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KR-20260068007-A - Cytochrome P450 2C19 fluorescently labeled human induced pluripotent stem cell line and uses thereof

KR20260068007AKR 20260068007 AKR20260068007 AKR 20260068007AKR-20260068007-A

Abstract

The present invention relates to a cytochrome P450 2C19 fluorescently labeled human induced pluripotent stem cell line and a method for screening nuclear receptor regulators using the same. The human induced pluripotent stem cell line for nuclear receptor regulatory factor screening prepared according to the present invention can effectively screen for substances capable of inducing nuclear receptor activity in a living cell state in liver organoids derived from pluripotent stem cell lines, and can be utilized for the discovery of imaging-based nuclear receptor regulators using high-content screening equipment.

Inventors

  • 박한진
  • 김혜민
  • 김지우
  • 조성예

Assignees

  • 한국화학연구원

Dates

Publication Date
20260513
Application Date
20251104
Priority Date
20241104

Claims (20)

  1. Human induced pluripotent stem cell line for nuclear receptor regulatory factor screening transformed with fluorescent protein-labeled cytochrome P450 2C19 (CYP2C19).
  2. In paragraph 1, The above human induced pluripotent stem cell line for nuclear receptor regulatory factor screening is prepared by transfecting a first vector expressing the guide RNA of SEQ ID NO. 1 and Cas9 protein, and a second vector containing a fluorescent protein.
  3. In paragraph 1, A human induced pluripotent stem cell line for nuclear receptor regulatory factor screening, wherein the above fluorescent protein is one or more selected from the group consisting of mScarlet, mCherry, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), luciferase, β-galactosidase, mPlum, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase.
  4. In paragraph 3, The above fluorescent protein is mScarlet, a human induced pluripotent stem cell line for nuclear receptor regulatory factor screening.
  5. In paragraph 1, The above human induced pluripotent stem cell line for nuclear receptor regulatory factor screening is a human induced pluripotent stem cell line that has increased expression of one or more selected from the group consisting of OCT4, SOX2, NANOG, TRA-1-60 positive cells and TRA-1-81 positive cells compared to a stem cell line not transformed with the above fluorescent protein-labeled cytochrome P450 2C19.
  6. In paragraph 1, The above-mentioned nuclear receptor is a pregnane X receptor (PXR) or a constitutive androstane receptor (CAR), a human induced pluripotent stem cell line for screening nuclear receptor regulators.
  7. A method for preparing a human induced pluripotent stem cell line for nuclear receptor regulatory factor screening, comprising the step of transforming a human induced pluripotent stem cell line with cytochrome P450 2C19 (CYP2C19) labeled with a fluorescent protein.
  8. In Paragraph 7, A manufacturing method comprising the step of transforming the above-mentioned guide RNA of SEQ ID NO. 1 and transfecting a first vector expressing Cas9 protein and a second vector containing a fluorescent protein.
  9. In Paragraph 7, A method for manufacturing, wherein the above fluorescent protein is one or more selected from the group consisting of mScarlet, mCherry, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), luciferase, β-galactosidase, mPlum, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase.
  10. In Paragraph 7, A method for manufacturing in which the above nuclear receptor is a pregnane X receptor (PXR) or a constitutive androstane receptor (CAR).
  11. (a) A step of differentiating the human induced pluripotent stem cell line for nuclear receptor regulatory factor screening of claim 1 into a liver organoid; (b) a step of treating the differentiated liver organoid with a candidate substance to measure the expression level of a fluorescent protein; and (c) a step of selecting a candidate substance in which the expression level of the fluorescent protein is changed in a liver organoid administered the candidate substance compared to a control group not administered the candidate substance; a nuclear receptor regulator screening method comprising: a step of screening a candidate substance in which the expression level of the fluorescent protein is changed in a liver organoid administered the candidate substance compared to a control group not administered the candidate substance.
  12. In Paragraph 11, A screening method wherein the above fluorescent protein is one or more selected from the group consisting of mScarlet, mCherry, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), luciferase, β-galactosidase, mPlum, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase.
  13. In Paragraph 11, A screening method in which the above human induced pluripotent stem cell line is prepared by transfecting a first vector expressing the guide RNA of SEQ ID NO. 1 and Cas9 protein and a second vector containing a fluorescent protein.
  14. In Paragraph 11, A screening method in which the liver organoid differentiated in step (a) above has increased expression of one or more selected from the group consisting of albumin, alpha-1 antitrypsin, hepatocyte nuclear factor 4 alpha, cytochrome P450 (CYP450), multidrug resistance protein 1, multidrug resistance-associated protein 2, alpha-fetoprotein, and bile salt export pump compared to the undifferentiated case.
  15. In Paragraph 14, A screening method in which the above cytochrome P450 is one or more selected from the group consisting of CYP3A4, CYP2C19, CYP1A1, CYP2C9, and CYP2B6.
  16. In Paragraph 11, A screening method in which the step of differentiating the human induced pluripotent stem cell line of (a) into a liver organoid includes the step of culturing in a medium that does not contain an extracellular matrix.
  17. In Paragraph 16, A screening method in which culture in a medium not containing the above extracellular matrix is carried out using a bioreactor.
  18. In Paragraph 11, A screening method comprising the step (c) of determining that the candidate substance is a nuclear receptor agonist when the expression level of the fluorescent protein is increased compared to a control group in which the candidate substance was not administered.
  19. In Paragraph 11, A screening method in which the nuclear receptor is a pregnane X receptor (PXR) or a constitutive androstane receptor (CAR).
  20. In Paragraph 11, The above screening method is a screening method performed by high-content screening analysis.

Description

Cytochrome P450 2C19 fluorescently labeled human induced pluripotent stem cell line and uses thereof The present invention relates to a cytochrome P450 2C19 fluorescently labeled human induced pluripotent stem cell line and a method for screening nuclear receptor regulators using the same. Nuclear receptors are a group of proteins that act as transcription factors through ligand binding to regulate the expression of various genes, playing a crucial role in maintaining metabolic homeostasis in the body. The nuclear receptor protein superfamily is a group composed of ligand-dependent transcription factors, including steroid hormone receptors, thyroid hormone receptors, and vitamin D3 receptors. Among nuclear receptors, constitutive androstane receptors (CARs) and pregnane X receptors (PXRs) are primarily expressed in hepatocytes and intestinal epithelium and are known as representative nuclear receptors involved in detoxification and excretion by recognizing xenobiotics or endobiotics. CARs and PXRs influence the in vivo concentration, metabolic rate, and efficacy of drugs by regulating the transcriptional activity of drug-metabolizing enzymes and drug transport genes, such as CYP2B6, CYP3A4, CYP2C19, UGT1A1, and MRP2, respectively. Furthermore, recent studies report that these receptors are involved in various physiological pathways beyond simple detoxification responses, including lipid metabolism, glucose metabolism, bile acid homeostasis, and cholesterol regulation. Therefore, the discovery of regulatory factors capable of controlling the activity of CARs and PXRs is very important not only for ensuring pharmacometabolic safety but also for the development of treatments for metabolic and liver diseases. However, conventional research on CAR and PXR has primarily relied on non-selective ligands, and has suffered from low selectivity and poor reproducibility of evaluation results due to cross-activation between the two receptors. In particular, CARs have high constitutive activity, and PXRs have flexible binding pockets that can accommodate compounds of various structures, which presents limitations such as the difficulty in distinguishing accurate functional characteristics with existing screening systems. Therefore, there is a growing need to develop an efficient screening system capable of rapidly and quantitatively identifying regulators that can specifically bind to CAR and PXR or regulate their activity. Figure 1 shows a schematic diagram of the vector used to produce a stem cell line transformed from human induced pluripotent stem cells with CYP2C19 labeled with a fluorescent protein. Figure 2 shows the PCR analysis results to identify the selected transformed cell line (CYP2C19-mScarlet cell line) and the sequence analysis results to confirm the targeted integration of the introduced gene. Figure 3 shows the stem cell morphology and karyotype of the CYP2C19-mScarlet cell line of the present invention. Figure 4 shows the results of the expression analysis of OCT4, SOX2, and NANOG in the nuclei of the CYP2C19-mScarlet cell line of the present invention (A), and the results of the flow cytometry analysis to confirm the expression levels of TRA-1-60 positive cells and TRA-1-81 positive cells (B). Figure 5 shows the differentiation ability of the CYP2C19-mScarlet cell line of the present invention into the ectoderm, mesoderm, and endoderm. Figure 5A shows the results of confirming changes in the expression of stem cell markers (OCT4, SOX2, NANOG), ectoderm markers (PAX6, OTX2), mesoderm markers (EOMES, α-SMA), and endoderm markers (AFT, GATA4) in the CYP2C19-mScarlet cell line and embryos (EB) formed therefrom; Figure 5B shows the results of confirming the expression of ectoderm markers (TUBB3, NESTIN), mesoderm markers (DESMIN, α-SMA), and endoderm markers (AFP and SOX17) in embryos formed from the CYP2C19-mScarlet cell line. Figure 6 shows the approximate process of preparing a liver organoid from the CYP2C19-mScarlet cell line of the present invention and screening drugs using it. Figure 7 shows the confirmation of CYP2C19-mScarlet expression in liver organoids prepared from the CYP2C19-mScarlet cell line of the present invention. Figure 8 shows the expression levels of liver marker genes and drug transporter genes in liver organoids prepared from the CYP2C19-mScarlet cell line of the present invention. Figure 9 shows the expression levels of liver function indicators ALB, AAT, and HNF4A in liver organoids prepared from the CYP2C19-mScarlet cell line of the present invention. Figure 10 shows the comprehensive evaluation of liver function of liver organoids prepared from the CYP2C19-mScarlet cell line of the present invention. Figure 10A shows the results of comparing the secretion levels of liver function indicators ALB, AAT, and urea in liver organoids with those of liver endoderm organoids (HEO); Figure 10B shows the results of confirming the albumin expression level through FACS flow cytometry anal