Stable passage guinea pig pluripotent stem cell and preparation method thereof

Abstract

The invention provides guinea pig pluripotent stem cells capable of being stably passaged in vitro and a method for constructing pluripotent stem cells capable of being stably passaged from guinea pig embryos.

Description

Technical Field

[0001] The present invention relates to the field of stem cells, in particular to guinea pig-derived pluripotent stem cells and a preparation method and a culture reagent thereof. Background Art

[0002] Guinea pigs (Cavia porcellus) are closer to humans than mice in terms of reproductive development, organ structure, immune response, etc. Before the 1990s, they were one of the most widely used animal models for disease simulation and physiological development research, such as infectious diseases, preclinical studies of asthma, chronic obstructive pulmonary disease and arrhythmia. However, as embryonic stem cell lines and pluripotent stem cell lines that can be stably propagated in vitro are gradually established in other animals (such as mice, rats, pigs, humans, etc.), the difficulty of modeling and engineering transformation of these animals with stable stem cell lines is greatly reduced, while guinea pigs have been forced to gradually withdraw from the stage of mainstream animal models because they have not been able to establish stem cell lines (including embryonic stem cells and pluripotent stem cells) that can be stably propagated (Non-patent Document 1). Our research found that the sensitivity of guinea pig stem cells to important signal pathways is different from that of other rodents such as mice, but closer to humans. Therefore, the methods and reagents for preparing and culturing embryonic stem cells and pluripotent stem cells of rodents such as mice are often observed to be unqualified when used in guinea pigs.

[0003] Prior art has reported that guinea pig iPS cells (giPS) (non-patent literature 2) are obtained by using exogenous gene reprogramming, but the document has been withdrawn, and the reprogramming method cannot reliably prepare guinea pig pluripotent stem cells. Moreover, the reprogramming method inevitably has the risk of random integration of viral vectors into chromosomes and introduction of exogenous reprogramming factors. Some research groups have reported that iPS cells have defects in reprogramming errors and genomic instability (non-patent literature 3 to 5), and these defects will likely cause the clinical treatment potential of iPS cells to be limited. In addition, the method for inducing pluripotent stem cells by reprogramming also inevitably has common problems such as impure genetic background, low induction efficiency, long cycle, and low cell purity.

[0004] Pluripotent stem cells of mammals (e.g., primates or rodents) have the ability to differentiate into various tissues and organs in vivo and in vitro, and can be infinitely expanded in vitro. Therefore, they have always been regarded as important donor cells for tissue and organ regeneration. According to the degree of pluripotency, pluripotent stem cells can be further divided into primitive state, and the primed state. It is generally believed that naive pluripotent stem cells originate from pre-implantation embryos and not only have properties similar to the inner cell mass (ICM) of pre-implantation embryos but also have the ability to differentiate into primordial germ cells (PGCs). Therefore, when naive pluripotent stem cells are injected into blastocysts, chimeric animals with germline transmission ability can be generated, which is a crucial property for constructing animal models. Primed cells generally originate from post-implantation embryos, mainly the epiblast (EPI) cells formed after implantation. This type of pluripotent stem cell has lost the ability to form primordial germ cells, so its pluripotency is lower than that of naive pluripotent stem cells (Non-Patent Document 6 and Non-Patent Document 7). The culture conditions of naive PSCs show species specificity, and the developmental window for forming pluripotency is short. In contrast, primed PSCs can maintain a longer developmental period, and the self-renewal performance has more similarities among species.

[0005] It has been reported that epiblast pluripotent stem cell lines (Epiblast Stem Cell, namely EpiSC, called mouse mEpiSC and pig pgEpiSc for the two species respectively) have been established using the epiblast cells of pre-gastrula embryos of mice and pigs. Their transcriptome characteristics are similar to those of EPI cells at the gastrula stage and belong to primed pluripotent stem cells (Non-Patent Document 8 and Non-Patent Document 9). It has also been reported that there are differences between mouse pluripotent stem cells and human pluripotent stem cells in terms of the main signal pathway dependence for maintaining pluripotency and differentiation potential. For example, mice mainly rely on the ActA / SMADs signaling pathway to maintain Nanog expression, while humans rely on both the FGF / MEK and ActA / SMADs dual signaling pathways (Non-Patent Document 10); human primed pluripotent stem cells can be directly induced into trophoblast stem cells, while mouse cells are generally considered not to have this ability (Non-Patent Document 11). Our research found that guinea pig pluripotent stem cells have properties more similar to human pluripotent stem cells than mice.

[0006] The prior art has never reported a pluripotent stem cell line that can be stably passaged in vitro and successfully established from guinea pig embryos, nor has it reported research on distinguishing the naive and primed properties of guinea pig pluripotent cell lines.

[0007] References

[0008] Non-patent literature:

[0009] Non-Patent Document 1: Maxeiner S et al., Of mice and men - and guinea pigs? Ann Anat. 2021 Nov; 238: 151765.

[0010] Non-Patent Document 2: Yuehong Wu et al., Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts, Mol Med Rep. 2017 Jun; 15(6): 3690–3698;

[0011] Non-Patent Document 3: Gore, A. et al., Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63–67 (2011);

[0012] Non-Patent Document 4: Article Hussein, S.M. et al., Copy number variation and selection during reprogramming to pluripotency. Nature 471, 58–62 (2011);

[0013] Non-Patent Document 5: Article Lister, R. et al., Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011);

[0014] Non-Patent Document 6: Hackett JA et al., Regulatory Principles of Pluripotency: From the Ground State Up[J]. Cell Stem Cell, 2014, 15(4): 416-430;

[0015] Non-Patent Document 7: Nichols J et al., Naive and Primed Pluripotent States[J]. Cell Stem Cell, 2009, 4(6): 487-492;

[0016] Non - Patent Document 8: Kojima Y et al., The Transcriptional and Functional Properties of Mouse Epiblast Stem Cells Resemble the Anterior Primitive Streak[J]. Cell Stem Cell, 2014, 14(1): 107 - 120;

[0017] Non - Patent Document 9: Minglei Zhi et al., Generation and characterization of stable pig pregastrulation epiblast stem cell lines, Cell Research volume 32, pages 383–400(2022);

[0018] Non - Patent Document 10: Boris Greber et al., Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell. 2010 Mar 5; 6(3): 215 - 26;

[0019] Non - Patent Document 11: Sergey Viukov et al., Human primed and PSCs are both able to differentiate into trophoblast stem cells. Stem Cell Reports. 2022 Nov 8; 17(11): 2484 - 2500. Summary of the Invention

[0020] Through in-depth research, the inventors of the present application have for the first time discovered that the epiblast cells of early-stage implanted embryos of guinea pigs (especially embryos at 9.5 to 11.5 days) can be used to construct a pluripotent stem cell line (Guinea pig epiblast stem cells, GpEpiSC) that can be stably passaged in vitro. The resulting GpEpiSC cells exhibit the ability to proliferate infinitely and the potential to differentiate into cells of the three germ layers, and can still maintain a stable compact colony morphology and normal karyotype after multiple passages, have the ability to form teratomas, and show transcriptome characteristics close to the naive state, thus obtaining a pluripotent stem cell line that can be stably maintained in the naive state. Therefore, the GpEpiSC cells disclosed herein can be used as biological and medical research tools, providing a cell model that is closer to humans than other rodent pluripotent stem cells such as mice and rats.

[0021] Throughout the entire process of preparing and passaging GpEpiSC cells in this application, there is no need to introduce any exogenous genetic material (that is, no heterologous genes are introduced, no gene editing is performed, etc.), maintaining the genetic background purity and traceability of the cell line and avoiding the risks caused by impure genetic backgrounds (such as breakthroughs caused by random insertion of exogenous genes).

[0022] The GpEpiSC cells disclosed herein or the GpEpiSC cells prepared by the methods disclosed herein have all the common characteristics known for pluripotent stem cells, including the characteristics of infinite proliferation and the ability to differentiate into cell lineages of the three germ layers (ectoderm, endoderm, and mesoderm), and can be stably passaged under in vitro culture conditions. Therefore, the GpEpiSC cells disclosed herein or the GpEpiSC cells prepared by the methods disclosed herein can be used as cell factories in the biomedical industry like other known rodent pluripotent stem cell lines or non-rodent mammalian pluripotent stem cell lines, and have broad industrial application values. For example, they may be precisely gene-edited multiple times, thereby generating various gene-modified cell models; or be subjected to directed chemical induction or reprogramming induction to produce the expected differentiated cell populations.

[0023] Therefore, on the one hand, the present invention provides isolated guinea pig pluripotent stem cells, which are derived from the epiblast of guinea pig embryos, can be stably passaged at least 3 times, and have the potential to differentiate into endoderm, ectoderm, and mesoderm cells.

[0024] For example, in different embodiments, the isolated guinea pig pluripotent stem cells can be stably passaged for at least 5 generations, at least 8 generations, at least 10 generations, at least 15 generations, at least 20 generations, at least 25 generations, at least 30 generations, at least 35 generations, at least 40 generations, at least 45 generations, at least 50 generations, at least 55 generations, at least 60 generations, at least 65 generations, at least 70 generations, at least 75 generations, at least 80 generations, at least 85 generations, at least 90 generations, at least 95 generations, at least 100 generations, and the like.

[0025] In some possible embodiments, the guinea pig pluripotent stem cells are primed pluripotent stem cells. State pluripotent stem cells or formative state pluripotent stem cells.

[0026] In some possible embodiments, the guinea pig pluripotent stem cells are derived from the epiblast of a guinea pig embryo with a gestation period of less than E12.5, preferably from the epiblast of a guinea pig embryo with a gestation period of E9.5 to E12.5, and more preferably from the epiblast of a guinea pig embryo with a gestation period of E9.5 to E11.5. For example, in some specific embodiments, the guinea pig pluripotent stem cells can be derived from any one or more of the epiblast of a guinea pig embryo with a gestation period of E9.5, the epiblast of a guinea pig embryo with a gestation period of E9.8, the epiblast of a guinea pig embryo with a gestation period of E10, the epiblast of a guinea pig embryo with a gestation period of E10.5, the epiblast of a guinea pig embryo with a gestation period of E11, the epiblast of a guinea pig embryo with a gestation period of E11.2, the epiblast of a guinea pig embryo with a gestation period of E11.5, the epiblast of a guinea pig embryo with a gestation period of E12, and the epiblast of a guinea pig embryo with a gestation period of E12.5. For example, in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E9.5 to E10; in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E10 to E10.5; in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E10.5 to E11; in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E11 to E11.5; in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E11.5 to E12; in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E12 to E12.5; in some specific embodiments, the guinea pig pluripotent stem cells can be derived from the epiblast of a guinea pig embryo with a gestational period of E10.5 to E11.5; and so on.

[0027] In some possible embodiments, the guinea pig pluripotent stem cells have an activated FGF signaling pathway and / or ActA signaling pathway, preferably having both an activated FGF signaling pathway and ActA signaling pathway. In some possible embodiments, the guinea pig pluripotent stem cells have an inhibited or turned-off Wnt signaling pathway.

[0028] In some possible embodiments, the guinea pig pluripotent stem cells express any one selected from Nanog, Pou5F1, and Sox2. In some embodiments, the guinea pig pluripotent stem cells express Nanog, Pou5F1, and Sox2 simultaneously. In some embodiments, the guinea pig pluripotent stem cells express ETV4 and / or ZNF281. In some embodiments, the guinea pig pluripotent stem cells substantially do not express EOMES and / or OTX2.

[0029] In some possible embodiments, the guinea pig pluripotent stem cells maintain a normal karyotype; and / or, compared with guinea pig unipotent cells or guinea pig differentiated cells (such as guinea pig fibroblasts), the gene expression levels of one of the following functions are downregulated: endosome to lysosome transport, centrosome cycle, chromatin organization, neural tube closure, glycolytic process, epithelial cell differentiation, muscle cell development, neuronal action potential propagation, positive regulation of cell migration, negative regulation of canonical, Wnt signaling pathway, or bone morphogenesis; and / or

[0030] The guinea pig pluripotent stem cells have up-regulated gene expression levels of one of the following functions compared to guinea pig unipotent cells or guinea pig differentiated cells (such as guinea pig fibroblasts): mRNA splicing via spliceosome, rRNA processing, regulation of cell cycle, nucleosome mobilization.

[0031] Another aspect of the present invention also provides a genetically modified guinea pig pluripotent stem cell, which is obtained by genetically modifying the above-mentioned guinea pig pluripotent stem cell.

[0032] In some embodiments, the genetic modification includes genome editing, preferably including nucleic acid fragment deletion, gene modification, gene knockout, alteration of gene product expression, repair of mutations, insertion of polynucleotides, single-base mutations, or any combination thereof.

[0033] Another aspect of the present invention also provides an isolated cell population comprising the above-mentioned guinea pig pluripotent stem cells.

[0034] In some possible embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or about 100% of the cells in the cell population are the above-mentioned guinea pig pluripotent stem cells.

[0035] Another aspect of the present invention also provides a monoclonal cell line isolated from the above-mentioned guinea pig pluripotent stem cells.

[0036] Another aspect of the present invention also provides the use of the above-mentioned guinea pig pluripotent stem cells, the above-mentioned cell population, and the above-mentioned monoclonal cell line for producing transgenic animals or performing gene editing.

[0037] Another aspect of the present invention also provides a method for generating guinea pig pluripotent stem cells that can be stably passaged in vitro for at least 3 generations, comprising the following steps:

[0038] (1) Obtaining a cell mass of epiblast (EPI) that is substantially separated from the visceral endoderm (VE) from a guinea pig embryo;

[0039] (2) Inoculating the cell mass separated in step (1) onto a pre-prepared feeder layer;

[0040] (3) Cultivate until the cell mass produces an outgrowth. After separating the outgrowth from the feeder layer, obtain smaller cell masses from the outgrowth again, inoculate the smaller cell masses onto a fresh feeder layer, and complete one passage;

[0041] (4) After at least 3 passages, preferably 3 - 5 passages, more preferably 5 passages according to step (3), digest the outgrowth into single cells and passage; and

[0042] Optionally, (5) Obtain single cell clones from the passaged cells in step (4) to establish a guinea pig pluripotent stem cell line.

[0043] In some possible embodiments, the guinea pig embryo in step (1) is a guinea pig embryo with a gestation period of less than E12.5, preferably between E9.5 and E12.5, more preferably between E9.5 and E11.5.

[0044] In some possible embodiments, step (1) obtains cell masses by mechanical and / or enzymatic digestion. In some specific embodiments, optionally, after separating the epiblast from the visceral endoderm with a metal tool, directly divide the epiblast into masses. The metal tool is preferably a wire or forceps; or optionally, remove the extraembryonic ectoderm, turn the guinea pig embryo over to expose the epiblast, and digest with trypsin for 2 - 3 minutes.

[0045] In some possible embodiments, the culture medium used for inoculation in step (2) and the culture medium used for inoculation within 10 passages in step (4) contain 5 - 15 μM, preferably 8 - 12 μM, more preferably 9 - 10 μM of a Rock signaling pathway inhibitor.

[0046] In some possible embodiments, the culture medium used for cultivation in step (3) and the culture medium used for inoculation after 10 passages in step (4) contain 0 - 5 μM, preferably 2 - 3 μM of a Rock signaling pathway inhibitor.

[0047] In some possible embodiments, the Rock signaling pathway inhibitor is preferably selected from at least one of the following: Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, and more preferably Y-27632.

[0048] In some possible embodiments, the culture medium used in steps (2) to (4) further contains an FGF signaling pathway agonist and / or a Wnt signaling pathway inhibitor. Preferably, the FGF signaling pathway agonist is FGF2. In some embodiments, the Wnt signaling pathway inhibitor is an inhibitor of the canonical Wnt signaling pathway. In some embodiments, the Wnt signaling pathway inhibitor is an inhibitor of the non-canonical Wnt signaling pathway. In a preferred embodiment, the Wnt signaling pathway inhibitor is a combination of an inhibitor of the canonical Wnt signaling pathway and an inhibitor of the non-canonical Wnt signaling pathway. In some embodiments, the Wnt signaling pathway inhibitor is selected from IWR-1-endo, IWP2, or XAV939, or any combination thereof. Preferably, the content of the FGF signaling pathway agonist is 5 to 50 ng / ml, more preferably 10 to 40 ng / ml, and further preferably 20 to 30 ng / ml. Preferably, the content of the Wnt signaling pathway inhibitor or its combination is 1 to 6 μM, 1.5 to 5.5 μM, 2 to 5 μM, 2.1 to 5 μM, 2.2 to 5 μM, 2.3 to 5 μM, 2.4 to 5 μM, 2.5 to 5 μM, and more preferably 2 to 5 μM of IWR-1-endo, IWP2, or XAV939, or 2.5 to 5 μM of IWR-1-endo, IWP2, or XAV939, or a combination of 2.5 μM of IWR-1-endo and 100 nM of IWP2, or a combination of 2 μM of XAV939 and 100 nM of IWP2.

[0049] In some possible embodiments, when single-cell passage is performed in step (4), the passage is carried out once every 3 to 4 days, and the passage ratio is 1:5 to 1:10.

[0050] In some possible embodiments, the guinea pig pluripotent stem cells prepared by the above method can be stably passaged in vitro for at least 3 generations, preferably at least 5 generations, more preferably at least 10 generations, further preferably at least 20 generations, still further preferably at least 40 generations, and most preferably at least 70 generations.

[0051] For example, in some specific embodiments, the guinea pig pluripotent stem cells prepared by the above method can be stably passaged in vitro for at least 15 generations, at least 18 generations, at least 20 generations, at least 25 generations, at least 28 generations, at least 30 generations, at least 35 generations, at least 38 generations, at least 40 generations, at least 45 generations, at least 50 generations, at least 55 generations, at least 60 generations, at least 65 generations, at least 68 generations, at least 70 generations, at least 72 generations, at least 75 generations, at least 78 generations, at least 80 generations, at least 85 generations, at least 90 generations, at least 95 generations, at least 100 generations, at least 120 generations, at least 150 generations, at least 180 generations, at least 200 generations, at least 250 generations, at least 300 generations, at least 350 generations, at least 400 generations, at least 450 generations, at least 500 generations, at least 600 generations, at least 700 generations, at least 800 generations, or at least 900 generations, and so on.

[0052] On the other hand, the present invention also provides the use of a Rock signaling pathway inhibitor in the separation, preparation, culture and / or maintenance of guinea pig pluripotent stem cells that can be stably passaged for at least 3 generations. The Rock signaling pathway inhibitor is preferably selected from at least one of the following: Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, and more preferably Y-27632.

[0053] In some possible embodiments, the working concentration of the Rock signaling pathway inhibitor is 1 to 20 μM, preferably 2 to 15 μM, more preferably 2 to 10 μM, and most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM. In some possible embodiments, the Rock signaling pathway inhibitor is also used in combination with an FGF signaling pathway agonist and / or a Wnt signaling pathway inhibitor. Optionally, the agonist of the FGF signaling pathway is FGF2. Preferably, the Wnt signaling pathway inhibitor is selected from IWR-1-endo, IWP2 or XAV939, or any combination thereof. In some possible embodiments, the content of the FGF signaling pathway agonist is 5 to 50 ng / ml, more preferably 10 to 40 ng / ml, and further preferably 20 to 30 ng / ml. In some possible embodiments, the content of the Wnt signaling pathway inhibitor or its combination is 1 to 6 μM, 1.5 to 5.5 μM, 2 to 5 μM, 2.1 to 5 μM, 2.2 to 5 μM, 2.3 to 5 μM, 2.4 to 5 μM, 2.5 to 5 μM, more preferably 2 to 5 μM of IWR-1-endo, IWP2 or XAV939, or 2.5 to 5 μM of IWR-1-endo, IWP2 or XAV939, or a combination of 2.5 μM of IWR-1-endo and 100 nM of IWP2, or a combination of 2 μM of XAV939 and 100 nM of IWP2.

[0054] Another aspect of the present invention also provides a medium for separating, preparing, culturing and / or maintaining guinea pig pluripotent stem cells that can be stably passaged for at least 3 generations, which comprises a basal medium, a stem cell growth factor, a Rock signaling pathway inhibitor, an agonist of the FGF signaling pathway and a Wnt signaling pathway inhibitor.

[0055] In some possible embodiments, the basal medium is selected from DMEM / F12 or Neurobasal. In some possible embodiments, the stem cell nutrient factors are selected from N2 serum-free additive, B27 serum-free additive, GlutaMax, MEM NEAA, or BSA, or any combination thereof. In some possible embodiments, the Rock signaling pathway inhibitor is selected from at least one of the following: Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, preferably Y-27632. In some possible embodiments, the agonist of the FGF signaling pathway is FGF2. In some possible embodiments, the Wnt signaling pathway inhibitor is selected from IWR-1-endo, IWP2, or XAV939, or any combination thereof.

[0056] In some possible embodiments, the content of the basal medium is 50% (v / v) DMEM / F12 and 50% (v / v) neurobasal. Optionally, the content of the stem cell nutrient factors is 0.5% to 1% (v / v) N2 serum-free additive and 0.5% to 1% (v / v) B27 serum-free additive, 1% (v / v) GlutaMax, 1% (v / v) MEM NEAA, 1% (v / v) penicillin-streptomycin solution, 0.1 mM β-mercaptoethanol, 0 to 1 mg / ml BSA fraction V. Optionally, the content of the Rock signaling pathway inhibitor is 0 to 15 μM, 0 to 10 μM or 2 to 10 μM Y-27632. Optionally, the content of the FGF signaling pathway agonist is 5 ng / ml to 50 ng / ml, more preferably 10 to 40 ng / ml, and further preferably 20 to 30 ng / ml FGF2. Optionally, the content of the Wnt signaling pathway inhibitor is 1 to 6 μM, 1.5 to 5.5 μM, 2 to 5 μM, 2.1 to 5 μM, 2.2 to 5 μM, 2.3 to 5 μM, 2.4 to 5 μM, 2.5 to 5 μM, more preferably 2 to 5 μM IWR-1-endo, IWP2 or XAV939, or 2.5 to 5 μM IWR-1-endo, IWP2 or XAV939, or a combination of 2.5 μM IWR-1-endo and 100 nM IWP2, or a combination of 2 μM XAV939 and 100 nM IWP2.

[0057] Further, the medium contains DMEM / F12, neurobasal, N2 serum-free additive, B27 serum-free additive, GlutaMax, MEM NEAA, penicillin-streptomycin solution, β-mercaptoethanol, Y-27632, FGF2, IWR-1-endo and IWP2.

[0058] In some possible embodiments, every 500 ml of the medium contains 237.5 mL DMEM / F12, 237.5 mL neurobasal, 2.5 ml N2 serum-free additive, 5 ml B27 serum-free additive, 1% (v / v) GlutaMax, 1% (v / v) MEM NEAA, 1% (v / v) penicillin-streptomycin solution, 0.1 mM β-mercaptoethanol, 1 mg / ml BSA fraction V, 20 ng / ml FGF2, 2.5 μM IWR-1-endo, 100 nM IWP2, 2 μM or 10 μM Y-27632. Description of the Drawings

[0059] Figure 1 Shows an example of a guinea pig embryo from E9.5 to E12.5 during pregnancy. The position of the epiblast is schematically shown below the dotted line, and the scale bar in the figure is 200 μm;

[0060] Figure 2 A shows a guinea pig embryo under high magnification and marks three lineages; Figure 2 B shows pushing out the hypoblast (visceral endoderm, VE) outward with forceps to expose the epiblast; Figure 2 C shows the separated epiblast in a disc shape; Figure 2 D shows the outgrowth obtained by mechanically fragmenting the epiblast with forceps and inoculating it onto the feeder layer, which has not been passaged; Figure 2 E shows the outgrowth obtained by dispersing the epiblast into small cell clusters using a glass capillary in combination with enzymatic digestion and then inoculating it onto the feeder layer, which has not been passaged. The scale bar in the figure is 200 μm.

[0061] Figure 3 Is a schematic flow chart for isolating and culturing guinea pig pluripotent stem cells GpEpiSC according to the present application. EPGS represents extraplacental giant cells, EXE represents extraembryonic ectoderm, VE represents visceral endoderm, and EPI represents epiblast;

[0062] Figure 4 Shows an overview of the growth of outgrowths from guinea pig embryos and the isolation and culture of pluripotent stem cells GpEpiSC obtained according to the exemplary embodiments and comparative examples of the present application. The upper figure shows the separated epiblast, and its smooth outer boundary indicates a clear separation from the visceral endoderm. The lower figure shows the outgrowth generated in vitro from the epiblast. The boundary of the P0 outgrowth is circled with a dotted line in the low magnification (5x) image. The high magnification (20x) image more clearly shows the boundary of the outgrowth and cell morphology, and cell clusters are clearly visible. The scale bar is 100 μm;

[0063] Figure 5 Shows the cell growth curve (5A), doubling time (5B), and single cell cloning efficiency (5C) of GpEpiSC (two cell lines derived from E10.5 each) according to the exemplary embodiments of the present application. In the figure, the error bars represent ±SD (n = 3, independent experiments), n.s. represents P≥0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0064] Figure 6 Shows the results of the morphology, AP staining, and immunofluorescence of OCT4, SOX2, and NANOG of clones of GpEpiSC at different passage numbers. The scale bar in the figure is 200 μm;

[0065] Figure 7Shows the karyotype analysis results of GpEpiSC (male) at passage 73 according to an exemplary embodiment of the present application (31 pairs of autosomes and 1 pair of sex chromosomes XY);

[0066] Figure 8 Shows the adherent survival rate and proliferation rate of GpEpiSC according to an exemplary embodiment of the present application. Figure 8 A shows the results of cell adhesion tests under the conditions of the presence of Y27632, FGF2, PD0325901, and / or AZD4547, and three different cell lines at passages 22, 25, and 32 were tested for each condition respectively. Figure 8 B shows the cell proliferation curves of GpEpiSC treated with different concentrations of FGF2, and three different cell lines derived from E10.5 were tested for each concentration. In the figure, the error bars represent ±SD (n = 3, independent experiments), n.s. represents P≥0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0067] Figure 9 Shows the changes in the expression levels of NANOG and POU5F1 of GpEpiSC according to an exemplary embodiment of the present application, and cell lines derived from E10.5 at passages 50 to 54 were used for the test. In the figure, the error bars represent ±SD (n = 3, independent experiments), n.s. represents P≥0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0068] Figure 10A Shows the transcriptome analysis results of GpEpiSC (passage 36) according to an exemplary embodiment of the present application, indicating that the Wnt inhibitors IWR1 and IWP2 affect the overall transcriptome of GpEpiSC;

[0069] Figure 10B Shows Figure 10A the GO analysis results of the differentially expressed genes in

[0070] Figure 11A Shows the morphological and OCT4, SOX2 immunofluorescence results of the GpEpiSC clones of the present application after removing the Wnt inhibitor IWR1 or IWP2, or both, from the culture medium. The scale bar in the figure is 200 μm;

[0071] Figure 11B To show Figure 23A the bar chart of the average fluorescence intensities of OCT4 and SOX2 in . The results of OCT4 were counted from 34 cells, and the results of SOX2 were counted from 36 cells. The error bars represent ±SD, ****P<0.0001;

[0072] Figure 11C Results of qPCR quantitative analysis of the selective pluripotency marker gene and the differentiation marker gene. In the figure, the error bars represent ±SD (n = 3, independent experiments), n.s. represents P≥0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, FRP represents the FRP medium, and FR, FP, and F represent the media after removing IWP2, removing IWR1, or removing both from the FRP medium, respectively;

[0073] Figure 11D Bar graph showing the TPM values of the typical pluripotency genes and the typical differentiation genes, respectively. In the figure, FRP represents the FRP medium, and FR, FP, and F represent the media after removing IWP2, removing IWR1, or removing both from the FRP medium, respectively. In the figure, the error bars represent ±SD (n = 3, independent experiments), n.s. represents P≥0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0074] Figure 12 Shows the pluripotency verification results of GpEpiSC according to an exemplary embodiment of the present application. The scale bar in the figure is 100 μm;

[0075] Figure 13 Shows the hematoxylin and eosin (H&E) staining results of teratoma sections of GpEpiSC with lower passage numbers and higher passage numbers. N represents neurons, C represents chondrocytes, EN represents endodermal epithelium, and NR represents neural rosettes;

[0076] Figure 14 Results of the detection of the expression levels of pluripotency genes, mesoderm, endoderm, and ectoderm differentiation genes in GpEpiSC with different passage numbers (P24, P44, P85). The cell lines used were all derived from E10.5. The values shown are the TPM values (transcripts per million) of RNA-seq;

[0077] Figure 15 Shows the immunofluorescence staining results of the stem cell surface markers TRA-1-81 and SSEA1 at passage 36;

[0078] Figure 16 Shows the immunofluorescence staining results of H3K27me3 in the GpEpiSC cell line (female) at passage 36;

[0079] Figure 17Shows the PCA analysis results of the following cell lines: GpEpiSC cell lines (P24, P24, and P22) derived from E10.5 embryos, EPI cells isolated from E9.5, E10.5, E11.5, and E12.5, and embryonic fibroblast gpEF clones (P1) derived from guinea pig E23.5 embryos;

[0080] Figure 18 For Figure 17 Correction analysis results of GpEpiSC cells, EPI cells isolated from E9.5, E10.5, E11.5, and E12.5 embryos, and gpEF cells in

[0081] Figure 19 To present Figure 17 Heatmap results of specific markers of EPI, TE, and PE of the cells in

[0082] Figure 20 For Figure 17 Transcriptome heatmap analysis results of GpEpiSC and EPI in

[0083] Figure 21A For Figure 17 Heatmap results of GpEpiSC cells, EPI cells isolated from E9.5, E10.5, E11.5, and E12.5 embryos in

[0084] Figure 21B Is a heatmap of ATAC-seq, divided into three categories: gpEF specific (gpEF), GpEpiSC specific (GpEpiSC), and overlapping sites (overlap). The number of target sequences is shown in parentheses. One GpEpiSC line (P44) derived from E10.5 and one gpEF clone (P1) derived from E23.5 were detected respectively;

[0085] Figure 21C Shows the top ten motif analysis results of gpEG classification and GpEpiSC classification in ATAC-seq;

[0086] Figure 21DTriplots showing transcription factors conserved (TPM > 1) (left panel) and representative transcription factors (right panel) among three species (mouse, human, and guinea pig). Genes with similar expression levels are shaded. Coordinate values represent the percentage of gene expression in the three species, with red for guinea pig, blue for mouse, and green for human. Mouse results are from four independent experiments of GSE127925, human results are from two independent experiments of GSE144994, and GpEpiSC (P44) results are from two independent experiments;

[0087] Figure 21E MultiOmics results showing chromatin accessibility, epigenetic status, and expression levels of common pluripotency genes (such as Nanog and OCT4), primed state genes expressed in GpEpiSC (such as ETV4 and ZNF281), and primed state genes not expressed in GpEpiSC (such as OTX2 and EOMES). For CUT&Tag, one EpiSC line (P33) from E10.5 and one gpEF clone (P1) from E23.5 were used; for ATAC-seq, one EpiSC line (P44) from E10.5 and one gpEF clone (P1) from E23.5 were used; for RNA-seq, one EpiSC line (P24) from E10.5 and one gpEF clone (P1) from E23.5 were used;

[0088] Figure 22A Shows that the GpEpiSC of the present application was passaged at least 10 times (40 days) under feeder-free in vitro culture conditions; the scale bar in the figure is 200 μm;

[0089] Figure 22B qPCR quantitative analysis results of the expression levels of Oct4, Sox2, and Nanog of the GpEpiSC (derived from E10.5) of the present application under different culture conditions (feeder layer condition, feeder-free condition (Matrigel), and the condition of being feeder-free first and then moved back to the feeder layer). In the figure, the error bars represent ±SD (n = 3, independent experiments), and n.s. represents P ≥ 0.05;

[0090] Figure 23A Shows the transcriptional status of genes containing H3K4me3, H3K27me3, or a binary domain (H3K4me3 and H3K27me3). Genes with TPM ≥ 1 are considered expressed, and genes with TPM < 1 are considered not expressed. The X-axis of the bar chart shows the average TPM of all genes (expressed and unexpressed);

[0091] Figure 23B to 23DThe top 10 GO functional analysis results of genes containing H3K4me3, H3K27me3, or a binary domain (H3K4me3 and H3K27me3) are shown separately;

[0092] Figure 23E The MultiOmics results showing the epigenetic status and expression levels of H3K4me3-specific genes (MYCN, SALL4, and TGFRB1), H3K27me3-specific genes (NKX2-3, RUNX3, and PAX1), or binary genes (CBX2, CBX8, CBX4, WNT6, and BMP7). Detailed implementation manners

[0093] Definitions

[0094] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein will prevail over any dictionary or external definition.

[0095] As used herein, the terms "comprising" or "including" mean that a sequence, composition, and method include the recited components or steps, but do not exclude other components or steps. "Consisting essentially of", when used in defining a composition and method, shall mean excluding any other components or other steps that are clearly important for the technical effects it should achieve. "Consisting of" shall mean excluding other components and steps not mentioned.

[0096] Unless otherwise specified, the operating methods specifically adopted in this application (including: preparation processes, experimental procedures, detection means, etc.) adopt conventional biochemical experiments, cell biology experiments, molecular biology experiments, gene editing (e.g., recombinant DNA technology), zoological experiments and conventional techniques in related fields in the technical field. These techniques have been well described in the existing literature. For details, see Molecular Cloning: a Laboratory Manual, 4th Edition, by Sam brook et al., Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Press, updated irregularly; Embryonic Stem Cell Protocols, 3rd Edition, by Kursad Turksen et al., Springer-Verlag, 2016; Essentials of Laboratory Animal Science: Principles and Practices, by P. Nagarajan et al., Springer-Verlag, 2021; and Handbook of Laboratory Animal Science: Essential Principles and Practices, 4th Edition, by Jann Hau et al., CRC Press, 2021.

[0097] As used herein, the term "guinea pig-derived" or similar expressions may mean an individual, organ, tissue derived from, (directly or indirectly) isolated from guinea pigs (Cavia porcellus), or an existing cell line of guinea pigs (e.g., fibroblasts isolated from guinea pig embryos, which can be expressed as "guinea pig-derived fibroblasts"), or may also indicate an immortalized cell line obtained by modification, mutation, and culture after isolating cells from guinea pigs (e.g., guinea pig colon cancer cell line MC38 or its artificially gene-edited cells, or guinea pig pancreatic cancer cell line pan02 or its artificially gene-edited cells, can all be expressed as "guinea pig-derived cells"), or may also indicate cells obtained by fusing cells obtained from the guinea pig species with guinea pig cells or cells of other species (e.g., guinea pig fusion cells containing polyploid chromosome sets, or hybrid cells formed by fusing guinea pig cells and mouse cells, can both be expressed as "guinea pig-derived cells").

[0098] As used herein, the term "stem cell" refers to a cell that can remain undifferentiated in culture for an extended period of time (e.g., totipotent, pluripotent or multipotent stem cells), until it is induced to differentiate into other cell types with specific, specialized functions (e.g., fully differentiated specialized cells). The term "pluripotent stem cell" refers to a cell that can differentiate into all three embryonic germ layers (i.e., ectoderm, endoderm, and mesoderm) or remain undifferentiated, and the term or a derivative term indicating its cell line may refer to a pluripotent cell or cell population derived from at least one stem cell (e.g., embryonic epiblast cells or embryonic ectoderm cells) isolated from a single embryo (e.g., an implanted embryo of a guinea pig or an in vitro embryo of a guinea pig).

[0099] As used herein, the term "differentiation" or a similar expression refers to the generation of a cell type that is more specialized than the cell type from which it is derived, indicating a change from a relatively normalized to a specific type in the context of development, and thus the term can refer to a partially or terminally differentiated cell type, e.g., adult stem cells, pancreatic cells, adipocytes, etc. The term "substantially undifferentiated" refers to a stem cell population in a cell culture that contains at least 50% (preferably at least 60%, more preferably at least 70%, 80%, 90% or more) undifferentiated stem cells, and one or more markers (e.g., cell surface markers such as SSEA-4, or pluripotent stem cell transcription factor markers such as Oct-4, or telomerase reverse transcriptase and alkaline phosphatase, etc.) can be used to detect or evaluate the differentiated / undifferentiated state of the cells. Other techniques (e.g., transcriptome techniques) can also be used alone or in combination for such detection or evaluation.

[0100] As used herein, the "growth state" of a cell refers to the proliferation rate and differentiation state of the cell. The "infinitely proliferating cell" as used herein refers to a cell that has been altered by chemical, genetic, and / or recombinant means and thus has the ability to grow after an infinite number of divisions in culture.

[0101] As used herein, the terms "feeder (cells)", "feeder", or similar expressions refer to cells produced in vitro and co-cultured with target cells (e.g., desired pluripotent stem cells), e.g., it can represent some specific cells (e.g., granulosa cells, fibroblasts, etc.), optionally a cell monolayer obtained after treatment with a mitosis blocker. Optionally, stem cells can be cultured without feeder cells.

[0102] As used herein, the terms "maintained" and "maintenance" refer to substantially undifferentiated cell renewal, i.e., a substantially quiescent cell population that does not show differentiation during such a quiescent period.

[0103] As used herein, the term "naïve" or "naïve state" pluripotent stem cells or similar expressions refer to pre-implantation primitive epiblast epiblast cells that are captured and immortalized in vitro. Such cells have the most similar gene expression profile and the same epigenetic characteristics as the pre-implantation epiblast, including two active X chromosomes, DNA demethylation status, etc. Under appropriate culture conditions, naïve pluripotent stem cells can be passaged indefinitely in vitro and retain the ability to differentiate into all adult tissue cells, including germline cells, after injection into recipient blastocysts (Non-Patent Document 7).

[0104] As used herein, the term "primed" or "primed state" pluripotent stem cells or similar expressions refer to cells that are more transcriptionally similar to the late gastrula-stage epiblast. Such cells upregulate lineage-specific genes while retaining core pluripotency genes, have increased heterogeneity, and can better respond to external inductive differentiation signals. Primed pluripotent stem cells still have the ability to form teratomas, but can hardly integrate into the inner cell mass of blastocysts and cannot differentiate into germline cells (Non-Patent Document 7).

[0105] As used herein, the term "epiblast" refers to a group of cells directly produced after the differentiation of the inner cell mass of the blastocyst, and can also be called "primitive ectoderm". During mammalian embryonic development, the inner cell mass of the blastocyst differentiates and separates to form two types of cells: Cuboidal cells stratify towards the ventral side, away from the embryonic pole, and are connected to the blastocoel, becoming the hypoblast (also known as primitive endoderm); the remaining inner cell mass cells are located between the hypoblast and the polar trophoblast, and the cells are columnar, called the epiblast. During the process of differentiating into the gastrula, the migrating epiblast cells will transform from epithelial cells into mesenchymal cells, lose cell-cell adhesion (i.e., lose the cadherin E-cadherin), stratify with the epiblast and migrate along the dorsal surface of the epiblast until they pass through the primitive streak and replace the hypoblast to become the embryonic endoderm. Then, the migrating epiblast cells move through the primitive streak and spread between the endoderm and the remaining epiblast to form the mesoderm. After the formation of the mesoderm reaches its limit, the remaining epiblast is defined as the ectoderm. Therefore, the epiblast finally forms the three germ layers of the ectoderm, mesoderm and endoderm during gastrulation.

[0106] As used herein, the term "En days" (n is a number), "embryonic day", "gestational age" or similar expressions refer to the number of days of development of a fertilized egg or embryo after mating of a female experimental animal. Specifically, unless otherwise specified, in this article, the specific time of the day when a vaginal plug is visible in a female guinea pig is counted as E0.5 (day), and then accumulated day by day (for example, in this case, the second day / the next natural day is E1.5). For example, a female adult guinea pig with a vaginal opening is caged with a male adult guinea pig, and the vaginal plug is checked the next morning to see if it is visible. If a vaginal plug is seen, one o'clock in the afternoon (13:00) of the same day is recorded as E0.5 (day).

[0107] As used herein, the term "FGF" signaling pathway refers to the fibroblast growth factor family, including at least 28 members. Homologs of these members in various organisms have been reported. FGF family members play an important role in various cell processes such as mitosis, differentiation, migration and cell survival, and can participate in the regulation of embryonic development and morphogenesis by regulating cell proliferation, differentiation and migration.

[0108] As used herein, the term "ActA" signaling pathway refers to the activin A (ActA) signaling pathway. Activin A is a member of the transforming growth factor β (TGF-β) superfamily, which first binds to the type II activin receptor (ActIIRA or ActRIIB), then recruits and phosphorylates the type I activin receptor (ActRI), and signals through the SMAD2 / 3 protein to regulate a variety of functions, including inflammation, fibrosis, and tumorigenesis. ActA and TGF-β share the same signaling pathway at the Smad2 / 3 / 4 level, which can regulate a variety of biological functions including hematopoietic cell proliferation, neuronal differentiation, pituitary hormone secretion, and tissue repair.

[0109] As used herein, the term "Wnt" signaling pathway mainly refers to the canonical Wnt signaling pathway mediated by β-catenin, and the components involved mainly include the secretory protein Wnt family, the transmembrane receptor Frizzled family, CK1, Deshevelled, GSK3, APC, Axin, β-Catenin, and the transcription factor TCF / LEF family. The Wnt / β-catenin pathway is known to regulate the pluripotent differentiation of stem cells, the development and regeneration of organs.

[0110] As used herein, the term "Rock" signaling pathway is an abbreviation for the Rho-associated coiled-coil-containing protein kinase signaling pathway. The ROCK signaling pathway plays an important role in regulating cell morphology, motility, proliferation and apoptosis.

[0111] As used herein, the term "A depends on B" or similar expressions mean that two elements are functionally, physiologically, or biologically relevant. The presence, activation, or functioning of the dependent entity (B) enables the object (A) to have certain functions, at least exhibit some physiological activities, or render a biological effect, while excluding, removing, inhibiting, or reducing the dependent entity (B) will cause the object (A) to lose certain functions, at least weaken some physiological activities, or undergo degenerative / recessive / degradation in biological terms. As a non-limiting example, X cells depend on the activation of the Wnt signaling pathway, which can mean that under the condition of activation of the Wnt signaling pathway (for example, adding a ligand or agonist of the Wnt signaling pathway to the cell culture), X cells can maintain a normal culture morphology (for example, adherent production, stable subculture and proliferation, etc.), can activate transcription factors downstream of β-catenin (for example, TCF, etc.), or can activate the biological effects of continuous self-renewal and growth mediated by the Wnt signaling pathway, etc.; in the case of inhibiting or downregulating the Wnt signaling pathway, X cells may exhibit an abnormal cell culture morphology, be unable to activate transcription factors downstream of β-catenin, be unable to exhibit some biological effects (for example, in the case where X cells are stem cells, X cells undergo differentiation), and even exhibit at least some characteristics of apoptosis, pyrotosis, ferroptosis, necrosis / necroptosis, autophagy, cell death.

[0112] As used herein, the terms "significantly increase / enhance / upregulate" and "significantly decrease / reduce / inhibit / downregulate" mean that there is a statistical difference between the indicated objects under preset hypothesis testing conditions (for example, p ≤ 0.05 or 95% confidence interval), and do not specifically refer to the magnitude or level of difference between the indicated objects.

[0113] As used herein, the term "fibroblast" refers to cells that differentiate from mesenchymal cells during embryogenesis. Typically, fibroblasts exist in their mature form, fibrocytes. A detailed description of fibroblasts can be found in the prior art literature: Yang Guiran et al, Biological characteristics and differentiation potential of fibroblasts, Chinese Journal of Tissue Engineering Research, 2020; 24(13): 2114-2119. DOI: 10.3969 / j.issn.2095-4344.2052.

[0114] As used herein, the term "unipotent cell", "unipotent stem cell" or similar expressions refer to a type of stem cell that further differentiates from pluripotent stem cells and can only differentiate into one type or two closely related types of APSC pluripotent cells. For example, neural stem cells can differentiate into various types of nerve cells, and skin germinal layer cells are all unipotent stem cells.

[0115] As used herein, the term "stemness" refers to the ability of isolated and cultured guinea pig pluripotent stem cells to proliferate while still maintaining undifferentiated characteristics and having developmental pluripotency in function. For specific properties, reference can be made to relevant literature in the art (e.g., Anna M Wobus et al., Embryonic stem cells: prospects for developmental biology and cell therapy, Physiol Rev. 2005 Apr; 85(2): 635-78. doi: 10.1152 / physrev.00054.2003, and the references cited therein). The detection or characterization of stemness can be judged qualitatively or quantitatively according to relevant knowledge in the art. For example, stemness can be evaluated by detecting cell surface markers of stem cells, the levels of relevant transcription factors, transcriptome profiles, etc. For example, one or more stem cell-specific markers (such as SSEA-4, etc.) can be detected to determine whether stemness is maintained. Those skilled in the art can select specific markers according to common general knowledge (e.g., select marker molecules in the following literature and the references cited therein: Wenxiu Zhao et al., Embryonic Stem Cell Markers, Molecules. 2012 Jun; 17(6): 6196–6246, DOI: 10.3390 / molecules17066196). For example, those skilled in the art can evaluate stemness according to the stem cell transcriptome structure listed in the prior art literature. Without limitation, transcriptome results with at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% similarity are judged to maintain stemness (e.g., compared with the RNA-seq results listed in the following literature and the references cited therein: Shengyong Yu et al., Nat Cell Biol. 2020 Jun; 22(6): 651-662. doi: 10.1038 / s41556-020-0516-x).

[0116] As used herein, the term "gene expression" refers to the transcription of the gene and the proper translation from the resulting mRNA transcript to protein. Thus, it should be clear from the context that the expression of a protein from a coding sequence is the result of transcription and translation of that coding sequence. "Inhibition / reduction / downregulation of gene expression" refers to a lack (or an observable decrease) in the level of the protein and / or mRNA product derived from the target gene. For example, it can represent a decrease in the transcription level, an increase in the level of ubiquitination and degradation, etc., and can be extended to represent the blockade or inhibition of the biological effect pathway it exerts, etc. "Enhancement / augmentation / upregulation of gene expression" can represent the opposite meaning. The results of downregulation or upregulation can be confirmed by examining the extrinsic properties of the cell or organism (as introduced in the examples below) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, microarray gene expression monitoring, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0117] As used herein, the term "passage" refers to harvesting (recovering) cells that are already in a cultured state, usually after dilution, and then subculturing some or all of the recovered cells into one or more culture vessels. The cells after the above operations can be referred to as "passage cells". Therefore, "passage cells" clearly have a different meaning from "primary cells", which usually refer to the cells present in a cell suspension isolated from a target animal tissue, including the cells attached to a culture substrate such as a culture dish or a culture flask before the first passage. "Primary cells" can usually also be referred to as "generation 0" cells. And "passage cells", "cell strains" or "cell lines" refer to the cells at all steps in subsequent culture processes. Usually, passage is performed when the cells reach a certain density in the culture, and the number of subcultures can be used as the passage number of the passage cells. For example, the passage number of cells subcultured 10 times is 10 and can be referred to as the 10th generation cells. "Passage" usually includes adding or replacing a certain amount (e.g., all or half) of fresh culture medium. According to some embodiments of the present application, passage is performed by mechanical passage. As used herein, the term "mechanical dissociation" refers to separating a pluripotent stem cell mass into several cell masses or single cells by using physical forces rather than enzymatic activity. For mechanical dissociation, a pluripotent stem cell pellet (obtained by cell centrifugation) or a separated pluripotent stem cell mass can be separated by pipetting up and down in a small amount of culture medium (e.g., 0.2 to 1 ml). According to some embodiments of the present invention, passage is performed under conditions of enzymatic digestion / dissociation. As used herein, the terms "enzymatic digestion", "enzymatic digestion / dissociation / separation" refer to the process of using biological enzymes (such as collagenase, protease, etc.) to break down extracellular links (such as the extracellular matrix) to complete cell separation / assist in cell separation. It should be noted that the enzymatic digestion process is not absolutely without the participation of mechanical forces. During the enzymatic digestion process, there may be mechanical forces (e.g., the flow of liquid) to help complete this process.

[0118] As used herein, the terms "stable passage", "passage stability", or similar expressions may indicate that characteristics such as the phenotype, cell morphology, function, physiological activities, and genetics of cells remain stable over a period of time (e.g., within a certain number of passages). Passage stability can be characterized and judged from one or more aspects. For example, the chromosomal karyotype of cells remains stable after multiple passages, the culture morphology, proliferation rate, and number of divisions of cells do not change significantly, the specific surface markers of cells do not change significantly, the physiological activities of cells remain basically stable, and there are no significant mutations in the genome of cells (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the genome has no non-synonymous mutations). It should be particularly noted that "stable passage" does not mean that cells do not undergo any changes, but can indicate that all aspects of cells remain relatively stable or stably over a long period. For example, homologous recombination and significant changes in cell morphology may occur during mitosis of cells, and those skilled in the art can judge that the cells are still in a stable state based on the physiological activities and functions of the cells. For pluripotent stem cells, the stable expression levels of pluripotency markers (such as Pou5f1, Sox2, Nanog) are one of the important signs that they can be stably passaged.

[0119] As used herein, "genome editing" refers to a reverse genetics method (optionally using an engineered nuclease) that cuts at a desired position in the genome and generates a specific double-strand break, which is then repaired by endogenous cellular processes (e.g., homologous recombination (HR), homology-directed repair (HDR), and non-homologous end joining (NHEJ)). In double-strand breaks, NHEJ directly ligates the DNA ends, while HDR uses a homologous sequence as a template to regenerate the missing DNA sequence at the break point. Genome editing can employ homologous recombination, ZFN and TALEN endonuclease technologies, CRSPR / cas9 technology, or can be based on recombinant adeno-associated virus (rAAV)-based genome engineering. Specifically, genome editing can include nucleic acid fragment deletion, gene modification, gene knockout, altering the expression of gene products, repairing mutations, inserting polynucleotides, single-base mutations, or any combination thereof.

[0120] As used herein, the term "serum-free" means without human or animal serum. It should be noted that the function of serum in a culture protocol is to provide an environment similar to that present in vivo (i.e., in the organism from which the cells are derived, e.g., the blastocyst of an embryo) for the cultured cells. However, the use of serum from animal sources (e.g., mammalian livestock, e.g., bovine serum) or human sources (human serum) is limited by the significant differences in serum composition between individuals and the risk of having heterologous contaminants (if using serum from other species).

[0121] It should be noted that the expressions "first", "second" or their similar expressions used herein and in the text of this article are only intended to distinguish two said elements in a specific category, and do not indicate the importance, order, etc. of the said elements; the "first" and "second" elements can be the same or different referents / concepts. For example, in some cases, the "first sequence" and the "second sequence" are only used to represent two different sequences; for another example, in some cases, the "first label" and the "second label" can represent the same label (for example, polyA) or different labels (for example, independently polyA or polyG). Those skilled in the art can understand that the "first", "second" or their similar expressions can be used interchangeably.

[0122] It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of two or more cells, or the entire culture of cells. Unless expressly stated or obvious from the context, the term "or" as used herein is understood to be inclusive. Unless otherwise defined herein or in the remainder of the specification hereinafter, 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 invention belongs.

[0123] Unless expressly stated or obvious from the context, the term "about" as used herein should be understood to be within the normal tolerances in the art, for example, within two standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% of the said value. Unless otherwise obvious from the context, all numerical values provided herein are modified by the term "about".

[0124] The term "genetically engineered" or "engineered" refers to methods of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a part thereof or inserting a coding region or a part thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct incorporated into the cell genome, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0125] As used herein, the term "treatment" refers to therapeutic treatment and prophylactic or preventive or deterrent measures, wherein the aim is to prevent or slow down (mitigate) an undesired pathologic change or condition. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to: alleviation of symptoms, diminishment of the extent of a disease, delay or slowing of disease progression, amelioration or palliation of a disease state, and remission (whether partial or total), whether detectable or undetectable.

[0126] As used herein, the term "therapeutically effective amount" means an amount of a compound of the present invention that can: (i) treat or prevent a disease or condition described herein, (ii) ameliorate or eliminate one or more diseases or conditions described herein, or (iii) prevent or delay the onset of one or more symptoms of a disease or condition described herein.

[0127] As used herein, the term "genome" or "genomic DNA" refers to the heritable information of a host organism. The genomic DNA includes all the genetic material of a cell or organism, including nuclear DNA (chromosomal DNA), extrachromosomal DNA, and organelle (e.g., mitochondrial) DNA. Preferably, the term "genome" or genomic "DNA" refers to nuclear chromosomal DNA.

[0128] As used herein, when the term "recombinant" is used to describe an organism or cell (e.g., a microorganism), it is used to indicate that the organism or cell contains at least one "transgene", "transgenic", or "recombinant" polynucleotide as described hereinafter.

[0129] As used herein, a polynucleotide that is "foreign" to an individual organism is a polynucleotide that has been introduced into the organism by any means other than by sexual hybridization.

[0130] The present invention will be further described below by way of specific embodiments, which are not intended to limit the present invention. Those skilled in the art can make various modifications or adjustments according to the teachings of the present invention, which do not depart from the spirit and scope of the present invention.

[0131] Examples

[0132] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention, rather than for limiting the scope of the present invention. The following embodiments provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0133] In the following examples, the experimental methods are conventional methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources, unless otherwise specified. In the following examples, if the operating temperature is not specified, it means at room temperature. In the following examples, if not otherwise specified, the nucleotide sequences described are considered to be in the 5' to 3' direction, and the amino acid sequences described are considered to be in the amino to carboxyl terminal direction.

[0134] Reagents, instruments, cell lines, and experimental methods used

[0135] Guinea pigs : Hartley strain, obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were carried out in accordance with the animal protection guidelines of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences.

[0136] Culture medium:

[0137] Table 1

[0138]

[0139] FRP + 10 μ M Y medium : The final concentration of Y-27632 is 10 μM, and the others are the same as the FRP basal medium.

[0140] Collagenase IV : Obtained from Worthington Biochemical, catalog number LS004186;

[0141] Accutase : Obtained from Sigma-Aldrich, catalog number A6964;

[0142] Actvin A : Obtained from PeproTech, catalog number 120-14E-1000;

[0143] PD0325901 : A MEK inhibitor, synthesized in the laboratory;

[0144] AZD4547 : An FGFR family inhibitor, obtained from TargetMol, catalog number T1948;

[0145] A83-01 : An inhibitor of TGF-β type I receptors ALK5, ALK4, and ALK7, obtained from TargetMol, catalog number T3031;

[0146] AP staining: Use the Vector alkaline phosphatase staining kit (source: Vector Laboratories, SK-5100). According to the kit instructions, discard the original cell culture medium, wash three times with DPBS (source: eLGbio), and then perform staining. The staining results were observed and photographed using an optical microscope.

[0147] Quantitative PCR:

[0148] In some embodiments, quantitative PCR (Q-PCR) detection (or fluorescence real-time quantitative detection) was performed according to the methods listed in "Molecular Cloning Experiments", and the primers used are shown in Table 2 below.

[0149] Table 2:

[0150]

[0151] Karyotype analysis:

[0152] Before karyotype analysis, add colchicine (purchased from Guangzhou Dahui Biotechnology Co., Ltd...) to the cell culture medium to a final concentration of 1%, and incubate the cells at 37°C for 2 hours. Then, digest the GpEpiSC cell clusters into single cells with Accutase, centrifuge, and collect the precipitate. Resuspend the cells in 0.075M KCl (Sigma, P5405) hypotonic solution and incubate at 37°C for 15 minutes. Pre-fix at room temperature for 3 minutes with freshly prepared fixative (methanol: glacial acetic acid = 3:1), then fix at 37°C for 40 minutes with the fixative, and repeat 2 times. Drop the fixed GpEpiSCs cell suspension onto a pre-cooled glass slide, dry at room temperature, and age in an oven overnight. Then, treat with pre-warmed 0.025% trypsin (Gibco, 25300120) for 2 min, wash 2 times with PBS, and stain with 10% Giemsa staining solution (Guangzhou Dahui Biotechnology Co., Ltd.) for 3 minutes.

[0153] Transcriptome analysis:

[0154] After extracting RNA by Trizol, the transcriptome analysis was entrusted to Guangzhou Heqin Biotechnology Co., Ltd. It used the novaseq6000 high-throughput sequencing platform for PE150 sequencing to obtain raw data in FASTA format. Then, the raw data was aligned with the guinea pig reference transcriptome Cavpor3.0 (obtained from the NCBI public database: https: / / www.ncbi.nlm.nih.gov / datasets / taxonomy / 10141 / ) using the RSEM and Bowtie2 software, and an expression matrix was obtained through TPM normalization. The R package mFuzz was used for grouping to obtain differential expression, the pheatmap function was used to draw a heatmap, and the online website DAVID (https: / / david.ncifcrf.gov / home.jsp) was used for GO analysis. For PCA analysis, the corrplot of the R package was used to calculate the correlation coefficient to obtain the principal component values, and then the ggpubr and ggrepel of the R package were used for visualization. For the ternary plot drawing, the overlapping transcription factors in the epiblast cells of mice, humans, and guinea pigs, that is, the TPM mean values (1237 genes), were used to generate the ternary plot using the ggtern of the R package.

[0155] ATAC-seq and its analysis

[0156] The ATAC-seq was constructed using the Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme, TD711). The sequencing platform was novaseq6000 and the sequencing protocol was PE150. The ATAC-seq data was mapped according to the guinea pig genome mCavpor4.1 (NCBI).

[0157] ChIP-Seq

[0158] According to the manufacturer's instructions, the ChIP-seq was constructed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904). The ChIP-seq data was mapped according to the guinea pig genome mCavpor4.1 (NCBI).

[0159] Binary analysis

[0160] Based on the CUT tag data of H3K4me4 and H3K27me3, the corresponding genes of H3K4me3-only, H3K27me3, H3K4me3 and H3K27me3 were obtained respectively. Then, the TPM values of RNA-seq corresponding to the gene were obtained. If the TPM value is greater than 1, it is considered expressed; if the TPM value is less than 1, it is considered not expressed. According to the obtained data, plotting was performed using GraphPad Prism 9. The guinea pig Org.Db annotation file was obtained using the R package AnnotationHub, and then enrichment analysis was performed using the R package clusterProfiler. Immunofluorescence staining:

[0161] Primary antibody:

[0162] Sox2: Rabbit monoclonal Sox2 (D6D9) Rabbit mAb: Obtained from cell signaling, catalog number 3579s;

[0163] POU5F1: Mouse monoclonal anti-Oct-3 / 4 (C-10), obtained from Santa Cruz Biotechnology, catalog number sc-5279;

[0164] NANOG: Rabbit polyclonal anti-human Nanog, obtained from PeproTech, catalog number 500-P236;

[0165] Secondary antibody:

[0166] Alexa Fluor 488 goat anti-rabbit IgG, obtained from invitrogen, catalog number A11008;

[0167] Alexa Fluor 488 goat anti-mouse IgG, obtained from invitrogen, catalog number A11001.

[0168] Example 1:

[0169] Adult female guinea pigs with vaginal openings were caged with adult male guinea pigs, and plugs were checked the next morning. If a plug was seen, 1 pm on the same day was recorded as E0.5. Figure 1 The changes of guinea pig embryos during pregnancy from E9.5 to E12.5 were continuously shown. Guinea pigs at E9.5, E10.5 or E11.5 were humanely sacrificed with carbon dioxide, and the uterus was removed and placed in PBS. The uterine wall was torn open with fine forceps, and the decidua was peeled off from the uterine wall and transferred to fresh PBS. Under a stereomicroscope, the decidua was halved with fine forceps, and a guinea pig embryo with a long strip tubular structure was visible ( Figure 2),Remove the embryo with forceps (E10.5 to E11.5) or a stainless-steel needle (E9.5) and separate it from the visceral endoderm. Then transfer it to FRP + 10 μM Y.

[0170] Comparative Example 1:

[0171] Obtain embryos from guinea pigs at E12.5 of gestation by the exact same method as in Example 1, and transfer the embryos to FRP + 10 μM Y in the same way.

[0172] Example 2:

[0173] According to the "Preparation of Embryonic Fibroblasts and Feeder Cells" described in the "Experimental Manual for Mouse Embryo Manipulation" (Chemical Industry Press, Third Edition, 2006), prepare mouse embryonic fibroblast feeder cells (MEF) from embryos of ICR strain mice at 13.5 days of gestation. Seed the MEF cells at a density of 3 - 4x10 4 / cm 2 in a 48-well plate. Replace the medium of the seeded MEF cells with FRP + 10 μM Y medium at 500 μl / well, and equilibrate it in a cell culture incubator with 5% O2 and 5% CO2.

[0174] Figure 3 Schematically shows the isolation and culture process of GpEpiSC. Specifically, under a stereomicroscope, use an extremely fine tungsten wire for the E9.5 guinea pig embryos obtained in Example 1, use fine forceps for the E10.5 and E11.5 guinea pig embryos obtained in Example 1, and use fine forceps for the E12.5 guinea pig embryos obtained in Comparative Example 1 to separate the embryonic epiblast (EPI) from the visceral endoderm (VE). Then, tear the EPI into small pieces with fine forceps, or treat it with an enzyme mixture (Accutase: TrypLE = 1:1) for 5 minutes, and then disperse it into small cell clumps using a fine-drawn glass capillary with a diameter in the range of 50 to 100 μm, and seed it onto the pre-prepared MEF (treated with mitomycin C).

[0175] Culture the cell clumps seeded onto the MEF in a cell culture incubator with 5% O2 and 5% CO2 in FRP basal medium supplemented with 2 μM Y-27632. After 24 hours, replace half of the medium with FRP medium, and after 48 hours, replace all the medium with FRP medium. After culturing for another 3 - 5 days, it was observed that cell clumps obtained from E9.5 to E11.5 guinea pig embryos in Example 1 and from E12.5 guinea pig embryos in Comparative Example 1 both obtained obvious outgrowths, and there was little morphological difference. The exemplary results of the above guinea pig embryos at different gestational periods are shown in Figure 4 .

[0176] Alternatively, Y-27632 can also be omitted from the basal medium and instead administered only at a concentration of 10 μM during passage, without affecting cell morphology or growth characteristics. Subsequently, the cells are cultured in an incubator with 5% O2 and 5% CO2. After 24 hours, half of the medium is replaced with fresh FRP medium (with or without Y-27632 added as needed). Starting from 48 hours, the medium is changed daily, but without adding Y-27632.

[0177] After discarding the medium, the cells are washed once with DMEM / F12 and then digested with collagenase IV at a working concentration of 1 mg / ml in DMEM / F12 for 15 - 20 minutes to separate the outgrowth from the feeder cells. Then, the separated outgrowth is digested with the undiluted Accutase purchased from Sigma Aldrich for 15 to 30 seconds to form smaller cell clumps. The small clumps are seeded onto fresh feeder cells supplemented with an appropriate amount of FRP + 10 μM Y medium to complete one passage.

[0178] During the first 3 - 5 passages, it is preferred to enzymatically separate the compact outgrowth from the feeder cells and differentiated cells using collagenase IV treatment, and then briefly treat it with Accutase for about 2 - 3 minutes to disperse it into small cell clumps, which are re-seeded onto fresh feeder cells. After 3 - 5 passages, single-cell passage can be conveniently carried out by Accutase digestion (about 1 minute). The passaged GpEpiSCs are maintained on MEF feeder cells in FRP medium under the conditions of 37 °C, 5% O2, and 5% CO2, and the medium is changed daily. The dilution ratio for cell passage is 1:5 to 1:10, and passage is carried out every 3 - 4 days. During early passage (<10 passages), adding 10 μM Y-27632 can promote cell attachment and survival. When the cells can complete more than 15 passages in total, a pluripotent stem cell line capable of stable passage is considered to be obtained.

[0179] Table 3 below shows the repeated preparation of GpEpiSCs cells using embryos with different gestation days from different guinea pigs. As can be seen from the table below, guinea pig embryos with gestation days from E9.5 to E12.5 can all be used to prepare guinea pig pluripotent stem cells that can be stably passaged. However, as can be seen from Table 3, although the embryos of guinea pigs with gestation days from E9.5 to E12.5 can all easily produce outgrowths, and the success rates are all close to 100%, there are significant differences in the success rates of the outgrowths from embryos of different gestation days when used to prepare guinea pig pluripotent stem cells that can be stably passaged. Among them, using guinea pig embryos with gestation days from E9.5 to E11.5 can easily and repeatedly obtain pluripotent stem cells (19 out of 22 embryos obtained pluripotent stem cells that could be stably passaged at least 15 times). In contrast, among 8 guinea pig embryos with a gestation day of E12.5, only the cells obtained from 1 embryo could be passaged more than 15 times. It was also observed in the experiment that the outgrowths produced by E12.5 embryos generally differentiated after 1 to 3 passages, and the success rate of obtaining pluripotent stem cells that could be stably passaged was low. This may be because most guinea pig embryo cells have exited the pluripotent state since this time point of E12.5.

[0180] Table 3:

[0181] Gestational age in days Number of embryos collected Number of embryos with outgrowths obtained Number of embryos from which GpEpiSCs cell lines were established E9.5 8 7 5 E10.5 7 7 7 E11.5 7 7 7 E12.5 8 8 1

[0182] Example 3:

[0183] For the E10.5 guinea pig embryos obtained in Example 1, after excising the extraembryonic ectoderm (ExE), the part of the embryo containing the EPI was turned over to expose the EPI, and then transferred to TrypLE express (purchased from Gibco, catalog number: 12604021) for digestion at room temperature for 2 - 3 minutes. The digestion solution was transferred to FRP + 10 μM Y medium, and the EPI was blown into small pieces with a mouth pipette and a glass needle with a diameter of 50 - 70 μM and separated from the VE. Be careful not to digest the EPI into single cells. The separated EPI cell mass was inoculated onto MEF prepared in advance by the same method as in Example 2. Then, according to the same method and conditions as in Example 2, the separated EPI mass was cultured, and the outgrowths were separated, digested, and passaged in the same way.

[0184] Using the method of Example 3, guinea pig pluripotent stem cells GpEpiSC were repeatedly prepared from 5 guinea pig embryos with a gestation day of E10.5 respectively. These cells can be stably passaged more than 20 generations under the condition of using FRP medium for passage.

[0185] Example 4:

[0186] By the same method as in Example 3, GpEpiSCs cells were prepared from guinea pig embryos at E10.5. The GpEpiSCs cells that had been passaged 36 and 91 times respectively were seeded at a density of 1x10 5 / well on the feeder layer cells prepared as in Example 2 (Day 0, D0). The medium used for seeding was FRP + 10 μM Y. Thereafter, the medium was changed daily with FRP medium. Every 3 - 4 days, the cells were digested into single cells with Accutase and passaged at a ratio of 1:5 to 1:10. During the culture and passage process, the cells were counted every 24 hours, and the cell growth curve was measured and shown in Figure 5 A. Each data point in the figure is the average of three repeated experiments.

[0187] According to Figure 5 the results of the time-dependent change in the number of cells in A, the doubling time of GpEpiSCs cells was calculated using the following formula: Doubling time = 24 × [lg2 / (lgN t – lgN0)], where 24 is the cell culture time (unit: hours), N t is the number of cells at the fourth day (D4), and N0 is the number of cells at the second day (D2). The calculation results of the doubling time are shown in Figure 5 B. For a more specific method, see Generation and characterization ofstable pig pregastrulation epiblast stem cell lines Cell Res.2022Apr;32(4):383 - 400, the relevant content of which is incorporated herein by reference.

[0188] As can be seen from Figure 5 A and 5B, the doubling time of the GpEpiSCs cell line disclosed in this article is approximately 18 - 19 hours.

[0189] We further evaluated the single-cell cloning efficiency of the guinea pig pluripotent stem cells disclosed herein. After digesting the GpEpiSCs cells passaged 22, 24, and 104 times into single cells with Accutase enzyme respectively, they were seeded at a density of 500 cells / well on the feeder cells prepared in a 12-well cell culture plate in advance. For the cells at P24, two media were added to 1 ml / well: 1) FRP medium and 2) FRP + 10 μM Y medium, and for the cells at P22 and P104, FRP + 10 μM Y medium at 1 mL / well was used; there were three replicates for each medium condition. From the next day, the medium was changed daily with FRP medium. On the 8th day, the monoclonal colonies obtained were stained with AP and counted. The single-cell cloning efficiency was calculated by the formula: single-cell cloning efficiency = number of colonies / 500 x 100%, and is shown in Figure 5 Figures 5C and 5D.

[0190] Figure 5 Figure 5C shows that the passage number does not affect the colony formation efficiency of the GpEpiSCs cells of the present invention, and it can still remain above 40% after more than 100 passages, indicating that the GpEpiSCs cells have a stable amplification ability. Figure 5 Figure 5D shows that there is a significant difference in the single-cell cloning efficiency when using the two media, which are approximately 28% (FRP medium) and approximately 41% (FRP + 10 μM Y) respectively. The single-cell cloning efficiency mainly affects the efficiency of cell gene editing. The cells after gene editing need to be cultured into clones from single cells to obtain a pure genotype. Therefore, the higher the single-cell cloning efficiency, the higher the gene editing efficiency will be. Figure 5 Figure 5C shows that the single-cell cloning efficiency of GpEpiSCs is significantly affected by the concentration of Y27632. However, even when the concentration of Y27632 in the medium is low (2 μM), the GpEpiSCs cells disclosed herein can still achieve a relatively high single-cell cloning efficiency (about 28%). The higher cell line establishment ability indicates that the guinea pig pluripotent stem cells disclosed herein are very suitable as the starting cells for gene editing.

[0191] We also observed the passage stability of GpEpiSCs in an extended time window. Specifically, by the same method as in Example 3, GpEpiSCs cells were prepared from guinea pig embryos at E10.5, and the guinea pig pluripotent stem cells were seeded at 1 x 10 5Inoculate at a density of cells per pore on the feeder cells prepared as in Example 2, using FRP + 10 μM Y medium during inoculation. Then change the medium daily (using FRP + 10 μM Y medium for the first 10 passages and FRP medium thereafter). Digest into single cells with Accutase enzyme every 3 - 4 days and passage at a ratio of 1:5 to 1:10. Perform morphology, alkaline phosphatase (AP) staining, and immunofluorescence of pluripotency markers on the cells at passage 44 (P44) and passage 102 (P102), and the results are shown in Figure 6 . The results of karyotype analysis on the cells at passage 73 (P73) are shown in Figure 7 .

[0192] As can be seen from Figure 6 , the GpEpiSC cells disclosed herein are dense monolayer cell colonies under the microscope, with uniform cell morphology, cloned bulges, clear edges, and high refractive index, presenting the morphological characteristics of typical pluripotent stem cell colonies. In addition, the cell colonies formed by GpEpiSC cells all show positive red results in AP staining (red). High-level expression of AP is one of the typical characteristics of embryonic stem cells. Positive AP staining indicates that the GpEpiSC cells composing the colony are all undifferentiated stem cells. The cells composing the colony can detect the expression of core pluripotency markers such as OCT4 (POU5F1), SOX2, and NANOG, indicating that these cells can all maintain pluripotency well during passage.

[0193] As can be seen from Figure 7 , the GpEpiSC disclosed herein still maintains a stable normal chromosome karyotype after at least 70 consecutive passages, and no obvious aberrations are observed.

[0194] Thus, it can be seen that the GpEpiSC cells disclosed herein are pluripotent stem cell lines that can maintain stable morphological, physiological, and biochemical characteristics during long-term culture and passage.

[0195] Example 5

[0196] Identifying naive and primed pluripotent stem cells by the opening and closing of intracellular pluripotency-related pathways is one of the current common practices. Generally speaking, the typical characteristics of primed pluripotent stem cells are that the FGF and ActA signaling pathways are open or activated, and at the same time, the WNT signaling pathway is closed or inhibited.

[0197] Using the same method as in Example 3, prepare GpEpiSCs cells from E10.5 guinea pig embryos. Digest the GpEpiSCs cells at a certain number of passages (P22, P25, P32 respectively) into single cells with Accutase enzyme, and use 1x10 5Inoculate at a density of cells per well in a 24-well plate coated with Matrigel, and culture for 24 hours under the following 5 reagent combinations added to the culture medium:

[0198] 1) N2B27

[0199] 2) N2B27 + 10 μM Y27632;

[0200] 3) N2B27 + 10 μM Y27632 + 20 ng / ml FGF2;

[0201] 4) N2B27 + 10 μM Y27632 + 20 ng FGF2 + 1 μM PD0325901;

[0202] 5) N2B27 + 10 μM Y27632 + 20 ng FGF2 + 0.5 μM AZD4547.

[0203] Among them, the composition of N2B27 culture medium (500 ml) is: 237.5 mL DMEM / F12, 237.5 mL neurobasal, 2.5 ml N2 serum-free additive, 5 ml B27 serum-free additive, 1% GlutaMax, 1% MEM NEAA, 1% penicillin-streptomycin solution, 0.1 mM β-mercaptoethanol. The sources of each component are the same as those of the FRP culture medium.

[0204] After 24 hours, discard the culture medium and the non-adherent cells therein, digest the adherent cells into single cells with Accutase enzyme, and count. According to the formula: Adhesion rate = number of cells after 24 hours / 1 x 10 5 , calculate the adhesion rate of each group. Each experiment in each group was repeated 3 times, and the average value is shown in Figure 8 A. Taking the adhesion rate of group 1) as 100%, the ordinate shows the adhesion rate of groups 2) to 5) relative to group 1).

[0205] After digesting the same GpEpiSCs cells into single cells with Accutase enzyme, at a density of 1 x 10 5Inoculate at a density of cells per well into a 24-well plate pre-coated with feeder cells. When inoculating, use the following 4 kinds of culture media respectively: 1) FRP + 10 μM Y medium, without adding FGF2; 2) FGF2 at 5 ng / ml, and the other components are the same as those in FRP + 10 μM Y medium; 3) FGF2 at 10 ng / ml, and the other components are the same as those in FRP + 10 μM Y medium; 4) FRP + 10 μM Y medium (with FGF2 at 20 ng / ml). Starting from the first day (D1) after inoculation, change the medium with FRP medium every day. Digest the cells into single cells and count them on the second day (D2) and the third day (D3) after inoculation. The results are shown in Figure 8 B.

[0206] From Figure 8 the adherent survival rates of GpEpiSCs shown in A under different culture conditions, it can be seen that the FGF2 signaling pathway can promote the adhesion, survival and proliferation of GpEpiSCs. And exogenous addition of FGF2 can cooperate with exogenous ROCK inhibitors (such as Y27632) to increase the adherent survival rate of cells. However, the promoting effect of FGF2 is offset by exogenous addition of FGF inhibitors or MEK inhibitors. Figure 8 Figure B further shows the proliferation rate of GpEpiSCs under different FGF2 concentration conditions. It can be seen that the cell proliferation rate of GpEpiSCs shows an obvious FGF2 concentration dependence. As the FGF2 concentration in the culture medium increases, the proliferation rate of GpEpiSCs cells also increases proportionally, and the two show a positive correlation. It has been reported that FGF2 is beneficial to the cell proliferation of naive human PSCs, but no supportive effect has been observed on mouse EpiSCs. The results of this example show that the activation of the FGF2 pathway is beneficial to maintaining the self-renewal of GpEpiSCs cells.

[0207] Using the same method as in this example, inoculate GpEpiSCs cells into a 24-well plate pre-coated with Matrigel, and use FRP medium supplemented with 10 ng / ml Actvin A. When the cells grow to a density of 70 - 80%, discard the original medium, wash once with DPBS (eLGbio, EH80028), and then change to the following 6 groups of different culture media respectively:

[0208] 1) N2B27 + 20 ng / ml FGF2 + 10 ng / ml Actvin A ( Figure 9 "FA" in

[0209] 2) N2B27 + 10 ng / ml Actvin A ( Figure 9 "A" in

[0210] 3) N2B27 + 10 ng / ml Activin A + 1 μM PD0325901 ( Figure 9 in “A-PD” in

[0211] 4) N2B27 + 20 ng / ml FGF2 ( Figure 9 in “F” in

[0212] 5) N2B27 + 20 ng / ml FGF2 + 1 μM A83 - 01 ( Figure 9 in “F - A83” in

[0213] 6) N2B27 + 1 μM PD0325901 + 1 μM A83 - 01 ( Figure 9 in “PD - A83” in

[0214] After culturing for 6 hours in an incubator at 37 °C with 5% CO2, the cells were harvested and RNA was extracted for Q - PCR. After treatment with different inhibitors for 6 hours, the expression levels of NANOG and POU5F1 in the cells were measured. Each experiment was repeated 3 times, and the results are shown in Figure 9 .

[0215] As Figure 9 can be seen, inhibiting the ActA signaling pathway and the FGF2 signaling pathway can both rapidly down - regulate the expression level of NANOG in GpEpiSCs cells, especially the effect of inhibiting the ActA signaling pathway is more obvious. Moreover, there is a synergistic effect in reducing the Nanog expression level between inhibiting the ActA signaling pathway and the FGF2 signaling pathway. This indicates that the transcription of Nanog in GpEpiSCs is directly regulated by both the FGF2 and Activin A signaling pathways, and their promoting effects on Nanog transcription are additive or even synergistic. Nanog is known to be an important pluripotency factor directly related to the maintenance and self - renewal of stem cell pluripotency. Therefore, the pluripotency of GpEpiSCs cells is also regulated by both the FGF2 and Activin A signaling pathways.

[0216] This is also consistent with the culture conditions of GpEpiSCs cells with and without feeder layers that we observed. When culturing GpEpiSCs cells using feeder layers, since feeder layer cells can secrete Activin A (average concentration is about 10 ng / ml) under the stimulation of FGF2, there is no need to add Activin A in the medium containing FGF2; while when culturing without feeder layer cells, adding about 10 ng / ml of Activin A is beneficial to maintaining the pluripotency of GpEpiSCs cells. Figure 22A and 22BIt is shown that under the culture conditions with the addition of 10 ng / ml Actvin A, GpEpiSCs cells can be passaged at least 10 times or more under feeder-free conditions, and no obvious changes are observed in the morphology of the clones and the expression levels of pluripotency markers.

[0217] The same GpEpiSCs were seeded on feeder cells and cultured under the following 4 conditions respectively:

[0218] 1) FRP (IWR1 + , IWP2 + );

[0219] 2) FR (removing IWP2 from FRP; IWR1 + , IWP2 - );

[0220] 3) FP (removing IWR1 from FRP; IWR1 - , IWP2 + );

[0221] 4) F (removing both IWR1 and IWP2 from FRP; IWR1 - , IWP2 - ).

[0222] Passaging was performed once every four days. On the day of passaging, FRP + 10 μM Y medium was used, and FRP medium was used on the remaining days. However, the entire culture process conformed to the above 4 cases of Wnt inhibitors. When reaching the third passage (after 12 days of treatment), the clones were separated from the feeder cells with collagenase IV, and the RNA of the cloned cells was extracted. Guangzhou Heqin Biotechnology Co., Ltd. was commissioned to perform sequencing using the novaseq6000 high-throughput sequencing platform. Transcriptome analysis was performed on the sequencing results. Each experiment was repeated twice, and the results are shown in Figures 10 and 11.

[0223] IWR1 is an inhibitor known to block canonical WNT signaling by stabilizing the deconstruction complex member AXIN2, and IWP2 is a non-canonical WNT signaling inhibitor known to specifically inhibit Porcn-mediated WNT palmitoylation to prevent WNT processing and secretion. Figure 10A And 10B It is shown that after culturing GpEpiSCs for 12 days under the conditions of using no canonical WNT inhibitor IWR1 (group 2), or no non-canonical WNT inhibitor IWP2 (group 3), or neither of them (group 4), the development- and differentiation-related genes in the transcriptome are all upregulated, and the upregulation effect is more obvious when removing both inhibitors. This indicates that the long-term maintenance of GpEpiSC cell pluripotency also depends on the inhibition of the WNT signaling pathway. Figure 11A And11B It was shown that in the case of removing IWR1 or IWP2, obvious flattening of cell morphology was observed, and the immunofluorescence intensity of OCT4 and SOX2 decreased, suggesting downregulation of pluripotency marker expression; under the culture conditions of completely removing WNT inhibitors, the above-mentioned morphological and immunofluorescence changes were more significant. Figure 11C and 11D The downregulation of pluripotency marker expression and the upregulation of development-related gene expression were further verified by the results of RNA-seq and qPCR. The gene expression changes reflected by the RNA-seq and qPCR results were consistent with the above-mentioned changes in cell morphological phenotypes and the GO analysis results in Figure 10, all suggesting that WNT signal transduction plays an important role in regulating guinea pig cell differentiation and pluripotency maintenance.

[0224] In short, maintaining GpEpiSC cells requires the activated FGF and ActA signaling pathways and does not require the activated WNT signaling pathway, proving that it belongs to naïve pluripotent stem cells.

[0225] Example 6:

[0226] The pluripotency of stem cells is mainly manifested as the ability of unlimited proliferation and differentiation into the three germ layer cell lineages.

[0227] Using the same method as in Example 3, GpEpiSC pluripotent stem cells were prepared from guinea pig embryos at E10.5 of pregnancy. Immunofluorescence staining of SOX2, NANOG, and POU5F1 was performed on GpEpiSCs cells passaged to the 23rd passage (P23), 44th passage (P44), and 48th passage (P48), respectively. Specifically, after washing the cells twice with PBS, they were fixed with 4% paraformaldehyde (Jingxin Bio, wj0012) at room temperature for 30 minutes. Then, they were washed with PBS and blocked and permeabilized with 1.5% BSA containing 0.1% Triton X-100 at room temperature for 40 minutes. After that, the cells were incubated overnight at 4°C with the primary antibody solution diluted with 3% BSA. After washing three times with PBS, they were incubated with the corresponding diluted secondary antibody solution at room temperature for 1 hour. Then, they were washed four times with PBS and stained with DAPI (sigma, D9542) solution diluted 3000 times with PBS at room temperature for 1 minute. Observation and photography were carried out through a fluorescence microscope. The results are shown in Figure 12 . Similarly, immunofluorescence staining of the stem cell surface markers TRA-1-81 and SSEA1 was performed on GpEpiSCs cells passaged to the 36th passage (P36) (from E10.5 embryos), and the results are shown in Figure 15 . Figure 12 and 15It is shown that the GpEpiSC cells disclosed in this article can still stably and highly express key pluripotency factors POU5F1, SOX2, NANOG, TRA-1-81, and SSEA1 after multiple passages, indicating their potential for infinite proliferation. It is also shown that the culture method disclosed in this article can effectively and stably maintain the pluripotency of guinea pig pluripotent stem cells.

[0228] Using the same method as in Example 3, GpEpiSC pluripotent stem cells were prepared from guinea pig embryos at E10.5 of pregnancy. Teratoma experiments were performed using GpEpiSCs cells passaged to the 22nd generation (P22). Specifically, after digesting approximately 1x10 7 GpEpiSCs cells into single cells, they were resuspended in freshly prepared 50% Matrigel solution (Matrigel:DMEM / F12 = 1:1, kept on ice) to obtain a 250 μl suspension. 120 μl of the suspension was subcutaneously injected into the nape of NOD / SCID IL2rg- / - immunodeficient mice (obtained from the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, see Chinese Patent No.: ZL201310229629.9). The mice were sacrificed after 77 days, and the teratomas were removed and fixed with 4% paraformaldehyde (Jingxin Bio, wj0012), and then entrusted to the pathology laboratory of the animal house of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences for embedding, sectioning, hematoxylin-eosin staining, and slide preparation. With the assistance of specialized technicians, each germ layer and tissue were identified, and the results are shown in Figure 13 .

[0229] As Figure 13 can be seen, the GpEpiSC cells disclosed in this article have the ability to differentiate into three germ layers in vivo (ectoderm - nerve cells; mesoderm - cartilage tissue; endoderm - primitive gut epithelial tissue), and this ability is still well maintained after multiple passages.

[0230] Using the same method as in Example 3, GpEpiSC pluripotent stem cells were prepared from guinea pig embryos at E10.5 of pregnancy. Transcriptome analysis was performed on GpEpiSCs cells passaged to the 22nd generation (P22), 44th generation (P44), and 85th generation (P85) respectively. The results are shown in Figure 14 . Figure 14 It is shown that during the long-term culture of GpEpiSC cells, they can continuously express pluripotency genes at the transcriptional level, while stably maintaining a low level of expression of transcripts related to tridermal differentiation.

[0231] In addition, one strain of GpEpiSC pluripotent stem cells was prepared from guinea pig embryos at E11.5 and E12.5 of pregnancy, respectively. Immunofluorescence staining for H3K27me3 was performed on two strains of GpEpiSCs cells passaged to the 40th generation (P40) to evaluate the methylation status of histones, and the results are shown in Figure 16 (i.e., the article Figure 1 G). H3K27me3 is an epigenetic marker that is usually associated with the inactivated X chromosome in female cells (Qiao et al., 2020). Figure 16 It was shown that there was a single intense focus in the nuclei of GpEpiSCs from female guinea pigs.

[0232] Example 7:

[0233] This example confirmed the similarity between the GpEpiSC cells of the present invention and the epiblast (EPI) cells occurring in vivo.

[0234] GpEpiSC cells were prepared from E10.5 embryos according to the method described in Example 3, and corresponding EPI cells were prepared from embryos at E9.5, E10.5, E11.5, and E12.5, respectively. Guinea pig embryonic fibroblasts (gpEF) were prepared from E23.5 embryos as somatic cell controls. The transcriptome data of GpEpiSCs were subjected to correlation and principal component analysis (PCA) with the transcriptome data of EPI cells and gpEF cells, and the results are shown in Figures 17 - 20 .

[0235] Figure 17 and 18 showed that the GpEpiSC cells disclosed herein were closely similar to in vivo EPI cells from E10.5 and E11.5, but were significantly different from gpEF cells. Figure 19 showed that high expression levels of EPI-related genes were observed in both GpEpiSC cells and in vivo EPI cells, enabling clear cell identity discrimination between the two and the primitive endoderm (PE) and trophectoderm (TE). Figure 20 Heat map analysis of

[0236] Example 8:

[0237] This example confirmed that the pluripotency of the GpEpiSC cells disclosed herein conforms to the characteristics of naive pluripotency.

[0238] Transcriptome analysis was performed on the GpEpiSC cells (derived from E10.5 embryos) and EPI cells (derived from E9.5, E10.5, E11.5, and E12.5 embryos) prepared in Example 7, and the results are shown in Figure 21A . The results of the heatmap analysis showed that both EPI cells and GpEpiSC cells isolated from E9.5 to E12.5 embryos mainly expressed naive and common pluripotency genes; among these cells, the primitive pluripotency genes were hardly expressed. This indicates that the GpEpiSC cells disclosed herein have the characteristics of the naive state in the pluripotency state.

[0239] Using the gpEF cells prepared in Example 7 as somatic cell controls, chromatin accessibility analysis of GpEpiSC cells was performed by ATAC-seq, and the specific peak classification and corresponding highly expressed genes in the two types of cells were identified in detail, and the results are shown in Figure 21B and 21C . The results showed that in somatic cells, the dominant transcription factor motifs located in open chromatin sites mainly belonged to the AP-1 family, and this result was consistent with the findings in mice and humans. At the same time, enriched OCT4-SOX2-NANOG, POU family, RFX, and ZFP281 (ZNF281) motifs were also observed in GpEpiSC, and these genes all play key roles in maintaining and identifying stem cell identity. In particular, the ZNF281 gene is generally recognized as effective for maintaining and inducing naive epiblast stem cells in humans and mice (Fidalgo et al., 2016).

[0240] In this example, the consistency of the transcription factor expression profiles of GpEpiSC cells with naive embryonic stem cells (hESC and mEpiSC) of humans and mice was further compared. A ternary plot was obtained based on the transcriptome data of GpEpiSC cells, human naive embryonic stem cells (hESC), and mouse naive embryonic stem cells (mEpiSC), and the results are shown in Figure 21D . It can be seen that there is a high-density central region among the 1237 transcription factors shared among the three species, indicating the consistency and strong conservation of the expression of transcription factors in these species. Figure 21D (Left) shows that multiple cross-species co-expressed pluripotency factors and naive pluripotency factors (such as OCT4, ETV4, ETV5, and ZNF281) were identified in the central region, while transcription factors (such as EOMES and OTX2, etc.) distributed outside the central dense region showed species-specific expression patterns ( Figure 21D (Right)).

[0241] gpEF cells were also used as somatic cell controls, and the epigenetic characteristics of GpEpiSC cells were further characterized and identified through histone modification data (H3K4me3, H3K27me3, and H3K27ac), ATAC-seq, and RNA-seq. The results are shown in Figure 21E . The results showed that in GpEpiSC cells, key genes maintaining pluripotency (such as OCT4 and NANOG), as well as important genes related to naive pluripotency (such as ETV4 and ZNF281, etc.) all presented an open chromatin structure, marked by active histone modification, indicating that they were all in an actively transcribed state in GpEpiSC cells, consistent with their important roles in pluripotency. In contrast, some naive pluripotency genes expressed in other species (such as EOMES and OTX2) were observed to be mainly modified by inhibitory H3K27me3, indicating that their transcription in GpEpiSC was inhibited.

[0242] Example 9:

[0243] This example found that development-related genes in GpEpiSC cells were subject to binary modification.

[0244] The trilineage differentiation ability of stem cells is subject to complex epigenetic regulation, and the core of the regulation is binary chromatin modification, that is, genes crucial for differentiation simultaneously have activating (H3K4me3) and inhibitory (H3K27me3) marks (Macrae et al., 2022). This dual modification state ensures that development-related genes can be rapidly activated after receiving differentiation cues, while maintaining the stem cell identity and the preparatory state for specific development.

[0245] According to the same method described in Example 3, CUT&Tag of H3K4me3 and H3K27me3 was analyzed using two GpEpiSC cell lines (P33 and P38) derived from E10.5, and RNA-seq assays were performed using two GpEpiSC cell lines (both P24) derived from E10.5 to present the global distribution of H3K4me3 and H3K27me3 marks. The results are shown in Figure 23A . The combined analysis results of RNA-seq showed that 73.7% of the genes corresponding to the single H3K4me3 marker sites were detected to be expressed (TPM > 1), and 34.7% and 61.5% of the genes corresponding to the single H3K27me3 marker sites and binary marker sites were expressed, respectively. Moreover, the average gene expression level of the single H3K4me3 marker was also higher than that of the single H3K27me3 marker and binary marker genes (Tpm were 61.7, 15.3, and 40.5, respectively).

[0246] GO (Gene Ontology) functional enrichment analysis further elucidated the different roles of these epigenetic states. Genes with the single H3K4me3 mark were mainly associated with processes essential for stem cell function, such as cell division and cell cycle regulation ( Figure 23B ); while genes with the single H3K27me3 mark and bivalent-marked genes were enriched for functions related to neural development and embryonic morphogenesis, which are normally dormant in undifferentiated stem cells ( Figure 23C and 23D ).

[0247] Visualization of these genes highlighted the differences in chromatin modification patterns. Using the same method as described in Example 3, CUT&Tag was analyzed using one GpEpiSC cell line (P33) derived from E10.5 and one gpEF cell line (P1) derived from E23.5, and RNA-seq assays were performed using one GpEpiSC cell line (P24) derived from E10.5 and one gpEF cell line (P1) derived from E23.5. The results are shown in Figure 23E . Genes with the H3K4me3 mark showed concentrated peaks ( Figure 23E , left), genes with the H3K27me3 mark showed a broader distribution, usually spanning extensive genomic regions ( Figure 23E , middle), while co-localization of the two modifications was observed for bivalent-marked genes, indicating that they are in a stable state and can be further activated or repressed ( Figure 23E , right). For example, CBX2 / 4 / 8 have both H3K27me3 and H3K4me3 modifications, and these three genes have been reported to play important roles in germ layer lineage differentiation during mouse and human embryonic development (Vandamme et al., 2011). The above conservation of bivalent-marked genes was also observed in the guinea pig GpEpiSC cells disclosed herein.

Claims

1. Isolated guinea pig pluripotent stem cells, which are derived from the epiblast of guinea pig embryos, can be stably passaged for at least 3 generations, and have the potential to differentiate into endoderm, ectoderm and mesoderm cells; Optionally, the stem cells are primed pluripotent stem cells, which can preferably also be transformed into primitive pluripotent stem cells or formative pluripotent stem cells; Optionally, the stem cells are derived from the epiblast of a guinea pig embryo at a gestational period of less than E12.5, preferably between E9.5 and E12.5, and more preferably between E9.5 and E11.5; Optionally, the stem cells have an activated FGF signaling pathway and / or ActA signaling pathway, preferably have both an activated FGF signaling pathway and an activated ActA signaling pathway; Optionally, the stem cells have an inhibited or shut-down Wnt signaling pathway; Optionally, the stem cells express any one selected from Nanog, Pou5F1, Sox2, TRA-1-81 and SSEA1, preferably simultaneously express Nanog, Pou5F1 and Sox2, more preferably simultaneously express Nanog, Pou5F1, Sox2, TRA-1-81 and SSEA1; Preferably, the stem cells further express ETV4 and / or ZNF281; More preferably, the stem cells do not substantially express EOMES and / or OTX2; Optionally, the chromosomes of the stem cells maintain a normal karyotype; and / or, relative to guinea pig multipotent cells or guinea pig differentiated cells (preferably guinea pig fibroblasts), the gene expression level of one of the following functions is downregulated: endosome to lysosome transport, centrosome cycle, chromatin organization, neural tube closure, glycolytic process, epithelial cell differentiation, muscle cell development, neuronal action potential propagation, positive regulation of cell migration, negative regulation of canonical, Wnt signaling pathway, or bone morphogenesis; and / or, relative to guinea pig multipotent cells or guinea pig differentiated cells (preferably guinea pig fibroblasts), the gene expression level of one of the following functions is upregulated: mRNA splicing by spliceosome via spliceosome), rRNA processing, regulation of cell cycle, and nucleosome mobilization.

2. A genetically modified guinea pig pluripotent stem cell obtained by genetically modifying the guinea pig pluripotent stem cell according to claim 1; Optionally, the genetic modification comprises genome editing, preferably comprising nucleic acid fragment deletion, gene modification, gene knockout, altered expression of gene products, repair mutation, polynucleotide insertion, single base mutation or any combination thereof.

3. An isolated cell population comprising the guinea pig pluripotent stem cells of claim 1 or 2, preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or about 100% of the cells in the cell population are the guinea pig pluripotent stem cells of claim 1 or 2.

4. A monoclonal cell line isolated from the guinea pig pluripotent stem cells according to claim 1 or 2.

5. Use of the guinea pig pluripotent stem cells according to claim 1 or 2, the cell population according to claim 3, or the monoclonal cell line according to claim 4 for producing transgenic animals or performing gene editing.

6. A method for producing guinea pig pluripotent stem cells that can be stably propagated in vitro for at least 3 generations, comprising the following steps: (1) Obtaining epiblast (EPI) cell masses that are substantially separated from visceral endoderm (VE) from guinea pig embryos; (2) culturing the cell clumps separated in step (1) until the cell clumps produce outgrowths, separating the outgrowths, obtaining smaller cell clumps from the outgrowths, and inoculating the smaller cell clumps to complete one passage; (3) after at least 3, preferably 3 to 5, more preferably 5 times of passaging according to step (2), digesting the outgrowth into single cells and passaging; Optionally, before outgrowths are generated, the cell clumps separated in step (1) are inoculated on a feeder layer prepared in advance; Optionally, the subculturing in step (2) is to inoculate the smaller cell aggregates onto a feeder layer; Optionally, obtaining single cell clones from the passaged cells of step (4) to establish a guinea pig pluripotent stem cell line; Optionally, the guinea pig embryo in step (1) is a guinea pig embryo with a gestation period below E12.5, preferably E9.5 to E12.5, and more preferably E9.5 to E11.5; Optionally, the step (1) is to obtain cell aggregates by mechanical and / or enzymatic digestion. Preferably, the epiblast is directly divided into masses after the epiblast is separated from the visceral endoderm by a metal tool, and the metal tool is preferably a metal wire or forceps; or Preferably, the extraembryonic ectoderm is removed, the guinea pig embryo is turned over to expose the epiblast, and then digested with trypsin for 2 to 3 minutes; Optionally, the culture medium used for inoculation within 10 passages in step (2) and step (3) contains 5 to 15 μM, preferably 8 to 12 μM, more preferably 9 to 10 μM of Rock signaling pathway inhibitor; Optionally, after 10 passages in step (2) and step (3), the culture medium used for inoculation contains 0 to 5 μM, preferably 2 to 3 μM, of a Rock signaling pathway inhibitor; Optionally, the Rock signaling pathway inhibitor is preferably selected from at least one of the following: Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152dihydrochloride, Azaindole1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, more preferably Y-27632; Optionally, the culture medium used in steps (2) and (3) further contains an FGF signaling pathway agonist and / or a Wnt signaling pathway inhibitor; Preferably, the FGF signaling pathway agonist is FGF2; Preferably, the Wnt signaling pathway inhibitor is selected from IWR-1-endo, IWP2 or XAV939, or any combination thereof; Preferably, the content of the FGF signaling pathway agonist is 5 to 50 ng / ml, more preferably 10 to 40 ng / ml, and further preferably 20 to 30 ng / ml; Preferably, the content of the Wnt signaling pathway inhibitor or a combination thereof is 1 to 6 μM, 1.5 to 5.5 μM, 2 to 5 μM, 2.1 to 5 μM, 2.2 to 5 μM, 2.3 to 5 μM, 2.4 to 5 μM, 2.5 to 5 μM, more preferably 2 to 5 μM IWR-1-endo, IWP2 or XAV939, or 2.5 to 5 μM IWR-1-endo, IWP2 or XAV939, or a combination of 2.5 μM IWR-1-endo and 100 nM IWP2, or a combination of 2 μM XAV939 and 100 nM IWP2; Optionally, when single cell passaging is performed in step (3), the cells are passaged once every 3 to 4 days, and the passaging ratio is 1:5 to 1:

10.

7. The guinea pig pluripotent stem cells prepared according to the method of claim 6, which can be stably propagated in vitro for at least 3 generations, preferably at least 5 generations, more preferably at least 10 generations, further preferably at least 20 generations, further preferably at least 40 generations, and most preferably at least 70 generations.

8. Use of a Rock signaling pathway inhibitor for isolating, preparing, culturing and / or maintaining guinea pig pluripotent stem cells capable of stable passage for at least 3 generations, wherein the Rock signaling pathway inhibitor is preferably selected from at least one of the following: Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, and more preferably Y-27632; Optionally, the working concentration of the Rock signaling pathway inhibitor is 1 to 20 μM, preferably 2 to 15 μM, more preferably 2 to 10 μM, most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM; Optionally, the Rock signaling pathway inhibitor is also used in combination with an FGF signaling pathway agonist and / or a Wnt signaling pathway inhibitor. Preferably, the agonist of the FGF signaling pathway is FGF2; Preferably, the Wnt signaling pathway inhibitor is selected from IWR-1-endo, IWP2 or XAV939, or any combination thereof. Preferably, the content of the FGF signaling pathway agonist is 5 to 50 ng / ml, more preferably 10 to 40 ng / ml, and further preferably 20 to 30 ng / ml; Preferably, the content of the Wnt signaling pathway inhibitor or its combination is 1 to 6 μM, 1.5 to 5.5 μM, 2 to 5 μM, 2.1 to 5 μM, 2.2 to 5 μM, 2.3 to 5 μM, 2.4 to 5 μM, 2.5 to 5 μM, more preferably 2 to 5 μM IWR-1-endo, IWP2 or XAV939, or 2.5 to 5 μM IWR-1-endo, IWP2 or XAV939, or a combination of 2.5 μM IWR-1-endo and 100 nM IWP2, or a combination of 2 μM XAV939 and 100 nM IWP2.

9. A culture medium for isolating, preparing, culturing and / or maintaining guinea pig pluripotent stem cells capable of stable passage for at least 3 generations, comprising a basal culture medium, a stem cell trophic factor, a Rock signaling pathway inhibitor, an FGF signaling pathway agonist and a Wnt signaling pathway inhibitor, Optionally, the basal medium is selected from DMEM / F12 or Neurobasal; Optionally, the stem cell trophic factor is selected from N2 serum-free supplement, B27 serum-free supplement, GlutaMax, MEMNEAA or BSA, or any combination thereof; Optionally, the Rock signaling pathway inhibitor is selected from at least one of the following: Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152dihydrochloride, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, preferably Y-27632; Optionally, the agonist of the FGF signaling pathway is FGF2; Optionally, the Wnt signaling pathway inhibitor is selected from IWR-1-endo, IWP2 or XAV939, or any combination thereof; Optionally, the basal medium contains 50% (v / v) DMEM / F12 and 50% (v / v) neurobasal; Optionally, the content of the stem cell trophic factor is 0.5% to 1% (v / v) N2 serum-free supplement and 0.5% to 1% (v / v) B27 serum-free supplement, 1% (v / v) GlutaMax, 1% (v / v) MEM NEAA, 1% (v / v) penicillin-streptomycin solution, 0.1 mM β-mercaptoethanol, 0 to 1 mg / ml BSA fraction V; Optionally, the Rock signaling pathway inhibitor is present in an amount of 0 to 15 μM, 0 to 10 μM, or 2 to 10 μM Y-27632; Optionally, the content of the FGF signaling pathway agonist is 5 ng / ml to 50 ng / ml, more preferably 10 to 40 ng / ml, further preferably 20 to 30 ng / ml FGF2; Optionally, the content of the Wnt signaling pathway inhibitor is 1 to 6 μM, 1.5 to 5.5 μM, 2 to 5 μM, 2.1 to 5 μM, 2.2 to 5 μM, 2.3 to 5 μM, 2.4 to 5 μM, 2.5 to 5 μM, more preferably 2 to 5 μM IWR-1-endo, IWP2 or XAV939, or 2.5 to 5 μM IWR-1-endo, IWP2 or XAV939, or a combination of 2.5 μM IWR-1-endo and 100 nM IWP2, or a combination of 2 μM XAV939 and 100 nM IWP2; Optionally, the culture medium comprises DMEM / F12, neurobasal, N2 serum-free supplement, B27 serum-free supplement, GlutaMax, MEM NEAA, penicillin-streptomycin solution, β-mercaptoethanol, Y-27632, FGF2, IWR-1-endo, and IWP2; Optionally, the culture medium contains 237.5 mL DMEM / F12, 237.5 mL neurobasal, 2.5 ml N2 serum-free supplement, 5 ml B27 serum-free supplement, 1% (v / v) GlutaMax, 1% (v / v) MEM NEAA, 1% (v / v) penicillin-streptomycin solution, 0.1 mM β-mercaptoethanol, 1 mg / ml BSA fraction V, 20 ng / ml FGF2, 2.5 μM IWR-1-endo, 100 nM IWP2, 2 μM or 10 μM Y-27632 per 500 ml.

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