO:75 - 76.

[0033] Preferably, the kit further contains primers for detecting reference genes, and the reference genes are the GAPDH gene and the ACTB gene.

[0034] Preferably, the primer sequences for detecting reference genes are as follows: the primer sequences for the GAPDH gene are shown in SEQ ID NO: 77-78; the primer sequences for the ACTB gene are shown in SEQ ID NO: 79-80.

[0035] In recent years, islet transplantation, as an emerging treatment method for diabetes, has achieved certain success in clinical applications. However, the severe shortage of donor islets has limited the further popularization of this treatment method. Therefore, developing technical methods to generate a large number of insulin-expressing β-cells in vitro has clinical application value. There are mainly two methods. The first is the directed induction and differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to obtain functional islet cells. The second is to use cells from pancreatic tissue as a source, and transdifferentiate pancreatic duct cells or islet α-cells into β-cells. Nevertheless, the SC-islets generated in vitro are still not completely equivalent to donor islets in terms of function and transcription. Compared with β-cells from donor islets, SC-β cells from in vitro sources lack or have low expression of many important mature genes, such as MAFA. After transplantation, the function and quantity of INS secreted by SC-β cells increase. The reason for this increase is not yet clear, and it may be related to the differentiation of progenitor cells, the remodeling of the graft, and the fact that the mature state of the cells is not determined by the in vitro conditions of their origin. However, the further evaluation of these transplanted cells is limited to immunostaining and functional evaluation of in vitro transplantation.

[0036] In the embodiments of the present invention, by using the above method, with the help of multiplex amplicon sequencing technology, the gene expression changes of SC-islets generated in vitro and transplanted SC-islet cells compared with primary islets are quickly detected. This helps to understand and promote the current directed differentiation technology to develop new cell therapies for diabetes, and provides guidance for islet cell transplantation research and regenerative medicine applications.

[0037] By implementing the above technical solutions, the present invention has the following advantages: The present invention can quickly and efficiently obtain the expression of target genes by screening genes related to the stem cells to be evaluated, designing primers for the screened target genes, performing multiplex PCR amplification, and performing high-throughput sequencing on the PCR products, thereby helping to quickly and accurately evaluate the differentiation state of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG Figure 1 is a comparison curve graph of the uniformity before and after the optimization of the primer concentration in Example 2;

[0039] FIG Figure 2 is a statistical analysis bar graph of the detection of the reads of the target gene in each sample. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] In combination with the following specific embodiments, the present invention will be further described in detail. The protection scope of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and are subject to the protection scope of the appended claims. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no special limitations.

[0041] Example 1

[0042] Preliminary construction of a methodology for detecting the expression of stem cell differentiation-related genes by multiplex amplicon sequencing

[0043] I. Selection of target genes

[0044] The following genes related to stem cell differentiation or islet cell characteristic genes were selected for detection by searching relevant literature. The information list of the selected target genes is shown in Table 1.

[0045] Table 1 Information list of target genes

[0046] Gene geneID Gene geneID Gene geneID Gene geneID FEV 54738 INS 3630 MEIS2 4212 HSF4 3299 ONECUT2 9480 MAFA 389692 NROB1 190 ELF3 1999 zkscan1 7586 IAPP 3375 ATF4 468 HSF2 3298 stat1 6772 FOSB 2354 CEBPD 1052 IRF1 3659 ARID3A 1820 HOPX 84525 DDIT3 1649 UCN3 114131 RFX6 222546 NR4A1 3164 JUNB 3726 ngn3 50674 pax4 5078 SAMD11 148398 EGR1 1958 MNX1 3110 neurod1 4760 SIX3 6496 SIX2 10736 ZNF358 140467 TPH1 7166 ATF3 467 TSC22D1 8848 LMX1A 4009 FOS 2353 ARNTL2 56938

[0047] The reference genes will be screened from the 4 genes shown in Table 2 below:

[0048] Table 2 List of reference genes

[0049] Gene geneID GAPDH 2597 SDHA 6389 ACTB 60 18S rRNA /

[0050] II. Design and synthesis of primers

[0051] According to the conserved regions of the genes, the primer sequences were designed using the primer-probe design software Primer Express 3.0.1. The primer positions spanned different exons. The length range of the specific primers with adapters was 47bp - 62bp, and the amplicon length was controlled within the range of greater than 75bp and less than 300bp. The difference in the TM values of the upstream and downstream primers was basically within 0℃ - 3℃. The primers had good specificity through UCSCblast. The primer gene sequences of each gene are shown in Table 3 below.

[0052] Table 3 Primer sequence table of target genes and reference genes

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The primers of each target gene in Table 1 above have no primer dimers detected by multiple primer dimer analysis. Analysis website: https: / / www.thermofisher.cn / cn / zh / home / brands / thermo-scientific / molecular-biology / molecular-biology-learning-center / molecular-biology-resource-library / thermo-scientific-web-tools / multiple-primer-analyzer.html.

[0059] III. Detection sample test of multiple amplicon sequencing process

[0060] 1. Extract the RNA of the normal islet cell sample B_0903islet using the QIAGEN RNeasy Universal Kits. Detect the concentration and purity of the RNA using a spectrophotometer (Thermo Fisher), ensuring that the RNA concentration is greater than 50 ng / μl, the A260 / A280 is between 1.9 - 2.1, and the A260 / A230 is greater than 1.7. Standardize the concentration of the sample to be tested to 50 ng / μl.

[0061] 2. After centrifuging the dry powder of the primers for all target genes and reference genes, add the corresponding volume of Nuclease-free Water to dilute them to 100 μM, and then dilute them to 10 μM. Mix the upstream and downstream primers of all genes in a 1:1 ratio to form Primer-Mix.

[0062] 3. cDNA synthesis

[0063] 3.1. Prepare the reaction system

[0064] Prepare the reaction system according to Table 4 below:

[0065] Table 4 Reaction system

[0066]

[0067] Gently pipette up and down at least 10 times to thoroughly mix, and then briefly centrifuge to collect the solution at the bottom of the tube.

[0068] 3.2. Reaction program

[0069] Incubate the 20 μl reaction mix in a thermal cycler according to the following steps:

[0070]

[0071] 4. PCR1

[0072] 4.1 Prepare the PCR1 reaction on ice according to Table 5 below:

[0073] Table 5 PCR1 Reaction System

[0074]

[0075] 4.2 Pipette up and down at least 10 times, gently mix, and then briefly centrifuge to collect the solution at the bottom of the tube.

[0076] 4.3 Set the lid temperature to ≥100 °C in the thermal cycler to incubate the reaction, and perform PCR under the cycling conditions shown in Table 6 below:

[0077] Table 6 PCR1 Reaction Conditions

[0078]

[0079]

[0080] 5. PCR2 (Amplified Library with Index)

[0081] 5.1 Mark the pre-arranged Index number on each sample tube.

[0082] 5.2 Prepare the PCR Master Mix on ice according to the system composition shown in Table 7 below:

[0083] Table 7 PCR2 Reaction System

[0084]

[0085] 5.3 For each reaction, add 39 μl of the PCR Master Mix to a clean 0.2 ml PCR tube.

[0086] 5.4 Add 1 μl of the PCR product from PCR1 to each PCR tube.

[0087] 5.5 Add 5 μl of the Index Primer Mix to each sample. Mix well and briefly centrifuge to collect at the bottom.

[0088] 5.6 Set the lid temperature to ≥100 °C in the thermal cycler to incubate the reaction, and perform PCR under the cycling conditions shown in Table 8 below:

[0089] Table 8 PCR2 Reaction Conditions

[0090]

[0091] 5.7 After the PCR reaction is completed, briefly centrifuge and immediately proceed to the next purification step.

[0092] 6. AMPure XP Magnetic Bead Purification

[0093] 6.1 Place the AMPure XP magnetic beads at room temperature for more than 30 min and mix well before use.

[0094] 6.2 Add 75 ul of AMPure XP magnetic beads (1.5 times the volume) to each tube of the sample, pipette and mix 10 times, and let stand at room temperature for 5 min.

[0095] 6.3 Place the sample tube on the magnetic stand and let stand for 5 min until the liquid is clear.

[0096] 6.4 While the sample tube is still on the magnetic stand, carefully remove the supernatant, avoiding sucking up the magnetic beads.

[0097] 6.5 While the sample tube is still on the magnetic stand, wash the magnetic beads with 200 ul of 70% ethanol, let stand for 30 s, and then remove the ethanol.

[0098] 6.6 Repeat step 5 once.

[0099] 6.7 Remove the sample tube, briefly centrifuge, place the sample tube back on the magnetic stand, let stand for 30 s, and then remove the remaining ethanol.

[0100] 6.8 Open the lid and let dry at room temperature for 5 min to completely evaporate the ethanol.

[0101] 6.9 While the sample tube is still on the magnetic stand, add 20 ul of water to each tube.

[0102] 6.10 Remove the sample tube, pipette and mix, and then let stand at room temperature for 2 min.

[0103] 6.11 Place the sample tube on the magnetic stand and let stand for 3 min until the liquid is clear.

[0104] 6.12 Transfer 18 ul of the supernatant to a new 1.5 ml centrifuge tube.

[0105] 7. Qubit Detection of Library Concentration

[0106] Use the dsDNA HS Assay Kit for Qubit (Yeasen) reagent and Qubit 3.0 instrument to detect the library concentration.

[0107] 8. Library Quality Control

[0108] Use an Agilent 4150 analyzer, High Sensitivity D1000 chip, and reagent to detect the library fragment size. The results are shown in Table 9.

[0109] Table 9 Detection Results of Library Fragment Sizes

[0110]

[0111] 9. Sequencing on the Machine

[0112] Sequencing was performed on the Illumina HiSeq X Ten 2x 75, with a target sequencing volume of 0.5G. The sequencing experiment operation was carried out according to the operation manual provided by the manufacturer for sequencing on the machine.

[0113] 10. Analysis of the Results of the Data Retrieved from the Machine

[0114] The experimental data were the results of two replicates. The detection of reads of each gene is shown in Table 10:

[0115] Table 10 Detection of Reads of Each Gene

[0116]

[0117]

[0118] Result Analysis: 1. The detection of the reference gene SDHA was too low; 2. The proportion of the reference gene 18S rRNA was too large, resulting in a low detection of other genes. 3. The detection of the target genes DDIT3, SAMD11, SIX3, FOS, MEIS2, JUNB, and EGR1 was low.

[0119] Example 2

[0120] Optimization of the Process for Detecting the Expression of Genes Related to Stem Cell Differentiation by Multiplex Amplicon Sequencing 1

[0121] The results in Example 1 showed that among the four reference genes GAPDH, SHDA, ACTB, and 18S rRNA, the detection results of SDHA and 18S rRNA were too low and too high, respectively, and they were not suitable as reference genes for detection. Therefore, SDHA and 18S rRNA were removed, and two reference genes, GAPDH and ACTB, were retained. In addition, the detection of the target genes DDIT3, SAMD11, SIX3, FOS, MEIS2, JUNB, and EGR1 was low. Therefore, suitable primers were screened through ordinary PCR and gel electrophoresis tests. After screening, the primer sequences of these 7 target genes were adjusted as shown in Table 11:

[0122] Table 11 Adjusted Primer Sequence Table for Some Target Genes

[0123]

[0124]

[0125] Perform the normal islet cell sample B_0903islet test according to the experimental steps in Example 1. The detection of reads of each gene in the off-machine data results is shown in Table 12:

[0126] Table 12 Detection of reads of each gene

[0127]

[0128]

[0129] Result analysis: After adjustment, each target gene was detected normally. The relatively low detection of some genes was because these genes were low-expression genes, such as LMX1A and NR4A1.

[0130] Example 3

[0131] Process optimization 2 for detecting the expression of stem cell differentiation-related genes by multiplex amplicon sequencing

[0132] The results in Example 2 showed that after adjustment and optimization, each target gene was detected normally, but the uniformity of gene detection was poor. Therefore, in this example, the concentration of the primer pool was adjusted, and the optimal primer pool ratio was finally obtained, as shown in Table 13 below:

[0133] Table 13 Optimal primer pool concentration ratio

[0134] Gene Name Primer Concentration Added Volume Gene Name Primer Concentration Added Volume NR4A1 10uM 4ul IAPP 10uM 2ul CEBPD 10uM 4ul UCN3 10uM 2ul MAFA 10uM 4ul DDIT3 10uM 2ul SIX2 10uM 4ul ELF3 10uM 2ul HSF4 10uM 4ul zkscan1 10uM 2ul EGR1 10uM 1ul SIX3 10uM 2ul TSC22D1 10uM 4ul MEIS2 10uM 2ul INS 10uM 1ul HOPX 10uM 2ul ARNTL 10uM 4ul ngn3 20uM 2ul FOS 10uM 2ul ZNF358 10uM 2ul JUNB 10uM 2ul MNX1 10uM 2ul FOSB 30uM 2ul ONECUT2 10uM 2ul SAMD11 10uM 2ul RFX6 10uM 2ul IRF1 20uM 2ul neurod1 10uM 2ul ATF4 10uM 2ul ARID3A 10uM 2ul ATF3 20uM 2ul FEV 10uM 2ul HSF2 60uM 2ul stat1 10uM 2ul ACTIN 10uM 2ul pax4 10uM 2ul GAPDH 10uM 2ul TPH1 10uM 2ul NROB1 20uM 2ul LMX1A 10uM 2ul

[0135] Perform the normal islet cell sample B_0903islet test according to the experimental steps in Example 1. The detection of reads of each gene in the off-machine data results is shown in Table 14:

[0136] Table 14 Detection of reads of each gene

[0137] SampleID R2104018 FEV 1903 ONECUT2 4450 zkscan1 39820 stat1 67 ARID3A 1526 RFX6 2157 pax4 129 neurod1 2574 TPH1 379 LMX1A 1 INS 116097 MAFA 1198 IAPP 109683 FOSB 6745 HOPX 7257 NR4A1 54 SAMD11 361620 SIX3 12737 ATF3 138671 FOS 722225 MEIS2 7849 NROB1 30220 ATF4 241235 CEBPD 10011 DDIT3 131409 JUNB 412908 EGR1 79033 SIX2 672 TSC22D1 2837 ARNTL 1545 HSF4 2501 ELF3 16000 HSF2 166866 IRF1 37854 UCN3 78261 ngn3 240 MNX1 3237 ZNF358 4920 GAPDH 134250 ACTB 118752

[0138] Statistical analysis showed that the uniformity after optimization was significantly better than that before optimization. The uniformity results are shown in the appendix Figure 1 .

[0139] Example 4

[0140] Detection and comparison of cells after stem cell differentiation with normal islet cells and negative cells

[0141] Extract the RNA of negative cell (before treatment) samples adsc-HFF, cell samples B_DFZ10906 after stem cell differentiation treatment, and normal islet cell samples B_0903islet using the QIAGEN RNeasy Universal Kits. Detect the concentration and purity of the RNA using a spectrophotometer (Thermo Fisher). Ensure that the RNA concentration is greater than 50 ng / ul, the A260 / A280 is between 1.9 - 2.1, and the A260 / A230 is greater than 1.7. Standardize the concentration of the sample to be tested to 50 ng / ul.

[0142] Centrifuge the dry powder of the primers for all target genes and reference genes, and then add the corresponding volume of Nuclease-free Water to dilute them to 100 uM, and then further dilute them to 10 uM. Mix the upstream and downstream primers of all genes according to the optimized primer pool ratio to form Primer-Mix.

[0143] Conduct tests according to the subsequent experimental steps in Example 1. The detection and relative expression levels of the target gene reads in each sample in the off-machine data results are as shown in Table 15 below:

[0144] Table 15 Detection and relative expression levels of target gene reads in each sample

[0145]

[0146]

[0147] Statistical analysis of gene expression is shown in the appendix Figure 2 .

[0148] Result analysis: Compared with the negative cell sample adsc-HFF before treatment, the expression of most islet cell characteristic genes in the cell sample B_DFZ10906 after differentiation treatment tends to be that of normal islet cells, indicating that the stem cells have started to differentiate after differentiation treatment. In addition, the expression of a part of the genes is higher than that of normal islet cells, which may be caused by the stimulation of differentiation treatment. The overall results show that the differentiation state of stem cells can be quickly and accurately evaluated through the multiplex amplicon sequencing we constructed.

Claims

1. A method for evaluating the differentiation state of stem cells, characterized in that, it comprises the following steps: S1: Extract the RNA of the stem cells to be tested; S2: Synthesize cDNA; S3: Add primers for detecting target genes for the first round of multiplex PCR amplification. The primers for detecting target genes are as follows: The primers for the FEV gene are shown as SEQ ID NO:1~2; The primers for the ONECUT2 gene are shown in SEQ ID NO: 3 - 4; the primers for the zkscan1 gene are shown in SEQ ID NO: 5 - 6; the primers for the stat1 gene are shown in SEQ ID NO: 7 - 8; the primers for the ARID3A gene are shown in SEQ ID NO: 9 - 10; the primers for the RFX6 gene are shown in SEQ ID NO: 11 - 12; the primers for the pax4 gene are shown in SEQ ID NO: 13 - 14; the primers for the neurod1 gene are shown in SEQ ID NO: 15 - 16; the primers for the TPH1 gene are shown in SEQ ID NO: 17 - 18; the primers for the LMX1A gene are shown in SEQ ID NO: 19 - 20; the primers for the INS gene are shown in SEQ ID NO: 21 - 22; the primers for the MAFA gene are shown in SEQ ID NO: 23 - 24; the primers for the IAPP gene are shown in SEQ ID NO: 25 - 26; the primers for the FOSB gene are shown in SEQ ID NO: 27 - 28; the primers for the HOPX gene are shown in SEQ ID NO: 29 - 30; the primers for the NR4A1 gene are shown in SEQ ID NO: 31 - 32; the primers for the SAMD11 gene are shown in SEQ ID NO: 33 - 34; the primers for the SIX3 gene are shown in SEQ ID NO: 35 - 36; the primers for the ATF3 gene are shown in SEQ ID NO: 37 - 38; the primers for the FOS gene are shown in SEQ ID NO: 39 - 40; the primers for the MEIS2 gene are shown in SEQ ID NO: 41 - 42; the primers for the NROB1 gene are shown in SEQ ID NO: 43 - 44; the primers for the ATF4 gene are shown in SEQ ID NO: 45 - 46; the primers for the CEBPD gene are shown in SEQ ID NO: 47 - 48; the primers for the DDIT3 gene are shown in SEQ ID NO: 49 - 50; the primers for the JUNB gene are shown in SEQ ID NO: 51 - 52; the primers for the EGR1 gene are shown in SEQ ID NO: 53 - 54; the primers for the SIX2 gene are shown in SEQ ID NO: 55 - 56; the primers for the TSC22D1 gene are shown in SEQ ID NO: 57 - 58; the primers for the ARNTL2 gene are shown in SEQ ID NO: 59 - 60; the primers for the HSF4 gene are shown in SEQ ID NO: 61 - 62; the primers for the ELF3 gene are shown in SEQ ID NO: 63 - 64; the primers for the HSF2 gene are shown in SEQ ID NO: 65 - 66; the primers for the IRF1 gene are shown in SEQ ID NO: 67 - 68; the primers for the UCN3 gene are shown in SEQ ID NO: 69 - 70; the primers for the ngn3 gene are shown in SEQ ID NO: 71 - 72; the primers for the MNX1 gene are shown in SEQ ID NO: 73 - 74;The primers of ZNF358 gene are shown in SEQ ID NO:75~76; S4: Add adapter sequences for the second round of PCR reaction; S5: Purify with magnetic beads; S6: Perform sequencing analysis and evaluate the differentiation state of stem cells according to the sequencing results.

2. The method according to claim 1, characterized in that, the stem cells are pancreatic islet stem cells.

3. The method according to claim 1, characterized in that, when adding primers for detecting target genes in step S2, primers for detecting internal reference genes are also added. The internal reference genes are the GAPDH gene and the ACTB gene.

4. The method according to claim 3, characterized in that, the primer sequences for detecting internal reference genes are as follows: The primers for the GAPDH gene are shown as SEQ ID NO:77~78; The primers for the ACTB gene are shown as SEQ ID NO:79~80.

5. The method according to claim 1, characterized in that, the primers for detecting target genes are configured into a primer pool solution according to the following ratio: In step S3, 2 μL of the primer pool solution is added to every 20 μL of the cDNA solution.

6. A kit for evaluating the differentiation state of stem cells, characterized in that, the kit contains primers for detecting target genes: The primers for the FEV gene are shown as SEQ ID NO:1~2; The primers for the ONECUT2 gene are shown in SEQ ID NO: 3-4; the primers for the zkscan1 gene are shown in SEQ ID NO: 5-6; the primers for the stat1 gene are shown in SEQ ID NO: 7-8; the primers for the ARID3A gene are shown in SEQ ID NO: 9-10; the primers for the RFX6 gene are shown in SEQ ID NO: 11-12; the primers for the pax4 gene are shown in SEQ ID NO: 13-14; the primers for the neurod1 gene are shown in SEQ ID NO: 15-16; the primers for the TPH1 gene are shown in SEQ ID NO: 17-18; the primers for the LMX1A gene are shown in SEQ ID NO: 19-20; the primers for the INS gene are shown in SEQ ID NO: 21-22; the primers for the MAFA gene are shown in SEQ ID NO: 23-24; the primers for the IAPP gene are shown in SEQ ID NO: 25-26; the primers for the FOSB gene are shown in SEQ ID NO: 27-28; the primers for the HOPX gene are shown in SEQ ID NO: 29-30; the primers for the NR4A1 gene are shown in SEQ ID NO: 31-32; the primers for the SAMD11 gene are shown in SEQ ID NO: 33-34; the primers for the SIX3 gene are shown in SEQ ID NO: 35-36; the primers for the ATF3 gene are shown in SEQ ID NO: 37-38; the primers for the FOS gene are shown in SEQ ID NO: 39-40; the primers for the MEIS2 gene are shown in SEQ ID NO: 41-42; the primers for the NROB1 gene are shown in SEQ ID NO: 43-44; the primers for the ATF4 gene are shown in SEQ ID NO: 45-46; the primers for the CEBPD gene are shown in SEQ ID NO: 47-48; the primers for the DDIT3 gene are shown in SEQ ID NO: 49-50; the primers for the JUNB gene are shown in SEQ ID NO: 51-52; the primers for the EGR1 gene are shown in SEQ ID NO: 53-54; the primers for the SIX2 gene are shown in SEQ ID NO: 55-56; the primers for the TSC22D1 gene are shown in SEQ ID NO: 57-58; the primers for the ARNTL2 gene are shown in SEQ ID NO: 59-60; the primers for the HSF4 gene are shown in SEQ ID NO: 61-62; the primers for the ELF3 gene are shown in SEQ ID NO: 63-64; the primers for the HSF2 gene are shown in SEQ ID NO: 65-66; the primers for the IRF1 gene are shown in SEQ ID NO: 67-68; the primers for the UCN3 gene are shown in SEQ ID NO: 69-70; the primers for the ngn3 gene are shown in SEQ ID NO: 71-72; the primers for the MNX1 gene are shown in SEQ ID NO: 73-74;The primers of ZNF358 gene are shown in SEQ ID NO:75~76.; 7. The kit according to claim 6, characterized in that, the kit also contains primers for detecting internal reference genes. The internal reference genes are the GAPDH gene and the ACTB gene.

8. The kit according to claim 7, characterized in that, the primer sequences for detecting internal reference genes are as follows: The primer sequence for the GAPDH gene is shown as SEQ ID NO:77~78; The primer sequence for the ACTB gene is shown as SEQ ID NO:79~80.

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