A primer probe composition for detecting ipsc residues in ipsc-derived mesenchymal stem cells and application thereof

Abstract

The present application relates to a primer probe composition for detecting iPSC residues in iPSC-derived mesenchymal stem cells and its application, and belongs to the technical field of pluripotent stem cell residue detection in biological products. The present application screens the genes CRIPTO and ESRP1 which are highly expressed in iPSC and not expressed in iPSC-derived mesenchymal stem cells as biomarkers, which have high specificity. By combining the expression of the two genes and through relative quantitative analysis, the residual amount of iPSC in the sample can be more accurately determined. Compared with the judgment by the expression of a single gene, the use of two gene combinations has higher specificity and sensitivity, and is more scientific and practical. The present application also designs a primer probe combination for the biomarker, and screens a primer probe combination with high specificity and sensitivity, which has a detection sensitivity of 0.0005%, ensuring the safety of the product and reducing the corresponding clinical risk.

Description

Technical Field

[0001] This invention belongs to the field of pluripotent stem cell residue detection technology in biological products, and relates to a primer and probe composition for detecting iPSC residues in iPSC-derived mesenchymal stem cells and its application. Background Technology

[0002] MSCs face significant challenges in the industrialization stage, such as limited seed cell sources and the tendency for cells to undergo replicative senescence during production, hindering large-scale, multi-batch production and resulting in poor drug-likeness. Although umbilical cord-derived MSCs have low initial cell passages and strong proliferative capacity, they still suffer from poor consistency between MSCs from different umbilical cord sources, and their cell characteristics and functions are significantly influenced by the donor, thus limiting their drug-likeness.

[0003] With the development of the cell therapy industry, especially stem cell therapy, an increasing number of iPSC-derived cells have entered or are about to enter clinical trials. Under specific culture conditions, iPSCs can efficiently differentiate into MSCs, iPSC-derived mesenchymal stem cells, or iMSCs for short. Their cellular characteristics and functions are very similar to UC-MSCs, exhibiting advantages such as low immunogenicity and strong anti-inflammatory and repair capabilities, showing high similarity. Furthermore, because iPSCs are derived from iPSCs, a high-quality, stable cell bank can be established under strict GMP conditions, providing a large number of highly consistent starting cells for MSC production, demonstrating great promise in the industry. However, since iPSCs can theoretically expand indefinitely, they possess tumorigenic characteristics, posing a risk of tumor formation after injection into the body. Moreover, because cell therapy products are live cells, especially those derived from iPSC-induced differentiation, such as iMSCs mentioned above, and are ultimately formulated as live cells, terminal sterilization is impossible. Therefore, to ensure the safety of the final cell therapy product and avoid additional risks due to iPSC residues, it is essential to test the residual amount of iPSCs in the product.

[0004] Currently, the most commonly used methods are flow cytometry, cell culture, and nucleic acid detection. Flow cytometry primarily detects iPSC-specific surface marker antigens using flow cytometry. However, this method has low sensitivity and is affected by factors such as antibody dosage, staining specificity, cell number and activity, and parameter adjustments. Furthermore, it cannot detect small amounts of residual iPSCs in high-dose products. Cell culture typically involves seeding target cells into a stem cell culture system. After several days of culture, the presence of pluripotent stem cell clones is observed under high magnification. If clones appear, the number of clones is counted, and the residual amount is calculated based on the number of seeded cells. However, this method is complex and time-consuming, and is significantly affected by experimental procedures and culture conditions.

[0005] Nucleic acid detection methods detect residual iPSCs by measuring the expression levels of iPSC-specific genes in cell products. While these methods are characterized by their short processing time and high sensitivity, they are unsuitable for iPSC residue detection due to the significant differences in gene expression profiles among different derived cell types. Commonly used pluripotent stem cell marker genes such as SOX2, NANOG, and OCT4 are also expressed at low levels in some differentiated cells. Therefore, there is an urgent need to develop a method for detecting residual iPSCs in specific cell types derived from iPSCs through a particular differentiation pathway, such as iMSCs, to provide a reasonable basis for quality control testing of cell therapy products. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a primer-probe composition for detecting iPSC residues in iPSC-derived mesenchymal stem cells and its application.

[0007] The objective of this invention can be achieved through the following methods:

[0008] In a first aspect, the present invention provides the application of a biomarker in detecting iPSC residues in iPSC-derived mesenchymal stem cells, wherein the biomarker is a combination of the genes CRIPTO and ESRP1.

[0009] As one embodiment of the present invention, the nucleotide sequences of the genes CRIPTO and ESRP1 are shown in SEQ ID NO. 7-8, respectively. In some embodiments, the biomarker further includes the internal reference gene GAPDH.

[0010] In a second aspect, the present invention provides a primer-probe composition for detecting residual iPSCs in iPSC-derived mesenchymal stem cells, the primer-probe composition comprising:

[0011] The primer-probe composition for detecting CRIPTO includes:

[0012] The nucleotide sequence of the upstream primer CRIPTO-rt-F is shown in SEQ ID NO.1, nucleotide sequence: 5'-TGGCCCGCTTCTCTTACAGT-3';

[0013] The nucleotide sequence of the downstream primer CRIPTO-rt-R is shown in SEQ ID NO.2, nucleotide sequence: 5'-GGTATCCCCGAGATGGACGA-3';

[0014] The nucleotide sequence of the probe CRIPTO-rt-P is shown in SEQ ID NO.3, nucleotide sequence: 5'-CCAGCCCGGCAACTAATCCCA-3';

[0015] The primer-probe composition for detecting ESRP1 includes:

[0016] The nucleotide sequence of the upstream primer ESRP1-rt-F is shown in SEQ ID NO.4, nucleotide sequence: 5'-CTGGCACGGTGGTCAGAATG-3';

[0017] The nucleotide sequence of the downstream primer ESRP1-rt-R is shown in SEQ ID NO.5, nucleotide sequence: 5'-TGGCCTGGTCATTTGTGTGTA-3';

[0018] The nucleotide sequence of the probe ESRP1-rt-P is shown in SEQ ID NO.6, nucleotide sequence: 5'-ATCCTCGGTTGCATACTGGTAACCT-3'.

[0019] As one embodiment of the present invention, the 5' end of the probe CRIPTO-rt-P or the probe ESRP1-rt-P is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quencher group; wherein, the fluorescent reporter group is selected from at least one of FAM, HEX, TET, VIC, JOE, Cy3, Cy5, and the fluorescent quencher group is selected from at least one of BHQ1, BHQ2, BHQ3, TEMRA, MGB.

[0020] Thirdly, the present invention provides the use of the primer-probe composition in the preparation of a product for detecting residual iPSCs in iPSC-derived mesenchymal stem cells.

[0021] Fourthly, the present invention provides a kit for detecting residual iPSCs in iPSC-derived mesenchymal stem cells, the kit comprising the primer-probe composition described above.

[0022] As one embodiment of the present invention, the kit detects the residual amount of iPSC in iMSCs derived from iPSC by qPCR and / or digital PCR.

[0023] As one embodiment of the present invention, the kit further includes a primer and probe composition for detecting the internal reference gene GAPDH and cDNA standards.

[0024] As one embodiment of the present invention, the primer and probe composition of the internal reference gene GAPDH includes the following:

[0025] The nucleotide sequence of the upstream primer GAPDH-rt-F is shown in SEQ ID NO.9, nucleotide sequence: 5'-CAGAGATGATGACCCTTTTGGCT-3';

[0026] The nucleotide sequence of the downstream primer GAPDH-rt-R is shown in SEQ ID NO.10, nucleotide sequence: 5'-TCCAAAATCAAGTGGGGCGA-3';

[0027] The nucleotide sequence of the probe GAPDH-rt-P is shown in SEQ ID NO.11. The nucleotide sequence is 5'-AAATGAGCCCCAGCCTTCTCCA-3'. The 5' end of the probe contains a FAM fluorescent label, and the 3' end contains a BHQ1 label.

[0028] As one embodiment of the present invention, the cDNA standard is composed of cDNA prepared from UC-MSC samples containing (different proportions) iPSCs through RNA extraction and reverse transcription.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The genes screened in this invention are selected from transcriptome data. Using high-throughput sequencing and bioinformatics analysis, three types of cells were analyzed. Genes that are highly expressed in iPSCs but not expressed in MSCs were selected as detection genes. After detailed experimental verification, the two optimal genes CRIPTO and ESRP1 were selected as detection targets, which have high specificity.

[0031] 2. The primer and probe combination for detection in this invention preferably uses intron-spanning sequences during primer and probe design and screening to ensure high specificity. At the same time, genomic DNA is removed during sample processing to reduce the impact of genomic DNA on the detection results.

[0032] 3. When using commonly used quantitative real-time PCR for detection, this invention achieves a detection sensitivity of 0.0005%, meaning it can still detect one iPSC out of 200,000 MSCs. This extremely high sensitivity meets the safety requirements of the cell therapy industry, ensuring the safety of iPSC-derived mesenchymal stem cell products and reducing corresponding clinical risks. Using more advanced digital PCR for detection will result in even higher sensitivity.

[0033] 4. The standard provided by this invention can be used to establish a standard curve to quantify the iPSCs that may be present in the sample. By combining the expression of the two genes and further performing relative quantitative analysis, and comparing with the reference standard, the residual amount of iPSCs in the sample can be determined more accurately. Compared with judging by the expression of a single gene, the determination using the combination of two genes has higher specificity and sensitivity, and is more scientific and practical. Attached Figure Description

[0034] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0035] Figure 1 This is a heatmap of 36 genes obtained from screening three groups of cells in Example 1;

[0036] Figure 2 The amplification curves of the 11 candidate genes and internal reference genes in iPSC samples in Example 2 are shown.

[0037] Figure 3 The amplification curves of the 11 candidate genes and internal reference genes in UC-MSCs are shown in Example 2.

[0038] Figure 4 The amplification curves of LIN28A in iPSC and UC-MSC samples in Example 2 are shown.

[0039] Figure 5 The amplification curves of CRIPTO in iPSC and UC-MSC samples in Example 2 are shown.

[0040] Figure 6 The amplification curves of SLC7A3 in iPSC and UC-MSC samples in Example 2 are shown.

[0041] Figure 7 The amplification curves of ESRP1 in iPSC and UC-MSC samples in Example 2 are shown.

[0042] Figure 8 The amplification curves of CRIPTO in samples with different iPSC contents are shown in Example 3.

[0043] Figure 9 The amplification curves of ESRP1 in samples with different iPSC contents are shown in Example 3.

[0044] Figure 10 The amplification curve of CRIPTO in the low-abundance iPSC sample in Example 3;

[0045] Figure 11 The amplification curve of ESRP1 in the low-abundance iPSC sample in Example 3;

[0046] Figure 12 The amplification curve of GAPDH in the low-abundance iPSC sample in Example 3;

[0047] Figure 13 The standard curve was plotted in Example 3 based on the iPSC content and the relative expression level of CRIPTO in the sample.

[0048] Figure 14The amplification curves of CRIPTO for the five reference samples and blank control in Example 4 are shown.

[0049] Figure 15 The amplification curves of ESRP1 for the five reference samples and blank control in Example 4 are shown.

[0050] Figure 16 The amplification curves of CRIPTO, ESRP1, and GAPDH in the sample to be tested in Example 4 are shown.

[0051] Figure 17 The amplification curves of CRIPTO, ESRP1, and GAPDH for the positive control of the test sample in Example 4 are shown. Detailed Implementation

[0052] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0053] The main experimental materials used in the embodiments of this invention are as follows:

[0054] The RNA extraction kit was purchased from Tiangen Biotech Co., Ltd., catalog number: DP419.

[0055] The RNA reverse transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: R333-01.

[0056] The probe-based qPCR Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: Q513-02.

[0057] Example 1: Screening for specifically highly expressed genes in iPSCs and designing and synthesizing primers and probes.

[0058] Both iPSCs and iMSCs express a large number of genes. In order to screen out genes that are specifically highly expressed in iPSCs but not in MSCs, three groups of cell samples were collected. The first group was the starting cells iPSCs, the second group was iMSCs derived from iPSCs through induced differentiation. To ensure that the final cells received did not contain iPSCs, quercetin was added to the culture medium during differentiation culture to promote apoptosis of any remaining iPSCs. The third group was MSCs derived from umbilical cord (purchased from a cell bank). The construction method of the second group of iMSCs refers to patent CN117448267B, and the steps are as follows: S1: Human pluripotent stem cells are induced to form neural crest cells; a hard base is prepared so that the base stiffness is 500-3000 kPa; the supernatant of human pluripotent stem cells is aspirated, and a specific neural crest cell induction culture medium is added for culture; signal pathway regulators are added to the specific neural crest cell induction culture medium: BMP inhibitor, GSK-3 inhibitor, and one of the following three: Activin, Nodal, and TGFb inhibitor; wherein the human pluripotent stem cells are commercial human induced pluripotent stem cells iPSCs; S2: Neural crest cell expansion culture; a soft base is prepared so that the base stiffness is 1-10. The specific neural crest cell induction differentiation medium was removed by kPa. The neural crest cells were digested with EDTA or Accutase and resuspended in a soft-based culture plate. Neural crest cell expansion medium was added for expansion culture for 7 days. bFGF, EGF and one of the following three inhibitors were added to the expansion medium: Activin, Nodal and TGFb inhibitor. During this period, the neural crest cells were cryopreserved using cryopreservation solution. S3: The expanded neural crest cells differentiated into mesenchymal stem cells, thus obtaining iMSC samples.

[0059] Three replicate samples were prepared for each group of cells. After the samples were collected, RNA was extracted and the transcriptome sequencing was performed by a third-party company. After a series of bioinformatics analyses, the gene expression profiles of each sample were obtained. The results showed that the samples had good reproducibility and the expression profiles of iMSCs were highly similar to those of UC-MSCs.

[0060] Gene expression profiling was performed using the following criteria: FPKM(iPSC) ≥ 50 and FPKM(iMSC) ≤ 1. The heatmap of the 36 selected genes is shown below. Figure 1As shown in the results, genes that simultaneously meet the above two screening criteria are almost not expressed in UC-MSCs, indicating that iMSCs have similar characteristics to natural UC-MSCs. Further, a selection of genes suitable for qPCR detection was chosen from the obtained gene list, preferably those with large FPKM (iPSC) and FPKM (iMSC) and FPKM (UC-MSC) values ​​of 0 or close to 0. The final selected genes are as follows: LIN28A, POU5F1B, CLDN6, CRIPTO, EPCAM, DMKN, SLC7A3, RARRES2, ESRP1, PTPRZ1, and USP44.

[0061] Based on the gene name, the gene transcript sequence was downloaded from the NCBI website. The sequence characteristics of different transcripts were compared, and the fragment expressed in all transcripts was selected as the target sequence. Primers and probes were then designed based on the sequence characteristics. In order to improve specificity and reduce interference from genomic DNA, the target sequence must span introns, and the probe is preferably a sequence that can span introns.

[0062] Primer and probe sequences for detecting LIN28A are shown in SEQ ID NO. 12, 13, and 14; primer and probe sequences for detecting POU5F1B are shown in SEQ ID NO. 15, 16, and 17; primer and probe sequences for detecting CLDN6 are shown in SEQ ID NO. 18, 19, and 20; primer and probe sequences for detecting CRIPTO are shown in SEQ ID NO. 1, 2, and 3; primer and probe sequences for detecting EPCAM are shown in SEQ ID NO. 21, 22, and 23; primer and probe sequences for detecting DMKN are shown in SEQ ID NO. 24, 25, and 26; primer and probe sequences for detecting SLC7A3 are shown in SEQ ID NO. 27, 28, and 29; primer and probe sequences for detecting RARRES2 are shown in SEQ ID NO. 30, 31, and 32; and primer and probe sequences for detecting ESRP1 are shown in SEQ ID NO. 14, ...20. As shown in NO.4, 5 and 6, the primer and probe sequences for detecting PTPRZ1 are shown in SEQ ID NO.33, 34 and 35, and the primer and probe sequences for detecting USP44 are shown in SEQ ID NO.36, 37 and 38.

[0063] To further compare gene expression levels among different samples, a set of primers and probes were also designed for the internal reference gene GAPDH, as shown in sequences SEQ ID NO. 9, 10 and 11.

[0064] Example 2: Specificity of primer-probe composition for detecting candidate genes

[0065] In the development of iMSC products, to minimize the impact of culture medium components on cells and improve product safety, quercetin is not used in the process. Therefore, to detect the amplification effect and specificity of primers and probes, iPSCs and UC-MSCs with gene expression profiles similar to iMSCs were selected as test samples. The candidate primer-probe combinations were tested to detect the expression of candidate genes in the two cell types. The genes detected were: LIN28A, POU5F1B, CLDN6, CRIPTO, EPCAM, DMKN, SLC7A3, RARRES2, ESRP1, PTPRZ1, USP44, and the internal control gene GAPDH.

[0066] iPSCs and UC-MSCs were collected separately, and RNA was extracted using an RNA extraction kit. After elution with RNase-free H2O, the concentration was measured using a micro spectrophotometer. The RNA was then used for subsequent cDNA synthesis experiments or stored at -80℃. The cDNA reaction system was prepared according to Table 1 below, with 500 ng of RNA used for each sample.

[0067] Table 1 Reverse transcription system

[0068]

[0069] After preparation, mix thoroughly by pipetting, then briefly centrifuge to ensure all reaction solutions are at the bottom of the PCR tube, and perform reverse transcription according to the procedure in Table 2 below.

[0070] Table 2 Reverse Transcription Procedure

[0071]

[0072] The cDNA obtained from reverse transcription can be directly used to prepare the qPCR reaction system. The primer-probe combination was tested using a probe method. The qPCR reaction system is shown in Table 3, with 0.5 μl of cDNA added to each well. Two replicate wells were set up for each reaction. Additionally, a negative control was set up for each primer-probe combination, with no template added and only ddH2O added to 20 μl.

[0073] Table 3. Probe-based qPCR reaction system

[0074]

[0075] qPCR reactions and signal acquisition were performed using the Bio-Rad CFX Connect PCR instrument, and data processing and analysis were performed using the accompanying software. The reaction program was set according to Table 4.

[0076] Table 4. Probe-based qPCR reaction procedure

[0077]

[0078] The results show that all the genes tested were highly expressed in iPSCs. Five genes, namely LIN28A, CRIPTO, SLC7A3, ESRP1 and PTPRZ1, did not show obvious amplification signals in UC-MSCs. The specific detection results (Ct values) are shown in Table 5. Figure 2 These are the amplification curves of 11 candidate genes and the internal reference gene GAPDH in iPSC samples; Figure 3 These are the amplification curves of 11 candidate genes and the internal reference gene GAPDH in UC-MSCs; Figure 4 These are the amplification curves of LIN28A in iPSC and UC-MSC samples; Figure 5 These are the amplification curves of CRIPTO in iPSC and UC-MSC samples; Figure 6 These are the amplification curves of SLC7A3 in iPSC and UC-MSC samples; Figure 7 These are the amplification curves of ESRP1 in iPSC and UC-MSC samples.

[0079] The above results indicate that the five genes LIN28A, CRIPTO, SLC7A3, ESRP1, and PTPRZ1 have high expression specificity, consistent with the transcriptome results. They are expressed only in iPSCs and not in MSCs, making them more suitable for iPSC residue detection.

[0080] Table 5. Expression detection of candidate genes and internal reference genes in iPSCs and UC-MSCs (Ct values)

[0081]

[0082] Note: "N / A" indicates that the fluorescence value of the amplified product is below the threshold and there is no obvious amplification curve.

[0083] Example 3: Specificity and sensitivity of the primer-probe composition for detecting iPSC residues

[0084] Based on the results in Example 2, four genes, LIN28A, CRIPTO, SLC7A3, and ESRP1, were selected for subsequent detection. The internal reference gene was still GAPDH. The sensitivity of the primer and probe composition of these genes to iPSC residues in MSCs was tested.

[0085] iPSCs and UC-MSCs were collected separately, counted using a cell counter, and then the samples were prepared according to the proportions shown in Table 6 below. RNA was extracted, reverse transcribed into cDNA, and the expression of each gene in samples with different iPSC ratios was detected. The reverse transcription system was prepared according to Table 1, with the amount of RNA starting in the 20 μl reverse transcription system increased to 1 μg. cDNA synthesis was performed according to the reverse transcription program in Table 2.

[0086] Table 6. Reference table for cell sample preparation with different iPSC ratios.

[0087]

[0088] For the five prepared cDNA samples, the reaction systems were prepared according to the qPCR system in Table 7, with two replicate wells for each reaction. After preparation, qPCR reactions and detections were performed according to the reaction system in Table 8, with the cycle number set to 45.

[0089] Table 7 qPCR reaction system

[0090]

[0091] Table 8. Probe-based qPCR reaction procedure

[0092]

[0093] The results (Ct values) for each reaction well are shown in Table 9. The amplification curves of CRIPTO for all samples are shown in [Table 9]. Figure 8 The amplification curves of ESRP1 in all samples are shown below. Figure 9 The results show that CRIPTO and ESRP1 (exon sequences shown in SEQ ID NO. 7-8, respectively) have stronger specificity than other genes. Even with increased cDNA starting amount, UC-MSC samples still showed negative results, indicating that the primer-probe combination for CRIPTO and the primer-probe combination for ESRP1 have higher specificity.

[0094] Table 9. Expression (Ct values) of candidate genes in cell samples with different iPSC contents.

[0095]

[0096] To further investigate the minimum detection capability of CRIPTO and ESRP1, three types of UC-MSC samples containing 0.005%, 0.001%, and 0.0005% iPSC were prepared. The reverse transcription system was prepared according to Table 1, with the RNA starting amount increased to 1 μg in the 20 μl reverse transcription system. cDNA synthesis was performed according to the reverse transcription program in Table 2, with the reverse transcription time extended to 20 minutes. Using these three newly prepared cDNAs as templates, and cDNA from the UC-MSC sample with 0.01% iPSC as a positive control, and reaction wells without any template as negative controls, the expression of CRIPTO, ESRP1, and GAPDH was detected. When preparing the reaction system, the cDNA volume was increased to 2 μl / well. qPCR detection was performed according to the reaction program in Table 8. Specific detection results for each reaction well are shown in Table 10, and the amplification curves of CRIPTO for all samples are shown in Table 10. Figure 10 The amplification curve of ESRP1 is shown in the figure. Figure 11 The amplification curve of GAPDH is shown in the figure. Figure 12 The results show that CRIPTO and ESRP1 expression can still be detected even when the iPSC content is as low as 0.001%, and CRIPTO expression can still be detected even when the content is as low as 0.0005%, indicating that detecting the expression of CRIPTO and ESRP1 is highly sensitive in determining whether iPSC residues are present.

[0097] Table 10. Expression of candidate genes in cell samples with low iPSC abundance (Ct values)

[0098]

[0099] Three sets of CRIPTO data were selected from the table above, with iPSC contents of 0.005%, 0.001%, and 0.0001%, respectively. Relative quantitative analysis was performed using the expression results of the GAPDH internal reference. A sample with 0.005% iPSC content was used as a control for normalization. A standard curve was plotted using iPSC content and the relative expression level of CRIPTO. Figure 13 As shown in the results, R 2 >0.99, meets the requirements.

[0100] To further screen primer and probe compositions for qPCR detection with high sensitivity and specificity against CRIPTO and ESRP1, two additional primer and probe combinations were designed for CRIPTO and ESRP1, respectively. The second set of primers and probe sequences for detecting CRIPTO are shown in SEQ ID NO. 39, 40, and 41, with the original primer and probe composition labeled as CRIPTO-1 and the newly designed primer and probe composition labeled as CRIPTO-2. The second set of primers and probe sequences for detecting ESRP1 are shown in SEQ ID NO. 42, 43, and 44, with the original primer and probe composition labeled as ESRP1-1 and the newly designed primer and probe composition labeled as ESRP1-2.

[0101] Three types of UC-MSC samples containing 0.005%, 0.001%, and 0.0005% iPSC, and a pure UC-MSC sample were selected as samples for detecting the two newly designed primer-probe combinations. Water was used as a negative control. The qPCR reaction system and procedure were consistent with the detection of the low-abundance samples. Two biological replicates were used for each reaction well. The reaction results (Ct values) are shown in Table 11. As can be seen from the table, the newly designed primer-probe combination for CRIPTO did not show superior specificity compared to the original primer-probe combination. Amplification was observed in one well of the UC-MSC sample. Although the Ct value was >40, the sensitivity was also inferior to the original primer-probe combination. The newly designed primer-probe combination for ESRP1 showed no significant difference in specificity compared to the original primer-probe combination, and no significant advantage in sensitivity was observed either.

[0102] Table 11 Comparison of the specificity and sensitivity of different primer-probe combinations for target gene amplification

[0103]

[0104] Example 4: Detection of iPSC residues in iMSC using the product of the present invention

[0105] Based on the results of Examples 2 and 3, it can be seen that the primer-probe combination for detecting CRIPTO and the primer-probe combination for detecting ESRP1 have high specificity and high sensitivity for the detection of iPSC. Furthermore, the performance of the original primer-probe combination for detecting CRIPTO and ESRP1 is higher than that of the newly designed primers and probes. Therefore, the original primer-probe combination was still used for subsequent experiments. That is, the primer-probe sequences for detecting CRIPTO are shown in SEQ ID NO.1, 2 and 3, and the primer-probe sequences for detecting ESRP1 are shown in SEQ ID NO.4, 5 and 6.

[0106] Therefore, to facilitate the detection of iPSC presence and residual iPSC levels in differentiated cells, a detection product was prepared. This product contains a primer and probe composition for detecting CRIPTO (nucleotide sequences shown in SEQ ID NO. 1, 2, and 3), a primer and probe composition for detecting ESRP1 (nucleotide sequences shown in SEQ ID NO. 4, 5, and 6), and a primer and probe composition for detecting GAPDH (nucleotide sequences shown in SEQ ID NO. 9, 10, and 11). To accommodate more types of instruments, all three probes in the product are modified with 5'-FAM and 3'-BHQ1. In addition, the product includes five reference samples, which are cDNA samples from UC-MSC cell samples with different iPSC contents. The specific composition is shown in Table 12.

[0107] Table 12 Ingredients of Five Reference Products

[0108]

[0109] Preparation method of reference material: iPSC and UC-MSC cell samples were collected separately, and cell counts were performed. Then, samples were prepared according to the proportions in Table 12, RNA was extracted, and the concentration was determined. cDNA was synthesized by adding 1 μg RNA to a 20 μl reverse transcription system. Primers, probes and cDNA in the product were stored at -20℃.

[0110] According to the standard for determining iPSC residue in this product: the GAPDH expression in the five reference samples was normal, and the expression of CRIPTO and ESRP1 was detected in reference samples 1, 2, 3 and 4, showing a certain trend. The expression of CRIPTO and ESRP1 was not detected in reference sample 5. The expression of GAPDH, CRIPTO and ESRP1 was not detected in the blank control, indicating that the test quality control was qualified. Next, the iPSC residue level is determined based on the expression of each gene in the sample. If both CRIPTO and ESRP1 are negative, it can be directly determined that no iPSC residue was detected. If only one of CRIPTO and ESRP1 has a Ct value or both have a Ct value, further relative quantitative analysis is required. The calculated relative expression level is compared with reference sample 4. If the relative expression level of the sample is lower than that of reference sample 4, the iPSC residue level is determined to be <0.001%. If the relative expression levels of only two genes are higher than those of reference sample 4, the iPSC residue level can be directly determined to be >0.001%. If the relative expression level of only one gene is higher than that of reference sample 4, a retest is required, and duplicate wells should be added. The final determination is based on the results of the second test.

[0111] The prepared product was used for safety testing of iMSCs generated during the process development stage. An iMSC derived from iPSCs, at passage 7 (close to the passage of the final cell preparation), was selected. After digestion, the cells were counted using a cell counter. Another tube of iPSCs was collected and counted similarly, preparing two samples: one as the test sample containing only 10... 6 One iMSC contains 100 iMSCs and the other contains 0.01% iPSCs. Mixing 100 iPSCs and 999,900 iMSCs together creates the positive control for the sample to be tested.

[0112] RNA was extracted from both samples according to the instructions of the RNA extraction kit. After determining the concentration, cDNA was synthesized by adding 1 μg of RNA to a 20 μl reverse transcription system. The amount of cDNA used in the qPCR reaction system for both samples was consistent with the reference samples: 2 μl of cDNA was added to a 20 μl qPCR system. Amplification and detection were performed according to the qPCR reaction procedure in Table 8. Five reference samples, the test sample, and the positive control of the test sample were tested, with H2O used as a blank control. The target genes detected included CRIPTO, ESRP1, and GAPDH. The specific results (Ct values) of each reaction are shown in Table 13. The amplification curves of CRIPTO for the five reference samples and the blank control are shown in Table 13. Figure 14 The amplification curves of ESRP1 for the five reference samples and the blank control are shown in the figure. Figure 15 The amplification curves of CRIPTO, ESRP1, and GAPDH in the sample to be tested are shown in the figure. Figure 16 The amplification curves of CRIPTO, ESRP1, and GAPDH in the positive control of the test sample are shown in the figure. Figure 17 The results show that the expression of all genes in the reference sample is normal. The expression of the internal control in the test sample is normal, but CRIPTO and ESRP1 expression were not detected. Furthermore, the expression of the internal control GAPDH, CRIPTO, and ESRP1 was detected in both the test sample and the positive control. Therefore, there is no need to calculate the relative expression levels or plot the standard curve. Combined with the results of the reference sample, it can be concluded that no iPSC residue was detected in the test sample, or the content was lower than that of reference sample 4 (0.001%). The industry requirement is that the iPSC residue level should be lower than 0.001%, indicating that the iPSC residue level in this test sample meets the requirements.

[0113] Table 13 Expression results (Ct values) of each gene in the test sample and the reference sample.

[0114]

[0115] In summary, the detection method provided by this invention can detect iPSC residues. The presence or absence of iPSC residues in the test cells can be determined based on the expression of CRIPTO and ESRP1 genes. Furthermore, based on the detection results of the five reference samples provided by this invention, the range of iPSC residue levels in the test sample can be determined. The detection method of this invention has high specificity for iPSC detection and high sensitivity for detecting iPSC residues. It can stably detect samples containing as low as 0.001% iPSC residues, with a minimum detection limit of 0.0005%. Moreover, using the product of this invention for sample detection offers advantages such as simple and rapid operation and high detection sensitivity, providing results in just 3-5 hours. It has broad application prospects and practical value in the field of stem cell therapy.

[0116] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The use of a primer-probe composition in the preparation of a product for detecting residual iPSCs in iPSC-derived mesenchymal stem cells, characterized in that, The primer-probe composition comprises: The primer-probe composition for detecting CRIPTO includes: The nucleotide sequence of the upstream primer CRIPTO-rt-F is shown in SEQ ID NO.1; The nucleotide sequence of the downstream primer CRIPTO-rt-R is shown in SEQ ID NO.2; The nucleotide sequence of the probe CRIPTO-rt-P is shown in SEQ ID NO.3; The primer-probe composition for detecting ESRP1 includes: The nucleotide sequence of the upstream primer ESRP1-rt-F is shown in SEQ ID NO.4; The nucleotide sequence of the downstream primer ESRP1-rt-R is shown in SEQ ID NO.5; The nucleotide sequence of probe ESRP1-rt-P is shown in SEQ ID NO.6; The nucleotide sequences of genes CRIPTO and ESRP1 are shown in SEQ ID NO.7-8, respectively.

2. The application according to claim 1, characterized in that, The probe CRIPTO-rt-P or probe ESRP1-rt-P is modified with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end; wherein the fluorescent reporter group is selected from at least one of FAM, HEX, TET, VIC, JOE, Cy3, and Cy5, and the fluorescent quencher group is selected from at least one of BHQ1, BHQ2, BHQ3, TEMRA, and MGB.

3. The application of a kit in the preparation of a product for detecting residual iPSCs in iPSC-derived mesenchymal stem cells, characterized in that, The kit contains the primer and probe composition as described in claim 1 or 2.

4. The application according to claim 3, characterized in that, The kit detects residual iPSCs in iPSC-derived mesenchymal stem cells using qPCR and / or digital PCR.

5. The application according to claim 3, characterized in that, The kit also includes a primer and probe composition for detecting the internal reference gene GAPDH and cDNA standards.

6. The application according to claim 5, characterized in that, The primer and probe composition for the internal reference gene GAPDH includes the following: The nucleotide sequence of the upstream primer GAPDH-rt-F is shown in SEQ ID NO.9; The nucleotide sequence of the downstream primer GAPDH-rt-R is shown in SEQ ID NO.10; The nucleotide sequence of the probe GAPDH-rt-P is shown in SEQ ID NO.

11. The probe has a FAM fluorescent label at its 5' end and a BHQ1 label at its 3' end.

7. The application according to claim 5, characterized in that, The cDNA standard is composed of cDNA prepared from UC-MSC samples containing different proportions of iPSCs through RNA extraction and reverse transcription.

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