A general method for detecting pluripotent stem cell contamination based on genes

Abstract

The application relates to the technical field of gene detection, in particular to a pluripotent stem cell differentiation residual detection reagent, method and application based on genes. The application can realize detection of pluripotent stem cell residues in various induced differentiation cells, tissues or organoids based on a universal Marker gene combined with an internal reference gene.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to a universal detection method for pluripotent stem cell residues based on genes and application thereof. BACKGROUND

[0002] Pluripotent stem cells (PSC) are the collective term for induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC), which have the ability of unlimited proliferation and differentiation into various types of functional cells. Since the advent of pluripotent stem cells, the technology has been continuously developed, and for different indications, there are now a variety of hPSC-derived induced differentiation cell products entering IND declaration.

[0003] The clinical application of human pluripotent stem cell (hPSC)-derived induced differentiation cell products faces a greater risk of tumorigenicity compared to adult-derived cell products. The tumorigenicity risk of hPSC-derived induced differentiation cell products mainly comes from the following three aspects: 1) the residual hPSC in hPSC-derived induced differentiation cell products, which can form teratomas in vivo; 2) the accumulation of genetic mutations of hPSC during culture and induction differentiation may cause cancer or disease; 3) hPSC-derived induced differentiation cell products may contain precursor cells with strong proliferative capacity, or the product itself is a precursor cell, such as neural stem cells, which may expand massively and form tumors after being transplanted into the body. Therefore, it is necessary to establish a rapid and highly sensitive detection method for hPSC residues to ensure that the residual amount of hPSC in functional cell products is within a safe range and improve the safety of hiPSC induced differentiation products.

[0004] Currently, there are several methods for detecting residual hPSCs: 1) In vivo detection of residual hPSCs, which involves transplanting the test cells into immunodeficient mice and observing tumor formation. However, this method requires a long observation period of at least 6 months. 2) Flow cytometry, which uses flow cytometry antibodies against hPSC-specific markers (e.g., SSEA4 and TRA-1-60) to detect residual hPSCs. However, this method has low sensitivity (0.1%) and is sensitive to cell quantity and antibody dosage / activity, making standardization difficult. 3) Culture method, which uses hPSC culture conditions to culture the test cells for 10-14 days. Undifferentiated hPSCs form visible clones after culture, which are then stained with alkaline phosphatase. Before culture, residual hPSCs in the test cells can also be screened using magnetic beads or enriched by flow cytometry. This method can effectively detect residual hPSCs, but it requires 10-14 days of detection time and may induce false negatives by differentiating residual hPSCs from differentiated cells. 4) PCR, including RT-qPCR and digital PCR (ddPCR), is a faster, more sensitive, and relatively simpler method compared to flow cytometry and culture methods, and it holds promise for quantitative analysis. It detects residual hPSCs by detecting the expression of hPSC-specific genes. Before PCR detection, residual hPSCs in the test cells can be screened using magnetic beads or enriched by flow cytometry to improve detection sensitivity.

[0005] Genes used for detecting hPSC residues in induced functional cells must meet the following two conditions: ① the gene is specifically and extensively expressed in hPSCs; ② the gene is not expressed or is expressed in trace amounts in induced differentiated cells or adult human functional cells. Current methods use different marker genes for different cell types, resulting in significant differences in sensitivity. Commonly used hPSC-specific genes include SOX2, POU5F1, ESRG, LIN28A, NANOG, and TDGF1, but these genes are not necessarily universally applicable to induced differentiation cell products derived from different types of hPSCs. For example, Sox2 is a common hPSC-specific gene, but it is also expressed in neural stem cells. Therefore, Sox2 is not suitable for detecting residual hPSCs in induced neural stem cells or neural progenitor cells. In addition, existing technology can detect LIN28A by RT-qPCR to detect residual hPSCs in hPSC-induced differentiated retinal pigment epithelial cells (RPE). This method has been applied to patients, but studies have shown that LIN28A is expressed in induced hepatocytes, endothelial cells, and pancreatic islet bodies. Therefore, LIN28A is not suitable for detecting residual hPSCs in these functional cells. Currently, patent publication number CN110573607A discloses a method for detecting residual undifferentiated PSCs in cell cultures of pluripotent stem cells (PSCs). This method includes cell culture; pluripotency identification of the cultured cells, mainly using qRT-PCR to detect the genes LIN28 (Lin28A), OCT4 (POU5F1), SOX2, FOXD3, NANOG, PODXL, REX1 (ZFP42), SSEA1 (FUT4), DPPA2, and DPPA3; and comparing the pluripotency markers of reference cells (MSCs) and the cultured cells to determine whether there are no residual undifferentiated PSCs in the cultured cells or whether the proportion of residual undifferentiated PSCs in the cultured cells is lower than the known proportion of PSCs in MSCs. As can be seen, this method involves multiple pluripotency genes, some of which are significantly expressed in certain specific somatic cells. For example, LIN28A can be detected in liver and endothelial cells induced by hiPSC differentiation, therefore it cannot be used to detect hiPSC residues in such induced functional cells. SOX2 is expressed in nerve cells, therefore it is not suitable for detecting hPSC residues in hPSC-induced nerve cells. Patent publication number CN114150074A discloses the application of TDGF1 in detecting pluripotent stem cell residues in human pluripotent stem cell-derived functional cell products; however, TDGF1 has low sensitivity in certain induced differentiated cell types, therefore it is not suitable as a universal marker gene for detecting hiPSC residues.

[0006] In view of the above, this application is hereby submitted. Invention Overview

[0007] To address the aforementioned technical challenges, this application has identified universal marker genes applicable to all types of hPSC-derived induced differentiation cell products through research and screening. These genes are suitable for detecting hPSC residues in cells from various germ layers (endoderm, mesoderm, and ectoderm) derived from hPSCs. Furthermore, this set of universal marker genes can be used to rapidly validate the most sensitive marker genes in previously undefined induced differentiation cell products.

[0008] Therefore, this application includes at least the following objectives:

[0009] The primary objective of this application is to find a universal product for detecting residual differentiation of pluripotent stem cells across all differentiation types;

[0010] The second objective of this application is to find a method for detecting pluripotent stem cell differentiation residues in all differentiation types;

[0011] The third objective of this application is to find a method for detecting pluripotent stem cell differentiation residues during specific cell differentiation processes.

[0012] To achieve the above objectives, this application proposes the following specific technical solutions:

[0013] This 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 levels of any one, multiple, or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 in a sample.

[0014] All differentiation types include three types: differentiation into endoderm, mesoderm, and / or ectoderm.

[0015] In some aspects, the differentiated cells include, but are not limited to: neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, morphological endoderm cells, hepatic progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0016] In some respects, the product includes, but is not limited to, the form of a reagent kit or a system device; preferably, it is in the form of a reagent kit.

[0017] In some respects, any one of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can serve as an independent indicator for the detection of residual differentiation in universal pluripotent stem cells. Based on the experimental data of this application, any one of these genes can serve as a universal marker gene for detecting residual differentiation; therefore, detecting any one of them is sufficient to achieve the purpose of this application.

[0018] In some respects, any two, three, four, five, six, or seven of the aforementioned MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can be used as combined indicators for the detection of residual differentiation in universal pluripotent stem cells. In other respects, all of the aforementioned MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can be used as combined indicators for the detection of residual differentiation in universal pluripotent stem cells. It is understood that, under objective conditions, the reliability of the corresponding detection results will be higher as the number of detection marker genes increases.

[0019] In some respects, the level includes nucleic acid level or protein level; the nucleic acid level or protein level includes, but is not limited to, the abundance or concentration of nucleic acid or protein.

[0020] Furthermore, the nucleic acid level includes the DNA level or the RNA level.

[0021] More preferably, the nucleic acid level is obtained through sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biomolecular mass spectrometry technology, or chromatography technology.

[0022] More preferably, the methods for obtaining nucleic acid levels include, but are not limited to, any of the following: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, RNA blotting, nucleic acid mass spectrometry, or liquid chromatography.

[0023] More preferably, the protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, biomolecular mass spectrometry technology, or chromatography technology.

[0024] More preferably, the protein level acquisition method includes, but is not limited to, any of the following methods: amino acid sequencing, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, proteometry, or liquid chromatography.

[0025] In some respects, in addition to the aforementioned universal marker genes, the product may also include detection agents or components for obtaining levels of other biomarkers known in the prior art, to achieve more powerful detection and evaluation.

[0026] In some respects, the product may also include sample processing reagents, which may include nucleic acid extraction reagents, etc.

[0027] 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;

[0028] Preferably, the internal reference genes include any one, multiple, or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1, or ABRAXAS2.

[0029] Furthermore, the plurality can be two, three, four, five, six, seven, eight, nine, or ten, depending on the specific circumstances.

[0030] In some respects, the pluripotent stem cells described above are mammalian pluripotent stem cells; preferably, they are human pluripotent stem cells.

[0031] In practice, more applications involve residual detection targeting specific differentiated cells. Therefore, some specific embodiments of this application also include residual detection products for specific cells, such as:

[0032] In some respects, 1) when detecting induced differentiated cardiomyocytes, the universal marker genes in the detection product particularly include MIR302CHG and ESRG, and the internal reference genes include CDKN2AIP and RWDD4;

[0033] In some respects, 2) when testing for induced differentiated mesenchymal stem cells, the universal marker gene in the test product particularly includes TDGF1, and the internal reference genes include MAP3K7 and PDE12;

[0034] In some respects, 3) when detecting induced differentiated cells or natural killer cells, the universal marker gene in the detection product particularly includes ESRG, and the internal reference genes include TTF1 and ABRAXAS2;

[0035] In some respects, 4) when detecting dopaminergic neural progenitor cells for induced differentiation, the universal marker gene in the detection product particularly includes MIR302CHG, and the internal reference genes include C8orf76 and SMG8.

[0036] This application also provides the use of detection agents or components for obtaining levels of any one, multiple, or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 in samples in the detection of differentiation residues in all differentiation types of pluripotent stem cells; or in the preparation of a universal pluripotent stem cell differentiation residue detection kit.

[0037] This application also provides the application of any one, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 as single or combined biomarkers in the differentiation residues of universal pluripotent stem cells.

[0038] It is understood that in the aforementioned applications, the detection scenarios of this application are mostly conducted in vitro to evaluate the quality of induced stem cell products. Therefore, this application does not fall under the category of disease diagnosis.

[0039] All differentiation types include three types: differentiation into endoderm, mesoderm, and / or ectoderm.

[0040] In some aspects, the differentiated cells include, but are not limited to: neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, morphological endoderm cells, hepatic progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0041] In some aspects, the detection can be performed at the nucleic acid level (including DNA or RNA level) or the protein level. Further, the nucleic acid (including DNA or RNA) level or the protein level includes, but is not limited to, the abundance or concentration of nucleic acids (DNA or RNA) or proteins. More preferably, the nucleic acid level is obtained using sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biomolecular mass spectrometry, or chromatography technology; even more preferably, the method for obtaining the nucleic acid level includes, but is not limited to, any of the following: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization, gel electrophoresis, RNA blotting, nucleic acid mass spectrometry, or liquid chromatography. More preferably, the protein level is obtained using sequencing technology, immunoassay, electrophoresis, biomolecular mass spectrometry, or chromatography technology; even more preferably, the method for obtaining the protein level includes, but is not limited to, any of the following: amino acid sequencing, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, proteometry, or liquid chromatography.

[0042] In some aspects, the detection may also include the detection of internal reference genes. Further, 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 genes may be two, three, four, five, six, seven, eight, nine, or ten, depending on the specific circumstances.

[0043] In practice, more applications involve residual detection targeting specific differentiated cells. Therefore, some specific embodiments of this application also include residual detection products for specific cells, such as:

[0044] In some respects, 1) when detecting induced differentiated cardiomyocytes, the universal marker genes in the detection product particularly include MIR302CHG and ESRG, and the internal reference genes include CDKN2AIP and RWDD4;

[0045] In some respects, 2) when testing for induced differentiated mesenchymal stem cells, the universal marker gene in the test product particularly includes TDGF1, and the internal reference genes include MAP3K7 and PDE12;

[0046] In some respects, 3) when detecting induced differentiated cells or natural killer cells, the universal marker gene in the detection product particularly includes ESRG, and the internal reference genes include TTF1 and ABRAXAS2;

[0047] In some respects, 4) when detecting dopaminergic neural progenitor cells for induced differentiation, the universal marker gene in the detection product particularly includes MIR302CHG, and the internal reference genes include C8orf76 and SMG8.

[0048] In some respects, any of the above-described pluripotent stem cells are mammalian pluripotent stem cells; preferably, they are human pluripotent stem cells.

[0049] This application also provides a method for detecting residual differentiation of universal pluripotent stem cells, which includes the step of obtaining the level of any one, multiple or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in the sample; any detection based on this step is within the scope of this application.

[0050] Furthermore, the method may specifically include the following two steps:

[0051] (i) Obtain the levels of the universal marker genes in the sample to be tested;

[0052] (ii) Comparison with the levels of universal marker genes in the test sample and the control sample; wherein a significant difference in the levels of the universal marker genes between the test sample and the control sample indicates the presence of residual pluripotent stem cell differentiation in the test sample; or,

[0053] (ii) Compare with a set absolute threshold; wherein, a difference in absolute value between the level of the test sample and the threshold indicates the presence of pluripotent stem cell differentiation residues in the test sample.

[0054] Furthermore, the induced differentiated cells may be derived from various germ layers, including but not limited to: neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, morphological endoderm cells, liver progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0055] In some aspects, any one of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can serve as an independent indicator for the detection of residual differentiation in universal pluripotent stem cells. According to experimental data from this application, any one of these genes can serve as a universal marker gene for detecting residual differentiation; therefore, detecting any one of them is sufficient to achieve the purpose of this application. In some aspects, any two, three, four, five, six, or seven of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can serve as combined indicators for the detection of residual differentiation in universal pluripotent stem cells. In some aspects, all of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can serve as combined indicators for the detection of residual differentiation in universal pluripotent stem cells. Under normal circumstances, the reliability of the detection results will be higher as the number of marker genes increases.

[0056] In some aspects, the detection process further includes the detection of internal reference gene levels; preferably, the internal reference genes include any one, multiple, or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1, or ABRAXAS2; furthermore, the multiple genes can be two, three, four, five, six, seven, eight, nine, or ten, which can be selected according to the specific circumstances.

[0057] This application also provides a universal marker gene for detecting residual differentiation of pluripotent stem cells, wherein the universal marker gene includes any one, multiple or all of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1.

[0058] This application also provides a method for detecting residual pluripotent differentiation in induced differentiated cells for which no marker gene has been identified. This method includes: based on universal marker genes, screening for the most sensitive or relatively superior universal marker genes through downstream validation as the detection genes for residual pluripotent differentiation in the induced differentiated cells; wherein the universal marker genes are any one, multiple, or all of the following: MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1.

[0059] The beneficial technical effects of this application are as follows:

[0060] 1) Most existing marker genes are not applicable to all types of hPSC-derived induced differentiation cell products, and are only suitable for detecting residual hPSCs in single germ layer cells (endoderm, mesoderm, ectoderm) derived from hPSCs. This application expands the transcriptome sequencing sample size to include hiPSCs, ESCs, and 17 types of induced differentiation cells, including neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, fixed endoderm cells, hepatic progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, and intermediate-terminal cells. The study investigated neutrophils, eosinophils, megakaryocytes, etc. Through qPCR detection and statistical validation, a set of universal candidate marker genes compatible with endoderm, mesoderm, and ectoderm-directed differentiation cell products was identified: MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1. This addresses the current issue of insufficient universality of marker genes. The detection sensitivity of the universal marker genes screened in this application is 0.05-0.001%.

[0061] 2) This application provides a list of candidate marker genes for hiPSC-induced differentiation cell products for which marker genes have not yet been identified. Through downstream validation, the most sensitive marker gene can be selected as the gene for this type, shortening the gene screening time, reducing the difficulty, and improving the method's fault tolerance. It satisfies both versatility and sensitivity requirements. The method described in this application is characterized by high efficiency, reliability, strong versatility, and wide applicability. It can be applied to the quality monitoring of numerous hPSC-induced differentiation cell products, providing reliable cell products for clinical use.

[0062] 3) Currently, there is a lack of internal reference genes for the entire differentiation stage in this field. This application has identified several specific high-sensitivity internal reference genes and their combinations through screening. Correspondingly, several high-sensitivity differentiation residue detection methods for specific differentiated cells have also been established. Based on the aforementioned marker gene combination screening, several specific high-sensitivity internal reference genes and their combinations have been identified, achieving high-sensitivity (up to 0.0001%) residue detection. Attached Figure Description

[0063] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0064] Figure 1 Stability of candidate internal reference genes for iDAp differentiation direction;

[0065] Figure 2 Stability of candidate internal reference genes for iCM differentiation direction;

[0066] Figure 3 Stability of candidate internal reference genes for iMSC differentiation direction;

[0067] Figure 4 Stability of candidate internal reference genes for iNK differentiation direction;

[0068] Figure 5 Expression levels of candidate marker genes in transcriptome sequencing;

[0069] Figure 6 A histogram of expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSC and iCM (normalized at CDKN2AIP level);

[0070] Figure 7 Expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSC and iMSC (normalized at CSNK1G3 level).

[0071] Figure 8 Expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSC and iNK (normalized at DCPS level).

[0072] Figure 9 Expression levels of candidate universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 in hiPSC and iDAp (normalized at C8orf76 level);

[0073] Figure 10 Sensitivity of candidate universal marker genes in iDAp;

[0074] Figure 11 Detecting the sensitivity of candidate universal marker genes in iCM;

[0075] Figure 12 Sensitivity of candidate universal marker genes in iMSCs;

[0076] Figure 13 The sensitivity of candidate universal marker genes in iNK was tested. Invention Details

[0077] This application discloses an application for detecting residual differentiation of pluripotent stem cells based on genes. Those skilled in the art can implement this application by referring to the content herein. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within this application. The methods and applications of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the preparation methods and applications described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0078] The following basic terms or definitions are provided merely to aid in understanding this application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this application are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are still set forth to better explain this application.

[0079] As used in this application, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted 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 only of those embodiments.

[0080] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0081] The terms "approximately" and "generally" in this application refer to an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effects of the mentioned features. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0082] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described herein can be implemented in a different order than that described or illustrated herein.

[0083] Reference will now be made to detailed embodiments of this application, one or more of which are described below. Each example is provided for explanation and not for limitation. It will be apparent to those skilled in the art that various modifications and variations can be made to this application without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments. Therefore, this application is intended to cover such modifications and variations falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of this application are disclosed in or will be apparent from the following detailed description. Those skilled in the art will understand that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of this application.

[0084] Detailed description of the invention in this application

[0085] 1. Universal marker genes

[0086] This application uses universal marker genes for the detection of residual differentiation in pluripotent stem cells. By expanding the sample size of transcriptome sequencing, this application includes hiPSCs, ESCs, and 17 types of cells that can be induced to differentiate, such as neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, morphological endoderm cells, liver progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc. By using qPCR detection and statistical validation, candidate marker genes suitable for use in products of induced differentiation of cells in the endoderm, mesoderm, and ectoderm directions were finally identified. These universal genes include MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1, thus solving the problem of insufficient universality of current marker genes.

[0087] It is understood that any of the aforementioned single genes can be used for the detection of undifferentiated pluripotent stem cell differentiation residues, thus serving as a single biomarker. When any gene (or its expression) is detected in a differentiated cell system, it can be used to evaluate the presence of undifferentiated pluripotent stem cell residues in the 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.

[0088] Of course, these genes can also be used in combination for the detection of pluripotent stem cell differentiation residues. It is understandable that when multiple genes, such as two, three, four, five, six, seven or eight, are detected together, the sensitivity and accuracy of the detection can be improved.

[0089] Therefore, the universal marker gene in this application can be a single gene or a combination of multiple genes.

[0090] 2. Products

[0091] This application relates to products for detecting residual differentiation of all differentiation types of pluripotent stem cells, which include detection agents or components for obtaining levels of one, more, or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 in samples.

[0092] The “all differentiation types” mentioned in the text include the three differentiation types that differentiate into endoderm, mesoderm and / or ectoderm.

[0093] In some aspects, the differentiated cells include, but are not limited to: neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, morphological endoderm cells, hepatic progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0094] In some embodiments, any one of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can serve as an independent indicator for the detection of residual differentiation in universal pluripotent stem cells. Based on the experimental data of this application, any one of these genes can serve as a universal marker gene for detecting residual differentiation; therefore, detecting any one of them is sufficient to achieve the purpose of this application.

[0095] In other 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 indicator for the detection of residual differentiation of universal pluripotent stem cells.

[0096] In other embodiments, all of the MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 are used as a combined indicator for the detection of residual differentiation in universal pluripotent stem cells. It is understood that, under objective conditions, the reliability of the detection results will be higher as the number of universal marker genes detected increases.

[0097] In this text, "detection reagent" typically refers to the reagent used for detection, such as the MIR302CH gene detection reagent, which refers to a reagent capable of detecting the MIR302CH gene. "Level" in this text usually refers to the abundance or concentration of an indicator, such as nucleic acid level, which refers to the abundance or concentration of nucleic acids. Therefore, a gene-level detection reagent refers to a detection reagent that can directly obtain the abundance or concentration of a specific gene in a sample. Additionally, "component" in this text is distinct from the direct means of obtaining the detection reagent; it typically refers to components that indirectly obtain gene-level information from a sample (e.g., obtaining indicators of a specific gene in a pre-tested sample through a computer program).

[0098] In some implementations, the nucleic acid levels described herein include DNA levels or RNA levels.

[0099] When the detection reagent is used for nucleic acid detection, it is understood that there are various nucleic acid detection methods in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biomolecular mass spectrometry technology, or chromatography technology. All of these technologies can be used in this application. In some specific embodiments of this application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization, gel electrophoresis, RNA blotting, nucleic acid mass spectrometry, or liquid chromatography.

[0100] Therefore, for example, in some specific 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 through sequencing, and then evaluates the level of the corresponding gene based on the sequencing results. In other specific 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 the corresponding gene's DNA or RNA is detected through amplification.

[0101] When the detection reagent is used to detect proteins, it is understood that there are various protein detection methods in the art, including but not limited to sequencing technology, immunoassay technology, electrophoresis technology, biomolecular mass spectrometry technology, or chromatography technology, all of which can be used in this application; in some specific embodiments of this application, including but not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, proteometry, or liquid chromatography.

[0102] Therefore, for example, in some specific embodiments, the detection agent can be an immunoassay reagent, such as an antibody reagent, which achieves the detection of protein levels through an immune reaction between the antibody and the gene-expressed protein.

[0103] The products described herein include, but are not limited to, reagent kits and system devices.

[0104] In some embodiments, the product is in the form of a kit. It will be understood that such a kit, in addition to including the aforementioned detection reagents or detection components, may further include instructions for predicting results based on the detected levels.

[0105] In some embodiments, the kits described herein include carriers, packaging, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, etc., each containing one of the independent elements to be used in the method. The kits of this application may include the containers described above, as well as one or more other containers containing substances required from a commercial end-user perspective, including buffers, diluents, filters, and packaging instructions with usage instructions.

[0106] In some embodiments, the kit may further include sample processing reagents, which may include at least one of sample lysis reagents, sample purification reagents, and sample extraction reagents.

[0107] 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 the sample;

[0108] For example, the specific internal reference genes for screening described in this application specification include any one, multiple, or all of C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1, or ABRAXAS2.

[0109] In some preferred embodiments, the plurality can be two, three, four, five, six, seven, eight, nine, or ten, depending on the specific circumstances.

[0110] In some embodiments, the product also includes a detection agent or component for obtaining levels of other markers known in the prior art, to enable more robust detection and evaluation.

[0111] In addition, considering that more applications of this application are for residual detection of specific differentiated cells, this application also designs another type of "non-general" kit, such as a kit for detecting residual cells of specific differentiated cells, as described in some specific embodiments of this application:

[0112] 1) When testing induced differentiated cardiomyocytes, this kit contains assays for the universal marker genes MIR302CHG and ESRG, as well as assays for the internal control genes CDKN2AIP and RWDD4;

[0113] 2) When testing for induced differentiated mesenchymal stem cells, this kit contains a detection agent for the universal marker gene TDGF1, as well as for the internal reference genes MAP3K7 and PDE12;

[0114] 3) When detecting induced differentiated natural killer cells, this kit contains a detection agent for the universal marker gene ESRG, as well as detection agents for the internal control genes TTF1 and ABRAXAS2;

[0115] 4) When testing for induced differentiated dopaminergic neural progenitor cells, this kit contains assays for the universal marker gene MIR302CHG, as well as for the internal reference genes C8orf76 and SMG8.

[0116] In some specific implementations, the aforementioned four kits are all ddPCR kits, which overcome the current shortcomings: the current ddPCR method is mainly based on absolute quantification and does not use internal reference genes to correct gene expression levels, resulting in insufficient reproducibility of results.

[0117] In addition to the components described above, the kit will further include instructions for implementing the subject method. These instructions may exist in various forms within the subject kit, including one or more forms. One possible form of these instructions is as printed information on a suitable medium or substrate, such as one or more sheets of paper with the information printed on them, which are included in the kit packaging as inserts, etc.

[0118] 3. Application

[0119] The application of this application includes at least the following:

[0120] 1) Application of detection agents or components that obtain the levels of one, more or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 in samples in the detection of universal pluripotent stem cell differentiation residues.

[0121] 2) The use of detection agents that obtain the levels of any one, multiple, or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 in the sample in the preparation of a universal pluripotent stem cell differentiation residue detection kit.

[0122] 3) The application of any one, multiple or all of the genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN or POU5F1 as universal markers for detecting residual differentiation of pluripotent stem cells.

[0123] It is understandable that the testing scenarios in this application are mostly in vitro tests, used to evaluate the quality of induced stem cell products, and therefore generally do not involve disease detection applications.

[0124] As described in the product description above, in some embodiments, the detection can be performed at the nucleic acid level (including the DNA or RNA level) or the protein level. Furthermore, the nucleic acid or protein level includes, but is not limited to, the abundance or concentration of nucleic acids or proteins.

[0125] In some preferred embodiments, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biomass spectrometry technology, or chromatography technology; more preferably, the method for obtaining the nucleic acid level 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 method, RNA blotting method, nucleic acid mass spectrometry, or liquid chromatography.

[0126] In some preferred embodiments, the protein level is obtained using sequencing, immunoassay, electrophoresis, mass spectrometry, or chromatography. More preferably, the protein level acquisition method includes, but is not limited to, any of the following: amino acid sequencing, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, immunochromatography, radioimmunoassay, immunohistochemistry, Western blotting, flow cytometry, gel electrophoresis, proteometry, or liquid chromatography.

[0127] In some implementations, the detection may further include detection of internal reference genes;

[0128] 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 genes can be two, three, four, five, six, seven, eight, nine, or ten, and can be selected according to the specific circumstances.

[0129] 4. Detection Method

[0130] The core steps of the universal pluripotent stem cell differentiation residue detection method of this application are: obtaining the levels of any one, multiple, or all of the universal marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 in the sample; it is understood that any detection method based on or including this step is within the scope of this application.

[0131] In some implementations, the method may specifically include the following two steps:

[0132] (i) Obtain the levels of the universal marker genes in the sample to be tested;

[0133] (ii) Comparison with the levels of universal marker genes in the test sample and the control sample; wherein a significant difference in the levels of the universal marker genes between the test sample and the control sample indicates the presence of residual pluripotent stem cell differentiation in the test sample; or,

[0134] (ii) Compare with a set absolute threshold; wherein, a difference in absolute value between the level of the test sample and the threshold indicates the presence of pluripotent stem cell differentiation residues in the test sample.

[0135] In some specific implementations, a set value for the level of any one or more of the aforementioned genes can be provided. This set value can be determined based on the gene levels in the test sample and the control sample. For example, the average gene levels of a suitable number of control samples can be selected, or a reasonable fold can be set based on this average value, such as 0.9, 0.8, 0.7, 0.6, 0.5, etc. When the gene level in the test sample is higher than this set value, it is judged that pluripotent stem cell residues exist. It is understood that the set value determined based on the average value, or a fold of the average value, needs to have good classification significance. The known samples can be tested using common statistical test methods based on the classification of the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment criterion.

[0136] Given that the aforementioned marker genes are universal marker genes, the method of this application can be applied to the detection of various germ layer-induced differentiated cells, including but not limited to: neural stem cells, neural progenitor cells, neural progenitor cells, neurons, astrocytes, oligodendrocytes, retinal pigment epithelial cells, morphological endoderm cells, liver progenitor cells, hepatocytes, pancreatic islet progenitor cells, islet cells, alveolar epithelial cells, renal tubular glomerular cells, cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, mesenchymal stromal cells, natural killer cells, T lymphocytes, macrophages, neutrophils, eosinophils, megakaryocytes, etc.

[0137] As previously stated, during the detection process, any one of MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, or POU5F1 can serve as an independent indicator for the detection of residual differentiation in universal pluripotent stem cells. According to the experimental data of this application, any one of these genes can serve as a universal marker gene for detecting residual differentiation; therefore, detecting any one of them is sufficient to achieve the purpose of this application. In some embodiments, any two, three, four, five, six, or seven of the following genes can serve as combined indicators for the detection of residual differentiation in universal pluripotent stem cells. In other embodiments, all of the following genes can serve as combined indicators for the detection of residual differentiation in universal pluripotent stem cells. Under normal circumstances, the reliability of the detection results will be higher as the number of marker genes increases.

[0138] As mentioned above, the detection level of this 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 methods are the same as above.

[0139] In some embodiments, the detection process further includes the detection of internal reference gene levels; 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; furthermore, the multiple genes may be two, three, four, five, six, seven, eight, nine, or ten, which can be selected according to the specific circumstances.

[0140] The pluripotent stem cells mentioned in this article include mammalian pluripotent stem cells; human pluripotent stem cells are preferred.

[0141] In practice, many applications involve the detection of residual cells in specific differentiated cells. Therefore, this application also relates to some detection methods for specific differentiated cells, as detailed in some specific embodiments of this application:

[0142] In some implementations, 1) when the induced differentiated cells are cardiomyocytes, the genes targeted in the detection method are: marker genes, particularly including MIR302CHG and ESRG, and internal reference genes including CDKN2AIP and RWDD4;

[0143] In some embodiments, 2) when the induced differentiated cells are mesenchymal stem cells, the genes targeted in the detection method are: marker genes, especially TDGF1, and internal reference genes, including MAP3K7 and PDE12.

[0144] In some implementations, 3) when the induced differentiated cells are detected as natural killer cells, the genes targeted in the detection method are: marker genes, especially ESRG, and internal reference genes, including TTF1 and ABRAXAS2.

[0145] In some embodiments, 4) when the induced differentiated cells are detected as dopaminergic neural progenitor cells, the genes targeted in the detection method are: marker genes, particularly including MIR302CHG, and internal reference genes including C8orf76 and SMG8.

[0146] Therefore, based on these specific implementation methods, this application also provides a ddPCR method for detecting hiPSC residues in hPSC-induced differentiated cardiomyocyte (iCM) products. The marker genes are MIR302CHG and ESRG, and the internal reference genes are CDKN2AIP and RWDD4. The sensitivity can reach 0.005%, i.e., within 1×10⁻⁶ m / s².6 Fifty PSCs were detected in iCM cells. The method includes the following steps: detecting the expression levels of MIR302CHG and ESRG genes in the test sample, wherein the expression levels of MIR302CHG and ESRG genes are normalized by the geometric square root of the expression levels of CDKN2AIP and RWDD4 genes, and the relative expression levels of MIR302CHG and ESRG genes after normalization indicate the level of residual pluripotent stem cells in the test sample. This application also provides a ddPCR method for detecting residual hPSCs in hPSC-induced differentiated mesenchymal stem cells (iMSC) products. The marker gene is TDGF1, and the internal reference genes are MAP3K7 and PDE12, with a sensitivity of 0.005%, i.e., within 1×10 6 Fifty PSCs were detected in MSCs. The method includes the following steps: detecting the expression level of the TDGF1 gene in the test sample, wherein the expression level of the TDGF1 gene is normalized by the geometric square root of the expression levels of the MAP3K7 and PDE12 genes, and the relative expression level of the TDGF1 gene after normalization indicates the level of residual pluripotent stem cells in the test sample. This application also provides a ddPCR method for detecting residual hPSCs in hPSC-induced natural killer cell (iNK) products. The marker gene is ESRG, and the internal reference genes are TTF1 and ABRAXAS2, with a sensitivity of 0.0001%, i.e., within 1×10⁻⁶ mcg / mcg. 6 One PSC was detected in iNK. The method includes 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 ABRAXAS2 gene, and the relative expression level of the ESRG gene after normalization indicates the level of residual pluripotent stem cells in the test sample. This application also provides a ddPCR method for detecting residual hPSCs in hPSC-induced differentiation dopaminergic neural progenitor cells (imDAP) products. The marker gene is MIR302CHG, and the internal reference genes are C8orf76 and SMG8, with a sensitivity of 0.001%, i.e., within 1×10 6 Ten pluripotent stem cells (PSCs) were detected by iDAp. The method includes the following steps: detecting the expression level of the MIR302CHG gene in the test sample, wherein the expression level of the MIR302CHG gene is normalized by the geometric square root of the expression levels of the TTF1 and ABRAXAS2 genes, and the relative expression level of the MIR302CHG gene after normalization indicates the level of residual pluripotent stem cells in the test sample. In the aforementioned method, this application proposes a dual internal reference gene, which has better stability than a single internal reference gene, thus improving the stability and reproducibility of the method.

[0147] This application discloses a method for detecting residual pluripotency differentiation in induced differentiated cells for which marker genes have not yet been identified. Such methods generally include: using universal marker genes as a basis, and through downstream validation, screening out the most sensitive or relatively superior universal marker genes as the detection genes for residual pluripotency differentiation in the induced differentiated cells; in this application, the universal marker genes are MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1.

[0148] In some specific embodiments, this method involves a set of candidate marker genes that can be used to detect hiPSC residues in hiPSC-induced differentiation cell products using ddPCR. This set of marker genes allows for rapid verification of the most sensitive marker genes in previously undefined hiPSC-induced differentiation cell products, accelerating research progress and improving product safety. The method includes the following steps: detecting the expression levels of eight candidate marker genes in the sample to be tested; determining the gene with the highest sensitivity as the optimal marker gene using ddPCR; and the expression level of the optimal marker gene indicates the level of hiPSC residues in this type of induced differentiation cell. In this application, there are no special restrictions on the sequences of the candidate marker genes; sequences derived from MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 genes can be used. It is evident that in this application, by screening for candidate marker genes common to all three germ layers, genes that may be expressed in trace amounts in other cell types or exhibit significant differences in expression in some PSCs are excluded. Then, the expression levels of candidate genes in hPSC-derived induced differentiation cell products were examined. The results showed that the aforementioned universal candidate marker genes were not expressed or expressed at very low levels in hPSC-derived induced differentiation cell products. Further sensitivity testing revealed that the optimal marker genes for different induced differentiation cell types could be found within the universal candidate marker genome. Therefore, by detecting the sensitivity of the aforementioned eight candidate marker genes in different hPSC-derived induced differentiation cell products, the optimal marker genes for detecting hPSC residues in different products can be screened.

[0149] The following detailed description of the specific implementation examples will fully elaborate on this application. Example

[0150] Sources of reagents and raw materials:

[0151] The RNA reverse transcription kit was purchased from Takara; the ddPCR kit was purchased from Bio-Rad.

[0152] The human induced pluripotent stem cells were obtained from Dongfang Hospital, Anhui ZhongSheng Suyuan Biotechnology Co., Ltd., Zhejiang Huode Biotechnology Co., Ltd., and Nanjing Aierpu Regenerative Medicine Technology Co., Ltd.

[0153] The mesenchymal stem cells and natural killer cells obtained from hPSC induced differentiation came from Anhui Zhong Sheng Su Yuan Biotechnology Co., Ltd., the cardiomyocytes came from Nanjing Aierpu Regenerative Medicine Technology Co., Ltd., and the dopamine precursor cells came from Zhejiang Huode Biotechnology Co., Ltd.

[0154] The primers involved in the examples were synthesized at Kunshan Pronop Biotechnology Co., Ltd., and the specific primer and probe sequences are as follows:

[0155]

[0156]

[0157] Example 1: Screening for internal reference genes suitable for detecting residual hPSCs in dopamine precursor cells (iDAp), mesenchymal stem cells (iMSC), cardiomyocytes (iCM), and natural killer cells (iNK) derived from hPSCs.

[0158] To ensure the stability of the method, this application utilizes transcriptome sequencing data of hiPSCs and their induced differentiation cells from public and laboratory databases. The databases were used to screen candidate internal control genes that were stably expressed during differentiation. Based on the specific performance of the internal control genes in the databases, the screening criteria were set as the top ten genes with a cv value less than 0.3 for their TPM values. Furthermore, since this application focuses on residual detection, the TPM value was set to be below 50 to accommodate subsequent experimental procedures. Through the aforementioned screening process, this application obtained candidate internal control genes that met the requirements, as detailed in Tables 1.1-1.4.

[0159] Table 1.1 Applicable internal reference genes for ectoderm that meet the screening criteria

[0160]

[0161]

[0162] Table 1.2 Applicable internal reference genes for iCM that meet the screening criteria

[0163] 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

[0164] Table 1.3 Applicable internal reference genes for iMSCs that meet the screening criteria

[0165] 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

[0166] Table 1.4 Applicable internal reference genes for iNK that meet the screening criteria

[0167] 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

[0168] Subsequently, the CT values ​​of candidate internal reference genes in iPSC and iDAp / iCM / iMSC / iNK were detected by RT-qPCR. The reaction system is shown in Table 1.5, and the reaction procedure is shown in Table 1.6. The internal reference gene screening software NormFinder was then used to further screen for stably expressed internal reference genes, with lower S values ​​indicating more stable surface gene expression.

[0169] Table 1.5 Reaction System

[0170]

[0171]

[0172] Table 1.6 Reaction Procedure

[0173]

[0174] Specific results are as follows Figures 1-4 As shown in the NormFinder results, the optimal single internal reference for iDAp differentiation is C8orf76, and the optimal dual internal references are C8orf76 and SMG8; the optimal single internal reference for iCM is CDKN2AIP, and the optimal dual internal references are CDKN2AIP and RWDD4; the optimal single internal reference for iMSC is CSNK1G3, and the optimal dual internal references are MAP3K7 and PDE12; the optimal single internal reference for iNK is DCPS, and the optimal dual internal references are TTF1 and ABRAXAS2. Among these, the dual internal reference combination exhibits better stability than the single internal reference gene.

[0175] Example 2: Screening of universal marker genes

[0176] To address the issue of insufficient universality of marker genes, the applicant utilized public domain databases and transcriptome sequencing data of hiPSCs and their derivatives collected in the laboratory (data from healthy individuals). These derivatives included hepatocytes, endodermal cells, endocrine progenitor cells, and pancreatic islet cells derived from the endoderm; cardiac progenitor cells, cardiomyocytes, mesenchymal stem cells, and natural killer cells derived from the mesoderm; and neural stem cells, neural progenitor cells, dopaminergic neurons, spinal motor neurons, astrocytes, and retinal pigment epithelial cells derived from the ectoderm. DESeq2 was used to screen for candidate marker genes common to all hiPSC-derived cell types.

[0177] The specific screening results are shown in Table 2.1. Fifteen marker genes that overlapped across all three germ layers were identified: C13orf42, PRDM14, MIR302CHG, ESRG, FOXD3-AS1, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, POU5F1, XCAT, ZSCAN10, LINC00458, and L1TD1. XCAT, ZSCAN10, LINC00458, and L1TD1 were excluded due to significant differences in expression across different iPSC strains. Figure 5 ).

[0178] Table 2.1 Common candidate marker genes for trilaminar differentiation

[0179]

[0180]

[0181] Subsequently, the applicant validated the actual expression levels of candidate marker genes in hiPSC-induced differentiated dopamine precursor cells, mesenchymal stem cells, cardiomyocytes, and natural killer cells. Specifically, the applicant first extracted total mRNA from iPSC, iCM, iMSC, iNK, and iDAp cell samples using the Trizol method; then, using a Takara RNA reverse transcription kit (PrimeScript), the mRNA was expressed at a rate of 500 ng / mL. TM RT Master Mix (#RR036A) was used to reverse transcribe mRNA into cDNA, and finally, the cDNA was generated according to the Takara qPCR kit (TB). Premix Ex (#RR420L) was used to detect the expression of C13orf42, PRDM14, MIR302CHG, ESRG, FOXD3-AS1, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 genes in iPSC, iCM, iMSC, iNK, and iDAp using RT-qPCR. The reaction system and procedure were the same as in Table 1.5 and Table 1.6 above. The 96 software calculates the relative expression levels of candidate marker genes based on the internal reference genes selected above.

[0182] Specific test results are as follows: Figures 6-9As shown in the RT-qPCR results, candidate marker genes FOXD3-AS1, C13orf42, and PRDM14 were excluded due to low expression levels, making them unsuitable as marker genes. MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, and POU5F1 were highly expressed in hiPSCs from different sources, but almost not expressed in induced differentiation iCM, iMSC, iNK, and iDAp. Therefore, these genes have the potential to serve as universal marker genes for detecting residual differentiation of pluripotent stem cells, and further evaluation of their sensitivity is needed.

[0183] Example 3: Sensitivity of detecting candidate marker genes in iDAp

[0184] To simulate the detection of hPSC residues during cell production, this application uses iPSC as a positive control and iDAp as a negative control, accurately aspirating 1×10⁻⁶ hPSCs. 6 One positive cell was placed in a 1.5 mL centrifuge tube, and 1 mL of Trizol was added. The mixture was then thoroughly mixed by pipetting. The Trizol lysis buffer containing the positive cells was further serially diluted with Trizol. 100 μL of the positive control cell lysis buffer containing Trizol was added to 900 μL of the negative control cell lysis buffer. The final mixing ratios are shown in Table 3.1. RNA was extracted and reverse transcribed into cDNA.

[0185] Table 3.1 Cell Sample Preparation

[0186]

[0187] The sensitivity of 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 4 times.

[0188] Table 3.2 ddPCR reaction system

[0189]

[0190]

[0191] Table 3.3 ddPCR reaction procedure

[0192]

[0193] The results were analyzed using relative quantification, as follows: The copy number results of all genes detected in one experiment were compiled, and the geometric mean of the C8orf76 and SMG8 gene copy numbers in each sample was calculated. The relative expression level of the candidate marker gene was obtained by dividing the candidate marker gene copy number by the geometric mean of the internal reference genes. The statistical difference between the relative expression levels of each cell mixture and iDAp was determined using a t-test to assess the sensitivity of the marker gene. The ddPCR calculation results are shown in Table 3.4.

[0194] Table 3.4 shows the calculation results of hPSC residual detection in iDAp induced by hPSC, using the MIR302CHG gene as an example.

[0195]

[0196]

[0197] Note: The quality control standard is that the number of positive droplets in the reverse transcription blank control group is less than 3.

[0198] For details of all candidate marker gene screening results, please see [link to full list]. Figure 10 The detection results showed that the gene with the highest sensitivity in iDAp was MIR302CHG, with a sensitivity of 0.001%, and it could detect up to 1×10⁻⁶ cells / day. 6 Each iDAp is divided into 10 hiPSCs.

[0199] Example 4: Sensitivity of detecting candidate marker genes in iCM

[0200] The method is the same as in Example 3. To simulate the detection of hPSC residues during cell production, this application uses iPSC as a positive control and iCM as a negative control. 1×10⁻⁶ hPSCs are accurately aspirated. 6 One positive cell was placed in a 1.5 mL centrifuge tube, and 1 mL of Trizol was added. The mixture was then thoroughly mixed by pipetting. The Trizol lysis buffer containing the positive cells was further serially diluted with Trizol. 100 μL of the positive control cell lysis buffer containing Trizol was added to 900 μL of the negative control cell lysis buffer. The final mixing ratios are shown in Table 3.1. RNA was extracted and reverse transcribed into cDNA.

[0201] The sensitivity of candidate marker genes was determined by ddPCR. The reaction system is shown in Table 3.2 and Table 3.3. Each sample was tested 4 times.

[0202] The results were analyzed using relative quantification, as follows: The copy number results of all genes detected in one experiment were compiled, and the geometric mean of the copy numbers of the CDKN2AIP and RWDD4 genes in each sample was calculated. The relative expression level of the candidate marker gene was obtained by dividing the candidate marker gene copy number by the geometric mean of the internal reference genes. The statistical difference between the relative expression levels of each cell mixture and iDAp was determined using a t-test to assess the sensitivity of the marker gene.

[0203] Candidate marker gene screening results are shown in Figure 11 The detection results showed that the genes with the highest sensitivity in iCM were MIR302CHG and ESRG, both with a sensitivity of 0.005%, and could be detected at 1×10⁻⁶. 6 The iCM distinguishes 50 hiPSCs.

[0204] Example 5: Sensitivity of detecting candidate marker genes in iMSCs

[0205] The method is the same as in Example 3. To simulate the detection of hPSC residues in cell production, this invention uses iPSC as a positive control and iMSC as a negative control. 1×10⁻⁶ hPSCs are accurately aspirated. 6 One positive cell was placed in a 1.5 mL centrifuge tube, and 1 mL of Trizol was added. The mixture was then thoroughly mixed by pipetting. The Trizol lysis buffer containing the positive cells was further serially diluted with Trizol. 100 μL of the positive control cell lysis buffer containing Trizol was added to 900 μL of the negative control cell lysis buffer. The final mixing ratios are shown in Table 3.1. RNA was extracted and reverse transcribed into cDNA.

[0206] The sensitivity of candidate marker genes was determined by ddPCR. The reaction system is shown in Table 3.2 and Table 3.3. Each sample was tested 4 times.

[0207] The results were analyzed using relative quantification, as follows: The copy number results of all genes detected in one experiment were compiled, and the geometric mean of the copy numbers of MAP3K7 and PDE12 genes in each sample was calculated. The relative expression level of the candidate marker gene was obtained by dividing the candidate marker gene copy number by the geometric mean of the internal reference gene. The statistical difference between the relative expression level of each cell mixture and iDAp was determined using a t-test to assess the sensitivity of the marker gene.

[0208] Candidate marker gene screening results are shown in Figure 12 The detection results showed that TDGF1 was the gene with the highest sensitivity in iMSCs, with a sensitivity of 0.005%, and it could detect up to 1×10⁻⁶ cells / mL. 6 The iMSC distinguishes 50 hiPSCs.

[0209] Example 6: Sensitivity of candidate marker genes in iNK

[0210] The method is the same as in Example 3. To simulate the detection of hPSC residues during cell production, this invention uses iPSC as a positive control and iNK as a negative control. 1×10⁻⁶ hPSCs are accurately aspirated. 6 Place 100 positive cells into a 1.5 mL centrifuge tube, add 1 mL of Trizol, and mix thoroughly by pipetting. Further serially dilute the Trizol lysis buffer containing positive cells with Trizol. Pipette 100...

[0211] μL of positive control cell lysis buffer containing Trizol was added to 900 μL of negative control cell lysis buffer. The final mixing ratio is shown in Table 3.1. RNA was extracted and reverse transcribed into cDNA.

[0212] The sensitivity of candidate marker genes was determined by ddPCR. The reaction system is shown in Table 3.2 and Table 3.3. Each sample was tested 4 times.

[0213] The results were analyzed using relative quantification, as follows: The copy number results of all genes detected in one experiment were compiled, and the geometric mean of the TTF1 and ABRAXAS2 gene copy numbers in each sample was calculated. The relative expression level of the candidate marker gene was obtained by dividing the candidate marker gene copy number by the geometric mean of the internal reference gene. The statistical difference between the relative expression levels of each cell mixture and iDAp was determined using a t-test to assess the sensitivity of the marker gene.

[0214] Candidate marker gene screening results are shown in Figure 13 The detection results showed that the gene with the highest sensitivity in iCM was ESRG, with a sensitivity of 0.0001%, and it could be detected at 1×10⁻⁶. 6 In iNK, one hiPSC is distinguished.

[0215] Furthermore, although the most sensitive marker genes were identified in Examples 3-6 above, from... Figures 10-13 The results also show that each gene screened in this application (MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN, POU5F1) has sufficient sensitivity for detecting various germ layer differentiated cells, with a minimum sensitivity of 0.5%, which can be used for residual detection in practice.

[0216] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. Use of a detection agent or component for obtaining the levels of all of the general marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN and POU5F1 in a sample in the detection of pluripotent stem cell differentiation residual in multiple differentiation types; the detection agent or component further comprises a detection agent or component for obtaining the levels of internal reference genes in the sample; the internal reference genes comprise C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 and ABRAXAS2; the multiple differentiation types comprise three differentiation types of differentiation into endoderm, mesoderm and ectoderm; and the pluripotent stem cells are human pluripotent stem cells.

2. A product for detecting the presence of undifferentiated pluripotent stem cells of a plurality of differentiation types, characterized in that, A detection agent or component for obtaining the levels of all of the general marker genes MIR302CHG, ESRG, LINC00678, LNCPRESS1, NANOG, TDGF1, VRTN and POU5F1 in a sample; the detection agent or component further comprises a detection agent or component for obtaining the levels of internal reference genes in the sample; the internal reference genes comprise C8orf76, SMG8, CDKN2AIP, RWDD4, CSNK1G3, MAP3K7, PDE12, DCPS, TTF1 and ABRAXAS2; the multiple differentiation types comprise three differentiation types of differentiation into endoderm, mesoderm and ectoderm; and the pluripotent stem cells are human pluripotent stem cells.

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