CN116925999A - Method for rapidly identifying iPSC and EPSC - Google Patents

Method for rapidly identifying iPSC and EPSC Download PDF

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CN116925999A
CN116925999A CN202310941706.7A CN202310941706A CN116925999A CN 116925999 A CN116925999 A CN 116925999A CN 202310941706 A CN202310941706 A CN 202310941706A CN 116925999 A CN116925999 A CN 116925999A
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赵谦
顾雨春
卢惠迪
吴理达
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Chengnuo Regenerative Medical Technology Beijing Co ltd
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Abstract

The application discloses a method for rapidly identifying iPSC and EPSC, which creatively applies mitomycin to EPSC residue detection in the iPSC of an EPSC source for the first time, and obtains a better detection effect.

Description

Method for rapidly identifying iPSC and EPSC
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to a method for rapidly identifying iPSC and EPSC.
Background
The induced pluripotent stem cells (Induced pluripotent stem cell, iPSC) were initially a pluripotent stem cell similar to embryonic stem cells in morphology, gene and protein expression, epigenetic modification, cell multiplication capacity, embryoid body and tumor-like capacity, differentiation capacity and the like, obtained by transferring a combination of four transcription factors (Oct 4, sox2, klf4 and c-Myc) into adult somatic cells using viral vectors in 2006 by a japanese scientist mountain extension (Shinya Yamanaka). The iPSC is obtained by reprogramming somatic cells, and is not limited by ethics and immune rejection, so that the iPSC becomes a seed cell with great interest, and the iPSC has wide application prospect in the aspects of disease model construction, drug screening and cell treatment, but the problems of high iPSC cell heterogeneity, unstable differentiation efficiency, low somatic cell maturity based on iPSC differentiation and the like still exist at present.
Expansion of pluripotent stem cells (Expanded potential stem cell, EPSC) was originally Deng Hongkui by chinese scientist using the addition of protein factor Lif and three compounds in iPSC medium in 2017: chir99021, (S) - (+) -Dimethindene maleate and Minocyline hydrochlorid induce a new type of multifunctional stem cells which can develop into extraembryonic tissue and are widely paid attention to due to the high cloning efficiency of single cells, efficient chimeric ability and extraembryonic tissue development ability. While ipscs can only be developed and differentiated to form ectodermal, mesodermal and endodermal tissues and cells, EPSCs can be differentiated to form extraembryonic tissues, and have stronger differentiation potential than ipscs. In addition, compared with iPSC, the functional cells formed by EPSC differentiation are closer to somatic cells, the efficiency of in vitro gene editing is higher, the genome is more stable in the passage process, and the functional cells become an important cell platform for the research and development of pluripotent stem cells.
The EPSC is first converted into iPSC in the process of differentiating into most somatic cells, and the traditional method for converting the EPSC into the iPSC is to convert all EPSC cells into the iPSC by default after the culture of the iPSC for about 48 hours by using an iPSC culture medium (such as E8 culture medium), but the differentiation mode is suitable for small-scale culture, is limited to be used in a laboratory range, and can not judge whether the EPSC is completely differentiated (namely whether the residual of the EPSC exists in the differentiated iPSC cells) or not, and if the EPSC is incompletely differentiated, the residual EPSC in the differentiated iPSC cells can be caused, so that the subsequent differentiation efficiency of the iPSC is further weakened. The transformation methods used in the current large-scale culture process have no clear identification criteria as to whether EPSC is completely transformed into iPSC.
At present, related researches or reports of applying mitomycin to EPSC and iPSC identification are not seen, and the application creatively applies mitomycin to EPSC residue detection in the EPSC-derived iPSC for the first time, and obtains better detection effect.
Disclosure of Invention
In order to solve the technical blank that whether the EPSC residue detection method exists in the EPSC source iPSC is not yet available in the field, the application aims to provide a detection method for identifying whether the EPSC is completely converted into the iPSC for the field.
The above object of the present application is achieved by the following technical solutions:
the first aspect of the application provides the use of a cell G1 phase blocker to identify EPSC and iPSC;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin.
Further, the mitomycin acts as a block to the growth of EPSCs in the G1 phase.
Further, the mitomycin is toxic to EPSC fines in S-phase and G2-phase.
Further, the mitomycin was toxic to ipscs.
Further, the mitomycin is used at a concentration of 0.1-1.0 μg/mL;
preferably, the mitomycin is used at a concentration of 0.3-0.6 μg/mL;
more preferably, the mitomycin is used at a concentration of 0.3 μg/mL or 0.6 μg/mL;
most preferably, the mitomycin is used at a concentration of 0.3 μg/mL.
Further, the treatment time of the mitomycin is 24-120 hours;
preferably, the treatment time of the mitomycin is 24-72 hours;
more preferably, the mitomycin is treated for 24 hours or 72 hours;
most preferably, the mitomycin is treated for 24 hours.
The inventors of the present application have unexpectedly found that EPSC and iPSC have a significant difference in cell cycle in a preliminary experimental study, wherein EPSC has a G1 phase checkpoint, whereas iPSC corresponds to an inner cell mass without a G1 phase checkpoint, and based on this difference in cell cycle regulation between EPSC and iPSC, the present application provides the use of cell G1 phase blockers in identifying EPSC and iPSC.
In some embodiments, the cell G1 phase blocker is not limited to the agents of the application as described above, as long as the agents act to block the growth of EPSCs in the G1 phase, act to poison EPSCs in the S phase and G2 phase, and act to poison iPSCs are within the scope of the application. In a specific embodiment of the application, the cell G1 phase blocker is preferably mitomycin.
In some embodiments, the mitomycin is used to interfere with the EPSC differentiation product, and can act on the G1 phase checkpoint of the EPSC to affect the cell cycle, block the cells in the G1 phase, and have toxic effects on the EPSC in other phases, so that as the action time of the mitomycin is prolonged, the EPSC in the G1 phase is partially stopped, and the cell ratio in the G1 phase is gradually increased. Whereas mitomycin-treated ipscs all entered apoptosis without G1 phase checkpoint, they were identified according to their different manifestations of ipscs and EPSCs under mitomycin.
If all cells in the differentiation product are detected to be dead cells after mitomycin treatment, the EPSC is completely differentiated into iPSC, namely, only the iPSC in the differentiation product is shown; if viable cells are detected in the differentiation product after mitomycin treatment, the presence of EPSC residues in the differentiation product, i.e., the simultaneous presence of iPSC and EPSC in the differentiation product, is indicated, wherein a higher detected cell viability indicates a lower EPSC differentiation efficiency and a lower detected cell viability indicates a higher EPSC differentiation efficiency.
In the present application, the cell cycle is also called cell proliferation phase, which is the whole process of the cell from the completion of one division to the completion of the next division, mainly the replication and synthesis of genetic material DNA, and then the cell cycle is completed by entering the division phase to complete proliferation. According to the different biochemical characteristics of the cell proliferation cycle, the cell proliferation cycle is divided into four continuous periods. Namely, the G1 phase (prophase DNA synthesis), the S phase (prophase DNA synthesis), the G2 phase (prophase DNA synthesis), and the M phase (mitosis).
Stage G1: the stages in preparation for the next step of DNA synthesis include RNA and nucleoprotein synthesis, deoxynucleotide, thymus kinase formation, etc. Whether a cell enters the G1 phase after the last cell proliferation is completed depends on many factors, such as whether it is affected by exogenous growth or other factors associated with proliferation. Studies have shown that: if the cells lack growth factors or nutrients after entering the G1 phase, they may become blocked and the G1 phase is prolonged, and the G1 phase is called the G0 phase. The length of the G1 phase of different cells varies from 4-5 hours to several days, the G1 phase of the cells with vigorous proliferation is short, and the time of the declining G1 phase is long.
S phase: after the G1 phase is completed smoothly, the cell enters into the S phase to complete the synthesis of DNA and histone. At the end of S phase, the DNA content doubled. This period is also a period in which DNA is extremely vulnerable because double strands are separated into single strands at the time of DNA synthesis, and the structure is unstable and easy to mutate. The S-phase time varies little from cell to cell, on average about 6-8 hours.
Stage G2: it is the later stage of DNA synthesis, the preparation for mitosis. During this period, DNA synthesis is terminated, and RNA and proteins, including tubulin and maturation-promoting factors, are synthesized in large quantities. The period of time is short and constant, about 2-5 hours.
M phase: also known as the mitosis phase, is the end of the cell cycle when a cell will successfully segregate from one cell into two daughter cells. It is a continuous process, generally 1-2 hours, with the need for going through the front, middle, back and end stages.
In a second aspect of the application, a test method is provided for identifying whether EPSC is fully converted to iPSC.
Further, the method comprises the following steps: treating a cell differentiation product obtained when EPSC is converted into iPSC by using a cell G1 phase retarder, and detecting the survival state of cells in the cell differentiation product;
preferably, if no cells in the cell differentiation product survive, it indicates that EPSC are all converted to iPSC;
preferably, if there is still cell survival in the cell differentiation product, it indicates that there is a residual of EPSC cells, EPSC not completely converted to iPSC;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin.
Further, the mitomycin is used at a concentration of 0.1-1.0 μg/mL;
preferably, the mitomycin is used at a concentration of 0.3-0.6 μg/mL;
more preferably, the mitomycin is used at a concentration of 0.3 μg/mL or 0.6 μg/mL;
most preferably, the mitomycin is used at a concentration of 0.3 μg/mL.
Further, the treatment time of the mitomycin is 24-120 hours;
preferably, the treatment time of the mitomycin is 24-72 hours;
more preferably, the mitomycin is treated for 24 hours or 72 hours;
most preferably, the mitomycin is treated for 24 hours.
Further, the cell differentiation product was expressed as 1X 10 4 -5×10 4 Individual cells/squareInoculating the seeds in an iPSC culture medium in a centimeter manner;
preferably, the cell differentiation product is present in an amount of 1.2X10 4 -3.4×10 4 Inoculating cells per square centimeter;
preferably, the iPSC medium is STEM CELL TeSR TM -E8 TM 。
In some embodiments, the cell differentiation product refers to a cell differentiation product obtained by differentiating EPSC into iPSC, detecting whether EPSC is completely converted into iPSC based on the difference of EPSC and iPSC in cell cycle regulation, and identifying according to the survival state of cells in the cell differentiation product after mitomycin treatment. If no cells survive in the mitomycin treated cell differentiation product, it is indicated that EPSCs are all converted to iPSCs; if there is cell survival in the cell differentiation product, it indicates that there is residual EPSC, EPSC is not completely converted into iPSC, and the conversion time needs to be prolonged, so that residual EPSC is prevented from weakening the subsequent differentiation efficiency of iPSC.
In some embodiments, the cell G1 phase blocker is not limited to the agents of the application as described above, as long as the agents act to block the growth of EPSCs in the G1 phase, act to poison EPSCs in the S phase and G2 phase, and act to poison iPSCs are within the scope of the application. In a specific embodiment of the application, the cell G1 phase blocker is preferably mitomycin.
In some embodiments, the method of detecting the survival status of a cell in a cell differentiation product includes electron microscopy (observation of cell morphology and internal structure by electron microscopy to determine whether the cell is dead), staining (observation of the cell after staining with a stain to determine whether the cell is dead), in vitro cell culture (culture of the cell in an in vitro environment to determine whether the cell is dead), electrophysiological analysis (by measuring cell membrane potential to determine whether the cell is dead), biochemical analysis (detection of specific cell markers in the cell to determine whether the cell is dead using biochemical methods), fluorescent detection (detection of active substances within the cell using fluorescent markers to determine whether the cell is dead).
In some embodiments, the method of detecting the survival state of cells in a cell differentiation product is not limited to the methods listed in the present application, and any method capable of identifying the survival state of cells in a cell differentiation product is within the scope of the present application.
The third aspect of the application provides the use of a cell G1 phase blocker in EPSC residue detection in EPSC-derived iPSCs;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin;
most preferably, the detection is carried out using the method of the second aspect of the application;
most preferably, if no cells survive in the cell differentiation product, this indicates that there is no EPSC remaining;
most preferably, the presence of EPSC residue is indicated if there is still cell survival in the cell differentiation product.
In some embodiments, EPSC residues in EPSC-derived ipscs are detected based on differences in cell cycle regulation between EPSC and iPSC, and identified based on the survival status of cells in mitomycin treated cell differentiation products. If no cells survive in the mitomycin treated cell differentiation product, this indicates no EPSC remains; if the cell differentiation product has survival cells, the EPSC residues are shown, the EPSC is not completely converted into the iPSC, the conversion time is also required to be prolonged, and the residual EPSC is prevented from weakening the subsequent differentiation efficiency of the iPSC cells.
In some embodiments, the cell G1 phase blocker is not limited to the agents of the application as described above, as long as the agents act to block the growth of EPSCs in the G1 phase, act to poison EPSCs in the S phase and G2 phase, and act to poison iPSCs are within the scope of the application. In a specific embodiment of the application, the cell G1 phase blocker is preferably mitomycin.
In a fourth aspect, the application provides a kit for detecting EPSC residues in an EPSC-derived iPSC.
Further, the kit comprises a cell G1 phase blocker;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin;
preferably, the kit also comprises an iPSC culture medium;
more preferably, the iPSC medium is STEM CELL TeSR TM -E8 TM 。
Further, the kit further comprises a substrate suitable for culturing a cell differentiation product obtained when EPSCs differentiate into ipscs, preferably the substrate is a plastic culture vessel (e.g., a petri dish), a multi-well plate or a flask.
Further, the kit includes instructions for using the kit, which describe how to use the kit to detect whether EPSC residues are present in ipscs derived from EPSCs.
Compared with the prior art, the application has the advantages and beneficial effects that:
the application firstly uses mitomycin in the identification of EPSC and iPSC, and provides a detection method for identifying whether EPSC is completely converted into iPSC based on mitomycin for the first time, the method has the advantages of high detection efficiency, low cost, high accuracy, simple operation, strong universality and wide application range, and provides a new thought for the detection of EPSC residues in the iPSC of EPSC sources in the field. The method is suitable for quality monitoring of the iPSC product obtained by EPSC differentiation, provides reliable cell products for clinic, can greatly shorten the time for EPSC residue detection, accelerates the preparation process of the cell products, and has good application prospect.
Drawings
FIG. 1 is a graph showing the results of the growth states of EPSC and iPSC under the action of mitomycin (0.6. Mu.g/mL) at the same concentration, wherein, in the graph A: EPSC+0.6. Mu.g/mL mitomycin, 20X; b, drawing: ipsc+0.6 μg/mL mitomycin, 20×;
FIG. 2 is a graph showing the results of the growth states of EPSC and iPSC under the action of mitomycin at different concentrations, wherein, the graph A: EPSC+0.3. Mu.g/mL mitomycin, 20X; b, drawing: EPSC+0.6. Mu.g/mL mitomycin, 20X; c, drawing: ipsc+0.3 μg/mL mitomycin, 20×; d, drawing: ipsc+0.6 μg/mL mitomycin, 20×; FIG. 3 is a graph showing the effect of mitomycin on EPSC cell cycle, wherein, panel A: EPSC control (without mitomycin treatment) for 24h; b, drawing: EPSC 0.3 μg/mL mitomycin treatment group for 24h; c, drawing: iPSC control (without mitomycin treatment) for 24h; d, drawing: iPSC 0.3 μg/mL mitomycin treatment group for 24h; e, drawing: cell cycle outcome statistics;
FIG. 4 is a statistical plot of the effect of mitomycin treatment at 0.3 μg/mL for 24h on EPSC cell cycle arrest.
Detailed Description
The inventor of the application unexpectedly found that EPSCs and iPSCs have obvious differences in cell cycle in early experimental study, wherein EPSCs have G1 phase checkpoints, while iPSCs corresponding to inner cell clusters do not have G1 phase checkpoints, and based on the differences of EPSCs and iPSCs in cell cycle regulation, the application provides a detection method for identifying whether EPSCs are completely converted into iPSCs, and the method utilizes mitomycin to interfere with EPSC differentiation products, and distinguishes the EPSCs and the iPSCs according to different performances of the EPSCs under the action of mitomycin. The application uses mitomycin for identifying EPSC and iPSC for the first time, and obtains unexpected technical effects.
After treatment with mitomycin, mitomycin can act on the G1 phase checkpoint of EPSCs, affect the cell cycle, block cells in the G1 phase, and have toxic effects on EPSCs in other phases, so that with the prolonged action time of mitomycin, the phenomenon of growth arrest of part of EPSC cells in the G1 phase occurs, and the cell proportion in the G1 phase also gradually increases. Whereas ipscs all entered apoptosis under the action of mitomycin due to the absence of G1 phase checkpoint.
If all cells in the differentiation product are detected to be dead cells after mitomycin treatment, the EPSC is completely differentiated into iPSC; if living cells are detected in the differentiation product, this indicates that EPSC remains in the differentiation product. A higher detected cell viability indicates a lower EPSC differentiation efficiency, and a lower detected cell viability indicates a higher EPSC differentiation efficiency.
Unless defined otherwise, all technical and scientific terms used in the context of the present application have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application, as well as some of the terminology will be interpreted as follows.
The term "induced pluripotent stem cells (Induced pluripotent stem cell, iPSC)" refers to stem cells having totipotency or multipotency obtained from certain adult cells (e.g., fibroblasts) by artificially inducing the expression of certain genes. In some methods known in the art, ipscs can be obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Transfection may be achieved by viral transduction using viruses such as retroviruses or lentiviruses. In some methods, the transfected genes may include the transcription factors Oct4, sox2, klf4, and c-Myc, although simultaneous transfection of other genes may potentially increase the efficiency of induction. In other methods, lentiviral systems can be used to transform somatic cells using Oct4, sox2, nanog, and Lin28 genes. Genes that induce expression in ipscs include (but are not limited to) Oct-3/4; some members of the Sox gene family (e.g., sox l, sox2, sox3, and Sox 15); some members of the Klf family (e.g., klf1, klf2, klf4, and Klf 5), some members of the Myc family (e.g., C-Myc, L-Myc, and N-Myc), nanog, lin28, tert, fbx15, ERas, ECAT15-1, ECAT15-2, tcl1, beta-Catenin, ECAT1, esg1, dnmt3L, ECAT, gdf3, fth117, sal14, rex1, UTF1, stilla, stat3, grb2, prdm14, nr5a1, nr5a2, or E-cadherein, or any combination thereof. Various reagents for preparing ipscs, such as reprogramming vectors, expression cassettes, media, etc., and even commercial ipscs, are currently commercially available.
The term "expanded pluripotent stem cells (Expanded Potential Stem Cells, EPSC)", also known as pluripotent expanded stem cells, expanded pluripotent stem cells, have the property of being "naive" or in the ground state and have the great potential to differentiate into extra-embryonic cell lines (trophoblasts and extra-embryonic endoderm in the yolk sac) and resident embryonic cells derived from the cell population inside the blastocyst. EPSCs are not only capable of developing into any type of cell, but also have developmental potential beyond Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (ipscs). EPSCs are useful in scientific research and development such as disease modeling, therapeutic screening, toxicity testing, genetic disease research, and reproductive biology research. EPSCs can be used to produce functional cells in vitro, and currently EPSCs have been successfully differentiated into a variety of cell types such as pancreatic cells, neurons, T cells, and the like.
The term Mitomycin (Mitomycin C) is an antitumor drug separated from a culture solution of actinomycetes, is effective on various solid tumors, and is one of the commonly used periodic nonspecific drugs. Structurally, the three effective groups of benzoquinone, uratam and ethylene imine are provided. Acting after intracellular activation by reductase, DNA can be depolymerized while antagonizing DNA replication. Also has inhibitory effect on RNA and protein synthesis at high concentrations. Mainly acts on late G1 and early S phases. Also has effect under acidic and hypoxic conditions.
The term "arrest", also known as "cell cycle arrest", requires the coordination of a large number of intracellular and extracellular signals, and in the absence of an appropriate signal, cells will not be able to pass from one stage to the next, a phenomenon known as cell cycle arrest. Cell cycle arrest helps maintain gene stability and cell cycle regulated gene mutations play an important role in the development of tumors. When the cell cycle is normal, if damage occurs to the DNA, the cell cycle stops at the corresponding checkpoint and the cell cycle arrest provides additional time for the cell to repair the damage, thereby reducing the occurrence of mutations and avoiding the generation of tumors.
The term "medium" refers to a cell culture environment. The culture medium is typically an isotonic solution and may be liquid, gel-like or semi-solid, for example, to provide a matrix for cell adhesion or support. As used herein, the medium may include components of nutritional, chemical, and structural support necessary to culture the cells.
The application is further illustrated below in conjunction with specific examples, which are intended to illustrate the application and are not to be construed as limiting the application. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the application, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
Example 1 identification procedure of residual EPSC in iPSC
1. Experimental materials
iPSC medium: STEM CELL TeSR TM -E8 TM Catalog#05990；
Cell cycle detection kit: biyun Tian Biotechnology C1052.
2. Experimental method
(1) Transformation of EPSC cell culture into iPSC cell Using iPSC Medium
The specific experimental method is as follows: EPSC cells were grown at 1X 10 using EPSC medium 4 -5×10 4 Inoculating individual CELLs/square centimeter on a culture plate coated with matrigel, and replacing the iPSC culture medium the next day, wherein the iPSC culture medium used in the application is STEM CELL TeSR TM -E8 TM . And after the cells are completely grown up for 2-3 days, passaging, and obtaining the iPSC cells obtained by EPSC transformation after passaging.
(2) Identifying whether EPSC remains in the transformed iPSC cells
Killing the iPSC cells obtained by transformation by adopting mitomycin, detecting the survival state of the cells, and when all the cells are killed, indicating that EPSCs are all transformed into iPSC; when the cells survive, the EPSC is not completely converted into the iPSC, and the conversion time is further prolonged, so that the residual EPSC is prevented from influencing the subsequent differentiation efficiency of the iPSC.
EXAMPLE 2 fumbling of mitomycin concentrations
1. Experimental method
(1) Effect of mitomycin at the same concentration on EPSC and iPSC growth status
EPSC and iPSC were treated with mitomycin at the same concentration (0.6. Mu.g/mL), respectively, as follows: 2X 10 4 EPSCs and iPSCs were seeded per square centimeter in matrigel coated plates, and mitomycin was added at a stock concentration of 25mg/mL the next day when the stock was changed. Cells were changed every day, and fresh mitomycin was added during the change. Co-cultivation was carried out for 72 hours.
(2) Effect of different concentrations of mitomycin on EPSC, iPSC growth status
EPSC and iPSC were treated with mitomycin at different concentrations (10. Mu.g/mL, 30. Mu.g/mL, 60. Mu.g/mL), respectively, as follows: 2X 10 4 The EPSCs and iPSCs were seeded per square centimeter in matrigel coated plates, and mitomycin at the different concentrations described above was added the next day after the plating, and all cells died after 24 hours.
(3) Effect of different concentrations of mitomycin on EPSC, iPSC growth status
The EPSC and the iPSC were treated with mitomycin at different concentrations (0.3. Mu.g/mL, 0.6. Mu.g/mL, 1. Mu.g/mL), respectively, as follows: 2X 10 4 And inoculating EPSC and iPSC into a culture plate coated with matrigel, adding mitomycin with different concentrations in the next day of liquid exchange, and adding new mitomycin in the process of liquid exchange every day, and culturing for 72 hours.
(4) Effect of different concentrations of mitomycin on EPSC, iPSC growth cycle
EPSC and iPSC were treated with mitomycin at different concentrations (0.3. Mu.g/mL, 0.6. Mu.g/mL), respectively, withThe body test method is as follows: 2X 10 4 The EPSC and the iPSC are inoculated to a culture plate coated by matrigel per square centimeter, mitomycin with the stock solution concentration of 25mg/mL, the final concentration of 0.3 mug/mL and 0.6 mug/mL is respectively added before the liquid change in the next day, and the cell culture plate is placed into an incubator for culturing for 1.5 hours. After 1.5h the medium containing mitomycin was aspirated and fresh cell medium was replaced.
2. Experimental results
The results of the growth states of EPSC and iPSC under the action of mitomycin with the same concentration (0.6 mug/mL) are shown in figures 1A-B, and the results show that the growth states of EPSC and iPSC are quite obviously different under the action of mitomycin with the same concentration, which indicates that the mitomycin can be used for identifying the EPSC and the iPSC.
However, the high concentration of mitomycin, both EPSC and iPSC, has a deleterious effect and the activity of the cells is greatly affected, so this example has been developed for the concentration of mitomycin used. At this stage, the designed gradient had 10. Mu.g/mL, 30. Mu.g/mL, 60. Mu.g/mL, and the mitomycin concentrations described above were found to be significant in cell toxicity.
The mitomycin concentration was then reduced and a gradient of 0.3. Mu.g/mL, 0.6. Mu.g/mL, 1. Mu.g/mL was designed. At these concentrations, ipscs perform consistently and die. While EPSCs did not differ significantly from one concentration group to another after 24h of mitomycin exposure, EPSCs from the mitomycin-treated group at 0.3. Mu.g/mL and 0.6. Mu.g/mL still had cell growth after 72h of mitomycin exposure, while EPSCs from the 1. Mu.g/mL experimental group all died, as shown in FIGS. 2A-D, indicating that mitomycin at 0.3. Mu.g/mL or 0.6. Mu.g/mL treatment time for 72h could be used for effective discrimination of EPSCs and iPSCs.
Example 3 Effect of mitomycin on EPSC cell cycle
1. Experimental method
Mitomycin can act on the G1 phase checkpoint of EPSC, and the effect of mitomycin on EPSC cell cycle is observed by detection of cell cycle as follows: cells were fixed with absolute ethanol for 2 hours after digestion, DNA was stained with Propidium Iodide (PI), and the fluorescence intensity of PI was detected by flow cytometry to determine cell cycle.
2. Experimental results
The results showed that with increasing culture time after mitomycin action, the cell fraction in G1 phase increased and the cell fractions in S and G2 phases decreased (see FIGS. 3A-E), indicating that mitomycin retarded the growth of EPSC cells in G1 phase, and the cells with G1 fraction increased from 22.4% to 32.7%, increased by about 45% and had a deleterious effect on EPSC cells in S and G2 phases. Whereas iPSC was treated for the same time, it was not found that the G1 phase was prolonged, and mitomycin at a concentration of 0.3. Mu.g/mL gave the best effect of blocking the EPSC cell cycle (see FIG. 4), and the G1 phase cell ratio was increased from about 20% to about 60%, so that the present application chose 0.3. Mu.g/mL for the experiment.
Example 4 optimized protocol for optimal identification of iPSC and EPSC
The transformed iPSC was inoculated with 1.2w-3.4w per square centimeter, and inoculated in iPSC medium (STEM CELL TeSR TM -E8 TM Catalog # 05990), after 24 hours of mitomycin addition at 0.3 μg/mL, the survival status of the cells was checked by flow cytometry, and if no cells survived, it indicated that all EPSC had been converted to iPSC, and if there were still cells survived, it indicated that there was still residual EPSC cells, and it was also necessary to extend the transformation time of iPSC, preventing EPSC residues from weakening the subsequent differentiation efficiency of iPSC cells.
The above description of the embodiments is only for the understanding of the method of the present application and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present application without departing from the principle of the application, and these improvements and modifications will fall within the scope of the claims of the application.

Claims (10)

1. The application of cell G1 phase retarder in identifying EPSC and iPSC;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin.
2. The use according to claim 1, wherein the mitomycin acts as a block for the growth of EPSC in G1 phase.
3. The use according to claim 2, wherein the mitomycin is toxic to EPSC in S-phase and G2-phase.
4. The use according to claim 3, wherein the mitomycin is toxic to ipscs.
5. A test method for identifying whether EPSC is fully converted to iPSC, said method comprising the steps of: treating a cell differentiation product obtained when EPSC is converted into iPSC by using a cell G1 phase retarder, and detecting the survival state of cells in the cell differentiation product;
preferably, if no cells in the cell differentiation product survive, it indicates that EPSC are all converted to iPSC;
preferably, if there is still cell survival in the cell differentiation product, it indicates that there is a residual of EPSC cells, EPSC not completely converted to iPSC;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin.
6. The method of claim 5, wherein the mitomycin is used at a concentration of 0.1-1.0 μg/mL;
preferably, the mitomycin is used at a concentration of 0.3-0.6 μg/mL;
more preferably, the mitomycin is used at a concentration of 0.3 μg/mL or 0.6 μg/mL;
most preferably, the mitomycin is used at a concentration of 0.3 μg/mL.
7. The method of claim 6, wherein the mitomycin is treated for 24 to 120 hours;
preferably, the treatment time of the mitomycin is 24-72 hours;
more preferably, the mitomycin is treated for 24 hours or 72 hours;
most preferably, the mitomycin is treated for 24 hours.
8. The method of claim 5, wherein the cell differentiation product is present in an amount of 1X 10 4 -5×10 4 Inoculating individual cells per square centimeter into an iPSC culture medium;
preferably, the cell differentiation product is present in an amount of 1.2X10 4 -3.4×10 4 Inoculating cells per square centimeter;
preferably, the iPSC medium is STEM CELL TeSR TM -E8 TM 。
9. Application of cell G1 phase retarder in EPSC residue detection in EPSC-derived iPSC;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin;
most preferably, the detection is performed using the method of any one of claims 5-8;
most preferably, if no cells survive in the cell differentiation product, this indicates that there is no EPSC remaining;
most preferably, the presence of EPSC residue is indicated if there is still cell survival in the cell differentiation product.
10. A kit for detecting EPSC residues in EPSC-derived ipscs, the kit comprising a cell G1 phase blocker;
preferably, the cell G1 phase blocker comprises any one or more of mitomycin, neofomycin, epirubicin, doxorubicin, tunicamycin, vincristine, sodium valproate, nituzumab, 5-fluorouracil, cisplatin, fluoxetine, sorafenib, indomethacin, rebaudinib, arbelide, pip Bai Xili, paclitaxel, epigallocatechin gallate, hydrogen peroxide, octreotide, X-ray irradiation;
more preferably, the cell G1 phase blocker is mitomycin;
preferably, the kit also comprises an iPSC culture medium;
more preferably, the iPSC medium is STEM CELL TeSR TM -E8 TM 。
CN202310941706.7A 2023-07-28 2023-07-28 Method for rapidly identifying iPSC and EPSC Pending CN116925999A (en)

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