WO2023025302A1 - 诱导全能性干细胞及其制备方法 - Google Patents

诱导全能性干细胞及其制备方法 Download PDF

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WO2023025302A1
WO2023025302A1 PCT/CN2022/115235 CN2022115235W WO2023025302A1 WO 2023025302 A1 WO2023025302 A1 WO 2023025302A1 CN 2022115235 W CN2022115235 W CN 2022115235W WO 2023025302 A1 WO2023025302 A1 WO 2023025302A1
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stem cells
cells
cell
mouse
pluripotent stem
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French (fr)
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丁胜
刘康
胡妍妍
杨媛媛
谭彭丞
马天骅
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Tsinghua University
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Priority to EP22860648.9A priority patent/EP4394030A4/en
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Definitions

  • the invention relates to a combination of small molecule reprogramming reagents for inducing totipotent stem cells, a method for preparing induced totipotent stem cells (ciTotiSC), and induced totipotent stem cells.
  • mammalian embryos begins with the fertilized egg formed by the combination of oocyte and sperm, and then develops into two-cell, four-cell, eight-cell, morula and other stages through cleavage.
  • mice only fertilized eggs and blastomeres at the two-cell stage have the potential for both intraembryonic and extraembryonic development, and have the ability to independently develop into a complete individual, that is, totipotency, which is called totipotent cells (totipotent cell).
  • totipotency which is called totipotent cells (totipotent cell).
  • the embryo develops, totipotent cells differentiate into two cell types, the inner cell mass (ICM) and the trophoblast cell (TE), and the embryo at this time is called a blastocyst.
  • ICM inner cell mass
  • TE trophoblast cell
  • the trophoblast cells are located on the outside of the blastocyst stage embryo and can develop into extraembryonic parts such as the placenta.
  • Trophoblast cells play an important role in the further differentiation of the inner cell mass.
  • the cells of the inner cell mass further differentiate into primitive endoderm (primitive endoderm, PE) and epiblast (epiblast, EPI).
  • PE primary endoderm
  • epiblast epiblast
  • the primitive endoderm further develops into an extraembryonic tissue—the yolk sac
  • the epiblast further develops into various tissues and organs of the fetus, and finally develops into a complete fetus.
  • epiblast cells can only develop into the intraembryonic part, they have lost the ability to develop into the extraembryonic part, and have pluripotency rather than the totipotency of both intraembryonic and extraembryonic development, so they are called pluripotent.
  • Cell pluripotent cell
  • ESCs Embryonic stem cells cultured from the inner cell mass are also pluripotent and can differentiate into all intraembryonic tissues and proliferate indefinitely in vitro.
  • mice pluripotent embryonic stem cell line mESC
  • all cultured mouse embryonic stem cells are in a pluripotent state. People have been trying to obtain stem cells with higher developmental potential in vitro. However, so far, inducing and culturing in vitro totipotent stem cells that are molecularly and functionally similar to totipotent embryonic cells in vivo remains a great challenge.
  • RNA-seq RNA sequencing
  • the inventors found that the totipotent blastomere-like cells are related to totipotent
  • the fertilized egg is not close to the second cell, but is closer to the cells of the post-implantation embryo at a later stage of development, so it cannot be considered as a totipotent cell.
  • the totipotent blastomere-like cells have not been proven to have the ability to independently develop into mouse embryos, and then produce a complete life individual, and do not meet the strict definition of totipotent cells (can independently develop into a complete life body), and therefore cannot be defined as totipotent stem cells.
  • a totipotent cell should meet one or more of the following, preferably two, more preferably all three: 1) the cell is transcriptionally identical to the totipotent embryonic cell, that is, the fertilized egg and the second cell Similar; 2) Further, the cells have bidirectional developmental potential to differentiate into various cell types inside and outside the embryo; 3) Further and most strictly, one or one type of cell can develop into a complete embryo or a living individual.
  • pluripotent stem cells can be induced into totipotent stem cells (referred to herein as ciTotiSC, i.e. chemical induced totipotent stem cells, chemically induced totipotent stem cells, or induced totipotent stem cells for short), and the induction is very fast and effective.
  • ciTotiSC i.e. chemical induced totipotent stem cells, chemically induced totipotent stem cells, or induced totipotent stem cells for short
  • the present invention provides a kind of composition, it comprises:
  • GSK-3 inhibitors One or more of the following: GSK-3 inhibitors, IKK signaling pathway inhibitors, HDAC inhibitors, histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators agent.
  • the invention provides a kit comprising:
  • GSK-3 inhibitors One or more of the following: GSK-3 inhibitors, IKK signaling pathway inhibitors, HDAC inhibitors, histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators agent.
  • the present invention provides (a) RA signaling pathway activator; and (b) one or more of the following: GSK-3 inhibitor, IKK signaling pathway inhibitor, HDAC inhibitor, histone A A base transferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cell metabolism regulator for producing induced pluripotent stem cells.
  • the RA signaling pathway activator is selected from TTNPB, Tretionin/RA/ATRA, AM580, Taza, 9-cis-RA, Acitretin, CD437, Tamibarotene, Tazarotene, Small molecules of the same pathway such as retinoic acid, Isotretinoin, Acitretin sodium, ch55 and AC55649.
  • the GSK-3 inhibitor is selected from the same pathway small molecules such as 1-Azakenpaullone, AZD2858, CHIR99021 and AZD1080.
  • the IKK signaling pathway inhibitor is selected from small molecules of the same pathway such as WS6, sc-514, PF184, and IKK16.
  • the HDAC inhibitor is selected from small molecules of the same pathway such as Trichostatin A (TSA), Valproic acid (VPA), Vorinostat (SAHA) and Entinostat (MS-275) .
  • TSA Trichostatin A
  • VPA Valproic acid
  • SAHA Vorinostat
  • MS-275 Entinostat
  • the histone methyltransferase inhibitor is selected from the same pathways such as BIX 01294, 3-deazaneplanocin A (DZNeP) HCl, A-366, UNC0638 and SGC 0946 Small molecule.
  • the Src kinase inhibitor is selected from the same pathways such as Dasatinib (BMS-354825), WH-4-023, Ponatinib (AP24534), Bosutinib (SKI-606) and the like Small molecule.
  • the cAMP activator is selected from the same pathway small molecules as Colforsin (Forskolin, HL 362) and 8-Br-cAMP.
  • the cell metabolism regulator is selected from cell metabolism regulators such as 2-Deoxy-D-glucose (2-DG), sodium acetate, L-sodium lactate, and D-ribose agent.
  • 2-DG 2-Deoxy-D-glucose
  • sodium acetate sodium acetate
  • L-sodium lactate L-sodium lactate
  • D-ribose agent D-ribose agent
  • the present invention provides a culture medium comprising the composition described herein.
  • the medium comprises basal medium.
  • the basal medium is selected from common basal mediums such as DMEM, Knockout DMEM, RPMI 1640 and DMEM/F12.
  • the present invention provides a method for producing induced pluripotent stem cells, the method comprising culturing pluripotent stem cells in the medium described herein, thereby producing the induced pluripotent stem cells.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • the method comprises reprogramming non-pluripotent cells into pluripotent stem cells.
  • the non-pluripotent cells are selected from somatic cells and/or adult stem cells.
  • said reprogramming non-pluripotent cells into pluripotent stem cells comprises expressing in said non-pluripotent cells selected from Oct4, Sox2, Klf4 and c - one or more reprogramming factors in Myc.
  • the cultured pluripotent stem cells are subjected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20 days.
  • the invention provides a culture comprising the medium described herein and pluripotent stem cells.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • the invention provides a culture comprising the medium described herein and totipotent stem cells.
  • the totipotent stem cells are induced totipotent stem cells, preferably manufacturable according to the methods described herein.
  • the present invention provides an induced totipotent stem cell characterized by one or more of the following:
  • the induced pluripotent stem cells can be produced by the methods described herein.
  • the present invention provides an induced totipotent stem cell, which can be produced by a method according to any one of the preceding claims.
  • the invention provides an organism produced from an induced pluripotent stem cell as described herein, preferably wherein said organism is a rodent or a mammal.
  • the present invention provides an organoid produced from the induced pluripotent stem cells described herein.
  • the present invention provides a tissue produced from an induced pluripotent stem cell as described herein, preferably said tissue is blood.
  • the present invention provides a differentiated cell differentiated from an induced pluripotent stem cell as described herein, preferably wherein said differentiated cell is a blood cell or an immune cell, such as a T cell or NK cell.
  • Figure 1.1 Subculture of mouse induced totipotent stem cells (ciTotiSC) of the present invention.
  • FIG. 1.2 Highly expressed totipotent marker genes of mouse induced totipotent stem cells (ciTotiSC) of the present invention.
  • FIG. 2.1 Gene Set Enrichment Analysis (GSEA) in mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) of the present invention (upper panel);
  • GSEA Gene Set Enrichment Analysis
  • ciTotiSC Mouse induced totipotent stem cells (ciTotiSC) of the present invention and mouse pluripotent embryonic stem cells (mESC), totipotent blastomere-like cells (TBLC), totipotent stem cell-like cells (TLSCs), and expanded potential stem cells (EPSC)
  • mESC mouse pluripotent embryonic stem cells
  • TBLC totipotent blastomere-like cells
  • TSCs totipotent stem cell-like cells
  • EPC expanded potential stem cells
  • high expression of maternal genes ZGA genes, totipotency genes, low expression of pluripotency-specific genes (below);
  • Figure 2.2 Cluster analysis of the whole transcriptome level of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) of the present invention;
  • Figure 2.3 Principal component analysis (PCA) of the whole transcriptome level of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) of the present invention;
  • PCA Principal component analysis
  • FIG. 2.4 Mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) and totipotent blastomere-like cells (TBLC) of the present invention, as well as the specific expression of each stage of embryonic development of normal mouse embryos Gene set enrichment analysis (GSEA);
  • ciTotiSC mouse induced totipotent stem cells
  • mESC mouse pluripotent embryonic stem cells
  • TBLC totipotent blastomere-like cells
  • FIG. 1 Mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (ESC) and totipotent blastomere-like cells (TBLC) of the present invention, as well as single-cell RNA sequencing (scRNA) of normal mouse embryos at various stages -seq) UMAP analysis;
  • ciTotiSC mouse induced totipotent stem cells
  • ESC mouse pluripotent embryonic stem cells
  • TBLC totipotent blastomere-like cells
  • scRNA single-cell RNA sequencing
  • FIG. 2.6 Chromatin accessibility sequencing (ATAC-seq) analysis of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) of the present invention
  • FIG. 2.7 Chromatin accessibility sequencing (ATAC-seq) analysis of specific sites of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) of the present invention;
  • FIG. 2.8 RRBS analysis of the genome methylation level of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC);
  • Figure 2.9 Permethylation principal component analysis of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC) based on RRBS data;
  • Figure 2.10 Analysis of methylation levels near specific sites in the genome of mouse induced totipotent stem cells (ciTotiSC) and mouse pluripotent embryonic stem cells (mESC);
  • FIG.11 Analysis of the metabolome of mouse induced totipotent stem cells (ciTotiSC);
  • Figure 3.1 Schematic flow chart of trophectoderm stem cell differentiation experiment
  • FIG. 3.2 RT-qPCR detection of transcription of mouse trophectoderm stem cell-specific genes in mouse induced totipotent stem cells (ciTotiSC), mouse embryonic embryonic stem cells (mESC) and mouse potential expanded pluripotent stem cells (mEPSC) ;
  • FIG. 3.3 Immunofluorescence staining detection of expression of mouse trophectoderm stem cell-specific proteins in mouse induced totipotent stem cells (ciTotiSC), mouse embryonic embryonic stem cells (mESC) and mouse potential expanded pluripotent stem cells (mEPS) ;
  • FIG 3.4 Expression analysis of pluripotent genes and pluripotent genes of different generations (P1-P8) totipotent mouse induced totipotent stem cells (ciTotiSC) after being replaced with mESC medium (2i/LIF);
  • Figure 4.1 Immunofluorescence staining analysis of embryoid body (EB) derived from mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic embryonic stem cells (mESC);
  • EB embryoid body
  • ciTotiSC mouse induced totipotent stem cells
  • mESC mouse embryonic embryonic stem cells
  • Figure 4.3 Analysis of the three germ layer differentiation ability of teratoma derived from mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic embryonic stem cells (mESC);
  • Figure 4.4 Analysis of teratoma extraembryonic lineage differentiation ability of mouse induced totipotent stem cells (ciTotiSC);
  • Figure 5.1 Schematic diagram of chimera experiment flow
  • Figure 5.2 Chimerism of mouse totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) at E4.5;
  • Figure 5.3 Chimerism statistics of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) in the inner cell mass (ICM) and trophectoderm (TE);
  • FIG 5.4 Confirmation of classical marker staining of chimeric embryonic trophectoderm (TE) in mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC);
  • TE chimeric embryonic trophectoderm
  • ciTotiSC mouse induced totipotent stem cells
  • mESC mouse embryonic stem cells
  • FIG. 6.1 Immunofluorescent staining analysis of in vivo development of chimeric embryos of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) to E7.5;
  • Figure 7.1 Chimerism analysis of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) chimeric embryos developed to E12.5 in vivo;
  • Figure 7.2 Flow cytometry analysis of the chimeric ratio of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) in each tissue at E12.5;
  • Figure 7.3 Immunofluorescent staining of E12.5 chimeric placenta frozen sections of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) to analyze the co-localization of chimeric cells and placental extraembryonic lineage markers CK8 and proliferin analyze;
  • Figure 7.4 Analysis of chimerism of mouse induced totipotent stem cells (ciTotiSC) and mouse embryonic stem cells (mESC) in three embryonic germ layers (middle, endoderm, and ectoderm) at E12.5;
  • FIG. 8.1 Detection of the developmental potential of a single mouse induced totipotent stem cell (ciTotiSC);
  • FIG. 8 Cell type analysis of mouse induced totipotent stem cells (ciTotiSC) E12.5 embryonic chimerism;
  • FIG. 8 Mouse induced totipotent stem cells (ciTotiSC) have the ability to chimerize to the reproductive ridge and produce healthy chimeric offspring;
  • FIG. 8 Induced blastocysts obtained from mouse induced totipotent stem cells (ciTotiSC);
  • FIG. 8.5 Mouse blastocysts induced by mouse induced totipotent stem cells (ciTotiSC) have three cell lineages of normal blastocysts in vivo;
  • FIG. 8 Mouse blastocysts induced by mouse induced totipotent stem cells (ciTotiSC) can develop post-implantation in vitro;
  • FIG. 8.7 Mouse blastocysts induced by mouse induced totipotent stem cells (ciTotiSC) implanted in the mouse uterus and further developed;
  • FIG. 1 The role of Dux and p53 in the induction of totipotent stem cells (ciTotiSC);
  • FIG.2 2C:tdTomato+ cell ratio of mouse totipotent stem cells (ciTotiSC) induced by various small molecule combinations;
  • FIG.3 2C: tdTomato+ and OCT4 expression tests of various common basal media used to induce mouse totipotent stem cells (ciTotiSC);
  • FIG. 10 Effects of various small molecules on gene expression in the induction of totipotent cells.
  • the present invention realizes the induction of pluripotent stem cells to produce pluripotent stem cells meeting one or more conditions in the generally accepted definition.
  • the totipotent stem cells cultured by the inventors showed 1) transcriptome characteristics similar to those of mouse embryonic zygotes and blastomeres at the two-cell stage, with changes in chromatin accessibility, DNA methylation levels, and cell metabolism to a level similar to that of totipotent cells in vivo. 2) Experiments of direct differentiation of monolayer cells in vitro, differentiation of embryoid bodies in suspension and differentiation of teratoma in vivo have proved that the totipotent stem cells cultured by the inventors have the ability to differentiate to some extraembryonic cells, which is not available in pluripotent stem cells. ability.
  • the in vivo chimerism experiment also strongly proves that the totipotent stem cells cultured by the inventors have high-efficiency bidirectional developmental potential to intraembryonic and extraembryonic tissues.
  • the induced pluripotent stem cells can be independently induced to develop into mouse blastocysts in vitro, and can correctly express mouse blastocyst marker genes.
  • the induced blastocysts can exhibit a series of characteristics after implantation in vitro; the induced blastocysts can also be implanted into the uterus to continue to develop after being transplanted into mice. Therefore, the mouse induced pluripotent stem cells have the potential to independently develop into a complete life without going through the traditional sperm-egg combination.
  • compositions and methods contemplated herein enable the production of compliant totipotent stem cells suitable for industrial and clinical use.
  • RNA Interference Technology from Basi c 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., NY); Gene Transfer Vectors For Mammalian Cells (
  • the term "about” or “approximately” refers to a change of up to 15%, 10%, or 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in quantity, level, value, amount, frequency, percentage, dimension, size, volume, weight or length.
  • the term "about” or “approximately” refers to ⁇ 15%, ⁇ 10%, ⁇ 9% around a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length , ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% amount, level, value, amount, frequency, percentage, scale, size, amount , weight or length range.
  • the term “substantially/essentially” means about 90%, 91%, compared to a reference amount, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater in quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length.
  • the term “substantially the same” refers to a quantity, level, value, amount, frequency, A percentage, measure, size, amount, weight, or length range.
  • the term "substantially free” when used to describe a composition such as a cell population or a culture medium means free of a specified substance, for example 95% free, 96% free, 97% free, 98% free A composition that is free, 99% free of the specified substance, or is undetectable as measured by conventional means.
  • a similar meaning applies to the term “absent” when referring to the absence of a particular substance or component of the composition.
  • the term “substantial” refers to an amount, level, value, amount, frequency, percentage, dimension, size, amount, weight or length range that is readily detectable by one or more standard methods.
  • the terms “not-appreciable” and “unappreciable” and equivalents mean an amount, level, value, amount, frequency, percentage that is not readily detectable or detectable by standard methods , scale, size, volume, weight, or length range. In one embodiment, an event is not substantial if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less.
  • Consisting of means including, but limited to, anything following the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
  • Consisting essentially of is meant to include any of the elements listed after the phrase “consisting essentially of” and is limited to activities or actions specified in the disclosure that do not interfere with or contribute to the listed elements other elements. Thus, the phrase “consisting essentially of” is to indicate that the listed elements are required or mandatory, but that no other elements are optional, and depending on whether they affect the activities or actions of the listed elements and may or may not exist.
  • references to "one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “an embodiment,” “another embodiment,” or “further embodiments” A combination thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.
  • the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment.
  • the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
  • ex vivo generally refers to activities that take place outside a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside the living organism, preferably with minimal changes from natural conditions.
  • "ex vivo" procedures involve living cells or tissues obtained from an organism and cultured in a laboratory apparatus, usually under sterile conditions, and usually for several hours or up to about 24 hours, but including up to 48 or 72 hours, depending on circumstances. In certain embodiments, such tissues or cells can be harvested and frozen, then thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than several days using living cells or tissues are generally considered "in vitro," although in certain embodiments the term is used interchangeably with ex vivo.
  • in vivo generally refers to activities that occur within an organism.
  • reprogramming or “dedifferentiation” or “increasing the potential of a cell” or “increasing developmental potential” refers to a method or process that increases the potential of a cell or dedifferentiates a cell into a less differentiated state.
  • a cell with increased cellular potential has more developmental plasticity (ie, differentiates into more cell types) than the same cell in a non-reprogrammed state.
  • a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.
  • reprogramming comprises reprogramming pluripotent stem cells to totipotent stem cells.
  • reprogramming comprises reprogramming non-pluripotent stem cells to pluripotent stem cells.
  • reprogramming comprises reprogramming non-pluripotent stem cells to pluripotent stem cells.
  • the term "potency" refers to the sum of all developmental options available to a cell (ie, developmental potential).
  • cellular potential is a continuum ranging from the most plastic cells, i.e. totipotent stem cells, which have the most developmental potential, to the least plastic cells, i.e. terminally differentiated cells, which have the least developmental potential.
  • the continuum of cellular potential includes, but is not limited to, totipotent, pluripotent, multipotent, oligopotent, unipotent, and terminally differentiated cells.
  • embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).
  • the term "totipotent” means that the cells meet one or more, preferably two, and more preferably all three of the following: 1) the cells are transcriptionally identical to totipotent embryonic cells, i.e. fertilized eggs and The two cells are similar; 2) further, the cells have the potential to develop into both intraembryonic and extraembryonic cell types; 3) further and most strictly, one or one cell can develop into a complete embryo or a living individual.
  • the induced pluripotent stem cells of the present invention exhibit 1) transcriptome characteristics similar to mouse embryonic zygotes and two-cell stage blastomeres, in terms of chromatin accessibility, DNA methylation level and cell metabolism are transformed to a level similar to that of totipotent cells in vivo. 2) Experiments of direct differentiation of monolayer cells in vitro, differentiation of embryoid bodies in suspension and differentiation of teratoma in vivo have proved that the totipotent stem cells cultured by the inventors have the ability to differentiate to some extraembryonic cells, which is not available in pluripotent stem cells. ability.
  • the in vivo chimerism experiment also strongly proves that the totipotent stem cells cultured by the inventors have high-efficiency bidirectional developmental potential to intraembryonic and extraembryonic tissues.
  • the induced pluripotent stem cells can be independently induced to develop into mouse blastocysts in vitro, and can correctly express mouse blastocyst marker genes.
  • the induced blastocysts can exhibit a series of characteristics after implantation in vitro; the induced blastocysts can also be implanted into the uterus to continue to develop after being transplanted into mice. Therefore, the mouse induced pluripotent stem cells have the potential to independently develop into a complete life without going through the traditional sperm-egg combination.
  • Totipotency can be determined, in part, by evaluating totipotency characteristics of cells.
  • Totipotent features include, but are not limited to: (1) totipotent stem cell morphology; (2) increased transcription of pluripotent transcriptional markers, such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763; (3 ) reduced transcription of pluripotency transcriptional markers, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB; (4) ability to differentiate into intraembryonic cell types; (5) ability to differentiate into extraembryonic cell types; (6) The ability to develop into an independent individual.
  • Induced pluripotent stem cells of the invention can be characterized by one or more of these characteristics. These pluripotent characteristics of the induced totipotent stem cells of the invention can be compared with natural or artificial totipotent stem cells and/or natural or artificial pluripotent stem cells, for example, with the natural and/or compared with embryonic stem cells or induced pluripotent stem cells; or compared with control totipotent and/or pluripotent stem cells lacking the same culture conditions.
  • a given culture condition results in increased or decreased transcription of a transcriptional marker, which may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9 compared to an appropriate control ,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.
  • a transcriptional marker which may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9 compared to an appropriate control ,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.
  • gene expression or “gene transcription” refers to the relative level of expression/transcription and/or expression/transcription pattern of a gene in a biological sample such as a totipotent cell or a population of cells comprising totipotent cells.
  • the totipotent cells are induced totipotent stem cells.
  • detecting expression/transcription means determining the amount or presence of an RNA transcript of a gene or its expression product.
  • Methods for detecting gene expression/transcription i.e., gene expression/transcription profiling, including polynucleotide-based hybridization analysis methods, polynucleotide-based sequencing methods, immunohistochemical methods, and proteomics-based methods method.
  • the methods generally detect the expression/transcript product (eg, mRNA) of a gene of interest.
  • PCR-based methods such as reverse transcription PCR (RT-PCR) (Weis et al., TIG8:263-64, 1992), and array-based methods such as microarrays (Schena et al., Science 270:467 -70, 1995).
  • RT-PCR reverse transcription PCR
  • array-based methods such as microarrays (Schena et al., Science 270:467 -70, 1995).
  • “Adherent” refers to the attachment of cells to a container, eg, to a sterile plastic (or coated plastic) cell culture dish or flask, in the presence of an appropriate medium. Certain types of cells cannot be maintained or grow in culture unless they adhere to the cell culture vessel. Certain classes of cells (“non-adherent cells”) are maintained and/or proliferated in culture without attachment.
  • Cell culture refers to the maintenance, growth and/or differentiation of cells in an in vitro setting.
  • Cell culture medium refers to nutritional compositions for growing cell cultures.
  • “Culture” or “cell culture” refers to a cultured substance, such as a cell, and/or a medium in which a cultured substance, such as a cell, is present.
  • “Cultivate” refers to the maintenance, propagation (growth) and/or differentiation of cells outside a tissue or organism, eg, in sterile plastic (or coated plastic) cell culture dishes or flasks. “Cultivation” may utilize a culture medium as a source of nutrients, hormones, and/or other factors that help to propagate and/or maintain cells.
  • dissociated cells refer to cells that have been substantially separated or purified from other cells or surfaces (eg, the surface of a culture plate).
  • cells can be dissociated from animals or tissues by mechanical or enzymatic methods.
  • cells aggregated in vitro can be dissociated from each other enzymatically or mechanically, for example by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters.
  • adherent cells are dissociated from a culture plate or other surface. Dissociation may thus involve disrupting the interaction of cells with the extracellular matrix (ECM) and substrate (eg, culture surface), or disrupting the ECM between cells.
  • ECM extracellular matrix
  • substrate eg, culture surface
  • the term “enrich” refers to increasing the amount of a specified component in a composition, such as a composition of cells, and "enriched" when used to describe a composition of cells, such as a population of cells, is refers to a population of cells that has a proportionally increased amount of a specified component compared to the proportion of such component in the population of cells prior to enrichment.
  • a composition such as a cell population
  • can be enriched for a target cell type i.e., cells with a specified characteristic
  • target cell type i.e., cells with a specified characteristic
  • Cell populations can be enriched for target cell types by cell selection and sorting methods known in the art.
  • the population of cells is enriched by sorting or selection methods.
  • the method of enriching with respect to the target cell population enriches the cell population by at least about 20% with respect to the target cell population, thereby meaning that the enriched cell population contains more cells than the cell population before the cell population was enriched. Proportional to approximately 20% more target cell types.
  • the method of enriching the target cell population proportionally enriches the cell population by at least about 30+%, 40+%, 50+%, 60+%, 70+%, 80% relative to the target cell population %, 85%, 90%, 95%, 97%, 98%, or 99%, or at least about 98%, or in particular embodiments, about 99%.
  • a population of cells is enriched for the amount of totipotent cells or cells exhibiting characteristics of totipotency.
  • the population of cells undergoing reprogramming is enriched for target cells with pluripotency characteristics, such as the expression of pluripotency markers, including but not limited to MERVL, Zscan4c, Zscan4d , Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763.
  • the enriched cells comprise a distinct gene or protein expression profile, such as cell surface expression of one or more pluripotency markers such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763 .
  • the population of cells 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.
  • the method of enriching a population of cells for pluripotent cells comprises sorting the population of cells based on cell surface expression of pluripotency markers such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763 , and fractions of cells expressing such markers are collected to obtain a cell population enriched for totipotent cells.
  • pluripotency markers such as MERVL, Zscan4c, Zscan4d, Zscan4f, Zfp352, Tcstv1, Tcstv3, Teme92, Gm6763 , and fractions of cells expressing such markers are collected to obtain a cell population enriched for totipotent cells.
  • cell populations are sorted based on cell surface expression of pluripotent cell markers such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, and depleting such cell populations to obtain cells enriched in pluripotent cells population, making the cell population rich in totipotent cells.
  • pluripotent cell markers such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, and depleting such cell populations to obtain cells enriched in pluripotent cells population, making the cell population rich in totipotent cells.
  • feeder cell is used to describe one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow because the feeder cell
  • the cells provide growth factors and nutrients to support the second cell type.
  • Feeder cells are optionally from a different species than the cells they support.
  • certain types of human cells including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts.
  • feeder cells can often be inactivated by irradiation or treatment with anti-mitotic agents such as mitomycin C to prevent them from outgrowing the cells they support.
  • one particular feeder cell type may be human feeder cells, such as human skin fibroblasts.
  • Another feeder cell type may be mouse embryonic fibroblasts (MEFs).
  • a “feeder-free” (FF) environment refers to an environment such as a cell culture or culture medium that is substantially free of feeder cells and/or has not been preconditioned by feeder cell culture.
  • Preconditioned medium refers to medium that is harvested after the feeder cells have been cultured in the medium for a period of time, such as at least one day. Preconditioned media contain a number of mediator substances, including growth factors and cytokines secreted by feeder cells grown in the media.
  • Genomic stability refers to the ability of cells to faithfully replicate DNA and maintain the integrity of the DNA replication process.
  • the terms “genomically stable cells” and “cells with genomic stability” refer to cells that exhibit a certain frequency of mutations and chromosomal abnormalities such as translocations, aneuploidies, copy number variations, and duplications , said frequency being substantially similar to the frequency of mutations and chromosomal abnormalities relative to normal human cells.
  • “Ingredient” means any compound or other material, whether chemical or biological in origin, that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation.
  • component means any compound or other material, whether chemical or biological in origin, that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation.
  • component means any compound or other material, whether chemical or biological in origin, that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation.
  • component nutrient
  • “ingredient” are used interchangeably.
  • Conventional ingredients for cell culture media may include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like.
  • Other components that promote and/or maintain ex vivo or in vitro cell culture can be selected by those of ordinary skill in the art as needed for the desired effect.
  • Isolate means to separate and collect a composition or material from its natural environment, eg, separation of individual cells or cell cultures from a tissue or body.
  • a cell population or composition is substantially free of cells and materials with which it is associated in nature.
  • isolated or purified or “substantially pure” with respect to a target cell population means at least about 50%, at least about 75%, at least about 85% of the target cells making up the total cell population , at least about 90%, and in particular embodiments, at least about 95% pure cell population.
  • the purity of a cell population or composition can be assessed by appropriate methods well known in the art.
  • a substantially pure population of totipotent cells refers to at least about 50%, at least about 75%, at least about 85%, at least about 90%, and in particular embodiments, at least about A population of cells that is about 95%, and in certain embodiments, about 98% pure.
  • Passage refers to the act of subdividing and plating cells onto multiple cell culture surfaces or vessels when the cells have proliferated to the desired extent. In some embodiments, “passaging” refers to subdividing, diluting, and plating cells. When cells are passaged from a primary culture surface or vessel to a subsequent set of surfaces or vessels, the subsequent culture may be referred to herein as “subculture” or “first passage” or the like. Each subdivision and plating into a new culture vessel is considered a passage.
  • Platinum refers to placing one or more cells into a culture vessel such that the cells adhere to and spread on the cell culture vessel.
  • Pluripotency factor refers to an agent that, alone or in combination with other agents, increases the developmental potential of a cell to the extent of pluripotency.
  • Pluripotency factors include, but are not limited to, polynucleotides, polypeptides and small molecules capable of increasing the developmental potential of a cell to the extent of pluripotency.
  • Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.
  • Totipotency factor refers to an agent that, alone or in combination with other agents, increases the developmental potential of a cell to the extent of pluripotency.
  • Totipotency factors include, but are not limited to, polynucleotides, polypeptides and small molecules capable of increasing the developmental potential of a cell to the extent of totipotency.
  • Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.
  • Proliferation refers to the property of a cell dividing into two substantially equal cells or a population of cells increasing in number (eg, to replicate).
  • Propagation refers to growing (eg, replicating via cell proliferation) cells outside a tissue or body, eg, in sterile containers, eg, plastic (or coated plastic) cell culture dishes or flasks.
  • Primary culture refers to cells, tissues and/or cultures in which isolated cells are placed in a first culture vessel with culture medium. However, said cells, tissue and/or culture may be maintained and/or may be proliferated as long as said cells, tissue and/or culture remain in said first container. Such cells, tissues and/or cultures are referred to as primary cultures.
  • small molecule reprogramming agent or "small molecule reprogramming compound” are used interchangeably herein to refer to small molecules that, alone or in combination with other factors, can increase the developmental potential of a cell.
  • 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 may be used as a source of small molecules in certain embodiments.
  • one or more cells can be cultured, dissociated and passaged using the compositions and methods contemplated herein.
  • single cells are cultured, dissociated and passaged using the compositions and methods contemplated herein.
  • a cell population or plurality of cells is cultured, dissociated and passaged using the compositions and methods contemplated herein.
  • Starting cells suitable for particular embodiments may be derived from essentially any suitable source, heterogeneous or homogeneous with respect to cell type or state of pluripotency. Suitable such cells include fetal cells and adult cells. In addition, suitable said cells may be mammalian in origin, eg from rodents, cats, dogs, pigs, goats, sheep, horses, cattle, or primates, eg humans. In one embodiment, the cells are human cells.
  • the cells may be somatic cells, non-pluripotent, incompletely or partially pluripotent stem cells, pluripotent cells, oligopotent cells, unipotent cells, terminally differentiated cells, or a mixed population of cells comprising any combination of the foregoing.
  • Pluripotent cells suitable for use in certain embodiments include, but are not limited to, naturally occurring stem cells, embryonic stem cells, or iPSCs.
  • a "mixed" cell population is one that has varying degrees of developmental potential.
  • a mixed population of cells may comprise cells undergoing reprogramming such that the mixed population comprises pluripotent cells, partially pluripotent cells, and non-pluripotent cells such as fully differentiated cells, such as somatic cells.
  • pluripotent stem cells are used to induce totipotent stem cells described herein.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • the pluripotent stem cells are reprogrammed from non-pluripotent stem cells.
  • the non-pluripotent stem cells are selected from somatic and/or adult stem cells.
  • reprogramming non-pluripotent cells into pluripotent stem cells comprises expressing one or more reprogramming agents selected from Oct4, Sox2, Klf4, and c-Myc in said non-pluripotent cells factor.
  • the starting cell population is selected from adult or neonatal stem/progenitor cells.
  • the starting population of stem/progenitor cells is selected from the group consisting of: mesoderm stem/progenitor cells, endoderm stem/progenitor cells, and ectoderm stem/progenitor cells.
  • mesoderm stem/progenitor cells include, but are not limited to: mesoderm 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, kidney stem/progenitor cells, osteoblast stem/progenitor cells, chondrocyte stem/progenitor cells, etc.
  • HSC hematopoietic Stem/progenitor cells
  • 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.
  • endoderm stem/progenitor cells include, but are not limited to, hepatic stem/progenitor cells, pancreatic stem/progenitor cells, epithelial stem/progenitor cells, and the like.
  • the starting cell population can 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, fat cells, kidney cells, lung cells, chondrocytes, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, endothelial cells, etc.
  • a culture platform that can be used to induce pluripotency and induce pluripotent stem cells
  • the present invention provides a culture platform that can be used to induce pluripotency and induce pluripotent stem cells, which employs specific small molecule reprogramming reagents.
  • the invention provides a composition comprising:
  • GSK-3 inhibitors One or more of the following: GSK-3 inhibitors, IKK signaling pathway inhibitors, HDAC inhibitors, histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators agent.
  • the invention provides a kit comprising:
  • GSK-3 inhibitors One or more of the following: GSK-3 inhibitors, IKK signaling pathway inhibitors, HDAC inhibitors, histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators agent.
  • the present invention provides (a) RA signaling pathway activators; and (b) one or more of the following: GSK-3 inhibitors, IKK signaling pathway inhibitors, HDAC inhibitors, histone A A base transferase inhibitor, a Src kinase inhibitor, a cAMP activator, and a cell metabolism regulator for producing induced pluripotent stem cells.
  • RA (Retinoic acid, Retinoic acid) signaling pathway activator may be various agents capable of activating RA pathway.
  • Exemplary RA signaling pathway activators include, but are not limited to, TTNPB, Tretionin/RA/ATRA, AM580, Taza, 9-cis-RA, Acitretin, CD437, Tamibarotene, Tazarotene, retinoic acid, Isotretinoin, Acitretin sodium, ch55 and AC55649.
  • the RA signaling pathway activator is selected from TTNPB, Tretionin/RA/ATRA, AM580, Taza, 9-cis-RA, Acitretin, CD437, Tamibarotene, Tazarotene, retinoic acid, Isotretinoin, Acitretin sodium, ch55 and AC55649.
  • the RA signaling pathway activator is TTNPB, as shown in the following formula.
  • GSK-3 (glycogen synthase kinase-3, Glycogen synthase kinase-3) inhibitors may be various agents capable of inhibiting GSK-3.
  • GSK-3 inhibitors include, but are not limited to, 1-Azakenpaullone, AZD2858, CHIR99021 and AZD1080.
  • the GSK-3 inhibitor is selected from 1-Azakenpaullone, AZD2858, CHIR99021 and AZD1080.
  • the GSK-3 inhibitor is 1-Azakenpaullone, represented by the formula below.
  • the IKK (I ⁇ B kinase/NF- ⁇ B, IKK/NF- ⁇ B)) signaling pathway inhibitor may be various 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.
  • the IKK signaling pathway inhibitor is selected from WS6, sc-514, PF184, and IKK16.
  • the IKK signaling pathway inhibitor is WS6, as shown in the following formula.
  • HDAC (Histone deacetylase, Histone deacetylase) inhibitors may be various agents capable of inhibiting HDAC.
  • Exemplary HDAC inhibitors include, but are not limited to, Trichostatin A (TSA), Valproic acid (VPA), Vorinostat (SAHA), and Entinostat (MS-275).
  • TSA Trichostatin A
  • VPA Valproic acid
  • SAHA Vorinostat
  • MS-275 Entinostat
  • the HDAC inhibitor is selected from Trichostatin A (TSA), Valproic acid (VPA), Vorinostat (SAHA), and Entinostat (MS-275).
  • Histone methyltransferase inhibitors can be a wide variety of agents capable of inhibiting histone methyltransferases.
  • Exemplary histone methyltransferase inhibitors include, but are not limited to, BIX 01294, 3-deazaneplanocin A(DZNeP)HCl, A-366, UNC0638, and SGC 0946.
  • the histone methyltransferase inhibitor is selected from BIX 01294, 3-deazaneplanocin A(DZNeP)HCl, A-366, UNC0638, and SGC0946.
  • Src kinase inhibitors can be a wide variety of agents capable of inhibiting Src.
  • Exemplary Src kinase inhibitors include, but are not limited to, Dasatinib (BMS-354825), WH-4-023, Ponatinib (AP24534), Bosutinib (SKI-606).
  • the Src kinase inhibitor is selected from Dasatinib (BMS-354825), WH-4-023, Ponatinib (AP24534), Bosutinib (SKI-606).
  • 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, HL 362) and 8-Br-cAMP.
  • the cAMP activator is selected from Colforsin (Forskolin, HL 362) and 8-Br-cAMP.
  • Regulators of cellular metabolism can be a wide variety of agents capable of modulating cellular metabolism.
  • Exemplary regulators of cell metabolism include, but are not limited to, 2-Deoxy-D-glucose (2-DG), sodium acetate, sodium L-lactate, and D-ribose.
  • the modulator of cellular metabolism is selected from 2-Deoxy-D-glucose (2-DG), sodium acetate, sodium L-lactate, and D-ribose.
  • the amount of small molecule reprogramming reagents in the compositions, kits, media or cultures of the invention can vary and can be optimized for specific culture conditions, including the specific molecules and combinations used, the cells cultured in the media types, and specific applications.
  • the small-molecule reprogramming agent is present in the composition, kit, culture medium of the invention at a concentration sufficient to induce pluripotency, improve reprogramming efficiency, increase or maintain cell potential, or induce or maintain ground state pluripotency. medium or culture.
  • the RA signaling pathway activator 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.
  • the RA signaling pathway activator is dosed at 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 ⁇ M or higher or the concentration of any two of the foregoing values in the range of composition exists in the
  • the RA signaling pathway activator is present in the composition, kit, medium or culture of the present invention at a concentration of 0.05-5 ⁇ M, preferably 0.1-1 ⁇ M, more preferably 0.2 ⁇ M.
  • TTNPB is present in the composition, kit, medium or culture of the invention at a concentration of 0.2 ⁇ .
  • a GSK-3 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.
  • the GSK-3 inhibitor is dosed at 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 ⁇ M or higher or the concentration of any two of the foregoing values in the range of composition exists in the combination of the present invention
  • the GSK-3 inhibitor is present in the composition, kit, medium or culture of the invention at a concentration of 0.5-10.0 ⁇ M, preferably 2.0-3.0 ⁇ M, more preferably 2.5 ⁇ M.
  • 1-Azakenpaullone is present in the composition, kit, medium or culture of the invention at a concentration of 2.5 ⁇ .
  • the 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.
  • the IKK signaling pathway inhibitor is dosed at 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 ⁇ M or higher or the concentration of any two of the foregoing values in the range of composition exists in the combination
  • the IKK signaling pathway inhibitor is present in the composition, kit, medium or culture of the present invention at a concentration of 0.1-10.0 ⁇ M, preferably 0.3.0-1 ⁇ M, more preferably 0.5 ⁇ M.
  • WS6 is present in the composition, kit, medium or culture of the invention at a concentration of 0.5 ⁇ .
  • an HDAC 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.
  • the HDAC inhibitor is dosed at 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 ⁇ M or higher or the concentration of any two of the aforementioned values in the composition of the present invention, kits, media or cultures.
  • the histone methyltransferase 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 .
  • the histone methyltransferase inhibitor is dosed at 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 ⁇ M or higher or any two of the foregoing values in the range of concentrations present in this In the composition, kit, medium or culture of the invention.
  • a Src kinase 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.
  • the Src kinase inhibitor is dosed at 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 ⁇ M or higher or the concentration of any two of the aforementioned values in the composition of the present invention , kits, media or cultures.
  • a cAMP activator 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.
  • the cAMP activator is dosed at 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 ⁇ M or higher or the concentration of any two of the aforementioned values in the composition of the present invention, kits, media
  • a modulator of cellular metabolism 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.
  • the modulator of cellular metabolism is dosed at 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 ⁇ M or higher or the concentration of any two of the aforementioned values in the composition of the present invention , kits, media or cultures.
  • compositions, kits, media or cultures of the invention are listed in Table 1.
  • composition, kit or use according to the present invention comprises or more preferably consists of the following: RA signaling pathway activator, GSK-3 inhibitor and IKK signaling pathway inhibitor.
  • the RA signaling pathway activator is TTNPB
  • the GSK-3 inhibitor is 1-Azakenpaullone
  • the IKK signaling pathway inhibitor is WS6.
  • the RA signaling pathway activator is 0.2 ⁇ M TTNPB
  • the GSK-3 inhibitor is 2.5 ⁇ M 1-Azakenpaullone
  • the IKK signaling pathway inhibitor is 0.5 ⁇ M WS6.
  • the invention provides a culture medium comprising a composition described herein.
  • the composition comprises: (a) RA signaling pathway activator; and (b) one or more of the following: GSK-3 inhibitor, IKK signaling pathway inhibitor, HDAC inhibitor, histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and regulators of cell metabolism.
  • media of the invention comprise basal media.
  • basal media include, but are not limited to, DMEM, Knockout DMEM, RPMI 1640, and DMEM/F12.
  • the basal medium is selected from DMEM, Knockout DMEM, RPMI 1640, and DMEM/F12.
  • the culture medium of the invention contains cytokines and/or growth factors. In specific embodiments, the culture medium of the invention is substantially free or free of cytokines and/or growth factors. In certain embodiments, the culture medium contains one or more supplements including, but not limited to, serum, extracts, growth factors, hormones, cytokines, and the like.
  • the 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-beta), vascular endothelial growth factor (VEGF), transferrin, various interleukins (eg, IL-1 to E-18) , various colony-stimulating factors (such as granulocyte/macrophage colony-stimulating factor (GM-CSF)), various interferons (such as IFN- ⁇ ), and other cytokines that have effects on stem cells such as stem cell factor (SCF) and Erythropoietin (IL), IGF-
  • cytokines are commercially available, for example, from R&D Systems Minneapolis, Minn, and can be native or recombinant.
  • growth factors and cytokines may be added at concentrations envisioned herein for small molecule reprogramming agents.
  • Any suitable vessel or cell culture vessel can be used as a support for cell culture in basal media and/or cell culture supplements.
  • a matrix coating on the support is not necessary.
  • coating the surface of a culture vessel with an attachment-promoting matrix e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.
  • an attachment-promoting matrix e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.
  • Suitable matrices for culturing and passaging cells include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, naturally occurring cell line production Mixtures of substrates such as Matrigel TM and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminated monolayers.
  • the present invention provides a method of producing induced pluripotent stem cells, the method comprising culturing cells in the medium described herein, thereby producing the induced pluripotent stem cells.
  • the medium comprises a composition described herein.
  • the composition comprises: (a) RA signaling pathway activator; and (b) one or more of the following: GSK-3 inhibitor, IKK signaling pathway inhibitor, HDAC inhibitor, Histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators.
  • Cells used as starting material in the methods of the invention can be a wide variety of cells as described herein.
  • the methods of the invention may start from pluripotent cells, such as pluripotent stem cells, such as induced pluripotent stem cells, or the methods of the invention may start from non-pluripotent cells, such as non-pluripotent stem cells, such as somatic cells.
  • the methods of the invention comprise culturing pluripotent stem cells in a medium as described herein, thereby producing said induced pluripotent stem cells.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • the methods of the invention comprise culturing non-pluripotent stem cells in a medium as described herein, thereby producing said induced pluripotent stem cells.
  • the method comprises reprogramming non-pluripotent cells into pluripotent stem cells.
  • the non-pluripotent cells are selected from somatic cells and/or adult stem cells.
  • the reprogramming of non-pluripotent cells into pluripotent stem cells includes expressing one or more of Oct4, Sox2, Klf4 and c-Myc in the non-pluripotent cells reprogramming factor.
  • the cultured pluripotent stem cells are subjected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.
  • the invention provides a culture comprising the medium described herein and pluripotent stem cells.
  • the medium comprises a composition described herein.
  • the composition comprises: (a) RA signaling pathway activator; and (b) one or more of the following: GSK-3 inhibitor, IKK signaling pathway inhibitor, HDAC inhibitor, Histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators.
  • the cells contained in the cultures described herein can be a wide variety of cells as described herein.
  • the cells may be starting cells for culturing or inducing 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.
  • the cells may be intermediate or final cells cultured or induced as described herein.
  • the intermediate cells may be cells with various developmental potentials that differ from the starting and final cells.
  • the final cells may be totipotent stem cells as described herein.
  • a culture according to the invention comprises a medium as described herein and pluripotent stem cells.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • a culture according to the invention comprises a medium as described herein and totipotent stem cells.
  • the totipotent stem cells are induced totipotent stem cells.
  • the totipotent stem cells can be produced by the methods described herein.
  • totipotent cells in mammals only exist in embryos in the early stages of development.
  • mice only the zygote and the blastomeres at the two-cell stage have totipotency, and this totipotency will gradually be lost as the embryo develops.
  • the strictest definition of totipotency means that one or one type of cell can develop into a complete embryo or living individual.
  • a broader definition of totipotency means that the cell has the potential to develop into both intraembryonic and extraembryonic cell types. So far, scientists have created mouse stem cell lines with a pluripotent state, but these cells can only develop into cells of the intraembryonic component and do not have the potential to develop into cells of the extraembryonic type.
  • the researchers jointly set a standard for assessing whether cells are truly totipotent; together, they identified four criteria for murine stem cell lines to be pluripotent: 1) the transcriptome properties of these cells or The gene expression profile needs to be closer to the cells of early totipotent embryos than to the cells of later stage embryos; 2) In vitro can differentiate into extraembryonic cell lineages, and then differentiate into extraembryonic cell types; 3) After in vitro induced development , these cells can form blastocysts, and can simulate some early embryonic development events; 4) inject these cells into early mouse embryos, these cells can participate in the differentiation of embryos and extraembryos, and differentiate into normal expression of corresponding genes The cell type of the marker (aka intraembryonic extraembryonic mosaicism).
  • the totipotent stem cells cultured by the inventors showed 1) transcriptome characteristics similar to those of mouse embryonic zygotes and blastomeres at the two-cell stage, with changes in chromatin accessibility, DNA methylation levels, and cell metabolism to a level similar to that of totipotent cells in vivo. 2) Experiments of direct differentiation of monolayer cells in vitro, differentiation of embryoid bodies in suspension and differentiation of teratoma in vivo have proved that the totipotent stem cells cultured by the inventors have the ability to differentiate to some extraembryonic cells, which is not available in pluripotent stem cells. ability.
  • the in vivo chimerism experiment also strongly proves that the totipotent stem cells cultured by the inventors have high-efficiency bidirectional developmental potential to intraembryonic and extraembryonic tissues.
  • the induced pluripotent stem cells can be independently induced to develop into mouse blastocysts in vitro, and can correctly express mouse blastocyst marker genes.
  • the induced blastocysts can exhibit a series of characteristics after implantation in vitro; the induced blastocysts can also be implanted into the uterus to continue to develop after being transplanted into mice. Therefore, the mouse induced pluripotent stem cells have the potential to independently develop into a complete life without going through the traditional sperm-egg combination.
  • the present invention provides an induced totipotent stem cell, which can be produced by the methods described herein.
  • the method comprises culturing the cells in a medium described herein, thereby producing the induced pluripotent stem cells.
  • the medium comprises a composition described herein.
  • the composition comprises: (a) RA signaling pathway activator; and (b) one or more of the following: GSK-3 inhibitor, IKK signaling pathway inhibitor, HDAC inhibitor, Histone methyltransferase inhibitors, Src kinase inhibitors, cAMP activators, and cell metabolism regulators.
  • Induced pluripotent stem cells produced using the culture platform described herein can be characterized in a variety of ways.
  • Induced pluripotent stem cells may exhibit high expression of pluripotency marker genes and repetitive sequences (e.g. MuERVL, Zscan4, ZFP352, Tstv3, Gm6763) compared to starting cells such as embryonic stem cells (mESC), which means Pluripotent embryonic stem cells undergo a cell fate transition to totipotent stem cells.
  • mESC embryonic stem cells
  • the expression level of one or more of the pluripotency marker gene and the repeat sequence in the induced pluripotent stem cell is 1, 2, 3, 4, 5, 6, 7, 8 compared to the starting cell ,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 .
  • transcriptome sequencing and single-cell RNA sequencing (scRNA-seq) can also be used to analyze the changes in transcription levels after initial cells such as embryonic stem cells acquire pluripotency .
  • This may include analysis of the enrichment of pluripotency marker genes and pluripotency marker genes in starting cells such as embryonic stem cells and induced pluripotent stem cells (GSEA analysis).
  • GSEA analysis induced pluripotent stem cells
  • Cluster analysis can be used to analyze the similarity of starting cells such as embryonic stem cells and induced pluripotent stem cells according to the present invention with the whole transcriptome level of various stages of embryonic development. It can be found that the induced pluripotent stem cells according to the present invention can approach totipotent one-cell and two-cell stage embryos at the whole transcriptome level, so they are totipotent at the transcription level.
  • the induced pluripotent stem cells according to the present invention can be between the totipotent one-cell and two-cell stage embryos in terms of developmental stage, while the starting cells such as embryonic stem cells are more Blastocysts approaching later stages of development.
  • the enrichment of induced pluripotent stem cells according to the present invention relative to starting cells such as embryonic stem cells can be analyzed by analyzing the specifically expressed gene sets of embryonic development fertilized egg, 2-cell stage, 4-cell stage, 8-cell stage, and 16-cell stage , fertilized eggs and pluripotency marker gene sets specifically expressed at the 2-cell stage can be significantly enriched in the induced pluripotent stem cells according to the present invention.
  • UMAP analysis can be performed on the single-cell RNA sequencing (scRNA-seq) results of induced pluripotent stem cells according to the present invention and control cells, and induced pluripotent stem cells according to the present invention can be very close to pluripotent 2-cell embryos, and Highly expressed pluripotent marker genes, low expressed pluripotent marker genes, and pluripotent transcriptome characteristics.
  • scRNA-seq single-cell RNA sequencing
  • ATAC-seq Assay for Transposase-Accessible Chromatin using sequencing
  • Chromatin accessibility indicates activation or repression of genes.
  • the induced pluripotent stem cells according to the present invention can show a closed peak and an open peak at 5 kb near the transcription start site (TSS) in an open or closed state similar to the 2-cell embryo (2C) stage.
  • TSS transcription start site
  • the induced pluripotent stem cells according to the present invention can display more important pluripotent genes and the vicinity of reverse transcription elements, such as Zscan4c, Zscan4d, Zscan4f, ZFP352, MERVL, etc. High openness, while classic pluripotency genes, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, etc., can be closed. This means that induced pluripotent stem cells according to the present invention can have chromatin accessibility similar to that of totipotent 2-cell stage embryos.
  • RRBS Reduced representation bisulfite sequencing
  • Pluripotent stem cells do not have the ability to differentiate into extraembryonic cells.
  • commercial trophoblast stem cells TSC
  • TSC trophoblast stem cells
  • CDX2 trophectoderm stem cell-specific protein
  • the induced pluripotent stem cells according to the present invention can be efficiently induced to significantly express CDX2, and down-regulate the expression of Oct4, thus having the ability to differentiate into extraembryonic part, ie, trophectoderm stem cells.
  • RT-qPCR can be used to analyze the transcription of trophectoderm stem cell-specific genes in the induced pluripotent stem cells according to the present invention during the process of induction into trophectoderm stem cells.
  • the induced pluripotent stem cells according to the present invention can gradually increase the transcription of trophectoderm stem cell-specific marker genes during the induction process, including but not limited to CDX2, Elf5, TFAP2C and Esx1, similar to trophoblast stem cells (TSC) as a positive control.
  • CDX2 is a classical mouse trophectoderm stem cell-specific gene.
  • CDX2-positive cells (representing extraembryonic cells, indicating the differentiation of pluripotent stem cells into extraembryonic cell types) can be detected by immunofluorescence staining, so as to determine the ability of inducing pluripotent stem cells to differentiate into embryoid bodies according to the present invention.
  • the embryoid body differentiation experiment proves that the induced pluripotent stem cells according to the present invention have in vitro the ability to differentiate to the extraembryonic part, ie, trophectoderm stem cells, which pluripotent stem cells do not possess.
  • the teratoma assay is a classic assay to examine the ability of cells to randomly differentiate into the three endoembryonic germ layers and extraembryonic lineages.
  • the ability of inducing pluripotent stem cells to differentiate into teratomas according to the present invention can be determined by observing the results of tissue sections under a microscope to find specific tissue structures of triple germ layers and extraembryonic cell lineages.
  • the teratoma differentiation experiment proves that the induced pluripotent stem cells according to the present invention have the ability to differentiate into extraembryonic cells in vivo, which pluripotent stem cells do not possess.
  • induced pluripotent stem cells according to the invention have the developmental potential to differentiate into both intraembryonic and extraembryonic cell types.
  • the blastocysts of Rosa26-tdTomato mice can be used to establish fluorescent cell lines of mouse induced pluripotent stem cells or mouse embryonic stem cells (mESC), which are used for injection of 8-cell embryos, so that the stem cells can stably express tdTomato fluorescence. If the stem cells injected into the embryo develop with the embryo and differentiate into the intraembryonic or extraembryonic part, fluorescence can be observed in the corresponding intraembryonic or extraembryonic part.
  • mESC mouse embryonic stem cells
  • induced totipotent stem cells can be chimerized into the inner cell mass (ICM) of the embryo and the extraembryonic trophectoderm (TE) ) both, as determined by tdTomato-positive cell mosaicism. Differentiation to extraembryonic cell types was further confirmed by the colocalization of tdtomato fluorescence with CDX2 fluorescence.
  • ICM inner cell mass
  • TE extraembryonic trophectoderm
  • induced pluripotent stem cells according to the present invention In in vivo development experiments of chimeric embryos using induced pluripotent stem cells according to the present invention (for example, to E4.5, E7.5 and E12.5), co-localization of tdtomato fluorescence and CDX2 fluorescence can be observed. It is confirmed that the induced pluripotent stem cells according to the present invention can participate in the development of trophectoderm (TE) and have the ability to differentiate to the extraembryonic part.
  • TE trophectoderm
  • ELF5 extraembryonic ectoderm
  • EPI embryonic ectoderm
  • EPC placental cone
  • ELF5 extraembryonic ectoderm
  • ELF5 extraembryonic ectoderm
  • the induced pluripotent stem cells according to the present invention can be chimerized to embryos (Em), extraembryonic tissue placenta ( Pl) and yolk sac (Yo). It is proved that after embryo implantation, the induced pluripotent stem cells according to the present invention can still have the ability to participate in the development of extraembryonic tissue of the embryo.
  • the ability of the induced pluripotent stem cells according to the present invention to independently develop into blastocysts can be examined using the culture conditions used for inducing blastocysts.
  • Blastocysts induced from induced pluripotent stem cells according to the present invention can have highly similar morphological characteristics to normal blastocysts.
  • blastocysts induced from induced pluripotent stem cells according to the present invention can have three cell lineages of normal blastocysts in vivo, demonstrating that induced pluripotent stem cells according to the present invention can be efficiently induced into structurally correct Blastocysts with correct gene expression.
  • Blastocysts induced from induced pluripotent stem cells according to the present invention can be further cultured in vitro to produce a three-dimensional structure similar to that of post-implantation embryos (e.g. E4.5-E5.5 stage), including Germ layer (stain positive for SOX17) surrounded by ectoderm (stain positive for TFAP2C) and EPI (stain positive for Oct4).
  • Germ layer stain positive for SOX17
  • ectoderm stain positive for TFAP2C
  • EPI stain positive for Oct4
  • the blastocysts induced from the induced pluripotent stem cells according to the present invention can be implanted into the uterus for further development in vivo, and can trigger decidualization after implantation in the uterus and continue to grow.
  • the induced pluripotent stem cells according to the present invention can be characterized by one or more of the above characteristics. Such characterizations can be performed using methods described herein or known to those skilled in the art.
  • the induced pluripotent stem cells according to the present invention can be used in various applications expected in scientific research, industry and clinical practice.
  • a wide variety of products can be produced from the differentiation of induced pluripotent stem cells of the invention, which can be used, for example, to build models, study targets, develop surrogates, and other potential therapeutic or diagnostic applications.
  • the induced pluripotent stem cells according to the present invention can be used to induce the production of organisms.
  • the organism can be used for potential scientific research, treatment and diagnosis applications such as constructing disease models.
  • the invention provides an organism produced from the induced pluripotent stem cells described herein.
  • the organism may be a eukaryote, including but not limited to animals, plants, fungi, and other eukaryotes known in the art.
  • the animals may include, but are not limited to, mammals, such as primates, such as humans, non-human primates, non-primates, bovines, equines, ovines, porcines, canines, Lagodae, lagodae, rodents such as monkeys, cows, sheep, pigs, dogs, rabbits, rats or mice.
  • mammals such as primates, such as humans, non-human primates, non-primates, bovines, equines, ovines, porcines, canines, Lagodae, lagodae, rodents such as monkeys, cows, sheep, pigs, dogs, rabbits, rats or mice.
  • the organism is a rodent or a mammal.
  • the mammal is not a human.
  • the plant may be a monocot or a dicot, and may be a crop or cereal plant such as cassava, corn, sorghum, soybean, wheat, oat or rice.
  • the plants may also be algae, trees or produce plants, fruits or vegetables (for example, trees such as citrus trees, such as orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as Almond or walnut or pistachio trees; nightshades; Brassica; Lactuca; spinach; capsicum; cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).
  • the induced pluripotent stem cells according to the present invention can be used to induce the production of organoids.
  • the organoids can be used for constructing disease models, transplantation therapy or other potential scientific research, treatment and diagnosis applications.
  • the invention provides an organoid produced from the induced pluripotent stem cells described herein.
  • the organoid may be an organoid including, but not limited to, the following organs:
  • the musculoskeletal system including the human skeleton, such as bones, wrist, clavicle, femur, fibula, humerus, mandible, metacarpal, metatarsal, ossicles, patella, phalanges, radius, skull, tarsus, tibia, ulna, ribs, spine, pelvis , sternum, cartilage; joints, such as fibrous joints, cartilaginous joints, synovial joints; muscular system, such as muscles, tendons, diaphragm;
  • Circulatory system including cardiovascular system, such as peripheral blood supply (arteries, veins, lymphatic vessels), heart; lymphatic system, primary (bone marrow, thymus), secondary (spleen, lymph nodes), gum lymphatic system;
  • cardiovascular system such as peripheral blood supply (arteries, veins, lymphatic vessels), heart; lymphatic system, primary (bone marrow, thymus), secondary (spleen, lymph nodes), gum lymphatic system;
  • Nervous system including brain, such as hindbrain (medulla, pons, cerebellum), midbrain, forebrain (diencephalon (retina, optic nerve) brain, limbic system), spinal cord, nerves, sensory system (ear, eye); epidermal system ;
  • Epidermal system including skin, subcutaneous tissue, breast (mammary gland);
  • Immune system including myeloid cells, lymphocytes;
  • Respiratory system including upper respiratory tract (nose, pharynx, larynx), lower respiratory tract (trachea, bronchi, lungs);
  • Digestive system including mouth (saliva, tongue), upper gastrointestinal tract (oropharynx, hypopharynx, esophagus, stomach), lower gastrointestinal tract (small intestine, appendix, large intestine, rectum, anus), accessory digestive glands (liver, biliary tract , pancreas);
  • Urinary system including genitourinary system, kidneys, ureters, bladder, urethra;
  • Reproductive system including female reproductive system (uterus, vagina, vulva, ovary, placenta), male reproductive system (scrotum, penis, prostate, testis, seminal vesicle);
  • Endocrine system including pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland, and pancreatic islets.
  • the induced pluripotent stem cells according to the present invention can be used to induce tissue.
  • the tissue can be used to construct disease models, transplant therapy or other potential scientific research, treatment and diagnosis applications.
  • the invention provides a tissue produced from the induced pluripotent stem cells described herein.
  • Such tissues include, but are not limited to, animal tissues and plant tissues.
  • the animal tissue includes, but is not limited to, epithelial tissue, muscle tissue, nervous tissue, and connective tissue.
  • the connective tissue includes, but is not limited to, proper connective tissue, including loose connective tissue (cellular tissue), dense connective tissue, adipose tissue, reticular connective tissue, elastic connective tissue; bone or cartilage tissue; blood; and lymph. Blood is the fluid tissue that circulates through the cardiovascular system.
  • Lymph is the fluid flowing in the lymphatic vessels, which is formed by the flow of tissue fluid into the lymph. The lymph eventually drains into the veins. Lymph contains lymphocytes, and the composition of lymph is different under different physiological conditions.
  • the tissue is blood.
  • the induced pluripotent stem cells according to the present invention can be used to induce differentiated cells.
  • the differentiated cells can be used to construct disease models, transplant therapy or other potential scientific research, treatment and diagnosis applications.
  • the invention provides a differentiated cell differentiated from an induced pluripotent stem cell as described herein.
  • the differentiated cells may be cells of any organ or tissue mentioned above.
  • the cells are immune cells, more preferably T cells or NK cells.
  • the cells are neural cells, more preferably neurons or glial cells.
  • the cells are blood cells, more preferably red blood cells or white blood cells.
  • Example 1 Induction and preliminary identification of mouse induced pluripotent stem cells
  • mouse pluripotent stem cells after being treated with small molecule reprogramming reagents, have the characteristics of totipotent cells highly similar to mouse fertilized eggs or embryonic cells at the two-cell stage. Such stem cells are called by the inventors Induced totipotent stem cells for mice.
  • mouse pluripotent embryonic stem cells or mouse induced pluripotent stem cells both purchased from commercial channels or established by themselves through standardized experiments
  • mouse pluripotent embryonic stem cells or mouse induced pluripotent stem cells both purchased from commercial channels or established by themselves through standardized experiments
  • 0.05% ( Volume ratio) trypsinized to become suspended single cells and passaged (one generation is enough) at a ratio of about 1:10 to inoculate in totipotent stem cell medium (based on commercialized mouse pluripotent stem cell culture
  • Additional small molecule reprogramming reagents were added to the base.
  • the commercial pluripotent stem cell basal medium used here contained: Knockout DMEM basal medium, and 5% KSR, 1% N2, 0.2% chemically defined lipid concentrate (CDL), 1% GlutaMAX TM (L -glutamine substitute), 1% double antibody (penicillin/streptomycin), 1% non-essential amino acid, 0.1mM ⁇ -mercaptoethanol, 50ng/ml Sodium L-ascorbyl-2-phosphate and 1000U/mL mouse leukemia Inhibitor factor (mLIF) (Yang, Y. et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 169, 243-257.e225(2017).).
  • CDL chemically defined lipid concentrate
  • GlutaMAX TM L -glutamine substitute
  • double antibody penicillin/streptomycin
  • non-essential amino acid 0.1mM ⁇ -mercaptoethanol
  • Various small molecule reprogramming agents are described in the Detailed Description section herein. Some combinations tested are listed in Table 1.
  • Some of the small molecule reprogramming reagents were also individually replaced and tested for performance, as shown in Figure 9.4. It should be noted that the induction ratio of totipotent stem cells is not the only factor for evaluating the combination.
  • the inventors selected the optimal combination, that is, the combination of three small molecule compounds TTNPB, 1-Azakenpaullone and WS6, for other experiments. Add 2.5 ⁇ M 1-Azakenpaullone, 0.5 ⁇ M WS6, and 0.2 ⁇ M TTNPB to the aforementioned commercial pluripotent stem cell basal medium to obtain the pluripotent stem cell medium used below.
  • the inventors also tested various basal media (including DEME, Knockout DMEM, RPMI 1640, and DMEM/F12) to determine whether their type had an effect on the induction of totipotent stem cells. As shown in Figure 9.3, no significant difference was seen in the 2C::td (2C:tdTomato) and OCT4 fluorescence assays. It can be seen that the type of basal medium has no substantial effect on the induction of totipotent stem cells described herein. In other experiments, Knockout DMEM was used.
  • basal media including DEME, Knockout DMEM, RPMI 1640, and DMEM/F12
  • mouse pluripotent embryonic stem cells gradually acquire mouse totipotent stem cell properties, which are characterized below .
  • the expression of the pluripotency gene marker Oct4 in the obtained mouse totipotent stem cells was down-regulated, and the mouse totipotent stem cells could be maintained and subcultured for more than 10 passages without losing their totipotency and maintaining a good clonal morphology ( Figure 1.1) .
  • Dux and p53 are required for totipotent stem cell (ciTotiSC) induction.
  • the inventors knocked out Dux or p53 in mESCs.
  • loss of Dux significantly decreased the proportion of MERVL+ cells in mESCs (Fig. 9.1a), with little increase in TAW-induced MERVL+ cells (see Fig. 9b below).
  • TAW was consistently unable to induce pluripotent genes such as Dux, Zscan4, Zfp352, and Tcstv3 (Fig. 9.1c).
  • p53 knockdown also resulted in a decrease in the percentage of MERVL+ cells in TAW-treated or non-TAW-treated mESCs (Fig. 9.1a and 9.1b). Further analysis revealed that p53 knockdown also reduced the expression of pluripotency marker genes MERVL, Dux, Zscan4, Zfp352, and Tcstv3 compared with TAW-induced WT cells (Fig. 9.1d). Together these lines of evidence suggest that the induction of totipotent stem cells (ciTotiSC) is partially dependent on p53.
  • ciTotiSC totipotent stem cells
  • RT-qPCR reactions were carried out in Bio-Rad CFX384 Real-Time PCR System. The data results were analyzed and graphed in Prism 8 software.
  • mice pluripotent embryonic stem cells displayed pluripotent marker genes and repetitive sequences (MuERVL, Zscan4, ZFP352, Tstv3, Gm6763 ), which means that mouse pluripotent embryonic stem cells have undergone a cell fate transition to mouse totipotent stem cells (Figure 1.2).
  • Example 2 Molecular Biological Identification of Mouse Induced Totipotent Stem Cells: Passed Transcriptome profiles of mouse induced pluripotent stem cells analyzed by RNA-seq and scRNA-seq
  • RNA-seq transcriptome sequencing
  • scRNA-seq single-cell RNA sequencing
  • mice pluripotent embryonic stem cells mESC
  • mouse induced pluripotent stem cells Enrichment GSEA analysis
  • mESC mouse pluripotent embryonic stem cells
  • GSEA mouse induced pluripotent stem cells Enrichment
  • mouse pluripotent embryonic stem cells are more gene-wide transcriptome level Close to the mouse 3.5 day (E3.5) embryonic cell mass (ICM) with pluripotency, so it exhibits a pluripotent state at the transcriptional level; while mouse induced pluripotent stem cells are at the gene-wide transcriptome level It is closer to mouse 1-cell and 2-cell stage embryos with totipotency, and thus presents a totipotent state at the transcriptional level (Fig. 2.2).
  • mouse induced pluripotent stem cells are between mouse 1-cell and 2-cell stage embryos with totipotency, while mouse pluripotent embryonic stem cells (mESC) and mouse expanded potential stem cells (EPS) are closer to mouse blastocysts at a later stage of development (Fig. 2.3).
  • mESC mouse pluripotent embryonic stem cells
  • EPS mouse expanded potential stem cells
  • the expression of induced pluripotent stem cells in mice is relatively different from that in mice. Based on the enrichment of embryonic stem cells, the inventors further confirmed that the pluripotent marker genes specifically expressed in fertilized eggs and 2-cell stage were significantly enriched in mouse induced pluripotent stem cells (Fig. 2.4).
  • mice By inducing totipotent stem cells and totipotent blastomere-like cells in mice (TBLC, Shen, H. et al. Mouse 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).) UMAP analysis of single-cell RNA sequencing (scRNA-seq) results found that mouse induced totipotent stem cells are very close to totipotent 2-cell embryos in mice (shown in Figure 2.5 TPSC and Late 2C), and high expression of marker genes of pluripotency, low expression of marker genes of pluripotency, with characteristics of totipotent transcriptome; while totipotent blastomere-like cells (TBLC, Shen, H. et al.
  • ATAC-seq Assay for Transposase-Accessible Chromatin using sequencing
  • mouse induced totipotent stem cells mouse pluripotent embryonic stem cells
  • mouse 2-cell stage embryos mouse Chromatin accessibility of cell clumps within the blastocyst stage.
  • mice induced totipotent stem cells had a closed peak and an open peak at 5 kb near the transcription start site (TSS) that were similar to mouse 2-cell embryo (2C) stage Similar open or closed state; while mouse pluripotent embryonic stem cells (mESC) are more similar to mouse blastocyst inner cell mass (ICM) (Fig. 2.6).
  • TSS transcription start site
  • mESC mouse pluripotent embryonic stem cells
  • mouse induced pluripotent stem cells near some important pluripotent genes and reverse transcription elements, such as Zscan4c, Zscan4d, Zscan4f, ZFP352, MERVL, etc., showed higher Open; while classic pluripotency genes, such as POU5f1, ZFP42, NANOG, KLF4, ESRRB, etc., are closed ( Figure 2.7).
  • mice induced pluripotent stem cells have similar chromatin accessibility to totipotent 2-cell embryos in mice.
  • RRBS Reduced representation bisulfite sequencing
  • mice pluripotent embryonic stem cells The inventors found that the overall methylation level of mouse pluripotent embryonic stem cells is 23.9%, which is close to the E6.5-E7.5 period after implantation of mouse embryos (the overall methylation levels are 23.2% and 26.6% respectively) ; while the methylation level of mouse induced pluripotent stem cells was significantly reduced to 12.1%, similar to the fertilized egg, 2-cell and 4-cell stages before embryo implantation in mice (the overall methylation levels were 15.4%, 13.2%, respectively. and 14.8%) (Figure 2.8).
  • mice induced totipotent stem cells and mouse totipotent embryos were all hypomethylated Methylation state, while no decrease in methylation was found in mouse pluripotent embryonic stem cells and post-implantation E6.5-E7.5 embryos (Fig. 2.10).
  • ciTotiSC totipotent stem cells
  • pluripotent stem cells ciTotiSC
  • 2C embryos were more inclined to utilize one-carbon metabolism and reduction-state-related pathways for metabolism
  • pluripotent mESCs and blastocysts had higher levels of purine metabolism and mitochondrial tricarboxylic acid (TCA) cycle metabolites, exhibiting a higher oxidation state (Fig. 2.11 bottom).
  • TCA mitochondrial tricarboxylic acid
  • Example 3 Differentiation ability of mouse induced totipotent stem cells to trophoblast cells in vitro Analysis
  • Mouse pluripotent stem cells do not have the ability to differentiate into trophoblast cells.
  • Figure 3.1 shows mouse induced totipotent stem cells, pluripotent embryonic stem cells (mESC, Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519-523, doi: 10.1038/nature06968 (2008).) and potential expansion of pluripotent stem cells (mEPS, Yang, Y. et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 169, 243-257.e225 (2017).) Cultured in trophectoderm stem cells (TSC) Schematic illustration of the differentiation of basal trophoblast stem cells.
  • TSC trophectoderm stem cells
  • mice induced totipotent stem cells of the present invention have the ability to differentiate to the extraembryonic part, ie, trophoblast stem cells, which pluripotent stem cells do not possess.
  • Mouse induced pluripotent stem cells, embryonic stem cells (mESC) and potential expanded pluripotent stem cells (mEPS) cultured on feeder cells (mouse embryonic fibroblasts, which are widely used in stem cell culture) were digested with 0.05% trypsin ), spread the digested cells on a 0.3% gelatin-coated cell culture plate, collect the suspended cells half an hour later, and make the feeder layer cells adhere to the cell culture plate to remove the feeder layer cells.
  • Mouse induced pluripotent stem cells, embryonic stem cells (mESC) and potential expanded pluripotent stem cells (mEPS) were inoculated in the mouse feeder layer at a density of 1 ⁇ 105 cells per well of a 12-well plate.
  • Stem Cell Culture Medium (detailed below). The medium was changed once a day. 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), and commercial trophoblast stem cells (TSC) were used as positive control. Immunofluorescent staining was performed on day 12 to detect the expression of a mouse trophectoderm stem cell-specific protein (CDX2) (Fig. 3.3).
  • CDX2 mouse trophectoderm stem cell-specific protein
  • TSC mouse trophoblast stem cells
  • RPMI 1640 basal medium Gibco, C11875500BT
  • 20% fetal bovine serum 1X GlutaMAX (L-glutamine substitute)
  • 1% double antibody penicillin/streptomycin
  • non-essential amino acids 1X sodium pyruvate (Gibco, 11360070)
  • 1 mM ⁇ -mercaptoethanol 25 ng/ml FGF4 (R&D systems, 235-F4) and 1 ⁇ g/ml heparin (Sigma-Aldrich, H3149).
  • CDX2 is a classic mouse trophoblast stem cell-specific gene. The expression of its protein level was detected by immunofluorescence, and combined with the change of Oct4, a marker of mouse pluripotent stem cells, to determine whether the lineage of mouse trophoblast stem cells had occurred. differentiation.
  • mouse induced pluripotent stem cells could significantly express CDX2 after being induced to differentiate, and down-regulate the expression of Oct4, so they had the ability to differentiate to the extraembryonic part, ie, trophoblast stem cells; while embryonic stem cells ( Although mESC) and potential-expanded pluripotent stem cells (mEPS) can also down-regulate the expression of Oct4 gene after induction and differentiation, they hardly express CDX2, so they do not have the ability to differentiate into extraembryonic part, that is, trophoblast stem cells.
  • mESC embryonic stem cells
  • mEPS potential-expanded pluripotent stem cells
  • totipotent stem cells of different generations (P1-P8) can gradually differentiate into pluripotent embryonic stem cells (rESC ciTotiSC ) after being replaced with mESC medium (2i/LIF), which is reflected in the expression of totipotency genes.
  • rESC ciTotiSC pluripotent embryonic stem cells
  • mESC medium 2i/LIF
  • Blocking add blocking solution (10% donkey serum + 1% BSA + 0.3% Triton-X100, diluted in DPBS) for blocking at room temperature for 1 hour, and wash 3 times with DPBS;
  • the primary antibody is mouse anti-CDX2 (1:150, BioGenex, MU392A-UC). Dilute the antibody with DPBS containing 1% BSA according to the required concentration, incubate at room temperature for 2 hours or overnight at 4°C, wash with DPBS for 5 minutes x 3 times;
  • Secondary antibody incubation the secondary antibody was Donkey anti Mouse 555 (1:500, Life Technology, A-31570). Dilute the corresponding fluorescently labeled secondary antibody with DPBS containing 1% BSA at a ratio of 1:1000, and incubate at room temperature in the dark for 1 hour;
  • mouse induced pluripotent stem cells gradually activated trophoblast stem cell-specific marker genes during the induction and differentiation process, including CDX2, Elf5, TFAP2C and Esx1, which were similar to trophoblast stem cells (TSC) as a positive control.
  • TSC trophoblast stem cells
  • mESC embryonic stem cells
  • mEPS potential-expanding pluripotent stem cells
  • Example 4 Comparison of Mouse Induced Totipotent Stem Cells and Mouse Embryonic Stem Cells (mESC) for embryoid body differentiation in vitro and teratoma differentiation capacity in vivo
  • mice induced pluripotent stem cells and mouse embryonic stem cells After digesting mouse induced pluripotent stem cells and mouse embryonic stem cells (mESC) with 0.05% trypsin, spread them on a 0.3% gelatin-coated cell culture plate, collect the suspension cells half an hour later, and make the feeder layer cells Adhere to the cell culture plate to remove the feeder cells.
  • mESC mouse embryonic stem cells
  • Mouse induced pluripotent stem cells or embryonic stem cells (mESC) that had been removed from the feeder layer were resuspended in mouse embryoid body formation medium (Knockout DMEM basal medium, further Add 10% fetal bovine serum (FBS), 1% GlutaMAX TM (L-glutamine substitute), 1% double antibody (penicillin/streptomycin), 1% non-essential amino acid, 0.1 mM ⁇ -mercaptoethanol). Then the embryoid body formation experiment was carried out by the hanging drop method: the hanging drop was carried out on the cover of the 10cm culture dish, and every 20ul of the cell mixture was one hanging drop, which was placed in a 5% CO2, 37°C incubator for cultivation. Two days later, the cells in the hanging drop were collected and placed in a 6-well low-adsorption culture plate to continue culturing, and embryoid bodies were harvested on days 0, 3, and 6 for immunofluorescence staining identification.
  • CDX2-positive cells (representing extraembryonic trophoblast cells, indicating that totipotent stem cells can develop into extraembryonic cell types) could be detected in embryoid bodies derived from mouse induced pluripotent stem cells on day 6. differentiation), while no CDX2-positive cells were detected in embryoid bodies derived from mouse embryonic stem cells (mESCs) (indicating that pluripotent stem cells did not differentiate to extraembryonic cell types) (Fig. 4.1).
  • mESCs mouse embryonic stem cells
  • Fig. 4.1 mouse embryonic stem cells
  • CDX2 is a classical mouse trophoblast stem cell-specific gene. Embryoid body differentiation experiments have proved that mouse induced pluripotent stem cells have the ability to differentiate to the extraembryonic part, ie, trophoblast stem cells, which pluripotent stem cells do not possess in vitro.
  • the teratoma assay is a classic assay to examine the ability of cells to randomly differentiate into the three endoembryonic germ layers and extraembryonic lineages.
  • mouse induced totipotent stem cells or mouse embryonic stem cells (mESC) under the condition of feeder cell culture were digested into single cells with 0.05% trypsin-EDTA, the mouse induced totipotent stem cells or mouse embryonic stem cells ( mESC) were resuspended in the culture medium, spread it on a 0.3% gelatin-coated cell culture plate, incubated in a 37° C. incubator for 30 minutes, and removed the feeder layer cells.
  • the resuspended cells were injected subcutaneously into the groin of the hind legs of immunodeficient SCID mice, and the number of injected cells per mouse was about 1.0 ⁇ 10 6 .
  • mice Five weeks after subcutaneous injection of the cells, the mice were killed by neck dislocation, the teratomas were taken out from the subcutaneous area, fixed with 4% paraformaldehyde solution, paraffin sectioned and stained with HE.
  • mice mouse induced pluripotent stem cells or mouse embryonic stem cells (mESC) into the subcutaneous of immunodeficiency SCID mice, and both cell lines could form teratomas. After paraffin sections and HE staining of these teratomas, the typical organizational structure of ectoderm (left column), mesoderm (middle column) and endoderm (right column) could be observed under the microscope. This also shows that both mouse induced pluripotent stem cells and mouse embryonic stem cells (mESC) have the ability to differentiate into three germ layers in embryos ( Figure 4.3).
  • mouse induced pluripotent stem cells have the ability to differentiate into extraembryonic cells, which pluripotent stem cells do not possess.
  • Example 5 In vivo chimerism experiment of mouse induced totipotent stem cells (E4.5 embryos in vitro fetal period)
  • Rosa26-tdTomato mouse blastocysts were used to establish mouse induced pluripotent stem cell lines and mouse pluripotent embryonic stem cell lines (mESC) with red fluorescent markers for injection into 8-cell stage embryos. If the stem cells injected into mouse 8-cell embryos developed and differentiated to the intraembryonic or extraembryonic part, red fluorescence (tdTomato) could be observed in the corresponding intraembryonic or extraembryonic part.
  • the two cell lines were respectively digested into single cells with 0.05% trypsin-EDTA, resuspended with the corresponding medium, spread on 0.3% gelatin-coated cell culture plates, and incubated in a 37°C incubator for 30 minutes, remove the feeder cells. Cells were resuspended in the corresponding medium after collection.
  • mice The purchased commercial ICR female mice were subjected to superovulation and mated with commercial ICR male mice in cages. After 1.5 days, 8-cell stage embryos were collected from the oviducts of successful mating female mice. Chimeric embryos were obtained by injecting 5-10 feeder-depleted mouse induced pluripotent stem cells or mouse embryonic stem cells (mESCs) into each 8-cell embryo.
  • mESCs mouse embryonic stem cells
  • mice induced totipotent stem cell chimeric embryos were placed in KSOM medium, And resume culture in 5% CO2, 37 °C incubator for 1-2 hours, then transplant it into the uterus of pseudopregnant ICR female mice 0.5 days after mating with ligated ICR male mice for further development.
  • mouse induced pluripotent stem cells can be chimerized to both the inner cell mass (ICM) of the embryo and the trophectoderm (TE) outside the embryo;
  • Mouse embryonic stem cells (mESC) can only chimerize into the inner cell mass (ICM) of the embryo, but fail to chimerize into the extraembryonic trophectoderm (TE) ( Figure 5.2).
  • Mouse induced pluripotent stem cells chimerism to both TE and ICM: 18/21 (85.7%); chimerism to TE only: 2/21 (9.5%); chimerism to ICM only: 1/21 (4.8%) . That is, mouse induced pluripotent stem cells have dual developmental potentials within the embryo (inner cell mass) and outside the embryo (trophectoderm).
  • Mouse embryonic stem cells chimerism to both TE and ICM: 0/21 (0%); chimerism to TE only: 0/21 (0%); chimerism to ICM only: 21/21 (100% ). That is, mouse pluripotent stem cells only have the ability to develop in the embryo (inner cell mass).
  • 5.3CDX2 is a classic marker of trophectoderm (TE).
  • the inventors further confirmed whether the tdtomato fluorescence-labeled mouse induced totipotent stem cells chimerized into trophectoderm (TE) could express CDX2, a key marker of trophectoderm, by CDX2 immunofluorescence staining.
  • the staining results showed (Fig. 5.4), the cells labeled with tdtomato fluorescence could express CDX2 at the same time.
  • mouse induced pluripotent stem cells are indeed involved in the development of trophectoderm (TE) in E4.5-day chimeric embryos, that is, they have the ability to differentiate to the extraembryonic part.
  • Example 6 In vivo development of mouse induced totipotent stem cell chimeric embryos to E7.5
  • E7.5 embryonic stem cells
  • mice induced pluripotent stem cells could express the embryonic epiblast marker OCT4 and the extraembryonic (embryonic cone EPC and extraembryonic ectoderm EXE) marker ELF5.
  • OCT4 embryonic epiblast marker
  • EXE extraembryonic ectoderm
  • Example 7 In vivo development of mouse induced totipotent stem cell chimeric embryos to E12.5
  • mice Thirteen days after the chimeric embryos obtained by injecting mouse induced pluripotent stem cells or mouse embryonic stem cells (mESC) into embryos at the 8-cell stage were transplanted into pseudopregnant mice, the embryos that developed into E12.5-E13.5 mice after transplantation were isolated Embryo (Em), placenta (Pl) and yolk sac (Yo). See Figure 5.1 for the experimental scheme.
  • mouse induced pluripotent stem cells can be chimerized into mouse embryos (Em) and extraembryonic tissues placenta (Pl) and yolk sac (Yo) in a high proportion ( Figure 7.1 ). This experiment proves that mouse induced pluripotent stem cells still have the ability to participate in the development of embryos and extraembryonic tissues (placenta and amniotic membrane) at E12.5-13.5 days of mouse embryo implantation.
  • mice induced pluripotent stem cells In order to further analyze the ratio of tdTomato-positive cells derived from mouse induced pluripotent stem cells to the intraembryonic/extraembryonic parts, the inventors digested mouse embryos (Em), extraembryonic placenta (Pl) and yolk sac (Yo) respectively. ) and analyze the chimerism ratio of mouse induced pluripotent stem cells (which are tdTomato positive cells) in each tissue by flow cytometry.
  • Em mouse embryos
  • Pl extraembryonic placenta
  • Yo yolk sac
  • mice embryonic (Em) cells with collagenase IV with 1 U/ml of DNase
  • add 1 U/ml DNase with collagenase IV digest the yolk sac (Yo) cells in a 37°C incubator for 5 minutes, and then digest them with TrypLE for 3 minutes.
  • the digestion reaction was terminated with DPBS+10% fetal bovine serum of 3 times the enzyme volume. After centrifugation at 800 rpm for 5 minutes, the cell pellet was resuspended with DPBS, the cells were filtered with a 70 ⁇ m cell mesh, and the single-cell filtrate was collected. Transfer the cell filtrate to a flow analysis tube, and analyze or sort on the BD FACS Aria III according to the experimental requirements.
  • mice embryonic stem cell (mESC) injection group tdTomato-positive cells can only be detected in mouse embryos (Em), while the presence of tdTomato-positive cells can hardly be detected in extraembryonic tissue placenta (Pl) and yolk sac (Yo);
  • mESC mouse embryonic stem cell
  • a high proportion of tdTomato positive cells could be detected in mouse embryo (Em), extraembryonic tissue placenta (Pl) and yolk sac (Yo).
  • mouse induced pluripotent stem cells can efficiently chimerize to mouse embryo (Em), extraembryonic tissue placenta (Pl) and yolk sac (Yo) (Fig. 7.2).
  • tdTomato-positive cells could not be detected in the placenta in the non-injected group as a negative control; mouse embryonic stem cell (mESC) injected group could only detect the presence of tdTomato-positive cells in the Laby area of the placenta.
  • mESC mouse embryonic stem cell
  • mice induced totipotent stem cell injection groups and mouse embryonic stem cell (mESC) injection groups could be chimerized to various tissues of the fetus (including Including brain, heart and liver), but the mouse induced totipotent stem cell injection group had higher chimerism efficiency (Fig. 7.4).
  • Example 8 Mouse induced pluripotent stem cells develop independently into mouse embryos and life individual
  • a single mouse induced totipotent stem cell (ciTotiSC) has bidirectional chimerism to extraembryonic cells
  • ciTotiSC totipotent stem cells
  • the inventors injected a single tdtomato-labeled mESC or ciTotiSC 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 could only chimerize to the embryonic ICM. In contrast, totipotent stem cells (ciTotiSC) had chimerism for both ICM and TE.
  • the inventors confirmed that a single totipotent stem cell (ciTotiSC) could indeed develop into the extraembryonic TE lineage and correctly express the lineage marker CDX2 (Fig. 8.1, top).
  • the inventors transplanted 8-cell embryos injected with unipotent stem cells (ciTotiSC) or mESCs into the oviducts of pseudopregnant mother mice, and further observed the chimerism of E6.5-E7.5.
  • ciTotiSC single totipotent stem cell
  • EPI intraembryonic lineage
  • E and EPC extraembryonic lineage
  • mESCs were only chimerized to OCT4+EPI (Fig. 8.1 bottom).
  • ciTotiSC 8.2 Induced totipotent stem cells
  • ciTotiSC totipotent stem cells
  • scRNA-seq scRNA-seq to analyze tdTomato+ cells derived from totipotent stem cells (ciTotiSC) in chimeric placenta and yolk sac.
  • ciTotiSC totipotent stem cell
  • ciTotiSC totipotent stem cell
  • yolk sac cell types such as visceral yolk sac cells (Apoa4+Fxyd2+ Entpd2+), spongy trophoblast (Tpbpa+Rhox9+) and syncytiotrophoblast (Itm2a+), cells of embryonic origin including erythrocytes, macrophages and monocytes (Fig. 8.2).
  • Totipotent stem cells have the ability to chimerize to the germline ridge and generate healthy chimeric offspring (Fig. 8.3).
  • mice induced totipotent stem cells cultured under the condition of feeder cells were digested into single cells with 0.05% trypsin-EDTA, resuspended with the totipotent stem cell culture medium described in Example 1, and transferred to a 0.3% gelatin-coated
  • the 6-well plate was incubated in a 37°C incubator for 30 minutes to remove the feeder cells.
  • Mouse induced pluripotent stem cells were collected, resuspended in the medium used to induce mouse blastocysts, and filtered through a 40 ⁇ m filter to remove impurities and incompletely digested cell clumps.
  • AggreWell 400 (STEMCELL Technologies, 34415) was pretreated according to the instructions.
  • mice induced pluripotent stem cells were planted in one well of a 6-well plate AggreWell 400 (which contains 1,200 chambers) and placed in the medium for inducing mouse blastocysts for culture. Centrifuge the AggreWell 400 culture plate at 100g for 3 minutes to settle the cells, and then place the culture plate in a 37°C incubator for 4-5 days to obtain induced mouse blastocysts. The inventor observed that the induced blastocysts had highly similar morphological characteristics to normal blastocysts (Fig. 8.4). Statistics show that the efficiency of induced blastocysts from mouse induced pluripotent stem cells is about 70% (Fig. 8.4).
  • 2mM Y-27632 (only added on the first day), 12.5ng/mL rhFGF4 (R&D, 235F4025), 0.5mg/mL Heparin (Sigma-Aldrich, H3149), 3mM GSK3 inhibitor CHIR99021 (Reagents Direct, 27-H76), 5ng/mL BMP4 (Proteintech, HZ-1040), and 0.5mM A83-01.
  • mice blastocysts induced by mouse induced totipotent stem cells have three cell lineages of normal mouse blastocysts.
  • Normal mouse late blastocyst (E4.5) mainly contains three cell lineages: inner cell mass (ICM, specifically expressing OCT4), trophectoderm (TE, specifically expressing CDX2), and primitive endoderm (PrE, specifically expressing Sexual expression of SOX17).
  • ICM inner cell mass
  • TE trophectoderm
  • PrE primitive endoderm
  • mice induced pluripotent stem cells can be efficiently induced into mouse blastocysts with correct structure and gene expression patterns.
  • the inventors used an in vitro embryo culture system to further culture induced blastocysts derived from mouse induced totipotent stem cells, and found that induced blastocysts could produce a cylindrical structure after in vitro culture.
  • the ectoderm (stained positive for TFAP2C) and EPI (stained positive for Oct4) were surrounded by endoderm (stained positive for SOX17) as two hemispheres, similar to postimplantation embryos at E4.5-E5.5 in mice (Fig. 8.6).
  • the specific culture method is as follows: the induced blastocysts were picked out with a mouth pipette, washed twice in IVC-1 medium (Cell Guidance Systems, M11), and then transferred to u-Slide 8-wells supplemented with IVC-1 medium Board (ibidi, 80826). Approximately 20–30 induced blastocysts are placed in one well of a u-Slide 8-well plate. After induction of blastocyst adherence, the medium was replaced with IVC-2 (Cell Guidance Systems, M12). About 2-4 days later, the post-implantation embryo-like structure appeared, which was fixed with 4% PFA at room temperature for 15 minutes, and then the next step of immunofluorescence staining analysis was performed.
  • the accepted standard to verify whether the blastocyst is really functional is to transplant the blastocyst obtained in vitro into the uterus of a pseudopregnant mouse to observe whether it can implant and further develop into a fetus.
  • the inventors transplanted the induced blastocyst into the uterus of a 2.5-day-old pseudopregnant mouse, and at 7.5dpc, decidua implantation sites could be formed in the uterus of the induced blastocyst transplanted mouse.
  • the size of the decidua induced by blastocysts varies, but most of them are similar to normal mouse decidua, and some are slightly smaller.
  • TTNPB, 1-AKP, WS6 and mLIF are all very important for inducing pluripotent stem cells, reflected in the expression of pluripotency genes, maternal genes and ZGA genes Significantly upregulated (Fig. 10a).
  • the RA agonist TTNPB could directly activate a large number of RAR-binding motif-enriched totipotent, maternal and ZGA genes (Fig. 10d–g).
  • TTNPB or another widely used RA agonist, Trans-RA displayed similar induction effects on pluripotent genes, further confirming the specificity of the RA signaling pathway in the induction of pluripotent cells, whereas in the presence of the RA antagonist AGN193109, Totipotency genes could not be induced (Fig. 10e,f). These results confirm the central role of the retinoic acid signaling pathway in the establishment of pluripotency in cells.
  • 1-Azakenpaullone (1-AKP) is a selective GSK3 ⁇ and CDK1/cyclinB dual inhibitor. Studies have shown that 1-AKP treatment induces specific Wnt signaling downstream gene expression and G2/M arrest concurrently. GO analysis consistently showed that specific Wnt signaling-targeted genes were not upregulated after withdrawal of 1-AKP in totipotent stem cell (ciTotiSC) culture conditions (Fig. 10h). At the same time, the inventors also observed that the G2 phase of the cell cycle was prolonged after the induction of totipotent stem cells (ciTotiSC), which was consistent with the difference between 2C early embryos and blastocysts (Fig. 10i).
  • ciTotiSC totipotent stem cell
  • the G2 phase prolongation of totipotent stem cells was reduced when 1-Azakenpaullone was withdrawn from the culture conditions of totipotent stem cells (ciTotiSC), which means that 1-Azakenpaullone maintained the full position of totipotent stem cells (ciTotiSC) to a certain extent.
  • a sex-related feature—prolonged G2 phase (Fig. 10i).
  • WS6 is an IKK-NF- ⁇ B inhibitor previously shown to promote post-mitotic cell proliferation.
  • ERVs endogenous retroviruses
  • dsRNA double-stranded RNA
  • dsDNA double-stranded DNA
  • WS6 may play a role in promoting and stabilizing the induction of totipotent stem cells (ciTotiSC) partly by inhibiting the NF- ⁇ B-mediated immune response triggered by ERV activation (Fig. 10k, j).

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WO2025207779A1 (en) * 2024-03-28 2025-10-02 Pioneer Hi-Bred International, Inc. Kinase inhibitors for plant cell reprogramming

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