WO2023036166A1 - 猪多能干细胞培养基及其用途 - Google Patents

猪多能干细胞培养基及其用途 Download PDF

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WO2023036166A1
WO2023036166A1 PCT/CN2022/117466 CN2022117466W WO2023036166A1 WO 2023036166 A1 WO2023036166 A1 WO 2023036166A1 CN 2022117466 W CN2022117466 W CN 2022117466W WO 2023036166 A1 WO2023036166 A1 WO 2023036166A1
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component
concentration
medium
pgepiscs
cells
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韩建永
郅明雷
张金颖
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中国农业大学
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/50Fibroblast growth factors [FGF]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues

Definitions

  • the invention relates to the field of biotechnology, in particular to a pig pluripotent stem cell culture medium and its application.
  • epiblast cells are an important source of pluripotent stem cells (PSCs), including those from Mouse embryonic stem cells (Embryonic stem cells, ESCs) from the epiblast (Boroviak et al., 2014; Evans and Kaufman, 1981; Martin, 1981; Ying et al., 2008) and epiblast derived from later stages of development ( Epiblast stem cells (EpiSCs) (Bao et al., 2009; Brons et al., 2007; Tesar et al., 2007).
  • PSCs pluripotent stem cells
  • pigs are more advanced in embryonic development (Kobayashi et al., 2017; Zhu et al., 2021), anatomy (Niu et al., 2017; Yue et al., 2021) and physiology (Yan et al., 2021). al., 2018) are very similar to humans, therefore, stable porcine PSCs derived from epiblast cells should be an excellent model for understanding the properties of human PSCs, potentially providing valuable information for modeling human development (Xu et al ., 2020; Yan et al., 2018).
  • porcine PSC lines isolated from ICM or different stages of epiblast cells (Alberio et al. al., 2010; Choi et al., 2019; Gao et al., 2019; Haraguchi et al., 2012; Hou et al., 2016; Notarianni et al., 1990; Park et al., 2013; Vassiliev et al ., 2010; Yuan et al., 2019; Zhang et al., 2019).
  • the inventors collected porcine preimplantation embryos and performed scRNA-seq on all stages (embryos per day during E0-E14) to comprehensively analyze the molecular basis of early development and pluripotency changes in porcine embryos. Subsequently, based on the analysis results, the inventors developed a medium (termed 3i/LAF) that easily supported the establishment of a stable porcine E8-10 pre-gastrulation epiblast stem cell line (termed pgEpiSCs).
  • pgEpiSCs have the molecular characteristics of pluripotent and pregastrifying epiblast (Epiblast) cells, exhibit a raised dome morphology, express pluripotency markers, remain stable for more than 200 passages, and have Efficient teratoma formation and ability to differentiate into different cell types.
  • Epiblast pluripotent and pregastrifying epiblast
  • the inventors also achieved successive rounds of gene editing using pgEpiSCs, followed by nuclear transfer using gene-edited donor cells, and successfully generated homozygous edited piglets.
  • the present invention provides culture medium, it comprises:
  • a second component is selected from WH-4-023, A419259;
  • a third component, the third component is selected from fibroblast growth factors.
  • the medium further comprises:
  • the fourth component, the fourth component is selected from CHIR99021, WNT3a;
  • a fifth component selected from members of the TGF-beta superfamily
  • the sixth component, the sixth component is LIF.
  • the second component is WH-4-023.
  • the third component is selected from FGF2, FGF1.
  • the third component is FGF2.
  • the third component is recombinant human FGF2.
  • the fourth component is CHIR99021.
  • the fifth component is selected from Activin A, Nodal.
  • the fifth component is Activin A.
  • the fifth component is recombinant human Activin A.
  • the sixth component is selected from recombinant human LIF, recombinant mouse LIF.
  • the sixth component is recombinant human LIF.
  • the concentration of the first component is 0.1-10 ⁇ M, such as 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.7 ⁇ M, 0.9 ⁇ M, 1.1 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.7 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.3 ⁇ M, 3.5 ⁇ M, 3.7 ⁇ M , 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.3 ⁇ M, 4.5 ⁇ M, 4.7 ⁇ M, 4.9 ⁇ M, 5.0 ⁇ M, 5.1 ⁇ M, 5.3 ⁇ M, 5.5 ⁇ M, 5.7 ⁇ M, 5.9 ⁇ M, 6.0 ⁇ M, 6.1 ⁇ M, 6.3 ⁇ M, 6.5 ⁇ M, 6.7 ⁇ M,
  • the concentration of the first component is 0.9-3 ⁇ M.
  • the concentration of the first component is 1-3 ⁇ M, such as 1 ⁇ M, 1.1 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.7 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M or 3.0 ⁇ M, or 1-1.1 ⁇ M, 1.1-1.3 ⁇ M, 1.3-1.5 ⁇ M, 1.5-1.7 ⁇ M, 1.7-1.9 ⁇ M, 1.9-2.0 ⁇ M , 2.0-2.1 ⁇ M, 2.1-2.3 ⁇ M, 2.3-2.5 ⁇ M, 2.5-2.7 ⁇ M, 2.7-2.9 ⁇ M, or 2.9-3.0 ⁇ M.
  • the concentration of the first component is 2.5 ⁇ M.
  • the concentration of the second component is 3nM-30 ⁇ M, such as 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 0.01 ⁇ M, 0.03 ⁇ M, 0.05 ⁇ M, 0.07 ⁇ M, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.3 ⁇ M, 3.5 ⁇ M, 3.7 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.3 ⁇ M, 3n
  • the concentration of the second component is 0.01-5 ⁇ M, such as 0.01 ⁇ M, 0.03 ⁇ M, 0.05 ⁇ M, 0.07 ⁇ M, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.3 ⁇ M, 3.5 ⁇ M, 3.7 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.3 ⁇ M, 4.5 ⁇ M, 4.7 ⁇ M , 4.9 ⁇ M or 5.0
  • the concentration of the second component is 1 ⁇ M.
  • the concentration of the third component is 0.01-100 ng/mL, such as 0.01 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL mL, 0.6ng/mL, 0.7ng/mL, 0.8ng/mL, 0.9ng/mL, 1ng/mL, 1.5ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL , 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 14ng/mL, 15ng/mL, 16ng/mL, 17ng/mL, 18ng/mL, 19ng /mL, 20ng/mL, 25ng/mL, 30ng/mL, 35ng/mL, 40ng/mL,
  • the concentration of the third component is 1-100 ng/mL.
  • the concentration of the third component is 10 ng/mL.
  • the concentration of the fourth component is 0.0025nM-3 ⁇ M, such as 0.0025nM, 0.005nM, 0.01nM, 0.015nM, 0.02nM, 0.025nM, 0.03nM, 0.035nM, 0.04nM, 0.045nM , 0.05nM, 0.1nM, 0.15nM, 0.2nM, 0.25nM, 0.3nM, 0.35nM, 0.4nM, 0.45nM, 0.5nM, 1nM, 1.5nM, 2nM, 2.5nM, 3nM, 4nM, 5nM, 6nM, 7nM , 8nM, 9nM, 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.15 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M,
  • the concentration of the fourth component is 0.01-3 ⁇ M, such as 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.15 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M or 3.0 ⁇ M, or 0.01-0.05 ⁇ M, 0.05-0.1 ⁇ M, 0.1-0.15 ⁇ M, 0.15-0.2 ⁇ M, 0.2-0.3 ⁇ M, 0.3-0.4 ⁇ M, 0.4 -0.5 ⁇ M, 0.5-0.7 ⁇ M, 0.7-0.9 ⁇
  • the concentration of the fourth component is 1 ⁇ M.
  • the concentration of the fifth component is 0.01-100 ng/mL, such as 0.01 ng/mL, 0.5 ng/mL, 1 ng/mL, 1.5 ng/mL, 2 ng/mL, 2.5 ng/mL, 3ng/mL, 3.5ng/mL, 4ng/mL, 4.5ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 14ng/mL, 15ng/mL, 16ng/mL, 17ng/mL, 18ng/mL, 19ng/mL, 20ng/mL, 21ng/mL, 22ng/mL, 23ng/mL, 24ng/mL, 25ng/mL mL, 26ng/mL, 27ng/mL, 28ng/mL, 29ng/mL, 30ng/mL, 31ng/mL,
  • the fifth component is at a concentration of 25 ng/mL.
  • the concentration of the sixth component is 0.01-100 ng/mL, such as 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 0.7 ng/mL, 1 ng/mL , 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 14ng /mL, 15ng/mL, 16ng/mL, 17ng/mL, 18ng/mL, 19ng/mL, 20ng/mL, 21ng/mL, 22ng/mL, 23ng/mL, 24ng/mL, 25ng/mL, 26ng/mL , 27ng/mL, 28ng/mL, 29ng/mL, 30ng/mL, 31ng/mL, 32
  • the concentration of the sixth component is 1-100 ng/mL.
  • the sixth component is at a concentration of 10 ng/mL.
  • the concentration ratio of the fourth component to the first component is 25:1-1:25, such as 25:1, 24:1, 23:1, 22:1, 21:1 1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:1 6.
  • the concentration ratio of the fourth component to the first component is 2:3-1:3.
  • the concentration ratio of the fourth component to the first component is 1:2-1:3.
  • the medium comprises:
  • concentrations of the first component, the second component, the third component, the fourth component, the fifth component, and the sixth component all refer to the concentration of each component in the culture medium. Final concentration.
  • the culture medium further comprises: a seventh component, the seventh component being a ROCK inhibitor.
  • a seventh component being a ROCK inhibitor.
  • ROCK inhibitors such as Y-27632 can promote the proliferation of pgEpiSCs.
  • the seventh component is Y-27632.
  • the concentration of the seventh component is 0.01-50 ⁇ M, such as 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 0.5 ⁇ M, 0.7 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.1 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M ⁇ M, 1.7 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.3 ⁇ M, 2.5 ⁇ M, 2.7 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.3 ⁇ M, 3.5 ⁇ M, 3.7 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.3 ⁇ M, 4.5 ⁇ M, 4.7 ⁇ M, 4.9 ⁇ M, 5.0 ⁇ M, 5.1 ⁇ M, 5.3 ⁇ M, 5.5 ⁇ M, 5.7 ⁇ M, 5.9 ⁇ M, 6.0 ⁇ M, 6.1 ⁇ M, 6.3 ⁇ M, 6.5 ⁇ M, 6.7 ⁇ M, 6.9 ⁇ M, 7.0 ⁇ M , 7.1 ⁇ M, 0.01
  • the concentration of the seventh component is 0.01-20 ⁇ M, such as 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 0.5 ⁇ M, 0.7 ⁇ M, 0.9 ⁇ M , 1 ⁇ M, 1.1 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.7 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.3 ⁇ M, 2.5 ⁇ M, 2.7 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.3 ⁇ M, 3.5 ⁇ M, 3.7 ⁇ M , 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.3 ⁇ M, 4.5 ⁇ M, 4.7 ⁇ M, 4.9 ⁇ M, 5.0 ⁇ M, 5.1 ⁇ M, 5.3 ⁇ M, 5.5 ⁇ M, 5.7 ⁇ M, 5.9 ⁇ M, 6.0 ⁇ M, 6.1 ⁇ M, 6.3 ⁇ M, 6.5 ⁇ M, 6.7 ⁇ M, 6.9 ⁇ M, 7.0 ⁇ M,
  • the concentration of the seventh component is 0.01-10 ⁇ M, such as 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 0.5 ⁇ M, 0.7 ⁇ M, 0.9 ⁇ M , 1 ⁇ M, 1.1 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.7 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.3 ⁇ M, 2.5 ⁇ M, 2.7 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.3 ⁇ M, 3.5 ⁇ M, 3.7 ⁇ M , 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.3 ⁇ M, 4.5 ⁇ M, 4.7 ⁇ M, 4.9 ⁇ M, 5.0 ⁇ M, 5.1 ⁇ M, 5.3 ⁇ M, 5.5 ⁇ M, 5.7 ⁇ M, 5.9 ⁇ M, 6.0 ⁇ M, 6.1 ⁇ M, 6.3 ⁇ M, 6.5 ⁇ M, 6.7 ⁇ M, 6.9 ⁇ M, 7.0 ⁇ M
  • concentration of the seventh component refers to the final concentration of the component in the culture medium.
  • the medium further comprises: an eighth component, the eighth component being a basal medium.
  • the basal medium is a basal medium for culturing mammalian (preferably porcine) pluripotent stem cells.
  • the basal medium comprises basal medium, N2supplement, B27supplement, non-essential amino acids, ⁇ -mercaptoethanol, knockout serum replacement, and any one selected from GlutaMAX and glutamine.
  • the basal medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, ⁇ -mercaptoethanol, knockout serum replacement, and GlutaMAX.
  • the basal medium comprises minimal medium, N2 supplement, B27 supplement, non-essential amino acids, ⁇ -mercaptoethanol, knockout serum replacement, ascorbic acid, GlutaMAX, and penicillin-streptomycin.
  • the minimal medium is selected from DMEM/F12, Neurobasal, DMEM, KO-DMEM, RPMI1640, MEM, mTeSR1, or any combination thereof.
  • the minimal medium is selected from DMEM/F12, Neurobasal, or combinations thereof.
  • the minimal medium is DMEM/F12 and Neurobasal.
  • the volume fraction of the basic medium is 1%-99%, such as 1%, 3%, 5%, 7%, 9%, 10%, 11%, 13%, 15%, 17% %, 19%, 20%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 45.5%, 46%, 47%, 48%, 49%, 50%, 51%, 53%, 55%, 57%, 59%, 60%, 61%, 63% , 65%, 67%, 69%, 70%, 71%, 73%, 75%, 77%, 79%, 80%, 81%, 83%, 85%, 87%, 89%, 90%, 91 %, 93%, 95%, 97% or 99%, or such as 1%-3%, 3%-5%, 5%-7%, 7%-9%, 9%-11%, 11%-13 %, 13%-15%, 15%-17%, 17%-19%
  • the minimal medium has a volume fraction of 91%.
  • the volume fraction of DMEM/F12 is 1%-99%, such as 1%, 3%, 5%, 7%, 9%, 10%, 11%, 13%, 15%, 17% %, 19%, 20%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 45.5%, 46%, 47%, 48%, 49%, 50%, 51%, 53%, 55%, 57%, 59%, 60%, 61%, 63% , 65%, 67%, 69%, 70%, 71%, 73%, 75%, 77%, 79%, 80%, 81%, 83%, 85%, 87%, 89%, 90%, 91 %, 93%, 95%, 97% or 99%, or such as 1%-3%, 3%-5%, 5%-7%, 7%-9%, 9%-11%, 11%-13 %, 13%-15%, 15%-17%, 17%, 1
  • the volume fraction of DMEM/F12 is 45%-50% (such as 45.5%, 46%, 46.5%).
  • the volume fraction of Neurobasal is 1%-99%, such as 1%, 3%, 5%, 7%, 9%, 10%, 11%, 13%, 15%, 17%, 19%, 20%, 21%, 23%, 25%, 27%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 40%, 41%, 42%, 43% , 44%, 45%, 45.5%, 46%, 47%, 48%, 49%, 50%, 51%, 53%, 55%, 57%, 59%, 60%, 61%, 63%, 65 %, 67%, 69%, 70%, 71%, 73%, 75%, 77%, 79%, 80%, 81%, 83%, 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%, or such as 1%-3%, 3%-5%, 5%-7%, 7%-9%, 9%-11%, 11%-13%, 13%-15%, 15%-17%, 17%-19%, 19%-20
  • the volume fraction of Neurobasal is 45%-50% (eg, 45.5%, 46%, 46.5%).
  • the volume fraction of the N2supplement is 0.002%-10%, such as 0.002%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.13%, 0.15%, 0.17%, 0.2%, 0.25%, 0.3% , 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.1%, 1.3%, 1.5 %, 1.7%, 1.9%, 2.0%, 2.1%, 2.3%, 2.5%, 2.7%, 2.9%, 3.0%, 3.1%, 3.3%, 3.5%, 3.7%, 3.9%, 4.0%, 4.1%, 4.3%, 4.5%, 4.7%, 4.9%, 5.0%, 5.1%, 5.3%, 5.
  • the volume fraction of the N2supplement is 0.5%.
  • the volume fraction of the B27supplement is 0.002%-20%, such as 0.002%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.13%, 0.15%, 0.17%, 0.2%, 0.25%, 0.3% , 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.1%, 1.3%, 1.5 %, 1.7%, 1.9%, 2.0%, 2.1%, 2.3%, 2.5%, 2.7%, 2.9%, 3.0%, 3.1%, 3.3%, 3.5%, 3.7%, 3.9%, 4.0%, 4.1%, 4.3%, 4.5%, 4.7%, 4.9%, 5.0%, 5.1%, 5.3%, 5.
  • the volume fraction of the B27 supplement is 1%.
  • the volume fraction of the non-essential amino acids is 0.01%-10%, such as 0.01%, 0.05%, 0.1%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% %, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.1%, 1.3%, 1.5%, 1.7%, 1.9%, 2.0%, 2.1%, 2.3%, 2.5%, 2.7%, 2.9%, 3.0%, 3.1%, 3.3%, 3.5%, 3.7%, 3.9%, 4.0%, 4.1%, 4.3%, 4.5%, 4.7%, 4.9% , 5.0%, 5.1%, 5.3%, 5.5%, 5.7%, 5.9%, 6.0%, 6.1%, 6.3%, 6.5%, 6.7%, 6.9%, 7.0%, 7.1%, 7.3%, 7.5%, 7.7 %, 7.9%, 8.0%, 8.1%, 8.3%, 8.5%, 8.7%, 8.9%, 9.0%, 9.1%, 9.3%, 9.5%, 9.7%, 9.9%, 9.9%,
  • the volume fraction of the non-essential amino acids is 1%.
  • the concentration of ⁇ -mercaptoethanol is 0.01mM-1mM, for example, 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1 mM, 0.11mM, 0.12mM, 0.13mM, 0.14mM, 0.15mM, 0.16mM, 0.17mM, 0.18mM, 0.19mM, 0.2mM, 0.21mM, 0.22mM, 0.23mM, 0.24mM, 0.25mM, 0.26mM, 0.27mM, 0.28mM, 0.29mM, 0.3mM, 0.31mM, 0.32mM, 0.33mM, 0.34mM, 0.35mM, 0.36mM, 0.37mM, 0.38mM, 0.39mM, 0.4mM, 0.41mM, 0.42mM, 0.43mM , 0.44mM, 0.45mM, 0.41mM,
  • the concentration of ⁇ -mercaptoethanol is 0.1 mM.
  • the volume fraction of the knockout serum replacement is 0.01%-50%, such as 0.01%, 0.05%, 0.1%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% %, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.1%, 1.3%, 1.5%, 1.7%, 1.9%, 2.0%, 2.1%, 2.3%, 2.5%, 2.7%, 2.9%, 3.0%, 3.1%, 3.3%, 3.5%, 3.7%, 3.9%, 4.0%, 4.1%, 4.3%, 4.5%, 4.7%, 4.9% , 5.0%, 5.1%, 5.3%, 5.5%, 5.7%, 5.9%, 6.0%, 6.1%, 6.3%, 6.5%, 6.7%, 6.9%, 7.0%, 7.1%, 7.3%, 7.5%, 7.7 %, 7.9%, 8.0%, 8.1%, 8.3%, 8.5%, 8.7%, 8.9%, 9.0%, 9.1%, 9.3%, 9.5%, 9.7%, 9.9%, 10%
  • the volume fraction of the knockout serum replacement is 5%.
  • the concentration of ascorbic acid is 1 ⁇ g/mL-5000 ⁇ g/mL, such as 1 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g /mL, 40 ⁇ g/mL, 41 ⁇ g/mL, 42 ⁇ g/mL, 43 ⁇ g/mL, 44 ⁇ g/mL, 45 ⁇ g/mL, 46 ⁇ g/mL, 47 ⁇ g/mL, 48 ⁇ g/mL, 49 ⁇ g/mL, 50 ⁇ g/mL, 51 ⁇ g/mL , 52 ⁇ g/mL, 53 ⁇ g/mL, 54 ⁇ g/mL, 55 ⁇ g/mL, 56 ⁇ g/mL, 57 ⁇ g/mL, 58 ⁇ g/mL, 59 ⁇ g/mL, 60 ⁇ g/mL, 65 ⁇ g/mL, 70
  • the ascorbic acid is at a concentration of 50 ⁇ g/mL.
  • the volume fraction of GlutaMAX or glutamine is 0.01%-10%, such as 0.01%, 0.1%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% %, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.1%, 1.3%, 1.5%, 1.7%, 1.9%, 2.0%, 2.1%, 2.3%, 2.5%, 2.7%, 2.9%, 3.0%, 3.1%, 3.3%, 3.5%, 3.7%, 3.9%, 4.0%, 4.1%, 4.3%, 4.5%, 4.7% , 4.9%, 5.0%, 5.1%, 5.3%, 5.5%, 5.7%, 5.9%, 6.0%, 6.1%, 6.3%, 6.5%, 6.7%, 6.9%, 7.0%, 7.1%, 7.3%, 7.5 %, 7.7%, 7.9%, 8.0%, 8.1%, 8.3%, 8.5%, 8.7%, 8.9%, 9.0%, 9.1%, 9.3%, 9.5%, 9.7%
  • the volume fraction of GlutaMAX or glutamine is 0.5%.
  • the volume fraction of the penicillin-streptomycin is 0.01%-20%, such as 0.01%, 0.1%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% , 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.1%, 1.3%, 1.5%, 1.7%, 1.9%, 2.0%, 2.1 %, 2.3%, 2.5%, 2.7%, 2.9%, 3.0%, 3.1%, 3.3%, 3.5%, 3.7%, 3.9%, 4.0%, 4.1%, 4.3%, 4.5%, 4.7%, 4.9%, 5.0%, 5.1%, 5.3%, 5.5%, 5.7%, 5.9%, 6.0%, 6.1%, 6.3%, 6.5%, 6.7%, 6.9%, 7.0%, 7.1%, 7.3%, 7.5%, 7.7% , 7.9%, 8.0%, 8.1%, 8.3%, 8.5%, 8.7%, 8.9%, 9.0%, 9.1%, 9.3%, 9.5%, 9.7%, 9.9%, 10%,
  • the volume fraction of penicillin-streptomycin is 1%.
  • the volume ratio of the DMEM/F12 and the Neurobasal is 5:1-1:5, such as 5:1, 4:1, 3:1, 2:1, 1:1, 1:1 2. 1:3, 1:4 or 1:5, or 5:1-4:1, 4:1-3:1, 3:1-2:1, 2:1-1:1, 1:1 -1:2, 1:2-1:3, 1:3-1:4 or 1:4-1:5.
  • the volume ratio of the DMEM/F12 and the Neurobasal is 1:1.
  • concentration of each specific component in the above-mentioned eighth component all refers to the final concentration of each specific component in the medium.
  • volume fraction of each specific component in the above-mentioned eighth component refers to the volume of the specific component/total volume of the culture medium.
  • each 500 mL of culture medium comprises:
  • each component of the above-mentioned culture medium or specific components in each component are reagents routinely used by those skilled in the art, and can be obtained commercially.
  • the nonessential amino acids comprise glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.
  • the non-essential amino acids comprise:
  • the concentration of each component of the non-essential amino acid refers to the concentration of each specific component in the non-essential amino acid.
  • the present invention provides a method for preparing mammalian pluripotent stem cells, comprising:
  • Epiblast embryonic embryonic epiblast
  • Epiblast mammalian embryonic epiblast
  • the mammal is a pig.
  • the mammalian embryonic epiblast is an E8 to E10 (eg, E8, E9 or E10) mammalian embryonic epiblast.
  • the method is performed in the presence of feeder cells.
  • the feeder cells are selected from mouse embryonic fibroblasts or STO cells.
  • the feeder cells are mouse embryonic fibroblasts.
  • the feeder cells are arrested mouse embryonic fibroblasts.
  • the feeder cells are mitomycin C-treated mouse embryonic fibroblasts.
  • the density of the feeder cells is 10 4 cells/well to 10 ⁇ 10 5 cells/well.
  • the density of the feeder cells is 2 ⁇ 10 4 cells/well to 2 ⁇ 10 5 cells/well.
  • the culture vessel is a 12-well culture plate.
  • the method is at a temperature of 37-39°C (preferably 37°C), an oxygen concentration of 5%-22% (preferably 5%), and a carbon dioxide concentration of 4%-6% (preferably 5%) , under the condition of 100% humidity.
  • the replacement frequency of the medium is every 10-48 hours (preferably 10-24 hours, more preferably 12 hours).
  • the present invention provides a method for cultivating mammalian pluripotent stem cells and/or maintaining the pluripotency of mammalian pluripotent stem cells; which includes:
  • the mammal is a pig.
  • the mammalian pluripotent stem cells are porcine embryonic pre-gastrulation epiblast stem cells.
  • the method is performed in the presence of feeder cells.
  • the feeder cells are selected from mouse embryonic fibroblasts or STO cells.
  • the feeder cells are mouse embryonic fibroblasts.
  • the feeder cells are arrested mouse embryonic fibroblasts.
  • the feeder cells are mitomycin C-treated mouse embryonic fibroblasts.
  • the density of the feeder cells is 3 ⁇ 10 4 cells/cm 2 to 10 ⁇ 10 5 cells/cm 2 .
  • the density of the feeder cells is 5 ⁇ 10 4 cells/cm 2 .
  • the method is at a temperature of 37-39°C (preferably 38.5°C), an oxygen concentration of 5%-22% (preferably 20%), and a carbon dioxide concentration of 4%-6% (preferably 5%) carried out under the conditions.
  • the replacement frequency of the medium is every 10-48 hours (preferably 10-24 hours, more preferably 12 hours).
  • Figure 1-1 Lineage segregation and tracking of pluripotency changes during embryonic development
  • G Expression changes of JAK/STAT3, Activin/Nodal, FGF/ERK and Wnt/ ⁇ -catenin signaling pathway-related genes from morula (EM and pre-ICM), ICM, epiblast and ectoderm cells from E4 to E14
  • EM and pre-ICM morula
  • ICM ICM
  • epiblast ectoderm cells from E4 to E14
  • the gradient from blue to red on the right indicates the low expression to high expression of the gene, respectively.
  • a gradient from green to red at the top of the heatmap indicates Changes in formative and primed pluripotent states.
  • Figure 1-2 Lineage segregation and tracking of pluripotency changes during embryonic development
  • A Summary of single-cell transcriptome sequencing sample information.
  • Pig embryos collected from Embryonic day (Embryonic day, E) 0 to E14 were used for sc-RNA sequence analysis.
  • There are a total of 16 developmental stages including, oocyte (E0), zygote (E1), 2C (2-cell embryo, E2), 4C (4-cell embryo, E3), 8C (8-cell embryo, E3) EM (early morula, E4), LM (late morula, E5), EB (early blastocyst, E6), LB (late blastocyst, E7), HB (hatching blastocyst, E8) EBi (early bilaminar embryo, E9), LBi (late bilaminar embryo, E10), PPS (pre-primitive stripe embryo, E11), EPS (early primitive stripe embryo, E12), PS (primitive stripe embryo, E13), LPS (late primitive stripe embryo, E14)
  • Figure 1-3 Lineage segregation and tracking of pluripotency changes during embryonic development
  • A Networks of differentially expressed genes (DEGs) in each cell type compared to other cell types. Different colors indicate different cell types. Circles represent collections of DEGs. Each cell type is connected to its DEGs by lines within the network.
  • DEGs differentially expressed genes
  • FIG. B shows the GO functional enrichment analysis of 1,735 Naive pluripotency-related genes, 1,117 Formaive pluripotency-related genes and 1,289 Primed pluripotency-related genes in Figure 3-2-1. Displays the 20 top functional items of the Metascape (https://metascape.org) abstract gene set, and the numbers after each item represent the hit genes in the total genes of the item.
  • SOX1 is a neuroectoderm marker
  • T is a mesoderm marker
  • GATA6 is an endoderm marker
  • nuclear is a DAPI marker.
  • Scale bar 200 ⁇ m.
  • Figure 3-2 Characteristics of SNVs and Short InDels ( ⁇ 30bp) in pgEpiSC cell lines in different passages of the same cell line and between different cell lines.
  • NJ Neighbor-joining
  • Figure 3-3 The effect of different culture conditions on pgEpiSC preparation.
  • Figure 3-4A The effect of adjusting the concentration of CHIR99021 in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper panel represents before AP staining, and the lower panel represents after AP staining.
  • Figure 3-4B The effect of adjusting the concentration of IWR-1-endo in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper panel represents before AP staining, and the lower panel represents after AP staining.
  • Figure 3-4C The effect of adjusting the concentration of WH-4-023 in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper panel represents before AP staining, and the lower panel represents after AP staining.
  • Figure 3-4D The effect of adjusting the concentration of recombinant human Activin A in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper figure shows before AP staining, and the lower figure shows after AP staining.
  • Figure 3-4E The effect of adjusting the concentration of recombinant human FGF-basic in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper panel shows before AP staining, and the lower panel shows after AP staining.
  • Figure 3-4F Effects of adjusting the concentration of recombinant human LIF in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro.
  • the upper panel shows before AP staining, and the lower panel shows after AP staining.
  • Figure 3-4G The effect of replacing CHIR99021 with WNT3a in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper panel shows before AP staining, and the lower panel shows after AP staining.
  • Figure 3-4H Effect of replacing WH-4-023 with A419259 in 3i/LAF medium on the pluripotency of pgEpiSCs in vitro, the upper panel represents before AP staining, and the lower panel represents after AP staining.
  • Figure 3-4I The effect of replacing recombinant human Activin A with Nodal in the 3i/LAF medium on the pluripotency of pgEpiSCs in vitro.
  • the upper panel shows before AP staining, and the lower panel shows after AP staining.
  • Figure 4B The dot plot of classical marker genes of TE, hypoblast and epiblast during pig embryonic development.
  • the color gradient represents the average expression level, and the point size corresponds to the percentage of cells expressing the signature gene in TE, hypoblast and epiblast cell populations.
  • Figure 4C PCA plots of pgEpiSCs and epiblast or ectoderm cells from E7-E14. Each dot represents a single cell in prebed embryonic cells, and asterisks represent a single cell in pgEpiSCs. Colors indicate embryo age and passage of pgEpiSCs.
  • Figure 4D The Spearman correlation coefficient is based on the average expression level of specifically expressed genes in each epiblast or ectoderm cell from E7 to E14, which is related to the regulation of pluripotency and epithelial cell differentiation.
  • Fig. 4E Violin plot showing expression levels (log2(TPM/10+1)) of canonical pluripotent genes in E7-E14 and low- and high-passage pgEpiSCs according to scRNA-seq data. Each dot represents a cell.
  • Figure 4F formative and conventional hPSCs; formative and primed mPSCs; and a plot of PCA results for pgEpiSCs based on the set of uniquely expressed genes for each PSC. Colors represent pluripotency status. Triangles represent pigs, squares represent humans, and circles represent rats.
  • Figure 4G In hPSCs, formative hPSCs, and conventional hESCs (left) and Expression levels of uniquely expressed genes identified in mESCs, formative mPSCs, and primed mEpiSCs (right) compared to those in pgEpiSCs. The listed genes are highly expressed in pgEpiSCs.
  • Figure 5A The resolution of each Hi-C map is 100 kb intrachromosomally and 1 mb interchromosomally, respectively. Examples of cross-sections of pgEpiSCs-1-B and pEF-1-G nuclei, colored by eusomes (left) or polychromosomal admixture index (reflecting chromosome diversity as measured by Shannon's index) (right) (Tan et al., 2018).
  • Figure 5B Probability of extensive polychromosomal confounding of 18 autosomes (smoothed by 1mb window) in pgEpiSCs (green) and pEFs (red) at 100 kb resolution (average of 16 Hi-C maps per cell type ) (Tan et al., 2018).
  • Figure 5C Example of a contact map at 100 kb resolution for chromosome 18 pgEpiSCs-1-B (upper half) and pEF-1-G (lower half).
  • Figure 5D Degree of disorder in chromatin structure (quantified with Von Neumann entropy) (Lindsly et al., 2021) at 100 kb resolution each of 16 Hi-C maps of pgEpiSCs (green) and pEFs (red).
  • Figures 5E-5K Schematic representation of PEIs for OTX2(E), LIN28A(F), NANOG(G), PRDM14(H), SALL4(I), UTF1(J) and ZFP42(K).
  • Top panel Hi-C map of the region near the center of the gene transcription start site ( ⁇ 250kb).
  • Middle panel 3D models of promoters (blue balls) and their enhancers (red and green balls represent super-enhancers and regular enhancers, respectively).
  • Bottom panel RNA-seq profile. A Benjamini-Hochberg adjusted FDR was calculated.
  • Figure 6A Schematic diagram of generation of cloned piglets using pgEpiSCs as donors, through nuclear transfer experiments, and multiple gene sequential editing strategies.
  • Figure 6B Morphology and fluorescence detection of GFP-pgEpiSC clones, scale bar, 200 ⁇ m.
  • FIG. 6C Identification of NANOG-tdTomato knock-in by PCR.
  • GN represents GFP-positive NANOG-tdTomato knock-in pgEpiSC.
  • Figure 6D Expression of NANOG-tdTomato knock-in reporter gene in GFP-labeled pgEpiSCs. Scale bar, 100 ⁇ m.
  • Figure 6E Loss of tdTomato expression after differentiation of NANOG-tdTomato reporter pgEpiSCs. Scale bar, 200 ⁇ m.
  • Figure 6F Statistics of unedited, homozygous and heterozygous ratios of TYR gene C to T mutations in WT pgEpiSCs (1-pgEpiSCs) and genetically modified pgEpiSCs (1-GP-pgEpiSCs and 1-GN-pgEpiSCs) (left); Representative DNA sequencing analysis of TYR gene C to T mutation sites in wild-type, heterozygous, and homozygous pgEpiSCs (right).
  • Figure 6G Summary of pgEpiSC nuclear transfer experiments. The blastocyst rate was calculated using embryos retained before transfer. Fibroblasts were obtained from BAMA pigs.
  • FIG. 6H Top panel: Ear fibroblasts from WT pgEpiSCs cloned piglets and GFP-pgEpiSCs cloned piglets; Middle panel: 3 GNT-pgEpiSCs cloned piglets and their surrogate mothers; Bottom panel: Representative GNT-pgEpiSCs clones Piglets show GFP fluorescence, in contrast to WT piglets cloned from Bama pig fibroblasts.
  • Figure 6I Representative cloned piglets generated from WT pgEpiSCs as donor cells (left) exhibited black coat color, whereas representative cloned piglets generated from GNT-pgEpiSCs as donor cells (right) displayed white coat color albinism phenotype.
  • porcine embryo pregastrulation epiblast E10 embryo pregastrulation epiblast (Epiblast ) Stem cell lines (Pre-gastrulation epiblast stem cell lines, pgEpiSCs).
  • Epiblast E10 embryo pregastrulation epiblast
  • pgEpiSCs Pre-gastrulation epiblast stem cell lines
  • the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 by the method described; restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer.
  • restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer.
  • the pgEpiSC described in the following examples refers to a cell line established from E10 epiblast.
  • RNA-seq libraries were prepared by a modified Smart-seq2 protocol (Gao et al., 2018; Wang et al., 2018). Briefly, transfer single embryonic cells into the prepared lysis buffer containing the 8bp barcode. Then, reverse transcription (RT) mixture containing 4 U RNase inhibitor, 100 U SuperScript II reverse transcriptase (Invitrogen, 18064071), 1 mM dNTPs (TAKARA, 4019), 60 mM MgCl, and 3 ⁇ M RT primer and 10 ⁇ M TSO primer First-strand cDNA is transcribed and amplified.
  • RT reverse transcription
  • RNA-seq library was constructed according to the PCR library Amplification/Illumina series KAPA Hyper Prep kit (KAPA, KK8054). High-quality libraries were sequenced at 150 bp paired-end on Illumina Hiseq Xten (Novogene).
  • Cells were dissociated by Accutase (Gibco, A11105-01), counted using a hemocytometer, and seeded in triplicate onto pre-seeded 6-well plate feeder layers at densities of 100, 200, and 1,000 cells per well under pgEpiSCs culture conditions on the cells. Colonies were counted after 6 days using AP staining, and clonogenic efficiency was assessed as a percentage of the number of clones per number of cells plated.
  • 1% KaryoMAX Colcemid solution (Gibco, 15212012) was added to the pgEpiSCs medium, and the cells were incubated for 1 hour.
  • pgEpiSCs were digested into single cells by TrypLE TM Express (Gibco, 12605010) and collected by centrifugation. Resuspend pgEpiSCs with 0.075M KCl (sigma, P5405) hypotonic solution and incubate at 37 °C for 15 min. Then fix pgEpiSCs with methanol and acetic acid at a ratio of 3:1, and repeat this process 3 times.
  • the pgEpiSCs suspension was dropped onto a pre-cooled glass slide, dried thoroughly at room temperature, and then stained with 10% Giemsa staining solution (Sangon, E607314-0001) for 30 minutes. For each cell line, more than 30 cells in metaphase were examined.
  • Total DNA of pgEpiSCs was extracted using TIANamp Genomic DNA Kit (TIANGEN, DP304). After DNA extraction, 1 ⁇ g of genomic DNA was randomly fragmented with Covaris, and fragments of 200-400 bp were selected using Agencourt AMPure XP-Medium kit (BERCKMAN COULTER, A63880). Selected fragments are end-repaired and 3' adenylated, and adapters are ligated to the ends.
  • the products were amplified by PCR, and then the purified PCR products were heat-denatured into single strands and circularized by the splint oligonucleotide sequence. Format single-stranded circular DNA into the final library and verify it with quality control. The final validated library was sequenced by BGISEQ-500.
  • Alkaline phosphatase staining of pgEpiSC was based on the alkaline phosphatase detection kit (Millipore, SCR004).
  • pgEpiSCs were dissociated by Accutase (Gibco, A11105-01), separated from feeder cells using the differential attachment method, cultured on a 35mm low-attachment dish, and placed on a 50rpm horizontal shaker with the addition of 10% FBS (Gibco, 11960-044), 1% Penicillin streptomycin (Thermo Fisher Scientific, 15140-122) and 1% glutamine (Thermo Fisher Scientific, 35050-061) were cultured in DMEM (Gibco, 11960-044) for 5-7 days. Select regular spherical EBs and plate them on gelatin-coated plates in the same medium within 1 week, then fix and detect them using the same method as for immunofluorescence.
  • neural induction For neural induction, two days after passage of pgEpiSCs, replace the 3i/LAF medium with neural induction medium I (2.5 ⁇ M IWR-1-endo, 5 ⁇ M SB431542, and 10 ng/mL FGF2 in BM). After 2 days of culture, change the medium to Neural Induction Medium II (4 ⁇ M RA, 10 ng/mL FGF2 and 20 ng/mL Noggin in BM) and perform immunostaining 2 days later.
  • neural induction medium I 2.5 ⁇ M IWR-1-endo, 5 ⁇ M SB431542, and 10 ng/mL FGF2 in BM.
  • mesoderm induction medium II 3 ⁇ M IWR-1-endo, 5 ⁇ M CHIR99021, 20 ng/mL FGF2 in BM was subsequently added and immunostaining was performed 2 days later.
  • Teratomas were collected subcutaneously from nude mice, washed twice in PBS, and fixed with 4% PFA at 4 °C for 2 days.
  • Teratoma tissue was dehydrated with alcohol gradients (70%, 80%, 90%, 95% and 100%), each gradient for 1 hour, transferred to xylene and embedded in paraffin, and the sample was cut into a thickness of 5 ⁇ m , deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol.
  • the samples were then stained with hematoxylin (Sigma-Aldrich, MHS16) and eosin (Sigma-Aldrich, HT110116), and observed under a microscope (Leica, DM5500B).
  • RNA Preparation, Purified Cell/Bacteria Kit (TIANGEN, DP430), and then reverse-transcribed into cDNA using 5 ⁇ All-in-One RT Master Mix (Abm, G490).
  • PCR was performed on a LightCycler 480II real-time system (Roche) using 2x RealStar Green Power Mixture (GenStar, A311-05). Data were analyzed using the comparative CT (2 - ⁇ CT ) method. ⁇ CT was calculated using EF1A as an internal control. All experiments were performed in three biological replicates. Primers for quantitative real-time PCR are listed in the table of key resources.
  • the total protein in the cells was extracted with cell lysis buffer (Beyotime Biotechnology, P0013), and the nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, P0027) supplemented with protease and phosphatase inhibitors (Beyotime Biotechnology, P1050) was used to extract nuclear and cytoplasmic proteins. cytoplasmic protein.
  • concentration of extracted protein was measured using Bradford protein detection kit (Bio-red, 5000201). Equal amounts of proteins (15 ⁇ g) were separated by SDS-PAGE gel electrophoresis, and the proteins were transferred from the gel to Immobilon-P transfer membrane (Merck Millipore; pore size: 0.45 ⁇ m; IPVH00010).
  • Blots were blocked in 5% nonfat dry milk (Sangon Biotech, A600669-0250) in TBST (20 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20) for 1 hour at room temperature, and then mixed with antibodies in TBST. Dilute in 5% skimmed milk to 4°C overnight. The next day, the blot was washed 3 times with TBST for 5 min each, then incubated with HRP-conjugated secondary antibody diluted in 5% nonfat dry milk in TBST for 1 h at room temperature, and finally rinsed 3 times with TBST , 5 minutes each time.
  • Thermo Fisher Scientific, 34075 West Dura Extended Duration Substrate (Thermo Fisher Scientific, 34075) was developed, and the band intensity of the target protein was analyzed using CLINX chemiluminescence software.
  • the specific experimental methods and reagent formulations come from the general protocol of Western Blotting (Bio-red, bulletin 6376).
  • the specific experimental methods and reagent formulations come from the general procedures of Western Blotting (Bio-red, bulletin 6376).
  • Hi-C libraries were constructed separately (as technical replicates).
  • cells (5 x 10 6 ) were cross-linked with formaldehyde at a final concentration of 4% for 30 min at room temperature and then quenched with glycine at a final concentration of 0.25M/L.
  • centrifuge the mixture at 1,500 ⁇ g for 10 min at room temperature and combine the supernatant with lysis buffer and incubate on ice for 15 min.
  • the mixture was then centrifuged at 5,000 ⁇ g for 10 min at room temperature.
  • the precipitate was washed with NEBuffer 2.
  • DNA was purified using a Covaris S220 sonicator and sonicated for fragments of approximately 300-500 bp, then the ligated fragments were pulled down with Dynabeads TM -280 streptavidin (Invitrogen, 11206D), followed by end repair and add poly-A. Next, the adapters were ligated, and the DNA fragments were amplified by PCR for 8-10 cycles using the KAPA Hyper Prep Kit (Roche, KK8504).
  • fragments were then double-selected using AMPure XP Beads (Beckman, A63882) to isolate fragments between 300 and 800 bp that were ready to be sequenced on the DNBSEQ platform (BGI) to provide 100 bp paired-end reads.
  • AMPure XP Beads Beckman, A63882
  • the inventors performed ChIP-seq of H3K27ac (a typical histone mark for enhancers) in two biological replicates of pgEpiSCs and pEFs, 1 ⁇ 10 cells per sample. Cross-link the cells with formaldehyde at a final concentration of 1% for 10 min at room temperature and then quench with glycine. Cells were lysed with lysis buffer supplemented with protease inhibitor cocktail and 1 mM PMSF (1x final), then sonicated for fragments of approximately 200-500 bp using a Bioruptor.
  • pgEpiSCs can tolerate continuous gene editing
  • the inventors performed three gene editing experiments using different gene editing techniques in pgEpiSCs: 1) Stable transfection of the GFP-NLS cassette using the PiggyBac (PB) transposase tool; 2) NANOG-tdTomato reporter gene was knocked in (KI) by CRISPR/Cas9 system; 3) TYR gene point mutation was carried out by cytidine base editors (CBEs).
  • the inventor constructed the PB-CMV-EF1A-GFP-NLS plasmid (from Professor Wu Sen), and replaced the chicken ⁇ -actin promoter with the human elongation factor 1 ⁇ (Homo sapiianfactor1alpha, EF1A) promoter, And insert GFP-NLS at the end of EF1A promoter.
  • the inventors constructed a NANOG DNA donor vector backbone with four fragments, the left homology arm 3 ⁇ Flag, 3 ⁇ Flag-P2A-tdTomato-Loxp-Puro-Loxp and the right Homology arms, as previously described.
  • HiFi DNA Assembly Master Mix (NEB, E2621X). Target the NANOG sgRNA just before the stop codon site to knock out the donor fragment as a reporter gene. The annealed sgRNA sequence was cloned into the Bsa I digested pGL3-U6-sgRNA-PGK-puromycin vector (Addgene, 51133). Finally, the inventors knocked out the TYR gene using the AncBE4max plasmid. AncBE4max and pGL3-U6-sgRNA-EGFP vectors were obtained from Huang Xingxu's laboratory at ShanghaiTech University.
  • the sgRNA was synthesized by BGI with the ACCG sequence at the 5' end of the forward primer and the AAAC sequence at the 5' end of the reverse primer. The sgRNA was then annealed and cloned into the pGL3-U6-sgRNA-EGFP vector. Details of sgRNA sequences are provided in the key resource table.
  • pgEpiSCs were dissociated using Accutase Cell Dissociation Reagent (Gibco, A11105-01). For each electroporation, 5 ⁇ 10 5 cells were transfected using BTX ECM 2001 (Harvard Biosciences, Holliston, MA, USA) at 220V, 5 ms, 2 pulses.
  • BTX ECM 2001 Hard Biosciences, Holliston, MA, USA
  • For stable transfection of the GFP-NLS cassette use 1 ⁇ g of PBase helper plasmid and 3 ⁇ g of PB-CMV-EF1A-GFP-NLS donor plasmid (mass ratio 1:3) for electroporation.
  • NANOG-tdTomato reporter gene knock-in use 1 ⁇ g pST1374-NLS-flag-linker-Cas9 plasmid (Addgene, 44,758), 1 ⁇ g NANOG sgRNA plasmid and 1 ⁇ g NANOG HMEJ donor plasmid (1 ⁇ g per vector) for electroporation; for TYR gene point mutation , with 2 ⁇ g ancBE4max vector and 2 ⁇ g pGL3-U6-TYR sgRNA-GFP vector (mass ratio 1:1) for electroporation.
  • the electrotransfection buffer was provided by the Wu Sen Laboratory of the State Key Laboratory of Agricultural Biotechnology, China Agricultural University.
  • Primers were designed online using NCBI Primer BLAST and synthesized by BGI. GFP-positive cells were sorted using FACS (MoFlo XDP, Backman) and detected using a 488nm (710/50 bandpass filter) channel. To obtain NANOG-tdTomato-positive cells, select transfected cells with puromycin (0.3 ⁇ g/mL) and blasticidin (4 ⁇ g/mL), and pick and amplify GFP-positive clones. To identify base-edited cells, DNA was extracted using cell lysis buffer (Invitrogen, AM8723) as a template for PCR. PCR products were sequenced to confirm point mutations.
  • IVM stock solution M199 contains 0.1% L-cysteine (Sigma, C7352-25G), 5% FBS (Gibco, 10099141), 0.1% EGF (Sigma, E9644), 1% penicillin-streptomycin (Gibco, 15140122) and 10% porcine follicular fluid (follicular fluid was collected during oocyte collection, centrifuged and filtered, and then stored at -80°C).
  • the IVM stock solution was filtered with a 0.22 ⁇ m filter and stored at 4 °C for later use. Before use, add 1% GlutaMAX (Gibco, 35050061), 10IU/mL PMSG and 10IU/mL hCG. Porcine EpiSCs were differentiated in basal medium containing 10 ng/mL BMP4, 5 ⁇ M SB431542, and 10 ng/mL FGF2 for more than 1 week, and then used as donor cells for nuclear transfer. Mature oocytes in metaphase II were removed by micromanipulation in HM medium containing 7.5 ⁇ g/mL cytochalasin B.
  • Inject morphologically qualified donor cells into the perivitelline space and culture in fusion medium (0.3M/L mannitol, 1.0 mM/L CaCl2, 0.1 mM/L MgCl2, and 0.5 mM/L HEPES) using a BLS electrocytomanipulator. .
  • Oocytes were then incubated in PZM-3 for 15 min and the fusion ratio was assessed under a stereomicroscope. Place fifty to sixty fused embryos into a four-well Petri dish containing 500 ⁇ L of PZM-3 per well, and place them in PZM-3 at 38.5 °C in 5% CO , 5% O , and 90% N. Incubate and maintain maximum humidity. After 24 h, 150-250 ESCNT zygotes were surgically transplanted into surrogate mothers. The pregnancy status of the surrogate is determined by ultrasonography at 25-30 days. All cloned piglets gave birth spontaneously on days 114-120 of gestation.
  • the inventors performed scRNA-seq using a modified single-cell marker reverse transcription sequencing (STRT-seq) protocol (see STAR Methods) (Gao et al. , 2018; Wang et al., 2018), and finally obtained the data of 1,458 single cells retained after quality control of porcine oocytes and embryos sampled from E1 to E14 (Fig. 1-1A and Fig. 1- 2A).
  • the inventors identified cell types at different stages through Shared nearest neighbor (SNN) and t-distributed stochastic neighbor embedding (t-SNE) (Stuart et al., 2019) and described different lineage differentiation processes.
  • SNN Shared nearest neighbor
  • t-SNE t-distributed stochastic neighbor embedding
  • ICM and TE cells divided into two cell populations, and PDGFRA, NANOG, and SOX2 were upregulated in ICMs, while CDX2, DAB2, GATA2, and GATA3 were upregulated in TEs (Fig. 1-1C).
  • Heterogeneous expression of GATA6 (a hypoblast marker) and NANOG (an epiblast marker) was detected in the ICMs of E6 (Fig. 1-1D), marking the beginning of the second lineage separation (Plusa et al., 2008; Saiz et al., 2016).
  • GATA6 and NANOG positive cells separated into two populations (Fig. 1-1D), which indicated that the second lineage segregation ended and hypoblast cells (GATA4 + and GATA6 + ) and epiblast cells were established in the embryo (NANOG + and SOX2 + ) lineage.
  • GATA4 + and GATA6 + hypoblast cells
  • epiblast cells were established in the embryo (NANOG + and SOX2 + ) lineage.
  • the number of epiblast cells and hypoblast cells increased rapidly, and the hypoblast cells extended to form a complete cavity in the TE, called the original intestinal lumen (Oestrup et al., 2009); epiblast cell pluripotency marker genes NANOG, High expression of POU5F1 and SOX2 (Fig.
  • E4 early morula E5pre-ICMs
  • E6ICMs E7-E10epiblasts
  • E11-E14ectoderms embryonic stages were grouped into 36 clusters based on their gene expression trends (see STAR Methods).
  • E4 early morula to E7 epiblast representative Expression of pluripotent genes such as ESRRB, KLF4, LIFR, STAT3, TFCP2L1, and TBX3 was significantly reduced.
  • pluripotent genes such as ZIC5, ETV5, ZIC2, LEF1, BMP4, and LIN28B
  • ZIC5, ETV5, ZIC2, LEF1, BMP4, and LIN28B increased after E7
  • Formative marker genes such as TDGF1, DNMT3A, OTX2, KLF5, LIN28A and NODAL
  • TDGF1, DNMT3A, OTX2, KLF5, LIN28A and NODAL were highly expressed in the E7-E10 epiblast, while their expression was low in the E11-E13 ectoderm (Fig. 1-1F).
  • annotations of expression trends across the three pluripotency states were supported by pluripotency regulatory networks and enriched functional terms (Fig. 1-3A and Fig. 1-3B).
  • Example 1 On the basis of Example 1, the inventors concluded from scRNA-seq analysis that the establishment of pgEpiSCs should prevent embryonic gastrulation by using small molecule inhibitors related to WNT signaling, as well as using the TGF- ⁇ superfamily and FGF family cytokines to promote epiblast self-renewal. To achieve this goal in E10 epiblast cells, the inventors tested a variety of medium formulations combining various inhibitors and cytokines, and finally developed a combination of three inhibitors (CHIR99021, IWR1-endo and WH-4-023) and three cytokines (LIF, Activin A, and FGF2), which the inventors call "3i/LAF" medium (Fig. 2A).
  • three inhibitors CHIR99021, IWR1-endo and WH-4-023
  • three cytokines LIF, Activin A, and FGF2
  • pgEpiSCs retained their dome-shaped clonal morphology (Fig. 2G), AP positive (Fig. 2H), and normal karyotype (Fig. 3-2a).
  • pgEpiSCs expressed pluripotent stem cell markers such as POU5F1, NANOG, and SOX2 ( Figure 2I), and pluripotency surface markers including SSEA1, SSEA4, TRA-1-81, and TRA-1-60 ( Figure 2J) during long-term in vitro culture.
  • embryonic body (EB) differentiation assays showed that pgEpiSCs could differentiate into three germ layers after removal of inhibitors and cytokines in the medium ( Figure 2K).
  • pgEpiSCs were derived from the Epiblast of porcine E8-E10 embryos, and the Hypoblast and TE were removed mechanically as much as possible to ensure that the surface of the Epiblast did not adhere to the hypoblast or TE cells.
  • pgEpiSCs there are two ways to establish pgEpiSCs. 1 Treat the epiblast with TrypLE TM Express for 3mins, and then use a mouth pipette with a diameter of 40-50 ⁇ m to repeatedly blow and suck to disperse into small cell clusters.
  • cryopreservation After digesting into single cells according to the above method, collect the cells by centrifugation at 1000rpm for 5mins, remove the supernatant, and use freshly prepared pEpiSCs cell freezing solution (10% DMSO + 90% KOSR) pre-cooled at 4°C to gently Resuspend the cells, divide them into cryopreservation tubes according to the subculture ratio, label the cell information, transfer to a 4°C pre-cooled cell freezing box, and quickly transfer to a -80°C ultra-low temperature refrigerator, and transfer to liquid nitrogen after 24hrs Long-term preservation.
  • pEpiSCs cell freezing solution (10% DMSO + 90% KOSR) pre-cooled at 4°C
  • the components contained in basal medium (BM) and their respective contents or the final concentration in 500mL 3i/LAF medium are as follows: 227.5mL DMEM/F12 (Thermo Fisher 729Scientific, 10565- 018), 227.5mL Neurobasal (Thermo Fisher Scientific, 21103-049), 2.5mL N2supplement (Thermo Fisher Scientific, 17502-048), 5mL B27supplement (Thermo Fisher Scientific, 12587-010), 0.5% GlutaMAX (Thermo -061), 1% non-essential amino acid (Thermo Fisher 732 Scientific, 11140-050), 0.1mM ⁇ -mercaptoethanol (Thermo Fisher Scientific, 21985-023), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122) , 5% knockout serum replacement (KOSR, Thermo Fisher Scientific, A3181502, optional), and 50 ⁇ g/mL
  • each small molecule or cytokine in 500mL 3i/LAF medium is as follows: CHIR99021 (1 ⁇ M, Selleckchem, S1263), IWR-1-endo (2.5 ⁇ M, Selleckchem, S7086), WH-4-023 (1 ⁇ M, Selleckchem, S7565), recombinant human LIF (10ng/mL, PeproTech, 300-05), recombinant human Activin A ( 25ng/mL, Peprotech, 120-14E), and recombinant human FGF-basic (10ng/mL, Peprotech, 100-18B).
  • ROCK inhibitor Y-27632 In order to promote the proliferation of pgEpiSCs, you can choose to further add ROCK inhibitor Y-27632 to the previously configured 3i/LAF medium (passage, final concentration 10 ⁇ M; maintenance, final concentration 2 ⁇ M; Selleckchem, S1049).
  • the porcine pgEpiSCs were cultured with 3i/LAF medium under the conditions of 20% O 2 , 5% CO 2 , and 38.5°C.
  • PgEpiSCs were cultured on mouse embryonic fibroblast (MEF) feeder cells (5 ⁇ 10 4 cells/cm 2 ) treated with mitomycin C (Selleckchem, S8146). Change the medium every 12 hours and add fresh 3i/LAF medium.
  • MEF mouse embryonic fibroblast
  • pgEpiSC maintains long-term requirements for inhibitors and growth factors
  • the inventors based on the 3i/LAF medium configured in the above-mentioned point 2 and its culture method, and other conditions remain unchanged, and on the basis of removing small molecule inhibitors and cytokines in the culture solution one by one, tested each of the 3i/LAF medium. Factors required for long-term in vitro maintenance of pgEpiSCs.
  • EMT Epithelial-mesenchymal transition
  • IWR-1-endo directly resulted in unclear borders of pgEpiSC clones and resulted in significant downregulation of core pluripotency factors such as NANOG, POU5F1, SOX2, and REX1 ( Figure 3-1B).
  • CHIR99021 down-regulated the expression of cell proliferation-related genes such as LIN28A, C-MYC, ETV4, and ETV5 (Fig. 3-1D), suggesting that the proliferation of pgEpiSC was impaired.
  • High concentrations of CHIR99021 resulted in the downregulation of the pluripotency marker POU5F1 and the significant upregulation of the endoderm marker GATA6 (Fig. 3-1E), these results were further confirmed by immunofluorescence staining (Fig. and porcine PSCs are conserved.
  • Activin A a member of the TGF- ⁇ superfamily, resulted in decreased levels of the pluripotency marker NANOG after removal of cytokines in 3i/LAF (Fig. 3-1G and Fig. 3-1H).
  • TGF- ⁇ general inhibitor SB431542 to avoid the influence of feeder secretions
  • the addition of TGF- ⁇ general inhibitor SB431542 in the medium resulted in irregular clone morphology and a significant decrease in the level of NANOG (Fig. 3-1G and Fig. 3-1H).
  • SB431542 also resulted in a significant decrease in pluripotency markers such as POU5F1 and REX1 and a significant increase in BMP4 and BMP downstream transcription factors such as ID2 and ID3, which mediate the activation of circular bars during embryonic development. Induction (Kurek et al., 2015; Valdez Magana et al., 2014) (Fig. 3-1H).
  • pgEpiSCs When FGF2 was removed (with the addition of the ERK/MEK inhibitor PD0325901), pgEpiSCs could not proliferate or be passaged normally (Fig. Significantly reduced the proliferation ability of pgEpiSCs (Fig. 3-1K), low concentration of FGF2 is not conducive to cell proliferation, while high concentration of cells proliferates faster. It is noteworthy that the inventors found that LIF is not a necessary condition for maintaining the morphology of pgEpiSC clones (Fig. 3-1L), but the addition of the JAK1/2 inhibitor ruxolitinib made the clones flattened, and western blotting showed that phosphorylated STAT3 could only be detected in the presence of LIF (Fig. 3-1M), indicating that pgEpiSCs could respond to LIF Promotes pluripotency.
  • the inventors also conducted the following tests based on the 3i/LAF medium configured in the above-mentioned point 2 and its culture method, while keeping other conditions unchanged.
  • the concentration of CHIR99021 in the culture system affects the pluripotency and homogeneity of cells.
  • concentration of CHIR99021 was high, the expression of pluripotency gene POU5F1 was decreased, and the expression of lineage differentiation gene GATA6 was up-regulated, showing a heterogeneous expression pattern.
  • the expression levels of the mesoderm marker gene TBX3 and the endoderm gene ESRRB increased with increasing concentrations (as shown in Figure 3-3C and Figure 3-3D).
  • the inventor configured the 3i/LAF medium and its culture method based on the second point above, and adjusted the concentrations of small molecule inhibitors and cytokines in the 3i/LAF medium one by one or replaced the small molecule inhibitors and cytokines in the 3i/LAF medium with other conditions unchanged.
  • the effect of adjusted or replaced medium on the long-term maintenance of pluripotency of pgEpiSCs in vitro was studied.
  • Undifferentiated stem cells will express Ap at a high level. By staining fixed stem cells, differentiated cells are colorless, and undifferentiated cells appear purple or red.
  • Undifferentiated stem cells will express Ap at a high level. By staining fixed stem cells, differentiated cells are colorless, and undifferentiated cells appear purple or red.
  • Table B Component replacement and test results of small molecule inhibitors or cytokines in 3i/LAF medium
  • Example 3 Correlation of the transcriptome of pgEpiSC with pre-gastrulation Epiblast cells
  • transcriptome signature of pgEpiSCs we performed scRNA-seq on pgEpiSCs at passage 10 and passage 60 (i.e., low and high passages), and then compared the transcriptomes of pgEpiSCs with porcine embryonic single cells (from E0 to E14) transcriptomes for comparison.
  • tSNE visualization revealed that pgEpiSCs clustered individually into groups independently of cells at each stage of the embryo (from E0 to E14) ( Figure 4A).
  • Table 4-1 Representative genes with increased expression compared to conventional hESCs
  • Table 4-2 Representative genes with reduced expression compared to conventional hESCs
  • RPS Regulatory Potential Score
  • a total of 875 genes covariant in RPS and gene expression i.e., genes with higher RPS values were generally upregulated in pgEpiSCs compared with pEFs (log2fold change[FC]>1, FDR ⁇ 0.05)) were identified, of which 75 Genes are strongly expressed in pgEpiSCs (TPM>5 compared with TPM ⁇ 0.5 in pEFs), and representative covariant genes strongly expressed in pgEpiSCs are exemplified in Table 5.
  • OTX2 (as well as LIN28A, NANOG, PRDM14, SALL4, UTF1, and ZFP42) specifically interacted with enhancers in pgEpiSCs but absent in pEFs (Fig. 5E-5K).
  • Example 5 Serial gene editing of pgEpiSCs and production of cloned piglets
  • GN-pgEpiSC clones were screened based on NANOG-tdTomato fluorescence and then re-amplified in 3i/LAF medium (Figure 6D). Consistent with the known status of NANOG (a marker of pluripotency), no tdTomato reporter fluorescence was detected after experimental induction of differentiation in post-knockin edited pgEpiSCs ( Figure 6E).

Abstract

猪多能干细胞培养基及其用途,具体提供了培养基,其包含:第一组分,所述第一组分为IWR-1-endo;第二组分,所述第二组分选自WH-4-023、A419259;和第三组分,所述第三组分选自成纤维细胞生长因子。该培养基易于支持建立稳定的猪多能干细胞系,尤其是猪E8-10 pre-gastrulation上胚层(Epiblast干细胞系)(称为pgEpiSCs)。

Description

猪多能干细胞培养基及其用途 技术领域
本发明涉及生物技术领域,具体涉及猪多能干细胞培养基及其用途。
背景技术
在胚胎发育过程中,上胚层(epiblast)由胚胎的内细胞团(Inner cell mass,ICM)发育来的,并产生所有体细胞和生殖细胞谱系,从而形成正常的胚胎(Chazaud and Yamanaka,2016;Rossant and Tam,2018)。在多能性方面,上胚层(Epiblast)细胞是多能干细胞(Pluripotent stem cells,PSCs)的重要来源,包括来自
Figure PCTCN2022117466-appb-000001
上胚层(Epiblast)的小鼠胚胎干细胞(Embryonic stem cells,ESCs)(Boroviak et al.,2014;Evans and Kaufman,1981;Martin,1981;Ying et al.,2008)和来源于发育后期上胚层(Epiblast)的上胚层(Epiblast)干细胞(Epiblast stem cells,EpiSCs)(Bao et al.,2009;Brons et al.,2007;Tesar et al.,2007).最近,formative(或“intermediate”)多能干细胞成功地从小鼠前原肠上胚层(Epiblast)细胞中获得(Kinoshita et al.,2021;Wang et al.,2021;Yu et al.,2021)。人类传统的ESCs来源于胚泡的ICM,同时显示出与小鼠EpiSCs类似的primed多能性特征(Tesar et al.,2007;Thomson et al.,1998),具有
Figure PCTCN2022117466-appb-000002
或formative多能状态的人类PSCs也可以获得(Gafni et al.,2013;Kinoshita et al.,2021;Theunissen et al.,2014)。
与许多其他动物模型相比,猪在胚胎发育(Kobayashi et al.,2017;Zhu et al.,2021),解剖学(Niu et al.,2017;Yue et al.,2021)和生理学(Yan et al.,2018)方面与人类相比非常相似,因此,稳定的来自上胚层细胞的猪PSCs应该是了解人类PSCs特性的优秀模型,有可能为人类发育建模提供有价值的信息(Xu et al.,2020;Yan et al.,2018)。稳定的猪PSCs与精确的多基因编辑技术的结合将对生物医学研究和农业动物育种产生巨大影响(Navarro et al.,2019;Park et al.,2021;Whitworth et al.,2016;Zhao et al.,2019)。
令人意外的是,尽管科学家们自20世纪90年代以来进行了持久广泛的尝试,仍然没有从ICM或不同阶段的上胚层(Epiblast)细胞分离获得稳定的能够长期传代的猪PSC系(Alberio et al.,2010;Choi et al.,2019;Gao et al.,2019;Haraguchi et al.,2012;Hou et al.,2016;Notarianni et al.,1990;Park et al.,2013;Vassiliev et al.,2010;Yuan et al.,2019;Zhang et al.,2019)。有人提出,胚胎发生过程中的种间差异和早期胚胎发育过程中用来调节多能性的信号通路的物种间的差异可能用以解释数十年来人类和小鼠干细胞培养技术未 能用于猪的原因(Liu et al.,2021;Ramos-Ibeas et al.,2019)。
发明内容
在胚胎发生及其PSC建立的分子基础方面,猪的高分辨率转录组图谱研究远远落后于目前用于小鼠、人类和非人灵长类动物的。然而,基于单细胞RNA测序(scRNA-seq)技术的大规模研究已被用以描述小鼠、人类和猴子的早期胚胎,并追踪谱系发育轨迹和胚胎到干细胞的转变(Boroviak et al.,2014;Deng et al.,2014;Nakamura et al.,2016;Petropoulos et al.,2016;Pijuan-Sala et al.,2019;Stirparo et al.,2018;Tang et al.,2010;Yan et al.,2013;Zhou et al.,2019),目前由于胚胎阶段和细胞数量的限制,对猪胚胎的单细胞转录组研究并没有为植入前胚胎提供准确和高分辨率的转录组图谱(Cao et al.,2014;Kong et al.,2020;Liu et al.,2021;Ramos-Ibeas et al.,2019;Tam and Ho,2020;Wei et al.,2018)。
为此,发明人收集了猪胚胎植入前胚胎,并对所有阶段(E0-E14期间每天的胚胎)进行scRNA-seq,以全面分析猪胚胎早期发育和多能性变化的分子基础。随后,发明人根据分析结果开发了一种培养基(称为3i/LAF),该培养基易于支持建立稳定的猪E8-10pre-gastrulation上胚层(Epiblast)干细胞系(称为pgEpiSCs)。广泛的研究表明,这些pgEpiSCs具有多能性和原肠化前上胚层(Epiblast)细胞的分子特性,呈***的穹顶形态,表达多能性标记物,在200次以上传代中保持稳定,并具有高效畸胎瘤形成和向不同类型细胞分化的能力。发明人还利用pgEpiSCs实现了连续多轮的基因编辑,然后使用基因编辑过的供体细胞进行细胞核移植,成功地产生了纯合子编辑的仔猪。
有鉴于此,在本发明的第一方面,本发明提供了培养基,其包含:
第一组分,所述第一组分为IWR-1-endo;
第二组分,所述第二组分选自WH-4-023、A419259;
第三组分,所述第三组分选自成纤维细胞生长因子。
在一些实施方案中,所述培养基进一步包含:
第四组分,所述第四组分选自CHIR99021、WNT3a;
第五组分,所述第五组分选自TGF-β超家族成员;
第六组分,所述第六组分为LIF。
在一些实施方案中,所述第二组分为WH-4-023。
在一些实施方案中,所述第三组分选自FGF2、FGF1。
在一些实施方案中,所述第三组分为FGF2。
在一些实施方案中,所述第三组分为重组人FGF2。
在一些实施方案中,所述第四组分为CHIR99021。
在一些实施方案中,所述第五组分选自Activin A、Nodal。
在一些实施方案中,所述第五组分为Activin A。
在一些实施方案中,所述第五组分为重组人Activin A。
在一些实施方案中,所述第六组分选自重组人LIF、重组小鼠LIF。
在一些实施方案中,所述第六组分为重组人LIF。
在一些实施方案中,所述第一组分的浓度为0.1-10μM,例如0.1μM、0.2μM、0.3μM、0.4μM、0.5μM、0.7μM、0.9μM、1.1μM、1.3μM、1.5μM、1.7μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.3μM、3.5μM、3.7μM、3.9μM、4.0μM、4.1μM、4.3μM、4.5μM、4.7μM、4.9μM、5.0μM、5.1μM、5.3μM、5.5μM、5.7μM、5.9μM、6.0μM、6.1μM、6.3μM、6.5μM、6.7μM、6.9μM、7.0μM、7.1μM、7.3μM、7.5μM、7.7μM、7.9μM、8.0μM、8.1μM、8.3μM、8.5μM、8.7μM、8.9μM、9.0μM、9.1μM、9.3μM、9.5μM、9.7μM、9.9μM或10μM,或者0.1-0.2μM、0.2-0.3μM、0.3-0.4μM、0.4-0.5μM、0.5-0.7μM、0.7-0.9μM、0.9-1.1μM、1.1-1.3μM、1.3-1.5μM、1.5-1.7μM、1.7-1.9μM、1.9-2.0μM、2.0-2.1μM、2.1-2.3μM、2.3-2.5μM、2.5-2.7μM、2.7-2.9μM、2.9-3.0μM、3.0-3.1μM、3.1-3.3μM、3.3-3.5μM、3.5-3.7μM、3.7-3.9μM、3.9-4.0μM、4.0-4.1μM、4.1-4.3μM、4.3-4.5μM、4.5-4.7μM、4.7-4.9μM、4.9-5.0μM、5.0-5.5μM、5.5-6μM、6-6.5μM、6.5-7μM、7-7.5μM、7.5-8μM、8-8.5μM、8.5-9μM、9-9.5μM或9.5-10μM。
在一些实施方案中,所述第一组分的浓度为0.9-3μM。
在一些实施方案中,所述第一组分的浓度为1-3μM,例如1μM、1.1μM、1.3μM、1.5μM、1.7μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM或3.0μM,或者1-1.1μM、1.1-1.3μM、1.3-1.5μM、1.5-1.7μM、1.7-1.9μM、1.9-2.0μM、2.0-2.1μM、2.1-2.3μM、2.3-2.5μM、2.5-2.7μM、2.7-2.9μM或2.9-3.0μM。
在一些实施方案中,所述第一组分的浓度为2.5μM。
在一些实施方案中,所述第二组分的浓度为3nM-30μM,例如3nM、4nM、5nM、6nM、7nM、8nM、9nM、0.01μM、0.03μM、0.05μM、0.07μM、0.1μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.1μM、1.2μM、1.3μM、1.4μM、 1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.3μM、3.5μM、3.7μM、3.9μM、4.0μM、4.1μM、4.3μM、4.5μM、4.7μM、4.9μM、5.0μM、7.0μM、10μM、13μM、15μM、17μM、20μM、23μM、25μM、27μM或30μM,或者3-4nM、4-5nM、5-6nM、6-7nM、7-8nM、8-9nM、9-10nM、0.01-0.03μM、0.03-0.05μM、0.05-0.07μM、0.07-0.1μM、0.1-0.2μM、0.2-0.3μM、0.3-0.4μM、0.4-0.5μM、0.5-0.7μM、0.7-0.9μM、0.9-1.1μM、1.1-1.3μM、1.3-1.5μM、1.5-1.7μM、1.7-1.9μM、1.9-2.0μM、2.0-2.1μM、2.1-2.3μM、2.3-2.5μM、2.5-2.7μM、2.7-2.9μM、2.9-3.0μM、3.0-3.1μM、3.1-3.3μM、3.3-3.5μM、3.5-3.7μM、3.7-3.9μM、3.9-4.0μM、4.0-4.1μM、4.1-4.3μM、4.3-4.5μM、4.5-4.7μM、4.7-4.9μM、4.9-5.0μM、5.0-7.0μM、7-10μM、10-13μM、13-15μM、15-17μM、17-20μM、20-23μM、23-25μM、25-27μM或27-30μM。
在一些实施方案中,所述第二组分的浓度为0.01-5μM,例如0.01μM、0.03μM、0.05μM、0.07μM、0.1μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.3μM、3.5μM、3.7μM、3.9μM、4.0μM、4.1μM、4.3μM、4.5μM、4.7μM、4.9μM或5.0μM,或者0.01-0.03μM、0.03-0.05μM、0.05-0.07μM、0.07-0.1μM、0.1-0.2μM、0.2-0.3μM、0.3-0.4μM、0.4-0.5μM、0.5-0.7μM、0.7-0.9μM、0.9-1.1μM、1.1-1.3μM、1.3-1.5μM、1.5-1.7μM、1.7-1.9μM、1.9-2.0μM、2.0-2.1μM、2.1-2.3μM、2.3-2.5μM、2.5-2.7μM、2.7-2.9μM、2.9-3.0μM、3.0-3.1μM、3.1-3.3μM、3.3-3.5μM、3.5-3.7μM、3.7-3.9μM、3.9-4.0μM、4.0-4.1μM、4.1-4.3μM、4.3-4.5μM、4.5-4.7μM、4.7-4.9μM或4.9-5.0μM。
在一些实施方案中,所述第二组分的浓度1μM。
在一些实施方案中,所述第三组分的浓度为0.01-100ng/mL,例如0.01ng/mL、0.1ng/mL、0.2ng/mL、0.3ng/mL、0.4ng/mL、0.5ng/mL、0.6ng/mL、0.7ng/mL、0.8ng/mL、0.9ng/mL、1ng/mL、1.5ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、25ng/mL、30ng/mL、35ng/mL、40ng/mL、45ng/mL、50ng/mL、55ng/mL、60ng/mL、65ng/mL、70ng/mL、75ng/mL、80ng/mL、85ng/mL、90ng/mL、95ng/mL或100ng/mL,或者0.01-0.1ng/mL、0.1-0.2ng/mL、0.2-0.5ng/mL、0.5-1ng/mL、1-1.5ng/mL、1.5-2ng/mL、2-3ng/mL、3-4ng/mL、 4-5ng/mL、5-6ng/mL、6-7ng/mL、7-8ng/mL、8-9ng/mL、9-10ng/mL、10-11ng/mL、11-12ng/mL、12-13ng/mL、13-14ng/mL、14-15ng/mL、15-16ng/mL、16-17ng/mL、17-18ng/mL、18-19ng/mL、19-20ng/mL、20-25ng/mL、25-30ng/mL、30-35ng/mL、35-40ng/mL、40-45ng/mL、45-50ng/mL、50-55ng/mL、55-60ng/mL、60-65ng/mL、65-70ng/mL、70-75ng/mL、75-80ng/mL、80-85ng/mL、85-90ng/mL、90-95ng/mL或95-100ng/mL。
在一些实施方案中,所述第三组分的浓度为1-100ng/mL。
在一些实施方案中,所述第三组分的浓度10ng/mL。
在一些实施方案中,所述第四组分的浓度为0.0025nM-3μM,例如0.0025nM、0.005nM、0.01nM、0.015nM、0.02nM、0.025nM、0.03nM、0.035nM、0.04nM、0.045nM、0.05nM、0.1nM、0.15nM、0.2nM、0.25nM、0.3nM、0.35nM、0.4nM、0.45nM、0.5nM、1nM、1.5nM、2nM、2.5nM、3nM、4nM、5nM、6nM、7nM、8nM、9nM、0.01μM、0.05μM、0.1μM、0.15μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM或3.0μM,或者0.0025-0.005nM、0.005-0.01nM、0.01-0.015nM、0.015-0.02nM、0.02-0.025nM、0.025-0.03nM、0.03-0.035nM、0.035-0.04nM、0.04-0.045nM、0.045-0.05nM、0.05-0.1nM、0.1-0.15nM、0.15-0.2nM、0.2-0.25nM、0.25-0.3nM、0.3-0.35nM、0.35-0.4nM、0.4-0.45nM、0.45-0.5nM、0.5-1nM、1-1.5nM、1.5-2nM、2-2.5nM、2.5-3nM、3-4nM、4-5nM、5-6nM、6-7nM、7-8nM、8-9nM、9-10nM、0.01-0.05μM、0.05-0.1μM、0.1-0.15μM、0.15-0.2μM、0.2-0.3μM、0.3-0.4μM、0.4-0.5μM、0.5-0.7μM、0.7-0.9μM、0.9-1.1μM、1.1-1.3μM、1.3-1.5μM、1.5-1.7μM、1.7-1.9μM、1.9-2.0μM、2.0-2.1μM、2.1-2.3μM、2.3-2.5μM、2.5-2.7μM、2.7-2.9μM或2.9-3.0μM。
在一些实施方案中,所述第四组分的浓度为0.01-3μM,例如0.01μM、0.05μM、0.1μM、0.15μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM或3.0μM,或者0.01-0.05μM、0.05-0.1μM、0.1-0.15μM、0.15-0.2μM、0.2-0.3μM、0.3-0.4μM、0.4-0.5μM、0.5-0.7μM、0.7-0.9μM、0.9-1.1μM、1.1-1.3μM、1.3-1.5μM、1.5-1.7μM、1.7-1.9μM、1.9-2.0μM、2.0-2.1μM、2.1-2.3μM、2.3-2.5μM、2.5-2.7μM、2.7-2.9μM或2.9-3.0μM。
在一些实施方案中,所述第四组分的浓度为1μM。
在一些实施方案中,所述第五组分的浓度为0.01-100ng/mL,例如0.01ng/mL、0.5ng/mL、1ng/mL、1.5ng/mL、2ng/mL、2.5ng/mL、3ng/mL、3.5ng/mL、4ng/mL、4.5ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL、30ng/mL、31ng/mL、32ng/mL、33ng/mL、34ng/mL、35ng/mL、36ng/mL、37ng/mL、38ng/mL、39ng/mL、40ng/mL、41ng/mL、42ng/mL、43ng/mL、44ng/mL、45ng/mL、46ng/mL、47ng/mL、48ng/mL、49ng/mL、50ng/mL、55ng/mL、60ng/mL、65ng/mL、70ng/mL、75ng/mL、80ng/mL、85ng/mL、90ng/mL、95ng/mL或100ng/mL,或者0.01-0.5ng/mL、0.5-1ng/mL、1-2ng/mL、2-3ng/mL、3-4ng/mL、4-5ng/mL、5-6ng/mL、6-7ng/mL、7-8ng/mL、8-9ng/mL、9-10ng/mL、10-11ng/mL、11-12ng/mL、12-13ng/mL、13-14ng/mL、14-15ng/mL、15-16ng/mL、16-17ng/mL、17-18ng/mL、18-19ng/mL、19-20ng/mL、20-21ng/mL、21-23ng/mL、23-25ng/mL、25-27ng/mL、27-29ng/mL、29-30ng/mL、30-31ng/mL、31-33ng/mL、33-35ng/mL、35-37ng/mL、37-39ng/mL、39-40ng/mL、40-41ng/mL、41-43ng/mL、43-45ng/mL、45-47ng/mL、47-49ng/mL、49-50ng/mL、50-55ng/mL、55-60ng/mL、60-65ng/mL、65-70ng/mL、70-75ng/mL、75-80ng/mL、80-85ng/mL、85-90ng/mL、90-95ng/mL或95-100ng/mL。
在一些实施方案中,所述第五组分的浓度为25ng/mL。
在一些实施方案中,所述第六组分的浓度为0.01-100ng/mL,例如0.01ng/mL、0.05ng/mL、0.1ng/mL、0.5ng/mL、0.7ng/mL、1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL、30ng/mL、31ng/mL、32ng/mL、33ng/mL、34ng/mL、35ng/mL、36ng/mL、37ng/mL、38ng/mL、39ng/mL、40ng/mL、41ng/mL、42ng/mL、43ng/mL、44ng/mL、45ng/mL、46ng/mL、47ng/mL、48ng/mL、49ng/mL、50ng/mL、53ng/mL、55ng/mL、57ng/mL、60ng/mL、63ng/mL、65ng/mL、67ng/mL、70ng/mL、73ng/mL、75ng/mL、77ng/mL、80ng/mL、83ng/mL、85ng/mL、87ng/mL、90ng/mL、93ng/mL、95ng/mL、97ng/mL 或100ng/mL,或者0.01-0.05ng/mL、0.05-0.1ng/mL、0.1-0.5ng/mL、0.5-0.7ng/mL、0.7-1ng/mL、1-2ng/mL、2-3ng/mL、3-4ng/mL、4-5ng/mL、5-6ng/mL、6-7ng/mL、7-8ng/mL、8-9ng/mL、9-10ng/mL、10-11ng/mL、11-12ng/mL、12-13ng/mL、13-14ng/mL、14-15ng/mL、15-16ng/mL、16-17ng/mL、17-18ng/mL、18-19ng/mL、19-20ng/mL、20-21ng/mL、21-23ng/mL、23-25ng/mL、25-27ng/mL、27-29ng/mL、29-30ng/mL、30-31ng/mL、31-33ng/mL、33-35ng/mL、35-37ng/mL、37-39ng/mL、39-40ng/mL、40-41ng/mL、41-43ng/mL、43-45ng/mL、45-47ng/mL、47-49ng/mL、49-50ng/mL、50-53ng/mL、53-55ng/mL、55-57ng/mL、57-59ng/mL、59-60ng/mL、60-63ng/mL、63-65ng/mL、65-67ng/mL、67-69ng/mL、69-70ng/mL、70-73ng/mL、73-75ng/mL、75-77ng/mL、77-79ng/mL、79-80ng/mL、80-83ng/mL、83-85ng/mL、85-87ng/mL、87-90ng/mL、90-93ng/mL、93-95ng/mL、95-97ng/mL、97-99ng/mL或99-100ng/mL。
在一些实施方案中,所述第六组分的浓度为1-100ng/mL。
在一些实施方案中,所述第六组分的浓度10ng/mL。
在一些实施方案中,所述第四组分和所述第一组分的浓度比为25:1-1:25,例如25:1、24:1、23:1、22:1、21:1、20:1、19:1、18:1、17:1、16:1、15:1、14:1、13:1、12:1、11:1、10:1、9:1、8:1、7:1、6:1、5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17、1:18、1:19、1:20、1:21、1:22、1:23、1:24或1:25,或者25:1-23:1、23:1-21:1、21:1-20:1、20:1-19:1、19:1-17:1、17:1-15:1、15:1-13:1、13:1-11:1、11:1-10:1、10:1-9:1、9:1-7:1、7:1-5:1、5:1-3:1、3:1-1:1、1:1-1:3、1:3-1:5、1:5-1:7、1:7-1:9、1:9-1:10、1:10-1:11、1:11-1:13、1:13-1:15、1:15-1:17、1:17-1:19、1:19-1:20、1:20-1:21、1:21-1:23或1:23-1:25。
在一些实施方案中,所述第四组分和所述第一组分的浓度比为2:3-1:3。
在一些实施方案中,所述第四组分和所述第一组分的浓度比为1:2-1:3。
在一些实施方案中,所述培养基包含:
名称 浓度
CHIR99021 1μM
IWR-1-endo 2.5μM
WH-4-023 1μM
重组人Activin A 25ng/mL
重组人FGF2 10ng/mL
重组人LIF 10ng/mL
需要说明的是,上述第一组分、第二组分、第三组分、第四组分、第五组分、和第六组分的浓度均指的是各组分在培养基中的终浓度。
在一些实施方案中,所述培养基进一步包含:第七组分,所述第七组分为ROCK抑制剂。加入ROCK抑制剂例如Y-27632可以促进pgEpiSCs增殖。
在一些实施方案中,所述第七组分为Y-27632。
在一些实施方案中,所述第七组分的浓度为0.01-50μM,例如0.01μM、0.05μM、0.1μM、0.3μM、0.5μM、0.7μM、0.9μM、1μM、1.1μM、1.3μM、1.5μM、1.7μM、1.9μM、2.0μM、2.1μM、2.3μM、2.5μM、2.7μM、2.9μM、3.0μM、3.1μM、3.3μM、3.5μM、3.7μM、3.9μM、4.0μM、4.1μM、4.3μM、4.5μM、4.7μM、4.9μM、5.0μM、5.1μM、5.3μM、5.5μM、5.7μM、5.9μM、6.0μM、6.1μM、6.3μM、6.5μM、6.7μM、6.9μM、7.0μM、7.1μM、7.3μM、7.5μM、7.7μM、7.9μM、8.0μM、8.1μM、8.3μM、8.5μM、8.7μM、8.9μM、9.0μM、9.1μM、9.3μM、9.5μM、9.7μM、9.9μM、10μM、10.1μM、10.3μM、10.5μM、10.7μM、10.9μM、11μM、11.1μM、11.3μM、11.5μM、11.7μM、11.9μM、12μM、12.1μM、12.3μM、12.5μM、12.7μM、12.9μM、13μM、13.1μM、13.3μM、13.5μM、13.7μM、13.9μM、14μM、14.1μM、14.3μM、14.5μM、14.7μM、14.9μM、15μM、15.5μM、16μM、16.5μM、17μM、17.5μM、18μM、18.5μM、19μM、19.5μM、20μM、20.5μM、21μM、21.5μM、22μM、22.5μM、23μM、23.5μM、24μM、24.5μM、25μM、25.5μM、26μM、26.5μM、27μM、27.5μM、28μM、28.5μM、29μM、29.5μM、30μM、30.5μM、31μM、31.5μM、32μM、32.5μM、33μM、33.5μM、34μM、34.5μM、35μM、35.5μM、36μM、36.5μM、37μM、37.5μM、38μM、38.5μM、39μM、39.5μM、40μM、40.5μM、41μM、41.5μM、42μM、42.5μM、43μM、43.5μM、44μM、44.5μM、45μM、45.5μM、46μM、46.5μM、47μM、47.5μM、48μM、48.5μM、49μM、49.5μM或50μM,或者0.01-0.05μM、0.05-0.1μM、0.1-0.5μM、0.5-1μM、1-1.5μM、1.5-2μM、2-2.5μM、2.5-3μM、3-3.5μM、3.5-4μM、4-4.5μM、4.5-5μM、5-5.5μM、5.5-6μM、6-6.5μM、6.5-7μM、7-7.5μM、7.5-8μM、8-8.5μM、8.5-9μM、9-9.5μM、9.5-10μM、10-10.5μM、10.5-11μM、11-11.5μM、11.5-12μM、12-12.5μM、12.5-13μM、13-13.5μM、13.5-14μM、14-14.5μM、14.5-15μM、15-20μM、20-23μM、23-25μM、25-27μM、27-30μM、30-33μM、33-35μM、35-37μM、37-40μM、40-43μM、43-45μM、45-47μM或47-50μM。
在一些实施方案中,所述培养基用于细胞传代时,所述第七组分的浓度为0.01-20μM,例如0.01μM、0.05μM、0.1μM、0.3μM、0.5μM、0.7μM、0.9μM、1μM、1.1μM、1.3μM、1.5μM、1.7μM、1.9μM、2.0μM、2.1μM、2.3μM、2.5μM、2.7μM、2.9μM、3.0μM、3.1μM、3.3μM、3.5μM、3.7μM、3.9μM、4.0μM、4.1μM、4.3μM、4.5μM、4.7μM、4.9μM、5.0μM、5.1μM、5.3μM、5.5μM、5.7μM、5.9μM、6.0μM、6.1μM、6.3μM、6.5μM、6.7μM、6.9μM、7.0μM、7.1μM、7.3μM、7.5μM、7.7μM、7.9μM、8.0μM、8.1μM、8.3μM、8.5μM、8.7μM、8.9μM、9.0μM、9.1μM、9.3μM、9.5μM、9.7μM、9.9μM、10μM、10.1μM、10.3μM、10.5μM、10.7μM、10.9μM、11μM、11.1μM、11.3μM、11.5μM、11.7μM、11.9μM、12μM、12.1μM、12.3μM、12.5μM、12.7μM、12.9μM、13μM、13.1μM、13.3μM、13.5μM、13.7μM、13.9μM、14μM、14.1μM、14.3μM、14.5μM、14.7μM、14.9μM、15μM、15.5μM、16μM、16.5μM、17μM、17.5μM、18μM、18.5μM、19μM、19.5μM或20μM,或者0.01-0.05μM、0.05-0.1μM、0.1-0.5μM、0.5-1μM、1-1.5μM、1.5-2μM、2-2.5μM、2.5-3μM、3-3.5μM、3.5-4μM、4-4.5μM、4.5-5μM、5-5.5μM、5.5-6μM、6-6.5μM、6.5-7μM、7-7.5μM、7.5-8μM、8-8.5μM、8.5-9μM、9-9.5μM、9.5-10μM、10-10.5μM、10.5-11μM、11-11.5μM、11.5-12μM、12-12.5μM、12.5-13μM、13-13.5μM、13.5-14μM、14-14.5μM、14.5-15μM或15-20μM,优选为10μM。
在一些实施方案中,所述培养基用于细胞维持时,所述第七组分的浓度为0.01-10μM,例如0.01μM、0.05μM、0.1μM、0.3μM、0.5μM、0.7μM、0.9μM、1μM、1.1μM、1.3μM、1.5μM、1.7μM、1.9μM、2.0μM、2.1μM、2.3μM、2.5μM、2.7μM、2.9μM、3.0μM、3.1μM、3.3μM、3.5μM、3.7μM、3.9μM、4.0μM、4.1μM、4.3μM、4.5μM、4.7μM、4.9μM、5.0μM、5.1μM、5.3μM、5.5μM、5.7μM、5.9μM、6.0μM、6.1μM、6.3μM、6.5μM、6.7μM、6.9μM、7.0μM、7.1μM、7.3μM、7.5μM、7.7μM、7.9μM、8.0μM、8.1μM、8.3μM、8.5μM、8.7μM、8.9μM、9.0μM、9.1μM、9.3μM、9.5μM、9.7μM、9.9μM或10μM,或者0.01-0.05μM、0.05-0.1μM、0.1-0.5μM、0.5-1μM、1-1.5μM、1.5-2μM、2-2.5μM、2.5-3μM、3-3.5μM、3.5-4μM、4-4.5μM、4.5-5μM、5-5.5μM、5.5-6μM、6-6.5μM、6.5-7μM、7-7.5μM、7.5-8μM、8-8.5μM、8.5-9μM、9-9.5μM或9.5-10μM,优选为2μM。
需要说明的是,上述第七组分的浓度指的是该组分在培养基中的终浓度。
在一些实施方案中,所述培养基进一步包含:第八组分,所述第八组分为基础培养基。
在一些实施方案中,所述基础培养基为用于培养哺乳动物(优选猪)多能干细胞的基础培养基。
在一些实施方案中,所述基础培养基包含基本培养基、N2supplement、B27supplement、非必需氨基酸、β-巯基乙醇、knockout serum replacement,和选自GlutaMAX、谷氨酰胺的任意一种。
在一些实施方案中,所述基础培养基包含基本培养基、N2supplement、B27supplement、非必需氨基酸、β-巯基乙醇、knockout serum replacement和GlutaMAX。
在一些实施方案中,所述基础培养基包含基本培养基、N2supplement、B27supplement、非必需氨基酸、β-巯基乙醇、knockout serum replacement、抗坏血酸、GlutaMAX和青霉素-链霉素。
在一些实施方案中,所述基本培养基选自DMEM/F12、Neurobasal、DMEM、KO-DMEM、RPMI1640、MEM、mTeSR1或其任意组合。
在一些实施方案中,所述基本培养基选自DMEM/F12、Neurobasal或其组合。
在一些实施方案中,所述基本培养基为DMEM/F12和Neurobasal。
在一些实施方案中,所述基本培养基的体积分数为1%-99%,例如1%、3%、5%、7%、9%、10%、11%、13%、15%、17%、19%、20%、21%、23%、25%、27%、29%、30%、31%、33%、35%、37%、39%、40%、41%、42%、43%、44%、45%、45.5%、46%、47%、48%、49%、50%、51%、53%、55%、57%、59%、60%、61%、63%、65%、67%、69%、70%、71%、73%、75%、77%、79%、80%、81%、83%、85%、87%、89%、90%、91%、93%、95%、97%或99%,或者如1%-3%、3%-5%、5%-7%、7%-9%、9%-11%、11%-13%、13%-15%、15%-17%、17%-19%、19%-20%、20%-21%、21%-23%、23%-25%、25%-27%、27%-29%、29%-30%、30%-31%、31%-33%、33%-35%、35%-37%、37%-39%、39%-40%、40%-41%、41%-42%、42%-43%、43%-44%、44%-45%、45%-45.5%、45.5%-46%、46%-47%、47%-48%、48%-49%、49%-50%、50%-51%、51%-53%、53%-55%、55%-57%、57%-59%、59%-60%、60%-61%、61%-63%、63%-65%、65%-67%、67%-69%、69%-70%、70%-71%、71%-73%、73%-75%、75%-77%、77%-79%、79%-80%、80%-81%、81%-83%、83%-85%、85%-87%、87%-89%、89%-90%、90%-91%、91%-93%、93%-95%、95%-97%或97%-99%。
在一些实施方案中,所述基本培养基的体积分数为91%。
在一些实施方案中,所述DMEM/F12的体积分数为1%-99%,例如1%、3%、5%、7%、9%、10%、11%、13%、15%、17%、19%、20%、21%、23%、25%、27%、29%、 30%、31%、33%、35%、37%、39%、40%、41%、42%、43%、44%、45%、45.5%、46%、47%、48%、49%、50%、51%、53%、55%、57%、59%、60%、61%、63%、65%、67%、69%、70%、71%、73%、75%、77%、79%、80%、81%、83%、85%、87%、89%、90%、91%、93%、95%、97%或99%,或者如1%-3%、3%-5%、5%-7%、7%-9%、9%-11%、11%-13%、13%-15%、15%-17%、17%-19%、19%-20%、20%-21%、21%-23%、23%-25%、25%-27%、27%-29%、29%-30%、30%-31%、31%-33%、33%-35%、35%-37%、37%-39%、39%-40%、40%-41%、41%-42%、42%-43%、43%-44%、44%-45%、45%-45.5%、45.5%-46%、46%-47%、47%-48%、48%-49%、49%-50%、50%-51%、51%-53%、53%-55%、55%-57%、57%-59%、59%-60%、60%-61%、61%-63%、63%-65%、65%-67%、67%-69%、69%-70%、70%-71%、71%-73%、73%-75%、75%-77%、77%-79%、79%-80%、80%-81%、81%-83%、83%-85%、85%-87%、87%-89%、89%-90%、90%-91%、91%-93%、93%-95%、95%-97%或97%-99%。
在一些实施方案中,所述DMEM/F12的体积分数为45%-50%(如45.5%、46%、46.5%)。
在一些实施方案中,所述Neurobasal的体积分数为1%-99%,例如1%、3%、5%、7%、9%、10%、11%、13%、15%、17%、19%、20%、21%、23%、25%、27%、29%、30%、31%、33%、35%、37%、39%、40%、41%、42%、43%、44%、45%、45.5%、46%、47%、48%、49%、50%、51%、53%、55%、57%、59%、60%、61%、63%、65%、67%、69%、70%、71%、73%、75%、77%、79%、80%、81%、83%、85%、87%、89%、90%、91%、93%、95%、97%或99%,或者如1%-3%、3%-5%、5%-7%、7%-9%、9%-11%、11%-13%、13%-15%、15%-17%、17%-19%、19%-20%、20%-21%、21%-23%、23%-25%、25%-27%、27%-29%、29%-30%、30%-31%、31%-33%、33%-35%、35%-37%、37%-39%、39%-40%、40%-41%、41%-42%、42%-43%、43%-44%、44%-45%、45%-45.5%、45.5%-46%、46%-47%、47%-48%、48%-49%、49%-50%、50%-51%、51%-53%、53%-55%、55%-57%、57%-59%、59%-60%、60%-61%、61%-63%、63%-65%、65%-67%、67%-69%、69%-70%、70%-71%、71%-73%、73%-75%、75%-77%、77%-79%、79%-80%、80%-81%、81%-83%、83%-85%、85%-87%、87%-89%、89%-90%、90%-91%、91%-93%、93%-95%、95%-97%或97%-99%。
在一些实施方案中,所述Neurobasal的体积分数为45%-50%(如45.5%、46%、 46.5%)。
在一些实施方案中,所述N2supplement的体积分数为0.002%-10%,例如0.002%、0.005%、0.01%、0.015%、0.02%、0.025%、0.03%、0.035%、0.04%、0.045%、0.05%、0.055%、0.06%、0.065%、0.07%、0.075%、0.08%、0.085%、0.09%、0.095%、0.1%、0.13%、0.15%、0.17%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、1.1%、1.3%、1.5%、1.7%、1.9%、2.0%、2.1%、2.3%、2.5%、2.7%、2.9%、3.0%、3.1%、3.3%、3.5%、3.7%、3.9%、4.0%、4.1%、4.3%、4.5%、4.7%、4.9%、5.0%、5.1%、5.3%、5.5%、5.7%、5.9%、6.0%、6.1%、6.3%、6.5%、6.7%、6.9%、7.0%、7.1%、7.3%、7.5%、7.7%、7.9%、8.0%、8.1%、8.3%、8.5%、8.7%、8.9%、9.0%、9.1%、9.3%、9.5%、9.7%、9.9%或10%,或者0.002%-0.05%、0.05%-0.1%、0.1%-0.15%、0.15%-0.2%、0.2%-0.25%、0.25%-0.3%、0.3%-0.35%、0.35%-0.4%、0.4%-0.45%、0.45%-0.5%、0.5%-0.55%、0.55%-0.6%、0.6%-0.65%、0.65%-0.7%、0.7%-0.75%、0.75%-0.8%、0.8%-0.85%、0.85%-0.9%、0.9%-0.95%、0.95%-1.0%、1.0%-1.1%、1.1%-1.3%、1.3%-1.5%、1.5%-1.7%、1.7%-1.9%、1.9%-2.0%、2.0%-2.1%、2.1%-2.3%、2.3%-2.5%、2.5%-2.7%、2.7%-2.9%、2.9%-3.0%、3.0%-3.1%、3.1%-3.3%、3.3%-3.5%、3.5%-3.7%、3.7%-3.9%、3.9%-4.0%、4.0%-4.1%、4.1%-4.3%、4.3%-4.5%、4.5%-4.7%、4.7%-4.9%、4.9%-5.0%、5.0%-5.5%、5.5%-6.0%、6.0%-6.5%、6.5%-7.0%、7.0%-7.5%、7.5%-8.0%、8.0%-8.5%、8.5%-9.0%、9.0%-9.5%或9.5%-10%。
在一些实施方案中,所述N2supplement的体积分数为0.5%。
在一些实施方案中,所述B27supplement的体积分数为0.002%-20%,例如0.002%、0.005%、0.01%、0.015%、0.02%、0.025%、0.03%、0.035%、0.04%、0.045%、0.05%、0.055%、0.06%、0.065%、0.07%、0.075%、0.08%、0.085%、0.09%、0.095%、0.1%、0.13%、0.15%、0.17%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、1.1%、1.3%、1.5%、1.7%、1.9%、2.0%、2.1%、2.3%、2.5%、2.7%、2.9%、3.0%、3.1%、3.3%、3.5%、3.7%、3.9%、4.0%、4.1%、4.3%、4.5%、4.7%、4.9%、5.0%、5.1%、5.3%、5.5%、5.7%、5.9%、6.0%、6.1%、6.3%、6.5%、6.7%、6.9%、7.0%、7.1%、7.3%、7.5%、7.7%、7.9%、8.0%、8.1%、8.3%、8.5%、8.7%、8.9%、9.0%、9.1%、9.3%、9.5%、 9.7%、9.9%、10%、10.1%、10.3%、10.5%、10.7%、10.9%、11%、11.1%、11.3%、11.5%、11.7%、11.9%、12%、12.1%、12.3%、12.5%、12.7%、12.9%、13%、13.1%、13.3%、13.5%、13.7%、13.9%、14%、14.1%、14.3%、14.5%、14.7%、14.9%、15%、15.5%、16%、16.5%、17%、17.5%、18%、18.5%、19%、19.5%或20%,或者0.002%-0.05%、0.05%-0.1%、0.1%-0.15%、0.15%-0.2%、0.2%-0.25%、0.25%-0.3%、0.3%-0.35%、0.35%-0.4%、0.4%-0.45%、0.45%-0.5%、0.5%-0.55%、0.55%-0.6%、0.6%-0.65%、0.65%-0.7%、0.7%-0.75%、0.75%-0.8%、0.8%-0.85%、0.85%-0.9%、0.9%-0.95%、0.95%-1.0%、1.0%-1.1%、1.1%-1.3%、1.3%-1.5%、1.5%-1.7%、1.7%-1.9%、1.9%-2.0%、2.0%-2.1%、2.1%-2.3%、2.3%-2.5%、2.5%-2.7%、2.7%-2.9%、2.9%-3.0%、3.0%-3.1%、3.1%-3.3%、3.3%-3.5%、3.5%-3.7%、3.7%-3.9%、3.9%-4.0%、4.0%-4.1%、4.1%-4.3%、4.3%-4.5%、4.5%-4.7%、4.7%-4.9%、4.9%-5.0%、5.0%-5.5%、5.5%-6.0%、6.0%-6.5%、6.5%-7.0%、7.0%-7.5%、7.5%-8.0%、8.0%-8.5%、8.5%-9.0%、9.0%-9.5%、9.5%-10%、10%-10.5%、10.5%-11%、11%-11.5%、11.5%-12%、12%-12.5%、12.5%-13%、13%-13.5%、13.5%-14%、14%-14.5%、14.5%-15%或15%-20%。
在一些实施方案中,所述B27supplement的体积分数为1%。
在一些实施方案中,所述非必需氨基酸的体积分数为0.01%-10%,例如0.01%、0.05%、0.1%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、1.1%、1.3%、1.5%、1.7%、1.9%、2.0%、2.1%、2.3%、2.5%、2.7%、2.9%、3.0%、3.1%、3.3%、3.5%、3.7%、3.9%、4.0%、4.1%、4.3%、4.5%、4.7%、4.9%、5.0%、5.1%、5.3%、5.5%、5.7%、5.9%、6.0%、6.1%、6.3%、6.5%、6.7%、6.9%、7.0%、7.1%、7.3%、7.5%、7.7%、7.9%、8.0%、8.1%、8.3%、8.5%、8.7%、8.9%、9.0%、9.1%、9.3%、9.5%、9.7%、9.9%或10%,或者0.01%-0.05%、0.05%-0.1%、0.1%-0.2%、0.2%-0.25%、0.25%-0.3%、0.3%-0.35%、0.35%-0.4%、0.4%-0.45%、0.45%-0.5%、0.5%-0.55%、0.55%-0.6%、0.6%-0.65%、0.65%-0.7%、0.7%-0.75%、0.75%-0.8%、0.8%-0.85%、0.85%-0.9%、0.9%-0.95%、0.95%-1.0%、1.0%-1.1%、1.1%-1.3%、1.3%-1.5%、1.5%-1.7%、1.7%-1.9%、1.9%-2.0%、2.0%-2.1%、2.1%-2.3%、2.3%-2.5%、2.5%-2.7%、2.7%-2.9%、2.9%-3.0%、3.0%-3.1%、3.1%-3.3%、3.3%-3.5%、3.5%-3.7%、3.7%-3.9%、3.9%-4.0%、4.0%-4.1%、4.1%-4.3%、4.3%-4.5%、4.5%-4.7%、4.7%-4.9%、4.9%-5.0%、5.0%-5.5%、5.5%-6.0%、6.0%-6.5%、 6.5%-7.0%、7.0%-7.5%、7.5%-8.0%、8.0%-8.5%、8.5%-9.0%、9.0%-9.5%或9.5%-10%。
在一些实施方案中,所述非必需氨基酸的体积分数为1%。
在一些实施方案中,所述β-巯基乙醇的浓度为0.01mM-1mM,例如,0.01mM、0.02mM、0.03mM、0.04mM、0.05mM、0.06mM、0.07mM、0.08mM、0.09mM、0.1mM、0.11mM、0.12mM、0.13mM、0.14mM、0.15mM、0.16mM、0.17mM、0.18mM、0.19mM、0.2mM、0.21mM、0.22mM、0.23mM、0.24mM、0.25mM、0.26mM、0.27mM、0.28mM、0.29mM、0.3mM、0.31mM、0.32mM、0.33mM、0.34mM、0.35mM、0.36mM、0.37mM、0.38mM、0.39mM、0.4mM、0.41mM、0.42mM、0.43mM、0.44mM、0.45mM、0.46mM、0.47mM、0.48mM、0.49mM、0.5mM、0.51mM、0.53mM、0.55mM、0.57mM、0.59mM、0.6mM、0.61mM、0.63mM、0.65mM、0.67mM、0.69mM、0.7mM、0.71mM、0.73mM、0.75mM、0.77mM、0.79mM、0.8mM、0.81mM、0.83mM、0.85mM、0.87mM、0.89mM、0.9mM、0.91mM、0.93mM、0.95mM、0.97mM、0.99mM或1mM,或者0.01-0.02mM、0.02-0.03mM、0.03-0.04mM、0.04-0.05mM、0.05-0.06mM、0.06-0.07mM、0.07-0.08mM、0.08-0.09mM、0.09-0.1mM、0.1-0.11mM、0.11-0.12mM、0.12-0.13mM、0.13-0.14mM、0.14-0.15mM、0.15-0.16mM、0.16-0.17mM、0.17-0.18mM、0.18-0.19mM、0.19-0.2mM、0.2-0.21mM、0.21-0.23mM、0.23-0.25mM、0.25-0.27mM、0.27-0.29mM、0.29-0.3mM、0.3-0.31mM、0.31-0.33mM、0.33-0.35mM、0.35-0.37mM、0.37-0.39mM、0.39-0.4mM、0.4-0.41mM、0.41-0.43mM、0.43-0.45mM、0.45-0.47mM、0.47-0.49mM、0.49-0.5mM、0.5-0.55mM、0.55-0.6mM、0.6-0.65mM、0.65-0.7mM、0.7-0.75mM、0.75-0.8mM、0.8-0.85mM、0.85-0.9mM、0.9-0.95mM或0.95-1mM。
在一些实施方案中,所述β-巯基乙醇的浓度为0.1mM。
在一些实施方案中,所述knockout serum replacement的体积分数为0.01%-50%,例如0.01%、0.05%、0.1%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、1.1%、1.3%、1.5%、1.7%、1.9%、2.0%、2.1%、2.3%、2.5%、2.7%、2.9%、3.0%、3.1%、3.3%、3.5%、3.7%、3.9%、4.0%、4.1%、4.3%、4.5%、4.7%、4.9%、5.0%、5.1%、5.3%、5.5%、5.7%、5.9%、6.0%、6.1%、6.3%、6.5%、6.7%、6.9%、7.0%、7.1%、7.3%、7.5%、7.7%、7.9%、8.0%、8.1%、8.3%、8.5%、8.7%、8.9%、9.0%、9.1%、9.3%、9.5%、9.7%、9.9%、10%、10.1%、10.3%、10.5%、10.7%、10.9%、11%、11.1%、11.3%、11.5%、 11.7%、11.9%、12%、12.1%、12.3%、12.5%、12.7%、12.9%、13%、13.1%、13.3%、13.5%、13.7%、13.9%、14%、14.1%、14.3%、14.5%、14.7%、14.9%、15%、15.5%、16%、16.5%、17%、17.5%、18%、18.5%、19%、19.5%、20%、20.5%、21%、21.5%、22%、22.5%、23%、23.5%、24%、24.5%、25%、25.5%、26%、26.5%、27%、27.5%、28%、28.5%、29%、29.5%、30%、35%、40%、45%或50%,或者0.01%-0.05%、0.05%-0.1%、0.1%-0.2%、0.2%-0.25%、0.25%-0.3%、0.3%-0.35%、0.35%-0.4%、0.4%-0.45%、0.45%-0.5%、0.5%-0.55%、0.55%-0.6%、0.6%-0.65%、0.65%-0.7%、0.7%-0.75%、0.75%-0.8%、0.8%-0.85%、0.85%-0.9%、0.9%-0.95%、0.95%-1.0%、1.0%-1.1%、1.1%-1.3%、1.3%-1.5%、1.5%-1.7%、1.7%-1.9%、1.9%-2.0%、2.0%-2.1%、2.1%-2.3%、2.3%-2.5%、2.5%-2.7%、2.7%-2.9%、2.9%-3.0%、3.0%-3.1%、3.1%-3.3%、3.3%-3.5%、3.5%-3.7%、3.7%-3.9%、3.9%-4.0%、4.0%-4.1%、4.1%-4.3%、4.3%-4.5%、4.5%-4.7%、4.7%-4.9%、4.9%-5.0%、5.0%-5.5%、5.5%-6.0%、6.0%-6.5%、6.5%-7.0%、7.0%-7.5%、7.5%-8.0%、8.0%-8.5%、8.5%-9.0%、9.0%-9.5%、9.5%-10%、10%-10.5%、10.5%-11%、11%-11.5%、11.5%-12%、12%-12.5%、12.5%-13%、13%-13.5%、13.5%-14%、14%-14.5%、14.5%-15%、15%-20%、20%-25%、25%-30%、30%-35%、35%-40%、40%-45%或45%-50%。
在一些实施方案中,所述knockout serum replacement的体积分数为5%。
在一些实施方案中,所述抗坏血酸的浓度为1μg/mL-5000μg/mL,例如1μg/mL、5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL、41μg/mL、42μg/mL、43μg/mL、44μg/mL、45μg/mL、46μg/mL、47μg/mL、48μg/mL、49μg/mL、50μg/mL、51μg/mL、52μg/mL、53μg/mL、54μg/mL、55μg/mL、56μg/mL、57μg/mL、58μg/mL、59μg/mL、60μg/mL、65μg/mL、70μg/mL、75μg/mL、80μg/mL、85μg/mL、90μg/mL、95μg/mL、100μg/mL、110μg/mL、120μg/mL、130μg/mL、140μg/mL、150μg/mL、160μg/mL、170μg/mL、180μg/mL、190μg/mL、200μg/mL、210μg/mL、220μg/mL、230μg/mL、240μg/mL、250μg/mL、260μg/mL、270μg/mL、280μg/mL、290μg/mL、300μg/mL、310μg/mL、320μg/mL、330μg/mL、340μg/mL、350μg/mL、360μg/mL、370μg/mL、380μg/mL、390μg/mL、400μg/mL、410μg/mL、420μg/mL、430μg/mL、440μg/mL、450μg/mL、460μg/mL、470μg/mL、480μg/mL、490μg/mL、500μg/mL、550μg/mL、600μg/mL、650μg/mL、700μg/mL、750μg/mL、800μg/mL、850μg/mL、900μg/mL、950μg/mL、1000μg/mL、2000μg/mL、3000μg/mL、 4000μg/mL或5000μg/mL,或者1-5μg/mL、5-10μg/mL、10-15μg/mL、15-20μg/mL、20-25μg/mL、25-30μg/mL、30-35μg/mL、35-40μg/mL、40-45μg/mL、45-50μg/mL、50-55μg/mL、55-60μg/mL、60-65μg/mL、65-70μg/mL、70-75μg/mL、75-80μg/mL、80-85μg/mL、85-90μg/mL、90-95μg/mL、95-100μg/mL、100-110μg/mL、110-120μg/mL、120-130μg/mL、130-140μg/mL、140-150μg/mL、150-170μg/mL、170-190μg/mL、190-200μg/mL、200-210μg/mL、210-230μg/mL、230-250μg/mL、250-270μg/mL、270-290μg/mL、290-300μg/mL、300-310μg/mL、310-330μg/mL、330-350μg/mL、350-370μg/mL、370-390μg/mL、390-400μg/mL、400-410μg/mL、410-430μg/mL、430-450μg/mL、450-470μg/mL、470-490μg/mL、490-500μg/mL、500-550μg/mL、550-600μg/mL、600-650μg/mL、650-700μg/mL、700-750μg/mL、750-800μg/mL、800-850μg/mL、850-900μg/mL、900-950μg/mL、950-1000μg/mL、1000-2000μg/mL、2000-3000μg/mL、3000-4000μg/mL或4000-5000μg/mL。
在一些实施方案中,所述抗坏血酸的浓度为50μg/mL。
在一些实施方案中,所述GlutaMAX或谷氨酰胺(优选GlutaMAX)的体积分数为0.01%-10%,例如0.01%、0.1%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、1.1%、1.3%、1.5%、1.7%、1.9%、2.0%、2.1%、2.3%、2.5%、2.7%、2.9%、3.0%、3.1%、3.3%、3.5%、3.7%、3.9%、4.0%、4.1%、4.3%、4.5%、4.7%、4.9%、5.0%、5.1%、5.3%、5.5%、5.7%、5.9%、6.0%、6.1%、6.3%、6.5%、6.7%、6.9%、7.0%、7.1%、7.3%、7.5%、7.7%、7.9%、8.0%、8.1%、8.3%、8.5%、8.7%、8.9%、9.0%、9.1%、9.3%、9.5%、9.7%、9.9%或10%,或者0.01%-0.1%、0.1%-0.2%、0.2%-0.25%、0.25%-0.3%、0.3%-0.35%、0.35%-0.4%、0.4%-0.45%、0.45%-0.5%、0.5%-0.55%、0.55%-0.6%、0.6%-0.65%、0.65%-0.7%、0.7%-0.75%、0.75%-0.8%、0.8%-0.85%、0.85%-0.9%、0.9%-0.95%、0.95%-1.0%、1.0%-1.1%、1.1%-1.3%、1.3%-1.5%、1.5%-1.7%、1.7%-1.9%、1.9%-2.0%、2.0%-2.1%、2.1%-2.3%、2.3%-2.5%、2.5%-2.7%、2.7%-2.9%、2.9%-3.0%、3.0%-3.1%、3.1%-3.3%、3.3%-3.5%、3.5%-3.7%、3.7%-3.9%、3.9%-4.0%、4.0%-4.1%、4.1%-4.3%、4.3%-4.5%、4.5%-4.7%、4.7%-4.9%、4.9%-5.0%、5.0%-5.5%、5.5%-6.0%、6.0%-6.5%、6.5%-7.0%、7.0%-7.5%、7.5%-8.0%、8.0%-8.5%、8.5%-9.0%、9.0%-9.5%或9.5%-10%。
在一些实施方案中,所述GlutaMAX或谷氨酰胺(优选GlutaMAX)的体积分数为0.5%。
在一些实施方案中,所述青霉素-链霉素的体积分数为0.01%-20%,例如0.01%、0.1%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、1.1%、1.3%、1.5%、1.7%、1.9%、2.0%、2.1%、2.3%、2.5%、2.7%、2.9%、3.0%、3.1%、3.3%、3.5%、3.7%、3.9%、4.0%、4.1%、4.3%、4.5%、4.7%、4.9%、5.0%、5.1%、5.3%、5.5%、5.7%、5.9%、6.0%、6.1%、6.3%、6.5%、6.7%、6.9%、7.0%、7.1%、7.3%、7.5%、7.7%、7.9%、8.0%、8.1%、8.3%、8.5%、8.7%、8.9%、9.0%、9.1%、9.3%、9.5%、9.7%、9.9%、10%、10.1%、10.3%、10.5%、10.7%、10.9%、11%、11.1%、11.3%、11.5%、11.7%、11.9%、12%、12.1%、12.3%、12.5%、12.7%、12.9%、13%、13.1%、13.3%、13.5%、13.7%、13.9%、14%、14.1%、14.3%、14.5%、14.7%、14.9%、15%、15.5%、16%、16.5%、17%、17.5%、18%、18.5%、19%、19.5%或20%,或者0.01-0.1%、0.1%-0.2%、0.2%-0.25%、0.25%-0.3%、0.3%-0.35%、0.35%-0.4%、0.4%-0.45%、0.45%-0.5%、0.5%-0.55%、0.55%-0.6%、0.6%-0.65%、0.65%-0.7%、0.7%-0.75%、0.75%-0.8%、0.8%-0.85%、0.85%-0.9%、0.9%-0.95%、0.95%-1.0%、1.0%-1.1%、1.1%-1.3%、1.3%-1.5%、1.5%-1.7%、1.7%-1.9%、1.9%-2.0%、2.0%-2.1%、2.1%-2.3%、2.3%-2.5%、2.5%-2.7%、2.7%-2.9%、2.9%-3.0%、3.0%-3.1%、3.1%-3.3%、3.3%-3.5%、3.5%-3.7%、3.7%-3.9%、3.9%-4.0%、4.0%-4.1%、4.1%-4.3%、4.3%-4.5%、4.5%-4.7%、4.7%-4.9%、4.9%-5.0%、5.0%-5.5%、5.5%-6.0%、6.0%-6.5%、6.5%-7.0%、7.0%-7.5%、7.5%-8.0%、8.0%-8.5%、8.5%-9.0%、9.0%-9.5%、9.5%-10%、10%-10.5%、10.5%-11%、11%-11.5%、11.5%-12%、12%-12.5%、12.5%-13%、13%-13.5%、13.5%-14%、14%-14.5%、14.5%-15%或15%-20%。添加所述青霉素-链霉素有利于防止细胞感染。
在一些实施方案中,所述青霉素-链霉素的体积分数为1%。
在一些实施方案中,所述DMEM/F12和所述Neurobasal的体积比为5:1-1:5,例如5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4或1:5,或者5:1-4:1、4:1-3:1、3:1-2:1、2:1-1:1、1:1-1:2、1:2-1:3、1:3-1:4或1:4-1:5。
在一些实施方案中,所述DMEM/F12和所述Neurobasal的体积比为1:1。
需要说明的是,上述第八组分中的各具体成分的浓度均指的是各具体成分在培养基中 的终浓度。另外,上述第八组分中的各具体成分的体积分数均指的是该具体成分的体积/培养基的总体积。
在一些实施方案中,每500mL的培养基中包含:
名称 浓度或体积或体积分数
CHIR99021 1μM
IWR-1-endo 2.5μM
WH-4-023 1μM
重组人Activin A 25ng/mL
重组人FGF2 10ng/mL
重组人LIF 10ng/mL
DMEM/F12 227.5mL
Neurobasal 227.5mL
N2 supplement 2.5mL
B27 supplement 5mL
GlutaMAX 体积分数0.5%
非必需氨基酸 体积分数1%
β-巯基乙醇 0.1mM
青霉素-链霉素 体积分数1%
knockout serum replacement 体积分数5%
抗坏血酸 50μg/mL
Y-27632(任选) 10μM或2μM
还需要说明的是,上述培养基的各组分或者各组分中的具体成分均是本领域技术人员常规使用的试剂,均可以市购获得。
在一些实施方案中,市购示例如下:
名称 品牌、货号
DMEM/F12 Thermo Fisher 729Scientific,10565-018
Neurobasal Thermo Fisher Scientific,21103-049
N2 supplement Thermo Fisher Scientific,17502-048
B27 supplement Thermo Fisher Scientific,12587-010
GlutaMAX Thermo Fisher Scientific,35050-061
非必需氨基酸 Thermo Fisher 732Scientific,11140-050
β-巯基乙醇 Thermo Fisher Scientific,21985-023
青霉素-链霉素 Thermo FisherScientific,15140-122
knockout serum replacement KOSR,Thermo Fisher Scientific,A3181502,optional
抗坏血酸 Sigma-Aldrich,A4544
CHIR99021 Selleckchem,S1263
IWR-1-endo Selleckchem,S7086
WH-4-023 Selleckchem,S7565
重组人LIF PeproTech,300-05
重组人Activin A Peprotech,120-14E
重组人FGF2 Peprotech,100-18B
Y-27632 Selleckchem,S1049
在一些实施方案中,所述非必需氨基酸包含甘氨酸、L-丙氨酸、L-天冬酰胺、L-天冬氨酸、L-谷氨酸、L-脯氨酸和L-丝氨酸。
在一些实施方案中,所述非必需氨基酸包含:
成分 浓度(mg/L) 浓度(mM)
甘氨酸 750.0 10.0
L-丙氨酸 890.0 10.0
L-天冬酰胺 1320.0 10.0
L-天冬氨酸 1330.0 10.0
L-谷氨酸 1470.0 10.0
L-脯氨酸 1150.0 10.0
L-丝氨酸 1050.0 10.0
需要说明的是,上表中,所述非必需氨基酸的各成分的浓度指的是各具体成分在所述非必需氨基酸中的浓度。
在本发明的第二方面,本发明提供了制备哺乳动物多能干细胞的方法,其包括:
1)提供哺乳动物胚胎上胚层(Epiblast)或其内细胞团;
2)利用前述的培养基培养所述哺乳动物胚胎上胚层(Epiblast)或其内细胞团,获得哺 乳动物多能干细胞。
在一些实施方案中,所述哺乳动物为猪。
在一些实施方案中,所述哺乳动物胚胎上胚层(Epiblast)是E8至E10(例如E8、E9或E10)的哺乳动物胚胎上胚层(Epiblast)。
在一些实施方案中,所述方法是在饲养细胞存在的条件下进行的。
在一些实施方案中,所述饲养细胞选自小鼠胚胎成纤维细胞或STO细胞。
在一些实施方案中,所述饲养细胞为小鼠胚胎成纤维细胞。
在一些实施方案中,所述饲养细胞为停止***的小鼠胚胎成纤维细胞。
在一些实施方案中,所述饲养细胞为丝裂霉素C处理的小鼠胚胎成纤维细胞。
在一些实施方案中,所述饲养细胞的密度为10 4个/孔~10×10 5个/孔。
在一些实施方案中,所述饲养细胞的密度为2×10 4个/孔~2×10 5个/孔。
在一些实施方案中,所述培养器皿为12孔培养板。
在一些实施方案中,所述方法是在温度为37-39℃(优选37℃),氧气浓度为5%-22%(优选5%),二氧化碳浓度为4%-6%(优选5%),湿度为100%的条件下进行的。
在一些实施方案中,所述方法中,所述培养基的更换频率为每10-48小时(优选10-24小时,更优选12小时)更换一次。
在本发明的第三方面,本发明提供了培养哺乳动物多能干细胞和/或维持哺乳动物多能干细胞多能性的方法;其包括:
1)提供哺乳动物多能干细胞;
2)利用前述的培养基培养所述哺乳动物多能干细胞。
在一些实施方案中,所述哺乳动物为猪。
在一些实施方案中,所述哺乳动物多能干细胞为猪胚胎原肠化前上胚层(Epiblast)干细胞。
在一些实施方案中,所述方法是在饲养细胞存在的条件下进行的。
在一些实施方案中,所述饲养细胞选自小鼠胚胎成纤维细胞或STO细胞。
在一些实施方案中,所述饲养细胞为小鼠胚胎成纤维细胞。
在一些实施方案中,所述饲养细胞为停止***的小鼠胚胎成纤维细胞。
在一些实施方案中,所述饲养细胞为丝裂霉素C处理的小鼠胚胎成纤维细胞。
在一些实施方案中,所述饲养细胞的密度为3×10 4个/cm 2~10×10 5个/cm 2
在一些实施方案中,所述饲养细胞的密度为5×10 4个/cm 2
在一些实施方案中,所述方法是在温度为37-39℃(优选38.5℃),氧气浓度为5%-22%(优选20%),二氧化碳浓度为4%-6%(优选5%)的条件下进行的。
在一些实施方案中,所述方法中,所述培养基的更换频率为每10-48小时(优选10-24小时,更优选12小时)更换一次。
附图说明
图1-1.胚胎发育过程中多能性变化的谱系分离和追踪
(A)从胚龄(Embryonic day,E)0至E14采集的猪胚胎进行形态学分析,用于sc-RNA序列分析。总共有16个发展阶段包括,***(E0)、受精卵(E1)、2C(2细胞期胚胎,E2),4C(4细胞期胚胎,E3),8C(8细胞期胚胎,E3)EM(桑椹胚早期,E4),LM(桑椹胚晚期,E5),EB(早期囊胚,E6)、LB(晚期囊胚,E7),HB(孵化囊胚,E8)EBi(早期bilaminar胚胎,E9),LBi(晚期bilaminar胚胎,E10),PPS(前原始条纹胚,E11),EPS(早期原始条纹胚,E12),PS(原始条纹胚,E13),LPS(晚期原始条纹胚,E14)。E0-E8比例尺,100μm;E9-E14比例尺,500μm。
(B)t-SNE图显示所有猪胚胎细胞的转录相似性;不同颜色的圆点代表胚胎期和发育阶段;背景颜色代表所指示的谱系;箭头表示已知的发育轨迹。
(C)E4EM、E5LM和E6EB期胚胎细胞的t-SNE图谱,箭头示ICM/TE谱系分离;小提琴谱图显示了主系分离标记基因。
(D)与C相似对应E6EB、E7LB和E8HB期胚胎细胞,箭头轨迹表明上胚层(Epiblast)/下胚层谱系分离。
(E)与C相似,对应E10LBi、E11PPS和E12EPS阶段的胚胎细胞,箭头轨迹表明外胚层/中胚层谱系分离。
(F)具有相似表达趋势的代表性基因簇显示了桑葚胚(EM和pre-ICM)、ICM、epiblast和ectoderm细胞
Figure PCTCN2022117466-appb-000003
formative和primed多能性基因在E4~E14期间的变化。用绿色、黄色和红色表示的基因名称分别代表可能的
Figure PCTCN2022117466-appb-000004
形成性和启动性多能基因(Kinoshita et al.,2021;Yu et al.,2021);其余的基因(黑色的名字)都是在集群中预测的。
(G)E4~E14期间,来自桑椹胚(EM和pre-ICM)、ICM、epiblast和ectoderm细胞的JAK/STAT3、Activin/Nodal、FGF/ERK和Wnt/β-catenin信号通路相关基因的表达变 化热图,右侧从蓝色到红色的梯度分别表示基因的低表达到高表达。热图顶部从绿色到红色的梯度表示
Figure PCTCN2022117466-appb-000005
formative和primed多能状态的变化。
图1-2.胚胎发育过程中多能性变化的谱系分离和追踪
(A)单细胞转录组测序样品信息汇总。从胚龄(Embryonic day,E)0至E14采集的猪胚胎用于sc-RNA序列分析。总共有16个发展阶段包括,***(E0)、受精卵(E1)、2C(2细胞期胚胎,E2),4C(4细胞期胚胎,E3),8C(8细胞期胚胎,E3)EM(桑椹胚早期,E4),LM(桑椹胚晚期,E5),EB(早期囊胚,E6)、LB(晚期囊胚,E7),HB(孵化囊胚,E8)EBi(早期bilaminar胚胎,E9),LBi(晚期bilaminar胚胎,E10),PPS(前原始条纹胚,E11),EPS(早期原始条纹胚,E12),PS(原始条纹胚,E13),LPS(晚期原始条纹胚,E14)。
(B)E3-E14天胚胎各阶段t-SNE图。不同颜色的点显示谱系分离。
(C)谱系特异性基因表达Heatmap。在每个细胞类型中,基因通过无监督层次聚类(Unsupervised Hierarchical Clustering,UHC)排序。给出了相关基因富集的具有代表性的功能类别。
图1-3.胚胎发育过程中多能性变化的谱系分离和追踪
(A)每个细胞类型与其他细胞类型相比的差异表达基因网络(DEGs)。不同的颜色表示不同的细胞类型。圆圈表示DEGs的集合。每一种细胞类型都通过网络内的线与它的DEGs相连接。
(B)展示了图3-2-1中1,735个Naive多能性相关基因,1,117个Formaive多能性相关基因和1,289个Primed多能性相关基因的GO功能富集分析。显示Metascape(https://metascape.org)摘要基因集的20个顶级功能项,每一项后的数字代表该项总基因中的命中基因。
图2.pgEpiSCs的分离和特征
(A)建立稳定的pgEpiSCs的策略。
(B)不同胚期的胚胎epiblast细胞和ectoderm细胞的衍生物的形态学比较。从左至右依次为E8、E10、E12胚盘(左)和衍生物(右)。E8和E10上胚层(Epiblast)细胞的生长呈球形,E12外胚层细胞的生长呈扁平状和不规则状。E8和E10胚盘的比例尺,100μm;E12胚盘的比例尺,400μm;所有外延的标尺,200μm。
(C)在3i/LAF培养基中建立不同胚期outgrowth细胞的生长效率和细胞系。
(D)pgEpiSCs细胞增殖曲线。初始细胞计数为2×10 5.
(E)pgEpiSCs倍增时间。
(F)pgEpiSCs单细胞克隆效率。
(G)低传代数和高传代数pgEpiSC克隆的形态。标尺,200μm.
(H)碱性磷酸酶(AP)染色法检测低传代和高传代pgEpiSC克隆。标尺,200μm.
(I)pgEpiSCs中多能性标记物POU5F1、NANOG和SOX2的免疫染色。DAPI染色细胞核.Scale bar,50μm.
(J)pgEpiSCs中多能性表面标记物SSEA1、SSEA4、TRA-1-81和TRA-1-60的免疫染色。DAPI用于细胞核染色。Scale bar,50μm.
(K)体外EB分化实验。外胚层神经特异性标记蛋白Tubulinβ-III,中胚层肌肉特异性标记蛋白α-SMA和内胚层特异性标记蛋白GATA6的免疫染色。DAPI染色细胞核。标尺,50μm.
(L)定向诱导分化后pgEpiSCs的免疫染色。SOX1为神经外胚层标记,T为中胚层标记,GATA6为内胚层标记,核为DAPI标记。标尺,200μm.
(M)体内畸胎瘤形成实验。苏木精和伊红染色源自pgEpiSCs的畸胎瘤。标尺,100μm.
误差为±SD(n=3个独立实验)。对于(G-M),在三个独立实验中得到了类似的结果。
图3-1.pgEpiSC体外维护需要3i/LAF培养条件
(A)比较不含CHIR99021、IWR-1-endo和WH-4-023的pgEpiSCs的形态和AP。scale bar,200μm.
(B)采用qRT-PCR对EMT(上)、多能性(中)和中胚层分化(下)中代表性多能标记基因的mRNA表达进行定量分析.
(C)能性标记基因POU5F1和中胚层/内胚层祖标记EOMES的免疫染色。细胞核用DAPI表示.Scale bar,100μm.
(D)qRT-PCR定量增殖相关基因的mRNA表达。
(E)不同浓度CHIR99021处理后pgEpiSCs中POU5F1和GATA6的相对表达变化.
(F)不同浓度CHIR99021处理的pgEpiSCs中POU5F1和GATA6的免疫染色。细胞核用DAPI表示。Scale bar,100μm.
(G)AP染色和免疫染色对未Activin A(Act A)或添加SB431542(SB43)的pgEpiSCs进行染色,并与对照组3i/LAF培养的pgEpiSCs进行比较。Scale bar,200μm.
(H)在不含Act A和/或添加SB43的培养基中,对多能基因和BMP4信号通路相关基 因的mRNA表达进行定量分析。
(I)pgEpiSCs加入或不加入FGF2或加入ERK抑制剂PD0325901培养的AP染色试验。Scale bar,200μm.
(J)有指示分子存在时pgEpiSCs的细胞存活和附着测试。
(K)不同浓度FGF2处理pgEpiSCs的细胞增殖曲线。
(L)AP染色检测pgEpiSCs与LIF或加入JAK1/2抑制剂ruxolitinib(RUXO)培养。Scale bar,200μm.
(M)Western blot观察pgEpiSCs在体外维持中的LIF功能.
For(B),(D),(E),(H),(J),and(K)error bars indicate±SD(n=3independent experiments),n.s.,P≥0.05;*,P<0.05;**,P<0.01,***,P<0.001.For(A),(C),(F),(G),(I),(L),and(M),similar results were obtained in three independent experiments.
图3-2.同细胞系不同代次和不同细胞系之间pgEpiSC细胞系SNVs和Short InDels(≤30bp)的特征。
(a)低代次与高代次pgEpiSCs的核型分析。每个细胞系在有丝***中期检测30个细胞。
(b)来自4个独立供体细胞系(A、B1、B2、B3)的19个pgEpiSC样品(每个样品测序深度为24.42×)全基因组重测序示意图。值得注意的是,三个独立供体细胞系(即B1、B2和B3)是全同胞。
(c)19个pgEpiSC细胞系的Neighbor-joining(NJ)***发育树。比例尺代表p距离。
(d,e)SNVs(d)和InDels(e)的数量和组成。相比之下,在同一代次但来自不同供体的pgEpiSCs之间存在大量基因突变(三个全同胞之间:~3.46M SNVs[Ts/Tv比率:~2.41]和~498.10K InDels;不同家庭之间:~5.71M SNVs(Ts/Tv比率:~2.41)和~816.72K InDels)。来自同一供体的pgEpiSCs在多次传代后的突变数量(~37.98K SNVs[Ts/Tv比率:~2.13]和~15.58K InDels)仅占一小部分(与两个全同胞相比,SNVs:~1.10%;InDels:~3.13%)。此外,与来自同一供体的pgEpiSCs在多次传代后的纯合子突变的比例相比(SNVs:~0.08%;InDels:~0.22%),来自不同供体的pgEpiSCs之间的纯合突变发生显著增加(3个全同胞之间:SNVs:~5.22%,InDels:~4.96%;不同家庭供体之间的SNVs和InDels分别为~16.67%和~16.02%)。
(f,g)不同基因组元件中SNVs(f)和InDels(g)的总结和注释。利用ANNOVAR软件包 对每个SNV和InDel基因进行位点注释。
图3-3.不同培养条件对pgEpiSC制备的影响。
(A)IWR-1-endo替换为XAV939后对细胞维持多能性的影响,AP阳性在传代8代左右丧失。
(B)IWR-1-endo替换为XAV939后对核心多能性因子OCT4(POU5F1)、多能性因子REX1,STELLA,ESRRB表达的影响。
(C)不同浓度CHIR99021对多能性基因POU5F1和谱系分化基因GATA6表达的影响。
(D)不同浓度CHIR99021对多能性基因POU5F1、谱系分化基因GATA6、中胚层标记基因TBX3及内胚层基因ESRRB表达的影响。
(E)CHIR99021和IWR-1-endo浓度比例对多能性因子Nanog表达的影响。
图3-4A:3i/LAF培养基中CHIR99021的浓度调整对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4B:3i/LAF培养基中IWR-1-endo的浓度调整对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4C:3i/LAF培养基中WH-4-023的浓度调整对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4D:3i/LAF培养基中重组人Activin A的浓度调整对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4E:3i/LAF培养基中重组人FGF-basic的浓度调整对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4F:3i/LAF培养基中重组人LIF的浓度调整对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4G:3i/LAF培养基中将CHIR99021替换为WNT3a对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4H:3i/LAF培养基中将WH-4-023替换为A419259对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图3-4I:3i/LAF培养基中将重组人Activin A替换为Nodal对pgEpiSCs体外多能性的影响,上图表示AP染色前,下图表示AP染色后。
图4A:利用猪着床前胚胎细胞(n=1458)和pgEpiSCs(n=196)的scRNA-seq数据绘制t-SNE图。集群是根据胚龄和pgEpiSCs的代次进行颜色编码的。圆圈部分是pre-gastrulation epiblast细胞和pgEpiSCs。
图4B:猪胚胎发育过程中TE、下胚层和上胚层(Epiblast)的经典标记基因点图。颜色梯度代表平均表达水平,点的大小对应TE、下胚层细胞和上胚层(Epiblast)细胞群体中表达特征基因的细胞百分比。
图4C:pgEpiSCs和E7~E14的epiblast或ectoderm细胞PCA图。每个点代表着床前胚胎细胞中的单个细胞,星号代表pgEpiSCs中的单个细胞。颜色表示胚龄和pgEpiSCs的代次。
图4D:Spearman相关系数是基于E7~E14各epiblast或ectoderm细胞中特异表达基因的平均表达水平,与多能性调控和上皮细胞分化有关。
图4E:根据scRNA-seq数据,小提琴图显示E7-E14和低代和高代pgEpiSCs的经典多能基因的表达水平(log2(TPM/10+1))。每个点代表一个细胞。
图4F:
Figure PCTCN2022117466-appb-000006
formative和conventional hPSCs;
Figure PCTCN2022117466-appb-000007
formative和primed mPSCs;以及pgEpiSCs的PCA结果图,其基于每种PSC的独特表达基因的集合。颜色代表多能性状态。三角形代表猪,正方形代表人,圆圈代表鼠。
图4G:在
Figure PCTCN2022117466-appb-000008
hPSCs,formative hPSCs,and conventional hESCs(左)和
Figure PCTCN2022117466-appb-000009
mESCs,formative mPSCs,and primed mEpiSCs(右)中鉴定的独特表达基因的表达水平与pgEpiSCs中的这些基因的表达水平的比较。列出的基因在pgEpiSCs中高度表达。
图5A:每个Hi-C图谱的分辨率分别为染色体内100kb和染色体间1mb。pgEpiSCs-1-B和pEF-1-G核的横切面示例,按常小体(左)或多染色体混合指数(反映由Shannon’s指数测量的染色体多样性)(右)着色(Tan et al.,2018).
图5B:在100kb分辨率的pgEpiSCs(绿色)和pEFs(红色)中,18个常染色体(被1mb窗口平滑)广泛的多染色体混染的概率(每种细胞类型的16个Hi-C图平均)(Tan et al.,2018).
图5C:18号染色体pgEpiSCs-1-B(上半部)和pEF-1-G(下半部)100kb分辨率的接触图示例。
图5D:染色质结构的无序程度(用Von Neumann熵定量)(Lindsly et al.,2021)在 16个Hi-C地图的每个100kb分辨率pgEpiSCs(绿色)和pEFs(红色)。
图5E-5K:OTX2(E)、LIN28A(F)、NANOG(G)、PRDM14(H)、SALL4(I)、UTF1(J)和ZFP42(K)的PEIs示意图。Top panel:基因转录起始位点中心附近区域的Hi-C图(±250kb)。Middle panel:启动子(蓝色球)及其增强子(红色球和绿色球分别代表超级增强子和规则增强子)的三维模型。Bottom panel:RNA-seq配置文件。计算了Benjamini-Hochberg调整后的FDR。
图6A:以pgEpiSCs为供体,通过细胞核转移实验,多基因连续编辑策略生成克隆仔猪示意图。
图6B:GFP-pgEpiSC克隆的形态以及荧光检测,比例尺,200μm。
图6C:通过PCR鉴定NANOG-tdTomato敲入。“GN”代表GFP阳性的NANOG-tdTomato敲入的pgEpiSC。
图6D:NANOG-tdTomato敲入报告基因在GFP标记的pgEpiSCs中的表达。比例尺,100μm。
图6E:NANOG-tdTomato报告基因pgEpiSCs分化后tdTomato表达缺失。比例尺,200μm。
图6F:WT pgEpiSCs(1-pgEpiSCs)和基因修饰的pgEpiSCs(1-GP-pgEpiSCs和1-GN-pgEpiSCs)中TYR基因C到T突变的未编辑比率、纯合子比率和杂合子比率的统计数据(左);野生型、杂合子以及纯合子pgEpiSCs中TYR基因C到T突变位点的代表性DNA测序分析(右)。
图6G:pgEpiSC核转移实验总结。用移植前保留的胚胎计算囊胚率。成纤维细胞来自巴马猪(BAMA pig)。
图6H:Top panel:WT pgEpiSCs克隆仔猪和GFP-pgEpiSCs克隆仔猪的耳朵成纤维细胞;Middle panel:3只GNT-pgEpiSCs克隆了小猪和它们的***母亲;Bottom panel:代表性的GNT-pgEpiSCs克隆仔猪显示GFP荧光,与从巴马猪成纤维细胞中克隆的WT仔猪相反。
图6I:以WT pgEpiSCs为供体细胞产生的代表性克隆仔猪(左)显示黑色毛色,而以GNT-pgEpiSCs为供体细胞产生的代表性克隆仔猪(右)显示白色毛色白化病的表型。
具体实施方式
下面详细描述本发明的实施例,下面描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
自20世纪90年代以来,研究人员一直在尝试建立猪的稳定的上皮细胞来源的干细胞系,但始终未能成功。发明人通过对猪胚胎从0天到14天的大规模单细胞转录组分析,开发了一种无血清的体外培养基,用于建立和维持稳定的猪E10胚胎原肠化前上胚层(Epiblast)干细胞系(Pre-gastrulation epiblast stem cell lines,pgEpiSCs)。在培养过程中,通过化学抑制wnt相关信号通路,pgEpiSCs在传代超过200次以上后仍保持多能性和正常核型。引人注目的是,超深原位Hi-C分析揭示了三维空间关联对pgEpiSCs多能性标记基因转录调控的功能影响。实际上,发明人证实了pgEpiSCs可以很好地耐受至少三轮连续的基因编辑,并通过体细胞核移植技术产生了克隆的基因编辑活仔猪。发明人的发现为人们期待已久的猪多能干细胞提供了希望,并为生物研究、畜牧业和再生生物医学开辟了新的途径。
下面结合具体实施例对本发明进行进一步的解释说明。
除非特别指明,本发明中所使用的分子生物学实验方法和免疫检测法,基本上参照J.Sambrook等人,分子克隆:实验室手册,第2版,冷泉港实验室出版社,1989,以及F.M.Ausubel等人,精编分子生物学实验指南,第3版,John Wiley&Sons,Inc.,1995中所述的方法进行;限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓,实施例以举例方式描述本发明,且不意欲限制本发明所要求保护的范围。
以下实施例中所述的pgEpiSC是指从E10上胚层(Epiblast)建立的细胞系。
实验方法
单细胞RNA文库的制备和测序
与之前的研究一样,单细胞RNA-seq文库,通过改良的Smart-seq2方案制备(Gao et al.,2018;Wang et al.,2018)。简单地说,将单个胚胎细胞转移到准备好的含有8bp bar code的裂解缓冲液中。然后,在含有4U RNA酶抑制剂、100U SuperScript II逆转录酶(Invitrogen,18064071)、1mM dNTPs(TAKARA,4019)、60mM MgCl 2和3μM RT引物和10μM TSO引物的逆转录(RT)混合物中逆转录合成并扩增第一链cDNA。PCR扩增后,用0.8×AMPure XP beads(Beckman,A63882)纯化产物。随后进行生物素PCR扩增。最后, 根据PCR library Amplification/Illumina series KAPA Hyper Prep kit(KAPA,KK8054)构建单细胞RNA-seq文库。高质量文库在Illumina Hiseq Xten(Novogene)上以150bp的配对端测序。
细胞生长曲线和种群倍增时间
猪EpiSCs在12孔板中培养。一式三份的细胞样品以每孔2×10 4个细胞的密度接种。每12小时计数一次细胞数。对于每个时间点,均消化细胞并使用Luna TM自动化细胞计数器进行计数,三次计数结果取平均值并作图。细胞倍增时间的计算如下:倍增时间(DT)=24×[lg2/(lgN t-lgN 0)],其中24是细胞培养时间(小时);N t是48小时培养的细胞数;N 0是24小时记录的细胞数。
单细胞克隆效率分析
通过Accutase(Gibco,A11105-01)将细胞解离,使用血细胞计数器计数,并在pgEpiSCs培养条件下以每孔100、200和1,000个细胞的密度一式三份接种到预接种的6孔板饲养层细胞上。6天后使用AP染色计数克隆,并以每接种的细胞数的克隆数的百分比来评估克隆形成效率。
核型分析
在进行核型分析之前,向pgEpiSCs培养基中加入1%KaryoMAX Colcemid溶液(Gibco,15212012),并将细胞孵育1小时。pgEpiSCs通过TrypLE TMExpress(Gibco,12605010)消化成单细胞,并通过离心收集。用0.075M KCl(sigma,P5405)低渗溶液重悬浮pgEpiSCs,并在37℃下孵育15分钟。然后用甲醇和乙酸按3:1的比例固定pgEpiSCs,重复此过程3次。将pgEpiSCs悬浮液滴到预冷的载玻片上,在室温下彻底干燥,然后用10%Giemsa染色液(Sangon,E607314-0001)染色30分钟。对于每个细胞系,检查了中期超过30个细胞。
全基因组测序
使用TIANamp基因组DNA试剂盒(TIANGEN,DP304)提取pgEpiSCs的总DNA。DNA提取后,用Covaris随机将1μg基因组DNA片段化,并使用Agencourt AMPure XP-Medium试剂盒(BERCKMAN COULTER,A63880)选择200-400bp的片段。将选定的片段末端修复并进行3'腺苷酸化,然后将衔接子连接至末端。
通过PCR扩增产物,然后将纯化的PCR产物热变性为单链,并通过夹板寡核苷酸序列环化。将单链环DNA格式化为最终文库,并通过质量控制进行验证。最终验证的文库通 过BGISEQ-500进行测序。
碱性磷酸酶(AP)染色
pgEpiSC的碱性磷酸酶染色基于碱性磷酸酶检测试剂盒(Millipore,SCR004)。
具体的实验步骤遵循试剂盒的说明。
免疫荧光分析
细胞用DPBS(Gibco,C14190500BT)洗涤并在室温下用4%多聚甲醛固定30分钟,然后再次用DPBS洗涤,在0.1%Triton X-100中通透20分钟,并用3%BSA封闭1小时。将细胞与用3%BSA稀释的一抗在4℃孵育过夜。然后将细胞用洗涤缓冲液(含有0.1%Triton X-100和0.1%Tween 20的DPBS)洗涤3次,每次3分钟。稀释第二抗体,并在室温下与洗涤缓冲液一起温育1小时,然后用洗涤缓冲液洗涤3次,持续5分钟,然后用DAPI(Roche Life Science,10236276001)将细胞核染色3分钟。
拟胚体分化
pgEpiSCs通过Accutase(Gibco,A11105-01)解离,使用差异附着法与饲养细胞分离,在35mm低附着皿上培养,50rpm水平摇床放置于添加10%FBS(Gibco,11960-044)、1%青霉素链霉素(Thermo Fisher Scientific,15140-122)和1%谷氨酰胺(Thermo Fisher Scientific,35050-061)的DMEM(Gibco,11960-044)中培养5-7天。选择规则的球形EB,并在相同的培养基中于1周内铺板在明胶包被的板上,然后使用与免疫荧光相同的方法进行固定和检测。
定向诱导分化
为了进行神经诱导,在pgEpiSCs传代后两天,将3i/LAF培养基替换为神经诱导培养基I(2.5μM IWR-1-endo,5μM SB431542和BM中的10ng/mL FGF2)。培养2天后,将培养基更换为神经诱导培养基II(4μM RA,10ng/mL FGF2和20ng/mL Noggin在BM中),并在2天后进行免疫染色。
对于内胚层诱导,在传代pgEpiSCs两天后,将3i/LAF培养基更换为10ng/mL BMP4、5μM SB431542和10ng/mL FGF2。传代后进行免疫染色。
对于中胚层诱导,在pgEpiSCs传代后两天,将3i/LAF培养基替换为中胚层诱导培养基I(10ng/mL BMP4、50ng/mL Activin A和BM中的20ng/mL FGF2)培养两天。随后加入中胚层诱导培养基II(3μM IWR-1-endo,5μM CHIR99021,20ng/mL FGF2在BM中),2天后进行免疫染色。
畸胎瘤形成
对于畸胎瘤形成分析,通过以1,000rpm离心5分钟收集大约1×10 7个解离的pgEpiSCs细胞,并将其皮下注射到BALB/c裸鼠的后颈中。喂养4-5周后可见畸胎瘤。
H&E分析
从裸鼠皮下收集畸胎瘤,在PBS中洗涤两次,并用4%PFA在4℃固定2天。畸胎瘤组织用酒精梯度(70%,80%,90%,95%和100%)脱水,每个梯度各1小时,转移到二甲苯中并用石蜡进行包埋,将样品切成5μm的厚度,在二甲苯中脱蜡,并用降低浓度的乙醇重新水化。然后将样品用苏木精(Sigma-Aldrich,MHS16)和曙红(Sigma-Aldrich,HT110116)染色,并在显微镜下(Leica,DM5500B)进行观察。
RT-qPCR
使用RNA制备、纯化细胞/细菌试剂盒(TIANGEN,DP430)提取pgEpiSCs的总RNA,然后使用5×多合一RT预混液(Abm,G490)反转录为cDNA。在LightCycler 480II实时***(Roche)上使用2×RealStar Green Power Mixture(GenStar,A311-05)进行PCR。使用比较CT(2 -ΔΔCT)方法分析数据。使用EF1A作为内部对照计算ΔCT。所有实验均进行了三个生物学重复。关键资源表中列出了用于定量实时PCR的引物。
蛋白质免疫印记
用细胞裂解缓冲液(Beyotime Biotechnology,P0013)提取细胞中的总蛋白,用补充了蛋白酶和磷酸酶抑制剂(Beyotime Biotechnology,P1050)的细胞核和细胞质蛋白提取试剂盒(Beyotime Biotechnology,P0027)提取核和细胞质蛋白。使用Bradford蛋白质检测试剂盒(Bio-red,5000201)测量提取蛋白质的浓度。通过SDS-PAGE凝胶电泳分离等量的蛋白质(15μg),并将蛋白质从凝胶中转移至Immobilon-P转移膜(Merck Millipore;孔径:0.45μm;IPVH00010)。室温下,在TBST(20mM Tris,pH 7.5;150mM NaCl;0.1%Tween 20)中的5%脱脂奶粉(Sangon Biotech,A600669-0250)中封闭印迹1小时,然后将其与将抗体在TBST中的5%脱脂奶粉中稀释至4℃过夜。第二天,将印迹用TBST漂洗3次,每次5分钟,然后在TBST中于5%的脱脂奶粉中稀释的HRP偶联二抗孵育,在室温下孵育1小时,最后用TBST漂洗3次,每次5分钟。将印迹用
Figure PCTCN2022117466-appb-000010
West Dura Extended Duration Substrate(Thermo Fisher Scientific,34075)进行显影,利用CLINX化学发光软件分析靶蛋白的条带强度。具体的实验方法和试剂配方来自于Western Blotting通用方案(Bio-red,bulletin 6376)。具体的实验方法和试剂配方来自Western Blotting的通用规程 (Bio-red,bulletin 6376)。
In situ Hi-C
根据先前发表的In situ Hi-C方法并进行了一些细微修改,发明人分别为四个pgEpiSC(生物重复)构建了四个Hi-C库(作为技术重复),为两个pEF(生物重复)分别构建了八个Hi-C库(作为技术重复)。
简而言之,将细胞(5×10 6)与终浓度为4%的甲醛在室温下交联30分钟,然后用终浓度为0.25M/L的甘氨酸淬灭。接下来,将混合物在室温下以1,500×g离心10分钟,并将上清液与裂解缓冲液合并,并在冰上孵育15分钟。然后将混合物在室温下以5,000×g离心10分钟。用NEBuffer 2洗涤沉淀物。将混合物与SDS合并,终浓度为0.1%,并在65℃下孵育10分钟,然后添加TritonX-100,终浓度为1%,并孵育在37℃下放置15分钟。细胞核被透化,并且DNA在37℃下用200单位的DpnII消化1小时。限制性片段突出端被填充并被生物素化的核苷酸标记,然后以小体积连接。交联反向后,使用Covaris S220超声仪纯化DNA并超声处理约300-500bp的片段,然后用Dynabeads TM-280链霉亲和素(Invitrogen,11206D)将连接的片段拉下,然后进行末端修复和加poly-A。接下来连接衔接子,并使用KAPA Hyper Prep Kit(Roche,KK8504)PCR扩增DNA片段8-10个循环。然后使用AMPure XP Beads(Beckman,A63882)对这些片段进行双倍选择,以分离300至800bp之间的片段,这些片段准备在DNBSEQ平台(BGI)上测序,以提供100bp的配对末端读段。
RNA测序
发明人收集并纯化了来自四个供体的pgEpiSCs(作为生物复制品)和来自两个供体的相同背面皮肤区域的pEF(作为生物复制品),每个复制品具有1×10 6个细胞。使用RNeasy Mini Kit(Qiagen,74106)分别提取了六个样品(四个pgEpiSC和两个pEF)的总RNA。发明人将rRNA耗竭方案(球蛋白-零金rRNA去除试剂盒,Illumina,GZG1224)与用于
Figure PCTCN2022117466-appb-000011
Figure PCTCN2022117466-appb-000012
Ultra TM定向RNA库制备试剂盒(NEB,E7420S)结合使用,构建了针对每个样本。使用Qubit dsDNA高灵敏度测定试剂盒(Invitrogen,Life Technologies,Q32851)对所有文库进行定量,并在HiSeq 4000平台(Illumina)上进行测序。
ChIP-seq
发明人对pgEpiSCs和pEFs进行了两个生物学复制,每个样品1×10 7个细胞,进行了H3K27ac(增强子的典型组蛋白标记)的ChIP-seq。在室温下,将细胞与终浓度为1%的甲 醛交联10分钟,然后用甘氨酸淬灭。用补充有蛋白酶抑制剂混合物和1mM PMSF(最终为1x)的裂解缓冲液裂解细胞,然后使用Bioruptor超声处理约200-500bp的片段。将20μL染色质保存在-20℃下用于输入DNA,并将100μL染色质在4℃下与5μg H3K27ac抗体(Abcam,ab4729)进行免疫沉淀。然后,添加30μL蛋白珠,并将样品进一步温育3小时。然后用20mM Tris/HCl(pH 8.1),50mM NaCl,2mM EDTA,1%Triton X-100、0.1%SDS洗涤小珠一次。用10mM Tris/HCl(pH 8.1),250mM LiCl,1mM EDTA,1%NP-40、1%脱氧胆酸处理两次;并用1×TE缓冲液(10mM Tris-Cl在pH 7.5。1mM EDTA)清洗两次。然后在300μL洗脱缓冲液(100mM NaHCO 3,1%SDS)中从磁珠上洗脱结合的材料,首先在65℃下用RNA酶A终浓度8μg/mL处理6小时,然后用蛋白酶K(最终浓度345μg/mL)在45℃下过夜。依照NEXTflex TMChIP-Seq试剂盒(Bioo Scientific,NOVA-5143-02)提供的协议,使用免疫沉淀的DNA来构建测序文库。所有文库均在HiSeq XTen(Illumina)平台上测序。
载体构建
为了测试pgEpiSCs是否可以忍受连续的基因编辑,发明人在pgEpiSCs中进行了三种使用不同基因编辑技术的基因编辑实验:1)使用PiggyBac(PB)转座酶工具稳定地转染GFP-NLS盒;2)通过CRISPR/Cas9***将NANOG-tdTomato报告基因敲入(KI);3)用胞苷碱基编辑器(CBEs)进行TYR基因点突变。
首先,为了获得GFP阳性细胞,发明人构建了PB-CMV-EF1A-GFP-NLS质粒(来自吴森教授),将鸡β-actin启动子替换为人延伸因子1α(Homo sapiianfactor1alpha,EF1A)启动子,并在EF1A启动子的末端***GFP-NLS。其次,为了获得NANOG-tdTomato KI细胞系,发明人构建了具有四个片段的NANOG DNA供体载体骨架,左同源臂3×Flag,3×标记-P2A-tdTomato-Loxp-Puro-Loxp和右同源臂,如前所述。使用
Figure PCTCN2022117466-appb-000013
HiFi DNA Assembly Master Mix(NEB,E2621X)。在终止密码子位点之前靶向NANOG sgRNA,以敲击供体片段作为报告基因。将退火的sgRNA序列克隆到经Bsa I消化的pGL3-U6-sgRNA-PGK-嘌呤霉素载体(Addgene,51133)中。最后,发明人使用AncBE4max质粒敲除TYR基因。AncBE4max和pGL3-U6-sgRNA-EGFP载体是从上海科技大学的黄兴许实验室获得的。SgRNA是由BGI合成的,在正向引物5'端具有ACCG序列,在反向引物5'端具有AAAC序列。然后将sgRNA退火并克隆到pGL3-U6-sgRNA-EGFP载体中。关键资源表中提供了sgRNA序列的详细信息。
细胞电转染
在电转染之前,使用Accutase Cell Dissociation Reagent(Gibco,A11105-01)将pgEpiSCs解离。对于每次电穿孔,使用BTX ECM 2001(哈佛生物科学,Holliston,MA,美国)以220V,5ms,2脉冲转染5×105个细胞。对于GFP-NLS盒稳定转染,使用1μg PBase辅助质粒和3μgPB-CMV-EF1A-GFP-NLS供体质粒(质量比为1:3)进行电穿孔。对于NANOG-tdTomato报告基因敲入,使用1μgpST1374-NLS-flag-linker-Cas9质粒(Addgene,44,758),1μgNANOG sgRNA质粒和1μgNANOG HMEJ供体质粒(每个载体1μg)进行电穿孔;对于TYR基因点突变,用2μg ancBE4max载体和2μgpGL3-U6-TYR sgRNA-GFP载体(质量比1:1)进行电穿孔。电转染缓冲液由中国农业大学农业生物技术国家重点实验室的吴森实验室提供。使用NCBI引物BLAST在线设计引物,并由BGI合成。使用FACS(MoFlo XDP,Backman)对GFP阳性细胞进行分选,并使用488nm(710/50带通滤光片)通道进行检测。为了获得NANOG-tdTomato阳性细胞,用嘌呤霉素(0.3μg/mL)和杀稻瘟素(4μg/mL)选择转染的细胞,并挑选并扩增GFP阳性克隆。为了鉴定碱基编辑的细胞,使用细胞裂解缓冲液(Invitrogen,AM8723)提取DNA作为PCR模板。对PCR产物进行测序以确认点突变。
猪pgEpiSCs克隆胚胎的产生
卵巢收集自北京附近的屠宰场。选择具有三层或四层卵丘细胞的***,并在IVM溶液中于38.5℃,100%湿度和5%CO 2下培养44小时。IVM母液M199(Sigma,M2154)包含0.1%L-半胱氨酸(Sigma,C7352-25G),5%FBS(Gibco,10099141),0.1%EGF(Sigma,E9644),1%青霉素-链霉素(Gibco,15140122)和10%的猪***(***采集过程中收集***,离心并过滤,然后储存在-80℃下)。制备后,IVM母液用0.22μm过滤器过滤,并保存在4℃下以备后用。在使用前,添加1%GlutaMAX(Gibco,35050061),10IU/mL PMSG和10IU/mL hCG。猪EpiSCs在含有10ng/mL BMP4、5μMSB431542和10ng/mL FGF2的基础培养基中分化超过1周,然后用作供体细胞进行核移植。通过在含7.5μg/mL胞松弛素B的HM培养基中进行显微操作,去除了处于中期II的成熟***。将形态学合格的供体细胞注入卵周隙,并使用BLS电细胞操纵器在融合培养基(0.3M/L甘露醇,1.0mM/L CaCl2、0.1mM/L MgCl2和0.5mM/L HEPES)。
然后将***在PZM-3中孵育15分钟,并在体视显微镜下评估融合比率。将五十至六十个融合胚放入每孔含500μL PZM-3的四孔培养皿中,并在38.5℃的PZM-3中于5% CO 2、5%O 2和90%N 2中培养,并保持最大湿度。24小时后,将150-250个ESCNT合子通过外科手术移植到***母亲中。在25-30天时通过超声检查来确定***的妊娠状态。所有克隆的仔猪均在妊娠第114-120天时自然分娩。
资源表
Figure PCTCN2022117466-appb-000014
Figure PCTCN2022117466-appb-000015
Figure PCTCN2022117466-appb-000016
Figure PCTCN2022117466-appb-000017
Figure PCTCN2022117466-appb-000018
Figure PCTCN2022117466-appb-000019
实施例1
1、scRNA-seq揭示猪胚胎发育过程中的谱系分离
为了揭示猪胚胎发育过程中谱系分离和多能性变化的分子基础,发明人使用改良的单细胞标记逆转录测序(STRT-seq)协议进行了scRNA-seq(见STAR Methods)(Gao et al.,2018;Wang et al.,2018),并最终筛选获得了从E1至E14取样的猪***和胚胎共1,458个经过质控后保留的单细胞的数据(图1-1A和图1-2A)。发明人通过共享最近邻算法(Shared nearest neighbor,SNN)和t分布随机近邻嵌入(t-distributed stochastic neighbor embedding,t-SNE)(Stuart et al.,2019)识别了不同阶段的细胞类型,并描述了不同的谱系分化过程。根据已知的分化和多能性标记基因,这些细胞在不同的胚胎时期被分类为特定的胚胎谱系(图1-1B和图1-2B)(Edgar et al.,2013;Nakamura et al.,2016;Ramos-Ibeas et al.,2019),通过特异性表达基因和共表达基因网络的功能富集,形成不同的基因表达特征的细胞群(图1-2C和图1-3A)。
发明人发现猪胚胎第一次谱系分离是在E5桑葚胚晚期开始的(图1-1C)。桑葚胚晚期细胞的两个亚群,被称为pre-ICM和pre-TE,表现出ICMs(如PDGFRA)或TEs(如DAB2)的经典前体标记基因的差异表达(Petropoulos et al.,2016;Wei et al.,2018;Wu et al.,2016)(图1-1C)。在E6早期囊胚期,ICM和TE细胞分成两个细胞群,在ICMs中上调PDGFRA、NANOG和SOX2,而在TEs中上调CDX2、DAB2、GATA2和GATA3(图1-1C)。在E6的ICMs中检测到GATA6(一种hypoblast标记物)和NANOG(一种epiblast标记物)的异质表达(图1-1D),标志着第二次谱系分离的开始(Plusa et al.,2008;Saiz et al.,2016)。
在E7囊胚晚期,GATA6和NANOG阳性细胞分为两个群体(图1-1D),结果表明,第二次谱系分离结束,在胚胎中建立了hypoblast细胞(GATA4 +和GATA6 +)和epiblast细胞 (NANOG +和SOX2 +)谱系。从E7到E10,epiblast细胞和hypoblast细胞的数量迅速增加,hypoblast细胞在TE内延伸形成一个完整的腔,称为原肠腔(Oestrup et al.,2009);epiblast细胞多能性标记基因NANOG、POU5F1和SOX2的高表达(图1-1D),直到中胚层在E11开始形成(以原肠化标记基因如LEF1、KDR、TBXT、HAND1和BMP4上调为标志)(图1-1E)。根据先前报道的组织学和形态学证据,发明人在E0-E14发育过程中观察到的分子遗传调控趋势与发育过程中特征明确的模式完全一致(Kobayashi et al.,2017;Oestrup et al.,2009)。这种一致性凸显了发明人单细胞转录组测序数据的代表性和实用性,该数据可用于支持应用生物技术探索和胚胎调控的基础研究。
2、追踪猪epiblast发育过程中的多能性变化
为了追踪猪epiblast发育过程中的多能性变化,并确定其对信号通路的影响,发明人通过对E4早期桑葚胚、E5pre-ICMs、E6ICMs、E7-E10epiblasts和E11-E14ectoderms中的11,113个DEGs进行配对比较,根据胚胎阶段的基因表达趋势将其分为36个集群(see STAR Methods)。有趣的是,从E4早期桑葚胚到E7epiblast,具有代表性的
Figure PCTCN2022117466-appb-000020
多能基因(如ESRRB、KLF4、LIFR、STAT3、TFCP2L1和TBX3)的表达显著降低。相比之下,经典primed多能基因(如ZIC5、ETV5、ZIC2、LEF1、BMP4和LIN28B)在E7后表达升高(图1-1F).formative标记基因(如TDGF1、DNMT3A、OTX2、KLF5、LIN28A和NODAL)在E7-E10上胚层(Epiblast)中表达量较高,而在E11-E13外胚层中表达量较低(图1-1F)。此外,对三种多能性状态的表达趋势的注释还得到了多能性调控网络和丰富的功能术语的支持(图1-3A和图1-3B)。发明人在E7上胚层(Epiblast)形成前观察到
Figure PCTCN2022117466-appb-000021
多能性的快速丧失—可能有助于解释前期研究中关于猪
Figure PCTCN2022117466-appb-000022
胚胎干细胞建立为什么失败的原因。此外,这些数据表明epiblast从E7到E10保持稳定的formative状态,并可能支持建立稳定的多能干细胞系。
发明人假设epiblast来源的干细胞系的分离建立需要多能性信号通路的稳定和分化信号的抑制。最后,发明人重点研究了4条信号通路:JAK/STAT3、Activin/Nodal、FGF/ERK和Wnt/β-catenin。发明人发现JAK/STAT3信号通路的中枢基因在E4早期桑葚胚到E6ICMs细胞中高度表达,但在E7epiblast形成后急剧下降(图1-1G),与
Figure PCTCN2022117466-appb-000023
多能性标记基因在猪epiblast发育过程中的表达模式一致(图1-1F).Activin A和FGF2受体在E7至E10的上胚层(Epiblast)中高度表达(图1-1G),这表明,猪上胚层(Epiblast)细胞的增殖和维持需要Activin A和FGF2的存在。有趣的是,发明人注意到Wnt/β-catenin信号活性在 从E10到E11的epiblast到ectoderm发育过渡过程中显著增加(图1-1G),这表明抑制Wnt/β-catenin信号通路可能是诱导和维持稳定的猪epiblast所必需的。
实施例2
1、用E10上胚层(Epiblast)细胞建立稳定的猪pgEpiSC系
(1)研究过程
在实施例1的基础上,发明人从scRNA-seq分析得出的结论表明,pgEpiSCs的建立应通过使用与WNT信号相关的小分子抑制剂来防止胚胎原肠化,以及使用TGF-β超家族和FGF家族细胞因子来促进上胚层(Epiblast)自我更新。为了在E10上胚层(Epiblast)细胞中实现这一目标,发明人测试了多种结合多种抑制剂和细胞因子的培养基配方,最终开发出一种由3种抑制剂(CHIR99021、IWR1-endo和WH-4-023)和3种细胞因子(LIF、Activin A和FGF2)组成的条件培养基,发明人称之为“3i/LAF”培养基(图2A)。在3i/LAF条件下,发明人发现E10期epiblast细胞的建立效率高于其他时期epiblast细胞(图2B和图2C)。来自E10epiblast的pgEpiSCs可以在单细胞水平上通过酶解传代,传代率为1:3-1:5/2~3天。pgEpiSCs增殖倍增时间约为16小时(图2D和图2E),单细胞集落形成效率约为33.83%(图2F).
发明人发现pgEpiSCs保留了其穹顶状克隆形态(图2G),AP阳性(图2H),和正常核型(图3-2a)。pgEpiSCs在长期体外培养过程中表达多功能干细胞标志物如POU5F1、NANOG和SOX2(图2I),以及多能性表面标记包括SSEA1、SSEA4、TRA-1-81和TRA-1-60(图2J)。重要的是,拟胚体(embryonic body,EB)分化试验表明,pgEpiSCs在去除培养基中的抑制剂和细胞因子后可以分化为三胚层(图2K)。定向诱导分化试验表明,pgEpiSCs在条件培养基中也能分化为预期的三胚层(图2L)。畸胎瘤形成试验证实,pgEpiSCs在体内发育成预期的三个胚层(图2M)。所有pgEpiSC系可以传代60次以上而不改变上述特性。通过比较同细胞系不同代次和不同供体细胞系之间pgEpiSC细胞系的核型、SNVs和Short InDels(≤30bp),发明人发现在培养过程中经过60次传代后,细胞系核型正常,且检测到的基因突变极少(图3-2b到图3-2g)。然后发明人随机选择两株细胞系进行长期维护能力测试,已传代至少216次。
这些结果表明,发明人成功建立了稳定的猪pgEpiSC系。
(2)具体步骤
1)pgEpiSCs来源于猪E8-E10胚胎的Epiblast,尽最大可能用机械法去除Hypoblast和TE,保证Epiblast表面不粘附hypoblast或者TE细胞。之后有两种方法可以建立pgEpiSCs,①用TrypLE  TM Express处理epiblast 3mins,然后用直径40-50μm的口吸管反复吹吸使其分散成小细胞团。之后将细胞团接种于添加750μL 3i/LAF培养基(注:3i/LAF培养基中添加10μM Y27632)的12孔的细胞培养皿(饲养层细胞密度约为2×10 5个细胞/孔)中,在37℃,湿度100%,5%CO 2,5%O 2(氧气浓度可选择低氧,也可用常氧)的培养箱中培养。②将整个Epiblast完整的接种于细胞培养皿中,相同条件下培养。两种方法均可在P2代通过消化传代获得稳定细胞系,相比之下,第①种方法效率更高。12hrs后,在不改变培养基的情况下再加入750μL 3i/LAF培养基,24hrs后,更换新鲜3i/LAF培养基(含有2μM Y27632)。之后每隔12hrs更换新鲜培养基,在克隆生长2-3天后,观察克隆形态,消化传代。
2)传代时,首先吸去培养基,用37℃温浴的DPBS清洗细胞表面,添加适量(根据孔板大小视情况而定,一般的,6孔板覆盖表面需要至少500μL,其余型号孔板以此类推)Accutase细胞解离试剂(Gibco,A11105-01),于37℃培养箱中消化3-5mins,见大部分克隆脱落,即可添加等量培养基终止消化,轻柔吹打10-15次,使细胞克隆充分分散,之后按照1:3-1:5的传代比例进行接种,传代时使用的3i/LAF培养基添加10μM Y27632。
3)稳定细胞系正常培养时,传代后12hrs更换新鲜培养基(视情况而定,若消化后并没有离心去除Accutase,则12hrs后更换新鲜培养基;若消化后已经离心去除Accutase,则24hrs后更换新鲜培养基),24hrs后再次更换培养基,之后每隔12hrs更换培养基,直至传代。
4)冻存时,按照上述方法消化成单细胞后,1000rpm 5mins离心收集细胞,去除上清液,使用4℃预冷的新鲜配制的pEpiSCs细胞冻存液(10%DMSO+90%KOSR)轻柔重悬细胞,按照可传代比例分装于冻存管中,标记细胞信息,转移至4℃预冷的细胞冻存盒中,并迅速转移至-80℃超低温冰箱中,24hrs后转移至液氮中长久保存。
5)解冻细胞时,将细胞从液氮中取出,用浮漂放于37℃水浴锅中,快速摇动,使其解冻,至冻存管中仅有很小一点冰晶未融化时,喷洒75%酒精消毒,在超净工作台中将细胞冻存液小心转移至装有3mL细胞培养液的15mL离心管中,1000rpm离心5mins,去除上清,轻柔重悬细胞,之后按照所需密度将细胞接种于饲养层细胞上。
2、“3i/LAF”培养基的具体制备及其应用
配置500mL 3i/LAF培养基时,基础培养基(BM)包含的成分及各自的含量或在500mL 3i/LAF培养基中的终浓度如下所示:227.5mL DMEM/F12(Thermo Fisher 729Scientific,10565-018),227.5mL Neurobasal(Thermo Fisher Scientific,21103-049),2.5mL N2supplement(Thermo Fisher Scientific,17502-048),5mL B27supplement(Thermo Fisher Scientific,12587-010),0.5%GlutaMAX(Thermo Fisher Scientific,35050-061),1%非必需氨基酸(Thermo Fisher 732 Scientific,11140-050),0.1mMβ-巯基乙醇(Thermo Fisher Scientific,21985-023),1%青霉素-链霉素(Thermo FisherScientific,15140-122),5%knockout serum replacement(KOSR,Thermo Fisher Scientific,A3181502,optional),以及50μg/mL抗坏血酸(Sigma-Aldrich,A4544)。为了配置3i/LAF培养基,需要进一步向前述基础培养基中添加小分子和细胞因子,各小分子或细胞因子在500mL 3i/LAF培养基中的终浓度如下所示:CHIR99021(1μM,Selleckchem,S1263),IWR-1-endo(2.5μM,Selleckchem,S7086),WH-4-023(1μM,Selleckchem,S7565),重组人LIF(10ng/mL,PeproTech,300-05),重组人Activin A(25ng/mL,Peprotech,120-14E),以及重组人FGF-basic(10ng/mL,Peprotech,100-18B)。
为了促进pgEpiSCs增殖,可以选择进一步向前述配置完成的3i/LAF培养基中添加ROCK抑制剂Y-27632(传代,终浓度10μM;维持,终浓度2μM;Selleckchem,S1049)。
在20%O 2、5%CO 2、38.5℃条件下将猪pgEpiSCs用3i/LAF培养基进行培养。
PgEpiSCs在丝裂霉素C(Selleckchem,S8146)处理的小鼠胚胎成纤维细胞(MEF)饲养细胞(5×10 4个/cm 2)上培养。每12小时更换一次培养基,加入新鲜的3i/LAF培养基。
3、pgEpiSC长期维持对抑制剂和生长因子的需求
发明人基于上述第2点配置的3i/LAF培养基及其培养方法,在其他条件不变,并逐个去除培养液中小分子抑制剂和细胞因子的基础上,测试了3i/LAF培养基中各因子对pgEpiSCs长期体外维持的需求。
发明人发现去除三种WNT信号通路相关抑制剂中的任何一种都会破坏所需的圆顶克隆形态,并削弱碱性磷酸酶(AP)染色信号强度(图3-1A);这也上调了上皮-间充质转化(EMT)相关基因,包括IGF2、SNAI2、SRC和WNT5A(图3-1B)。特别是,IWR-1-endo 的去除直接导致pgEpiSC克隆的边界不清晰,并导致核心多能性因子如NANOG、POU5F1、SOX2和REX1的显著下调(图3-1B)。
此外,去除IWR-1-endo或WH-4-023可导致pgEpiSCs中胚层和内胚层分化,表现为原肠形成标记基因BMP2、BMP4、EOMES和T的上调(图3-1B),并导致多能性因子POU5F1积累减少或异质性,同时促进中胚层和内胚层祖细胞标记物EOMES的表达(图3-1C)。CHIR99021在小鼠和人PSCs的多能性维持中具有双重作用,低浓度促进自我更新,高浓度促进分化。去除CHIR99021下调了LIN28A、C-MYC、ETV4、ETV5等细胞增殖相关基因的表达(图3-1D),提示pgEpiSC增殖受损。高浓度的CHIR99021导致多能性标记物POU5F1下调,内胚层标记物GATA6显著上调(图3-1E),免疫荧光染色进一步证实了这些结果(图3-1F),这表明CHIR99021在小鼠、人类和猪的PSCs中是保守的。
检测去除3i/LAF中的细胞因子后,TGF-β超家族成员Activin A的去除导致多能性标记物NANOG的水平降低(图3-1G和图3-1H)。进一步支持Activin A在pgEpiSCs长期培养中的作用,在培养基中加入TGF-β通用抑制剂SB431542(避免feeder分泌物的影响),导致克隆形态不规则,NANOG水平大幅下降(图3-1G和图3-1H)。值得注意的是,添加SB431542还导致多能性标记(如POU5F1和REX1)显著减少,BMP4和BMP下游转录因子(如ID2和ID3)显著增加,这些转录因子介导了胚胎发育过程中圆条的诱导(Kurek et al.,2015;Valdez Magana et al.,2014)(图3-1H)。
当FGF2被移除时(加入ERK/MEK抑制剂PD0325901),pgEpiSCs不能正常增殖或传代(图3-1I到图3-1K).发明人还注意到,在其他条件不变时,降低FGF2浓度显著降低了pgEpiSCs的增殖能力(图3-1K),低浓度的FGF2不利于细胞增殖,而高浓度的细胞增殖较快.值得注意的是,发明人发现LIF并不是维持pgEpiSC克隆形态的必要条件(图3-1L),但是加入JAK1/2抑制剂ruxolitinib会使克隆变得扁平,western blotting显示只有在LIF存在时才能检测到磷酸化的STAT3(图3-1M),说明pgEpiSCs能响应LIF的促进多能性的作用。
另外,发明人基于上述第2点配置的3i/LAF培养基及其培养方法,在其他条件不变的基础上,还进行了以下测试。
(1)其他条件不变,仅仅用XAV939替换IWR-1-endo
IWR-1-endo替换为XAV939后,细胞无法长期维持多能性,AP阳性在传代8代左右丧失(如图3-3A所示)。
IWR-1-endo替换为XAV939后,核心多能性因子OCT4(POU5F1)表达下调;多能性因子REX1,STELLA,ESRRB表达下调(如图3-3B所示)。
(2)其他条件不变,仅仅调整CHIR99021的浓度
培养体系中CHIR99021的浓度影响细胞的多能性和均质性。当CHIR99021浓度高时,多能性基因POU5F1表达降低,谱系分化基因GATA6表达上调,呈现异质性表达模式。中胚层标记基因TBX3及内胚层基因ESRRB表达量随浓度升高而表达增高(如图3-3C和图3-3D所示)。
(3)其他条件不变,仅仅调整CHIR99021和IWR-1-endo浓度比例
CHIR99021和IWR-1-endo浓度比例对多能性有影响,最适配比C/I≈1:2-1:3,多能性因子Nanog表达最高(如图3-3E所示)。
4、“3i/LAF”培养基的扩展研究
发明人基于上述第2点配置的3i/LAF培养基及其培养方法,在其他条件不变,并逐个调整3i/LAF培养基中小分子抑制剂和细胞因子的浓度或者替换3i/LAF培养基中小分子抑制剂和细胞因子的成分的基础上,研究了调整或替换后的培养基对pgEpiSCs长期体外维持多能性的影响。
(1)调整3i/LAF培养基中小分子抑制剂和细胞因子的浓度
培养基调整:相比于上述第2点配置的3i/LAF培养基及其培养方法,仅对3i/LAF培养基中小分子抑制剂或细胞因子的浓度分别进行了如下表A所示的单因素调整,其他条件保持不变。
实验方法:用调整后的培养基对pgEpiSCs进行培养,之后对培养的P1代(表示调整后传1代的pgEpiSCs)或P3代(表示调整后传3代的pgEpiSCs)的pgEpiSCs进行AP染色。AP染色的具体步骤详见前述实验方法部分。
实验原理:未分化的干细胞会高水平表达Ap。通过对固定的干细胞染色,分化的细胞无色,未分化的细胞呈现紫色或者红色。
实验结果:结果如下表A或图3-4A~3-4F所示。
表A:3i/LAF培养基中小分子抑制剂或细胞因子的浓度调整及测试结果
Figure PCTCN2022117466-appb-000024
Figure PCTCN2022117466-appb-000025
Figure PCTCN2022117466-appb-000026
从图3-4A~3-4F可以看出,对3i/LAF培养基中小分子抑制剂或细胞因子的浓度进行上述调整后获得的培养基依然可以长期维持pgEpiSCs的体外多能性。
(2)替换3i/LAF培养基中小分子抑制剂和细胞因子的成分
培养基调整:相比于上述第2点配置的3i/LAF培养基及其培养方法,仅对3i/LAF培养基中小分子抑制剂或细胞因子的成分分别进行了如下表B所示的单因素替换,其他条件保持不变。
实验方法:用调整后的培养基对pgEpiSCs进行培养,之后对培养的P1代(表示调整后传1代的pgEpiSCs)或P3代(表示调整后传3代的pgEpiSCs)的pgEpiSCs进行AP染色。AP染色的具体步骤详见前述实验方法部分。
实验原理:未分化的干细胞会高水平表达Ap。通过对固定的干细胞染色,分化的细胞无色,未分化的细胞呈现紫色或者红色。
实验结果:结果如下表B或图3-4G~3-4I所示。
表B:3i/LAF培养基中小分子抑制剂或细胞因子的成分替换及测试结果
Figure PCTCN2022117466-appb-000027
Figure PCTCN2022117466-appb-000028
从图3-4G~3-4I可以看出,对3i/LAF培养基中小分子抑制剂或细胞因子的成分进行上述替换后获得的培养基依然可以长期维持pgEpiSCs的体外多能性。
实施例3:pgEpiSC的转录组与原肠化前上胚层(Epiblast)细胞的相关性
为了研究pgEpiSCs的转录组特征,我们对第10代和第60代(即低代和高代)的pgEpiSCs进行了scRNA-seq,然后将pgEpiSCs的转录组与猪胚胎单细胞(从E0到E14)的转录组进行比较。tSNE可视化显示pgEpiSCs单独聚成一组,独立于胚胎各时期细胞(从E0到E14)(图4A)。谱系分离的marker基因表达水平表明,上胚层(Epiblast)特异性基因(NANOG、TDGF1、ETV4、GDF3和NODAL等)在pgEpiSCs中高度一致地表达(图4B),这表明pgEpiSCs维持了上胚层(Epiblast)细胞的转录组特性。主成分分析(PCA)使用分化和多能性DEGs显示pgEpiSCs聚集在E10上胚层细胞(图4C)。两两相关分析表明,低和高代次pgEpiSCs表现非常一致(r=0.97, P<2.2×10 -16,Spearman's rank correlation),共同显示与E10上胚层(Epiblast)最相似,相比其他胚胎时期的epiblast或ectoderm细胞(平均r=0.88,P<2.2×10到16,Spearman's rank correlation)(图4D)。此外,pgEpiSCs中典型的多能性和原肠化标记基因的表达水平表明它们与E10上胚层(Epiblast)最接近(图4E)。所有这些结果表明,pgEpiSCs具有与其起源E10上胚层(Epiblast)相似的转录组特征。
接下来,我们对pgEpiSCs的RNA-seq数据与人类
Figure PCTCN2022117466-appb-000029
传统的和formative PSCs以及小鼠
Figure PCTCN2022117466-appb-000030
primed和先前报道的formative PSCs进行了比较转录组分析(Guo et al.,2017;Ji et al.,2016;Kinoshita et al.,2021)。我们发现pgEpiSCs比
Figure PCTCN2022117466-appb-000031
PSCs更类似于formative和primed(或传统)PSCs(图4F)。重要的是,与conventional和
Figure PCTCN2022117466-appb-000032
hPSCs相比,pgEpiSCs表现出更强的formative hPSC特异性基因的表达(图4G)。与conventional hESC相比,表达增强的代表性基因如表4-1所示,表达降低的代表性基因如表4-2所示。
表4-1:与conventional hESC相比表达增加的代表性基因
Figure PCTCN2022117466-appb-000033
Figure PCTCN2022117466-appb-000034
表4-2:与conventional hESC相比表达减少的代表性基因
Figure PCTCN2022117466-appb-000035
Figure PCTCN2022117466-appb-000036
上述结果支持了pgEpiSCs细胞系的成功建立,同时也表明pgEpiSCs具有E10前原胚上皮细胞的特征和多能性。
实施例4:pgEpiSC的转录的空间调控特征
利用超深原位高通量染色质构象捕获(high-deep in situ high-throughput chromatin conformation capture,hic)测序技术,我们重建了pgEpiSCs和猪胚胎成纤维细胞(pEFs)的三维基因组结构,结合16次重复的数据,最终获得最大分辨率为300bp的图谱。我们发现pgEpiSCs的染色质空间可塑性高于pEFs(反映在pgEpiSCs的染色体混融程度低于pEFs:0.18/0.71,P<2.2×10 -16,Wilcoxon秩和检验)(图5A-5B)。基于高熵状态,pgEpiSCs的染色质更无序(1.57/1.00,P=5.63×10 -4,Wilcoxon秩和检验)(图5C-5D),这与之前对人类和老鼠的研究一致(Lindsly et al.,2021;Tan et al.,2018)。由此可见,我们在超深原位Hi-C分析中检测到的典型的松散调控架构,这可能由于pgEpiSCs建立多能性。
进一步,我们研究了相对松散的pgEpiSCs异染色质调控结构可能影响多能性的转录过程。我们发现在pgEpiSCs和PEF相比,有较少的启动子-增强子相互作用(PEIs)(pgEpiSCs,20,389个增强子分配给6,498个启动子)(PEF,30,852个增强子分配给7,823个启动子)。pgEpiSCs和pEFs之间具有明显的转录组差异,仅共享5,547个PEIs。我们计算了每个启动子的调控潜力得分(RPS),RPS是一种基于空间邻近性的指数,代表了多个增强子对给定基因的联合调控效果(Cao et al.,2017;Fulco et al.,2019;Whalen et al.,2016),对于给定的启动子,其RPS计算公式为:∑n(log10In),其中In是该启动子的PEI n的归一化相互作用强度(normalized interaction intensity)。共鉴定出875个RPS与基因表达的共变异基因(即,与pEFs相比,pgEpiSCs中具有较高RPS值的基因通常上调(log2fold change[FC]>1,FDR<0.05)),其中75个基因在pgEpiSCs中强表达(TPM>5与pEFs中TPM<0.5相比),表5中示例性给出了代表性在pgEpiSCs中强表达的共变异基因。
表5:代表性的在pgEpiSCs中强表达的共变异基因
Figure PCTCN2022117466-appb-000037
Figure PCTCN2022117466-appb-000038
Figure PCTCN2022117466-appb-000039
Figure PCTCN2022117466-appb-000040
此外,我们检测到OTX2(以及LIN28A、NANOG、PRDM14、SALL4、UTF1和ZFP42)在pgEpiSCs中与增强子特异性相互作用,而在pEFs中则是增强子不存在(图5E-5K)。
实施例5:pgEpiSCs的连续基因编辑以及克隆仔猪的生产
目前使用猪体细胞核移植的主要限制之一是体细胞供体细胞通常只能支持单轮基因组编辑(Yan et al.,2018)。为了测试pgEpiSCs是否能够忍受连续的基因组编辑,我们进行了多种形式的基因组操作的实验(图6A).
首先,我们稳定转染GFP-nls报告片段获得pgEpiSCs,流式细胞仪检测GFP阳性细胞率为21.27%(图6B).其次,利用这些GFP阳性细胞,我们进行了CRISPR/cas9介导的敲入,具体地将tdTomato报告基因盒***到NANOG基因座中,位于天然终止密码子的正 前面,称为GFP-NANOG-tdTomato pgEpiSCs(GN-pgEpiSCs)(图6C)。根据NANOG-tdTomato荧光筛选GN-pgEpiSC克隆,然后在3i/LAF培养基中重新扩增(图6D)。与NANOG(多能性标记物)的已知状态一致,在对敲入后编辑的pgEpiSCs进行实验诱导分化后,没有检测到tdTomato报告荧光(图6E)。对于第三个也是最终的基因组修饰,我们使用胞嘧啶碱基编辑器(CBEs)进行了c-t转换(Koblan et al.,2018;Komor et al.,2016)在TYR位点的停止密码子,诱导了一种已知的与猪毛色有关的白化病(Li et al.,2018;Xie et al.,2019)。对99个克隆进行测序分析,其中24.24%(24/99)为杂合子,3.03%(3/99)为纯合子(GNT-pgEpiSCs背景中的C-T碱基编辑称为GNT-pgEpiSCs)(图6F)。这些结果表明,pgEpiSCs能够耐受连续的基因组修饰,包括传统的转基因***、CRISPR/Cas9的精确敲入和CBEs的单碱基转换编辑。
然后,我们进行了细胞核移植试验,并专门使用野生型(WT)pgEpiSCs、GFP-pgEpiSCs和GNT-pgEpiSCs作为核供体细胞,以获得克隆胚胎,进一步混合移植200个WT pgEpiSCs和203个GFP-pgEpiSCs克隆胚胎,以及660个GNT-pgEpiSCs克隆胚胎(图6G)。我们最终获得了1只WT pgEpiSCs克隆仔猪,1只GFP-pgEpiSCs克隆仔猪,3只GNT-pgEpiSCs克隆仔猪(图6H)。基因编辑pgEpiSC的克隆效率与野生型pgEpiSC细胞相似,与成纤维细胞相当(图6G)。重要的是,GNT-pgEpiSCs克隆仔猪显示了预期的白化毛色表型(图6I)。这些结果表明,pgEpiSCs能够耐受连续的多基因编辑,具有生成复杂猪模型的潜力。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。

Claims (10)

  1. 培养基,其包含:
    第一组分,所述第一组分为IWR-1-endo;
    第二组分,所述第二组分选自WH-4-023、A419259;
    第三组分,所述第三组分选自成纤维细胞生长因子。
  2. 权利要求1的培养基,其中,所述培养基进一步包含:
    第四组分,所述第四组分选自CHIR99021、WNT3a;
    第五组分,所述第五组分选自TGF-β超家族成员;
    第六组分,所述第六组分为LIF。
  3. 权利要求1-2任一项的培养基,其中,所述培养基具有以下(1)至(5)中的一项或多项技术特征:
    (1)所述第二组分为WH-4-023;
    (2)所述第三组分选自FGF2、FGF1;
    优选地,所述第三组分为FGF2;
    更优选地,所述第三组分为重组人FGF2。
    (3)所述第四组分为CHIR99021;
    (4)所述第五组分选自Activin A、Nodal;
    优选地,所述第五组分为Activin A;
    更优选地,所述第五组分为重组人Activin A;
    (5)所述第六组分选自重组人LIF、重组小鼠LIF;
    更优选地,所述第六组分为重组人LIF。
  4. 权利要求1-3任一项的培养基,其中,所述培养基具有以下(1)至(7)中的一项或多项技术特征:
    (1)所述第一组分的浓度为0.1-10μM;
    优选地,所述第一组分的浓度为0.9-3μM;
    更优选地,所述第一组分的浓度为2.5μM;
    (2)所述第二组分的浓度为3nM-30μM;
    优选地,所述第二组分的浓度为0.01-5μM;
    更优选地,所述第二组分的浓度1μM;
    (3)所述第三组分的浓度为0.01-100ng/mL;
    优选地,所述第三组分的浓度为1-100ng/mL;
    更优选地,所述第三组分的浓度10ng/mL;
    (4)所述第四组分的浓度为0.0025nM-3μM;
    优选地,所述第四组分的浓度为0.01-3μM;
    更优选地,所述第四组分的浓度为1μM;
    (5)所述第五组分的浓度为0.01-100ng/mL;
    优选地,所述第五组分的浓度为25ng/mL;
    (6)所述第六组分的浓度为0.01-100ng/mL;
    优选地,所述第六组分的浓度为1-100ng/mL;
    更优选地,所述第六组分的浓度10ng/mL;
    (7)所述第四组分和所述第一组分的浓度比为25:1-1:25;
    优选地,所述第四组分和所述第一组分的浓度比为2:3-1:3。
  5. 权利要求1-4任一项所述的培养基,其中,所述培养基进一步包含:第七组分,所述第七组分为ROCK抑制剂;
    优选地,所述第七组分为Y-27632;
    优选地,所述第七组分的浓度为0.01-50μM;
    优选地,所述培养基用于细胞传代时,所述第七组分的浓度为0.01-20μM,优选为10μM;
    优选地,所述培养基用于细胞维持时,所述第七组分的浓度为0.01-10μM,优选为2μM。
  6. 权利要求1-5任一项所述的培养基,其中,所述培养基进一步包含:第八组分,所述第八组分为基础培养基;
    优选地,所述基础培养基为用于培养哺乳动物(优选猪)多能干细胞的基础培养基;
    更优选地,所述基础培养基包含基本培养基、N2 supplement、B27 supplement、非必需氨基酸、β-巯基乙醇、knockout serum replacement,和选自GlutaMAX、谷氨酰胺的任意一种;
    进一步优选地,所述基础培养基包含基本培养基、N2 supplement、B27 supplement、非必需氨基酸、β-巯基乙醇、knockout serum replacement和GlutaMAX;
    最优选地,所述基础培养基包含基本培养基、N2 supplement、B27 supplement、非必需氨基酸、β-巯基乙醇、knockout serum replacement、抗坏血酸、GlutaMAX和青霉素-链霉素;
    优选地,所述基本培养基选自DMEM/F12、Neurobasal、DMEM、KO-DMEM、RPMI1640、MEM、mTeSR1或其任意组合;
    优选地,所述基本培养基选自DMEM/F12、Neurobasal或其组合;
    优选地,所述基本培养基为DMEM/F12和Neurobasal。
  7. 权利要求6的培养基,其中,所述基础培养基具有以下(1)至(12)中的一项或多项技术特征:
    (1)所述基本培养基的体积分数为1%-99%,优选为91%;
    (2)所述DMEM/F12的体积分数为1%-99%,优选为45%-50%(如45.5%);
    (3)所述Neurobasal的体积分数为1%-99%,优选为45%-50%(如45.5%);
    (4)所述N2 supplement的体积分数为0.002%-10%,优选为0.5%;
    (5)所述B27 supplement的体积分数为0.002%-20%,优选为1%;
    (6)所述非必需氨基酸的体积分数为0.01%-10%,优选为1%;
    (7)所述β-巯基乙醇的浓度为0.01mM-1mM,优选为0.1mM;
    (8)所述knockout serum replacement的体积分数为0.01%-50%,优选为5%;
    (9)所述抗坏血酸的浓度为1μg/mL-5000μg/mL,优选为50μg/mL;
    (10)所述GlutaMAX或谷氨酰胺(优选GlutaMAX)的体积分数为0.01%-10%,优选为0.5%;
    (11)所述青霉素-链霉素的体积分数为0.01%-20%,优选为1%;
    (12)所述DMEM/F12和所述Neurobasal的体积比为5:1-1:5,优选为1:1。
  8. 制备哺乳动物多能干细胞的方法,其包括:
    1)提供哺乳动物胚胎上胚层(Epiblast)或其内细胞团;
    2)利用权利要求1-7任一项所述的培养基培养所述哺乳动物胚胎上胚层(Epiblast)或其内细胞团,获得哺乳动物多能干细胞;
    优选地,所述哺乳动物为猪;
    优选地,所述哺乳动物胚胎上胚层(Epiblast)是E8至E10(例如E8、E9或E10)的哺乳动物胚胎上胚层(Epiblast)。
  9. 培养哺乳动物多能干细胞和/或维持哺乳动物多能干细胞多能性的方法,其包括:
    1)提供哺乳动物多能干细胞;
    2)利用权利要求1-7任一项所述的培养基培养所述哺乳动物多能干细胞;
    优选地,所述哺乳动物为猪;
    优选地,所述哺乳动物多能干细胞为猪胚胎原肠化前上胚层(Epiblast)干细胞。
  10. 权利要求8-9任一项的方法,其中,所述方法是在饲养细胞存在的条件下进行的;
    优选地,所述饲养细胞选自小鼠胚胎成纤维细胞或STO细胞;
    优选地,所述饲养细胞为小鼠胚胎成纤维细胞;
    优选地,所述饲养细胞为停止***的小鼠胚胎成纤维细胞;
    优选地,所述饲养细胞为丝裂霉素C处理的小鼠胚胎成纤维细胞。
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