Compositions and methods for screening and target analysis of aging-related drugs

Abstract

A mammalian cell model that can be used for anti-aging drugs is provided. The mammalian-derived extraembryonic trophoblast cell model is an early mammalian extraembryonic stage. The cell differentiation model into extraembryonic trophoblast cells / organoids and the aging evaluation index system can be used in various anti-aging research and target testing, anti-aging drug discovery, screening, and identification, and research and development of innovative drugs / natural products, chemical products, and / or health foods and additives. This will help screen and identify many resistance targets. Candidate targets of aging can be further mechanized and functionally verified for future therapeutic applications. Screening for anti-aging drugs or natural products will create significant economic and social benefits.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of PCT application PCT / CN2022 / 121158, filed September 25, 2022, the entire contents of which are expressly incorporated herein by reference.

[0002] The present invention relates to the field of using cultured cells, particularly mammalian cells, and its applications in assessing mammalian cell senescence, evaluating and screening potential anti-senescence agents, and identifying target genes involved in senescence. [Background technology]

[0003] Aging is a natural process that occurs in various organisms and is influenced by factors, including environmental, genetic, and epigenetic factors, resulting in cell and tissue damage. Various human diseases, such as diabetes, neurodegenerative diseases, heart disease, vascular disease, and cancer, are associated with aging. Various other aging processes can be observed in various other mammals.

[0004] However, studying aging in mammals is time-consuming due to their long lifespans. Other organisms have been used to study aging (summarized in Table 1). Among the earliest and simplest model organisms used to study aging is the yeast, Saccharomyces cerevisiae. With lifespans ranging from days to weeks, the replicative and chronological lifespans of yeast have been monitored using two different models, each of which has revealed important insights into aging pathways and potential interventions. However, yeast models are not without limitations. While 90% of yeast genes have been characterized, only 30% of the genome is conserved in humans. Another challenge is pleiotropic drug resistance (PDR), which requires further validation of optimal dosages of bioactive compounds in animal models.

[0005] The nematode Caenorhabditis elegans is also a popular model organism for aging research. Its short lifespan and high genetic similarity with humans (60-80%) make it a good candidate for high-throughput screening of anti-aging compounds. However, C. elegans lacks crucial epigenetic changes, such as DNA methylation, that occur during the human aging process.

[0006] Another model organism for aging research, the fruit fly Drosophila melanogaster, provides insight into aging at the level of whole-organism physiology and behavior. Despite the advantages this model offers, such as low cost and ease of generating large populations, its small size and unknown reasons for mortality pose challenges to aging research. Furthermore, when conducting high-throughput drug screening in Drosophila, many drugs are not included in drug libraries, primarily because the solvent commonly used for drugs is DMSO, which is cytotoxic to Drosophila.

[0007] Mouse models have become a powerful tool in aging research because they offer a longer lifespan, a degree of genetic control, and a large amount of available baseline phenotypic data, albeit at a higher cost. However, they pose several limitations due to their different genetic background and physiological characteristics compared to humans.

[0008] Attempts have also been made to use non-canonical models, such as African turquoise killifish, birds, nonhuman primates, and dogs, each of which offers unique advantages. Due to their rapid reproduction, killifish age faster than other vertebrates by 4–6 months. Avian models have provided insights into multiple anti-aging mechanisms, given their remarkable resistance to harmful factors such as oxidative stress. Primates are evolutionarily closer to humans, allowing for the testing of interventions and compounds. Companion dogs share an environment with humans, and their genetics are more tractable than those of primates. Despite these features, these models still present significant challenges, including limited reference and homology to humans, cost, time, and ethical concerns.

[0009] Although various other animal models have been used to study aging (Table 1), their impact has been limited for several reasons. Model animals differ from humans in absorption, distribution, metabolism, and secretory functions, as well as in their proteomes and genomes. Therefore, many experimental results cannot be easily applied to humans.

[0010] In recent years, human cell lines, particularly primary cell lines such as fibroblasts and induced pluripotent stem cells (iPSCs), have attracted attention as models for studying aging due to their relevance to human aging and disease. In vitro cellular senescence has been demonstrated to share many characteristics with in vivo aging, such as loss of division potential and chromatin alterations. In vitro aging readouts have also improved significantly over the past few years, from a decline in cell replication, with varying accuracy depending on the method, to more systematic transcriptome-based approaches such as CultureAge, scAge, and AgeScore. Nevertheless, current applications have generally focused on specific aspects of aging, such as skin aging and vascular aging, rather than human aging in general. Mesenchymal stem cells (MSCs) are another cell type being explored for use in aging research. However, MSC differentiation phenotypes only partially overlap with natural aging.

[0011] The use of accelerated aging models offers certain advantages over natural aging models, such as time efficiency and the ability to study aging-related changes in a controlled environment. Although several artificially generated mouse strains are the most popular options for accelerated aging models, differences between mice and humans in various aspects, including physiological, metabolic, and environmental factors, have yet to be addressed.

[0012] While many promising drugs have been screened using these models, drug screening using only these models may leave some side effects unknown. For example, aspirin, ibuprofen, and celecoxib are drugs that have been shown to extend lifespan in some model organisms but not in humans. Aspirin, previously demonstrated in C. elegans, Drosophila, and male mice, has been shown to increase the risk of gastrointestinal bleeding, while ibuprofen and celecoxib, tested in yeast, worms, and flies, have been shown to have side effects such as major bleeding events and increased overall mortality in humans.

[0013] [Table 1-1] [Table 1-2] Chinese Patent Document No. CN107858330B discloses an in vitro method for screening anti-aging drugs using a mouse bone marrow hematopoietic stem / progenitor cell model. The method involves isolating hematopoietic stem / progenitor cells from mouse bone marrow and culturing the cells in a stem cell culture medium containing IL3, IL6, and SCF for 5 to 14 days. It has been reported that this method can rapidly obtain a large number of aged hematopoietic stem / progenitor cells, which can be used to screen anti-aging drugs. Because this method utilizes mouse bone marrow hematopoietic stem / progenitor cells for anti-aging drug screening, it is difficult to adapt to other mammals and humans. Furthermore, the method is complex to implement and the evaluation system is inefficient, resulting in uncertain screening results and preventing easy widespread application. Therefore, there remains a need to establish a more reliable, relevant, and human-specific system for evaluating cellular senescence, as well as methods for assessing senescence, evaluating and screening anti-aging drugs, and identifying candidate genes involved in senescence. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Chinese Patent No. CN107858330B Specification Summary of the Invention

[0015] The terms "invention," "the invention," "this invention," and "the present invention" as used in this disclosure are intended to broadly refer to all of the subject matter of this patent application and the claims that follow. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the claims of this patent that follow. The subject embodiments of the present invention are defined by the claims, not this Summary. This Summary is a high-level overview of various aspects of the invention and introduces some of the concepts described and illustrated in this disclosure and the accompanying drawings. This Summary is not intended to identify key or important features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any and all figures, and appropriate portions of each claim. This disclosure describes and refers to various embodiments of the present invention. No particular embodiment is intended to limit the scope of the invention. Rather, the embodiments merely provide non-limiting examples of various methods, compositions, kits, systems, and the like that are included within the scope of the present invention. Some embodiments of the present invention are summarized below, while other embodiments are described and illustrated elsewhere in this disclosure.

[0016] In some embodiments, provided herein is a senescence assessment system for mammalian cells comprising totipotent or pluripotent stem cells or early extraembryonic cells, wherein the totipotent or pluripotent stem cells or early extraembryonic cells optionally comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or syncytiotrophoblasts ("STBs").

[0017] In some embodiments, the totipotent or pluripotent stem cells or early extraembryonic cells are naturally occurring. In some embodiments, the totipotent or pluripotent stem cells or early extraembryonic cells are genetically modified.

[0018] In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells may comprise a reporter molecule, hi some embodiments, the reporter molecule is selected from the group consisting of physically activated molecules and chemically activated molecules.

[0019] In any embodiment herein, totipotent or pluripotent stem cells or early extraembryonic cells can contain heterologous nucleic acid.In some embodiments, the heterologous nucleic acid is integrated into the genome of totipotent or pluripotent stem cells or early extraembryonic cells.In some embodiments, the heterologous nucleic acid is under the control of the promoter of an endogenous biomarker gene that encodes a biomarker that indicates the senescence or differentiation of totipotent or pluripotent stem cells or early extraembryonic cells toward trophoblast or STB.In some embodiments, the heterologous nucleic acid is introduced into totipotent or pluripotent stem cells or early extraembryonic cells by gene editing tools.

[0020] In any of the embodiments herein, the reporter molecule may be selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, tdTomato, photoconvertible fluorescent protein, bioluminescence, enzyme assay, antibody-based assay, chloramphenicol acetyltransferase, and biosensor.

[0021] In any of the embodiments herein, the system may include early extraembryonic cells. In some embodiments, the early extraembryonic cells are trophoblast stem cells ("TSCs") or trophoblast progenitor cells ("TPCs"). In some embodiments, the early extraembryonic cells are TSCs. In any of the embodiments herein, the early extraembryonic cells may be derived from totipotent stem cells, pluripotent stem cells, embryonic tissue, or placental tissue. In any of the embodiments herein, the early extraembryonic cells may be derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

[0022] In any of the embodiments herein, the endogenous biomarker may be selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-to-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion.

[0023] In some embodiments, the endogenous biomarkers are β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3 , CDKN1A, CDKN1C, CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, and SIRT family members.

[0024] In any of the embodiments herein, the system may comprise totipotent or pluripotent stem cells. In some embodiments, the system comprises totipotent stem cells. In some embodiments, the system comprises pluripotent stem cells. In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells may be derived from a human, a pig, a cow, a mouse, a rat, a bat, a rabbit, a dog, a cat, and a sheep.

[0025] In some embodiments, provided herein are methods of assessing the aging process in mammalian cells, the methods comprising subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate the totipotent or pluripotent stem cells or extraembryonic cells towards trophoblasts or STBs, and determining one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells.

[0026] In some embodiments, provided herein are methods for evaluating the anti-aging function of a candidate agent, the methods comprising: 1) subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the totipotent or pluripotent stem cells or early extraembryonic cells to differentiate toward trophoblasts, particularly STBs; 2) contacting the totipotent or pluripotent stem cells or early extraembryonic cells with a candidate agent before, during, or after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the totipotent or pluripotent stem cells or early extraembryonic cells to differentiate; and 3) assessing a change in one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells not contacted with the candidate agent.

[0027] In some embodiments, evaluating the anti-aging function of the candidate agent comprises assessing whether the candidate agent has anti-aging function, hi some embodiments, evaluating the anti-aging function of the candidate agent comprises determining an effective concentration of the candidate agent that has anti-aging function.

[0028] In some embodiments, provided herein are methods of screening candidate agents with anti-aging function, the methods comprising: 1) evaluating the anti-aging function of a plurality of candidate agents according to any of the methods of embodiments herein; and 2) identifying candidate agents with anti-aging function based on the ability of the candidate agents to cause a change in one or more characteristics of differentiation of totipotent or pluripotent stem cells or early extraembryonic cells into trophoblasts or STBs compared to totipotent or pluripotent stem cells or early extraembryonic cells without the candidate agent.

[0029] In any of the embodiments herein, the candidate agent may be selected from the group consisting of an antibody, a virus, a virus-like substance, a small molecule, a peptide, a polypeptide, DNA, mRNA, a guide RNA, a microRNA, an RNAi, an lncRNA, an siRNA molecule, and an antisense RNA. In any of the embodiments herein, the candidate agent may be a naturally occurring substance. In any of the embodiments herein, the candidate agent may be a nutritional supplement.

[0030] In some embodiments, provided herein are methods of identifying candidate genes involved in the aging process, the methods comprising: 1) subjecting a plurality of totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate the plurality of totipotent or pluripotent stem cells or early extraembryonic cells toward trophoblasts or STBs, wherein each of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprises an alteration in a candidate agent compared to a wild-type totipotent or pluripotent stem cell or early extraembryonic cell, and wherein at least two of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprise a different alteration; 2) determining one or more differentiation characteristics of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells; and 3) identifying the candidate genes involved in the aging process based on the ability of the alteration in the candidate gene to cause a change in the one or more differentiation characteristics of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells that do not comprise the corresponding alteration.

[0031] In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells may be naturally occurring. In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells may be genetically modified.

[0032] In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells may comprise a reporter molecule that indicates differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells toward STBs. In some embodiments, the reporter molecule is selected from the group consisting of physically activated molecules and chemically activated molecules.

[0033] In any of the embodiments described herein, the totipotent or pluripotent stem cells or early extraembryonic cells may comprise a heterologous nucleic acid encoding a reporter molecule that indicates differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells toward trophoblasts or STBs. In some embodiments, the heterologous nucleic acid is integrated into the genome of the totipotent or pluripotent stem cells or early extraembryonic cells. In some embodiments, the heterologous nucleic acid is under the control of a promoter of an endogenous biomarker gene that encodes a biomarker that indicates differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells toward STBs. In any of the embodiments described herein, the heterologous nucleic acid may be introduced into the totipotent or pluripotent stem cells or early extraembryonic cells by a gene editing tool. In any of the embodiments described herein, the reporter molecule may be selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, and tdTomato.

[0034] In any embodiment herein, the STB may be an early STB, a late STB, or a mature and senescent STB.

[0035] In any of the embodiments herein, the one or more differentiation characteristics may include 1) the presence or absence of a biomarker associated with totipotent or pluripotent stem cell or early extraembryonic cell differentiation, 2) the level of a biomarker associated with totipotent or pluripotent stem cell or early extraembryonic cell differentiation, 3) the secretion of a biomarker associated with totipotent or pluripotent stem cell or early extraembryonic cell differentiation, 4) cell morphology, 5) the rate of change to the differentiation state, 6) the properties of cell organelles, 7) the number of nuclei in the cell, and 8) the presence or absence of a reporter molecule.

[0036] In some embodiments, the one or more differentiation characteristics comprise organelle characteristics. In some embodiments, the organelles are selected from the group consisting of mitochondria, proteosomes, endoplasmic reticulum, Golgi apparatus, and nuclear envelope. In some embodiments, the organelle characteristics comprise organelle number, morphology, and function.

[0037] In any of the embodiments described herein, the totipotent or pluripotent stem cells or early extraembryonic cells can be early extraembryonic cells. In any of the embodiments described herein, the early extraembryonic cells can be derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells. In some embodiments, the one or more differentiation characteristics include the presence or absence of a biomarker associated with differentiation of the early extraembryonic cells.

[0038] In some embodiments, the biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion.

[0039] In some embodiments, the biomarkers are β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1 alpha, IL1 beta, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A , CDKN1C, CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, SIRT family members, and combinations thereof.

[0040] In any embodiment herein, the one or more differentiation hallmarks may include nucleotide excision repair (NER), base excision repair (BER), DNA mismatch repair (MMR), the Fanconi anemia pathway, homologous recombination (HR), non-homologous end joining (NHEJ), variant histones, insulin resistance, pro-inflammatory factors, the mTOR / AMPK pathway, mitophagy, a senescence-associated secretory phenotype, and / or molecular indicators of senescence.

[0041] In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells can be totipotent or pluripotent stem cells. In some embodiments, the totipotent or pluripotent stem cells are totipotent stem cells. In some embodiments, the totipotent or pluripotent stem cells are pluripotent stem cells.

[0042] In any of the embodiments herein, the one or more differentiation characteristics may include the presence or absence of a biomarker associated with totipotent or pluripotent stem cell differentiation.

[0043] In some embodiments, the biomarkers are β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1 alpha, IL1 beta, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A , CDKN1C, CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, SIRT family members, and combinations thereof.

[0044] In any of the embodiments herein, the biomarkers can be RNA molecules. In some embodiments, assessing changes in one or more differentiation characteristics comprises RNA sequencing, RT-qPCR, and / or in situ hybridization.

[0045] In any of the embodiments herein, the biomarkers can be protein molecules. In some embodiments, assessing changes in one or more differentiation characteristics comprises Western blot, ELISA, proteomics, and / or immunofluorescence.

[0046] In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells can be contacted with the candidate agent before being subjected to conditions for differentiation. In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells can be contacted with the candidate agent at the same time as being subjected to conditions for differentiation. In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells can be contacted with the candidate agent after being subjected to conditions for differentiation.

[0047] In any of the embodiments herein, the conditions for differentiation may include a cell culture medium containing DMEM / F12, β-mercaptoethanol, penicillin-streptomycin-glutamine, BSA, ITS-X, Y27632, forskolin, and KnockOut Serum Replacement.

[0048] In any of the embodiments herein, differentiation characteristics can be assessed at least one day after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions for differentiation.

[0049] In any of the embodiments herein, differentiation characteristics are assessed 2 to 8 days after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions for differentiation.

[0050] In any of the embodiments herein, the totipotent or pluripotent stem cells or early extraembryonic cells may be derived from human, pig, cow, mouse, rat, bat, rabbit, dog, cat, and sheep.

[0051] In any of the embodiments herein, the methods may further include determining the effect of the candidate agent on the viability of the totipotent or pluripotent stem cells or early extraembryonic cells, or cells differentiated therefrom.

[0052] In any of the embodiments herein, the methods may further include determining the effect of the candidate agent on the viability of the totipotent or pluripotent stem cells or early extraembryonic cells, or cells differentiated therefrom. [Brief explanation of the drawings]

[0053] [Figure 1A] 1A to 1Q show the establishment of TSC cells from proliferative stem cells (EPSCs) and the differentiation of STBs from TSCs. Figure 1A shows TSCs derived from M1-EPSCs. [Figure 1B] Figure 1 shows the morphology of established cell lines after monoclonal M1-TSC proliferation stabilized. [Figure 1C] It is shown that the TSCs obtained by this method expressed TSC markers (CDX2, GATA2, GATA3, CGA, KRT7, and ERVW). [Figure 1D] GATA2 and GATA3 expression in TSCs by immunofluorescence. [Figure 1E] 1 shows that STB differentiated from TSCs exhibited the expected cell morphology. [Figure 1F] Higher hCG expression in STB than in TSC. [Figure 1G] CGA and CGB3 are expressed at higher levels in STB than in TSC. [Figure 1H] Higher hCG expression in STB than TSC as demonstrated by ELISA. [Figure 1I] The contrasting morphologies of TSC and STB are shown. [Figure 1J] Differences in cell morphology and CGB expression between TSC and STB are shown. [Figure 1K] The STB markers SDC1, ERVW1, and CSH1 were significantly more expressed in STB at day 6. [Figure 1L] Higher hCG expression in STB than in TSC. [Figure 1M] This indicates that STB was in a growth-arrested state. [Figure 1N] RNA sequencing data show a decreased cell cycle score as TSCs differentiate towards the STB. [Figure 1O] STB showed significantly lower expression of the proliferation marker Ki67. [Figure 1P] STB showed significantly lower expression of the proliferation marker Ki67. [Figure 1Q] STB showed significantly higher expression of cell cycle inhibitors p27 and p38.

[0054] [Figure 2A] Figures 2A-2O illustrate the changes in various senescence-associated markers during trophoblast stem cell (TSC)-syncytiotrophoblast (STB) differentiation. Figure 2A shows changes in DNA repair (nucleotide excision repair, NER). [Figure 2B] Changes in DNA repair (base excision repair, BER) are shown. [Figure 2C] Shows changes in DNA repair (DNA mismatch repair, MMR). [Figure 2D] Shows alterations in DNA repair (Fanconi anemia pathway). [Figure 2E] Changes in DNA repair (homologous recombination, HR) are shown. [Figure 2F] Changes in DNA repair (non-homologous end joining, NHEJ) are shown. [Figure 2G] The changes in variant histone scores are shown. [Figure 2H] Indicates changes in incorrect nutrient sensing (insulin resistance). [Figure 2I] Changes in pro-inflammatory factors are shown. [Figure 2J] Shows altered nutrient sensing (AMPK signaling pathway). [Figure 2K] Shows altered nutrient sensing (mitophagy). [Figure 2L] The change in overall aging score is shown. [Figure 2M] Shows changes in epigenetics (aging epigenetics). [Figure 2N] Shows changes in senescence inhibition. [Figure 2O] Changes in linker histones are indicated. [Figure 2P] Shows changes in false nutrient sensing (insulin score).

[0055] [Figure 3A] 3A-3Y illustrate the changes in various biomarkers during TSC-STB differentiation. Figure 3A shows the differential expression levels of biomarkers in TSCs and STBs on days 2, 4, and 6, as measured by RNA sequencing. [Figure 3B] Differential expression levels of biomarkers in TSC and STB at days 2, 4, and 6 as measured by RNA sequencing are shown. [Figure 3C] Figure 1 shows the differential expression levels of biomarkers in TSC and STB as measured by RT-qPCR. [Figure 3D] Images of β-galactosidase staining of TSCs and STBs on days 2, 4, and 6 are shown. [Figure 3E] β-galactosidase-positive cells increase as TSCs differentiate toward the STB. [Figure 3F] This shows that γH2AX expression is higher in STB than in TSC. [Figure 3G] We show that p53 expression is higher in STB than in TSC. [Figure 3H] We show that gamma H2AX expression is higher in STB than in TSC. [Figure 3I] We show that p53 expression is higher in STB than in TSC. [Figure 3J] Figure 1 shows that mitochondrial mass is higher in STB than in TSC. [Figure 3K] We show that H3K9me3 expression is lower in STB than in TSC. [Figure 3L] We show that SETDB1 expression is lower in STB than in TSC. [Figure 3M]1 shows lower expression of SIRT1, SIRT3, and SIRT6 in STB than in TSC. [Figure 3N] Biomarkers with higher expression in STB than in TSC are shown. [Figure 3O] We show that the expression of HP1γ and lamin B1 decreased during TSC-STB differentiation, while CGA and CGB increased. [Figure 3P] We showed that the expression of nuclear lamin proteins, lamin A and lamin B, both decreased during TSC-STB differentiation. [Figure 3Q] Immunostaining images showing that HP1γ was more highly expressed in TSCs than in STB cells. [Figure 3R] Immunostaining images showing that lamin B1 is highly expressed in TSC and hCG is positively expressed in STB. [Figure 3S] We show that senescence-associated secretory phenotype (SASP), such as IL-6, IL-8, IL-1 alpha, IL-1 beta, and CCL2, were more highly expressed in STB than in TSC. [Figure 3T] The classical SASP factor IL-6 is shown co-stained with hCG in STB, which was much more highly expressed than in TSC. [Figure 3U] Expression of LTR5 and HERVK at different time points during TSC-STB differentiation is shown. [Figure 3V] 1 shows changes in HERVK expression during TSC-STB differentiation. [Figure 3W] The distribution of different classes of permeable elements (TEs) that are up- and down-regulated as TSCs differentiate towards the STB is shown. [Figure 3X] Changes in γH2AX expression during TSC-STB differentiation are shown. [Figure 3Y] We show that TRF1-γH2AX colocalization increases as TSCs differentiate toward the STB.

[0056] [Figure 4A]Figures 4A-4I show the results of testing the potential anti-aging effects of various drugs. Figure 4A shows the morphology of cells treated with rapamycin. [Figure 4B] Figure 1 shows that rapamycin reduced hCG expression compared to the control (DMSO). [Figure 4C] Figure 1 shows that cells treated with rapamycin showed significantly lower CGA and CGB3 expression. [Figure 4D] Figure 1 shows that cells treated with rapamycin exhibited significantly lower CKDKN1 and IL6 expression. [Figure 4E] 1 shows the morphology of cells treated with remdesivir, GC376, molnupiravir, rapamycin, INK128, and STM2457. [Figure 4F] β-hCG expression levels in cells treated with remdesivir, GC376, molnupiravir, rapamycin, INK128, and STM2457 are shown. [Figure 4G] Morphology of cells treated with nicotinamide mononucleotide (NMN), acarbose, spermidine, fisetin, and quercetin at either 1 μM or 10 μM. [Figure 4H] 1 shows CGA expression in cells treated with NMN, acarbose, spermidine, fisetin, and quercetin at either 1 μM or 10 μM. [Figure 4I] Figure 1 shows that β-hCG expression was reduced in cells treated with different concentrations of rapamycin, INK128, and fisetin.

[0057] [Figure 5A] Figures 5A-5H show the construction of a reporter cell line and its use in testing anti-aging drugs. Figure 5A shows the design of the PD31-CGA-H2B-GFP plasmid used to generate the reporter cell line. [Figure 5B] 1 shows the design of the U6-CGAgRNA-cas9 plasmid used to generate the reporter cell line. [Figure 5C] Genotyping of transfected cells is shown. [Figure 5D] Figure 1 shows that transfected cells expressed GFP upon differentiation into STBs. [Figure 5E] Figure 1 shows that transfected cells express more GFP as they differentiate towards STB. [Figure 5F] Figure 1 shows that transfected cells express more GFP as they differentiate towards STB. [Figure 5G] GFP signals from cells treated with various drugs are shown. [Figure 5H] GFP signals from cells treated with various drugs are shown.

[0058] [Figure 6] A schematic diagram of the workflow for using TSC-STB differentiation for drug screening is shown.

[0059] [Figure 7] Differentially expressed genes during TSC-STB differentiation are shown. DETAILED DESCRIPTION OF THE INVENTION

[0060] Stem cells and their derived differentiated cells hold significant promise in fields such as regenerative medicine, research into disease mechanisms, therapeutic decision-making, and drug screening. Traditionally, embryonic stem cells (ESCs) are derived from early embryos containing approximately 100 cells and generally lack the ability to generate extraembryonic tissues. Recently, it has been reported that proliferative stem cells (EPSCs), naive embryonic stem cells (NAESCs), and 8-cell-like totipotent stem cells (8CLCs) can differentiate into various tissue / cell types, including the three embryonic germ layers and the extraembryonic trophoblast. Due to their ability to differentiate into extraembryonic lineages, these cells can be induced to produce various early extraembryonic cell types, such as trophoblast stem cells (TSCs) and trophoblast progenitor cells. Trophoblast stem cells and trophoblast progenitor cells have also been isolated from placental tissue.

[0061] During mammalian embryonic development, the placenta undergoes developmental processes as the embryo develops and matures. The placenta primarily contains trophoblast cells, which can nurture and protect the fetus. Both types of trophoblast, the syncytiotrophoblast and the extravillous trophoblast, are derived from precursor cells called cytotrophoblasts (CTBs). In particular, cytotrophoblasts can undergo cell fusion to give rise to syncytiotrophoblasts (STBs), which have multiple nuclei in a single cell and can produce large amounts of human chorionic gonadotropin (hCG).

[0062] The generation of mature syncytiotrophoblast (STB) from cytotrophoblast (CTB) is accompanied by characteristics such as growth arrest, upregulation of β-galactosidase, upregulation of cell cycle regulatory genes (e.g., p16, p21, and p57), accumulation of heterochromatin, activation of mTOR, and telomere shortening.

[0063] Recent studies of embryonic stem cells suggest that mature STBs and EVTs can be produced in vitro by inducing TSCs to differentiate for approximately 8–10 days, suggesting that TSCs can be derived from very early stem cells such as trophoblasts from preimplantation embryos or placental tissue, or from human naive ESCs, primed ESCs, or proliferative stem cells (EPSCs). Human TSCs derived from these very early stem cells are highly similar in transcriptome and epigenetics to trophoblast precursor cells (CTBs) derived from placental tissue and are considered to be the in vitro counterpart of CTBs. Very early stem cells in animals such as pigs and cattle have also been shown to be capable of differentiating into trophoblast-like cells.

[0064] The inventions described herein relate, in part, to systems and methods for evaluating and assessing senescence in mammalian cells. The inventions described herein also relate, in part, to systems and methods for evaluating the potential anti-aging function of candidate drugs and screening such drugs in candidate drug libraries. Furthermore, the inventions described herein relate, in part, to systems and methods for identifying candidate genes involved in the aging process. The systems and methods disclosed herein provide a solution for the rapid evaluation and screening of drugs for anti-aging effects by exploiting the differentiation process of syncytiotrophoblasts (STBs) from early extraembryonic cells or stem cells. The systems and methods disclosed herein also enable large-scale, high-throughput screening of candidate drugs and candidate genes, and thus can be used to improve the efficiency of research into anti-aging substances and aging-related target genes.

[0065] In some embodiments, the invention described herein involves culturing early extraembryonic cells or stem cells under conditions that allow differentiation of the early extraembryonic cells or stem cells toward STB. In some cases, the cells become mature STB within 6 to 8 days and exhibit cellular changes consistent with the normal mammalian aging process, such as differential expression of specific senescence marker genes. Thus, the process of STB differentiation from early extraembryonic cells or stem cells mimics the normal mammalian aging process. In some cases, the early extraembryonic cells or stem cells contain reporter molecules that signal STB differentiation and / or cellular senescence. By observing the cellular changes and / or reporter molecules, the invention can be used to evaluate the effects of drugs on cellular senescence. The invention can also be used to screen candidate drug libraries for drugs with anti-aging effects. Furthermore, the invention can be used to identify target genes involved in senescence. For example, cells can be engineered such that expression of a reporter molecule signals the expression of a target gene during STB differentiation.

[0066] One aspect of the present invention relates to a system for assessing senescence of mammalian cells comprising totipotent or pluripotent stem cells or early extraembryonic cells, which optionally comprise a reporter molecule or heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or syncytiotrophoblasts ("STBs").

[0067] Another aspect of the invention relates to a method for assessing the aging process in mammalian cells, the method comprising subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs, and determining one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells.

[0068] Another aspect of the present invention relates to a method for evaluating the anti-aging function of a candidate agent, the method comprising: 1) subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the totipotent or pluripotent stem cells or early extraembryonic cells to differentiate towards trophoblasts, particularly STBs; 2) contacting the totipotent or pluripotent stem cells or early extraembryonic cells with a candidate agent before, during, or after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the totipotent or pluripotent stem cells or early extraembryonic cells to differentiate; and 3) assessing a change in one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells not contacted with the candidate agent.

[0069] Another aspect of the present invention relates to a method for screening candidate agents with anti-aging function, the method comprising: 1) evaluating the anti-aging function of a plurality of candidate agents; and 2) identifying candidate agents with anti-aging function based on the ability of the candidate agents to cause a change in one or more characteristics of differentiation of totipotent or pluripotent stem cells or early extraembryonic cells into trophoblasts or STBs compared to totipotent or pluripotent stem cells or early extraembryonic cells without the candidate agent.

[0070] Yet another aspect of the invention relates to a method for identifying candidate genes involved in the aging process, the method comprising: 1) subjecting a plurality of totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate the plurality of totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs, wherein each of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprises an alteration in a candidate agent compared to a wild-type stem cell or early extraembryonic cell, and at least two of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprise a different alteration; 2) determining one or more differentiation characteristics of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells; and 3) identifying the candidate genes involved in the aging process based on the ability of the alteration in the candidate gene to cause the change in the one or more differentiation characteristics of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells that do not comprise the corresponding alteration.

[0071] All publications, including patent documents, scientific articles, and databases, referred to herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including a patent, patent application, or scientific article, were specifically and individually indicated to be incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, publication, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0072] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0073] I. Definition Certain terms and concepts are described below, which, in conjunction with the remainder of this disclosure and the accompanying drawings, are intended to facilitate understanding of various embodiments of the present invention. These terms and concepts may be further clarified and understood based on accepted practice in the field of the invention and the descriptions provided throughout this disclosure and / or the accompanying drawings. Some other terms may be explicitly or implicitly defined in other sections of this disclosure and in the accompanying drawings, and may be used and understood based on accepted practice in the field of the invention and the descriptions provided throughout this disclosure and / or the accompanying drawings. Terms that are not explicitly defined may also be defined and understood based on accepted practice in the field of the invention and may be interpreted in the context of this disclosure and / or the accompanying drawings.

[0074] As used herein, the terms "a," "an," and "the" may refer to "one," "one or more," or "at least one," unless otherwise specified.

[0075] The term "about" or "approximately" is used herein to indicate that a value includes the inherent variation of the error of the device, the method used to determine the value, or simply the error tolerance of the value. For example, the term "about" or "approximately" can mean a ±1%, ±5%, ±10%, ±15%, or ±20% variation from a given value.

[0076] As used herein, the terms "isolating," "separating," or "purifying," and related terms, are not necessarily used to refer to the removal of all materials other than the component(s) of interest from a sample. Instead, in some embodiments, these terms are used to refer to procedures that enrich the amount of one or more components of interest relative to one or more other components present in a sample. In some embodiments, "isolation," "separation," or "purification" can be used to remove or reduce the amount of one or more components from a sample. For example, the phrase "isolated cells" can refer to cells that have been substantially separated or purified from other cells in a cell culture or organism.

[0077] The term "derived" and related phrases, when referring to cells or biological samples, indicate that the cells or sample were obtained at some point from the described source. For example, cells derived from an organism may represent primary cells (i.e., unmodified) obtained directly from an individual, or they may be modified or immortalized, for example, by the introduction of a recombinant vector, exposure to specific conditions, or culturing under specific conditions. In some cases, cells derived from a given source undergo cell division and / or differentiation such that the original cells no longer exist, but subsequent cells are understood to be derived from the same source. The terms "induce," "induction," and related terms and phrases may also be used in this disclosure to refer to the creation of a cell population, cell, or culture from a different starting or preceding cell population, cell, or culture. For example, the trophoblast stem cells (TSCs) described in this disclosure may be described as derived from proliferative stem cells (EPSCs).

[0078] When used in this disclosure to describe various embodiments of the present invention, the term "comprising" and related terms (e.g., "comprise," "comprises," etc.) are open-ended, meaning that they do not exclude additional elements, and are synonymous with the terms "including," "containing," or "having." When embodiments of the present invention are described using the term "comprising," it is intended to include embodiments in which the term "comprising" is replaced with the term "consisting of" or "consisting essentially of." In other words, descriptions of embodiments of the invention described in this disclosure using the term "comprising" and related terms also provide descriptions of related embodiments using "consisting of" or "essentially consisting of" instead of "comprising." The term "consisting of" excludes all elements (steps, components, etc.) not specified in the description. The term "consisting essentially of" is intended to exclude only elements not specified in the description that do not materially affect the basic and novel characteristics of the embodiment.

[0079] The term "lineage," when used in reference to cells, encompasses all stages of development of a cell type, from the most ancient precursor cell to a fully mature (specialized) cell.

[0080] In the context of cell culture, the term "dissociating" can refer to the process of isolating cells from other cells or from a surface, such as a culture plate surface. For example, cells can be dissociated from an organ or tissue by mechanical or enzymatic methods. In another example, cells that aggregate in vitro can be dissociated from each other. In yet another example, adherent cells are dissociated from a culture plate or other surface. Dissociation can include disrupting the extracellular matrix (ECM) and cellular interactions with the substrate (e.g., the culture surface) or disrupting the ECM between cells.

[0081] "Cell potency" refers to the ability of a cell to differentiate into other cell types. Cells can be referred to as pluripotent cells, multipotent cells (capable of differentiating into some, but not all, cell types, e.g., umbilical cord blood stem cells and mesenchymal stem cells), or oligopotent cells (capable of differentiating into some cell types, e.g., lymphocytes or vascular cells). Under current understanding, potency exists on a continuum. Thus, the boundaries between cell divisions based on potency can be fluid and are not necessarily definitive.

[0082] The term "induced pluripotent stem cells" (iPSCs) refers to pluripotent stem cells artificially derived from non-pluripotent cells. For example, human iPSCs are artificially derived from human non-pluripotent cells. iPSCs can be derived by introducing a specific set of pluripotency-associated genes, or products of "reprogramming factors," into a given cell type and / or by exposing non-pluripotent cells to specific conditions. Reprogramming factors are usually only transiently active until the cells acquire pluripotent properties.

[0083] "Adult stem cells," sometimes also called "somatic stem cells," are stem cells found in an organism among differentiated cells in a tissue or organ and can differentiate to yield some or all of the specialized cell types in the tissue or organ. Somatic stem cells can be propagated in culture. When differentiating into specialized cells, they usually generate intermediate cells called "precursor cells" or "progenitor cells." Somatic stem and progenitor cells can be described as "multipotent" or "oligopotent," depending on their degree of potency. Some examples of somatic stem cells are hematopoietic stem cells, which give rise to all blood cell types (erythrocytes, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, and macrophages); mesenchymal stem cells, including bone marrow stromal stem cells and skeletal stem cells, which can give rise to bone cells (osteoblasts and osteocytes), chondrocytes (chondrocytes), adipocytes (adipocytes), and stromal cells that support hematopoiesis; neural stem cells, which can give rise to nerve cells (neurons), astrocytes, and oligodendrocytes; epithelial stem cells of the mucosa of the digestive tract, which can give rise to absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells; and skin stem cells, which arise in the basal layer of the epidermis (which can give rise to keratinocytes) and at the base of hair follicles (which can give rise to both hair follicles and epidermis). Tissue-specific progenitor cells are cells lacking the ability to self-renew and which are committed to differentiate into cells of a specific organ or tissue. Certain somatic stem cell types can differentiate into cell types found in organs or tissues other than those predicted from the somatic stem cell's origin, a phenomenon called "transdifferentiation."

[0084] As used herein, the term "precursor cell" or "progenitor cell" refers to a cell that can normally differentiate to form one or more types of cells. A "precursor cell" or "progenitor cell" can be any cell in a cell differentiation pathway that can differentiate into a more mature cell. Precursor cells can be primary cells obtained from an organism, cells grown in culture, or cells derived from stem cells.

[0085] The term "placenta" and related terms refer to the mammalian temporary vascular organ that connects the umbilical cord of the developing fetus to the wall of the maternal uterus and mediates metabolic exchange between the fetal and maternal blood supplies through the association of placental tissue with the uterine mucosa.

[0086] The term "trophoblast" and related terms refer to all cells of the trophoblast lineage, which includes the extraembryonic lineage groups (cytotrophoblast, syncytiotrophoblast, intermediate trophoblast) and therefore do not directly contribute to the cells of the fetal body. The extraembryonic lineage consists of the chorion (a combination of trophoblast and underlying extraembryonic mesoderm), amnion, yolk sac, and allantois. In some contexts, the term "trophoblast" is also used to encompass trophectoderm.

[0087] The term "trophoblast stem cell" and related terms refer to cells of a subpopulation of trophoblast cells that have stem cell properties and the ability to differentiate into either syncytiotrophoblasts or extravillous trophoblasts by fusion.

[0088] The terms "cytotrophoblast," "cytotrophoblast cells," and related terms refer to a population of mononuclear cells within the placental villi that have stem cell and epithelial properties located just below the syncytiotrophoblast. These trophoblast progenitor cells have the ability to generate either syncytiotrophoblasts by fusion or extravillous trophoblasts that invade the endometrium and remodel maternal spiral arteries. In some contexts, the terms are used interchangeably with "trophoblast stem cells" and "early extraembryonic cells." In some cases, early extraembryonic cells are used to refer to cytotrophoblasts cultured in vitro.

[0089] The term "syncytiotrophoblast" and related terms refer to the multinucleated cells (which may also be described as multinucleated structures) that cover the surface of the placental villi. Syncytiotrophoblast is created by the fusion of underlying cytotrophoblasts and represents the fetal side of the maternal-fetal interface. A distinct early form of syncytiotrophoblast is formed by the fusion of trophectoderm cells in the blastocyst and facilitates implantation of the embryo into the maternal endometrium.

[0090] "Differentiation" is the process by which less specialized cells become more specialized cell types. For example, early development of multicellular animals is characterized by the rapid proliferation of embryonic cells, which then differentiate to produce the many specialized cell types that make up the tissues and organs of the multicellular animal. As cells differentiate, their proliferation rate typically slows. Some types of differentiated cells never divide again, but many differentiated cells can resume proliferation as needed to replace cells lost as a result of damage or cell death. Some cells divide continuously throughout life to replace cells that have a high turnover rate in adult multicellular animals. Examples of differentiated cells are fibroblasts, hepatocytes, cardiomyocytes, myoblasts, neurons, osteoclasts, and lymphocytes.

[0091] The term "modified cells" and related terms and phrases encompass all cells that are artificially modified or derived by any method compared to the original cell or cell from which they are derived. Modified cells can be produced from primary cells, secondary cells, stem cells, cultured cells, and / or other modified cells. Modifications include, but are not limited to, genetic modification or engineering, in which case the modified cells may be referred to as "genetically modified" or "genetically engineered." Genetic modification can be achieved by various methods that result in the incorporation of foreign or heterologous nucleic acid into the modified cell. Some examples of such methods are transduction with a virus or viral vector, or transfection of an isolated nucleic acid into cells through transient pores in the cell membrane. Other modifications include exposing the source cell to biological and non-biological molecules or factors or culture conditions. Some examples of modified cells are iPSCs, genetically modified cells, including those used in gene therapy; one example is gene-edited cells, such as those modified using CRISPR / Cas9, TALEN, or ZFN.

[0092] The terms "passage," "passaging," and related terms and phrases used in the context of cell culture refer to subculture, which typically involves transferring cells from a previous culture to fresh growth medium. Passage is performed to ensure the proliferation of cells in culture. Cell proliferation in culture decreases or stops when the volume of the culture vessel and / or medium required to support further cell growth is reduced. For example, cells in adherent culture may occupy all available substrate and have no space left to grow, while cells in suspension culture exceed the volume of the medium required to support further growth. To maintain cells in culture at a density optimal for continued growth and stimulate further proliferation, the culture must be expanded and supplied with fresh medium. To split a culture of adherent cells, such as a monolayer culture of cells, e.g., a culture of differentiated EPSCs described in this disclosure, the cells are first dissociated, e.g., by enzymatic dissociation. Enzymatic dissociation can be performed by removing the incubation medium from the plate, adding a buffer such as PBS and an enzymatic dissociation reagent such as Accutase, TrypLE, or Trypsin (e.g., available from Thermo Fisher Scientific) to the plate, incubating the cells with the buffer and dissociation reagent under appropriate conditions, and collecting the resulting dissociated cells by centrifugation, sedimentation, filtration, or other suitable method. The dissociated cells are transferred to a similar or equivalent reaction vessel, such as a flask, containing fresh medium at a lower cell density.

[0093] As used herein, "marker" refers to any molecule that can be observed or detected. For example, markers can include, but are not limited to, nucleic acids such as transcripts of a particular gene, polypeptide products of a gene, non-gene product polypeptides, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, or small molecules (e.g., molecules with a molecular weight of less than 10,000 AMU). When the presence or absence of an amount of a marker can be experimentally observed or detected, such marker or its amount can be described as "observable" or "detectable." The presence or absence of a marker as applied to embodiments of the present invention refers to the detectable presence or absence of a marker detected by an applicable method for detecting such marker, and can refer to a specific detectable or undetectable level of such marker. In other words, presence can refer to presence above a certain detectable level, while absence can refer to absence below a certain detectable level, not necessarily a zero detection level. For most markers described herein, the symbols provided are those developed and / or recognized by the HUGO Gene Nomenclature Committee of the European Bioinformatics Institute.

[0094] In the context of an observable or detectable marker, such as a marker for cell development or differentiation, "expression" refers to the production of a gene product (which may be a nucleic acid, such as RNA, or a protein) as well as the level or amount of the gene product produced. Thus, determining the expression of a particular marker refers to detecting either the relative or absolute amount of the marker being expressed (which may mean detecting RNA or protein expression), or simply detecting the presence or absence of the marker (which may mean detecting RNA or protein expression). A marker can be said to be "detectably expressed" if expression of RNA or protein corresponding to the marker is detected. Expression of a particular marker can be determined by detecting the presence or absence of the marker in a cell, cell culture, or cell population. Expression of a particular marker can also be determined by measuring the level at which the marker is present in a cell, cell culture, or cell population. Quantitative, qualitative, or semi-quantitative techniques can be used to measure marker expression. For example, marker expression can be detected and / or quantified through the use of techniques that detect nucleic acids, such as PCR-based detection or RNA (e.g., real-time reverse transcriptase PCR), RNA sequencing (RNA-seq), or nucleic acid array-based techniques. In another example, immunochemistry can be used to detect and / or quantify marker proteins. For example, expression of marker gene products can be detected by using antibodies specific for the marker gene product of interest using Western blotting, immunofluorescence, flow cytometry analysis, etc. Various techniques for marker detection can be used in combination to effectively and appropriately characterize and identify cell types and determine both the amount and relative proportion of such markers in the cell type of interest. Expression of a particular marker can be determined by measuring the level of the marker present in cells of a cell culture or cell population compared to a standardized or normalized control marker.Identification and characterization of cells, cell cultures, or cell populations can be based on the expression of certain markers, or on different expression levels and patterns of multiple markers (including the absence, presence, high expression, or low expression of one or more markers). Also, certain markers can have transient expression, where the marker shows higher expression during one or more stages of the processes described herein and lower expression during other stage(s).

[0095] II. Mammalian cell aging evaluation system In one aspect, the application provides a senescence assessment system for mammalian cells, including stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells, wherein the stem cells or early extraembryonic cells optionally comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells toward trophoblasts or syncytiotrophoblasts ("STB"). In some embodiments, the totipotent or pluripotent stem cells or early extraembryonic cells are naturally occurring. In some embodiments, the totipotent or pluripotent stem cells or early extraembryonic cells are genetically modified. In some embodiments, the stem cells or early extraembryonic cells comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule. In some embodiments, the heterologous nucleic acid encoding the reporter molecule is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of senescence or differentiation of the stem cells or early extraembryonic cells toward STB. In some embodiments, the endogenous biomarkers are selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion. In some embodiments, the stem cells or early extraembryonic cells are derived from human, pig, cow, mouse, rat, rabbit, dog, cat, or sheep.

[0096] In one aspect, the present application provides a senescence assessment system for mammalian cells, including stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells, wherein the stem cells or early extraembryonic cells comprise a reporter molecule that indicates senescence or differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells toward trophoblasts or syncytiotrophoblasts ("STBs"). In some embodiments, the stem cells or early extraembryonic cells are derived from a human, pig, cow, mouse, rat, rabbit, dog, cat, or sheep.

[0097] In one aspect, the present application provides a system for assessing senescence in mammalian cells, including stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells, wherein the stem cells or early extraembryonic cells comprise a heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells toward trophoblasts or syncytiotrophoblasts ("STBs"). In some embodiments, the heterologous nucleic acid encoding the reporter molecule is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of senescence or differentiation of the stem cells or early extraembryonic cells toward trophoblasts or STBs. In some embodiments, the endogenous biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion. In some embodiments, the stem cells or early extraembryonic cells are derived from a human, pig, cow, mouse, rat, rabbit, dog, cat, or sheep.

[0098] In one aspect, the present application provides a system for assessing senescence in mammalian cells, including pluripotent stem cells, which optionally comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of totipotent or pluripotent stem cells toward syncytiotrophoblast ("STB"). In some embodiments, the pluripotent stem cells comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule. In some embodiments, the heterologous nucleic acid encoding the reporter molecule is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of differentiation of the pluripotent stem cells toward STB. In some embodiments, the endogenous biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion. In some embodiments, the pluripotent stem cells are derived from a human, pig, cow, mouse, rat, rabbit, dog, cat, or sheep.

[0099] In one aspect, the present application provides a system for assessing aging in mammalian cells, including trophoblast stem cells ("TSCs"), wherein the TSCs optionally comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates differentiation of the TSCs toward syncytiotrophoblasts ("STBs"). In some embodiments, the TSCs comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule. In some embodiments, the heterologous nucleic acid encoding the reporter molecule is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of differentiation of the TSCs toward STBs. In some embodiments, the endogenous biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, ineffective macroautophagy, deregulation of nutrient sensing, altered cell-to-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion. In some embodiments, the TSCs are derived from a human, pig, cow, mouse, rat, rabbit, dog, cat, or sheep.

[0100] 1. Reporter molecule and heterologous nucleic acid encoding the reporter molecule In some embodiments, the stem cells or early extraembryonic cells comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates differentiation of the engineered extraembryonic cells towards syncytiotrophoblasts ("STB").

[0101] In some embodiments, the stem cells or early extraembryonic cells comprise a reporter molecule. Examples of reporter molecules include physically activated molecules and chemically activated molecules. The reporter molecule can be fluorescent or non-fluorescent. Examples of physically activated molecules include, but are not limited to, GFP, RFP, mCherry, photoconvertible fluorescent proteins, and the like. Examples of chemically activated molecules include, but are not limited to: 1) enzyme-activated detector molecules, such as bioluminescence (e.g., luciferase) and enzyme assays (e.g., β-galactosidase, β-glucuronidase, and β-lactamase); 2) antibody-based assays, such as IF antibody assays; 3) chloramphenicol acetyltransferase; and 4) biosensors (e.g., probes).

[0102] In some embodiments, the stem cells or early extraembryonic cells contain heterologous nucleic acid encoding a reporter molecule. The heterologous nucleic acid can be integrated into the genome of the stem cells or early extraembryonic cells by techniques such as gene editing, as described in Section II.2. In some embodiments, the heterologous nucleic acid is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of senescence or differentiation of the stem cells or early extraembryonic cells toward trophoblasts or STBs. As a result, when the promoter is actively expressed in the cells, the reporter gene is also expressed and can be detected / measured. Reporter genes can produce proteins that have little apparent or immediate effect on the cell culture or organism. Ideally, they are not present in the native genome, allowing reporter gene expression to be isolated as a result of expression of the gene of interest. Reporter genes can be genetically integrated into the host DNA of individual cells.

[0103] A reporter gene replaces the stop codon of a gene of interest, creating a gene fusion that allows these genes to be expressed together with the gene of interest. To construct a reporter gene system, a segment of DNA encoding a flexible polypeptide linker region, such as T2A or an IRES, is typically inserted immediately before the reporter gene. This method is an example of the use of cis-acting elements, where two genes are under the same promoter element and transcribed into a single messenger RNA molecule. The mRNA is then translated into protein, and a linker region, such as T2A or an IRES, mediates co-translational cleavage. In this way, both proteins can properly fold into their active structures instead of becoming fusion proteins. The products of the reporter and gene of interest interfere with each other only minimally.

[0104] The reporter gene can also be under the control of a transcriptional regulatory complex (e.g., a promoter) that is inducible, where the transcriptional regulatory element responds to endogenous cellular signals (e.g., transcription factors and transcriptional regulatory complexes) or exogenous chemical or physical conditions that can initiate and regulate expression of the reporter gene.

[0105] Reporter systems typically contain two components: a specific gene and regulatory complex, and a specific substrate that interacts with the gene product. The reporter gene product is either a protein, i.e., an enzyme that catalyzes a chemical reaction, or a protein that fluoresces upon exposure to light. Examples of commonly used reporter system pairs include radionuclide-based pairs (e.g., HSV1-tk [herpes simplex virus type 1 thymidine kinase] and 124 / 131I-FIAU [5-iodo-2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyluracil] or 18F-FEAU [2'-deoxy-2'-18F-fluoro-5-ethyl-1-β-d-arabinosyluracil]), bioluminescent pairs (e.g., firefly luciferase [FLuc] and d-luciferin), and fluorescent pairs (e.g., green fluorescent protein [GFP] and activated blue light), as well as sensors that utilize fluorescence resonance energy transfer between two mutant GFP molecules.

[0106] In some embodiments, the reporter molecule is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, tdTomato, and photoconvertible fluorescent protein. When expressed, the reporter molecule can be detected using methods such as fluorescence microscopy and flow cytometry (see, e.g., Kremers et al., J Cell Sci. 2011, 124(2):157-160; Chudakov et al., Physiol Rev. 2010, 90(3):1103-63).

[0107] In some embodiments of the systems disclosed herein, stem cells or early extraembryonic cells can contain multiple reporter molecules or multiple heterologous nucleic acids encoding reporter molecules, or combinations thereof. For example, early extraembryonic cells used in the systems disclosed herein can contain a first heterologous nucleic acid encoding green fluorescent protein under the control of a promoter of gene A and a second heterologous nucleic acid encoding red fluorescent protein under the control of a promoter of gene B. Cell differentiation can be assessed by the intensities of green fluorescent protein and red fluorescent protein, which indicate the expression levels of gene A and gene B, respectively.

[0108] 2. Mammalian Cells for Assessing Senescence One embodiment of the present disclosure is a system for assessing aging in mammalian cells, including stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells. In some embodiments, the stem cells or early extraembryonic cells are naturally occurring.

[0109] The mammalian cell aging evaluation system may include stem cells capable of producing cells of the trophoblast lineage. The mammalian cell aging evaluation system may include totipotent or pluripotent stem cells. Totipotent stem cells are cells that have the ability to self-renew by division and develop into the three primary germ cell layers of the early embryo and into extraembryonic tissues such as the placenta. Totipotency exists transiently at the early zygote and two-cell embryo stages of development, then transitions to two distinct lineages: the embryonic cell lineage (inner cell mass, ICM) that forms the embryo proper, and the extraembryonic cell lineage (trophectoderm, TE) that forms placental tissue. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the ability to differentiate into all somatic cell types. Recent studies have shown that pluripotent stem cells can be programmed to produce extraembryonic cells such as trophoblast stem cells (Dong et al., 2020, eLife 9:e52504).

[0110] Methods for obtaining totipotent stem cells and induced pluripotent stem cells are known in the art. Totipotent stem cells can be obtained by methods such as transferring a somatic cell nucleus into an enucleated oocyte, also known as somatic cell nuclear transfer (SCNT). The SCNT procedure involves three major steps: enucleation, injection / fusion, and activation. After removing the egg cell nucleus, a donor cell nucleus is injected or fused with the enucleated oocyte, followed by activation of the reconstructed embryo. Successful cloning of more than 20 mammalian species using SCNT has been reported (Matoba et al., Cell Stem Cell. 2018 Oct 4;23(4):471-485). In addition to animal cloning, SCNT technology holds great potential for stem cell biology and human therapeutics. Similar to the derivation of embryonic stem cells (ESCs) from fertilized egg blastocysts, blastocysts generated by SCNT can be used to derive pluripotent stem cells.

[0111] Induced pluripotent stem cells (iPSCs) are typically derived by introducing a specific set of pluripotency-associated genes, or "reprogramming factors," into adult cell types. The original set of reprogramming factors (also called Yamanaka factors) is the genes Oct4 (Pou5f1), Sox2, cMyc, and Klf4. There are several methods for generating iPSCs, including retroviral or lentiviral-mediated gene transfer and chemical induction. Each of the pluripotency factors can also be replaced by the associated transcription factor, miRNA, or small molecule to generate iPSCs (Ghaedi et al., Methods Mol Biol. 2019;1576:55-92).

[0112] In some embodiments, the mammalian cell senescence evaluation system comprises early extraembryonic cells. In some embodiments, the early extraembryonic cells are trophoblast stem cells ("TSCs") or trophoblast progenitor cells ("TPCs"). In some embodiments, the early extraembryonic cells are TSCs. In some embodiments, the early extraembryonic cells are derived from totipotent stem cells, pluripotent stem cells, embryonic tissue, or placental tissue. In any of the embodiments herein, the early extraembryonic cells may be derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

[0113] In one specific embodiment, TSCs are derived from proliferative stem cells (EPSCs). EPSCs derived from cleavage-stage preimplantation embryos retain the developmental potential of both extraembryonic and embryonic cell lineages (Yang et al., 2017, Cell 169, 243-257.e25; Yang et al., 2017, Nature 550, 393-397; Ruan et al., 2022, Cell Reports Medicine 3, 100849; Gao et al., 2019, Nat. Cell Biol. 21, 687-699.). Methods for inducing EPSCs to differentiate into TSCs are known in the art. An exemplary protocol is described in Okae et al., 2018, Cell stem cell, 22(1), 50-63, the contents of which are incorporated herein in their entirety. In some embodiments, a method for inducing EPSCs to differentiate into TSCs includes culturing cells in a cell culture medium containing DMEM / F12, β-mercaptoethanol, FBS, penicillin-streptomycin, bovine albumin fraction V (BSA), insulin-transferrin-selenium-ethanolamine (ITS-X) supplement, 2-phospho-L-ascorbic acid (Vc), EGF, CHIR99021, A83-01, SB431542, valproic acid (VPA), and Y27632. In some embodiments, the cell culture medium comprises DMEM / F12 supplemented with 110 μM β-mercaptoethanol, 0.2% FBS, 0.5% penicillin-streptomycin, 0.3% BSA, 1× ITS-X supplement, 50.0 μg / mL Vc, 50.0 ng / mL EGF, 2.0 μM CHIR99021, 0.5 μM A83-01, 1.0 μM SB431542, 0.8 μM VPA, and 5.0 μM Y27632.

[0114] In some embodiments, TSCs are derived from naive pluripotent stem cells (e.g., naive embryonic stem cells). Naive pluripotent stem cells differ from primed pluripotent stem cells in that primed pluripotent stem cells are primed for lineage commitment. One type of naive pluripotent stem cell, naive embryonic stem cells (ESCs), readily differentiates into somatic or germ cell lineages but is impaired in its ability to form extraembryonic lineages such as the placenta or yolk sac. Recent studies have shown that human naive ESCs can transdifferentiate into cells that exhibit the cellular and molecular phenotype of human trophoblast stem cells (hTSCs) derived from human placenta or blastocysts. An exemplary protocol is described in Cinkornpumin et al., 2020, Stem Cell Rep, 15, 198-213, the contents of which are incorporated herein in their entirety.

[0115] In some embodiments, the stem cells or early extraembryonic cells are genetically modified. For example, in some embodiments, the stem cells or early extraembryonic cells contain heterologous nucleic acids, and the heterologous nucleic acids are introduced into the stem cells or early extraembryonic cells by gene editing tools. Examples of gene editing tools include, but are not limited to, (1) clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas), (2) transcription activator-like effector nucleases (TALENs), (3) zinc-finger nucleases (ZFNs), and (4) homing endonucleases or meganucleases.

[0116] 3. Endogenous biomarkers Many endogenous biomarkers may be suitable for the systems disclosed herein. In some embodiments, the endogenous biomarkers are selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, ineffective macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, senescence-associated secretory phenotypes, and stem cell exhaustion.

[0117] In some embodiments, the endogenous biomarkers are PEG3, DDX3Y, RPS4Y1, TXLNGY, KDM5D, USP9Y, THEM6, CAT, ZFY, LYNX1, EIF1AY, PLAC4, LAIR2, WIPF1, APOC3, LY6K, ZNF558, OAS3, LINC02055, LGALS14, ZNF826P, TTTY14, NLGN4Y, TM SB4Y, SEC14L4, LINC01446, ZFP41, LGALS13, ZNF257, STEAP4, GH2, MAG, LINC00278, TPTEP1, CD22, AIRE, CNN1, APOA5, ZFY-AS1, OAS2, ANOS2P, GNG4, CMKLR2, OXGR1, HOXB7, RLN2, WNT9B, LINC01949, COX20P1, SP ANXN5, NBPF6, PRKY, IGHV3-74, CSH2, UNC13A, BACE2, TMC1, LINC01090, CYP26A1, PSCA, PAEP, UTY, MIR4 482, ZNF835, RN7SL708P, ZNF717, WFDC21P, FLT4, NOC2LP1, NBPF4, LY6D, ZNF736P9Y, OR2C3, FAM167A, GA BRE, GYG2P1, ZNF630, HLF, KCNS3, MTNR1B, NPR, HOXC9, LINC02701, ZNF726, WSCD1, KRT78, ETV7, ZNF658, GPC4, NKAIN4, FLJ16779, RERG, BGN, DLX5, HTRA4, SCUBE1, LINC00323, LY6E, NCF1C, GJA3.

[0118] In some embodiments, the endogenous biomarkers are DDX3Y, PEG3, RPS4Y1, PLAC4, TXLNGY, KDM5D, CSH2, LGALS14, OAS3, OAS2, ZFY, GH2, WIPF1, HLF, LAIR2, USP9Y, STEAP4, THEM6, CAT, LYNX1, LY6K, APOC3, EIF1AY, ADAM12, CSH1, ZNF558, IFNL3, LGALS13, SCUBE1, SPDYE13, WNT9B, FLT4, LINC02055, CD22, TTTY14, ZFP41, IFIT2, MMEL1, XAF1, TMSB4Y, CMKLR2, LINC01446, KCNS3, GPC3, IFNL2, LAMA4, MAG, LINC00278, ABCB1, LINC01949, MU CL1, TPTEP1, SPANXN5, KCNJ2, HTRA4, ZNF826P, APOA5, AIRE, OASL, SPTA1, NLGN4Y, PDK4, PTPN22, GUCY 2F, PAEP, PSCA, HTRA1, OXGR1, ACVRL1, MAN1C1, ARNT2, H2BC8, SEC14L4, BGN, UNC5A, MIR100HG, NBPF6, I Selected from the group consisting of FIT3, GBP1, ZFY-AS1, NPR1.00, RLN2, BACE2, ZNF257, CGB2, IGHV3-74, NBPF4, LINC02701, IFNL1, GNG4, ZNF835, CNN1, FAM167A, GPR143P, KRT78, RN7SL708P, CGB1, LY6D, LINC02533, TMEM225B.

[0119] In some embodiments, the endogenous biomarkers are OAS2, PEG3, DDX3Y, OAS3, CSH2, RPS4Y1, GH2, CSH1, LAIR2, PLAC4, LGALS14, KDM5D, TXLNGY, THEM6, WIPF1, LYNX1, XAF1, IFNL3, ZFY, LAMA4, APOC3, ADAM12, USP9Y, STEAP4, HLF, IFNL2, SPDYE13, ZNF558, CAT, LY6K, EIF1AY, CD22, IFIT2, IFIT3, OAS1, SCUBE1, CSHL1, GPC3, LINC02055, WNT9B, CNN1, FLT4, BST2, SAMD9L, ZFP41, GBP1, CMKLR2, MUCL1, MX1, IFNL1, OASL, LINC01 446, SPANXN5, KCNJ2, ETV7, NLGN4Y, KCNS3, IFITM1, MAG, TPTEP1, TTTY14, LGALS13, MX2, KCNK3, SEM A3B, PDK4, HTRA1, APOA5, FLJ16779, IGHA1, TAC3, ZNF826P, GNG4, HTRA4, CYP26A1, SHFL, GPC4, TCAM 1P, WFDC21P, ITIH3, KRT78, SPDYE9, EPHB2, ANGPTL4, SPDYE11, PSAPL1, PSCA, IFIT1, IFI27, MYO1A, PAEP, LINC01949, LINC02533, FGF21, ESAM, SLC16A2, FN1, SAMD9, NBPF6, GTF2I-AS1.

[0120] In some embodiments, the endogenous biomarkers are OAS2, OAS3, PEG3, DDX3Y, LGALS14, RPS4Y1, PLAC4, CSH2, STEAP4, IFNL3, GH2, IFNL2, CSH1, KDM5D, HLF, USP9Y, LYNX1, LAIR2, TXLNGY, THEM6, ZFY, XAF1, IFIT2, ADAM12, WIPF1, APOC3, CAT, CNN1, ZNF558, EIF1AY, PDK4, IFIT3, IFNL1, MYO1A, SAMD9L, LY6K, SPANXN5, GPC3, CSHL1, OASL, OAS1, SPDYE13, FLJ16779, TAC3, PAPPA, ZFP41, WNT9B, NLGN4Y, IGHA1, GBP1, TRIM40, SPTA1, M X1, LINC01949, FLT4, KCNJ2, LINC02055, TTTY14, LINC02533, TPTEP1, NKAIN4, CMKLR2, KRT17P2, CCL 22, LINC01446, ETV7, SEMA3B, LAMA4, MT-TH, MMEL1, BST2, LGALS13, WFDC21P, GUCY2F, MUCL1, CXCL10 , FAM167A, ACVRL1, LINC00278, IFI27, SAMD9, IFITM1, SCEL, ZNF826P, CD22, AIRE, NCF1C, FN1, MAN1C1, CYP26A1, SHFL, DLGAP1, SLC16A2, KRT78, PSCA, UNC13A, APOA5, ANGPTL4, NBPF6, CPE.

[0121] In some embodiments, the endogenous biomarkers are MAGEA2B, MAGEA2, MAGEA12, CSAG1, MAGEB2, SH3BGRL, CSAG3, LINC02413, TSSC2, PTCHD1, HMGA2, ARHGEF9, GALNT14, FAM86GP, CSAG2, AJAP1, LPAR1, TLR4, DDX11L2, OR7E12P, CDCA7L, LINC01405, P I15, LINC02474, DCLK1, SNHG14, NDN, PRKCQ, PINCR, CDX1, IMPDH1P4, RPE65, UCP1, MSN, CHMP1B2P, SNTB1, BMP5, DPYSL4, FBXW12, STAMBPL1, A2M, KCNJ5, E2F6P4, ALOX12P2, NETO1, LRRTM4, KRT72, ATP8A1, PRKCQ-AS1, PCDHB2, CSAG4, AMER1, LINC01291, LINC01807, LINC02412, MIR2052HG, NCAM1, PCDHA4, MIEOV, CDH13, ALCAM, ARTN, PCDH A10, TRIM61, TEX41, PCDHA12, PTPRB, CYP2C8, PROM1, PCDHA11, FAM110C, TGFA, KRTAP2-3, ZNF57, NACAP8, IGSF1 , MPV17L, SIMC1, BUD13P1, NRROS, UGT1A6, STK32B, ROR2, HMGN5, LINC02617, LINC00379, WDR90, PLA2G4A, CASC9, CYP4F26P, TEX35, LINC02154, GSTT2B, IFFO1, CELF2, CHRM3, C1RL-AS1, RARRES1, RGPD2, HOMER2.

[0122] In some embodiments, the endogenous biomarkers are MAGEA12, CSAG1, MAGEA2B, MAGEA2, MAGEB2, SH3BGRL, LINC02413, TSSC2, CDX1, ARHGEF9, CHRM3, GALNT14, AJAP1, CSAG3, ALCAM, TLR4, TGFA, DDX11L2, OR7E12P, PTCHD1, CDCA7L, HMGA2, PI15, MYEOV, LI NC02474, PRKCQ-AS1, LINC02617, PINCR, HMGN5, FAM162B, NDN, EYA1, PRKCQ, LINC01405, PCDHA10, PCDHA12, RPE65 , ZNF57, CLDN10, SFTA1P, CHMP1B2P, SNTB1, C10orf82, DPYSL4, MSN, FBXW12, STAMBPL1, E2F6P4, FAM83A, LINC0112 7, NETO1, CSAG2, KRT72, ATP8A1, SNX18P7, CSAG4, COL19A1, LINC01807, LINC02458, LINC02412, MIR2052HG, NCAM1 , LPAR1, NECTIN1-DT, SLC7A11-AS1, KCTD15, LINC00379, ITGB1BP2, PROM1, UGT1A1, C1orf220, CA8, CDH13, ZSCAN1 6-AS1, RARRES1, TRIM61, ANKRD18B, EML5, PRKG2, PRPH2, FHL1, FAM86GP, RBP5, UGT1A6, BMP5, STK32B, CLEC20A, TEX35, GLUD2, CERS6, SNHG14, KLHL4, C1RL, PTPRB, LINC00668, CHST6, ITPRIPL1, GSTT2B, TERT, DCLK1.

[0123] In some embodiments, the endogenous biomarkers are MAGEA2B, MAGEA2, MAGEA12, TSSC2, CSAG1, MAGEB2, CSAG3, LINC02413, HMGA2, ARHGEF9, FAM86GP, CSAG2, AJAP1, GALNT14, OR7E12P, PTCHD1, LINC02474, DCLK1, PRKCQ-AS1, PINCR, SNHG14, FAM162B, PRK CQ, CYP4F26P, MSN, PCDHA10, EML5, LINC00379, PCDHA12, IMPDH1P4, CDX1, RPE65, ZNF57, CHMP1B2P, TLR4, PKIA, DS G2-AS1, UGT1A6, DDX11L2, LINC01405, BMP5, C10orf82, DPYSL4, STAMBPL1, WHAMMP2, HAPLN1, PTPRB, PLA2G4A, NETO 1, KRT72, ST13P20, PCDHB2, SNX18P7, HMGN5, MAP10, CSAG4, LINC02617, MIR3176, UCP1, LINC01807, NDN, LINC0241 2, MIR2052HG, NCAM1, CYP1A1, SH3BGRL, CELF2, ARMC3, UGT1A1, ZSCAN16-AS1, NR0B1, TRIM61, DIAPH2-AS1, SAPCD1, Selected from the group consisting of CYP2C8, PCDHA11, MRPS18AP1, PCDHB5, KRTAP2-3, NACAP8, CDKL2, FAM83A, DOC2GP, LPAR1, SNTB1, RARRES1, RBP5, LINC02575, DEPDC1, MECOM, KCNE3, ALCAM, ABHD12B, SSBP3-AS1, GLUD2, A2M, CHST6, HPDL, AK4, FGFBP1.

[0124] In some embodiments, the endogenous biomarkers are MAGEA2B, MAGEA2, MAGEA12, GALNT14, CSAG1, MAGEB2, LINC01405, CDX1, PTCHD1, ARHGEF9, AJAP1, LINC02413, TLR4, DDX11L2, OR7E12P, LINC02474, DCLK1, LINC02575, PRKCQ-AS1, CERS6, CHRM3, NDN, P RKCQ, CYP4F26P, TSSC2, PCDHA10, PIMREG, IMPDH1P4, LINC00379, RPE65, ZNF57, SH3BGRL, PINCR, SNTB1, UGT1A6, B MP5, C10orf82, DPYSL4, STAMBPL1, E2F6P4, PI15, PNPO, NETO1, LRRTM4, KRT72, ATP8A1, PCDHB2, SNX18P7, HMGN5, CS AG4, COL19A1, PROM1, LINC01807, LINC02412, MIR2052HG, NCAM1, NECTIN1-DT, TEX35, DMC1, LINC02617, FAM86GP, TERT, TRIM61, EML5, TEX41, PCDHA12, PCDHA11, CSAG3, TSPEAR-AS2, RARRES1, LINC00668, ZNF785, MPV17L, EVX1, FA Selected from the group consisting of M83A, CHMP1B2P, ABHD12B, PKIA, C1RL-AS1, STK32B, LINC02582, CTTNBP2, TGFA, CTAGE3P, SNORD99, AMER1, LINC01291, ITPRIPL1, FAM162B, MCM10, AK4, MSN, KRT3, CHST6, HHLA3-AS1, CSAG2, NR0B1, EMB, KCNE3, UCP1.

[0125] In some embodiments, the endogenous biomarkers are β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3 , CDKN1A, CDKN1C, CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, and SIRT family members.

[0126] In some embodiments of the present invention, the endogenous biomarker used is a gene whose expression increases during normal mammalian cell aging. In some embodiments, the endogenous biomarker used also shows increased expression in a mammalian cell aging evaluation system. For example, the expression of the endogenous biomarker increases as stem cells or early extraembryonic cells senesce or differentiate toward STB. As the expression of the endogenous biomarker increases, the expression of the reporter molecule also increases. The increased expression can be detected as described above, thus signaling STB senescence or differentiation.

[0127] In some embodiments, the endogenous biomarker used is a gene whose expression decreases during normal mammalian cell aging and in mammalian cell aging evaluation systems.For example, the expression of endogenous biomarker decreases as stem cells or early extraembryonic cells differentiate toward STB.As the expression of endogenous biomarker decreases, the expression of reporter molecule also decreases.Examples of such suitable endogenous biomarkers include HP1 gamma, lamin, and SIRT family members.

[0128] In some embodiments, the endogenous biomarker is a marker of genomic instability. In some embodiments, the endogenous biomarker is HP1 gamma, HP1 alpha, lamin A / C, lamin B1, or the SETDB1 / KAP1 complex.

[0129] In some embodiments, the endogenous biomarker is a marker of DNA damage. Suitable markers include, but are not limited to, γH2AX and p53. H2AX is a mammalian variant of histone belonging to the H2A family. Histones are proteins that constitute nucleosomes, the basic units of chromatin. When a double-strand break occurs in DNA, H2AX becomes rapidly phosphorylated at serine 139. This specific phosphorylation is referred to as "γ-phosphorylation," and the term "γH2AX" refers to the specific phosphorylation of histone H2AX at serine 139. γH2AX is an early sign of DNA damage induced by replication stall. p53 plays a prominent role as a facilitator of DNA repair by arresting the cell cycle, allowing time for repair mechanisms to restore genomic stability. It is also directly involved in DNA repair pathways (e.g., chromatin remodeling during nucleotide excision repair).

[0130] Furthermore, biomarkers can be markers of DNA repair. DNA damage can be caused by numerous factors but ultimately takes the form of chemical modifications to DNA bases, single-stranded DNA (ssDNA) or ssDNA breaks, or the presence of more severe DNA-DSBs. Repair of lesions generated by chemical modifications to DNA bases, such as oxidative lesions or bulky adducts, is rapidly processed by the nucleotide excision repair (NER) and base excision repair (BER) pathways. DNA can be damaged when cells are exposed to mutagens, which can distort the double-stranded structure or prevent correct transcription. Damaged DNA can be detected and repaired by the process of nucleotide excision repair (NER). Research has shown that all cells possess different types of glycosylases that can recognize damaged nucleotides. Glycosylases can specifically cleave the N-β-glycosidic bond on the damaged base, generating an abasic (AP) site. When an AP site is generated in a DNA molecule, an AP-endonuclease cleaves the AP site, generating a 3'OH and a 5'deoxyribophosphate (dRP) end and removing a small fragment of DNA containing the AP site. DNA polymerase I then synthesizes a new fragment, and finally, DNA ligase joins the two together into a new, repaired DNA strand. This process is called base excision repair (BER). It represents a DNA repair mechanism for damage. Markers associated with these cellular processes are listed in Table 2.

[0131] Biomarkers can also be selected from other pathways for DNA repair, including: 1) DNA mismatch repair (MMR), which can correct mismatches that occasionally arise during DNA replication and recombination, and mismatched substrates can be recognized and repaired by mismatch repair enzymes; 2) the Fanconi anemia signaling pathway, which is thought to be necessary for the effective repair of damaged DNA; 3) homologous recombination (HR), which provides high-fidelity, template-dependent repair or tolerance of complex DNA lesions, including DNA gaps, DNA double-strand breaks, and DNA interstrand crosslinks; and 4) non-homologous end joining (NHEJ), which repairs DNA double-strand breaks by direct ligation of DNA without the need for a homologous template.

[0132] [Table 2]

[0133] In some embodiments, the endogenous biomarker is a marker of epigenetic changes. During the aging process, large-scale epigenetic changes occur in response to both exogenous and endogenous stimuli. Examples of epigenetic-related markers include, but are not limited to, H3K9me3, H3K4me3, H3K9ac3, and linker histone H1. In some embodiments, the endogenous biomarker is H3K9me3.

[0134] In some embodiments, the endogenous biomarker is a differentiation marker. Examples of differentiation markers include, but are not limited to, CDX2, KRT7, HLA-G, ID2, CGA, CGB, ERVW family members, CSH1, SDC1, and PSG1. CGA and CGB are involved in hormonal processes and have been shown to increase in expression during STB differentiation (Marchand et al., Biology of Reproduction, Volume 84, Issue 6, June 1, 2011, Pages 1258-1271; Ruan et al., 2022, Cell Reports Medicine 3, 100849). TSC differentiation toward STB is primarily driven by human endogenous retroviruses (HERVs), specifically syncytin-1 (the product of the ERVW-1 gene) and syncytin-2 (ERVFRD-1), which are key drivers of CTB fusion. CSH1 (chorionic somatomammotropic hormone 1), SDC1 (syndecan 1), and PSG1 (pregnancy-specific beta-1-glycoprotein 1) are highly enriched in STB. The transcription factor CDX2 acts early in the process of blastocyst formation and plays an instructive role in trophoblast formation. KRT7, HLA-G, and ID2 are also markers of STB differentiation. Other suitable differentiation markers include DLX3, GATA3, DAB2, TEAD3, and TFAP2C. In some embodiments, the endogenous biomarker is CGA.

[0135] In some embodiments, the endogenous biomarker is an endogenous transposon element. Examples of endogenous transposon element markers include, but are not limited to, HERVK (human endogenous retrovirus K), pTBK1, and line 1. In some embodiments, the endogenous biomarker is HERVK. As reported in Liu et al., 2023, Cell 186, 287-304, upregulation of HERVK triggers young cells to enter cellular senescence. HERVK has been shown to be highly expressed in STB differentiation around day 2, which is the beginning of the TSC-STB aging process.

[0136] In some embodiments, the endogenous biomarker is a cell cycle-related marker. Examples of cell cycle-related markers include, but are not limited to, p27 and p38. P27 binds to and inhibits cyclin-CDK, causing cell cycle arrest, and has been shown to be expressed in differentiated, non-dividing STBs. P38 is essential for mediating the initiation of STB differentiation and is highly expressed early in TSC-STB differentiation.

[0137] In some embodiments, the endogenous biomarker is a telomere wear marker.Examples of cell cycle-related markers include but are not limited to TRF1.TRF1 encodes a telomere-specific protein, which is a component of telomere nucleoprotein complex.This protein exists at telomere throughout the cell cycle, and acts as an inhibitor of telomerase, acting in cis to limit the elongation of individual chromosome ends.

[0138] In some embodiments, the endogenous biomarker is a marker of cellular senescence. Cellular senescence is a process that globally regulates cell fate and can be considered a hallmark of aging. It is associated with multiple cellular and molecular changes and distinct phenotypic changes, including stable and generally irreversible growth arrest that is unresponsive to mitogenic stimuli. One characteristic feature of senescent cells is a temporal cascade of increased lysosomal activity, macromolecular damage, and the development of a complex senescence-associated secretory phenotype (SASP). Examples of markers of senescence include, but are not limited to, CDKN1A, CDKN2A, CDKN2B, members of the BCL2 family, NF-κB, FOXO3, and SMAD3, which play important roles in regulating senescence. For example, CDKN1A inhibits p21, a protein that can inactivate all CDKs. WAF1 / CIP1 NF-κB encodes NF-κB, which inhibits cell cycle progression. NF-κB is generally recognized as a key regulator of senescence due to its role in cellular senescence and inflammatory pathways. In some embodiments, a marker of senescence is senescence-associated β-galactosidase (SA-β-gal) activity.

[0139] In some embodiments, the endogenous biomarker is a marker of the senescence-associated secretory phenotype (SASP). The SASP is a phenotype associated with senescent cells; these cells secrete high levels of inflammatory cytokines, immune modulators, growth factors, and proteases. The SASP also contains enzymes, microRNAs, DNA fragments, chemokines, and other bioactive factors. The SASP is one of three major characteristics of senescent cells; the other two are arrest of cell proliferation and resistance to apoptosis. SASP expression is induced by several transcription factors, including C / EBPβ, the most important of which is NF-κB. SASP factors drive non-senescent cells into senescence, induce insulin resistance, and disrupt normal tissue function by producing chronic inflammation, inducing fibrosis, and inhibiting stem cells. Cell fusion, an essential physiological process for establishing and expanding STB, has recently been recognized as an additional trigger for cellular senescence. Senescence as a response to fusion may have evolved to arrest the growth of cells infected with fusogenic viruses (e.g., paralysis viruses), and it is important to note that cytotrophoblast fusion requires the retroviral fusogen syncytin-1 (the product of the ERVW-1 gene). Examples of markers of SASP include, but are not limited to, IL-6, IL-8, IL-1 alpha, IL-1 beta, CCL2, TIMP1, TIMP2, MCP1, and MIP3.

[0140] In some embodiments, the endogenous biomarker is a marker of deregulated nutrient sensing, such as insulin resistance and calorie restriction. Examples of markers of deregulated nutrient sensing include, but are not limited to, IGF-1, the PI3K / AKT / mTOR pathway, AMPK, PGC1 alpha, NAD+ / NADH, SIRT, and FOXO.

[0141] In some embodiments, the endogenous biomarker is a marker of chronic inflammation. Inflammation is known to be important in aging and aging-related diseases and has been implicated as a key aging mechanism. Examples of inflammatory markers include, but are not limited to, TNF, IL-6, IL-8, IL-10, IL-12, IL-18, hsCRP, IFN-γ, IL-1β, IL-1RA, MCP, MIP, SGP130, STNF-RI, STNF-RII, TGF-β1, and TRAIL. In some embodiments, the marker of inflammation is a pro-inflammatory cytokine, such as IL-6, IL-8, IL-10, IL-12, or IL-18.

[0142] Biomarkers can also be cell cycle markers. The cell cycle is a series of coordinated events leading to cell division that is important for both the development and survival of multicellular organisms. Stable cell cycle arrest, which marks the cell's inability to continue dividing, is essential and one of the defining characteristics of senescent cells. One example of a cell cycle marker is Ki67, a nuclear protein associated with cell proliferation and ribosomal RNA transcription, which is routinely used as a marker for proliferating cells.

[0143] Biomarkers can also be markers of loss of proteostasis: senescent cells accumulate damaged and misfolded proteins through impaired function of proteostasis mechanisms, leading to reduced cell viability and the development of protein misfolding diseases.

[0144] Biomarkers can also be markers of organelle dysfunction, such as mitochondrial dysfunction, proteosome dysfunction, endoplasmic reticulum dysfunction, Golgi apparatus dysfunction, and nuclear envelope dysfunction.

[0145] Biomarkers may also be markers of ineffective macroautophagy, a catabolic process in which a portion of the cytoplasm is trapped within double- or multi-membrane vesicles called autophagosomes and then delivered to lysosomes for bulk degradation. Macroautophagy has been shown to decrease with aging.

[0146] Biomarkers can also be markers of changes in intercellular communication. Intercellular communication refers to the various methods and structures that biological cells use to communicate with each other directly or through their environment. Different cell types use different proteins and mechanisms to communicate with each other using extracellular signaling molecules. Changes in intercellular communication are considered a hallmark of aging and can affect 1) the standard aging-associated secretory phenotype, 2) direct intercellular communication via gap junctions or tubule-like structures, and 3) long-distance communication involving extracellular vesicles and paracrine communication mediated by connexin-containing hemichannels.

[0147] Biomarkers can also be markers of stem cell exhaustion. The functional stem cell pool is usually depleted in aging animals. The depletion of the stem cell pool with aging can occur because these cells lose their autorenewal activity and exit the stem cell pool by terminal differentiation, or because they undergo apoptosis or senescence induced by exposure to cellular stress.

[0148] Indicators of the mTOR / AMPK pathway may also be suitable biomarkers for use in the methods disclosed herein. The mammalian / mechanistic target of rapamycin (mTOR) is a key component of cellular metabolism, integrating nutrient sensing with cellular processes that promote cell growth and proliferation. mTOR receives and integrates inputs from its upstream regulators, one of which is the AMPK pathway, which is sensitive to energy status.

[0149] III. Methods for assessing aging In one aspect, the present application provides a method for assessing the aging process in mammalian cells, the method comprising subjecting stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells to conditions that differentiate the stem cells or extraembryonic cells towards STB, and determining one or more differentiation characteristics of the stem cells or early extraembryonic cells.

[0150] In some embodiments, the method of assessing the aging process comprises subjecting stem cells to conditions that differentiate the stem cells toward STB. In some embodiments, the method of assessing the aging process comprises subjecting early extraembryonic cells to conditions that differentiate the early extraembryonic cells toward STB. In some embodiments, the cells of the present disclosure are pluripotent stem cells or toripotent stem cells. In some embodiments, the early extraembryonic cells are trophoblast stem cells ("TSCs") or trophoblast progenitor cells ("TPCs"). In some embodiments, the early extraembryonic cells are TSCs. In some embodiments, the early extraembryonic cells are derived from totipotent stem cells, pluripotent stem cells, embryonic tissue, or placental tissue. In any of the embodiments herein, the early extraembryonic cells may be derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

[0151] 1. Culture conditions that allow STB differentiation Cultured EPSCs can be induced to differentiate toward STBs. In some embodiments, EPSCs are contacted with Y27632 and SB431542. Y27632 is a cell-permeable, highly potent, and selective inhibitor of Rho-associated coiled-coil protein kinase. SB431542 is a TGF-beta inhibitor. In one specific embodiment, human EPSCs are dissociated with TrypLE and plated at 1 x 10 cells per well onto a 100x Geltrex-coated 6-well plate. 5Cells are seeded at a density of 1000 x g. The cells are cultured in 20% KSR medium supplemented with 10 μM Y27632 for 1 day (pretreatment). From day 2, 10 μM SB431542 is added to the 20% KSR medium to initiate differentiation. STB characteristics can be detected around day 7 or 8.

[0152] EPSCs and other stem cells can be induced to differentiate into TSCs, which can then be induced to differentiate into STBs. Methods for inducing EPSCs to differentiate into TSCs are described in Section II. 2. Methods for inducing TSCs to differentiate into STBs are known in the art (see, e.g., US20230220334A1). In some embodiments, conditions for STB differentiation include cell culture medium containing DMEM / F12, β-mercaptoethanol, penicillin-streptomycin, 7.5% BSA, ITS-X, Y27632, forskolin, and KnockOut Serum Replacement. In some embodiments, TSCs are cultured in syncytiotrophoblast medium (STBM) for about 6 days. STBM contains DMEM / F12 supplemented with 110 μM β-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, 1× ITS-X, 2.5 μM Y27632, 2 μM forskolin, and 4% KnockOut Serum Replacement. Cells become early syncytiotrophoblasts on days 2–3 and mature syncytiotrophoblasts around day 6.

[0153] 2. Characteristics of differentiation In some embodiments, the methods include determining one or more differentiation characteristics of the stem cells or early extraembryonic cells, including, but not limited to, 1) the presence or absence of a biomarker associated with early extraembryonic cell differentiation, 2) the level of a biomarker associated with early extraembryonic cell differentiation, 3) the secretion of a biomarker associated with early extraembryonic cell differentiation, 4) cell morphology, 5) the rate of change to a differentiation state, 6) the characteristics of cell organelles, and 7) the number of nuclei in the cell, and 8) the presence or absence of a reporter molecule.

[0154] In some embodiments, the biomarkers are β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1 C, CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, SIRT family members, and combinations thereof. Without wishing to be bound by any theory, genes such as OCT4, NANOG, SSEA-4, SOX2, REX1, and SALL4 may be considered markers of proliferative stem cells (EPSCs); genes such as SDC1, KLF5, TP63, TEAD4, TBX3, KRT7, GATA2, and GATA3 may be considered markers of TSCs, and genes such as CGB, CSH1, CGA, and ERVW1 may be considered markers of STBs.

[0155] In some embodiments, the differentiation characteristic comprises the presence or absence of a biomarker associated with early extraembryonic cell differentiation. In some embodiments, the biomarker is selected from the group consisting of OCT4, NANOG, SSEA-4, SOX2, REX1, and SALL4. In some embodiments, the differentiation characteristic comprises the level of a biomarker associated with early extraembryonic cell differentiation. In some embodiments, the biomarker is selected from the group consisting of SDC1, KLF5, TP63, TEAD4, TBX3, KRT7, GATA2, GATA3, CGB, CSH1, CGA, and ERVW1.

[0156] In some embodiments, the biomarker is a hormone. Syncytiotrophoblasts secrete large amounts of hormones, including metabolic proteins (leptin, adiponectin), peptide hormones (hCG, hPL, PGH), and steroid hormones (progesterone, estrogen). Human chorionic gonadotropin hormone (hCG) is composed of an α-subunit and a β-subunit, and β-hCG can be measured by ELISA. In some embodiments, the biomarker is human chorionic gonadotropin (hCG). In some embodiments, the biomarker is β-hCG.

[0157] Biomarkers can be detected or measured at the mRNA level or protein level. Various methods are available for detecting / measuring mRNA levels, including RNA sequencing, RT-qPCR, and in situ hybridization. Methods for detecting / measuring protein levels include Western blot, ELISA, proteomics, and immunofluorescence.

[0158] In addition to the presence, absence, and secretion of biomarkers, differentiation can also be characterized by cell morphology. Morphological characteristics of cells, such as cell volume, area, and thickness, can be determined using microscopy. TSC cells are characterized as mononuclear epithelial cells with well-defined cell borders and large nuclei, whereas STB cells are characterized as villous multinucleated entities.

[0159] In some embodiments, differentiation characteristics are assessed as a percentage change to a differentiation state. In certain embodiments, cells become early syncytiotrophoblasts around days 2-3 and mature syncytiotrophoblasts around day 6. The percentage change to a differentiation state can be determined as the time it takes for cells to become early syncytiotrophoblasts and / or mature syncytiotrophoblasts compared to days 2-3 and 6, respectively. The percentage change can be expressed as a percentage change, e.g., about 10% faster / slower, about 20% faster / slower, about 30% faster / slower, about 40% faster / slower, about 50% faster / slower, about 60% faster / slower, about 70% faster / slower, about 80% faster / slower, about 90% faster / slower, about 100% faster / slower, etc.

[0160] In some embodiments, differentiation hallmarks include organelle characteristics. In some embodiments, the organelle is selected from the group consisting of mitochondria, proteosomes, endoplasmic reticulum, Golgi apparatus, and nuclear envelope. In some embodiments, the organelle is mitochondria. As stem cells or early extraembryonic cells differentiate toward STB, mitochondrial characteristics (including number, morphology, and function) are expected to change. Mitophagy is expected to increase. Characteristics can be examined using methods such as mitochondrial labeling and / or electron microscopy imaging. The accumulation of damaged and misfolded proteins in senescent cells results from impaired proteosome function, which can be characterized by dysfunction of E3 ubiquitin ligases and chaperone family heat shock proteins (HSPs). Both the endoplasmic reticulum (ER) and Golgi apparatus are part of a membranous tubule network that plays a major role in ion homeostasis, lipid, and protein biosynthesis. Together with the proteosome, they maintain protein homeostasis (also known as proteostasis). Different types of stress, such as nutrient deprivation, viral infection, and hypoxia, can cause unfolded or misfolded proteins to accumulate and aggregate in these organelles, thus potentially being detrimental to cell survival. ER stress activates the ER transmembrane proteins PKR-like ER kinase (PERK), IRE1α, and ATF6α. The Golgi apparatus is important for bidirectional vesicular transport between the ER, proteosomes, and mitochondria. Golgi apparatus-associated molecules include Nir2, CERT, and oxysterol-binding protein (OSBP).

[0161] In some embodiments, differentiation characteristics include the number of nuclei in the cells. STB cells are multinucleated, and the number of nuclei is not consistent among all STB cells. The number of nuclei can be determined by DAPI or Hoechst staining and quantified by confocal microscopy.

[0162] In some embodiments, the differentiation characteristic comprises the presence or absence of a reporter molecule, as described in Section II.1.

[0163] IV. Methods for Evaluating and Screening Candidate Drugs One aspect of the present disclosure provides a method for evaluating the anti-aging function of a candidate drug, the method comprising: 1) subjecting stem cells (totipotent or pluripotent stem cells) or early extraembryonic cells to conditions that differentiate the stem cells or early extraembryonic cells toward trophoblasts, particularly STBs; 2) contacting the stem cells or early extraembryonic cells with a candidate drug before, during, or after subjecting the stem cells or early extraembryonic cells to conditions that differentiate the stem cells or early extraembryonic cells; and 3) assessing a change in one or more differentiation characteristics of the stem cells or early extraembryonic cells compared to stem cells or early extraembryonic cells not contacted with the candidate drug. In some embodiments, the candidate drug is selected from the group consisting of an antibody, a virus, a virus-like substance, a small molecule, a peptide, a polypeptide, DNA, mRNA, a guide RNA, a microRNA, an RNAi, an lncRNA, an siRNA molecule, and an antisense RNA. In some embodiments, the candidate drug is a naturally occurring substance. In some embodiments, the candidate drug is a nutritional supplement.

[0164] One aspect of the present disclosure provides a method for screening candidate drugs with anti-aging function, the method comprising: 1) evaluating the anti-aging function of a plurality of candidate drugs; and 2) identifying candidate drugs with anti-aging function based on the ability of the candidate drugs to cause a change in one or more differentiation characteristics of stem cells or early extraembryonic cells compared to stem cells or early extraembryonic cells without the candidate drug. In some embodiments, the candidate drug is selected from the group consisting of antibodies, viruses, virus-like agents, small molecules, peptides, polypeptides, DNA, mRNA, guide RNA, microRNA, RNAi, lncRNA, siRNA molecules, and antisense RNA. In some embodiments, the candidate drug is a naturally occurring substance. In some embodiments, the candidate drug is a nutritional supplement.

[0165] 1. Evaluation of anti-aging function In some embodiments, methods for evaluating the anti-aging function of a candidate agent include subjecting stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells to conditions that differentiate the stem cells or extraembryonic cells toward trophoblasts, particularly STBs as described in Section III.1. In some embodiments, the stem cells or early extraembryonic cells are contacted with the candidate agent before being subjected to conditions that allow for differentiation. In some embodiments, the stem cells or early extraembryonic cells are contacted with the candidate agent simultaneously as they are subjected to conditions that allow for differentiation. In some embodiments, the stem cells or early extraembryonic cells are contacted with the candidate agent after being subjected to conditions that allow for differentiation. For example, in some embodiments, the cells or early extraembryonic cells are subjected to conditions that allow for differentiation on day 1 and simultaneously contacted with the candidate agent. In some embodiments, the stem cells or early extraembryonic cells are subjected to conditions that allow for differentiation on day 0 and contacted with the candidate agent on day 2. In some embodiments, the stem cells or early extraembryonic cells are contacted with the candidate agent on day 0 and subjected to conditions that allow for differentiation on day 2.

[0166] In some embodiments, methods for evaluating the anti-aging function of a candidate agent include assessing a change in one or more differentiation characteristics of stem cells or early extraembryonic cells compared to stem cells or early extraembryonic cells not contacted with the candidate agent. Differentiation characteristics, as well as methods for detecting and / or measuring, are described in Section III.2. In some embodiments, the differentiation characteristics are assessed at least one day after subjecting the stem cells or early extraembryonic cells to differentiation conditions. In some embodiments, the differentiation characteristics are assessed 2-8 days after subjecting the stem cells or early extraembryonic cells to differentiation conditions.

[0167] In some embodiments, differentiation characteristics are assessed at least one day after subjecting the stem cells or early extraembryonic cells to differentiation conditions. In some embodiments, differentiation characteristics are assessed two to eight days after subjecting the stem cells or early extraembryonic cells to differentiation conditions. An agent that increases or decreases differentiation- or senescence-associated marker signals by about 20% or more compared to stem cells or early extraembryonic cells not contacted with the agent can be considered to have an anti-senescence effect. The agent can then be subjected to further validation and mechanistic investigation.

[0168] In some embodiments, evaluating the anti-aging function of the candidate agent comprises assessing whether the candidate agent has anti-aging function. In some embodiments, evaluating the anti-aging function of the candidate agent comprises determining an effective concentration of the candidate agent that has anti-aging function. In some embodiments, the method may further comprise contacting the stem cells or early extraembryonic cells with different concentrations of the candidate agent.

[0169] In one embodiment, a method for evaluating the anti-aging function of a candidate agent includes 1) subjecting TSCs to conditions that differentiate the TSCs toward STBs, 2) contacting the TSCs with a candidate agent while subjecting the TSCs to conditions that allow differentiation, and 3) assessing changes in the expression of hCG and / or differentiation / senescence markers in STBs compared to STBs that have not been contacted with the candidate agent. If cells contacted with the candidate agent secrete less hCG and / or differentiation / senescence markers compared to cells that have not been contacted with the candidate agent, the candidate agent can be predicted to have anti-aging function.

[0170] In one embodiment, a method for evaluating the anti-aging function of a candidate agent includes 1) subjecting TSCs to conditions that differentiate the TSCs toward STBs, 2) contacting the TSCs with different concentrations of a candidate agent while simultaneously subjecting the TSCs to conditions that allow differentiation, and 3) assessing changes in expression of hCG and / or differentiation / senescence markers in STBs that have been contacted with or not contacted with the candidate agent. An effective concentration of the candidate agent may be determined as the lowest concentration at which expression of hCG and / or differentiation / senescence markers is reduced by at least 20% compared to STBs that have not been contacted with the candidate agent.

[0171] 2. Evaluation of the impact on survival rate In some embodiments, the method further comprises determining the effect of the candidate agent on the viability of stem cells or early extraembryonic cells, or cells differentiated therefrom. Methods for assaying cell viability, including MTT assay, tetrazolium reduction, resazurin reduction, protease markers, and ATP detection, are known in the art. Cell viability can also be assessed by quantifying intensity / cell number using confocal microscopic imaging of Hoechst-stained live cells or fixed adherent cells.

[0172] 3. Exemplary Drug Screening Workflow The present invention provides a method for drug screening for potential anti-aging effects. In the exemplary workflow outlined in Figure 6, the method includes: 1) drug library preparation; 2) compound dilution; 3) TSC preparation; 4) TSC-STB differentiation; 5) high-content microscopy screening; 7) readout analysis, such as reporter molecule signals (fluorescent and / or non-fluorescent), hCG expression, biomarker analysis, and 9) single-cell RNA sequencing (scRNA-seq).

[0173] V. Methods for Identifying Candidate Genes 1. Engineered Cells In one aspect, a method for identifying candidate genes involved in the aging process is provided, the method comprising: 1) subjecting a plurality of stem cells (such as totipotent or pluripotent stem cells) or early extraembryonic cells to conditions that cause the plurality of stem cells or early extraembryonic cells to differentiate toward STB, wherein each of the plurality of stem cells or early extraembryonic cells comprises an alteration in a candidate agent compared to a wild-type stem cell or early extraembryonic cell, and at least two of the stem cells or early extraembryonic cells comprise a different alteration; 2) determining one or more differentiation characteristics of the stem cells or early extraembryonic cells; and 3) identifying the candidate genes involved in the aging process based on the ability of the alteration in the candidate gene to cause the alteration in one or more differentiation characteristics of the stem cells or early extraembryonic cells compared to stem cells or early extraembryonic cells that do not comprise the corresponding alteration. In some embodiments, the stem cells or early extraembryonic cells are genetically modified.

[0174] Suitable methods for genetically modifying stem cells or early extraembryonic cells are known in the art, and include clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homing endonucleases or meganucleases, and base editing. The result of the genetic modification can be gene knockout / knockin, gene mutation, or gene inversion.

[0175] CRISPR is a family of DNA sequences found in the genomes of prokaryotes, such as bacteria and archaea. These sequences originate from DNA fragments of bacteriophages that previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. CRISPR-Cas systems consist of CRISPR repeat-spacer arrays (which can be further transcribed into CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA)) and a set of CRISPR-associated (cas) genes encoding Cas proteins with endonuclease activity. CRISPR-Cas systems can be classified into two classes (class 1 and class 2), six types (I-VI), and several subtypes. Class 1 systems (types I, III, and IV) have multi-Cas protein effector complexes, while class 2 systems (types II, V, and VI) have single effector proteins. The type II CRISPR-Cas9 system derived from Streptococcus pyogenes (SpCas9) is one of the most well-characterized and most commonly used CRISPR-Cas systems. The key components of the CRISPR-Cas9 system are the RNA-guided Cas9 endonuclease and a single guide RNA (sgRNA). The Cas9 protein contains two nuclease domains, HNH and RuvC, each of which cleaves one strand of the target double-stranded DNA. The single guide RNA (sgRNA) is a simplified combination of the crRNA and tracrRNA. The Cas9 nuclease and sgRNA form the Cas9 ribonucleoprotein (RNP), which can bind to and cleave specific DNA targets. Furthermore, a protospacer adjacent motif (PAM) sequence is required for the Cas9 protein to bind to the target DNA.

[0176] ZFNs are fusions between custom-designed Cys2-His2 zinc finger proteins and the cleavage domain of the FokI restriction endonuclease. ZFNs function as dimers, with each monomer recognizing a specific "half-site" sequence (typically 9-18 base pairs (bps) of DNA) via its zinc finger DNA-binding domain.

[0177] TALENs are structurally similar to ZFNs. Both methods use Fokl nuclease to cleave DNA and require dimerization for function, but they differ in their DNA-binding domains. TALENs use transcription activator-like effectors (TALEs), tandem arrays of 33-35 amino acid repeats. The amino acid repeats have single-base recognition, thereby improving targeting ability and specificity compared to ZFNs.

[0178] Homing endonucleases, also known as meganucleases, are a group of naturally occurring enzymes that recognize and cleave long DNA sequences (14-40 bps). These enzymes typically exhibit excellent specificity due to the extensive sequence-specific contacts they make with their DNA substrates.

[0179] Base editing is a relatively new genome editing method derived from CRISPR-Cas9. Unlike traditional CRISPR systems, base editors (BEs) do not induce double-strand breaks in the genome. Base editing systems use a "catalytically inactive" Cas9 (dCas9) that cannot cut DNA fused to a bacterial enzyme called a DNA deaminase. Cytidine deaminase, which induces C-to-T substitutions, occurs naturally in bacteria, while adenine deaminase, which induces A-to-G substitutions, is engineered from bacterial enzymes specifically for base editing purposes. By fusing dCas9 to either cytidine deaminase (CBE) or adenine deaminase (ABE) and providing an sgRNA to direct it to the target sequence, researchers can introduce substitutions into DNA.

[0180] The invention will now be described by reference to the following examples, which are intended to be illustrative of embodiments of the invention and should not be construed as limiting the invention.

[0181] Illustrative Embodiments Embodiment 1. A senescence evaluation system for mammalian cells comprising totipotent or pluripotent stem cells or early extraembryonic cells, said totipotent or pluripotent stem cells or early extraembryonic cells optionally comprising a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of said totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or syncytiotrophoblasts ("STBs").

[0182] Embodiment 2. The mammalian cell aging evaluation system according to embodiment 1, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are naturally occurring.

[0183] Embodiment 3. The mammalian cell aging evaluation system according to embodiment 1, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are genetically modified.

[0184] Embodiment 4. The mammalian cell aging evaluation system according to any one of embodiments 1 to 3, wherein the totipotent or pluripotent stem cells or early extraembryonic cells contain a reporter molecule.

[0185] Embodiment 5. The mammalian cell aging evaluation system according to embodiment 4, wherein the reporter molecule is selected from the group consisting of physically activated molecules and chemically activated molecules.

[0186] Embodiment 6. A system for evaluating the aging of mammalian cells according to any one of embodiments 1 to 3, wherein the totipotent or pluripotent stem cells or early extraembryonic cells contain the heterologous nucleic acid.

[0187] Embodiment 7. The aging evaluation system for mammalian cells according to embodiment 6, wherein the heterologous nucleic acid is integrated into the genome of the totipotent or pluripotent stem cell or early extraembryonic cell.

[0188] Embodiment 8. The mammalian cell senescence evaluation system of embodiment 7, wherein the heterologous nucleic acid is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of senescence or differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell toward trophoblast or STB.

[0189] Embodiment 9. The mammalian cell aging evaluation system of embodiment 8, wherein the heterologous nucleic acid is introduced into the totipotent or pluripotent stem cells or early extraembryonic cells by a gene editing tool.

[0190] Embodiment 10. The mammalian cell aging evaluation system according to any one of embodiments 6 to 9, wherein the reporter molecule is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, tdTomato, photoconvertible fluorescent protein, bioluminescence, enzyme assay, antibody-based assay, chloramphenicol acetyltransferase, and biosensor.

[0191] Embodiment 11. A system for evaluating the aging of mammalian cells according to any one of embodiments 1 to 10, wherein the system comprises early extraembryonic cells.

[0192] Embodiment 12. The mammalian cell aging evaluation system of embodiment 11, wherein the early extraembryonic cells are trophoblast stem cells ("TSCs") or trophoblast progenitor cells ("TPCs").

[0193] Embodiment 13. The mammalian cell aging evaluation system according to embodiment 12, wherein the early extraembryonic cells are TSCs.

[0194] Embodiment 14. The mammalian cell aging evaluation system according to any one of embodiments 11 to 13, wherein the early extraembryonic cells are derived from totipotent stem cells, pluripotent stem cells, embryonic tissue, or placental tissue.

[0195] Embodiment 15. The mammalian cell aging evaluation system according to any one of embodiments 11 to 14, wherein the initial extraembryonic cells are derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

[0196] Embodiment 16. The aging evaluation system for mammalian cells according to any one of embodiments 8 to 15, wherein the endogenous biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, ineffective macroautophagy, deregulation of nutrient sensing, altered intercellular communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion.

[0197] Embodiment 17. The endogenous biomarkers are β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1C , CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, and SIRT family members.

[0198] Embodiment 18. A system for evaluating the aging of mammalian cells according to any one of embodiments 1 to 10, wherein the system comprises totipotent or pluripotent stem cells.

[0199] Embodiment 19. The aging evaluation system for mammalian cells according to embodiment 18, wherein the system comprises totipotent stem cells.

[0200] Embodiment 20. The mammalian cell aging evaluation system according to embodiment 18, wherein the system comprises pluripotent stem cells.

[0201] Embodiment 21. The mammalian cell aging evaluation system according to any one of embodiments 1 to 20, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are derived from humans, pigs, cows, mice, rats, bats, rabbits, dogs, cats, and sheep.

[0202] Embodiment 22. A method of assessing the aging process in mammalian cells, said method comprising subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate said totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs, and determining one or more differentiation characteristics of said totipotent or pluripotent stem cells or early extraembryonic cells.

[0203] Embodiment 23. A method for evaluating the anti-aging function of a candidate agent, the method comprising: 1) subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the totipotent or pluripotent stem cells or early extraembryonic cells to differentiate towards trophoblasts, in particular STBs; 2) contacting the totipotent or pluripotent stem cells or early extraembryonic cells with the candidate agent before, during, or after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to the conditions that cause the totipotent or pluripotent stem cells or early extraembryonic cells to differentiate; and 3) assessing a change in one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells that are not contacted with the candidate agent.

[0204] Embodiment 24 The method of embodiment 23, wherein evaluating the anti-aging function of the candidate agent comprises assessing whether the candidate agent has anti-aging function.

[0205] Embodiment 25 The method of embodiment 23, wherein evaluating the anti-aging function of the candidate agent comprises determining an effective concentration of the candidate agent having anti-aging function.

[0206] Embodiment 26. A method for screening candidate drugs with anti-aging function, the method comprising: 1) evaluating the anti-aging function of a plurality of candidate drugs according to the method of any one of embodiments 23 to 25; and 2) identifying the candidate drugs with anti-aging function based on the ability of the candidate drugs to cause a change in one or more characteristics of differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells into trophoblasts or STBs compared to the totipotent or pluripotent stem cells or early extraembryonic cells without the candidate drugs.

[0207] Embodiment 27. The method of any one of embodiments 23 to 26, wherein the candidate agent is selected from the group consisting of an antibody, a virus, a virus-like agent, a small molecule, a peptide, a polypeptide, DNA, mRNA, a guide RNA, a microRNA, RNAi, lncRNA, an siRNA molecule, and an antisense RNA.

[0208] Embodiment 28 The method of any one of embodiments 23 to 26, wherein the candidate agent is a naturally occurring substance.

[0209] Embodiment 29. The method of any one of embodiments 23 to 26, wherein the candidate agent is a nutritional supplement.

[0210] Embodiment 30. A method of identifying candidate genes involved in the aging process, the method comprising: 1) subjecting a plurality of totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the plurality of totipotent or pluripotent stem cells or early extraembryonic cells to differentiate toward trophoblasts or STBs, wherein each of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprises an alteration in a candidate agent compared to a wild-type totipotent or pluripotent stem cell or early extraembryonic cell, and wherein at least two of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprise a different alteration; 2) determining one or more differentiation characteristics of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells; and 3) identifying the candidate genes involved in the aging process based on the ability of the alteration in the candidate gene to cause a change in one or more differentiation characteristics of the plurality of totipotent or pluripotent stem cells or early extraembryonic cells compared to a totipotent or pluripotent stem cell or early extraembryonic cell that does not comprise the corresponding alteration.

[0211] Embodiment 31. The method of any one of embodiments 22 to 30, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are naturally occurring.

[0212] Embodiment 32 The method of any one of embodiments 22 to 30, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are genetically modified.

[0213] Embodiment 33. The method of any one of embodiments 22 to 32, wherein the totipotent or pluripotent stem cells or early extraembryonic cells comprise a reporter molecule indicative of differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards STBs.

[0214] Embodiment 34. The method of embodiment 33, wherein the reporter molecule is selected from the group consisting of physically activated molecules and chemically activated molecules.

[0215] Embodiment 35. The method of any one of embodiments 22 to 32, wherein the totipotent or pluripotent stem cells or early extraembryonic cells comprise a heterologous nucleic acid encoding a reporter molecule indicative of differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs.

[0216] Embodiment 36 The method of embodiment 35, wherein the heterologous nucleic acid is integrated into the genome of the totipotent or pluripotent stem cell or early extraembryonic cell.

[0217] Embodiment 37. The method of embodiment 36, wherein said heterologous nucleic acid is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of differentiation of said totipotent or pluripotent stem cells or early extraembryonic cells towards STB.

[0218] Embodiment 38. The method of any one of embodiments 35 to 37, wherein the heterologous nucleic acid is introduced into the totipotent or pluripotent stem cell or early extraembryonic cell by a gene editing tool.

[0219] Embodiment 39. The method of any one of embodiments 35 to 38, wherein the reporter molecule is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, and tdTomato.

[0220] Embodiment 40. The method of any one of embodiments 22 to 39, wherein the STB is early STB, late STB, or mature and senescent STB.

[0221] Embodiment 41. The method of any one of embodiments 22 to 40, wherein the one or more differentiation characteristics comprise: 1) the presence or absence of a biomarker associated with the differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell; 2) the level of a biomarker associated with the differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell; 3) the secretion of a biomarker associated with the differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell; 4) cell morphology; 5) the rate of change to a differentiation state; 6) the characteristics of cell organelles; 7) the number of nuclei in the cell; and 8) the presence or absence of a reporter molecule.

[0222] Embodiment 42. The method of embodiment 41, wherein the one or more differentiation characteristics include organelle properties.

[0223] Embodiment 43. The method of embodiment 42, wherein the organelle is selected from the group consisting of mitochondria, proteosomes, endoplasmic reticulum, Golgi apparatus, and nuclear envelope.

[0224] Embodiment 44. The method of embodiment 43, wherein the characteristics of the organelles include the number, morphology, and function of the organelles.

[0225] Embodiment 45. The method of any one of embodiments 22 to 44, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are early extraembryonic cells.

[0226] Embodiment 46. The method of embodiment 45, wherein the initial extraembryonic cells are derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

[0227] Embodiment 47 The method of embodiment 41, wherein said one or more differentiation characteristics comprises the presence or absence of a biomarker associated with differentiation of said early extraembryonic cells.

[0228] Embodiment 48. The method of embodiment 47, wherein the biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion.

[0229] Embodiment 49. The biomarkers are selected from the group consisting of β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1C, 49. The method of embodiment 48, wherein the IL-16 polypeptide is selected from the group consisting of CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1 gamma, HP1 alpha, LTR5, pTBK1, LINE1, HERVK, SIRT family members, and combinations thereof.

[0230] Embodiment 50. The method of any one of embodiments 47-49, wherein the one or more differentiation hallmarks comprise nucleotide excision repair (NER), base excision repair (BER), DNA mismatch repair (MMR), Fanconi anemia pathway, homologous recombination (HR), non-homologous end joining (NHEJ), variant histones, insulin resistance, pro-inflammatory factors, mTOR / AMPK pathway, mitophagy, a senescence-associated secretory phenotype, and / or molecular indicators of senescence.

[0231] Embodiment 51. The method of any one of embodiments 22 to 44, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are totipotent or pluripotent stem cells.

[0232] Embodiment 52 The method of embodiment 51, wherein the totipotent or pluripotent stem cells are totipotent stem cells.

[0233] Embodiment 53 The method of embodiment 51, wherein the totipotent or pluripotent stem cells are multipotent stem cells.

[0234] Embodiment 54. The method of any one of embodiments 51 to 53, wherein the one or more differentiation characteristics comprises the presence or absence of a biomarker associated with differentiation of the totipotent or pluripotent stem cell.

[0235] Embodiment 55. The biomarkers are selected from the group consisting of β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1C, 55. The method of embodiment 54, wherein the IL-16 polypeptide is selected from the group consisting of CDKN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1 gamma, HP1 alpha, LTR5, pTBK1, LINE1, HERVK, SIRT family members, and combinations thereof.

[0236] Embodiment 56. The method of any one of embodiments 47 to 50 and 54 to 55, wherein the biomarker is an RNA molecule.

[0237] Embodiment 57. The method of embodiment 56, wherein assessing changes in the one or more differentiation characteristics comprises RNA sequencing, RT-qPCR, and / or in situ hybridization.

[0238] Embodiment 58. The method of any one of embodiments 47 to 50 and 54 to 55, wherein the biomarker is a protein molecule.

[0239] Embodiment 59. The method of embodiment 58, wherein assessing changes in one or more differentiation characteristics comprises Western blot, ELISA, proteomics, and / or immunofluorescence.

[0240] Embodiment 60. The method of any one of embodiments 23-29 and 31-59, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are contacted with the candidate agent before being subjected to conditions for differentiation.

[0241] Embodiment 61. The method of any one of embodiments 23 to 29 and 31 to 59, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are contacted with the candidate agent simultaneously with being subjected to conditions for differentiation.

[0242] Embodiment 62. The method of any one of embodiments 23 to 29 and 31 to 59, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are contacted with the candidate agent after being subjected to conditions for differentiation.

[0243] Embodiment 63. The method of any one of embodiments 22 to 62, wherein the conditions for differentiation comprise a cell culture medium comprising DMEM / F12, β-mercaptoethanol, penicillin-streptomycin-glutamine, BSA, ITS-X, Y27632, forskolin, and KnockOut Serum Replacement.

[0244] Embodiment 64. The method of any one of embodiments 22 to 63, wherein the differentiation characteristics are assessed at least one day after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions for differentiation.

[0245] Embodiment 65. The method of embodiment 64, wherein the differentiation characteristics are assessed 2 to 8 days after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions for differentiation.

[0246] Embodiment 66. The method of any one of embodiments 22 to 65, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are derived from human, pig, cow, mouse, rat, bat, rabbit, dog, cat, and sheep.

[0247] Embodiment 67. The method of any one of embodiments 23-29 and 31-66, further comprising determining the effect of the candidate agent on the viability of the totipotent or pluripotent stem cells or early extraembryonic cells, or cells differentiated therefrom. [Example]

[0248] Example 1: Methods 1.1 Cell lines Proliferative stem cells (EPSCs) were derived from somatic cell lines, embryonic stem cells, or other stem cell lines. SNL76 / 7 cells were clonally derived from the mouse fibroblast STO cell line, which was transformed with neomycin resistance and the mouse LIF gene and established by Dr. Allan Bradley. Cells were expanded, treated with γ-irradiation, and used as feeder cells for EPSC culture. Reporter cell lines were constructed based on genome editing in normal EPSCs by transfection and purification of specific constructs. TSC cell lines were generated from different EPSC cell lines by differentiation in TSC medium (TSCM) and single-colony picking. Reporter TSC cell lines were differentiated from reporter EPSCs. STB cells were differentiated from TSC cell lines using STB medium (STBM). All cells were incubated at 37°C in a humidified incubator containing 5% CO2 and tested regularly weekly for mycoplasma using PlasmoTest (InvivoGen).

[0249] 1.2 Cell culture Human EPSC cells were maintained on SNL76 / 7 feeder layers in hEPSC medium (EPSCM) and passaged at a ratio of 1:5 to 1:10 using TrypLE™ Express Enzyme (1X). hEPSCM was an N2B27-based medium supplemented with four small molecules: 2.5 μM XAV939, 2.5 μM Endo-IWR-1, 1 μM CHIR99021, and 0.1 μM A419259. The components of N2B27-based medium are published in Geo et al. (2019) (Nat. Cell Biol. 21, 687-699).

[0250] 1.3 Establishment of human trophoblast stem cell line (hTSC) and differentiation of STB To generate hTSC cell lines, single-cell suspensions of hEPSCs were plated onto 6-well plates (1–2 × 10 cells / well) precoated with 10 mg / mL Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix or Matrigel. 3 The TSC cell lines were plated in 6-well plates (1 / well) and cultured in human trophoblast stem cell medium (hTSCM) for 7–10 days. Next, single colonies of differentiated cells were picked and expanded. The expanded cell lines were confirmed by trophoblast markers and RNA-Seq analysis. The established TSC cell lines were maintained in hTSCM medium and passaged every 3–5 days at a ratio of 1:4–1:5. hTSCM were grown in a DMEM / F12-based medium supplemented with 110 μM β-mercaptoethanol, 0.2% FBS, 0.5% penicillin-streptomycin, 0.3% BSA, 1× ITS-X supplement, 50.0 μg / mL Vc, 50.0 ng / mL EGF, 2.0 μM CHIR99021, 0.5 μM A83-01, 1.0 μM SB431542, 0.8 μM VPA, and 5.0 μM Y27632. Established TSC cells were plated at 1.0 × 10 cells per well onto 6-well plates precoated with 10 mg / mL Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix or Matrigel. 5 ~2×10 5 Cells were seeded and cultured for approximately 6 days in syncytiotrophoblast medium (STBM) and then induced to differentiate into STBs. STBM contains DMEM / F12 supplemented with 110 μM β-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, 1x ITS-X, 2.5 μM Y27632, 2 μM forskolin, and 4% KnockOut Serum Replacement. Cells become early syncytiotrophoblasts on days 2–3 and mature syncytiotrophoblasts around day 6.

[0251] 1.4 Protocol for Mechanism Testing During the TSC-STB Process ELISA: On days 3 and / or 6 of STB differentiation, the culture medium from each well was collected, and the supernatant was obtained by centrifugation. Sterile STBM was used as a blank control. To measure secreted β-hCG, ELISA was performed according to the manufacturer's specifications (Human-hCG-ELISA-Kit-protocol-book-v4-ab100533(website).pdf (abcam.com)). Specifically, 100 μl of TMB was added to the supernatant and blank control and incubated for 30 minutes. After that, stop solution was added, and measurements were performed at 450 nM using a spectrophotometer.

[0252] RT-qPCR: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's specifications. The extracted RNA was then reverse transcribed into cDNA using Fastking gDNA diselling RT SuperMix (Tiangen). Gene expression was measured using the primers listed in Table E1A with PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) and StepOnePlus™ Real-Time PCR (Applied Biosystems). Raw gene expression data were normalized to GAPDH using the ΔCt method. Statistical analysis was performed using one-tailed / two-tailed Student's t-tests in Prism 8 (GraphPad).

[0253] [Table E1A-1] [Table E1A-2]

[0254] Immunofluorescence staining (IF): Samples were fixed in 4% paraformaldehyde (Sigma Catalog P6148) for 15 minutes at room temperature, permeabilized with 0.3% Triton X-100 (Sigma Catalog T8787) for 10 minutes, and blocked with 5% donkey serum (Sigma Catalog D9663) and 1% BSA (Sigma Catalog A2153) in PBS for 0.5–1 hour. This was followed by overnight incubation with primary antibodies in a 4°C cold room. After aspirating the primary antibodies and washing three times (10 minutes each) in PBS, the cells were incubated with fluorophore-conjugated secondary antibodies for 1 hour at room temperature. After three additional washes in PBS, the cells were counterstained with 10 μg / mL DAPI (Thermo Fisher Scientific Catalog 62248) for 10 minutes to mark cell nuclei and imaged under a confocal microscope.

[0255] Western blot: Proteins were extracted using RIPA lysis (Thermofisher, catalog 89901) and Pierce Protease Inhibitor Mini Tablets (Thermofisher, catalog A32953). Protein concentrations were measured using Pierce™ BCA Protein Assay Kits (Thermofisher, catalog 23227). Samples containing a fixed amount of NuPAGE™ LDS Sample Buffer (4X) (Thermofisher, catalog NP0008) were then boiled at 70°C for 10 minutes. Equal amounts of the boiled samples were loaded and separated on a 12% polyacrylamide gel (Thermofisher, catalog NP0343BOX) and transferred to a PVDF membrane using a Bio-Rad Transblot Turbine system according to the manufacturer's instructions. Images were developed using a ChemiDoc Imaging System and processed with ImageJ 1.47v.The following primary antibodies were used: anti-GAPDH (thermosfisher, catalog AM4300, 1:10000 for Western blot), anti-Ki67 (thermofisher, catalog MA5-14520, 1:1000 for Western blot, 1:200 for IF), anti-HERVK-gag (Austral Biologicals, catalog HERM-1811-5, 1:200 for IF), anti-TRF1 (Abcam, catalog ab10579, 1:100 for IF), anti-ZO-1 (CST, catalog 13663, 1:200 for IF), anti-CGB3 (Abcam, catalog Ab131170, 1:200 for IF), anti-H3 (CST, catalog 44991, 1:2000 for Western blot), anti-H3K9me3 (Abcam, catalog ab8898, 1:100 0 for Western blot), anti-E-cadherin (CST, catalog 14472, 1:200 for IF), anti-p38MAPK (thermofisher, catalog 338700, 1:1000 for Western blot), anti-lamin B1 (thermofisher, catalog 702972, 1:1000 for Western blot, 1:200 for IF), anti-p27Kip1 (thermofisher, catalog PA527188, 1:1000 for Western blot) for Western blot), anti-Hp1 gamma (thermofisher, catalog MA3054, 1:1000 for Western blot, 1:300 for IF), anti-SIRT1 (CST, catalog 8469S, 1:1000 for Western blot), anti-SIRT6 (CST, catalog 12486S, 1:1000 for Western blot), anti-SIRT3 (thermofisher, catalog MA514910, 1:1000 for Western blot), anti-p53 (th thermofisher, catalog MA512557, 1:1000 for Western blot, 1:200 for IF), anti-gamma H2AX (CST, catalog 2577s, 1:1000 for Western blot, 1:200 for IF), anti-IL-6 (thermofisher, catalog MA523698, 1:50 for IF), anti-hCG (thermofisher, catalog 14-6508-82, 1:1000 for Western blot, 1:200 for IF).Secondary antibodies used: goat anti-mouse IgG (H+L) HRP (thermofisher, catalog 31430, 1:2000 for Western blot), goat anti-rabbit IgG (H+L) HRP (thermofisher, catalog 31460, 1:2000 for Western blot), goat anti-rabbit IgG (H+L) Alexa Fluor 647 (thermofisher, catalog A21244, 1:500 for IF), rabbit anti-mouse IgG (H+L) Alexa Fluor 647 (thermofisher, catalog A21239, 1:500 for IF), donkey anti-rabbit IgG (H+L) Alexa Fluor 488 (thermofisher, catalog A21206, 1:500 for IF), donkey anti-mouse IgG (H+L) Alexa Fluor 488 (thermofisher, catalog A21202, 1:500 for IF), donkey anti-mouse IgG (H+L) Alexa Fluor594 (thermofisher, catalog A21203, for 1:500 IF), donkey anti-rabbit IgG (H+L) AlexaFluor 594 (thermofisher, catalog A21207, for 1:500 IF).

[0256] Flow cytometry: Cells were digested with 0.25% trypsin / EDTA at 37°C for 2–3 min and dissociated into single cells by pipetting. Dissociated cells were filtered through a 40 mm nylon mesh (Kangning catalog 352235) to remove cell clumps. After centrifugation, cells were fixed using Fixation Medium (BD Cytofix, catalog 554655) according to the manufacturer's protocol. Washed cells were stored at 4°C in PBS supplemented with 2% FBS (Gibco catalog 10270) before flow cytometry analysis. All samples were assayed using an ACEA NovoCyte Quanteon™ microscope. FITC was detected using the 488 nm (530 / 30 bandpass filter) and 561 nm (610 / 20 bandpass filter) channels to exclude autofluorescence. DAPI-positive cells were detected using the 405 nm (445 / 45 bandpass filter) channel. FACS data was analyzed using FlowJo software.

[0257] RNA sequencing: RNA sequencing was performed at various time points. Adapter sequences and low-quality 3'-end sequences were removed using Cutadapt. Processed reads were mapped to the human hg38 genome assembly using hisat2. Gene annotations from Ensembl were used. Gene expression was quantified using FeatureCounts. Genes with an average count below 5 were filtered out. Transparent element annotations were obtained from the UCSC Genome Browser (RepeatMasker). TE expression was quantified using SQuIRE in "total" mode. Differentially expressed genes and TEs were analyzed using DESeq2. The R package Cluster Profiler was used for Gene Ontology (GO) and KEGG analysis. GSEA (Gene Set Enrichment Analysis) was performed using GSEApy, and gene sets were downloaded directly from https: / / www.gsea-msigdb.org. Bigwig files for RNA-seq signals were generated using bamCoverage in Deeptools, and IGV was used for visualization. For RNA-seq signals from endogenous retroviruses HERV-W, HERV-FRD, and HERV-K, GenBank accessions AY101582.1, BC068585.1, and N675077.1 were used, and reads were mapped and quantified using hisat2. For data deposited in E-MTAB-10429, processed count tables were used directly. Expression differences between TSC and STB (days 2, 4, 6, or 8) were calculated as log2 fold changes for each gene (adjusted p-value <0.01). The top 100 up-regulated and top 100 down-regulated genes in TSC and STB were selected after ranking genes by absolute log2 fold change.

[0258] 1.5 Drug screening protocol and cell viability validation Equal numbers of TSCs in a robust growth state were seeded (1.5 × 10 per well) in STBM with or without candidate drugs onto black polystyrene TC-treated 96-well microplates precoated with 10 mg / mL Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Matrigel). 4 ~2×10 4 Each screening microplate included three wells of controls (cells containing only 0.5% DMSO in 100 μL of STB growth medium) and three wells of positive controls (specific concentrations of several well-known anti-aging agents). Cells were cultured at 37°C, 5% CO2, and saturated humidity and allowed to differentiate into mature STBs in the medium for 6 days. Cell morphology was observed daily under a microscope. Reporter molecule signal readout was evaluated on days 2, 4, and 6. On day 6, cells in each well were fixed and stained with DAPI or Hoechst, and cell number and intensity (both reporter and nuclei) were assessed using high-content microscopy to assess drug effects and cell viability. Drugs, especially those affecting cell viability, are also tested at different concentrations and on different days during the STB differentiation process.

[0259] 1.6 Reporter cell lines The template plasmids pCD31-5HA-T2A-H2B-EGFP-loxp-EF1a-puro-loxp-3HA (SEQ ID NO: 1) and hSpCas9-2A-Puro(PX459) (Additional Gene ID 62988; SEQ ID NO: 2), CGA, CGB3, and ERVW1 plasmids were constructed for the reporter cell line. The complete sequences of human CGA, CGB3, and ERVW1 were obtained from the Ensembl genome browser (https: / / asia.ensembl.org / index.html). The gRNA for each gene was located 3' to the stop codon (less than 100 bp from the stop codon) and inserted into the U6-gRNA-Cas9 construct by digestion and T4 ligation. Homology arms (5HA and 3HA portions of each gene) (1000–1500 bp) were inserted 5′ of the stop codon and 3′ of the gRNA target site, respectively. Using homologous recombination (CloneExpress II One Step Cloning Kit), 5HA and 3HA were inserted into pCD31-5HA-T2A-H2B-EGFP-loxp-EF1a-puro-loxp-3HA to obtain the GA-T2A-H2B-GFP, CGB3-T2A-H2B-GFP, and ERVW1-T2A-H2B-GFP reporter constructs. The reporter plasmids for each gene and their corresponding gRNA constructs were combined and transfected into EPSCs. Puromycin selection was performed for 7 days, and surviving EPSC colonies were confirmed by genotyping for reporter insertions in the genome. The confirmed EPSC colonies were then expanded and induced to differentiate into TSC cell lines for further testing.

[0260] Example 2: Differentiation of STBs from EPSC-derived TSCs This example demonstrates that M1-EPSCs can be efficiently induced to differentiate into TSCs, and that TSCs can differentiate into STBs that express relevant markers.

[0261] As shown in Figure 1A, M1-EPSCs cultured in human trophoblast stem cell medium as described in Example 1 were induced to differentiate into multiple TSC single-cell clones. The morphology of the established cell line after monoclonal M1-TSC proliferation stabilized is shown (Figure 1B). Differentiation markers (CDX2, GATA2, GATA3, CGA, KRT7, and ERVW1) were expressed at significantly higher levels in TSCs compared with M1-EPSCs (Figure 1C). Immunofluorescence also showed that GATA2 and GATA3 were highly expressed in TSCs (Figure 1D).

[0262] M1-TSCs were then induced to differentiate into STBs in STBM as described in Example 1. The differentiated cells exhibited the expected morphology of STBs (Figures 1E, 1I, and 1J). hCG was expressed at higher levels in STBs than in TSCs, as shown by immunofluorescence (Figure 1F) and ELISA (Figure 1H). CGA and CGB3 levels were higher in STBs than in TSCs, as shown by RT-qPCR and immunofluorescence (Figures 1G, 1J, and 1L). STB markers SDC1, ERVW1, and CSH1 were significantly more highly expressed in STBs at day 6 (Figure 1K). Furthermore, STBs were in a growth-arrested state, as demonstrated by a low proliferation rate (Figure 1M), a low cell cycle score from RNA sequencing data (Figure 1N), low expression of the proliferation marker Ki67 (Figures 1O and 1P), and increased expression of the cell cycle inhibitors p27 and p38 (Figure 1Q).

[0263] Example 3: Senescence-associated characteristics during TSC-STB differentiation This example shows that various markers associated with aging exhibit patterns of change during TSC-STB differentiation (RNA-seq data) that mimic the normal aging process in humans (Table E2A).

[0264] [Table E2A]

[0265] *SIRT family members were increased at the RNA level in STB cells, which was inconsistent with normal aging, but Western blot analysis showed that they were decreased in STB cells compared with TSC cells, which was consistent with normal aging.

[0266] The level of NER gradually decreased from TSC to STB, suggesting a gradual decrease in repair capacity with STB maturation and senescence (Figure 2A). BER also decreased during the process of differentiation and maturation from TSC to STB, suggesting a decline in repair capacity (Figure 2B). MMR scores also decreased as TSC differentiated into STB, suggesting senescence (Figure 2C). As shown in Figure 2D, Fanconi anemia signaling pathway activity gradually decreased with TSC to STB differentiation, suggesting a decline in repair capacity. As shown in Figure 2E, homologous recombination decreased with TSC to STB differentiation, suggesting senescence. As shown in Figure 2F, non-homologous end joining (NHEJ) decreased with TSC to STB differentiation, suggesting senescence.

[0267] As shown in Figure 2G, the variant histone score decreased with differentiation of TSCs into STBs, suggesting senescence.

[0268] Insulin resistance, another indicator of cellular senescence, increased with differentiation of TSCs into STBs (Figure 2H), and the insulin score increased (Figure 2P).

[0269] The expression of proinflammatory factors gradually increased with the differentiation of TSCs into STBs, reaching the highest level on day 6, suggesting an increase in proinflammatory factors due to senescence ( Figure 2I ).

[0270] The mTOR / AMPK pathway can sense the energy demands of the microenvironment and regulate cellular metabolic pathways. With TSC differentiation to STB and aging, the mTOR / AMPK score decreased, indicating a reduction in cellular metabolic capacity and levels (Figure 2J).

[0271] Mitophagy is the selective removal of damaged mitochondria by autophagosomes and their subsequent catabolism by lysosomes. As shown in Figure 2K, mitophagy gradually increased with STB differentiation, suggesting an increase in senescent organelles.

[0272] Both the overall senescence score and the senescence-associated epigenetic score (including the linker histone score) increased, while scores related to the inhibition of senescence decreased (Figures 2L-2O).

[0273] Cells were further examined for senescence-associated secretory phenotype (SASP) and senescence-associated proteins. TSCs were derived from M1-EPSCs and induced to differentiate into STBs. During differentiation into STBs, cells were collected for transcriptome sequencing on days 2, 4, and 6. Sequencing data were analyzed as described in Example 1. RT-qPCR was performed at the beginning and end of differentiation as described in Example 1. Differentially expressed genes are shown in Figure 7. The top 100 up- and down-regulated genes in STB day 2 vs. TSCs, STB day 4 vs. TSCs, STB day 6 vs. TSCs, and STB day 8 vs. TSCs are shown in Table E2B.

[0274] As shown in Figures 3A-3C, 3N, and 3S, the senescence-associated secretory phenotype (SASP) and senescence-associated proteins showed differential expression levels in TSCs and STBs. In particular, BCL2, MCL1, CDKN1A, CDKN2A, APEX1, NFKB1, TP53, MMP3, MMP9, RB1, FOXO3, SMAD3, and CDKN2B were significantly more highly expressed in STBs than in TSCs. IL-6, IL-8, IL-1α, IL-1β, and CCL2 were also more highly expressed in STBs than in TSCs (Figures 3S-3T). The expression of beta-galactosidase, γH2AX, and p53 also increased as TSCs differentiated toward STBs (Figures 3D-3I). STBs were also shown to have increased mitochondrial mass, as indicated by Mito-tracker immunostaining (Figure 3J). Other biomarkers, such as H3K9me3, SETDB1 / KAP1 complex, HP1γ, and nuclear lamin proteins (lamin A, lamin B1, and lamin C), showed decreased expression as TSCs differentiated toward STBs (Figures 3K, 3O–3R), indicating increased genomic instability in STB cells compared with TSC cells. Classical sirtuin family members (SIRT1, SIRT3, and SIRT6), closely associated with longevity, increased at the RNA level (Table E2B) but decreased in STB cells (Figure 3M). In addition to increasing or decreasing trends, some biomarkers (such as LTR5 and HERVK) showed a more complex pattern of increased expression at day 2 during TSC-STB differentiation followed by a subsequent decrease (Figures 3U–3V). Pie charts were generated to show the classes of transposable element (TE) loci that were up- and downregulated after TSC differentiation. The size of each pie chart area is proportional to the number of TE loci (Figure 3W). TRF1, one of the molecules involved in telomere attrition, increased during STB differentiation and showed more colocalized foci with gamma-H2AX in STB than in TSC cells (Figures 3X–3Y).

[0275] [Table E2B-1] [Table E2B-2] [Table E2B-3] [Table E2B-4] [Table E2B-5]

[0276] Example 4: Effect of anti-aging agents on TSC-STB differentiation First, we tested the known anti-aging drug rapamycin. TSCs in good proliferative condition were plated at 1.0 × 10 cells per well in 24-well plates precoated with 10 mg / mL Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix or Matrigel. 4 ~2×10 4 Cells were seeded onto the wells. After 24 hours, three wells continued in TSCM, three wells were switched to STBM + DMSO, and three wells were switched to STBM + rapamycin (500 nM). The medium was changed every 2-3 days. On day 6, β-hCG was measured by ELISA as described in Example 1. The expression of senescence-associated marker genes (e.g., CGA and CGB3) was measured by RT-qPCR as described in Example 1.

[0277] The known anti-aging drug rapamycin reduced the expression levels of STBM hallmarks, SASP1L-6, and the DNA damage-related gene CDKN1A. Rapamycin at 500 nM did not affect cell viability or proliferation (Figure 4A). Cells treated with rapamycin showed significantly lower levels of β-hCG, CGA, CGB3, CDKN1A, and IL6 compared to the STBM + DMSO group (Figures 4B-4D).

[0278] In addition to rapamycin, several other drugs were also tested for their potential anti-aging effects. Figure 4E shows the morphology of cells treated with remdesivir, GC376, molnupiravir, rapamycin, INK128, and STM2457 during TSC-STB differentiation, where a delay in the appearance of multinucleated STB cells was observed. The ELISA results in Figure 4F show that cells treated with remdesivir, rapamycin, and INK128, which are known to have anti-aging effects, secreted significantly lower levels of β-hCG, suggesting that TSC-STB differentiation was inhibited or delayed. Cells treated with the antiviral drugs molnupiravir and GC376, and the Mettle3 inhibitor STM2457, showed similar or increased levels of β-hCG compared to the DMSO control group.

[0279] Next, we used this system to test other drugs. Nicotinamide mononucleotide (NMN), acarbose, spermidine, fisetin, and quercetin have been reported to have anti-aging effects. Figure 4G shows the morphology of cells treated with nicotinamide mononucleotide (NMN), acarbose, spermidine, fisetin, and quercetin during TSC-STB differentiation. NMN, acarbose, and fisetin reduced CGA expression at lower concentrations (1 μM) and increased it at higher concentrations (10 μM), suggesting that further screening could be performed at lower concentrations. On the other hand, spermidine and quercetin were shown to reduce CGA expression at both 1 μM and 10 μM, suggesting the need for further testing at higher concentrations and that their anti-aging mechanisms may differ from those of NMN, acarbose, and fisetin (Figure 4H). Western blot showed that certain concentrations of rapamycin, INK128, and fisetin could significantly decrease β-hCG expression ( Figure 4I ), further demonstrating the anti-aging effects of these drugs.

[0280] Example 5: Green fluorescent protein (GFP) reporter cell lines The PD31-CGA-H2B-GFP and hSp-CGAgRNA-Cas9-2A-Puro (PX459) plasmids were constructed as described in Example 1 (Figures 5A-5B) and transfected into EPSC cells. After puromycin selection, colonies were picked and expanded. The PD31-CGA-H2B-GFP insertions in both the adjacent 5HA and 3HA homology regions were confirmed through genotyping and sequencing. One colony showed band sizes to the right of both the adjacent 5HA and 3HA homology regions by electrophoresis (Figure 5C). EPSC colonies were differentiated into TSCs for stable cell lines, which were negative for GFP signal under a light microscope. However, after induction of STB differentiation, a strong GFP signal was detected, accompanied by a significant increase in CGA expression, as shown in Figures 5D-5F.

[0281] Next, we used the reporter cells to test various drugs. The GFP signal was reduced when treated with drugs known to have anti-aging effects, confirming that the GFP readout of reporter cells can be used as an evaluation criterion for testing potential anti-aging drugs (Figures 5G-5H).

[0282] In summary, the present invention successfully established stable trophoblast stem / progenitor cells and induced them to differentiate into STBs. The present invention also characterized changes during TSC-STB differentiation using cell morphology, biomarkers, secretory function (e.g., β-hCG), and bioinformatics, which can be used to assess aging. The utility of TSC-STB differentiation as a system for assessing aging was verified using the known anti-aging drug rapamycin. Rapamycin-treated cells showed delayed maturation and senescence in both cell morphology and function (e.g., β-hCG secretion), and significant changes in senescence-related biomarkers. Other anti-aging compounds (e.g., metformin, NMN, fisetin, and quercetin) also demonstrated the expected anti-aging effects in the TSC-STB system.

[0283] It will be appreciated that although the STBs in the above examples were derived from stem cells, they may also be derived from placental tissue.

[0284] Although the cells in the above examples were human cells, it will be appreciated that mammalian cells may also be used.

[0285] Although the cells in the above examples were in 2D culture, it will be appreciated that the cells may also be in 3D culture.

Claims

1. A senescence evaluation system for mammalian cells comprising totipotent or pluripotent stem cells or early extraembryonic cells, wherein the totipotent or pluripotent stem cells or early extraembryonic cells optionally comprise a reporter molecule or a heterologous nucleic acid encoding a reporter molecule, which indicates senescence or differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or syncytiotrophoblasts ("STBs").

2. The mammalian cell aging evaluation system according to claim 1 , wherein the totipotent or pluripotent stem cells or early extraembryonic cells are naturally occurring.

3. The mammalian cell aging evaluation system according to claim 1 , wherein the totipotent or pluripotent stem cells or early extraembryonic cells are genetically modified.

4. The mammalian cell aging evaluation system according to any one of claims 1 to 3, wherein the totipotent or pluripotent stem cells or early extraembryonic cells contain a reporter molecule.

5. The mammalian cell aging evaluation system according to claim 4 , wherein the reporter molecule is selected from the group consisting of physically activated molecules and chemically activated molecules.

6. The mammalian cell aging evaluation system according to any one of claims 1 to 3, wherein the totipotent or pluripotent stem cell or early extraembryonic cell comprises the heterologous nucleic acid.

7. The mammalian cell aging evaluation system according to claim 6 , wherein the heterologous nucleic acid is integrated into the genome of the totipotent or pluripotent stem cell or early extraembryonic cell.

8. The mammalian cell aging evaluation system of claim 7, wherein the heterologous nucleic acid is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of senescence or differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell toward trophoblast or STB.

9. The mammalian cell aging evaluation system of claim 8, wherein the heterologous nucleic acid is introduced into the totipotent or pluripotent stem cells or early extraembryonic cells by a gene editing tool.

10. The mammalian cell aging evaluation system according to any one of claims 6 to 9, wherein the reporter molecule is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, tdTomato, photoconvertible fluorescent protein, bioluminescence, enzyme assay, antibody-based assay, chloramphenicol acetyltransferase, and biosensor.

11. The system for evaluating aging in mammalian cells according to any one of claims 1 to 10, wherein the system comprises early stage extraembryonic cells.

12. The mammalian cell aging evaluation system according to claim 11, wherein the early extraembryonic cells are trophoblast stem cells ("TSCs") or trophoblast progenitor cells ("TPCs").

13. The mammalian cell aging evaluation system according to claim 12, wherein the early extraembryonic cells are TSCs.

14. The mammalian cell aging evaluation system according to any one of claims 11 to 13, wherein the early extraembryonic cells are derived from totipotent stem cells, pluripotent stem cells, embryonic tissue, or placental tissue.

15. The system for evaluating the aging of mammalian cells according to any one of claims 11 to 14, wherein the initial extraembryonic cells are derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

16. The mammalian cell aging evaluation system according to any one of claims 8 to 15, wherein the endogenous biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of protein homeostasis, telomere attrition, organelle dysfunction, ineffective macroautophagy, deregulation of nutrient sensing, altered intercellular communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion.

17. The endogenous biomarkers include β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1C, CD 17. The mammalian cell senescence evaluation system of claim 16, wherein the target gene is selected from the group consisting of KN2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, and SIRT family members.

18. The system for evaluating aging in mammalian cells according to any one of claims 1 to 10, wherein the system comprises totipotent or pluripotent stem cells.

19. The mammalian cell aging evaluation system according to claim 18 , wherein the system comprises totipotent stem cells.

20. The system for evaluating aging in mammalian cells according to claim 18 , wherein the system comprises pluripotent stem cells.

21. The mammalian cell aging evaluation system according to any one of claims 1 to 20, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are derived from a human, a pig, a cow, a mouse, a rat, a bat, a rabbit, a dog, a cat, or a sheep.

22. 1. A method for assessing the aging process in mammalian cells, the method comprising subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs, and determining one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells.

23. 1. A method for evaluating the anti-aging function of a candidate drug, comprising: 1) subjecting totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause differentiation of said totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts, in particular STBs; 2) contacting the totipotent or pluripotent stem cells or early extraembryonic cells with the candidate agent before, during, or after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions that cause the differentiation of the cells; and 3) assessing a change in one or more differentiation characteristics of the totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells not contacted with the candidate agent.

24. 24. The method of claim 23, wherein evaluating the anti-aging function of the candidate agent comprises assessing whether the candidate agent has anti-aging function.

25. 24. The method of claim 23, wherein evaluating the anti-aging function of the candidate agent comprises determining an effective concentration of the candidate agent having anti-aging function.

26. A method for screening a candidate drug having anti-aging function, comprising: 1) evaluating the anti-aging function of a plurality of candidate drugs according to the method of any one of claims 23 to 25; and 2) identifying the candidate agent having anti-aging function based on the ability of the candidate agent to cause a change in one or more characteristics of differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell into a trophoblast or STB compared to the totipotent or pluripotent stem cell or early extraembryonic cell without the candidate agent.

27. 27. The method of any one of claims 23 to 26, wherein the candidate agent is selected from the group consisting of an antibody, a virus, a virus-like, a small molecule, a peptide, a polypeptide, DNA, mRNA, a guide RNA, a microRNA, RNAi, LncRNA, an siRNA molecule, and an antisense RNA.

28. The method of any one of claims 23 to 26, wherein the candidate agent is a naturally occurring substance.

29. The method of any one of claims 23 to 26, wherein the candidate drug is a nutritional supplement.

30. 1. A method for identifying candidate genes involved in the aging process, comprising: 1) subjecting a plurality of totipotent or pluripotent stem cells or early extraembryonic cells to conditions that differentiate said plurality of totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs, wherein each of said plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprises an alteration in a candidate agent compared to a wild-type totipotent or pluripotent stem cell or early extraembryonic cell, and at least two of said plurality of totipotent or pluripotent stem cells or early extraembryonic cells comprise a different alteration; 2) determining one or more differentiation characteristics of said plurality of totipotent or pluripotent stem cells or early extraembryonic cells; and 3) identifying said candidate genes involved in the aging process based on the ability of said alterations in said candidate genes to cause an alteration in one or more differentiation characteristics of said plurality of totipotent or pluripotent stem cells or early extraembryonic cells compared to totipotent or pluripotent stem cells or early extraembryonic cells that do not contain the corresponding alterations.

31. The method of any one of claims 22 to 30, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are naturally occurring.

32. The method of any one of claims 22 to 30, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are genetically modified.

33. 33. The method of any one of claims 22 to 32, wherein the totipotent or pluripotent stem cells or early extraembryonic cells comprise a reporter molecule that indicates differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards STB.

34. 34. The method of claim 33, wherein the reporter molecule is selected from the group consisting of physically activated molecules and chemically activated molecules.

35. 33. The method of any one of claims 22 to 32, wherein the totipotent or pluripotent stem cells or early extraembryonic cells comprise a heterologous nucleic acid encoding a reporter molecule that is indicative of differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells towards trophoblasts or STBs.

36. 36. The method of claim 35, wherein the heterologous nucleic acid is integrated into the genome of the totipotent or pluripotent stem cell or early extraembryonic cell.

37. 37. The method of claim 36, wherein the heterologous nucleic acid is under the control of a promoter of an endogenous biomarker gene encoding a biomarker indicative of differentiation of the totipotent or pluripotent stem cell or early extraembryonic cell towards STB.

38. 38. The method of any one of claims 35 to 37, wherein the heterologous nucleic acid is introduced into the totipotent or pluripotent stem cells or early extraembryonic cells by means of a gene editing tool.

39. 39. The method of any one of claims 35 to 38, wherein the reporter molecule is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mCherry, and tdTomato.

40. 40. The method of any one of claims 22 to 39, wherein the STB is early STB, late STB, or mature and senescent STB.

41. the one or more differentiation characteristics 1) the presence or absence of a biomarker associated with differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells; 2) levels of biomarkers associated with differentiation of all said totipotent or pluripotent stem cells or early extraembryonic cells; 3) secretion of biomarkers associated with differentiation of the totipotent or pluripotent stem cells or early extraembryonic cells; 4) cell morphology, 5) rate of change relative to differentiation state; 6) characteristics of organelles; 7) the number of nuclei in the cell; and 8) The method of any one of claims 22 to 40, comprising the presence or absence of a reporter molecule.

42. 42. The method of claim 41, wherein the one or more differentiation characteristics comprises organelle properties.

43. 43. The method of claim 42, wherein the organelle is selected from the group consisting of mitochondria, proteosomes, endoplasmic reticulum, Golgi apparatus, and nuclear envelope.

44. 44. The method of claim 43, wherein the characteristics of the organelles include the number, morphology, and function of the organelles.

45. The method of any one of claims 22 to 44, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are early extraembryonic cells.

46. 46. ​​The method of claim 45, wherein the initial extraembryonic cells are derived from stem cells selected from the group consisting of embryonic stem cells, extraembryonic stem cells, proliferative stem cells (EPSCs), naive pluripotent stem cells, primed pluripotent stem cells, induced pluripotent stem cells, 2-cell-like cells, and 8-cell-like cells.

47. 42. The method of claim 41, wherein the one or more differentiation characteristics comprises the presence or absence of a biomarker associated with differentiation of the early extraembryonic cells.

48. 48. The method of claim 47, wherein the biomarker is selected from the group consisting of markers of genomic instability, epigenetic changes (such as DNA, RNA, and protein modifications), loss of proteostasis, telomere attrition, organelle dysfunction, disabled macroautophagy, deregulated nutrient sensing, altered cell-cell communication, cellular senescence, chronic inflammation, differentiation, endogenous transposon elements, cell cycle, and stem cell exhaustion.

49. The biomarkers include β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1C, CDK 49. The method of claim 48, wherein the target protein is selected from the group consisting of N2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, a SIRT family member, and a combination thereof.

50. 50. The method of any one of claims 47 to 49, wherein the one or more differentiation hallmarks comprise nucleotide excision repair (NER), base excision repair (BER), DNA mismatch repair (MMR), Fanconi anemia pathway, homologous recombination (HR), non-homologous end joining (NHEJ), variant histones, insulin resistance, pro-inflammatory factors, mTOR / AMPK pathway, mitophagy, a senescence-associated secretory phenotype, and / or molecular indicators of senescence.

51. 45. The method of any one of claims 22 to 44, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are totipotent or pluripotent stem cells.

52. 52. The method of claim 51, wherein the totipotent or pluripotent stem cells are totipotent stem cells.

53. 52. The method of claim 51, wherein the totipotent or pluripotent stem cells are pluripotent stem cells.

54. 54. The method of any one of claims 51 to 53, wherein the one or more differentiation characteristics comprises the presence or absence of a biomarker associated with differentiation of the totipotent or pluripotent stem cell.

55. The biomarkers include β-hCG, β-galactosidase, NANOG, OCT4, SOX2, CDX2, GATA2, GATA3, KRT7, TEAD4, TFAP2C, TP53, CGA, CGB, ERVW1, CSH1, SDC1, HLA-G, MMP3, MMP9, ITGB6, GABRP, MUC16, IL1α, IL1β, IL6, IL8, IL28A, TIMP1, TIMP2, MCP1, MIP3, CXCL1, CXCL8, TGF, CCL2, BCL2, BCL2L1, BCL2L2, APEX1, MCL1, RB1, FOXO3, SMAD3, CDKN1A, CDKN1C, CDK 55. The method of claim 54, wherein the target gene is selected from the group consisting of N2A, CDKN2B, NF-κB, PTGS2, PTGES2, γH2AX, p21, p27, p38, p53, p57, PD1, PD-L1, Ki67, TFR1, METTL3, METTL14, WTAP, YTHDC1, YTHDF1, FTO, ALKBH5, ALKBH1, H3K9me3, H3K4me3, H3K27me3, H3K9ac3, SETDB1, KAP1, lamin A, lamin B1, lamin C, HP1γ, HP1α, LTR5, pTBK1, LINE1, HERVK, a SIRT family member, and a combination thereof.

56. 56. The method of any one of claims 47 to 50 and 54 to 55, wherein the biomarker is an RNA molecule.

57. 57. The method of claim 56, wherein assessing changes in the one or more differentiation characteristics comprises RNA sequencing, RT-qPCR, and / or in situ hybridization.

58. 56. The method of any one of claims 47 to 50 and 54 to 55, wherein the biomarker is a protein molecule.

59. 59. The method of claim 58, wherein assessing changes in one or more differentiation characteristics comprises Western blot, ELISA, proteomics, and / or immunofluorescence.

60. 60. The method of any one of claims 23 to 29 and 31 to 59, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are contacted with the candidate agent before being subjected to conditions for differentiation.

61. 60. The method of any one of claims 23 to 29 and 31 to 59, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are contacted with the candidate agent simultaneously with being subjected to conditions for differentiation.

62. 60. The method of any one of claims 23 to 29 and 31 to 59, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are contacted with the candidate agent after being subjected to conditions for differentiation.

63. 63. The method of any one of claims 22 to 62, wherein the conditions for differentiation comprise a cell culture medium comprising DMEM / F12, β-mercaptoethanol, penicillin-streptomycin-glutamine, BSA, ITS-X, Y27632, forskolin, and KnockOut Serum Replacement.

64. 64. The method of any one of claims 22 to 63, wherein the differentiation characteristics are assessed at least one day after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions for differentiation.

65. 65. The method of claim 64, wherein the differentiation characteristics are assessed 2 to 8 days after subjecting the totipotent or pluripotent stem cells or early extraembryonic cells to conditions for differentiation.

66. 66. The method of any one of claims 22 to 65, wherein the totipotent or pluripotent stem cells or early extraembryonic cells are derived from human, pig, cow, mouse, rat, bat, rabbit, dog, cat, and sheep.

67. 67. The method of any one of claims 23 to 29 and 31 to 66, further comprising determining the effect of the candidate agent on the viability of the totipotent or pluripotent stem cells or early extraembryonic cells, or cells differentiated therefrom.

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