Gene-based universal detection method for residues of pluripotent stem cells

Abstract

The invention relates to the technical field of gene detection, in particular to a gene-based pluripotent stem cell differentiation residue detection reagent, a gene-based pluripotent stem cell differentiation residue detection method and application thereof. According to the application, on the basis of combination of the screened universal Marker gene and the reference gene, detection of pluripotent stem cell residues in various induced differentiation cells, tissues or organoids can be realized.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a universal gene-based detection method and application of pluripotent stem cell residues. Background Art

[0002] Pluripotent stem cells (PSC) are a general term for induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC), which have the ability to proliferate indefinitely and differentiate into various types of functional cells. Since the advent of pluripotent stem cells, the technology has continued to develop, and for different indications, a variety of hPSC-derived induced differentiation cell products have entered IND filing.

[0003] Compared with adult-derived cell products, the clinical application of induced differentiation cell products derived from human pluripotent stem cells (hPSCs) faces a greater risk of tumorigenicity. The tumorigenicity risk of induced differentiation cell products derived from hPSCs mainly comes from the following three aspects: 1) The residual hPSCs in induced differentiation cell products derived from hPSCs can form teratomas in vivo; 2) Gene mutations accumulated during the culture and induced differentiation of hPSCs may cause cancer or lesions; 3) The induced differentiation cell products derived from hPSCs may contain precursor cells with strong proliferation ability, or the products themselves are precursor cells, such as neural stem cells, which may proliferate in large quantities after being transplanted into the body to form tumors. Therefore, it is necessary to establish a rapid and highly sensitive hPSC residual detection method to ensure that the residual amount of hPSCs in functional cell products is within a safe range and improve the safety of hiPSC induced differentiation products.

[0004] At present, there are several methods for detecting hPSC residues: 1) In vivo detection of hPSC residues, that is, the cells to be tested are transplanted into immunodeficient mice to observe the tumor formation of the mice, but the cycle of this method requires at least 6 months of long-term observation. 2) Flow cytometry, that is, using flow cytometry antibodies of hPSC-specific markers (egSSEA4 and TRA-1-60) to detect the cells to be tested, which can detect the residues of hPSC. However, the sensitivity of this method is low at 0.1%, and this method is sensitive to the amount of cells and the amount / activity of antibodies used, and standardization is difficult. 3) Culture method, that is, using hPSC culture conditions, culturing the cells to be tested for 10-14 days, and the undifferentiated hPSCs are cultured to form clones visible to the naked eye, and alkaline phosphatase staining is applied. Before culture, the residual hPSCs in the cells to be tested can also be screened by magnetic beads or enriched by flow cytometry. This method can effectively detect hPSC residues, but this method requires 10 to 14 days of detection time, and there is a possibility of false negatives caused by differentiated cells inducing the differentiation of residual hPSCs. 4) PCR method, including RT-qPCR and digital PCR (ddPCR), is a method with short time, high sensitivity, relatively simple operation and the potential to achieve quantitative analysis compared to flow cytometry and culture methods. The purpose of hPSC residual detection is achieved by detecting the expression of hPSC-specific genes. Before PCR detection, the hPSCs remaining in the cells to be tested can also be screened by magnetic beads or enriched by flow cytometry to improve the detection sensitivity.

[0005] Genes used for detecting hPSC residues in induced functional cells need to meet the following two conditions: ① The gene is specifically expressed in large quantities in hPSCs; ② The gene is not expressed or expressed in very small amounts in induced differentiated cells or adult human functional cells. The current methods use different marker genes for different cells, and the sensitivity varies greatly. Commonly used hPSC-specific genes include SOX2, POU5F1, ESRG, LIN28A, NANOG, and TDGF1, etc. These genes are not necessarily universally applicable to different types of hPSC-derived induced differentiated cell products. For example, Sox2 is a common hPSC-specific gene, but it is also expressed in neural stem cells, so Sox2 is not suitable for detecting residual hPSCs in induced neural stem cells or neural precursor cells; in addition, existing technologies can detect LIN28A by RT-qPCR to detect residual hPSCs in retinal pigment epithelial cells (RPE) induced by hPSC differentiation. This method has been applied to patients, but studies have shown that LIN28A is expressed in induced liver cells, endothelial cells, and pancreatic islets. Therefore, LIN28A is not suitable for detecting residual hPSCs in these functional cells. At present, patent publication number CN110573607A discloses a method for detecting residual undifferentiated PSCs in a cell culture of differentiated pluripotent stem cells (PSCs), the method comprising culturing cells; identifying the pluripotency of the cultured cells, wherein the identification method mainly utilizes the qRT-PCR method to detect LIN28 (Lin28A), OCT4 (POU5F1), SOX2, FOXD3, NANOG, PODXL, REX1 (ZFP42), SSEA1 (FUT4), DPPA2 and DPPA3 genes, and compares the pluripotency markers in the reference cells (MSCs) and the cultured cells to determine whether there are no residual undifferentiated PSCs in the cultured cells or that the proportion of the residual undifferentiated PSCs in the cultured cells is lower than the known proportion of PSCs in MSCs. It can be seen that this method involves a variety of pluripotency genes, and some genes are significantly expressed in some special somatic cells. For example, LIN28A can be detected in liver and endothelial cells induced by hiPSC differentiation, so it cannot be used to detect hiPSC residues in such induced functional cells; SOX2 is expressed in neural cells, so it is not suitable for detecting hPSC residues in hPSC-induced neural cells. Patent publication number CN114150074A discloses a method for using TDGF1 in the detection of pluripotent stem cell residues in functional cell products derived from human pluripotent stem cells. However, TDGF1 has a low sensitivity in certain induced differentiated cell types, so it is not suitable as a universal marker gene for detecting hiPSC residues.

[0006] In view of this, this application is filed. SUMMARY OF THE INVENTION

[0008] In order to solve the above technical problems, the present application has screened and studied universal marker genes applicable to all types of hPSC-derived induced differentiation cell products, which are applicable to the detection of hPSC residues in each germ layer cell (endoderm, mesoderm, ectoderm) derived from hPSC. In addition, this group of universal marker genes can also be used to quickly verify the most sensitive marker genes in previously unknown induced differentiation cell products.

[0009] Therefore, this application includes at least the following objectives:

[0010] The first purpose of this application is to seek a universal product for residual detection of pluripotent stem cell differentiation in all differentiation types;

[0011] The second purpose of this application is to seek a method for detecting residual differentiation of pluripotent stem cells in all differentiation types;

[0012] The third objective of the present application is to seek a method for detecting the residual differentiation of pluripotent stem cells during a specific cell differentiation process.

[0013] To achieve the above objectives, this application proposes the following specific technical solutions:

[0014] The present application first provides a product for detecting residual differentiation of pluripotent stem cells in all differentiation types, comprising a detection agent or component for obtaining the level of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample.

[0015] The all differentiation types include three differentiation types into endoderm, mesoderm and / or ectoderm.

[0016] In some aspects, the differentiated cells include but are not limited to: neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, islet precursor cells, islet cells, alveolar epithelial cells, tubular glomerular cells, myocardial precursor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0017] In some aspects, the product includes, but is not limited to, a kit form or a system device form; preferably, the kit form.

[0018] In some aspects, any of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as an independent indicator for universal pluripotent stem cell differentiation residue detection. According to the experimental data of this application, any one of the genes in this application can be used as a universal differentiation residue detection Marke gene, so the purpose of this application can be achieved by detecting any one of them.

[0019] In some aspects, any two, three, four, five, six, or seven of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can be used as a joint indication for the detection of universal pluripotent stem cell differentiation residues. In other aspects, all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can be used as a joint indication for the detection of universal pluripotent stem cell differentiation residues. It can be understood that under objective conditions, as the number of detection marker genes increases, the reliability of the corresponding detection results will be higher.

[0020] In some aspects, the level comprises a nucleic acid level or a protein level; the nucleic acid level or protein level includes but is not limited to the abundance or concentration of a nucleic acid or a protein.

[0021] Furthermore, the nucleic acid level includes a DNA level or an RNA level.

[0022] Further preferably, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0023] More preferably, the method for obtaining nucleic acid levels includes but is not limited to any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0024] Further preferably, the protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0025] More preferably, the method for obtaining protein levels includes but is not limited to any of the following methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling or liquid chromatography.

[0026] In some aspects, in addition to the aforementioned universal marker genes, the product may also include detection agents or components for acquiring levels of other markers known in the prior art to achieve more powerful detection and evaluation.

[0027] In some aspects, the product may also include sample processing reagents, and the sample processing reagents may include nucleic acid extraction reagents and the like.

[0028] In some aspects, the detection agent or component further includes a detection agent or component for obtaining the level of an internal reference gene in a sample;

[0029] Preferably, the reference genes include any one, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2;

[0030] Furthermore, the plurality may be two, three, four, five, six, seven, eight, nine or ten, which can be selected according to specific circumstances.

[0031] In some aspects, the above-mentioned human pluripotent stem cells are mammalian pluripotent stem cells; preferably, they are human pluripotent stem cells.

[0032] In practice, more application scenarios are for residue detection of specific differentiated cells. Therefore, in some specific embodiments of the present application, residue detection products for specific cells are also included, such as:

[0033] In some aspects, 1) when detecting induced differentiated cardiomyocytes, in the detection product, the universal marker genes particularly include MIR302CHG and ESRG, and the reference genes include CDKN2AIP and RWDD4;

[0034] In some aspects, 2) when detecting induced differentiated cells, mesenchymal stem cells, in the detection product, the universal marker gene particularly includes TDGF1, and the internal reference genes include MAP3K7 and PDE12;

[0035] In some aspects, 3) when detecting induced differentiated cells and natural killer cells, in the detection product, the universal marker gene particularly includes ESRG, and the internal reference gene includes TTF1 and ABRAXAS2;

[0036] In some aspects, 4) when detecting induced differentiated dopaminergic neural precursor cells, in the detection product, the universal marker gene particularly includes MIR302CHG, and the internal reference genes include C8orf76 and SMG8.

[0037] The present application also provides the use of a detection agent or component for obtaining the level of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample in the detection of differentiation residues of all differentiation types of pluripotent stem cells; or its use in the preparation of a universal pluripotent stem cell differentiation residue detection kit.

[0038] The present application also provides the use of any, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 as a single or combined marker in the differentiation residues of universal pluripotent stem cells.

[0039] It can be understood that in the aforementioned applications, the detection scenarios of this application are mostly in vitro detection, which is used to evaluate the quality of induced stem cell products. Therefore, this application does not belong to the diagnosis application of the disease.

[0040] The all differentiation types include three differentiation types into endoderm, mesoderm and / or ectoderm.

[0041] In some aspects, the differentiated cells include but are not limited to: neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, islet precursor cells, islet cells, alveolar epithelial cells, tubular glomerular cells, myocardial precursor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0042] In some aspects, the detection can be performed at the nucleic acid level (including DNA level or RNA level) or protein level. Further, the nucleic acid (including DNA or RNA) level or protein level includes but is not limited to the abundance or concentration of nucleic acid (DNA or RNA) or protein. Further preferably, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology; further preferably, the nucleic acid level acquisition method includes but is not limited to any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography. Further preferably, the protein level is obtained by sequencing technology, immunotechnology, electrophoresis, biological mass spectrometry or chromatography technology; further preferably, the protein level acquisition method includes but is not limited to any of the following methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0043] In some aspects, the detection may also include detection of internal reference genes. Further, the internal reference genes include any, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2; wherein the multiple may be two, three, four, five, six, seven, eight, nine or ten, which can be selected according to the specific situation.

[0044] In practice, more application scenarios are for residue detection of specific differentiated cells. Therefore, in some specific embodiments of the present application, residue detection products for specific cells are also included, such as:

[0045] In some aspects, 1) when detecting induced differentiated cardiomyocytes, in the detection product, the universal marker genes particularly include MIR302CHG and ESRG, and the reference genes include CDKN2AIP and RWDD4;

[0046] In some aspects, 2) when detecting induced differentiated cells, mesenchymal stem cells, in the detection product, the universal marker gene particularly includes TDGF1, and the internal reference genes include MAP3K7 and PDE12;

[0047] In some aspects, 3) when detecting induced differentiated cells and natural killer cells, in the detection product, the universal marker gene particularly includes ESRG, and the internal reference gene includes TTF1 and ABRAXAS2;

[0048] In some aspects, 4) when detecting induced differentiated dopaminergic neural precursor cells, in the detection product, the universal marker gene particularly includes MIR302CHG, and the internal reference genes include C8orf76 and SMG8.

[0049] In some aspects, any of the above-mentioned pluripotent stem cells are mammalian pluripotent stem cells; preferably, they are human pluripotent stem cells.

[0050] The present application also provides a method for detecting universal pluripotent stem cell differentiation residues, which method comprises the steps of obtaining the levels of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample; any detection based on this step is within the scope of the present application.

[0051] Furthermore, the method may specifically include the following two steps:

[0052] (i) obtaining the level of the universal marker gene in the sample to be tested;

[0053] (ii) comparing the level of universal marker genes with that in a control sample; wherein a significant difference in the level of the universal marker genes between the test sample and the control sample indicates that there are residual pluripotent stem cell differentiation in the test sample; or,

[0054] (ii) comparing with a set threshold absolute amount; wherein, the difference in the level of the sample to be tested being higher than the threshold absolute amount indicates that there are residual pluripotent stem cell differentiation in the sample to be tested.

[0055] Furthermore, the induced differentiated cells may be from various germ layers, including but not limited to: neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, islet precursor cells, islet cells, alveolar epithelial cells, renal tubular and glomerular cells, myocardial precursor cells, myocardial cells, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0056] In some aspects, any of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as an independent indication for the detection of universal pluripotent stem cell differentiation residues. According to the experimental data of the present application, any one gene of the present application can be used as a universal differentiation residue detection Marke gene, so the purpose of the present application can be achieved by detecting any one. In some aspects, any two, three, four, five, six, or seven of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as a joint indication for the detection of universal pluripotent stem cell differentiation residues. In some aspects, all of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as a joint indication for the detection of universal pluripotent stem cell differentiation residues. It can be understood that, under objective conditions, as the number of detected marker genes increases, the reliability of the corresponding test results will be higher.

[0057] In some aspects, the detection process further includes detection of the level of internal reference genes; preferably, the internal reference genes include any one, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2; further, the multiple can be two, three, four, five, six, seven, eight, nine or ten, which can be selected according to the specific circumstances.

[0058] The present application also provides a universal marker gene for residual detection of pluripotent stem cell differentiation, wherein the universal marker gene includes any one, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1.

[0059] The present application also provides a method for detecting the residual pluripotent differentiation of induced differentiated cells whose marker genes have not yet been clearly identified, and such method comprises: based on universal marker genes, through downstream verification, screening the most sensitive or better universal marker genes as the residual pluripotent differentiation detection genes of the induced differentiated cells; the universal marker genes are any one, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1.

[0060] Beneficial technical effects of this application:

[0061] 1) Most of the existing marker genes are not suitable for all types of hPSC-derived induced differentiated cell products, and are only suitable for the detection of hPSC residues in single germ layer cells (endoderm, mesoderm, ectoderm) derived from hPSC. This application expands the sample size of transcriptome sequencing, including hiPSC, ESC and 17 types of induced differentiated cells, including neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, pancreatic islet precursor cells, pancreatic islet cells, alveolar epithelial cells, renal tubular glomerular cells, myocardial precursor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, leukocytes, leukocytes, mitochondria, leukocytes, cytokines ... granulocytes, eosinophils, megakaryocytes, etc.; through qPCR detection and statistical verification, a group of universal candidate marker genes compatible with endoderm, mesoderm and ectoderm induced differentiation cell products were determined: MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN and POU5F1, which solved the problem of insufficient universality of the current marker genes; the detection sensitivity of the universal marker genes screened in this application is 0.05-0.001%.

[0062] 2) This application can provide reference candidate marker genes for hiPSC induced differentiation cell products for which marker genes have not yet been determined, and through downstream verification, select the most sensitive marker gene as the gene of this type, shorten the time and reduce the difficulty of gene screening, improve the fault tolerance of the method, and meet both versatility and sensitivity. The method of this application is efficient, reliable, versatile, and has a wide range of applications. It can be applied to the quality monitoring of many hPSC induced differentiation cell products and provide reliable cell products for clinical use.

[0063] 3) Currently, this field lacks internal reference genes for the entire differentiation stage. This application has identified several groups of specific highly sensitive internal reference genes and their combinations through screening, and accordingly established several highly sensitive differentiation residue detection methods for specific differentiated cells. This method is based on the aforementioned marker gene combination screening to identify several groups of specific highly sensitive internal reference genes and their combinations, achieving high-sensitivity (up to 0.0001%) residue detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0065] Figure 1 Stability of candidate reference genes for iDAp differentiation direction;

[0067] Figure 2 Stability of candidate reference genes for iCM differentiation direction;

[0068] Figure 3 Stability of candidate reference genes for iMSC differentiation direction;

[0069] Figure 4 Stability of candidate reference genes for iNK differentiation direction;

[0070] Figure 5 Expression levels of candidate marker genes in transcriptome sequencing;

[0071] Figure 6 Bar graph of the expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSCs and iCMs (normalized at the CDKN2AIP level);

[0072] Figure 7 Expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSCs and iMSCs (normalized to CSNK1G3 levels);

[0073] Figure 8 Expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSCs and iNKs (normalized at DCPS levels);

[0074] Fig. 9 Expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSCs and iDAps (normalized at C8orf76 levels);

[0075] Fig.10 Detection of the sensitivity of candidate universal marker genes in iDAp;

[0076] Fig.11 The sensitivity of detecting candidate universal marker genes in iCM;

[0077] Fig.12Detect the sensitivity of candidate universal marker genes in iMSCs;

[0078] Fig.13 Detect the sensitivity of candidate universal marker genes in iNK. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present application discloses an application of gene-based residual detection of pluripotent stem cell differentiation. Those skilled in the art can refer to the content of this article to realize its application. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation methods and applications herein without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by ordinary technicians in the field to which this application belongs.

[0081] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0082] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0083] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0084] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10%, preferably ±5%, of the indicated value.

[0085] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0086] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. It will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment. Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0087] Detailed description of the invention of this application

[0088] 1. Universal marker genes

[0089] The present application provides universal marker genes for residual detection of pluripotent stem cell differentiation. The present application expands the sample size of transcriptome sequencing, including hiPSC, ESC and 17 types of induced differentiation cells, neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, pancreatic islet precursor cells, pancreatic islet cells, alveolar epithelial cells, renal tubular and glomerular cells, myocardial precursor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc. Through qPCR testing and statistical verification, we finally determined the candidate marker genes that are suitable for induced differentiation cell products in the endoderm, mesoderm, and ectoderm directions. These universal genes include MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1, thus solving the problem of insufficient universality of current marker genes.

[0090] It is understood that any single gene can be used for the detection of pluripotent stem cell differentiation residues, so that as a single marker, when any gene (or expression) is detected in a differentiated cell system, it can be used to evaluate the presence of undifferentiated pluripotent stem cell residues in differentiated cells. In some specific embodiments, the single gene is MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1; in some preferred embodiments, the single gene is LNCPRESS1.

[0091] Of course, these genes can also be used in combination for pluripotent stem cell differentiation residue detection. It can be understood that when multiple genes, such as two, three, four, five, six, seven or eight genes, are detected together, the detection sensitivity and accuracy can be improved.

[0092] Therefore, the universal marker gene of the present application can be a single gene or a combination of multiple genes.

[0093] 2. Products

[0094] The present application relates to products for detecting residual differentiation of pluripotent stem cells of all differentiation types, which contain detection agents or components for obtaining the levels of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample.

[0095] The “all differentiation types” mentioned herein include three differentiation types, namely, differentiation into endoderm, mesoderm and / or ectoderm.

[0096] In some aspects, the differentiated cells include but are not limited to: neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, islet precursor cells, islet cells, alveolar epithelial cells, tubular glomerular cells, myocardial precursor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0097] In some embodiments, any of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as an independent indicator for universal pluripotent stem cell differentiation residue detection. According to the experimental data of this application, any one of the genes in this application can be used as a universal differentiation residue detection Maker gene, so the purpose of this application can be achieved by detecting any one of them.

[0098] In other embodiments, any two, three, four, five, six, or seven of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can be used as a combined indicator for universal pluripotent stem cell differentiation residual detection.

[0099] In other embodiments, all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 are used as a joint indicator for the detection of universal pluripotent stem cell differentiation residues. It can be understood that under objective conditions, as the number of universal marker genes detected increases, the reliability of the corresponding test results will be higher.

[0100] The term "detection agent" in this article generally refers to a detection reagent, such as the MIR302CH gene detection agent, which refers to a reagent that can detect the MIR302CH gene. The "level" in this article generally refers to the abundance or concentration of an indicator, for example, the nucleic acid level refers to the abundance or concentration of nucleic acids. Therefore, a gene level detection agent refers to a detection reagent that can directly obtain the abundance or concentration of a gene in a sample. In addition, the "component" in this article is different from the direct acquisition method of the detection agent, which generally refers to the component that obtains the gene level in the sample by indirect means (for example, obtaining indicators of specific genes in samples that have been detected through computer programs).

[0101] In some embodiments, the nucleic acid levels described herein include DNA levels or RNA levels.

[0102] When the detection agent is detected at the nucleic acid level, it can be understood that there are many ways of nucleic acid detection in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0103] Therefore, for example, in some embodiments, the detection agent can be a sequencing reagent, such as a second-generation sequencing ("NGS") reagent, a third-generation sequencing reagent, etc. For example, the detection agent is a sequencing reagent, which detects the DNA or RNA of the aforementioned gene by sequencing, and then evaluates the corresponding gene level based on the sequencing result. In other embodiments, the detection agent is a PCR (especially ddPCR) primer reagent, wherein the primer reagent can bind to the target gene template sequence and achieve amplification, and the level of DNA or RNA of the corresponding gene is detected by amplification.

[0104] When the detection agent is used for detection at the protein level, it can be understood that there are many methods for protein detection in the art, including but not limited to sequencing technology, immunoassay, electrophoresis technology, biomass spectrometry technology or chromatography technology, all of which can be used in the present application; in some specific embodiments of the present application, including but not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0105] Therefore, for example, in some specific embodiments, the detection agent can be an immune reagent, such as an antibody reagent, which can detect protein levels through the immune reaction between the antibody and the gene expression protein.

[0106] The form of the product herein includes, but is not limited to, a kit form and a system device form.

[0107] In some embodiments, the product is in the form of a kit. It is understood that such a kit may further include instructions for predicting results based on the detected levels in addition to the aforementioned detection agents or detection components.

[0108] In some embodiments, the kits described herein include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each of which contains one of the individual elements to be used in the method. The kits of the present application may include a container as described above and one or more other containers containing materials required from a commercial end-user standpoint, including buffers, diluents, filters, and package inserts with instructions for use.

[0109] In some embodiments, the kit may further include a sample processing reagent, and the sample processing reagent may include at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0110] In some embodiments, the detection agent or component further includes a detection agent or component for obtaining the level of an internal reference gene in a sample;

[0111] For example, the specific reference genes for screening described in the specification of this application include any, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2;

[0112] In some preferred embodiments, the plurality may be two, three, four, five, six, seven, eight, nine or ten, which may be selected according to specific circumstances.

[0113] In some embodiments, the product further comprises a detection agent or component for acquiring the level of other markers known in the prior art to achieve more powerful detection and assessment.

[0114] In addition, considering that more application scenarios in the practice of this application are for residual detection of specific differentiated cells, this application also designs another "non-universal" kit, for example, this kit is a kit for residual detection of specific differentiated cells, such as described in some specific embodiments of this application:

[0115] 1) When detecting induced differentiated cardiomyocytes, the kit contains detection agents for universal marker genes MIR302CHG and ESRG, and detection agents for internal reference genes CDKN2AIP and RWDD4;

[0116] 2) When detecting induced differentiated cells, mesenchymal stem cells, the kit contains detection agents for the universal marker gene TDGF1, as well as for the internal reference genes MAP3K7 and PDE12;

[0117] 3) When detecting induced differentiated cell natural killer cells, the kit contains detection agents for the universal marker gene ESRG, as well as for the internal reference genes TTF1 and ABRAXAS2;

[0118] 4) When detecting induced differentiated dopaminergic neural precursor cells, the kit contains detection agents for the universal marker gene MIR302CHG, as well as for the internal reference genes C8orf76 and SMG8.

[0119] In some specific embodiments, the aforementioned four kits are all ddPCR kits, which overcome the current defects: the current ddPCR method is mainly based on absolute quantification, and does not cite internal reference genes for gene expression level correction, resulting in insufficient repeatability of the results.

[0120] In addition to the above components, the kit will further include instructions for implementing the subject method. These instructions can be present in the subject kit in a variety of forms, one or more of which can be present in the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, the paper being in the packaging of the kit in the form of a package insert, etc.

[0121] 3. Application

[0122] The application of this application includes at least:

[0123] 1) Use of a detection agent or component for obtaining the level of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample in a universal pluripotent stem cell differentiation residual detection;

[0124] 2) Use of a detection agent for obtaining the level of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample in the preparation of a universal pluripotent stem cell differentiation residual detection kit.

[0125] 3) Use of any, multiple or all of the genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 as universal markers for residual differentiation detection of pluripotent stem cells.

[0126] It can be understood that the detection scenarios of this application are mostly in vitro detection, which is used to evaluate the quality of induced stem cell products, and therefore usually does not involve disease detection applications.

[0127] As described above, in some embodiments, the detection can be performed at the nucleic acid level (including DNA level or RNA level) or protein level. Further, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein.

[0128] In some preferred embodiments, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology; further preferably, the nucleic acid level acquisition method includes but is not limited to any one of the following methods: gene sequencing method, polymerase chain reaction method, isothermal amplification reaction method, gene chip method, probe hybridization method, gel electrophoresis method, Northern blotting method, nucleic acid mass spectrometry or liquid chromatography.

[0129] In some preferred embodiments, the protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology. Further preferably, the protein level acquisition method includes but is not limited to any of the following methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0130] In some embodiments, the detection may also include detection of an internal reference gene;

[0131] Furthermore, the internal reference genes include any one, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2; wherein the multiple can be two, three, four, five, six, seven, eight, nine or ten, which can be selected according to the specific situation.

[0132] 4. Detection method

[0133] The core steps of the universal pluripotent stem cell differentiation residue detection method of the present application are: obtaining the levels of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in the sample; it can be understood that any detection method based on or including this step is within the scope of the present application.

[0134] In some embodiments, the method may specifically include the following two steps:

[0135] (i) obtaining the level of the universal marker gene in the sample to be tested;

[0136] (ii) comparing the level of universal marker genes with that in a control sample; wherein a significant difference in the level of the universal marker genes between the test sample and the control sample indicates that there are residual pluripotent stem cell differentiation in the test sample; or,

[0137] (ii) comparing with a set threshold absolute amount; wherein, the difference in the level of the sample to be tested being higher than the threshold absolute amount indicates that there are residual pluripotent stem cell differentiation in the sample to be tested.

[0138] In some specific embodiments, a set value for the level of any one or more of the aforementioned genes can be given, and the set value can be determined based on the levels of the genes in the test sample and the control sample, for example, the average value of the gene levels of the control sample of the appropriate sample number is selected, or a reasonable multiple is set based on the average value, such as 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, etc. When the gene level of the test sample is higher than the set value, it is judged that there are residual pluripotent stem cells. It is understandable that the set value determined based on the average value, or the multiple of the average value needs to have good classification significance, and the known samples can be tested by using a commonly used statistical test method through the classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment standard.

[0139] In view of the fact that the aforementioned marker genes are universal marker genes, the method of the present application can be applied to the detection in various germ layer induced differentiated cells, including but not limited to: neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, islet precursor cells, islet cells, alveolar epithelial cells, renal tubular and glomerular cells, myocardial precursor cells, myocardial cells, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0140] As mentioned above, during the detection process, any of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as an independent indication for the detection of universal pluripotent stem cell differentiation residues. According to the experimental data of this application, any one of the genes in this application can be used as a universal differentiation residue detection Marker gene, so the purpose of this application can be achieved by detecting any one of them. In some embodiments, any two, three, four, five, six, or seven of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as a combined indication for the detection of universal pluripotent stem cell differentiation residues. In other embodiments, all of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 can be used as a combined indication for the detection of universal pluripotent stem cell differentiation residues. It can be understood that, under objective conditions, as the number of detected marker genes increases, the reliability of the corresponding test results will be higher.

[0141] As mentioned above, the detection level of the present application includes nucleic acid level or protein level, etc.; the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein, and the specific means are the same as above.

[0142] In some embodiments, the detection process always further includes detection of the level of internal reference genes; in some preferred embodiments, the internal reference genes include any one, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2; further, the multiple can be two, three, four, five, six, seven, eight, nine or ten, which can be selected according to the specific circumstances.

[0143] The pluripotent stem cells mentioned herein include mammalian pluripotent stem cells; preferably human pluripotent stem cells.

[0144] In practice, more application scenarios are for specific differentiated cells for residue detection. Therefore, the present application also relates to some detection methods for specific differentiated cells, such as some specific implementation methods of the present application:

[0145] In some embodiments, 1) when the induced differentiated cells are detected as cardiomyocytes, the genes targeted in the detection method are: marker genes particularly include MIR302CHG and ESRG, and internal reference genes include CDKN2AIP and RWDD4;

[0146] In some embodiments, 2) when the induced differentiated cells are detected as mesenchymal stem cells, the genes targeted in the detection method are: marker genes particularly include TDGF1, and internal reference genes include MAP3K7 and PDE12;

[0147] In some embodiments, 3) when the induced differentiated cells are detected as natural killer cells, the genes targeted in the detection method are: marker genes particularly include ESRG, and internal reference genes include TTF1 and ABRAXAS2;

[0148] In some embodiments, 4) when the induced differentiated cells are detected as dopaminergic neural precursor cells, the genes targeted in the detection method are: marker genes particularly include MIR302CHG, and internal reference genes include C8orf76 and SMG8.

[0149] Therefore, based on these specific embodiments, the present application also provides a ddPCR method for detecting hiPSC residues in hPSC-induced differentiated cardiomyocyte (iCM) products. The marker genes are MIR302CHG and ESRG, the internal reference genes are CDKN2AIP and RWDD4, and the sensitivity can reach 0.005%, that is, at 1×106 50 PSCs were detected in iCM cells. The method includes the following steps: detecting the expression levels of MIR302CHG gene and ESRG gene in the sample to be tested, wherein the expression levels of MIR302CHG gene and ESRG gene are normalized by the geometric square root of the expression levels of CDKN2AIP gene and RWDD4 gene, and the relative expression levels of MIR302CHG gene and ESRG gene after normalization indicate the amount of residual pluripotent stem cells in the sample to be tested. The present application also provides a ddPCR method for detecting residual hPSC in hPSC-induced differentiated mesenchymal stem cell (iMSC) products. The marker gene is TDGF1, the internal reference genes are MAP3K7 and PDE12, and the sensitivity can reach 0.005%, that is, at 1×10 6 50 PSCs were detected in MSC. The method comprises the following steps: detecting the expression level of TDGF1 gene in the sample to be tested, wherein the expression level of TDGF1 gene is normalized by the geometric square root of the expression levels of MAP3K7 gene and PDE12 gene, and the relative expression level of TDGF1 gene after normalization indicates the residual amount of pluripotent stem cells in the sample to be tested. The present application also provides a ddPCR method for detecting the residual hPSC in the natural killer cell (iNK) product induced by hPSC differentiation. The marker gene is ESRG, the internal reference genes are TTF1 and ABRAXAS2, and the sensitivity can reach 0.0001%, that is, at 1×10 6 1 PSC was detected in the iNK of the test sample. The method comprises the following steps: detecting the expression level of the ESRG gene in the test sample, wherein the expression level of the ESRG gene is normalized by the geometric square root of the expression levels of the TTF1 gene and the ABRAXAS2 gene, and the relative expression level of the ESRG gene after normalization indicates the residual amount of pluripotent stem cells in the test sample. The present application also provides a ddPCR method for detecting the residual hPSC in the dopaminergic neural precursor cell (imDAP) product induced by hPSC differentiation. The marker gene is MIR302CHG, the internal reference genes are C8orf76 and SMG8, and the sensitivity can reach 0.001%, that is, at 1×10 6 10 PSCs were detected by each iDAp. The method comprises the following steps: detecting the expression level of the MIR302CHG gene in the sample to be tested, wherein the expression level of the MIR302CHG gene is normalized by the geometric square root of the expression levels of the TTF1 gene and the ABRAXAS2 gene, and the relative expression level of the MIR302CHG gene after normalization indicates the residual amount of pluripotent stem cells in the sample to be tested. In the aforementioned method, the present application proposes a dual internal reference gene, which has better stability than a single internal reference gene, thereby improving the stability and repeatability of the method.

[0150] The method of the present application for detecting the residual pluripotent differentiation of induced differentiated cells whose marker genes have not yet been clearly identified, this type of method basically includes: based on universal marker genes, through downstream verification, screening the most sensitive or better universal marker genes as the pluripotent differentiation residual detection genes of the induced differentiated cells; in the present application, the universal marker genes are MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN and POU5F1.

[0151] In some specific embodiments, this method is a group of candidate marker genes that can be used to detect hiPSC residual in hiPSC induced differentiation cell products by ddPCR. Through this group of marker genes, the most sensitive marker genes in previously unclear induced differentiation cell products can be quickly verified, accelerating research progress and improving product safety. The following steps are included: the expression levels of 8 candidate marker genes in the sample to be tested are detected, and the gene with the highest sensitivity is determined by ddPCR as the most suitable marker gene. The level of the expression of the most suitable marker gene indicates the level of hiPSC residual in this induced differentiation cell type. In this application, there is no special restriction on the sequence of the gene in the candidate marker gene, and the sequence derived from MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, POU5F1 genes can be used. It can be seen that in this application, by screening the candidate marker genes shared by the three germ layers, some genes that may be expressed in trace amounts in other cell types or expressed differently in some PSCs are excluded. Then, the expression levels of the candidate genes in the induced differentiated cell products derived from hPSC were detected. The results showed that the above-mentioned universal candidate marker genes were not expressed or expressed at very low levels in the induced differentiated cell products derived from hPSC. After sensitivity testing, the results showed that the most suitable marker genes in different induced differentiated cells can be found in the universal candidate marker gene set. Therefore, through the sensitivity testing of the above-mentioned 8 candidate marker genes in different induced differentiated cell products derived from hPSC, the most suitable marker genes for hPSC residual detection in different products can be screened.

[0152] The present application is fully described below in conjunction with specific implementation examples. Example

[0153] Sources of reagents and raw materials:

[0154] The RNA reverse transcription kit was purchased from Takara, and the ddPCR kit was purchased from Bio-Rad.

[0155] Human induced pluripotent stem cells came from Oriental Hospital, Anhui Zhongsheng Suyuan Biotechnology Co., Ltd., Zhejiang Hold Bioengineering Co., Ltd. and Nanjing Elp Regenerative Medicine Technology Co., Ltd.

[0156] Mesenchymal stem cells and natural killer cells obtained by hPSC-induced differentiation were from Anhui Zhongsheng Suyuan Biotechnology Co., Ltd., cardiomyocytes were from Nanjing Aierpu Regenerative Medicine Technology Co., Ltd., and dopamine precursor cells were from Zhejiang Huode Bioengineering Co., Ltd.

[0157] The primers involved in the examples were synthesized by Kunshan Pronopro Biotechnology Co., Ltd., and the specific primer probe sequences are as follows:

[0158]

[0159]

[0160] Example 1. Screening of reference genes suitable for detecting hPSC residuals in hPSC-derived dopamine precursor cells (iDAp), mesenchymal stem cells (iMSC), cardiomyocytes (iCM), and natural killer cells (iNK)

[0161] To ensure the stability of the method, this application uses transcriptome sequencing data of hiPSC and its induced differentiated cells in public databases and laboratory databases. The screening database includes screening out candidate internal reference genes that are stably expressed during the differentiation process, and according to the specific performance of the internal reference genes in the database, the screening criteria are set to genes with a cv value of less than 0.3 in the top ten TPM values. Because this application is a residual detection, the TPM value is set to less than 50 in consideration of subsequent experimental operations. Through the aforementioned screening process, this application obtains candidate internal reference genes that meet the requirements, as shown in Tables 1.1-1.4.

[0162] Table 1.1 Applicable reference genes for ectoderm that meet the screening criteria

[0163]

[0164]

[0165] Table 1.2 iCM applicable reference genes that meet the screening criteria

[0166] Gene TPM cv CDKN2AIP 12.93 0.20 FIP1L1 47.71 0.23 NKAPD1 20.01 0.23 GTF2B 19.77 0.24 DALRD3 32.22 0.24 RWDD4 15.15 0.25 RBM22 34.53 0.25

[0167] Table 1.3 Applicable reference genes for iMSCs that meet the screening criteria

[0168] Gene TPM cv CSNK1G3 33.34 0.24 PDE12 7.88 0.25 SMG8 19.60 0.27 NCK1 21.14 0.28 ABCF2 39.58 0.28 MAP3K7 45.01 0.28 DR1 21.33 0.28

[0169] Table 1.4 iNK applicable reference genes that meet the screening criteria

[0170] Gene TPM cv NDUFV3 14.71 0.17 DCPS 12.38 0.17 STAMBP 21.05 0.18 TTF1 20.51 0.18 MRPS17 27.66 0.18 ABRAXAS2 18.16 0.18

[0171] Subsequently, the present application used RT-qPCR detection to detect the CT values ​​of candidate reference genes in iPSC and iDAp / iCM / iMSC / iNK, respectively. The reaction system is shown in Table 1.5, and the reaction procedure is shown in Table 1.6. The reference gene screening software NormFinder was then used to further screen out the stably expressed reference genes, where the lower the S value, the more stable the surface gene expression.

[0172] Table 1.5 Reaction system

[0173]

[0174]

[0175] Table 1.6 Reaction procedure

[0176]

[0177] Specific results such as Figure 1-4 As shown in the figure, the NormFinder results show that the most suitable single internal reference for iDAp differentiation is C8orf76, and the double internal references are C8orf76 and SMG8; the most suitable single internal reference for iCM is CDKN2AIP, and the double internal references are CDKN2AIP and RWDD4; the most suitable single internal reference for iMSC is CSNK1G3, and the double internal references are MAP3K7 and PDE12; the most suitable single internal reference for iNK is DCPS, and the double internal references are TTF1 and ABRAXAS2. Among them, the stability of the double internal reference combination is better than that of the single internal reference gene.

[0178] Example 2: Screening of universal marker genes

[0179] In order to solve the problem of insufficient universality of marker genes, the applicant used the public group database and the transcriptome sequencing data of hiPSC and its derivatives collected by this laboratory (data from healthy individuals), including hiPSC and its derivatives, including endoderm-derived hepatocytes, definitive endoderm cells, endocrine progenitor cells, pancreatic islet cells, mesoderm-derived myocardial progenitor cells, cardiomyocytes, mesenchymal stem cells and natural killer cells, and ectoderm-derived neural stem cells, neural progenitor cells, dopaminergic neurons, spinal motor neurons, astrocytes and retinal pigment epithelial cells. DESeq2 was used to screen out candidate marker genes common to all hiPSC-derived cell types.

[0180] The specific screening results are shown in Table 2.1. 15 marker genes that overlapped in the three germ layer cells were screened out: C13orf42, PRDM14, MIR302CHG, ESRG, FOXD3-AS1, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, POU5F1, XCAT, ZSCAN10, LINC00458, L1TD1. Among them, XCAT, ZSCAN10, LINC00458 and L1TD1 were excluded because their expression differences in different iPSC lines were too large ( Figure 5 ).

[0181] Table 2.1 Candidate marker genes common to the three germ layers

[0182]

[0183]

[0184] Subsequently, the applicant verified the actual expression levels of candidate marker genes in dopamine precursor cells, mesenchymal stem cells, cardiomyocytes, and natural killer cells induced by hiPSC. Specifically, the applicant first used the Trizol method to extract total mRNA from iPSC, iCM, iMSC, iNK, and iDAp cell samples; according to the amount of 500ng RNA, the TakaraRNA reverse transcription kit (PrimeScript TM mRNA was reverse transcribed into cDNA using RT Master Mix, #RR036A, and then PCR was performed using the Takara qPCR kit (TB Premix Ex, #RR420L), the expression of C13orf42, PRDM14, MIR302CHG, ESRG, FOXD3-AS1, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, POU5F1 genes in iPSCs, iCMs, iMSCs, iNKs and iDAps was detected by RT-qPCR detection method, the reaction system was the same as Table 1.5 above, and the reaction procedure was the same as Table 1.6 above. The 96 software calculates the relative expression levels of candidate marker genes based on the internal reference genes screened above.

[0185] Specific test results such as Figure 6-9As shown in the figure, the results of RT-qPCR detection show that the candidate marker genes FOXD3-AS1, C13orf42, and PRDM14 are expressed at low levels and are not suitable as marker genes and are excluded. MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 are expressed in large quantities in hiPSCs from different sources, but are almost not expressed in induced differentiated iCMs, iMSCs, iNKs, and iDAps. Therefore, these genes have the potential to be used as universal marker genes for residual differentiation detection of pluripotent stem cells, and the sensitivity of these genes will be further evaluated in the future.

[0186] Example 3 Detection of sensitivity of candidate marker genes in iDAp

[0187] To simulate the detection of hPSC residues in cell production, this application uses iPSC as the positive control and iDAp as the negative control group. 1×10 6 Place positive cells in a 1.5 mL centrifuge tube, add 1 mL Trizol, and mix well by pipetting. Dilute the Trizol lysate containing positive cells with Trizol. Pipette 100 μL of positive control cell lysate containing Trizol and add it to 900 μL of negative control cell lysate. The final mixing ratio is shown in Table 3.1. Extract RNA and reverse transcribe it into cDNA.

[0188] Table 3.1 Cell sample preparation

[0189]

[0190] The sensitivity of the candidate marker genes was determined by ddPCR. The ddPCR reaction system is shown in Table 3.2, and the ddPCR reaction procedure is shown in Table 3.3. Each sample was tested four times.

[0191] Table 3.2ddPCR reaction system

[0192]

[0193]

[0194] Table 3. 3ddPCR reaction procedure

[0195]

[0196] The results were analyzed by relative quantification, as follows: the gene copy number results of all genes detected in an experiment were sorted out, the geometric mean of the copy number of C8orf76 gene and SMG8 gene in each sample was calculated, and the copy number of the candidate marker gene was divided by the geometric mean of the internal reference gene to obtain the relative expression of the candidate marker gene. The statistical difference between the relative expression of each cell mixture and iDAp was detected by t-test to determine the sensitivity of the marker gene. The ddPCR calculation results are shown in Table 3.4.

[0197] Table 3.4 shows the calculation results of hPSC residual detection in iDAp induced by hPSC differentiation, taking the MIR302CHG gene as an example.

[0198]

[0199]

[0200] Note: The quality control standard is that the number of positive droplets in the reverse transcription blank control group is less than 3.

[0201] The screening results of all candidate marker genes are shown in Fig.10 The test results showed that the gene with the highest sensitivity in iDAp was MIR302CHG, with a sensitivity of 0.001%, which was able to detect 6 Ten hiPSCs were differentiated from each iDAp.

[0202] Example 4: Detection of sensitivity of candidate marker genes in iCM

[0203] The method is the same as in Example 3. To simulate the detection of hPSC residues in cell production, the present application uses iPSC as a positive control and iCM as a negative control group. 1×10 6 Place positive cells in a 1.5 mL centrifuge tube, add 1 mL Trizol, and mix well by pipetting. Dilute the Trizol lysate containing positive cells with Trizol. Pipette 100 μL of positive control cell lysate containing Trizol and add it to 900 μL of negative control cell lysate. The final mixing ratio is shown in Table 3.1. Extract RNA and reverse transcribe it into cDNA.

[0204] The sensitivity of the candidate marker genes was determined by the ddPCR method. The reaction system is shown in Table 3.2 and the reaction system is shown in Table 3.3. Each sample was tested 4 times.

[0205] The results were analyzed by relative quantification, as follows: the gene copy number results of all genes detected in an experiment were sorted out, the geometric mean of the copy number of CDKN2AIP gene and RWDD4 gene in each sample was calculated, and the copy number of the candidate marker gene was divided by the geometric mean of the internal reference gene to obtain the relative expression of the candidate marker gene. The statistical difference between the relative expression of each cell mixture and iDAp was detected by t-test to determine the sensitivity of the marker gene.

[0206] The results of candidate marker gene screening are shown in Fig.11 The test results showed that the genes with the highest sensitivity in iCM were MIR302CHG and ESRG, both with a sensitivity of 0.005%, which could be detected in 1×10 6 Differentiate 50 hiPSCs in iCMs.

[0207] Example 5: Detection of sensitivity of candidate marker genes in iMSCs

[0208] The method is the same as that in Example 3. To simulate the detection of hPSC residues in cell production, the present invention uses iPSC as a positive control and iMSC as a negative control group. 1×10 6 Place positive cells in a 1.5 mL centrifuge tube, add 1 mL Trizol, and mix well by pipetting. Dilute the Trizol lysate containing positive cells with Trizol. Pipette 100 μL of positive control cell lysate containing Trizol and add it to 900 μL of negative control cell lysate. The final mixing ratio is shown in Table 3.1. Extract RNA and reverse transcribe it into cDNA.

[0209] The sensitivity of the candidate marker genes was determined by the ddPCR method. The reaction system is shown in Table 3.2 and the reaction system is shown in Table 3.3. Each sample was tested 4 times.

[0210] The results were analyzed by relative quantification, as follows: the gene copy number results of all genes detected in an experiment were sorted out, the geometric mean of the copy number of MAP3K7 gene and PDE12 gene in each sample was calculated, and the copy number of the candidate marker gene was divided by the geometric mean of the internal reference gene to obtain the relative expression of the candidate marker gene. The statistical difference between the relative expression of each cell mixture and iDAp was detected by t-test to determine the sensitivity of the marker gene.

[0211] The results of candidate marker gene screening are shown in Fig.12 The test results showed that the gene with the highest sensitivity in iMSCs was TDGF1, with a sensitivity of 0.005%, which could be detected in 1×10 6 Differentiate 50 hiPSCs from iMSCs.

[0212] Example 6: Detection of sensitivity of candidate marker genes in iNK

[0213] The method is the same as that in Example 3. To simulate the detection of hPSC residues in cell production, the present invention uses iPSC as a positive control and iNK as a negative control group. 1×10 6 Place 100 positive cells in a 1.5 mL centrifuge tube, add 1 mL Trizol, and mix well by pipetting. Dilute the Trizol lysate containing positive cells with Trizol.

[0214] μL of positive control cell lysate containing Trizol was added to 900 μL of negative control cell lysate. The final mixing ratio is shown in Table 3.1. RNA was extracted and reverse transcribed into cDNA.

[0215] The sensitivity of the candidate marker genes was determined by the ddPCR method. The reaction system is shown in Table 3.2 and the reaction system is shown in Table 3.3. Each sample was tested 4 times.

[0216] The results were analyzed by relative quantification, as follows: the gene copy number results of all genes detected in an experiment were sorted out, the geometric mean of the copy number of TTF1 gene and ABRAXAS2 gene in each sample was calculated, and the copy number of the candidate marker gene was divided by the geometric mean of the internal reference gene to obtain the relative expression of the candidate marker gene. The statistical difference between the relative expression of each cell mixture and iDAp was detected by t-test to determine the sensitivity of the marker gene.

[0217] The results of candidate marker gene screening are shown in Fig.13 The test results showed that the gene with the highest sensitivity in iCM was ESRG, with a sensitivity of 0.0001%, which could be detected in 1×10 6 Differentiate 1 hiPSC from iNK.

[0218] In addition, although the most sensitive marker genes were obtained in the above Examples 3-6, Figure 10-13 The results also show that each gene screened in this application (MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, POU5F1) can meet the time requirements in the detection sensitivity of various germ layer differentiated cells, and the minimum sensitivity can reach 0.5%, which can be used for residual detection in practice.

[0219] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a detection agent or component for obtaining the level of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample in the residual detection of pluripotent stem cell differentiation in all differentiation types.

2. A product for detecting residual differentiation of pluripotent stem cells in all differentiation types, characterized in that: Contains a detection agent or component for obtaining the level of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in a sample.

3. A method for detecting residual differentiation of pluripotent stem cells in all differentiation types, characterized in that: The steps include: obtaining the levels of any, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in the sample; Preferably, the method specifically comprises the following steps: (i) obtaining the level of the universal marker gene in the sample to be tested; (ii) comparing the level of universal marker genes with that in a control sample; wherein a significant difference in the level of the universal marker genes between the test sample and the control sample indicates that there are residual pluripotent stem cell differentiation in the test sample; or, (ii) comparing with a set threshold absolute amount; wherein, the difference in the level of the sample to be tested being higher than the threshold absolute amount indicates that there are residual pluripotent stem cell differentiation in the sample to be tested.

4. According to any one of claims 1-3, it is characterized in that All the differentiation types include three differentiation types of differentiation into endoderm, mesoderm and / or ectoderm; preferably, the cells differentiated by the differentiation types include but are not limited to: neural stem cells, neural progenitor cells, neural precursor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, definitive endoderm cells, liver precursor cells, hepatocytes, pancreatic islet precursor cells, pancreatic islet cells, alveolar epithelial cells, renal tubular and glomerular cells, myocardial precursor cells, myocardial cells, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, and megakaryocytes.

5. According to any one of claims 1-4, it is characterized in that: The level includes nucleic acid level or protein level; the nucleic acid level includes DNA or RNA level; preferably, the nucleic acid level is obtained by nucleic acid sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology; preferably; the protein level is obtained by sequencing technology, immunotechnology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

6. According to any one of claims 1-5, characterized in that: The detection agent or component further includes a detection agent or component for obtaining the level of an internal reference gene in a sample; preferably, the internal reference gene includes any one, multiple or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 or ABRAXAS2.

7. According to claim 6, it is characterized in that: 1) When inducing differentiated cells into cardiomyocytes, the universal marker genes include MIR302CHG and ESRG, and the internal reference genes include CDKN2AIP and RWDD4; 2) When the induced differentiated cells are mesenchymal stem cells, the universal marker gene includes TDGF1, and the internal reference genes include MAP3K7 and PDE12; 2) When the induced differentiated cells are natural killer cells, the universal marker gene includes ESRG, and the internal reference genes include TTF1 and ABRAXAS2; 2) When the induced differentiated cells are dopaminergic neural precursor cells, the universal marker gene includes MIR302CHG, and the internal reference genes include C8orf76 and SMG8.

8. According to any one of claims 1-7, characterized in that: The pluripotent stem cells are mammalian pluripotent stem cells; preferably, they are human pluripotent stem cells.

9. A universal marker gene for residual detection of pluripotent stem cell differentiation, characterized in that: The universal marker genes include any one, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1.

10. A method for detecting residual pluripotent differentiation of induced differentiated cells whose genes have not yet been clearly marked, characterized in that: The method comprises: based on universal marker genes, through downstream verification, selecting the most sensitive or better universal marker genes as the pluripotent differentiation residual detection genes of the induced differentiated cells; Preferably, the universal marker gene is any one, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1.

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