Induced totipotent stem cells and preparation method thereof

Abstract

The present invention relates to combinations of small molecule reprogramming agents for inducing the generation of totipotent stem cells, methods for preparing induced totipotent stem cells, and the induced totipotent stem cells generated therefrom.

Description

[Technical field]

[0001] The present invention relates to combinations of small molecule reprogramming agents for inducing the generation of totipotent stem cells, methods for generating induced totipotent stem cells (ciTotiSCs), and the induced totipotent stem cells produced by induction. [Background technology]

[0002] Mammalian embryo development begins with a zygote formed by the union of an oocyte and a sperm, which then develops into various cell stages, e.g., the 2-cell stage, the 4-cell stage, the 8-cell stage, etc., and then through embryonic cleavage to reach the morula. For example, in mice, only zygotes and two-cell stage blastomeres have the capacity for bidirectional intra- and extra-embryonic development and the capacity to form the entire organism, i.e., totipotency, and they are called totipotent cells. As embryo development progresses, the totipotent cells differentiate into two cell types: the inner cell mass (ICM) and the trophoblast (TE). The embryo at this stage is called a blastocyst.

[0003] TEs are located outside the embryo at the blastocyst stage and can develop into extraembryonic parts such as the placenta. TEs play an important role in the further differentiation of the ICM. The cells of the ICM further differentiate into primitive endoderm (PE) and epiblast (EPI). Among them, PEs further develop into extraembryonic tissues - the visceral yolk sac, while EPIs further develop into various tissues and organs of the fetus, and finally into a complete fetus. Since EPIs can only develop into embryonic parts and lose the ability to develop into extraembryonic parts, they possess pluripotency rather than totipotency for bidirectional embryonic / extraembryonic development and are therefore referred to as pluripotent cells. Embryonic stem cells (ESCs) derived from in vitro cultured ICM are also pluripotent and can differentiate into all embryonic tissues and proliferate indefinitely in vitro.

[0004] In 1981, Martin Evans and Matthew Kaufman of the University of Cambridge were the first to successfully isolate the first line of pluripotent mouse embryonic stem cells (mESCs) from mouse embryos, igniting a boom in embryonic stem cell research. In 2006, Shinya Yamanaka used a viral vector to transfect a combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) into somatic cells, thereby reprogramming highly differentiated somatic cells into pluripotent induced pluripotent stem cells (iPSCs), thus winning the Nobel Prize in 2012. In the four decades since pluripotent mESCs were established, all cultured mESCs have been in a pluripotent state. Attempts have been constantly made to obtain stem cells in vitro with higher developmental potential. However, to date, the in vitro induction and long-term culture of totipotent stem cells that resemble totipotent embryonic cells in vivo, both at the molecular level and in function, remains a major challenge. SHEN Hui et al. (Shen H, Yang M, Li S, et al. mouse totipotent stem cells captured and maintained through spliceosomal repression [J]. cells, 2021, 184(11): 2843-2859. e20) reported that suppressing the spliceosome in mouse ESCs promotes the transition from pluripotency to totipotency. Using the splicing inhibitor pladienolide B, the study achieves a novel cell type that is comparable to 2- and 4-cell blastomeres at the transcriptome level and called totipotent blastomere-like cells (TBLCs). Mouse chimera assays combined with RNA sequencing (RNA-seq) demonstrate that TBLCs have robust bidirectional developmental capacity to generate multiple intraembryonic and extraembryonic cell lineages. Mechanistically, spliceosome suppression leads to widespread splicing inhibition of pluripotent genes, while totipotent genes (containing several short introns) are efficiently spliced ​​and transcriptionally activated. However, in this study, the splicing inhibition technique to convert pluripotent stem cells into stem cells with a given totipotency requires multiple subculture steps and takes a significant period of time. In addition, by analyzing the scRNA-seq data of TBLCs and normal mouse embryos obtained in this study, we found that TBLCs are closer to cells of postimplantation embryos at later developmental stages than totipotent zygotes and 2-cell stage blastomeres. Therefore, TBLCs cannot be considered totipotent cells. More importantly, TBLCs have not yet been shown to have the ability to develop independently into mouse embryos and thus generate whole organisms. TBLCs do not meet the strict definition of totipotent cells (which can develop independently into whole organisms), and therefore cannot be defined as totipotent stem cells.

[0005] It is generally recognized in the art that a cell having totipotency should meet one or more, preferably two, and more preferably all three of the following: 1) the cell resembles totipotent embryonic cells, i.e., zygotes and two-cell blastomeres, on the transcriptional level; 2) the cell further has the ability to develop bidirectionally into both intraembryonic and extraembryonic cell types; and 3) even further, and most specifically, a cell is capable of developing into an entire embryo or organism. There remains a need in the art to induce more desirable totipotent stem cells, especially in a more rapid and efficient manner.The resulting induced totipotent stem cells meet the above definition of totipotency. Summary of the Invention

[0006] The present inventors have surprisingly discovered that by using a combination of specific compounds as an additive to the basal medium for pluripotent stem cells, pluripotent stem cells can be induced to totipotent stem cells (referred to herein as ciTotiSCs, i.e., "chemically induced totipotent stem cells," "chemically induced totipotent stem cells," or simply "induced totipotent stem cells"), and that the induction is very rapid and effective. Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) RA signaling pathway activator; (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. A composition comprising:

[0007] In another aspect, the present invention provides a method for producing a composition comprising: (a) RA signaling pathway activator; (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. A kit comprising: In another aspect, the present invention provides the use of (a) an RA signaling pathway activator, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator in producing induced totipotent stem cells.

[0008] In one or more of some embodiments of the above aspects, the RA signaling pathway activator is selected from small molecules involved in the same pathway, such as TTNPB, trethionine / RA / ATRA, AM580, Taza, 9-cis-RA, acitretin, CD437, tamibarotene, tazarotene, retinoic acid, isotretinoin, acitretin sodium, ch55, and AC55649.

[0009] In one or more of some embodiments of the above aspects, the GSK-3 inhibitor is selected from small molecules involved in the same pathway, such as 1-azakempaullone, AZD2858, CHIR99021, and AZD1080. In one or more of some embodiments of the above aspects, the IKK signaling pathway inhibitor is selected from small molecules involved in the same pathway, such as WS6, sc-514, PF184, and IKK16. In one or more of some embodiments of the above aspects, the HDAC inhibitor is selected from small molecules involved in the same pathway, such as trichostatin A (TSA), valproic acid (VPA), vorinostat (SAHA), and entinostat (MS-275). In one or more of some embodiments of the above aspects, the histone methyltransferase inhibitor is selected from small molecules involved in the same pathway, such as BIX01294, 3-Deazaneplanocin A (DZNeP) HCl, A-366, UNC0638, SGC0946, and the like. In one or more of some embodiments of the above aspects, the Src kinase inhibitor is selected from small molecules involved in the same pathway, such as dasatinib (BMS-354825), WH-4-023, ponatinib (AP24534), bosutinib (SKI-606), and the like. In one or more of some embodiments of the above aspects, the cAMP activator is selected from small molecules involved in the same pathway, such as colforsin (forskolin, HL362) and 8-Br-cAMP. In one or more of some embodiments of the above aspects, the cellular metabolism modulator is selected from cellular metabolism modulators such as 2-deoxy-D-glucose (2-DG), sodium acetate, sodium L-lactate, and D-ribose.

[0010] In another aspect, the present invention provides a culture medium comprising the composition described herein. In one or more of certain embodiments of the above aspects, the culture medium comprises a basal culture medium. In one or more of some embodiments of the above aspects, the basal medium is selected from common basal media such as DMEM, knockout DMEM, RPMI1640, and DMEM / F12.

[0011] In another aspect, the present invention provides a method of producing an induced totipotent stem cell, the method comprising culturing pluripotent stem cells in a culture medium described herein, thereby producing an induced totipotent stem cell. In one or more of several embodiments of the above aspects, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In one or more of several embodiments of the above aspects, the method comprises reprogramming a non-pluripotent cell into a pluripotent stem cell. In one or more of several embodiments of the above aspects, the non-pluripotent cells are selected from somatic cells and / or adult stem cells. In one or more of some embodiments of the above aspects, the step of reprogramming the non-pluripotent cell into a pluripotent stem cell comprises expressing in the non-pluripotent cell one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, and c-Myc. In one or more of some embodiments of the above aspects, culturing the pluripotent stem cells is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0012] In another aspect, the invention provides a culture comprising the culture medium described herein and pluripotent stem cells. In one or more of several embodiments of the above aspects, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In another aspect, the invention provides a culture comprising the culture medium described herein and totipotent stem cells. In one or more of several embodiments of the above aspects, the totipotent stem cells are induced totipotent stem cells, preferably produced by the methods described herein.

[0013] In another aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) increased transcription of one or more totipotent transcriptional markers selected from the group consisting of MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763; (b) decreased transcription of one or more pluripotency transcriptional markers selected from the group consisting of POU5f1, ZFP42, NANOG, KLF4, ESRRB, and (c) the ability to differentiate into extraembryonic cell type(s). The present invention provides induced totipotent stem cells characterized by one or more of the following: Preferably, the induced totipotent stem cells are producible by the methods described herein.

[0014] In another aspect, the present invention provides an induced totipotent stem cell producible by a method according to any one of the preceding claims. In another aspect, the invention provides an organism derived from the induced totipotent stem cells described herein, which is preferably a rodent or a mammal. In another aspect, the present invention provides organoids generated from the induced totipotent stem cells described herein. In another aspect, the present invention provides a tissue generated from the induced totipotent stem cells described herein, preferably blood. In another aspect, the invention provides a differentiated cell differentiated from an induced totipotent stem cell as described herein, the differentiated cell being preferably a blood cell or an immune cell, such as a T cell or a NK cell. [Brief description of the drawings]

[0015] [Figure 1-1] FIG. 1 shows the subculture of mouse induced totipotent stem cells (ciTotiSCs) of the present invention. [Figure 1-2] FIG. 1 shows highly expressed totipotency marker genes of mouse induced totipotent stem cells (ciTotiSCs) of the present invention. [Figure 2-1] Specific gene set enrichment analysis (GSEA) (upper panel) in mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) of the present invention. Mouse induced totipotent stem cells (ciTotiSC) of the present invention have higher expression levels of maternal genes, ZGA genes, and totipotency genes, and lower expression levels of pluripotency-specific genes, compared to mouse pluripotent embryonic stem cells (mESC), totipotent blastomere-like cells (TBLC), totipotent-like cells (TLSC), and expanded potential stem cells (EPSC) (lower panel). [Figure 2-2] FIG. 1 shows clustering analysis at the whole transcriptome level of mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs) of the present invention. [Figure 2-3]FIG. 1 shows a principal component analysis (PCA) at the whole transcriptome level of mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs) of the present invention. [Figure 2-4] FIG. 1 shows gene set enrichment analysis (GSEA) of different stages of embryonic development in mouse induced totipotent stem cells (ciTotiSCs), mouse pluripotent embryonic stem cells (mESCs), and totipotent blastomere-like cells (TBLCs) of the present invention, as well as in normal mouse embryos. [Figure 2-5] FIG. 1 shows single-cell RNA sequencing (scRNA-seq) UMAP analysis performed on mouse induced totipotent stem cells (ciTotiSCs) of the present invention, and mouse pluripotent embryonic stem cells (ESCs), and totipotent blastomere-like cells (TBLCs), as well as normal mouse embryos at various stages. [Figure 2-6] FIG. 1 shows Transposase-accessible chromatin sequencing (ATAC-seq) analysis of mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs) of the present invention. [Diagram 2-7] FIG. 1 shows site-specific transposase-accessible chromatin sequencing (ATAC-seq) analysis of mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs) of the present invention. [Figure 2-8] FIG. 1 shows RRBS analysis of genomic methylation levels in mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs). [Figure 2-9] FIG. 1 shows principal component analysis of global methylation for mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs) based on RRBS data. [Figure 2-10] FIG. 1 shows an analysis of methylation levels near specific sites in the genome of mouse induced totipotent stem cells (ciTotiSCs) and mouse pluripotent embryonic stem cells (mESCs). [Figure 2-11] FIG. 1 shows metabolomic analysis of mouse induced totipotent stem cells (ciTotiSCs). [Figure 3-1] FIG. 1 shows a schematic diagram of the experimental process of trophectoderm stem cell differentiation. [Figure 3-2] FIG. 1 shows detection of transcription of mouse trophectoderm stem cell-specific genes in mouse induced totipotent stem cells (ciTotiSCs), mouse embryonic stem cells (mESCs), and mouse potential-expanded pluripotent stem cells (mEPSCs) by RT-qPCR. [Figure 3-3] FIG. 1 shows detection of mouse trophectoderm stem cell-specific protein expression in mouse induced totipotent stem cells (ciTotiSCs), mouse embryonic stem cells (mESCs), and mouse potential-expanded pluripotent stem cells (mEPSCs) by fluorescent immunostaining. [Diagram 3-4] FIG. 13 shows the analysis of the expression of totipotency and pluripotency genes in mouse induced totipotent stem cells (ciTotiSCs) at different passages (P1-P8) after switching to mESC medium (2i / LIF). [Figure 4-1] FIG. 1 shows fluorescent immunostaining analysis of embryoid bodies (EBs) derived from mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs). [Figure 4-2] FIG. 13 shows statistics regarding the percentage of all CDX2 positive cells detected within embryoid bodies. [Figure 4-3] FIG. 1 shows an analysis of the three germ layer differentiation potential of teratomas derived from mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs). [Figure 4-4] FIG. 1 shows the analysis of the teratoma extraembryonic lineage differentiation potential of mouse induced totipotent stem cells (ciTotiSCs). [Figure 5-1] FIG. 1 shows a schematic diagram of the chimeric assay process. [Figure 5-2] FIG. 1 shows the chimeric potential of mouse totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs) at E4.5. [Figure 5-3] FIG. 1 shows statistics of chimerism ratio in mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs) within the inner cell mass (ICM) and trophectoderm (TE). [Figure 5-4] FIG. 1 shows confirmation of classical marker staining of chimeric embryonic trophectoderm (TE) in mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs). [Figure 6-1] FIG. 1 shows fluorescent immunostaining analysis in chimeric embryos consisting of mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs) developed to E7.5. [Figure 7-1] FIG. 1 shows chimera status analysis in chimeric embryos consisting of mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs) developed to E12.5. [Figure 7-2] FIG. 1 shows flow cytometry analysis of chimerism ratios for mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs) in each tissue at E12.5. [Figure 7-3] FIG. 13 shows fluorescent immunostaining of E12.5 chimeric placenta cryosections of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) to analyze co-localization of chimeric cells with placental extraembryonic cell lineage markers CK8 and proliferin. [Figure 7-4] FIG. 1 shows the analysis of chimeric potential of mouse induced totipotent stem cells (ciTotiSCs) and mouse embryonic stem cells (mESCs) in the three endodermal germ layers (mesoderm, endoderm, ectoderm) at E12.5. [Figure 8-1] FIG. 1 shows detection of developmental potential of single mouse induced totipotent stem cells (ciTotiSCs). [Figure 8-2] FIG. 1 shows an analysis of cell types formed by extra- and intra-embryonic chimerism at E12.5 in mouse induced totipotent stem cells (ciTotiSCs). [Figure 8-3] FIG. 1 shows that mouse induced totipotent stem cells (ciTotiSCs) have the ability to chimerize into the genital ridges and generate healthy chimeric offspring. [Figure 8-4] FIG. 1 shows induced blastocysts obtained from mouse induced totipotent stem cells (ciTotiSCs). [Figure 8-5] FIG. 1 shows that mouse blastocysts derived from mouse induced totipotent stem cells (ciTotiSCs) harbor the three cell lineages of normal blastocysts in vivo. [Figure 8-6] FIG. 1 shows that mouse blastocysts derived from mouse induced totipotent stem cells (ciTotiSCs) can develop after implantation in vitro. [Figure 8-7] FIG. 1 shows that mouse blastocysts derived from mouse induced totipotent stem cells (ciTotiSCs) are implanted into the mouse uterus and further developed in vivo. [Figure 9-1] FIG. 1 shows the role of Dux and p53 in the induction of totipotent stem cells (ciTotiSCs). [Figure 9-2] FIG. 1 shows the 2C:tdTomato+ cell ratio of mouse totipotent stem cells (ciTotiSCs) induced by various small molecule combinations. [Figure 9-3] FIG. 2C: tdTomato+ and OCT4 expression tests for various commonly used basal media used to induce and generate mouse totipotent stem cells (ciTotiSCs). [Figure 9-4] FIG. 1 shows the individual displacement of several small molecule reprogramming reagents and testing of their performance in mouse totipotent stem cell (ciTotiSC) derivation. [Figure 10] FIG. 1 shows the effect of various small molecules on gene expression in totipotent cell induction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] A. Overview Existing methods for the generation and maintenance of totipotent stem cells have not yet provided totipotent stem cells that meet one or more criteria of the accepted definition of totipotency, let alone totipotent stem cells for industrial or clinical applications. The present invention provides for the derivation of pluripotent stem cells to generate totipotent stem cells that meet one or more criteria of the accepted definition.

[0017] The totipotent stem cells induced and cultured by the present inventors 1) show similar transcriptome characteristics to those of mouse embryo zygotes and 2-cell stage blastomeres, and convert to similar levels of totipotency in vivo in terms of chromatin accessibility, DNA methylation level, and cell metabolic mode; 2) as demonstrated by in vitro direct differentiation of monolayer cells, differentiation of embryoid bodies in suspension, and in vivo teratoma differentiation assays, the totipotent stem cells induced and cultured by the present inventors have the ability to differentiate into extraembryonic cells that pluripotent stem cells do not possess. In addition, in vivo chimerism experiments also clearly support that the totipotent stem cells induced and cultured by the present inventors have the ability to develop into both intraembryonic and extraembryonic tissues with high efficiency and in both directions. 3) More importantly, the induced totipotent stem cells can be independently induced and developed into mouse blastocysts in vitro, and can correctly express mouse blastocyst marker genes. The induced blastocysts can exhibit a range of characteristics after embryo implantation when continuously cultured in vitro, and the induced blastocysts can also be transferred into the uterus for continued development after implantation into a mouse. Thus, the induced mouse totipotent stem cells have the capacity to develop independently into the native organism without going through the traditional sperm-oocyte binding process. Thus, the compositions and methods contemplated herein enable the generation of qualified totipotent stem cells suitable for industrial and clinical applications.

[0018] Unless otherwise specified, the practice of the present invention employs conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, cell biology, stem cell protocols, cell culture, and transgenic biology, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. See, for example, Sambrook, et al, Molecular Clocking: A Laboratory Manual (3 rdedition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2 ndEdition, 1989); Maniatis et al, Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Fire et al., RNA Interference Technology: From Basic Science to Drug Development (Cambridge University Press, Cambridge, 2005); Schepers, RNA Interference in Practice (Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology (DNA Press, 2003); Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology; Human Press, Totowa, NJ, 2004); Sohail, Gene Silencing by RNA Interference: Technology and Application (CRC, 2004); Clarke and Sanseau, microRNA: Biology, Function & Expression (Nuts & Bolts series; DNA Press, 2006); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. Blackwell, eds., 1986); Riott, Essential Immunology, 6. thEdition, (Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008); Hogan et al, Methods of Manipulating the Mouse Embryo (2. nd Edition, 1994); Nagy et al, Methods of Manipulating the Mouse Embryo (3 rd Edition, 2002), And The Zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4 th Ed., (University of Oregon Press, Eugene, OR, 2000). All publications, patents, and applications cited herein are hereby incorporated by reference in their entirety.

[0019] B. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined below.

[0020] As used herein, the articles "a," "an," and "the" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0021] The use of the alternative (eg, "or") should be understood to mean either, both, or any combination thereof. The term "and / or" should be understood to mean either or both of the alternatives. As used herein, the term "about" or "approximately" refers to a number, level, numerical value, amount, frequency, percentage, scale, size, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference number, level, numerical value, amount, frequency, percentage, scale, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" refers to a number, level, numerical value, amount, frequency, percentage, scale, size, amount, weight, or length range that is within ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference number, level, numerical value, amount, frequency, percentage, scale, size, amount, weight, or length.

[0022] As used herein, the terms "substantially" or "essentially" refer to a number, level, numerical value, quantity, frequency, percentage, scale, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more similar to a reference number, level, numerical value, quantity, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the term "substantially identical" refers to a number, level, numerical value, quantity, frequency, percentage, scale, size, amount, weight, or length range that is about the same as the reference number, level, numerical value, quantity, frequency, percentage, scale, size, amount, weight, or length range. As used herein, the term "substantially free of" when used to describe a composition, such as a cell population or culture medium, means a composition that does not contain the specified substance, e.g., does not contain 95%, 96%, 97%, 98%, 99%, etc. of the specified substance, or is undetectable when measured by conventional means. A similar meaning is applicable to the term "absent," which refers to the absence of a particular substance or component of a composition.

[0023] As used herein, the term "significant" refers to a number, level, value, amount, frequency, percentage, scale, size, quantity, weight, or length range that can be readily detected by one or more standard methods. The terms "unacceptable" and "non-significant" and equivalent expressions refer to a number, level, value, amount, frequency, percentage, scale, size, quantity, weight, or length range that cannot be readily detected or is undetectable by standard methods. In one embodiment, an event is not significant if it occurs at a frequency of less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, or less. Throughout this specification, unless otherwise required by context, the terms "comprising," "including," "containing," and "having" should be understood to imply the inclusion of a stated step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements. In certain embodiments, the terms "comprising," "including," and "containing" are used interchangeably.

[0024] The phrase "consisting of" means inclusive of and limited to any elements that follow the phrase "consisting of." Thus, the phrase "consisting of" implies that the listed elements are required or mandatory, and that no other elements may be present. The phrase "consisting essentially of" is meant to include any of the elements recited following the phrase "consisting essentially of," and is limited to those other elements that do not interfere with or contribute to the activity or action identified in this disclosure of the recited elements. Thus, the phrase "consisting essentially of" suggests that the recited elements are required or essential, but that other elements are not optional and may or may not be present depending on their effect on the activity or action of the recited elements.

[0025] Reference throughout this specification to "one embodiment," "one embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various parts throughout the specification do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0026] The term "ex vivo" generally refers to activities performed outside the body of an organism, such as experiments or measurements performed in / on living tissue in an artificial environment outside the body of an organism, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures involve live cells or tissues obtained from an organism and cultured, usually under sterile conditions, in laboratory equipment, typically for a few hours or up to about 24 hours (but including up to 48 or 72 hours, depending on the circumstances). In some embodiments, such tissues or cells can be collected and frozen, and then thawed for ex vivo processing. Although tissue culture experiments or procedures using live cells or tissues for more than a few days are generally considered to be "in vitro", in certain embodiments, the term can be used interchangeably with "ex vivo". The term "in vivo" generally refers to activities performed within a living organism.

[0027] As used herein, the terms "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refer to a method or process of increasing cell potential or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potential has greater developmental plasticity (i.e., can differentiate into more cell types) than the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state. In some embodiments, reprogramming includes reprogramming a pluripotent stem cell to a totipotent stem cell. In some embodiments, reprogramming includes reprogramming a non-pluripotent stem cell to a pluripotent stem cell. In some embodiments, reprogramming includes reprogramming a non-pluripotent stem cell to a pluripotent stem cell.

[0028] As used herein, the term "potency" refers to the sum of all developmental options available to a cell (i.e., developmental capacity). Those skilled in the art will recognize that cell potential is a continuum ranging from the most plastic cells (i.e., totipotent stem cells, which have the highest developmental capacity) to the least plastic cells (i.e., terminally differentiated cells, which have the lowest developmental capacity). The continuum of cell potential includes, but is not limited to, totipotent cells, pluripotent or multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells. As used herein, the term "pluripotency" refers to the ability of a cell to generate all lineages of an organism or organism (i.e., embryoid bodies). For example, embryonic stem cells are a class of pluripotent stem cells that have the ability to form cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0029] As used herein, the term "totipotent" refers to a cell that meets one or more, preferably two, and more preferably all three of the following criteria: 1) the cell resembles, with respect to the transcriptional level, a totipotent embryonic cell, i.e., a zygote and a two-cell blastomere; 2) the cell further has the ability to develop bidirectionally into both intraembryonic and extraembryonic cell types; and 3) even further and most specifically, a cell that can develop into a complete embryo or viable individual. For example, as demonstrated by the present invention, the induced totipotent stem cells of the present invention 1) exhibit similar transcriptome characteristics to those of mouse embryo zygotes and two-cell blastomeres, and are converted in vivo to levels similar to those of totipotent cells in terms of chromatin accessibility, DNA methylation level, and cell metabolic mode; 2) As demonstrated by in vitro direct differentiation of monolayer cells, differentiation of embryoid bodies in suspension, and teratoma differentiation in vivo assays, the totipotent stem cells induced and cultured by the present inventors have the ability to differentiate into extraembryonic cells that pluripotent stem cells do not possess. In addition, in vivo chimerism experiments also clearly support that the totipotent stem cells induced and cultured by the present inventors have the ability to develop into both intraembryonic and extraembryonic tissues in a highly efficient and bidirectional manner. 3) More importantly, the induced totipotent stem cells can be independently induced in vitro and developed into mouse blastocysts, and can correctly express mouse blastocyst marker genes. The induced blastocysts can exhibit a range of characteristics after embryo implantation when continuously cultured in vitro, and the induced blastocysts can also be transferred into the uterus for continued development after implantation into a mouse. Thus, the induced mouse totipotent stem cells have the capacity to develop independently into the native organism without going through the traditional sperm-oocyte binding process.

[0030] Totipotency can be determined in part by evaluating the totipotency characteristics of cells. Totipotency characteristics include, but are not limited to, (1) morphology of totipotent stem cells; (2) increased transcription of totipotency transcription markers, such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763, etc.; (3) decreased transcription of pluripotency transcription markers, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, etc.; (4) ability to differentiate into embryonic cell types; (5) ability to differentiate into extraembryonic cell types; and (6) ability to develop into independent individuals. The induced totipotent stem cells of the present invention can be characterized by one or more of these characteristics. Such totipotent characteristics of the induced totipotent stem cells of the present invention can be compared to natural or induced totipotent stem cells, and / or natural or induced pluripotent stem cells, for example, in comparison with natural totipotent stem cells of zygotes and / or two-cell blastomeres, and / or embryonic stem cells or induced pluripotent stem cells, or in comparison with control totipotent stem cells and / or pluripotent stem cells without using identical culture conditions.

[0031] In certain embodiments, the increase or decrease in transcription in a transcriptional marker caused by a given culture condition may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000% or more increase or decrease compared to an appropriate control.

[0032] As used herein, "gene expression" or "gene transcription" refers to the relative levels and / or expression / transcription patterns of gene expression / transcription in a biological sample, such as a totipotent cell or a cell population comprising a totipotent cell. In certain embodiments, the totipotent cell is an induced totipotent stem cell.

[0033] The present disclosure encompasses any method available in the art for detecting the expression / transcription of genes that characterize the cells of the present invention. As used herein, the term "detecting expression / transcription" refers to determining the amount or presence of the RNA transcript of a gene or its expression product. Methods for detecting gene expression / transcription, i.e., gene expression / transcription profile assay methods, include polynucleotide-based hybridization analysis, polynucleotide-based sequencing, immunohistochemistry, and proteomics-based methods. These methods generally detect the expression / transcription product (e.g., mRNA) of a gene of interest. In some embodiments, PCR-based methods, such as reverse transcription PCR (RT-PCR) methods (Weis et al., TIG8:263-64, 1992) and array-based methods, such as microarray methods (Schena et al., Science 270:467-70, 1995), are used. "Adherence" refers to the attachment of cells to a container in the presence of an appropriate culture medium, e.g., a sterile plastic (or coated plastic) cell culture dish or flask. Certain types of cells cannot be maintained or grown in culture unless they are attached to the cell culture container. Certain types of cells ("non-adherent cells") can be maintained and / or grown in culture without being attached to a wall.

[0034] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium", "culture media", "medium", "supplement", and "culture medium supplement" refer to nutritional compositions for culturing cell cultures. "Culture" or "cell culture" refers to the material being cultured, such as cells, and / or the culture medium containing the material being cultured, such as cells. "Culturing" refers to maintaining, replicating (growing), and / or differentiating cells outside a tissue or body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" can use culture medium as a source of nutrients, hormones, and / or other factors that promote cell replication and / or maintenance.

[0035] As used herein, "dissociated" cells refer to cells that are substantially separated or purified from other cells or surfaces (e.g., the surface of a culture plate). For example, cells may be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro may be enzymatically or mechanically dissociated from each other, for example, by dissociating into a suspension of clusters, single cells, or a mixture of single cells and clusters. In another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation may involve disrupting the interaction of cells with the extracellular matrix (ECM) and substrate (e.g., culture surface), or disrupting the ECM between cells.

[0036] As used herein, the term "enriched for" refers to increasing the amount of a given component in a composition, such as a cell composition, and when used to describe a composition, such as a cell population, "enriched" refers to a cell population having a proportionally increased amount of a given component compared to the proportion of such component in the cell population prior to enrichment. For example, a composition, such as a cell population, can be enriched for a target cell type (i.e., cells having a given characteristic), thus increasing the proportion or percentage of the target cell type compared to the proportion of target cells present in the cell population prior to enrichment. The cell population can be enriched for the target cell type by cell selection and sorting methods known in the art. In some embodiments, the cell population is enriched by a sorting or selection method. In particular embodiments, the method of enriching for a target cell population enriches the cell population for the target cell population by at least about 20%, meaning that the enriched cell population contains about 20% more of the target cell type proportionally than the cell population prior to enrichment. In one embodiment, the method of enrichment for the target cell population results in a cell population that is proportionally enriched for the target cell population by at least about 30+%, 40+%, 50+%, 60+%, 70+%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or at least about 98%, or in a particular embodiment, about 99%.

[0037] In certain embodiments, the cell population is enriched for the amount of totipotent cells or cells exhibiting characteristics of totipotency. In particular embodiments of the invention, the cell population that has undergone reprogramming is enriched for target cells that have characteristics of totipotency, such as expression of totipotency markers, including, but not limited to, MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763.

[0038] In certain embodiments, the enriched cells comprise a distinct gene or protein expression profile, such as cell surface expression of one or more totipotency markers, such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763, etc. In some embodiments, in one embodiment, the cell population comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% enriched cells, such as totipotent cells. Thus, in some embodiments, a method for enriching a cell population for totipotent cells comprises sorting the cell population based on cell surface expression of a totipotency marker, such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763, etc., and collecting the cell fraction expressing such markers to obtain a cell population enriched for totipotent cells. In other embodiments, the cell population is sorted based on cell surface expression of a pluripotency cell marker, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, etc., and depleting the cell population of such cells to obtain a cell population enriched for totipotent cells, thus enriching the cell population for totipotent cells.

[0039] As used herein, "feeder cells" or "feeders" are used to describe a type of cell that is co-cultured with a second type of cell and provides an environment in which the second type of cell can grow (because the feeder cells provide growth factors and nutrients to support the second type of cell). Feeder cells are optionally derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. When co-cultured with other cells, feeder cells can generally be inactivated by irradiation or treatment with antimitotic agents, such as mitomycin C, to prevent them from outgrowing the cells they support. Without being limited thereto, a particular feeder cell type can be a human feeder cell, such as a human dermal fibroblast. Another feeder cell type can be a mouse embryonic fibroblast (MEF). As used herein, a "feeder-free" (FF) environment refers to an environment that is substantially free of feeder cells and / or is not preconditioned by a feeder cell culture step, e.g., cell culture or culture medium. A "preconditioned" medium refers to medium that is harvested after feeder cells have been cultured in the medium for a period of time, e.g., at least one day. Preconditioned medium contains many media substances, including growth factors and cytokines secreted by feeder cells cultured in the medium.

[0040] Genomic stability refers to the ability of cells to faithfully replicate DNA and maintain the integrity of the DNA replication process.As used herein, the terms "genomically stable cell" and "cell with genomic stability" refer to cells that exhibit a certain frequency of mutations and chromosomal abnormalities (e.g., translocations, chromosomal aneuploidies, copy number variations, and duplications) that is substantially similar to the frequency of mutations and chromosomal abnormalities when compared with normal human cells.

[0041] "Component" refers to any compound or other substance, whether chemical or biological in origin, that can be used in cell culture media to maintain and / or promote cell growth and / or differentiation. The terms "component", "nutrient" and "ingredients" are used interchangeably. Conventional components for cell culture media can include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, etc. Other components for promoting and / or maintaining cell culture ex vivo or in vitro can be selected by the skilled artisan based on the requirements for the desired effect.

[0042] "Isolated" refers to the separation and collection of a composition or substance from its natural environment, such as the separation of individual cells or cell cultures from a tissue or organism. In one aspect, a cell population or composition is substantially free of cells and substances with which it is naturally associated. With respect to target cells contained in a whole cell population, "isolated" or "purified" or "substantially pure" means that the cell population is at least about 50%, at least about 75%, at least about 85%, at least about 90% pure, and in certain embodiments, at least about 95% pure. The purity of a cell population or composition can be assessed by suitable methods known in the art. For example, a substantially pure totipotent cell population means that with respect to totipotent cells contained in a whole cell population, the cell population is at least about 50%, at least about 75%, at least about 85%, at least about 90% pure, and in certain embodiments, at least about 95%, and in certain embodiments, at least about 98% pure.

[0043] "Passaging" refers to the act of subdividing cells when they have proliferated to a desired level and spreading them across multiple cell culture surfaces or vessels. In some embodiments, "passaging" refers to subdivision, dilution, and seeding of cells. When cells are passed from a primary culture surface or vessel to a subsequent population of surfaces or vessels, the subsequent culture may be referred to herein as a "subculture" or "first passage," or the like. Each act of subdividing and seeding into a new culture vessel is considered a passaging. "Seeding" refers to placing one or more cells into a culture vessel such that the cells attach to and spread throughout the cell culture vessel.

[0044] "Pluripotency factor" refers to a reagent that can increase the ability of a cell to develop to a pluripotent level, either alone or in combination with other reagents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the ability of a cell to develop to a pluripotent level. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents. "Totipotency factor" refers to a reagent that can increase the ability of a cell to develop to a degree of totipotency, either alone or in combination with other reagents. Totipotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the ability of a cell to develop to a degree of totipotency. Exemplary totipotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0045] "Proliferation" refers to the quality of a cell dividing into two substantially equal cells or increasing in number (eg, in the case of replication) of a population of cells. "Reproduction" refers to the growth of cells (e.g., reproduction via cell proliferation) outside a tissue or body, e.g., in a sterile container, such as a plastic (or coated plastic) cell culture dish or flask. "Primary culture" refers to a cell, tissue, and / or culture in which isolated cells are disposed in a first culture vessel having a culture medium. However, as long as the cell, tissue, and / or culture remains in the first vessel, the cell, tissue, and / or culture can be maintained and / or propagated. The cell, tissue, and / or culture is called a primary culture.

[0046] The terms "small molecule reprogramming agents" or "small molecule reprogramming compounds" are used interchangeably herein and refer to small molecules that can increase the developmental potential of cells, either alone or in combination with other factors. Small molecules include, but are not limited to, nucleic acids, peptidomimetics, peptoids, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be used as small molecule sources in certain embodiments.

[0047] C. Cell In particular embodiments, one or more cells may be cultured, dissociated, and passaged using the compositions and methods discussed herein. In one embodiment, a single cell is cultured, dissociated, and passaged using the compositions and methods discussed herein. In another embodiment, a cell population or a plurality of cells is cultured, dissociated, and passaged using the compositions and methods discussed herein. The starting cells suitable for use in particular embodiments can be derived from virtually any suitable source and can be heterogeneous or homogeneous with respect to cell type or totipotency state.Such suitable cells include fetal cells and adult cells.In addition, such suitable cells can be derived from mammalian origin, such as rodent, feline, canine, porcine, caprine, ovine, equine, bovine, or primate, such as human.In one embodiment, the cells are human cells.

[0048] The cells may be a mixed-species population of cells including somatic cells, non-pluripotent, incompletely or partially pluripotent stem cells, pluripotent cells, oligopotent cells, unipotent cells, terminally differentiated cells, or any combination of the above. Pluripotent cells suitable for use in particular embodiments include, but are not limited to, naturally occurring stem cells, embryonic stem cells, or iPSCs. A "mixed-species" population of cells is a collection of cells with different degrees of developmental potential. For example, a mixed-species population of cells may include cells that have undergone reprogramming such that the mixed-species population includes pluripotent cells, partially pluripotent cells, and non-pluripotent cells, such as fully differentiated cells, such as somatic cells. In some embodiments, pluripotent stem cells are used to derive the totipotent stem cells described herein. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are reprogrammed from non-pluripotent stem cells. In some embodiments, the non-pluripotent stem cells are selected from the group consisting of somatic cells and / or adult stem cells. In some embodiments, the step of reprogramming the non-pluripotent cell into a pluripotent stem cell comprises expressing in the non-pluripotent cell one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, and c-Myc.

[0049] In one embodiment, the starting cell population is selected from the group consisting of adult or neonatal stem / progenitor cells, hi a particular embodiment, the starting stem / progenitor cell population is selected from the group consisting of mesodermal stem / progenitor cells, endodermal stem / progenitor cells, and ectodermal stem / progenitor cells. Illustrative examples of mesodermal stem / progenitor cells include, but are not limited to, mesodermal stem / progenitor cells, endothelial stem / progenitor cells, bone marrow stem / progenitor cells, umbilical cord stem / progenitor cells, adipose tissue derived stem / progenitor cells, hematopoietic stem / progenitor cells (HSC), mesenchymal stem / progenitor cells, muscle stem / progenitor cells, renal stem / progenitor cells, osteoblast stem / progenitor cells, chondrocyte stem / progenitor cells, and the like. Illustrative examples of ectodermal stem / progenitor cells include, but are not limited to, neural stem / progenitor cells, retinal stem / progenitor cells, skin stem / progenitor cells, and the like. Illustrative examples of endodermal stem / progenitor cells include, but are not limited to, hepatic stem / progenitor cells, pancreatic stem / progenitor cells, epithelial stem / progenitor cells, and the like. In certain embodiments, the starting cell population may be a heterogeneous or homogeneous cell population selected from the group consisting of islet cells, CNS cells, PNS cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hematopoietic cells, bone cells, liver cells, adipocytes, kidney cells, lung cells, chondrocytes, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, endothelial cells, and the like.

[0050] D. Culture platforms that can be used to induce totipotency and derive totipotent stem cells There is an increasing demand for the generation of high quality totipotent cells in cell banking, disease modeling, and cell therapy applications. The present invention provides a culture platform that can be used to induce totipotency and derive totipotent stem cells using specific small molecule reprogramming agents. In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) RA signaling pathway activators, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. A composition comprising:

[0051] In another aspect, the present invention provides a method for producing a composition comprising: (a) RA signaling pathway activators, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. A kit comprising: In yet another aspect, the present invention provides (a) an RA signaling pathway activator, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator for use in generating induced pluripotent stem cells.

[0052] 1. RA signaling pathway activators The RA (retinoic acid) signaling pathway activator can be a variety of agents capable of activating the RA pathway. Exemplary RA signaling pathway activators include, but are not limited to, TTNPB, trethionine / RA / ATRA, AM580, Taza, 9-cis-RA, acitretin, CD437, tamibarotene, tazarotene, retinoic acid, isotretinoin, acitretin sodium, ch55, and AC55649. In some embodiments, the RA signaling pathway activator is selected from the group consisting of TTNPB, trethionine / RA / ATRA, AM580, Taza, 9-cis-RA, acitretin, CD437, tamibarotene, tazarotene, retinoic acid, isotretinoin, acitretin sodium, ch55, and AC55649. Preferably, in some embodiments, the RA signaling pathway activator is represented by the following formula:

[0053] [ka] The TTNPB is as shown in Figure 1. 2.GSK-3 inhibitors The GSK-3 (glycogen synthase kinase-3) inhibitor can be a variety of drugs capable of inhibiting GSK-3. GSK-3 inhibitors include, but are not limited to, 1-azakempaullone, AZD2858, CHIR99021, and AZD1080. In some embodiments, the GSK-3 inhibitor is selected from the group consisting of 1-azakempaullone, AZD2858, CHIR99021, and AZD1080. Preferably, in some embodiments, the GSK-3 inhibitor is represented by the following formula: [ka] The compound is 1-azakempauron, as shown in

[0054] 3.IKK signaling pathway inhibitors The IKK (IκB kinase / NF-κB, IKK / NF-κB) signaling pathway inhibitor can be a variety of agents capable of inhibiting the IKK signaling pathway. Exemplary IKK signaling pathway inhibitors include, but are not limited to, WS6, sc-514, PF184, and IKK16. In some embodiments, the IKK signaling pathway inhibitor is selected from the group consisting of WS6, sc-514, PF184, and IKK16. Preferably, in some embodiments, the IKK signaling pathway inhibitor has the following formula: [ka] The WS6 is shown in Figure 1.

[0055] 4.HDAC inhibitors The HDAC (histone deacetylase) inhibitor can be various drugs that have the ability to inhibit HDAC. Exemplary HDAC inhibitors include, but are not limited to, trichostatin A (TSA), valproic acid (VPA), vorinostat (SAHA), and entinostat (MS-275). In some embodiments, the HDAC inhibitor is selected from the group consisting of trichostatin A (TSA), valproic acid (VPA), vorinostat (SAHA), and entinostat (MS-275).

[0056] 5. Histone methyltransferase inhibitors The histone methyltransferase inhibitor can be a wide variety of drugs that have the ability to inhibit histone methyltransferase.Exemplary histone methyltransferase inhibitors include, but are not limited to, BIX01294, 3-deazaneplanocin A (DZNeP) HCl, A-366, UNC0638, and SGC0946.In some embodiments, the histone methyltransferase inhibitor is selected from the group consisting of BIX01294, 3-deazaneplanocin A (DZNeP) HCl, A-366, UNC0638, and SGC0946.

[0057] 6.Src kinase inhibitors The Src kinase inhibitor can be various drugs that have the ability to inhibit Src.Exemplary Src kinase inhibitors include, but are not limited to, dasatinib (BMS-354825), WH-4-023, ponatinib (AP24534), bosutinib (SKI-606).In some embodiments, the Src kinase inhibitor is selected from the group consisting of dasatinib (BMS-354825), WH-4-023, ponatinib (AP24534), bosutinib (SKI-606).

[0058] 7. cAMP Activators The cAMP activator can be a wide variety of agents capable of activating cAMP. Exemplary cAMP activators include, but are not limited to, olforsin (forskolin, HL362) and 8-Br-cAMP. In some embodiments, the cAMP activator is selected from the group consisting of olforsin (forskolin, HL362) and 8-Br-cAMP.

[0059] 8. Cellular metabolism modulators The cell metabolism modulator can be a wide variety of agents capable of regulating cell metabolism. Exemplary cell metabolism modulators include, but are not limited to, 2-deoxy-D-glucose (2-DG), sodium acetate, L-sodium lactate, and D-ribose. In some embodiments, the cell metabolism modulator is selected from the group consisting of 2-deoxy-D-glucose (2-DG), sodium acetate, L-sodium lactate, and D-ribose.

[0060] 9. Component quantities The amount of small molecule reprogramming agent in the compositions, kits, media, or cultures of the invention can vary and can be optimized based on the particular culture conditions, including the given molecules and combinations used, the type of cells cultured in the media, and the given application. In one embodiment, the small molecule reprogramming agent is present in the compositions, kits, cultures of the invention at a concentration sufficient to induce totipotency, improve reprogramming efficiency, increase or maintain the potency of cells, or induce or maintain basal state totipotency. In some embodiments, the RA signaling pathway activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the RA signaling pathway activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the RA signaling pathway activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. , 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above values. Preferably, the RA signaling pathway activator is present in the composition, kit, medium, or culture of the present invention at a concentration of 0.05 to 5 μM, preferably 0.1 to 1 μM, more preferably 0.2 μM. Most preferably, in one embodiment, TTNPB is present in the composition, kit, medium, or culture of the present invention at a concentration of 0.2 μM.

[0061] In some embodiments, the GSK-3 inhibitor is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the GSK-3 inhibitor is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the GSK-3 inhibitor is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. The GSK-3 inhibitor is present in the composition, kit, medium, or culture of the present invention at a concentration of 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above values. Preferably, the GSK-3 inhibitor is present in the composition, kit, medium, or culture of the present invention at a concentration of 0.5 to 10.0 μM, preferably 2.0 to 3.0 μM, more preferably 2.5 μM. Most preferably, in one embodiment, 1-azakempaullone is present in the composition, kit, medium, or culture of the present invention at a concentration of 2.5 μM.

[0062] In some embodiments, an IKK signaling pathway inhibitor is present in a composition, kit, medium, or culture of the invention in an amount or concentration sufficient to induce pluripotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the IKK signaling pathway inhibitor is present in an amount or concentration sufficient to induce pluripotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the IKK signaling pathway inhibitor is present in an amount or concentration sufficient to induce pluripotency, alone or in combination with other small molecule reprogramming agents. Preferably, the IKK signaling pathway inhibitor is present in the composition, kit, medium, or culture of the present invention at a concentration of 0.1 to 10.0 μM, preferably 0.3 to 1 μM, more preferably 0.5 μM. Most preferably, in one embodiment, WS6 is present in the composition, kit, medium, or culture of the present invention at a concentration of 0.5 μM.

[0063] In some embodiments, the HDAC inhibitor is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the HDAC inhibitor is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the HDAC inhibitor is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. The antibody is present in a composition, kit, medium, or culture of the present invention at a concentration of 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above numerical values.

[0064] In some embodiments, a histone methyltransferase inhibitor is present in a composition, kit, medium, or culture of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the ... concentration of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5. The antibody is present in a composition, kit, medium, or culture of the present invention at a concentration of 0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above numerical values.

[0065] In some embodiments, a Src kinase inhibitor is present in a composition, kit, medium, or culture of the invention in an amount or concentration sufficient to induce pluripotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the ... concentration of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2 , 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above numerical values.

[0066] In some embodiments, the cAMP activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the cAMP activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the cAMP activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the cAMP activator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. The antibody is present in a composition, kit, medium, or culture of the present invention at a concentration of 2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above numerical values.

[0067] In some embodiments, the cellular metabolism modulator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the cellular metabolism modulator is present in the compositions, kits, media, or cultures of the invention in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the cellular metabolism modulator is present in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. In some embodiments, the cellular metabolism modulator is present in an amount or concentration sufficient to induce totipotency, alone or in combination with other small molecule reprogramming agents. The antibody is present in a composition, kit, medium, or culture of the present invention at a concentration of 2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 μM or more, or a range consisting of any two of the above numerical values. In particular embodiments, preferred combinations of small molecule reprogramming reagents in the compositions, kits, media, or cultures of the present invention are listed in Table 1.

[0068] [Table 1]

[0069] Preferably, in one embodiment, the composition, kit or use according to the invention comprises, or preferably consists of, a RA signalling pathway activator, a GSK-3 inhibitor and an IKK signalling pathway inhibitor. More preferably, in one embodiment, the RA signaling pathway activator is TTNPB, the GSK-3 inhibitor is 1-azakempaullone, and the IKK signaling pathway inhibitor is WS6. Most preferably, in one embodiment, the RA signaling pathway activator is TTNPB at 0.2 μM, the GSK-3 inhibitor is 1-azakempaullone at 2.5 μM, and the IKK signaling pathway inhibitor is WS6 at 0.5 μM. E. Culture medium

[0070] In one aspect, the present invention provides a culture medium comprising the composition described herein, the composition comprising (a) anRA signaling pathway activator, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, an Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. In some embodiments, the medium of the present invention comprises a basal medium. Exemplary basal media include, but are not limited to, DMEM, knockout DMEM, RPMI1640, and DMEM / F12. In some embodiments, the basal medium is selected from the group consisting of DMEM, knockout DMEM, RPMI1640, and DMEM / F12. In particular embodiments, the culture medium of the present invention comprises cytokines and / or growth factors. In particular embodiments, the culture medium of the present invention is substantially free or completely free of cytokines and / or growth factors. In certain embodiments, the culture medium comprises one or more supplements, including, but not limited to, serum, extracts, growth factors, hormones, cytokines, etc.

[0071] In an illustrative embodiment, the culture medium comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor (iKTGF-β), vascular endothelial growth factor (VEGF), transferrin, various interleukins (e.g., IL-1 through E-18, etc.), various colony stimulating factors, such as granulocyte / macrophage colony stimulating factor (GM-CSF), various interferons, such as IFN-γ, and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo). These cytokines are commercially available, for example from R&D Systems Minneapolis, Minn, and can be native or recombinant. In certain embodiments, growth factors and cytokines can be added at concentrations discussed herein for small molecule reprogramming agents.

[0072] Any suitable vessel or cell culture vessel can be used as a support for culturing cells in basal medium and / or cell culture supplements. Matrix coating on the support is not required. However, coating the surface of the culture vessel with an anchoring-promoting matrix (e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.) can promote cell attachment and, in particular embodiments, enhance the effect of the medium and supplements disclosed herein. Suitable matrices for culturing and passaging cells are known in the art and include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, mixtures of matrices produced by naturally occurring cell lines, such as Matrigel™, and synthetic or artificial surfaces, such as polyamine monolayers and carboxyl-terminated monolayers.

[0073] F. Cell generation method In one aspect, the present invention provides a method of generating induced totipotent stem cells, comprising culturing cells in a medium as described herein, thereby generating induced totipotent stem cells. In some embodiments, the culture medium comprises a composition as described herein. In some embodiments, the composition comprises one or more of (a) an RA signaling pathway activator, and (b) a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. The cells used as starting material for the methods of the invention can be a variety of cells as described herein. For example, the methods of the invention can start with pluripotent cells, such as pluripotent stem cells, such as induced totipotent stem cells, or the methods of the invention can start with non-pluripotent cells, such as non-pluripotent stem cells, such as somatic cells.

[0074] In some embodiments, the methods of the invention comprise culturing pluripotent stem cells in a medium as described herein, thereby generating induced totipotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, the methods of the invention comprise culturing non-pluripotent stem cells in a medium as described herein, thereby generating induced totipotent stem cells. In some embodiments, the method comprises reprogramming a non-pluripotent cell into a pluripotent stem cell. In some embodiments, the non-pluripotent cells are selected from the group consisting of somatic cells and / or adult stem cells. In some embodiments, the step of reprogramming the non-pluripotent cell into a pluripotent stem cell comprises expressing in the non-pluripotent cell one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, and c-Myc. In some embodiments, culturing the pluripotent stem cells continues for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0075] G. Culture In one aspect, the present invention provides a culture comprising a culture medium and pluripotent stem cells as described herein. In some embodiments, the culture medium comprises the composition as described herein. In some embodiments, the composition comprises (a) an activator of RA signaling pathway, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cell metabolism modulator.

[0076] The cells contained in the cultures described herein can be various cells as described herein. For example, the cells can be initial cells for culturing or derivation as described herein, such as pluripotent cells, such as pluripotent stem cells, such as iPSCs, or non-pluripotent cells, such as non-pluripotent stem cells, such as somatic cells. Alternatively, the cells can be intermediate or final cells cultured or derived as described herein. The intermediate cells can be cells with different developmental capabilities than the initial cells and final cells. The final cells can be totipotent stem cells as described herein. In some embodiments, a culture according to the invention comprises a culture medium and pluripotent stem cells as described herein. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, a culture according to the invention comprises a culture medium and totipotent stem cells as described herein. In some embodiments, the totipotent stem cells are induced totipotent stem cells. Preferably, in some embodiments, the totipotent stem cells are generateable by the methods described herein.

[0077] H. Totipotent Cells and Their Characterization Recently, in a research report published in the international journal Nature Cell Biology (Posfai, E. et al. Evaluating totipotency using criteria of increasing stringency. Nature Cell Biology 23, 49-60, doi:10.1038 / s41556-020-00609-2(2021)), scientists from research institutions such as Karolinska Institutet (Sweden) have defined a golden standard for distinguishing between pluripotent and totipotent stem cells. Research suggests that in nature, mammalian totipotent cells are only present in the early developmental embryo. Correspondingly, in mice, only the zygote and two-cell stage blastomeres have totipotency, which is gradually lost as the embryo develops. The strictest definition of totipotency means that a cell can develop into an entire embryo or organism. A broader definition of totipotency means that a cell has bidirectional developmental capabilities toward both intraembryonic and extraembryonic cell types. To date, scientists have created mouse stem cell lines in a pluripotent state. However, such cells can only develop into cells of embryonic components and do not have the ability to develop into extraembryonic cell types.

[0078] In this study, the researchers set criteria to assess whether cells are truly totipotent through combinatorial testing. Overall, the researchers identified four criteria for a totipotent mouse stem cell lineage: 1) the transcriptomic signature or gene expression profile of such cells should resemble that of early totipotent embryos more than that of later embryos; 2) such cells can differentiate in vitro into extraembryonic cell lineages and thus extraembryonic cell types; 3) such cells can form blast-like cells through guided development in vitro and can simulate some early embryonic developmental events; and 4) such cells can participate in intraembryonic and extraembryonic differentiation and differentiate into cell types that normally express the corresponding genetic markers when injected into early mouse embryos (also known as embryonic-extraembryonic chimerism). The researchers then tested two mouse stem cell lines previously reported as possessing totipotency potential (L-EPSC and D-EPSC) and evaluated them using this gold standard, finding that neither of the lines met the criteria for totipotent stem cells.

[0079] The totipotent stem cells induced and cultured by the present inventors 1) exhibit similar transcriptome characteristics to those of mouse embryo zygotes and 2-cell blastomeres, and are converted to similar levels of totipotent cells in vivo in terms of chromatin accessibility, DNA methylation level, and cell metabolic mode; 2) As demonstrated by in vitro direct differentiation of monolayer cells, differentiation of suspension embryoid bodies, and teratoma differentiation in vivo assays, the totipotent stem cells induced and cultured by the present inventors have the ability to differentiate into extraembryonic cells that pluripotent stem cells do not possess. In addition, in vivo chimerism experiments also clearly support that the totipotent stem cells induced and cultured by the present inventors have the ability to develop into both intraembryonic and extraembryonic tissues with high efficiency and in both directions. 3) More importantly, the induced totipotent stem cells can be independently induced in vitro and developed into mouse blastocysts, and can correctly express mouse blastocyst marker genes. The induced blastocysts can exhibit a range of characteristics after embryo implantation when continuously cultured in vitro, and the induced blastocysts can also be transferred into the uterus for continued development after implantation into a mouse. Thus, the induced mouse totipotent stem cells have the capacity to develop independently into the native organism without going through the traditional sperm-oocyte binding process.

[0080] In one aspect, the present invention provides induced totipotent stem cells producible by the methods described herein. In some embodiments, the methods include culturing cells in a culture medium described herein, thereby producing induced totipotent stem cells. In some embodiments, the culture medium comprises a composition described herein. In some embodiments, the composition comprises one or more of (a) an activator of the RA signaling pathway, and (b) a GSK-3 inhibitor, an inhibitor of the IKK signaling pathway, an HDAC inhibitor, a histone methyltransferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator. The induced totipotent stem cells generated using the culture platform described herein can be characterized in a variety of ways.

[0081] The transcription of classical marker genes and repeats of totipotency (e.g., MERVL, Zscan4, ZFP352, Tcstv3, and Gm6763) in the obtained induced totipotent stem cells can be detected by RT-qPCR reaction. Compared with early cells, such as embryonic stem cells (mESCs), the induced totipotent stem cells according to the present invention can show high expression of totipotency marker genes and repeats (e.g., MuERVL, Zscan4, ZFP352, Tstv3, and Gm6763), which means that the pluripotent embryonic stem cells undergo a cell fate transition to totipotent stem cells. In some embodiments, the expression level of one or more of the totipotency marker genes and repeats in the induced totipotent stem cells is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000-fold or more, or within a range consisting of any two of the foregoing values, compared to the initial cells.

[0082] To further explore the molecular biology characteristics of induced totipotent stem cells, transcriptome sequencing (RNA-seq) and single cell RNA sequencing (scRNA-seq) can also be used to analyze the changes in transcription levels in early cells, such as embryonic stem cells, after obtaining totipotency. This may include enrichment analysis (GSEA analysis) of totipotency marker genes and pluripotency marker genes in early cells, such as embryonic stem cells, and induced totipotent stem cells. It can be found that the specific expression of totipotency marker genes, such as in 2-cell stage embryos, is significantly enriched in induced totipotent stem cells according to the present invention, while pluripotency marker genes are significantly enriched in early cells, such as embryonic stem cells.

[0083] Cluster analysis can be used to analyze the similarity at the whole transcriptome level among early cells, such as embryonic stem cells, at various stages of embryonic development, and among the induced totipotent stem cells according to the present invention, which can be found to be close to the totipotent 1-cell and 2-cell embryos at the whole transcriptome level, and thus in a totipotent state at the transcriptional level. Through principal component analysis (PCA) of the whole transcriptome, it can be determined that the induced totipotent stem cells of the present invention may be developmentally intermediate between totipotent one-cell and two-cell embryos, while earlier cells, such as embryonic stem cells, are closer to the blastocyst at a later developmental stage. By analyzing the enrichment of gene sets in the induced totipotent stem cells according to the present invention, in comparison with early cells, such as embryonic stem cells, for specific expression at the zygote, 2-cell stage, 4-cell stage, 8-cell stage, and 16-cell stage of embryonic development, it can be seen that the totipotency marker gene sets that are specifically expressed at the zygote and 2-cell stage are significantly enriched in the induced totipotent stem cells according to the present invention.

[0084] UMAP analysis can be performed on single-cell RNA sequencing (scRNA-seq) results of the induced totipotent stem cells according to the present invention and control cells. The induced totipotent stem cells according to the present invention closely resemble two-cell embryos with totipotency, and can show high expression of totipotency marker genes and low expression of pluripotency marker genes, and have a totipotency transcriptome signature.

[0085] To explore the chromatin accessibility characteristics of the induced totipotent stem cells according to the present invention, the induced totipotent stem cells according to the present invention can be analyzed by Transposase-Accessible Chromatin using sequencing (ATAC-seq). Chromatin accessibility indicates gene activation or repression. The induced totipotent stem cells according to the present invention may show 5 kb closed and open peaks (open or closed state similar to 2-cell embryonic (2C) stage) near the transcription start site (TSS). In addition, compared with early cells, such as embryonic stem cells, the induced totipotent stem cells according to the present invention may show a higher degree of openness near some important totipotency genes and reverse transcription elements, such as Zscan4c, Zscan4d, Zscan4f, ZFP352, MERVL, etc. Meanwhile, classical pluripotency genes, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, etc., can be in a closed state, which means that the induced totipotent stem cells according to the present invention can have chromatin accessibility similar to that of a totipotent two-cell stage embryo.

[0086] In order to detect the genomic methylation level of the induced totipotent stem cells according to the present invention, reduced representation bisulfite sequencing (RRBS) can be used to characterize the DNA methylome of the induced totipotent stem cells according to the present invention. Through global methylation principal component analysis of the RRBS sequencing results, it can be understood that the induced totipotent stem cells according to the present invention may be closer to the one-cell and two-cell embryos with totipotency in terms of developmental stage than early cells, such as embryonic stem cells. Pluripotent stem cells do not have the ability to differentiate into extraembryonic cells. To verify the ability of induced totipotent stem cells according to the present invention to differentiate into extraembryonic cell types, commercially available trophoblast stem cells (TSCs) can be used as a positive control to detect the specific protein expression of trophectoderm stem cells (CDX2) by fluorescent immunostaining. CDX2 is a classical specific gene of mouse trophectoderm stem cells. By combining with the change of pluripotent stem cell marker Oct4 and detecting its protein expression level by immunofluorescence, it can be determined whether differentiation into trophectoderm stem cell lineage has occurred. Induced totipotent stem cells according to the present invention can be effectively induced to significantly express CDX2, while the expression of Oct4 is downregulated, and therefore have the ability to differentiate into extraembryonic portion, i.e., trophectoderm stem cells.

[0087] The transcription of trophectoderm stem cell-specific genes occurring during induction of induced totipotent stem cells according to the present invention into trophectoderm stem cells can be analyzed by RT-qPCR. The induced totipotent stem cells according to the present invention may gradually increase trophectoderm stem cell-specific marker genes, including but not limited to CDX2, Elf5, TFAP2C, and Esx1, during the induction process, which is similar to the trophoblast stem cells (TSCs) used as a positive control. CDX2 is a classical specific gene of mouse trophectoderm stem cells. The ability of the induced totipotent stem cells according to the present invention to differentiate into embryoid bodies can be determined by detecting CDX2 positive cells (representing extraembryonic cells and suggesting the differentiation of totipotent stem cells into extraembryonic cell types) via fluorescent immunostaining, thereby determining the ability of the induced totipotent stem cells according to the present invention to differentiate into embryoid bodies. The embryoid body differentiation experiment demonstrates that the induced totipotent stem cells according to the present invention have the ability to differentiate into extraembryonic portions, i.e., trophectoderm stem cells in vitro (but not pluripotent stem cells).

[0088] Teratoma assay is a classical experiment to test the ability of cells to randomly differentiate into the three germ layers and extraembryonic lineages. The ability of the induced totipotent stem cells according to the present invention to differentiate into teratomas can be determined by observing tissue sections under a microscope to find the specific tissue structures of the three germ layers and extraembryonic lineages. The teratoma differentiation experiment demonstrates that the induced totipotent stem cells according to the present invention have the ability to differentiate into extraembryonic parts in vitro, which pluripotent stem cells do not possess.

[0089] Chimeric embryo development experiments can be used to demonstrate that the induced totipotent stem cells according to the present invention have the developmental ability to differentiate into both intraembryonic and extraembryonic cell types. Blastocysts from Rosa26-tdTomato mice can be used to establish fluorescent cell lines of mouse induced totipotent stem cells or mouse embryonic stem cells (mESCs) for injection into 8-cell stage embryos to obtain stem cells stably expressing tdTomato fluorescence. When the stem cells injected into the embryo develop with the embryo and differentiate into intraembryonic and extraembryonic parts, fluorescence can be observed in the corresponding intraembryonic and extraembryonic parts. In in vivo development experiments of chimeric embryos using induced totipotent stem cells (e.g., development to E4.5), the induced totipotent stem cells according to the present invention can be embedded into both the inner cell mass (ICM) inside the embryo and the trophectoderm (TE) outside the embryo, as determined by the chimerism of tdTomato-positive cells. Differentiation into extraembryonic cell types can also be further confirmed by co-localization of tdtomato fluorescence and CDX2 fluorescence. In the chimeric embryo in vivo expression experiments of the induced totipotent stem cells according to the present invention (e.g., development to E4.5, E7.5, and E12.5), the co-localization of tdtomato fluorescence and CDX2 fluorescence can be observed. It was confirmed that the induced totipotent stem cells according to the present invention can participate in the expression of trophectoderm (TE) and have the ability to differentiate into extraembryonic parts.

[0090] For further development of the chimeric embryo (e.g., E7.5), the ability of the induced totipotent stem cells according to the present invention to differentiate into extraembryonic parts can also be confirmed by fluorescent immunostaining of Oct4 (classical marker of embryonic ectoderm (EPI)), ELF5 (classical marker of placental cone (EPC)), and extraembryonic ectoderm (ExE). The induced totipotent stem cells according to the present invention can be embedded in embryonic epiblast (EPI), extraembryonic placental cone (EPC), and extraembryonic ectoderm (ExE). It has been demonstrated that the induced totipotent stem cells according to the present invention can still have the ability to participate in the development of the extraembryonic tissues of the embryo after embryo transfer. For further development of the chimeric embryo (e.g., E12.5), the induced totipotent stem cells according to the present invention can be embedded in the embryo (Em), extraembryonic tissue placenta (Pl), and yolk sac (Yo), which can also be confirmed by analyzing the chimerism of tdTomato positive cells. It has been demonstrated that the induced totipotent stem cells according to the present invention can still have the ability to participate in the development of the extraembryonic tissues of the embryo after embryo implantation.

[0091] The ability of the induced totipotent stem cells according to the present invention to develop independently into blastocysts can be tested under the culture conditions used to induce blastocysts. The blastocysts derived from the induced totipotent stem cells according to the present invention can have morphological characteristics very similar to those of normal blastocysts. The blastocysts derived from the induced totipotent stem cells according to the present invention can have the three cell lineages of normal blastocysts in vivo, as confirmed by fluorescent immunostaining, demonstrating that the induced totipotent stem cells according to the present invention can be effectively induced into blastocysts with proper structure and proper gene expression. Blastocysts derived from induced totipotent stem cells according to the present invention can be further cultured in vitro to generate a three-dimensional structure similar to a post-implantation embryo (e.g., stage E4.5-E5.5) comprising, as two hemispheres, ectoderm (staining positive for TFAP2C) and EPI (staining positive for Oct4) surrounded by endoderm (staining positive for SOX17). Blastocysts derived from the induced totipotent stem cells according to the present invention can be implanted in the uterus for further development in vivo, and can undergo a decidualization response and continue to grow after implantation in the uterus. As discussed above, induced totipotent stem cells according to the present invention may be characterized by one or more of the above properties. Such characterization may be performed using methods described herein or methods known to those skilled in the art.

[0092] I. Application The induced totipotent stem cells according to the present invention can be used for a wide range of desired applications in research, industry, and the clinic, for example, various products can be generated via differentiation of the induced totipotent stem cells of the present invention, and can be used, for example, to build models, test targets, develop surrogates, and for other potential therapeutic or diagnostic applications. The induced totipotent stem cells according to the present invention can be used to induce the generation of organisms. The organisms can be used for promising scientific, therapeutic, and diagnostic applications, such as constructing disease models. In one aspect, the present invention provides organisms derived from the induced totipotent stem cells described herein. The organisms can be eukaryotic organisms, including, but not limited to, animals, plants, fungi, and other eukaryotic organisms known in the art. The animals can include, but are not limited to, mammals, such as primates, such as humans, non-human primates, non-primates, bovine, equine, ovine, porcine, canine, lagomorphs, rodents, such as monkeys, bovine, ovine, porcine, canine, rabbit, rat, or mouse. Preferably, in some embodiments, the organism is a rodent or mammal. In some embodiments, the mammal is not a human.

[0093] The plant may be a monocot or dicot, or may be a crop or grain plant, such as cassava, corn, sorghum, soybean, wheat, oats, or rice. The plant may be an algae, a tree or a yielder plant, a fruit, or a vegetable (e.g., a tree, such as a citrus tree, such as an orange, grapefruit, or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree, such as an almond, walnut, or pistachio tree; a plant of the Solanum genus; a plant of the Brassica genus; a plant of the Lactuca genus; a plant of the Spinacia genus; a plant of the Capsicum genus; cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).

[0094] The induced totipotent stem cells according to the present invention can be used to induce the generation of organoids.The organoids can be used to construct disease models, transplantation therapy, or other promising scientific, therapeutic, and diagnostic applications.In one aspect, the present invention provides organoids derived from the induced totipotent stem cells described herein.The organoids can be derived from the following organs: the human skeleton, such as bones, carpals, clavicles, femur, fibula, humerus, jaw, metacarpals, metatarsals, ossicles, patella, phalanges, radius, skull, tarsus, tibia, ulna, ribs, spine, pelvis, sternum, cartilage, etc.; joints, such as fibrous joints, cartilaginous joints, and synovial joints; the musculoskeletal system, including muscles, tendons, diaphragm, etc.; Cardiovascular system, e.g. peripheral blood supply (arteries, veins, lymphatic vessels), heart, etc.; lymphatic system, circulatory system including primary (bone marrow, thymus), secondary (spleen, lymph nodes) and glymphatic systems; The nervous system, including the brain, e.g. hindbrain (medulla oblongata, pons, cerebellum), midbrain, forebrain (diencephalon (retina, optic nerves), cerebrum, limbic system), spinal cord, nerves, and sensory systems (ear, eye); The epidermal system, including the skin, subcutaneous tissue, and breasts (mammary glands); The immune system, including bone marrow cells and lymphocytes; The respiratory system, including the upper respiratory tract (nose, pharynx, larynx) and the lower respiratory tract (trachea, bronchi, lungs); Digestive system, including the mouth (salivary glands, tongue), upper digestive tract (oropharynx, laryngopharynx, esophagus, stomach), lower digestive tract (small intestine, appendix, large intestine, rectum, anus), and associated digestive glands (liver, bile duct, pancreas); Genitourinary system, the urinary system including the kidneys, ureters, bladder, and urethra; Reproductive system, including the female reproductive system (uterus, vagina, vulva, ovaries, placenta) and the male reproductive system (scrotum, penis, prostate, testes, seminal vesicles); The endocrine system, including the pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal glands, and pancreatic islets The organoid may be, but is not limited to, an organoid derived from.

[0095] The induced totipotent stem cells according to the present invention can be used to induce the generation of tissues. The tissues can be used to construct disease models, transplantation therapies, or other promising scientific, therapeutic, and diagnostic applications. In one aspect, the present invention provides tissues derived from the induced totipotent stem cells described herein. Tissues include, but are not limited to, animal tissues and plant tissues. Animal tissues include, but are not limited to, epithelial tissue, muscle tissue, nervous tissue, and connective tissue. Connective tissues include, but are not limited to, proper connective tissues, including loose connective tissues (loose tissues), dense connective tissues, adipose tissue, reticular connective tissue, and elastic connective tissue; bone or cartilage tissue; blood; and lymph. Blood is a liquid tissue that circulates within the cardiovascular system. Blood is composed of plasma and a variety of blood cells. Lymph is a liquid that flows through lymphatic vessels and is formed by the flow of tissue fluid into lymph. Lymph ultimately flows into the veins. Lymph contains lymphocytes, and the composition of lymph changes under different physiological conditions.Preferably, in some embodiments, the tissue is blood.

[0096] The induced totipotent stem cells according to the present invention can be used to induce the generation of differentiated cells. The differentiated cells can be used to establish disease models, transplantation therapies, or other promising scientific, therapeutic, and diagnostic applications. In one aspect, the present invention provides differentiated cells differentiated from the induced totipotent stem cells described herein. The differentiated cells can be cells derived from any organ or tissue previously described. Preferably, in some embodiments, the cells are immune cells, more preferably T cells or NK cells. Preferably, in some embodiments, the cells are neural cells, more preferably neurons or glial cells. Preferably, in some embodiments, the cells are blood cells, more preferably red blood cells or white blood cells.

[0097] All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application, or patent was specifically and individually indicated to be incorporated by reference herein. Although the present invention has been described in some detail in the form of embodiments and examples for the purpose of clarity of understanding, it is obvious to those skilled in the art that, based on the teachings of the present invention, certain changes and modifications thereto can be made without departing from the spirit or scope of the claims. The following examples are presented by way of demonstration and not by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to achieve substantially similar results. EXAMPLES

[0098] Example 1: Derivation and preliminary identification of mouse induced totipotent stem cells The present inventors have surprisingly found that mouse pluripotent stem cells have characteristics of totipotent cells very similar to mouse fertilized eggs or two-cell stage embryonic cells after treatment with small molecule reprogramming reagents, and such stem cells are referred to by the present inventors as mouse induced totipotent stem cells.

[0099] 1.1 Derivation of mouse totipotent stem cells When mouse pluripotent embryonic stem cells or mouse induced pluripotent stem cells (either commercially available or established by standardized experiments) were grown in standard medium to approximately 70% dish density, the cells were digested with 0.05% (v / v) trypsin to obtain single cells in suspension, which were passaged at a ratio of approximately 1:10 (one generation was sufficient) and seeded into totipotent stem cell medium (commercially available mouse pluripotent stem cell basal medium further supplemented with small molecule reprogramming reagents). The commercially available pluripotent stem cell basal medium used herein includes Knockout DMEM basal medium and 5% KSR, 1% N2, 0.2% chemically defined lipid concentrate (CDL), 1% GlutaMAX™ (L-glutamine substitute), 1% dual antibiotic (penicillin / streptomycin), 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 50 ng / ml L-ascorbyl-2-phosphate sodium, and 1000 U / mL murine leukemia inhibitory factor (mLIF) (Yang, Y. et al. Derivation of pluripotent stem cells with in vivo intra-embryonic and extra-embryonic potency. Cell 169, 243-257. e225 (2017)).

[0100] The inventors tested various combinations of small molecule reprogramming agents to determine their effects on induction of totipotent stem cells. Various small molecule reprogramming reagents are as described in the Detailed Description section of the present specification. Some of the combinations tested are listed in Table 1. The inventors tested the performance of various aspects of the combinations, including the induction ratio of totipotent stem cells, i.e., 2C:tdTomato cell ratio, as shown in Figure 9.2. As shown in Figure 9.4, some of such small molecule reprogramming reagents were individually substituted and their performance was also tested. It should be noted that the induction ratio of totipotent stem cells is not the only factor for evaluating the quality of the combination. After evaluating the performance in various aspects, the inventors selected the optimal combination, i.e., a combination of three small molecule compounds (TTNPB, 1-Azakenpaullone, and WS6), for use in further experiments. 2.5 μM 1-azakempauron, 0.5 μM WS6, and 0.2 μM TTNPB were added to the commercially available pluripotent stem cell basal medium described above to obtain the totipotent stem cell medium used below. We also tested various basal media (including DEME, knockout DMEM, RPMI1640, and DMEM / F12) to determine whether the type of basal medium has an effect on the induction of totipotent stem cells. As shown in Figure 9.3, no significant differences were observed in the 2C::td (2C:tdTomato) and OCT4 fluorescence assays. Thus, the type of basal medium has no substantial effect on the induction of totipotent stem cells as described herein. In other experiments, knockout DMEM was used.

[0101] Approximately 2 days after seeding in the totipotent stem cell medium, the mouse pluripotent embryonic stem cells gradually acquired the properties of mouse totipotent stem cells (characterized below). When the proliferation density of the mouse totipotent stem cells approached 70%, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3 to 1:5. The expression of the pluripotency gene marker Oct4 in the resulting mouse totipotent stem cells was downregulated, and the mouse totipotent stem cells could be maintained in subculture for more than 10 generations without losing their totipotency while maintaining good clonal morphology (Figure 1.1).

[0102] To investigate whether Dux and p53 are required for totipotent stem cell (ciTotiSC) induction, we knocked out either Dux or p53 in mESCs. As expected, depletion of Dux significantly reduced the percentage of MERVL+ cells in mESCs (Fig. 9.1a), but did not significantly increase TAW-induced MERVL+ cells (Fig. 9.1b). In Dux knockout cells, TAW was consistently unsuccessful in inducing totipotency genes such as Dux, Zscan4, Zfp352, and Tcstv3 (Fig. 9.1c). These results demonstrate that Dux is essential for the induction of MERVL+ciTotiSCs. Although to a lesser extent, p53 knockout also caused a reduction in the percentage of MERVL+ cells in TAW-treated or non-TAW-treated mESCs (Fig. 9.1a and Fig. 9.1b). Further analysis revealed that p53 knockout also reduced the expression of totipotency marker genes MERVL, Dux, Zscan4, Zfp352, and Tcstv3 compared to TAW-induced WT cells (Figure 9.1d). Collectively, such evidence suggests that induction of totipotent stem cells (ciTotiSCs) is partially dependent on p53. 1.2 Transcriptional analysis of totipotency marker genes in mouse ciTotiSCs by RT-PCR

[0103] 1) To detect the transcription of classical totipotency marker genes and repetitive sequences (MERVL, Zscan4, ZFP352, Tcstv3, Gm6763) in the obtained mouse ciTotiSCs, RT-qPCR reactions were performed using iQTM SYBR Green Supermix. RT-qPCR reactions were performed in a Bio-Rad CFX384 real-time PCR system. Data results were analyzed and graphed in Prism8 software. 2) qPCR reaction system Component Volume (μl) iQTM SYBR® Green supermix (2x) 5 Forward primer (10 pmol) 1 Reverse primer (10 pmol) 1 cDNA template 0.5 Deionized water 2.5 Total volume 10 3) qPCR primer sequences:

[0104] [Table 2] 4) qPCR reaction program

[0105] [Table 3] RT-qPCR analysis revealed that compared with mouse pluripotent embryonic stem cells (mESCs) used as starting cells, mouse ciTotiSCs showed significantly higher expression of totipotency marker genes and repeat sequences (MuERVL, Zscan4, ZFP352, Tstv3, Gm6763), implying that the cell fate of mouse pluripotent embryonic stem cells had undergone a transition to mouse totipotent stem cells (Figure 1.2). Example 2: Molecular characterization of mouse ciTotiSCs: Transcriptome characterization of mouse ciTotiSCs by RNA-seq and scRNA-seq To further explore the molecular biological characteristics of mouse induced pluripotent stem cells, we used transcriptome sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to analyze the changes at the transcriptome level after mouse pluripotent embryonic stem cells acquire totipotency.

[0106] By analyzing the enrichment of 266 totipotency marker genes specifically expressed in mouse 2-cell embryogenesis and 47 mouse pluripotency marker genes in mouse pluripotent embryonic stem cells (mESCs) and mouse ciTotiSCs (GSEA analysis), we found that such 2-cell embryogenesis specific totipotency marker genes are significantly enriched in mouse ciTotiSCs (Figure 2.1, top left panel), while mouse pluripotency marker genes are significantly enriched in mouse pluripotent embryonic stem cells (Figure 2.1, top right panel). Using transcriptome sequencing (RNA-seq) analysis, maternal genes, ZGA genes, and totipotency genes were found to be highly expressed in mouse totipotent stem cells, confirming that the cells are in a totipotent state (Figure 2.1, bottom panel).

[0107] Through whole-gene transcriptome-level clustering analysis of mouse pluripotent embryonic stem cells, mouse ciTotiSCs, and various stages of mouse embryonic development, we found that mouse pluripotent embryonic stem cells are closer to the pluripotent mouse day 3.5 (E3.5) embryonic inner cell mass (ICM) at the whole-genome transcriptome level, thus exhibiting a totipotent state at the transcriptional level, while mouse ciTotiSCs are closer to the totipotent mouse 1- and 2-cell stage embryos at the genome-wide transcriptome level, thus exhibiting a totipotent state at the transcriptional level (Figure 2.2).

[0108] Through whole-genome transcriptome principal component analysis (PCA), we further confirmed that mouse ciTotiSCs lie between mouse 1-cell and 2-cell embryos with totipotency, while mouse pluripotent embryonic stem cells (mESCs) and mouse expanded potential stem cells (EPSCs) are closer to mouse blastocysts at later stages of development (Figure 2.3). By analyzing gene sets specifically expressed in fertilized eggs, 2-cell, 4-cell, 8-cell, and 16-cell stages of early embryonic development in mice, and the enrichment of these gene sets in mouse ciTotiSCs compared with mouse pluripotent embryonic stem cells, respectively, we further confirmed that the expression of totipotency marker gene sets specific to the fertilized eggs and 2-cell stages is significantly enriched in mouse ciTotiSCs (Figure 2.4).

[0109] Single-cell RNA sequencing (scRNA-seq) results for mouse ciTotiSCs, totipotent blastomere-like cells (TBLC, Shen, H. et al. mice totipotent stem cells captured and maintained through spliceosomal repression. cell, doi:10.1016 / j.cell.2021.04.020 (2021)), and normal mouse embryos at various stages (Deng, Q., Ramskold, D., Reinius, B. & Sandberg, R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343, 193-196 (2014)), mouse ciTotiSCs were found to be very close to mouse totipotent 2-cell embryos (TPSCs and late 2C shown in Figure 2.5), with high expression of totipotent marker genes but low expression of pluripotent marker genes, and totipotent transcriptome characteristics, whereas totipotent blastomere-like cells (TBLC, Shen, H. et al. mice totipotent stem cells captured and maintained through spliceosomal repression. cell, doi:10.1016 / j.cell.2021.04.020 (2021)) did not resemble totipotent mouse zygotes and 2-cell embryos. Conversely, TBLCs were closer to embryos at later developmental stages, i.e., E4.5–E5.5 days after implantation (TBLC, and E4.5, E5.5 shown in Figure 2.5). In addition, totipotency marker genes are not significantly activated and pluripotency marker genes are not effectively repressed, and therefore totipotent blastomere-like cells cannot be considered as totipotent cells (Figure 2.5).

[0110] 2.2 Analysis of chromatin accessibility in mouse ciTotiSCs by ATAC-seq To explore the characteristics of chromatin accessibility in mouse ciTotiSCs, which suggests gene activation or repression, we used chromatin accessibility sequencing ATAC-seq (assay for transposase-accessible chromatin using sequencing) to analyze chromatin accessibility in mouse ciTotiSCs, mouse pluripotent embryonic stem cells, mouse 2-cell embryos, and mouse blastocyst inner cell mass.

[0111] The analysis revealed that compared with mouse pluripotent embryonic stem cells, mouse ciTotiSCs had 5 kb closed and open peaks near the transcription start site (TSS), with the open or closed state similar to that of mouse 2-cell embryo (2C) formation stage, while mouse pluripotent embryonic stem cells (mESCs) were more similar to mouse blastocyst inner cell mass (ICM) (Figure 2.6). In addition, compared with mouse pluripotent embryonic stem cells, mouse ciTotiSCs showed a higher level of openness near some important pluripotency genes and retroelements, such as Zscan4c, Zscan4d, Zscan4f, ZFP352, MERVL, while classical pluripotency genes, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, were in a closed state (Figure 2.7). These results suggest that mouse ciTotiSCs have chromatin accessibility similar to that of mouse totipotent two-cell embryogenesis in vivo. 2.3 Genomic methylation level analysis of mouse ciTotiSCs by RRBS. To detect the genomic methylation level of mouse ciTotiSCs, we used RRBS (reduced representation bisulfite sequencing) to characterize the genomic methylation levels of mouse ciTotiSCs and mouse pluripotent embryonic stem cells. We found that the global methylation level of mouse pluripotent embryonic stem cells was 23.9%, close to the E6.5-E7.5 period after mouse embryo implantation (global methylation levels were 23.2% and 26.6%, respectively), while the methylation level of mouse ciTotiSCs was significantly reduced to 12.1%, similar to the fertilized egg and the 2-cell and 4-cell stages of mouse embryos before implantation (global methylation levels were 15.4%, 13.2%, and 14.8%, respectively) (Figure 2.8).

[0112] By genome-wide principal methylation component (PCA) analysis of the RRBS sequencing results, the inventors reaffirmed that, compared with mouse pluripotent embryonic stem cells, mouse ciTotiSCs are closer to the totipotent mouse 1- and 2-cell stage embryos in terms of developmental stage (Figure 2.9). Further analysis of methylation levels near specific sites in the genome revealed that mouse ciTotiSCs and mouse totipotent embryos were hypomethylated near the Zscan4 gene family and at several totipotent repeat sequences on the X chromosome. In contrast, no reduction in methylation was found in either mouse pluripotent embryonic stem cells or mouse postimplantation E6.5-E7.5 embryos (Figure 2.10).

[0113] In addition to the analysis of the transcriptome and epigenome, we next determined the metabolome of totipotent stem cells (ciTotiSC), since cells in the totipotent state have unique metabolic characteristics. We found that the metabolites with the highest divergence between totipotent stem cells (ciTotiSC) and mESCs were very similar to those between totipotent 2C embryos and blastocysts (Figure 2.11, top). Further analysis revealed that totipotent stem cells (ciTotiSC) and 2C embryos are more likely to utilize pathways related to one-carbon metabolism and reductive states for metabolism, while pluripotent mESCs and blastocysts have higher levels of purine metabolism and mitochondrial tricarboxylic acid (TCA) cycle metabolites, indicating a more highly oxidative state (Figure 2.11, bottom).

[0114] Example 3: Analysis of the ability of mouse ciTotiSCs to differentiate into trophoblast cells in vitro Mouse pluripotent stem cells do not have the ability to differentiate into trophoblast cells. Figure 3.1 shows an experimental schematic for differentiation of mouse ciTotiSCs, pluripotent embryonic stem cells (mESCs, Ying, QL et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519-523, doi:10.1038 / nature06968 (2008)), and pluripotent potential-expanded stem cells (mEPSs, Yang, Y. et al. Derivation of pluripotent stem cells with in vivo intra-embryonic and extra-embryonic potency. Cell 169, 243-257. e225 (2017)) into trophoblast stem cells in trophectodermal stem cell (TSC) medium. This experiment verified that the mouse ciTotiSCs of the present invention have an extraembryonic portion, i.e., the ability to differentiate into trophoblast stem cells (but not pluripotent stem cells).

[0115] 0.05% trypsin was used to digest mouse ciTotiSCs, embryonic stem cells (mESCs), and pluripotent expanded stem cells (mEPSs) cultured on feeder cells (mouse embryonic fibroblasts, which are widely used in stem cell culture). The digested cells were spread on a cell culture plate coated with 0.3% gelatin, the suspended cells were collected after 30 minutes, and the feeder cells were allowed to adhere to the cell culture plate to remove the feeder cells. Mouse ciTotiSCs derived from feeder-depleted cells, embryonic stem cells (mESCs), and pluripotent expanded stem cells (mEPSs) were cultured at 1 × 10 cells per well of a 12-well plate. 5Cells were seeded at a density of 100x in mouse trophoblast stem cell medium (details below). Medium was changed once daily. Cells were harvested on days 0, 4, and 8 for RT-qPCR to detect the transcription of mouse trophoblast stem cell specific genes (CDX2, Elf5, Tfap2c, Esx1; Figure 3.2), using commercially available trophoblast stem cells (TSCs) as a positive control. Fluorescent immunostaining was performed on day 12 to detect the expression of mouse trophectoderm cell specific protein (CDX2) (Figure 3.3).

[0116] Mouse trophoblast stem cell (TSC) medium (Posfai, E. et al. Evaluating totipotency using criteria of increasing stringency. Nature Cell Biology 23, 49-60, doi:10.1038 / s41556-020-00609-2 (2021)): RPMI1640 basal medium (Gibco, C11875500BT), 20% fetal bovine serum, 1x GlutaMAX (L-glutamine substitute), 1% dual antibiotic (penicillin / streptomycin), 1% non-essential amino acids, 1x sodium pyruvate (Gibco, 11360070), 1mM β-mercaptoethanol, 25ng / ml FGF4 (R&D systems, 235-F4), and 1μg / ml heparin (Sigma-Aldrich, H3149). 3.1 Analysis of mouse trophoblast stem cell-specific protein CDX2 expression by fluorescent immunostaining CDX2 is a classical mouse trophoblast stem cell specific gene. To determine whether differentiation of the mouse trophoblast stem cell lineage occurred, its protein level expression was detected by immunofluorescence in conjunction with changes in a mouse pluripotent stem cell marker (Oct4).

[0117] Through this experiment (Figure 3.3), we found that mouse ciTotiSCs could have significant expression for CDX2 and downregulated expression for Oct4 after induced differentiation, thus mouse ciTotiSCs have the ability to differentiate into extraembryonic trophoblast stem cells, while embryonic stem cells (mESCs) and potential-expanded pluripotent stem cells (mEPSs) could also have downregulated Oct4 gene expression after induced differentiation, but these cells have little expression of CDX2, thus they do not have the ability to differentiate into extraembryonic trophoblast stem cells. The present inventors have demonstrated that totipotent stem cells (ciTotiSCs) of different passages (P1 to P8) were transformed into pluripotent embryonic stem cells (rESCs) after changing to mESC medium (2i / LIF). ciTotiSC ) (reflected by the downregulation of totipotency genes and upregulation of pluripotency genes, thereby mimicking normal embryonic development) (Figure 3.4). Immunofluorescence staining steps: Fixation: After the samples in the cell culture plate were washed with phosphate-buffered saline (DPBS), the cells were fixed using 4% paraformaldehyde solution, kept at 4°C for 30 min, and washed four times with DPBS. Blocking: Add blocking solution (10% donkey serum + 1% BSA + 0.3% Triton-X100 diluted in DPBS), block for 1 hour at room temperature, wash 3 times with DPBS.

[0118] Primary antibody incubation: The primary antibody was mouse anti-CDX2 (1:150, BioGenex, MU392A-UC). The antibody was diluted with DPBS containing 1% BSA according to the required concentration and incubated for 2 hours at room temperature or overnight at 4°C, and washed three times for 5 minutes with DPBS. Secondary antibody incubation: The secondary antibody was donkey anti-mouse 555 (1:500, Life technologies, A-31570). The corresponding fluorescently labeled secondary antibody was diluted 1:1000 in DPBS containing 1% BSA and incubated in the dark at room temperature for 1 hour. Nuclear staining: DAPI (nuclear dye, 4',6-diamidino-2-phenylindole dihydrochloride) was made 1 μg / ml in DPBS and incubated for 5 minutes at room temperature. Imaging: After washing with DPBS for 5 min x 3, the sections were placed under an Olympus inverted fluorescent microscope IX83 for observation and photography.

[0119] 3.2 Analysis of mouse trophoblast stem cell specific gene expression by RT-qPCR By RT-qPCR, we analyzed the transcription of mouse trophoblast stem cell-specific genes in mouse ciTotiSCs, embryonic stem cells (mESCs), and pluripotent potential-expanded stem cells (mEPSCs) on days 0, 4, and 8 during the induction period into mouse trophoblast stem cells. We found that mouse ciTotiSCs gradually activated trophoblast stem cell-specific marker genes, including CDX2, Elf5, TFAP2C, and Esx1, during the process of induced differentiation (similar to that of trophoblast stem cells (TSCs) used as a positive control). In contrast, in embryonic stem cells (mESCs) and pluripotent potential-expanded stem cells (mEPSCs), no significant transcription increase was detected in trophoblast stem cell-specific marker genes during the induction period (Figure 3.2).

[0120] Example 4: Comparison of the in vitro embryoid body differentiation and in vivo teratoma differentiation capacities of mouse ciTotiSCs and mouse embryonic stem cells (mESCs) 4.1 Comparison of the in vitro embryoid body differentiation capabilities of mouse ciTotiSCs and mouse embryonic stem cells (mESCs) Mouse ciTotiSCs and mouse embryonic stem cells (mESCs) were digested with 0.05% trypsin and then seeded onto 0.3% gelatin-coated cell culture plates. After 30 min, the suspension cells were harvested and the feeder cells were removed by allowing them to adhere to the cell culture plate. Feeder-depleted mouse ciTotiSCs or embryonic stem cells (mESCs) were cultured at 1 × 10 cells per ml in mouse embryoid body formation medium (Knockout DMEM basal medium further supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX™ (L-glutamine substitute), 1% dual antibiotics (penicillin / streptomycin), 1% non-essential amino acids, and 0.1 mM β-mercaptoethanol). 5 The cells were resuspended at a density of 10 μl per well. Then, the embryoid body formation experiment was carried out by hanging drop method: hanging drop was carried out on the lid of 10 cm culture dish, where each 20 μl of the cell mixture was a hanging drop, and it was placed in a 5% CO2, 37°C incubator for culturing. After 2 days, the hanging drop cells were collected and placed in a 6-well low-attachment culture plate for continuous culturing, and the embryoid bodies were taken on days 0, 3, and 6 for identification using fluorescent immunostaining.

[0121] Through immunofluorescence staining, we found that CDX2-positive cells (representing extraembryonic trophoblast cells, suggesting that totipotent stem cells can differentiate into extraembryonic cell types) could be detected in embryoid bodies derived from mouse ciTotiSCs on day 6, but CDX2-positive cells were not detected in embryoid bodies derived from mouse embryonic stem cells (mESCs) (suggesting that pluripotent stem cells did not differentiate into extraembryonic cell types) (Figure 4.1). By calculating the immunofluorescence staining results, we found that CDX2-positive cells were detected in all embryoid bodies derived from mouse ciTotiSCs on days 3 and 6 (6 / 6 on day 3 and 17 / 17 on day 6), but CDX2-positive cells were not detected in embryoid bodies derived from mouse embryonic stem cells (mESCs) (0 / 7 on day 3 and 0 / 21 on day 6) (Figure 4.2). CDX2 is a classical mouse trophoblast stem cell-specific gene. Embryoid body differentiation experiments demonstrated that mouse ciTotiSCs have the ability to differentiate into extraembryonic trophoblast stem cells (but not pluripotent stem cells) in vitro.

[0122] 4.2 Comparison of the in vivo teratoma differentiation ability of mouse ciTotiSCs and mouse embryonic stem cells (mESCs) The teratoma assay is a classical assay to test the ability of cells to randomly differentiate into the three endothelial germ layers and extraembryonic lineages.

[0123] Teratoma differentiation experimental procedure: Under feeder cell culture conditions, mouse ciTotiSC or mouse embryonic stem cells (mESC) were digested into single cells using 0.05% trypsin-EDTA, and then the cells were resuspended using mouse ciTotiSC medium or mouse embryonic stem cell (mESC) medium, respectively, and seeded onto 0.3% gelatin-coated cell culture plates and incubated in a 37°C incubator for 30 minutes to remove the feeder cells. The supernatant containing mouse ciTotiSC or mouse embryonic stem cell (mESC) single cells was collected and the cells were resuspended in DPBS. The resuspended cells were injected subcutaneously into the hind inguinal region of immunodeficient SCID mice, with the number of cells injected per mouse being approximately 1.0 × 10 6 There were 100 pieces. Five weeks after the subcutaneous injection of the cells, the mice were sacrificed by cervical dislocation, and the teratomas were removed from the subcutaneous tissue, fixed with 4% paraformaldehyde solution, and subjected to paraffin sectioning and HE staining. The tissue sections were examined under a microscope for tissue structures specific to the three intraembryonic germ layers and the extraembryonic cell lineages.

[0124] We subcutaneously injected mouse ciTotiSCs or mouse embryonic stem cells (mESCs) into immunodeficient SCID mice, and both cell lines were able to form teratomas. After paraffin sectioning and HE staining of the teratomas, typical ectoderm (left column), mesoderm (middle column), and endoderm (right column) histology could be observed under a microscope. This suggested that mouse ciTotiSCs and mouse embryonic stem cells (mESCs) have the ability to differentiate into three germ layers in the embryo (Figure 4.3). Next, the present inventors further observed whether HE-stained sections of teratomas formed by mouse ciTotiSCs or mouse embryonic stem cells (mESCs) had classical extraembryonic lineage tissue structures. Upon observation, it was found that classical extraembryonic lineage-placental giant cells could be observed in areas rich in internal hemorrhage in teratomas formed by induced totipotent stem cells in mice. Classical extraembryonic lineage-placental giant cells showed typical morphological characteristics such as having larger nuclei and larger cell volume, and expressed the placental giant cell marker gene PL-1 (Figure 4.4). In contrast, no extraembryonic lineage was observed in teratomas formed from mouse embryonic stem cells (mESCs). In vivo teratoma differentiation experiments demonstrated that mouse ciTotiSCs have the ability to differentiate into extraembryonic cells, whereas pluripotent stem cells do not.

[0125] Example 5: In vivo chimera assay of mouse ciTotiSCs (in vitro E4.5 embryonic stage) Schematic diagram of the chimeric assay process (Figure 5.1) Chimera assay steps: Rosa26-tdTomato mouse blastocysts were used to establish mouse induced totipotent stem cell lines and mouse pluripotent embryonic stem cell lines (mESCs) labeled with red fluorescence for injection into 8-cell stage embryos. If the stem cells injected into mouse 8-cell embryos developed with the embryo and differentiated into the corresponding intraembryonic or extraembryonic parts, red fluorescence (tdTomato) could be observed in the corresponding intraembryonic or extraembryonic parts. The two cell lines were digested into single cells using 0.05% trypsin-EDTA, resuspended in the corresponding medium, seeded on 0.3% gelatin-coated cell culture plates, and incubated in a 37°C incubator for 30 minutes to remove feeder cells. The cells were collected and resuspended in the corresponding medium.

[0126] After superovulation, commercially available ICR female mice were mated with commercially available ICR male mice. After 1.5 days, 8-cell stage embryos were collected from the oviducts of the successfully mated female mice. Chimeric embryos were obtained by injecting 5-10 mouse ciTotiSCs or mouse embryonic stem cells (mESCs) after depletion of feeder cells into each 8-cell stage embryo. In the mouse induced totipotent stem cell chimeric embryo E4.5 assay (Example 5, Figures 5.2, 5.3, and 5.4), injected 8-cell embryos were placed in KSOM medium and cultured for 48 hours in a 5% CO2, 37°C incubator before tdTomato positive cells were analyzed for chimerism in the inner cell mass (ICM) and trophectoderm (TE). In experiments to develop mouse induced totipotent stem cell chimeric embryos in vivo to E7.5 and E12.5 (Examples 6-7, Figures 6-7), the injected 8-cell stage embryos were placed in KSOM medium and collected for 1-2 hours of culture in a 5% CO2, 37°C incubator, and then implanted into the uterus of pseudopregnant ICR females 0.5 days after mating with ligated ICR males for further development.

[0127] 5.1 During early embryogenesis, the totipotent zygote gradually developed into the inner cell mass (ICM) inside the embryo and the trophectoderm (TE) outside the embryo. Mouse ciTotiSCs and mouse embryonic stem cells (mESCs) for 8-cell injection had stable expression of red fluorescence (tdTomato). By analyzing the chimerism of tdTomato-positive cells, we observed that mouse ciTotiSCs could chimerize into the inner cell mass (ICM) inside the embryo and the trophectoderm (TE) outside the embryo. However, mouse embryonic stem cells (mESCs) could only chimerize into the inner cell mass (ICM) of the embryo, but not into the trophectoderm (TE) outside the embryo (Figure 5.2).

[0128] 5.2 The chimerism ratios of mouse ciTotiSCs and mouse embryonic stem cells (mESCs) in the inner cell mass (ICM) and trophectoderm (TE) were calculated (Figure 5.3). Mouse ciTotiSC: Chimerized to both TE and ICM: 18 / 21 (85.7%); Chimerized only to TE: 2 / 21 (9.5%); Chimerism only to ICM: 1 / 21 (4.8%). That is, mouse ciTotiSCs had both intraembryonic (inner cell mass) and extraembryonic (trophectoderm) developmental capabilities. Mouse Embryonic Stem Cells (mESC): Chimerism to both TE and ICM: 0 / 21 (0%); Chimerized only to TE: 0 / 21 (0%); Chimerism was observed only in ICM: 21 / 21 (100%). That is, mouse pluripotent stem cells only had the ability to develop within the embryo (inner cell mass).

[0129] 5.3 CDX2 is a classical marker of trophectoderm (TE). We further confirmed whether tdtomato fluorescently labeled mouse ciTotiSCs chimerized into trophectoderm (TE) could express CDX2 (an important marker of trophectoderm) by using CDX2 fluorescent immunostaining. The staining results revealed that tdtomato fluorescently labeled cells could simultaneously express CDX2 (Figure 5.4). This further demonstrated that mouse ciTotiSCs are indeed involved in the development of trophectoderm (TE) in E4.5 chimeric embryos, i.e., they have the ability to differentiate into extraembryonic parts.

[0130] Example 6: In vivo development of mouse induced totipotent stem cell chimeric embryos to E7.5 Chimeric embryos derived from 8-cell stage embryos injected with mouse ciTotiSCs or mouse embryonic stem cells (mESCs) were implanted into pseudopregnant mice, and embryos that developed to E7.5 after implantation were isolated 7 days later. Embryos at this stage (E7.5) contained three parts: epiblast (EPI; after embryo implantation, EPI generated embryonic tissues containing three germ layers), placental cone (EPC), and extraembryonic ectoderm (ExE). The experimental protocol is shown in Figure 5.1. Fluorescent immunostaining was performed for embryonic epidermis (EPI) OCT4, a classical marker for placental cone (EPC), and extraembryonic ectoderm (ExE) by analyzing the chimerism of tdTomato-positive cells (Figure 6.1). We found that mouse ciTotiSCs could be almost chimerized with the whole E7.5 embryo, including the intraembryonic epiblast (EPI), extraembryonic placental cone (EPC), and extraembryonic ectoderm (ExE). In addition, we further found that tdTomato-positive cells derived from mouse ciTotiSCs could express the embryonic epiblast marker OCT4 and the extraembryonic (embryonic cone EPC and extraembryonic ectoderm EXE) marker ELF5 by fluorescent immunostaining. This experiment demonstrated that mouse ciTotiSCs still have the ability to participate in the development of intraembryonic and extraembryonic tissues at E7.5 days after mouse embryo implantation.

[0131] Example 7: In vivo development of mouse induced totipotent stem cell chimeric embryos to E12.5 Chimeric embryos obtained by injecting mouse ciTotiSCs or mouse embryonic stem cells (mESCs) into 8-cell stage embryos were implanted into pseudopregnant mice 13 days after which the implants were isolated and the chimeric embryos developed into E12.5-E13.5 mouse embryos (Em), placenta (Pl), and yolk sac (Yo). The experimental protocol is shown in Figure 5.1. By analyzing the chimerism of tdTomato-positive cells, we found that mouse ciTotiS could chimerize mouse embryos (Em) and extraembryonic tissues placenta (Pl) and yolk sac (Yo) at a high rate (Figure 7.1). This experiment demonstrated that mouse ciTotiSCs still had the ability to participate in the development of embryos and extraembryonic tissues (placenta and amnion) at E12.5-13.5 days after mouse embryo implantation.

[0132] To further analyze the proportion of tdTomato-positive cells derived from mouse ciTotiSCs relative to the chimerism of each part of embryonic / extraembryonic tissues, we digested mouse embryo (Em), extraembryonic tissue placenta (Pl), and yolk sac (Yo), respectively, and analyzed the chimeric ratio of mouse ciTotiSCs (tdTomato-positive cells) in each tissue by flow cytometry.

[0133] Flow cytometry steps: Before flow cytometry analysis of mouse embryos (Em), extraembryonic tissues placenta (Pl), and yolk sac (Yo), they were washed twice with DPBS and cut into small pieces of about 1 mm using micromanipulator scissors. Mouse embryonic (Em) cells were digested with collagenase IV (supplemented with 1 U / ml DNase) for 30 min in a 37°C incubator, followed by digestion with TrypLE for 5 min; placenta (Pl) cells were digested with Accutase for 10 min in a 37°C incubator; yolk sac (Yo) cells were digested with collagenase IV supplemented with 1 U / ml DNase for 5 min in a 37°C incubator, followed by digestion with TrypLE for 3 min. The digestion reaction was stopped with 3 enzyme volumes of DPBS + 10% fetal bovine serum. After centrifugation at 800 rpm for 5 min, the cell pellet was resuspended in DPB, the cells were filtered through a 70 μm cell mesh, and the single cell filtrate was collected. The cell filtrate was transferred to a flow assay tube for analysis or sorting on a BD FACS Aria III according to the experimental requirements.

[0134] Data was analyzed using FlowJo v10 software. Analysis revealed that in the non-injected group, tdTomato-positive cells were undetectable in mouse embryo (Em), extraembryonic tissue placenta (Pl), and yolk sac (Yo), which served as negative controls; in the mouse embryonic stem cell (mESC)-injected group, tdTomato-positive cells were only detectable in mouse embryo (Em), while tdTomato-positive cells were nearly undetectable in extraembryonic tissue placenta (Pl) and yolk sac (Yo); in the mouse induced totipotent stem cell-injected group, a high proportion of tdTomato-positive cells were detectable in mouse embryo (Em), extraembryonic tissue placenta (Pl), and yolk sac (Yo). This also suggested that mouse ciTotiSCs could efficiently chimerize into mouse embryo (Em), extraembryonic tissue placenta (Pl), and yolk sac (Yo) (Figure 7.2).

[0135] Immunohistochemical analysis of frozen sections of chimeric placentas revealed that the presence of tdTomato-positive cells was undetectable in the placenta of the non-injected group, which served as a negative control; in the mouse embryonic stem cell (mESC)-injected group, only a few tdTomato-positive cells were observed within the Laby region of the placenta (this region was part of the extraembryonic placenta, but a small number of intraembryonic cells were present), and the fluorescent signal of tdTomato could not be co-localized with the fluorescent signals of the placental extraembryonic lineage cell markers CK8 and proliferin; in the mouse induced totipotent stem cell-injected group, a high proportion of tdTomato-positive cells was observed in the extraembryonic part of the placenta (JZ and Laby regions), and the fluorescent signal of tdTomato could clearly be co-localized with the fluorescent signals of the placental extraembryonic lineage cell markers CK8 and proliferin (Figure 7.3).

[0136] Immunohistochemical analysis of the chimeric mouse embryos after cryosectioning revealed that both the mouse-derived totipotent stem cell-injected group and the mouse embryonic stem cell (mESC)-injected group could achieve high chimerism in various fetal tissues (including the brain, heart, and liver), but the mouse-derived totipotent stem cell-injected group had a higher chimerism efficiency (Figure 7.4). Example 8: Independent development of mouse ciTotiSCs into mouse embryos and organisms 8.1 Single mouse induced totipotent stem cells (ciTotiSCs) have bidirectional chimeric potential to develop into intraembryonic and extraembryonic cells.

[0137] To examine the developmental ability of the totipotent stem cells (ciTotiSCs) more closely, we injected single tdtomato-labeled mESCs or ciTotiSCs into mouse 8-cell embryos. After 48 hours of in vitro culture, the chimeric embryos developed to the late blastocyst stage (E4.5). As expected, mESCs only chimerized to the embryonic ICM. In contrast, the totipotent stem cells (ciTotiSCs) chimerized to both the ICM and TE. By further immunostaining for the TE-specific marker CDX2, we confirmed that the single totipotent stem cells (ciTotiSCs) could indeed develop to the extraembryonic TE lineage and correctly expressed the lineage marker CDX2 (Figure 8.1, top). In addition, we transplanted the 8-cell embryos injected with unipotent stem cells (ciTotiSCs) or mESCs into the oviducts of pseudopregnant female mice, and further observed the chimeras at E6.5-E7.5. We observed that a single totipotent stem cell (ciTotiSC) could chimerize to intraembryonic lineages (EPI) and extraembryonic lineages (ExE and EPC), which was confirmed by co-expression of tdTomato with OCT4 or ELF5. In contrast, mESCs were only chimerized to OCT4+EPI (Figure 8.1, bottom). These data demonstrated that a single totipotent stem cell (ciTotiSC) has chimeric ability to both intraembryonic and extraembryonic lineages, fulfilling the strict criteria for totipotency.

[0138] 8.2 Induced totipotent stem cells (ciTotiSCs) can develop chimerically into various cell types both inside and outside the E13.5 embryo To fully characterize the cell types developed from totipotent stem cells (ciTotiSCs) in extraembryonic tissues around E13.5, we used scRNA-seq to analyze tdTomato+ cells derived from totipotent stem cells (ciTotiSCs) in the placenta and yolk sac of chimeras. After aligning with extraembryonic lineage specific genes, we confirmed that cells derived from totipotent stem cells (ciTotiSCs) could develop into extraembryonic trophoblast and yolk sac cell types, such as visceral yolk sac cells (Apoa4+Fxyd2+Entpd2+), spongiotrophoblasts (Tpbpa+Rhox9+), and syncytiotrophoblasts (Itm2a+), and that cells of embryonic origin included erythrocytes, macrophages, and monocytes (Figure 8.2).

[0139] 8.3 Analysis of germline transmission ability of mouse ciTotiSCs The totipotent stem cells (ciTotiSCs) have the capacity to chimerize in the genital ridges and generate healthy chimeric offspring (Figure 8.3). 8.4 Induction of mouse ciTotiSCs and their independent development in vitro into mouse blastocysts

[0140] Mouse ciTotiSCs cultured under feeder cell conditions were digested into single cells using 0.05% trypsin-EDTA, resuspended in the totipotent stem cell medium described in Example 1, transferred to a 6-well plate coated with 0.3% gelatin, and incubated in a 37°C incubator for 30 minutes to remove feeder cells. Mouse ciTotiSCs were collected, resuspended in the medium used to induce mouse blastocysts, and filtered through a 40 μm filter to remove impurities and undigested cell clumps. AggreWell400 (STEMCELL Technologies, 34415) was pretreated according to the instructions. Approximately 6,000 mouse ciTotiSCs were seeded in one well of a 6-well plate of AggreWell400 (with 1,200 chambers) and cultured in the medium to induce mouse blastocysts. Induced mouse blastocysts can be obtained by centrifuging the AggreWell400 culture plate at 100g for 3 minutes to sediment the cells, and then placing the culture plate in a 37°C incubator for 4-5 days. We observed that the induced blastocysts had morphological characteristics very similar to those of normal blastocysts (Figure 8.4). Statistics revealed that the induction efficiency of induced blastocysts from mouse ciTotiSCs was about 70% (Figure 8.4).

[0141] Culture media for inducing mouse blastocysts (Li, R. et al. Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures. cell 179, 687-702 e618, doi:10.1016 / j.cell.2019.09.029 (2019)): 25% trophectoderm stem cell basal medium (RPMI1640 basal medium supplemented with 20% fetal bovine serum (FBS), 1x GlutaMAX (L-glutamine substitute), 1x sodium pyruvate (Gibco, 11360070), and 0.1 mM 2-mercaptoethanol), 25% N2B27 basal medium (DMEM / F-12 and Neurobasal 100 mM ethanol as basal medium ... Add 1:1 mixture of 0.5x N2 (17502-048), 0.5x B27 (17504-044), 1x non-essential amino acids, 1x GlutaMAX (L-glutamine substitute), 0.1 mM 2-mercaptoethanol, 0.1% BSA, or 5% KSR, and 50% KSOM (NaCl (95 mM), KCl (2.5 mM), KH2PO4 (0.35 mM) , MgSO4 (0.2 mM), NaHCO3 (25 mM), CaCl2 (1.71 mM), Na2-EDTA (0.01 mM), L-glutamine (1.0 mM), sodium lactate (10 mM), sodium pyruvate (0.2 mM), glucose (5.56 mM), essential amino acids (EAA; 10.0 mL / l), non-essential amino acids (NEAA; 5.0 mL / l), and BSA (4 g / l). The following items were also added: 2 mM Y-27632 (added only on day 1), 12.5 ng / mL rhFGF4 (R&D, 235F4025), 0.5 mg / mL heparin (Sigma-Aldrich, H3149), 3 mM GSK3 inhibitor CHIR99021 (Reagents Direct, 27-H76), 5 ng / mL BMP4 (Proteintech, HZ-1040), and 0.5 mM A83-01.

[0142] 8.5 Mouse blastocysts derived from mouse ciTotiSCs harbor the three cell lineages of normal mouse blastocysts A normal mouse late blastocyst (E4.5) contains primarily three cell lineages: the inner cell mass (ICM, which specifically expresses OCT4), the trophectoderm (TE, which specifically expresses CDX2), and the primitive endoderm (PrE, which specifically expresses SOX17). To detect whether the induced blastocysts derived from mouse ciTotiSCs have the three cell lineages of normal mouse blastocysts, we performed fluorescent immunostaining on blastocysts induced for 4 days. The staining revealed that the induced blastocysts could express the inner cell mass marker OCT4, the trophectoderm marker CDX2, and the primitive endoderm marker SOX17, and the expression pattern was consistent with that of normal mouse blastocysts. Statistics revealed that the percentage of induced blastocysts correctly expressing the three cell markers was about 82% (Figure 8.5). Thus, these results demonstrated that mouse ciTotiSCs could be efficiently induced into mouse blastocysts with the correct structure and gene expression pattern.

[0143] 8.6 Induced blastocysts derived from mouse ciTotiSCs can develop in vitro after implantation We further cultured the induced blastocysts derived from mouse ciTotiSCs using an in vitro embryo culture system, and found that the induced blastocysts could be cultured in vitro to generate cylindrical structures. The ectoderm (positively stained for TFAP2C) and EPI (positively stained for Oct4) formed as two hemispheres, surrounded by endoderm (positively stained for SOX17), similar to mouse postimplantation embryos at E4.5-E5.5 formation stage (Figure 8.6).

[0144] The specific culture method was as follows: the induced blastocysts were picked up using a mouth pipette, washed twice in IVC-1 medium (Cell Guidance Systems, M11), and then transferred to a u-Slide 8-well plate (ibidi, 80826) supplemented with IVC-1 medium. Approximately 20-30 induced blastocysts were placed in one well of the u-Slide 8-well plate. After inducing attachment of the blastocysts, the medium was changed to IVC-2 (Cell Guidance Systems, M12). After about 2-4 days, post-implantation embryo-like structures appeared, which were fixed with 4% PFA for 15 min at room temperature, and the next step (analysis by fluorescent immunostaining) was performed.

[0145] 8.7 In vivo implantation of induced blastocysts derived from mouse ciTotiSCs into mouse uterus for further development Currently, the widely accepted standard method to verify that blastocysts are truly functional is to transfer in vitro derived blastocysts into the uterus of pseudopregnant mice to determine whether the blastocysts can implant and develop into fetuses. To achieve this goal, we implanted induced blastocysts into the uterus of 2.5-day-old pseudopregnant mice. At 7.5 dpc, decidual implantation sites could be formed in the uterus of the induced blastocysts. The size of the decidua produced by the induced blastocysts varied, but most were similar to normal mouse decidua, and some were slightly smaller. Although the initial mouse ciTotiSCs carried the tdTomato red fluorescent marker, further sectioning of the decidual tissue revealed that embryonic structures within the decidua were positive for tdTomato, demonstrating that the structures were formed by the implantation of induced blastocysts derived from mouse ciTotiSCs into the uterus and further development there (Figure 8.7). These results demonstrated that induced blastocysts derived from mouse ciTotiSCs could undergo decidualization upon implantation in the uterus and further develop into certain embryonic structures.

[0146] Example 9: Effects of various small molecule reagents on gene expression By analyzing the expression of specific gene sets by RNA-seq data, it is quite clear that we found that TTNPB, 1-AKP, WS6, and mLIF are all important for the induction of totipotent stem cells, as reflected by the significant upregulation of totipotent, maternal, and ZGA genes (Figure 10a).

[0147] Among the three compounds, the RA agonist TTNPB could directly activate a large number of totipotent, maternal, and ZGA genes rich in RAR binding motifs (Fig. 10d-g). TTNPB or Trans-RA (another widely used RA agonist) showed similar induction effects on totipotent genes, further confirming the specificity of the RA signaling pathway in inducing totipotent cells, whereas pluripotent genes could not be induced in the presence of the RA antagonist AGN193109 (Fig. 10e, f). These results confirm the central role of the retinoic acid signaling pathway in establishing totipotency in cells.

[0148] 1-Azakenpaullone (1-AKP) is a selective dual inhibitor of GSK3β and CDK1 / cyclin B. The study revealed that treatment with 1-AKP simultaneously induced specific Wnt signaling downstream gene expression and G2 / M phase arrest. GO analysis consistently revealed that specific Wnt signaling target genes were not upregulated after 1-AKP was removed from the totipotent stem cell (ciTotiSC) culture conditions (Fig. 10h). Incidentally, we also observed an extension of the G2 phase of the cell cycle after totipotent stem cell (ciTotiSC) induction, consistent with the difference between early 2C embryos and blastocysts (Fig. 10i). Although the G2 phase extension of ciTotiSCs was decreased after 1-azakempaullone was removed from the ciTotiSC culture conditions, it suggests that 1-azakempaullone maintains some of the characteristics associated with poikilogenesis-G2 phase extension in ciTotiSCs (Figure 10i). These results suggested that the mechanism by which 1-azakempaullone promotes induction of totipotency is at least in part via activation of specific Wnt signaling and extension of the G2 phase of the cell cycle.

[0149] WS6 is an IKK-NF-κB inhibitor previously shown to promote postmitotic cell proliferation. GO analysis of differential genes showed that multiple innate immune-related pathways, especially NF-κB signaling, were enriched after WS6 ablation in totipotent stem cell (ciTotiSC) culture conditions (Fig. 10j). Interestingly, several previous studies revealed that expression of endogenous retroviruses (ERVs) can trigger NF-κB signaling via TLRs or cGAS recognition of ERV-derived cytoplasmic double-stranded RNA (dsRNA) or double-stranded DNA (dsDNA) (Chiappinelli et al. 2015; Lima-Junior et al. 2021; Mao et al. 2022). Consistent with that, when WS6 is removed, the expression of genes downstream of NF-κB signaling is also upregulated (Fig. 10k). Moreover, it has been reported that downregulation of NF-κB during zygotic development is important for the totipotent state, and activation of NF-κB is a trigger for subsequent development after ZGA (Akihiko Nishikimi, 1999; Paciolla et al. 2011). Thus, WS6 may play a role in promoting and stabilizing the induction of totipotent stem cells (ciTotiSCs) in part by inhibiting the NF-κB-mediated immune response triggered by ERV activation (Fig. 10k,j).

Claims

1. (a) an RA signaling pathway activator; (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, an Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator; A composition comprising:

2. (a) an RA signaling pathway activator; (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, an Src kinase inhibitor, a cAMP activator, and a cellular metabolism modulator; Kit including:

3. Use of (a) an RA signaling pathway activator, and (b) one or more of a GSK-3 inhibitor, an IKK signaling pathway inhibitor, an HDAC inhibitor, a histone methyltransferase inhibitor, an Src kinase inhibitor, a cAMP activator, and a cell metabolism modulator in producing induced totipotent stem cells.

4. The composition, kit, or use according to any one of claims 1 to 3, wherein the RA signaling pathway activator is selected from small molecules involved in the same pathway, such as TTNPB, tretionine / RA / ATRA, AM580, Taza, 9-cis-RA, acitretin, CD437, tamibarotene, tazarotene, retinoic acid, isotretinoin, acitretin sodium, ch55, and AC55649.

5. The composition, kit or use according to any one of claims 1 to 3, wherein the GSK-3 inhibitor is selected from small molecules involved in the same pathway, such as 1-azakempaullone, AZD2858, CHIR99021, AZD1080, etc.

6. The composition, kit, or use according to any one of claims 1 to 3, wherein the IKK signaling pathway inhibitor is selected from small molecules involved in the same pathway, such as WS6, sc-514, PF184, and IKK16.

7. The composition, kit, or use according to any one of claims 1 to 3, wherein the HDAC inhibitor is selected from small molecules involved in the same pathway, such as trichostatin A (TSA), valproic acid (VPA), vorinostat (SAHA), and entinostat (MS-275).

8. The composition, kit, or use according to any one of claims 1 to 3, wherein the histone methyltransferase inhibitor is selected from small molecules involved in the same pathway, such as BIX01294, 3-deazaneplanocin A (DZNeP) HCl, A-366, UNC0638, and SGC0946.

9. The composition, kit, or use according to any one of claims 1 to 3, wherein the Src kinase inhibitor is selected from small molecules involved in the same pathway, such as dasatinib (BMS-354825), WH-4-023, ponatinib (AP24534), bosutinib (SKI-606), etc.

10. The composition, kit or use according to any one of claims 1 to 3, wherein the cAMP activator is selected from small molecules involved in the same pathway, such as colforsin (forskolin, HL362) and 8-Br-cAMP.

11. The composition, kit, or use according to any one of claims 1 to 3, wherein the cellular metabolism modulator is selected from cellular metabolism modulators such as 2-deoxy-D-glucose (2-DG), sodium acetate, sodium L-lactate, and D-ribose.

12. A culture medium comprising the composition of claim 1.

13. 13. The culture medium of claim 12, comprising a basal culture medium.

14. The culture medium according to claim 13, wherein the basal culture medium is selected from common basal culture media such as DMEM, knockout DMEM, RPMI1640, and DMEM / F12.

15. A method for producing induced totipotent stem cells, comprising the step of culturing pluripotent stem cells in the culture medium of claim 12, thereby producing induced totipotent stem cells.

16. 16. The method of claim 15, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

17. 16. The method of claim 15, comprising reprogramming a non-pluripotent cell into a pluripotent stem cell.

18. 18. The method of claim 17, wherein the non-pluripotent cells are selected from somatic cells and / or adult stem cells.

19. 18. The method of claim 17, wherein the step of reprogramming a non-pluripotent cell into a pluripotent stem cell comprises expressing in the non-pluripotent cell one or more reprogramming factors selected from the group consisting of Oct4, Sox2, Klf4, and c-Myc.

20. 16. The method of claim 15, wherein culturing the pluripotent stem cells is carried out for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

21. A culture comprising the culture medium of claim 12 and pluripotent stem cells.

22. 22. The culture of claim 21, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

23. A culture comprising the culture medium of claim 12 and totipotent stem cells.

24. 24. The culture of claim 23, wherein the totipotent stem cells are induced totipotent stem cells, preferably produced by the method of claim 15.

25. The following: (a) increased transcription of one or more totipotent transcriptional markers selected from the group consisting of MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763; (b) reduced transcription of one or more pluripotency transcriptional markers selected from the group consisting of POU5f1, ZFP42, NANOG, KLF4, and ESRRB; and (c) the ability to differentiate into extraembryonic cell type(s); Induced totipotent stem cells characterized by one or more of: Preferably, an induced totipotent stem cell that can be produced by the method according to claim 15.

26. 16. An induced totipotent stem cell producible by the method of claim 15.

27. 27. An organism derived from the induced totipotent stem cell of claim 26, which is preferably a rodent or mammal.

28. 27. An organoid produced from the induced totipotent stem cell of claim 26.

29. 27. A tissue generated from the induced totipotent stem cells of claim 26, preferably blood.

30. A differentiated cell differentiated from the induced totipotent stem cell of claim 26, which is preferably a blood cell or an immune cell, such as a T cell or an NK cell.

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