Insulin resistance model

Abstract

Disclosed herein is an insulin resistance reporter for use in quantifying insulin response in living cells.These living cells can be stem cell compositions or their derivatives comprising insulin resistance reporters.Stem cell derivatives include, but are not limited to, insulin-responsive cells, tissues, or organoids, such as pancreatic, brain, fat, muscle, or liver cells, or tissues or organoids thereof.Also disclosed herein are methods for using the insulin resistance reporters and cells with these insulin resistance reporters as models for investigating insulin resistance, and methods for screening compounds that are potentially useful for treating diseases or disorders associated with insulin resistance.Cells comprising insulin resistance reporters can be hepatocytes or liver organoid compositions, which can be used to investigate hepatic insulin resistance, for example, as a result of non-alcoholic fatty liver disease or steatohepatitis.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 042,997, filed Jun. 23, 2020, which is hereby expressly incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research or Development The present invention was made with government support under Grant No. DK119982 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Reference to a Sequence Listing This application has been filed with an electronic sequence listing. The sequence listing is provided as a file named SeqListingCHMC63_032WO.TXT, created on Jun. 21, 2021 and last modified, with a size of 38,040 bytes. The information in the electronic sequence listing is hereby incorporated by reference in its entirety.

[0004] Technical Field Aspects of the present disclosure generally relate to insulin response reporters. These insulin response reporters can be used in cells such as stem cells, or derivatives of cells such as differentiated cells or organoids, for example, to detect insulin response or its presence or absence, or to screen for compounds that affect the insulin response of cells. The present disclosure also describes methods of operating insulin response reporters, as well as methods of making and using cells having insulin response reporters.

Background Art

[0005] Non-alcoholic fatty liver disease (NALFD) and associated metabolic syndromes continue to increase in the human population, leading to increased morbidity and mortality. Hepatic insulin resistance is often associated with NAFLD, which is a risk factor for comorbidities such as heart disease. In contrast to systemic insulin resistance, NAFLD can cause hepatic insulin resistance through increased hepatic fat accumulation, a highly variable, genetic and non-genetic-dependent adverse process. However, the precise genetic and molecular mechanisms linking hepatic fat accumulation and insulin resistance are not fully understood and are hampered by the lack of suitable human-based model systems. Therefore, there is a current need for robust and reliable models to study a wide range of liver disorders, including hepatic insulin resistance. Furthermore, there is a need for improved screening methods using human-based model systems to discover potential treatments for insulin resistance. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) U.S. Patent Application Publication No. 2018 / 0171302 (Patent Document 2) U.S. Patent Application Publication No. 2019 / 0298775 Specification (Non-patent literature) (Non-patent document 1) MCKIMPSON et al. "A fluorescent reporter assay of differential gene expression response to insulin in hepatocytes" Am J Physiol Cell Physiol, 15 May 2019, Vol. 317, pp C143-C151; abstract, pg C143, col 2, para 3, pg C144, col 2, para 3 [Overview of the Initiative]

[0006] Liver organoids containing insulin resistance reporters are disclosed herein. In some embodiments, the insulin resistance reporter is operably linked to an insulin-dependent gene of the liver organoid. The insulin-dependent gene may be any gene (and resulting protein) regulated by the effects of insulin or insulin signaling pathways occurring in living cells. In some embodiments, the insulin-dependent gene may be a gluconeogenic gene or a lipid biosynthesis gene. The insulin resistance reporter may be any one of the insulin resistance reporters disclosed herein.

[0007] Also disclosed herein are insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter. In some embodiments, the insulin resistance reporter is operably linked to an insulin-dependent gene in an insulin-responsive cell, tissue, or organoid. The insulin-responsive cell, tissue, or organoid may be any one or more cell types that exhibit a functional response to insulin or an insulin signaling pathway, or may include them. In some embodiments, the insulin-responsive cell, tissue, or organoid may be affected by diseases or disorders associated with a dysfunctional insulin response, such as insulin resistance. Expressing an insulin resistance reporter in an insulin-responsive cell, tissue, or organoid may be useful in detecting diseases or disorders associated with a dysfunctional insulin response, or in identifying molecules or compounds useful for treating diseases or disorders.

[0008] Insulin resistance reporters are also disclosed herein. In some embodiments, the insulin resistance reporter comprises one or more reporter genes adjacent to the 5' homologous and 3' homologous regions associated with the insulin-dependent gene. In some embodiments, these insulin resistance reporters are nucleic acids. In some embodiments, the insulin resistance reporter is intended to be integrated into the genome by the insulin-dependent gene, for example, by homologous recombination via the 5' homologous and 3' homologous regions associated with the insulin-dependent gene. In some embodiments, the insulin resistance reporter is integrated into insulin-responsive cells, tissues, or organoids. In some embodiments, the insulin resistance reporter is integrated into liver organoids, such as fatty liver organoids or fatty liver organoids.

[0009] Also disclosed herein are in vitro methods for screening candidate compounds for the treatment of diseases or disorders associated with insulin insufficiency. In some embodiments, the method includes contacting a liver organoid containing an insulin resistance reporter, or an insulin-responsive cell, tissue, or organoid containing an insulin resistance reporter, with a candidate compound, and observing improvement of diseases or disorders associated with insulin insufficiency in the liver organoid or insulin-responsive cell, tissue, or organoid.

[0010] Furthermore, stem cells containing insulin resistance reporters are disclosed herein. In some embodiments, the insulin resistance reporter is any one of the insulin resistance reporters disclosed herein. Furthermore, endoderm cells differentiated from any one of the stem cells disclosed herein are also disclosed herein. Furthermore, anterior foregut cells differentiated from any one of the stem cells disclosed herein are also disclosed herein. Furthermore, insulin-responsive cells, tissues, or organoids differentiated from any one of the stem cells disclosed herein are also disclosed herein. Furthermore, pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes differentiated from any one of the stem cells disclosed herein are also disclosed herein. Furthermore, liver organoids differentiated from any one of the stem cells, endoderm, or anterior foregut cells disclosed herein are also disclosed herein.

[0011] Also disclosed herein are in vitro methods for evaluating insulin resistance in insulin-responsive cells, tissues, or organoids, including an insulin resistance reporter. In some embodiments, the method comprises quantifying the baseline expression level of one or more reporter proteins of an insulin resistance reporter; contacting insulin-responsive cells, tissues, or organoids with insulin or its derivatives or mimics; quantifying the expression level of one or more reporter proteins after treatment; and determining that the insulin-responsive cells, tissues, or organoids exhibit insulin resistance based on the change or absence of the expression level of one or more reporter proteins.

[0012] Also disclosed herein are in vitro methods for screening compounds or compositions for treating insulin resistance. In some embodiments, the method includes contacting insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter with one or more fatty acids; quantifying the baseline expression level of one or more reporter proteins of the insulin resistance reporter; contacting insulin-responsive cells, tissues, or organoids with a compound or composition; quantifying the expression level of one or more reporter proteins after treatment; and determining whether the compound or composition can treat insulin resistance based on the change or absence of the expression level of one or more reporter proteins.

[0013] Also disclosed herein are compounds or compositions identified by any one of the screening methods disclosed herein. Also disclosed herein are pharmaceutical compositions comprising any one or more compounds or compositions identified by any one of the screening methods disclosed herein. Also disclosed herein are methods for treating insulin resistance in subjects requiring it by administering any one of the identified compounds or compositions to the subject.

[0014] Methods for monitoring the insulin response in a subject are also disclosed herein. In some embodiments, the method includes transplanting a liver organoid containing an insulin resistance reporter, or insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter, into a subject, and monitoring the expression of the insulin resistance reporter in the liver organoid or insulin-responsive cells, tissues, or organoids.

[0015] Exceptional embodiments of this disclosure are provided in embodiments numbered as follows: 1. Liver organoids, including insulin resistance reporters. 2. Any one of the above embodiments of a liver organoid, wherein the liver organoid is a human liver organoid (HLO). 3. A liver organoid according to any one of the embodiments described above, wherein the liver organoid is generated from induced pluripotent stem cells (iPSCs). 4. A liver organoid according to any one of the embodiments described above, wherein the liver organoid is generated from iPSCs derived from a human subject. 5. Liver organoids of Embodiment 3 or 4, in which iPSCs are first differentiated into anterior foregut cells. 6. Liver organoids of Embodiment 5, wherein anterior foregut cells are cryopreserved for a certain period and thawed before differentiating the anterior foregut cells into liver organoids. 7. A liver organoid of any one of the above embodiments, wherein the insulin resistance reporter comprises one or more nucleic acid sequences encoding a reporter protein and one or more nucleic acid sequences encoding self-cleaving peptides that isolate each of the one or more nucleic acid sequences encoding the reporter protein. 8. Any one of the above embodiments of a liver organoid, wherein the insulin resistance reporter comprises one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with a sequence encoding a reporter protein, and one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with a sequence encoding a self-cleaving peptide that separates each of the sequences encoding the reporter protein. 9. A liver organoid of any one of the above embodiments, wherein the reporter protein is a fluorescent protein. 10. A liver organoid of any one of the above embodiments, wherein the fluorescent protein is mScarlet. 11. A liver organoid of any one of the above embodiments, wherein the reporter protein is a luminescent protein. 12. A liver organoid of any one of the above embodiments, wherein the luminescent protein is luciferase. 13. Any one of the above embodiments of a liver organoid further comprising one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with sequences associated with insulin-dependent genes. 14. A liver organoid of any one of the embodiments described above, wherein the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. 15. A liver organoid of any one of the above embodiments, wherein the insulin-dependent gene is PCK1. 16. Insulin-dependent genes include PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, and CREB. 1、 A liver organoid selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC, which is one of the embodiments described above. 17. One or more nucleic acid sequences having homology with a sequence related to an insulin-dependent gene are adjacent to a sequence encoding a reporter protein and a sequence encoding a self-cleaving peptide, and the sequence having homology with the sequence related to the insulin-dependent gene acts as a homologous region for recombination into the genome of a liver organoid, the liver organoid of any one of the前述 embodiments. 18. The liver organoid of any one of the前述 embodiments, wherein an insulin resistance reporter is integrated into the genome of the liver organoid using a CRISPR nuclease. 19. The liver organoid of any one of the前述 embodiments, wherein the CRISPR nuclease is Cas9. 20. The liver organoid of any one of the前述 embodiments, wherein the liver organoid is a fatty liver organoid after treatment of the liver organoid with one or more fatty acids, and the fatty liver organoid exhibits insulin resistance. 21. The liver organoid of any one of the前述 embodiments, wherein the one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. 22. The insulin resistance reporter of any one of the前述 embodiments. 23. An in vitro method for evaluating the insulin resistance of the liver of a liver organoid comprising an insulin resistance reporter, comprising: quantifying the baseline expression level of one or more reporter proteins of the insulin resistance reporter; contacting the liver organoid with insulin or a derivative or mimetic thereof; quantifying the expression level of one or more reporter proteins after treatment; and determining that the liver organoid exhibits insulin resistance of the liver based on a change or absence of the expression level of one or more reporter proteins. 24. The method of Embodiment 23, further comprising contacting liver organoids with one or more of obeticholic acid (OCA), pioglitazone, or metformin, or any combination thereof. 25. The method of Embodiment 23 or 24, further comprising contacting liver organoids with one or more fatty acids before quantifying baseline expression levels. 26. Any one of embodiments 23 to 25, further comprising contacting the liver organoid with one or more fatty acids after quantifying baseline expression levels and before contacting the liver organoid with insulin or its derivatives or mimetic. 27. An in vitro method for screening compounds or compositions for treating insulin resistance of the liver caused by non-alcoholic fatty liver disease (NAFLD), Contacting liver organoids containing an insulin resistance reporter with one or more fatty acids, Quantifying the baseline expression levels of one or more reporter proteins of an insulin resistance reporter, Contacting liver organoids with a compound or composition, Quantifying the expression levels of one or more reporter proteins after treatment, A method comprising determining whether a compound or composition can treat hepatic insulin resistance based on changes in or absence of the expression level of one or more reporter proteins. 28. Any one of the embodiments 23 to 27, wherein the liver organoid is any one of the liver organoids from embodiments 1 to 21. 29. Any one of Embodiments 23 to 28, wherein the insulin resistance reporter is the insulin resistance reporter of Embodiment 22. 30. Any one of Embodiments 23 to 29, wherein one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. 31. Any one of embodiments 23 to 30, wherein the liver organoid is derived from a human subject requiring treatment for hepatic insulin resistance. 32. Stem cells, including insulin resistance reporters. 33. Any one of the above embodiments of stem cells, wherein the stem cell is an induced pluripotent stem cell (iPSC). 34. Stem cells derived from a human subject, one of any of the embodiments described above. 35. A stem cell from any one of the above embodiments, wherein the insulin resistance reporter comprises one or more nucleic acid sequences encoding a reporter protein and one or more nucleic acid sequences encoding self-cleaving peptides that isolate each of the one or more nucleic acid sequences encoding the reporter protein. 36. Any one of the above embodiments of a stem cell comprising: an insulin resistance reporter comprising one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with a sequence encoding a reporter protein; and one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with a sequence encoding a self-cleaving peptide that separates each of the sequences encoding the reporter protein. 37. A stem cell of any one of the above embodiments, wherein the reporter protein is a fluorescent protein. 38. A stem cell from any one of the above embodiments, wherein the fluorescent protein is mScarlet. 39. A stem cell of any one of the above embodiments, wherein the reporter protein is a luminescent protein. 40. A stem cell of any one of the above embodiments, wherein the luminescent protein is luciferase. 41. Any one of the above embodiments of a stem cell, further comprising one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence associated with an insulin-dependent gene. 42. A stem cell from any one of the embodiments described above, wherein the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. 43. A stem cell from any one of the embodiments described above, wherein the insulin-dependent gene is PCK1. 44. Insulin-dependent genes include PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, and CREB. 1、 A stem cell selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC, which is one of the embodiments described above. 45. One or more nucleic acid sequences homologous to sequences associated with insulin-dependent genes are adjacent to a sequence encoding a reporter protein and a sequence encoding a self-cleaving peptide, and the sequences homologous to sequences associated with insulin-dependent genes act as homologous regions for recombination into the genome of liver organoids, in any one of the above embodiments of stem cells. 46. ​​A stem cell in any one of the above embodiments, wherein an insulin resistance reporter is incorporated into the genome of the stem cell using a CRISPR nuclease. 47. A stem cell from any one of the above embodiments, wherein the CRISPR nuclease is Cas9. 48. Embryonic endoderm cells differentiated from any one of the stem cells in Embodiments 32 to 47. 49. Anterior foregut cells differentiated from any one of the stem cells in Embodiments 32 to 47. 50. Insulin-responsive cells, tissues, or organoids differentiated from any one of the stem cells in Embodiments 32 to 47. 51. An insulin-responsive cell, tissue, or organoid of Embodiment 50, wherein the insulin-responsive cell, tissue, or organoid includes pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes. 52. An insulin-responsive cell, tissue, or organoid of Embodiment 50 or 51, wherein the insulin-responsive cell, tissue, or organoid is a liver organoid. 53. Pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes differentiated from any one of the stem cells in Embodiments 32 to 47. 54. A liver organoid differentiated from any one of the stem cells in Embodiments 32 to 47, an endoderm cell of Embodiment 48, or an anterior foregut cell of Embodiment 49, wherein the liver organoid contains an insulin resistance reporter. 55. The liver organoid according to Embodiment 54, wherein the liver organoid is a fatty liver organoid obtained after treatment with one or more fatty acids, and the fatty liver organoid exhibits insulin resistance. 56. The liver organoid according to Embodiment 55, wherein one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. 57. An insulin resistance reporter according to any one of the embodiments described above. 58. An in vitro method for evaluating insulin resistance in insulin-responsive cells, tissues, or organoids, including an insulin resistance reporter, Quantifying the baseline expression levels of one or more reporter proteins of an insulin resistance reporter, Contacting insulin-responsive cells, tissues, or organoids with insulin or its derivatives or mimics, Quantifying the expression levels of one or more reporter proteins after treatment, A method comprising determining that insulin-responsive cells, tissues, or organoids exhibit insulin resistance based on changes in or absence of the expression level of one or more reporter proteins. 59. The method of Embodiment 58, further comprising contacting insulin-responsive cells, tissues, or organoids with one or more of obeticholic acid (OCA), pioglitazone, or metformin, or any combination thereof. 60. The method of Embodiment 58 or 59, further comprising contacting insulin-responsive cells, tissues, or organoids with one or more fatty acids before quantifying baseline expression levels. 61. Any one of embodiments 58 to 60, further comprising contacting insulin-responsive cells, tissues, or organoids with one or more fatty acids after quantifying baseline expression levels and before contacting the insulin-responsive cells, tissues, or organoids with insulin or its derivatives or mimetic. 62. An in vitro method for screening compounds or compositions for treating insulin resistance, Contacting insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter with one or more fatty acids, Quantifying the baseline expression levels of one or more reporter proteins of an insulin resistance reporter, Contacting insulin-responsive cells, tissues, or organoids with a compound or composition, Quantifying the expression levels of one or more reporter proteins after treatment, A method comprising determining whether a compound or composition can treat insulin resistance based on changes in or absence of the expression level of one or more reporter proteins. 63. Any one of embodiments 58 to 62, wherein the insulin-responsive cells, tissues, or organoids are any one of the insulin-responsive cells, tissues, or organoids described in the preceding embodiments. 64. Any one of embodiments 58 to 63, wherein the insulin resistance reporter is the insulin resistance reporter of embodiment 26. 65. Any one of embodiments 58 to 64, wherein one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. 66. Any one of embodiments 58 to 65, wherein insulin-responsive cells, tissues, or organoids are derived from a human subject requiring treatment for insulin resistance. 67. Any one of embodiments 58 to 66, wherein the insulin-responsive cells, tissues, or organoids are liver organoids, and the insulin resistance is hepatic insulin resistance. 68. The method of Embodiment 67, wherein insulin resistance of the liver is caused by non-alcoholic fatty liver disease (NAFLD). 69. A compound or composition identified by any one of the methods of Embodiments 62 to 68. 70. A pharmaceutical composition comprising the compound or composition of Embodiment 69 and at least one pharmaceutically acceptable diluent, excipient, or carrier. 71. A method for treating insulin resistance in a subject requiring the treatment, comprising administering the compound or composition of Embodiment 69 or the pharmaceutical composition of Embodiment 70 to the subject. 72. The method of Embodiment 71, wherein the compound or composition or pharmaceutical composition is administered enterally, orally, parenterally, intravenously, intraperitoneally, intramuscularly, or subcutaneously. 73. A composition identified by any one of embodiments 62 to 68 for use in the treatment of insulin resistance in subjects requiring treatment for insulin resistance. [Brief explanation of the drawing]

[0016] In addition to the features described herein, additional features and variations will readily become apparent from the following drawings and descriptions of exemplary embodiments. It should be understood that these drawings are illustrative of embodiments and are not intended to limit the scope.

[0017] [Figure 1A]A schematic diagram illustrating the formation of human liver organoids (HLOs) is shown. [Figure 1B] This shows an example of a bright-field image of HLO on day 20 of total culture. [Figure 1C] Examples of immunofluorescence images of HLOs at 20 days of total culture, stained with albumin (ALB), hepatocyte nuclear factor 4 alpha (HNF4), E-cadherin, and DAPI are shown. [Figure 2A] This document describes an embodiment of single-cell RNA sequencing (scRNA-seq) profiling of hepatocytes in HLO. The relative single-cell expression levels of characteristic hepatocyte markers, ALB, apolipoprotein (APOE), collagen type 1 alpha-1 (COL1A1), and platelet-derived growth factor receptor alpha (PDGFRA), are shown. [Figure 2B] This document illustrates the relative single-cell expression levels of characteristic markers for hepatic stellate cells, biliary tract cells, and cholangiocarcinoma, including ALB, retinol-binding protein 4 (RBP4), tryptophan 2,3-dioxygenase (TDO2), COL1A1, actin alpha 2 (ACTA2), bone morphogenetic protein 4 (BMP4), keratin 19 (KRT19), wingless form 6 (WNT6), and chromogranin A (CHGA). [Figure 3A] An embodiment of a schematic diagram of the liver insulin response is shown. [Figure 3B] This shows embodiments of the expression of receptor / signaling molecules necessary for the insulin response in HLO. The relative single-cell expression levels of insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), and insulin receptor (INSR) are shown. [Figure 3C] This document describes an embodiment of a candidate reporter gene for assaying insulin responsiveness. [Figure 4A] This shows an embodiment of AKT phosphorylation in response to insulin treatment in HLO. [Figure 4B]This document illustrates the suppression of gluconeogenesis regulatory gene expression in response to insulin treatment during HLO. The relative expression levels of forkheadbox O1 (FOXO1), CAMP response element-binding protein 1 (CREB1), and phosphoenolpyruvate carboxykinase 1 (PCK1) are shown. [Figure 4C] This document illustrates the induction of lipid biosynthesis regulatory gene expression in response to insulin treatment during HLO. The relative expression levels of sterol regulatory element binding protein 1c (SREBP1C), fatty acid synthase (FASN), ATP-citrate lyase (ACLY), and acetyl-CoA carboxylase (ACC) are shown. [Figure 5A] This document describes an embodiment of establishing a PCK1 reporter iPSC using CRISPR / Cas9 to visualize and quantify the insulin response. [Figure 5B] This document demonstrates an embodiment that confirms the incorporation of a reporter construct into an iPSC clone. [Figure 5C] This embodiment demonstrates that no changes in cell morphology or proliferation are observed after the incorporation of the PCK1 reporter construct. [Figure 6A] This document presents an embodiment of immunofluorescence imaging demonstrating the upregulation of PCK1-dependent mScarlet fluorescence (corresponding to gluconeogenesis) after cAMP treatment and the downregulation of PCK1-dependent fluorescence after insulin treatment in HLO. [Figure 6B] This document describes embodiments of upregulation of PCK1-dependent luciferase luminescence (corresponding to gluconeogenesis) after cAMP treatment and downregulation of PCK1-dependent luciferase luminescence after insulin treatment in HLO. [Figure 6C] This document describes an embodiment of detecting PCK1-luciferase luminescence in HLO using in vitro imaging. HLO was treated with ±cAMP and ±insulin to modulate gluconeogenesis and insulin stimulation. HLO that was not gene-edited with a PCK1 reporter did not show luminescence. [Figure 6D] Figure 6C shows an embodiment of the quantification of PCK1-luciferase luminescence in HLO detected by in vitro imaging. [Figure 6E] Embodiments are presented to test the effects of different insulin concentrations (no insulin control, 10 nM, 100 nM, or 1000 nM) on PCK1-luciferase luminescence in previously insulin-depleted HLOs. Luciferase activity of the PCK1 reporter decreased in response to insulin stimulation. [Figure 6F] This embodiment describes testing the effects of different insulin treatment times (no insulin control, 1 hour, 2 hours, or 3 hours) on PCK1-luciferase luminescence in previously insulin-depleted HLOs. Luciferase activity of the PCK1 reporter decreased after 1 hour of treatment. [Figure 6G] This describes an embodiment to confirm the effect of cAMP treatment on HLOs that had previously been insulin-depleted. HLOs were treated with cAMP for 24 hours or with insulin for 3 hours following 24 hours of cAMP, and then imaged. PCK1 reporter luciferase activity was increased by cAMP treatment and inhibited by insulin stimulation. [Figure 7A] A schematic diagram illustrating the induction of fatty liver HLO using oleic acid treatment is shown. [Figure 7B] This document illustrates an embodiment of immunofluorescence imaging showing lipid droplets and sustained PCK1 activation in fatty liver-induced HLO generated using oleic acid. [Figure 7C] This document describes an embodiment of quantification of triglycerides by NMR in control HLO or steatohepatitis HLO (sHLO) treated with 300 μM oleic acid. [Figure 7D] This document describes an example of gene expression analysis related to lipid droplet formation. Upregulation of DGAT1 and DGAT2, which catalyze the formation of triglycerides from diacylgycerol and acyl-CoA, was detected in sHLO. [Figure 7E] This document describes the analysis of pro-inflammatory cytokine gene expression and secretion in control HLO or sHLO. Gene expression of TNFa, TGFb, IL6, and IL8, as well as secretion of IL1b, were examined. These pro-inflammatory cytokines were uniformly upregulated in sHLO. [Figure 7F] This demonstrates an embodiment of PCK1-luciferase activity upregulated by sHLO, as detected by in vitro imaging. [Figure 7G] This document describes the quantification of PCK1 expression by luciferase luminescence (relative and absolute) and quantitative RT-PCR in fatty liver sHLO versus control HLO. sHLO showed increased PCK1 expression and activity. [Figure 7H] This document describes an embodiment of the quantification of glucose production in sHLO versus control HLO. Upregulation of PCK1 activity in sHLO was accompanied by increased glucose production. [Figure 7I] This document describes an embodiment of the analysis of the insulin signaling pathway in sHLO and control HLO. AKT phosphorylation was inhibited in sHLO. [Figure 7J] This document describes an embodiment of the analysis of insulin responsiveness in sHLO. Gene expression of downstream target genes in the insulin pathway was quantified. Insulin responsiveness to PCK1, CREB1, and FOXO1 was not suppressed in sHLO. [Figure 7K] This document describes an embodiment of the insulin response analysis of PCK1 in sHLO. PCK1 in sHLO did not respond to insulin. [Figure 7L] This document describes an embodiment of the analysis of the insulin response to gluconeogenesis in sHLO. Glucose production in sHLO was unaffected by insulin stimulation. [Figure 8A] This document presents representative immunofluorescence images and quantification embodiments of the reduction in fat accumulation in fatty liver HLO treated with obeticholic acid (OCA) compared to pioglitazone (PIO) and metformin (MET). [Figure 8B] This document describes the quantification of inflammation-related proteins in fatty liver HLO treated with OCA, PIO, and MET. The relative gene expression of tumor necrosis factor alpha (TNFα), nuclear factor kappa B subunit 1 (NFKB1), and nuclear factor kappa B subunit 2 (NFKB2) is shown. [Figure 8C]This describes an embodiment of insulin responsiveness recovery in fatty liver HLO after OCA treatment. [Figure 8D] This document illustrates the recovery of insulin responsiveness to representative genes involved in gluconeogenesis and lipid biosynthesis in fatty liver HLO after OCA treatment. Relative gene expression of FOXO1, CREB, SREBP1C, and FASN is shown. [Modes for carrying out the invention]

[0018] In normal cells, insulin stimulation generally slows the rate of gluconeogenesis and increases the rate of lipid biosynthesis. This occurs through the activation of a signaling pathway by insulin binding to insulin receptors on the cell surface, which leads to the phosphorylation of AKT. Conversely, decreased insulin levels and / or the presence of either cAMP or glucagon result in upregulation of gluconeogenesis. However, cells can develop insulin resistance, experiencing a homeostatic dysregulation of gluconeogenesis that is less easily disrupted by insulin. This phenomenon of insulin resistance is a significant medical burden affecting millions of individuals and is associated not only with prediabetes or type 2 diabetes but also with other metabolic disorders.

[0019] This disclosure relates, in general, to an insulin resistance reporter that can be expressed in cells to provide an efficient and reliable method for quantifying the intracellular insulin response and related gluconeogenesis and / or lipid biosynthesis. This provides a rapid process for, for example, evaluating the relative sensitivity of cells to insulin or screening compounds that affect the insulin sensitivity or resistance exhibited by cells. The cells may be derived from patients, opening up opportunities for personalized medicine.

[0020] In some embodiments, disclosed herein are insulin-responsive cells, tissues, or organoids comprising any one of the insulin resistance reporters disclosed herein. While all living cells use insulin, prominent cell types are those that consume relatively large amounts of energy, such as pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes, or cells that store and / or produce glucose. Developing methods for generating organoids, which are three-dimensional cellular structures that closely resemble the morphology of living organs, will also enable the study of tissues that more holistically represent their natural biological functions. It is conceivable that insulin resistance reporters may be manipulated against any of these cells, tissues, or organoids to investigate insulin function in these cell types.

[0021] Since the liver is the primary site of gluconeogenesis in mammals, diseases affecting the liver can lead to further complications involving insulin signaling pathways, gluconeogenesis, and lipid biosynthesis. The comorbidity of fatty liver (e.g., NAFLD or NASH) with diabetes is well recognized. Therefore, there is a high need to deepen our understanding of liver function in relation to these and other related diseases, as well as to develop models for diagnosing and treating these diseases.

[0022] Therefore, in some embodiments, the organoids are liver organoids such as human liver organoids (HLOs). HLOs can be generated from pluripotent stem cells (PSCs), and since PSC-derived organoids constitute non-hepatic liver tissue morphologies and additional cell types, including mesenchymal cells, astrocytes, biliary cells, and cholangiocarcinomas, the resulting liver organoids have proven superior to conventional two-dimensional or three-dimensional cultures of hepatocyte or adult stem cell-derived "organoids." Thus, these PSC-derived HLOs are excellent models for investigating metabolic, insulin regulation, and inflammation-related diseases. In some embodiments, these HLOs can be genetically engineered to express an insulin resistance reporter (such as any one of the insulin resistance reporters disclosed herein) and can be used as a model to reproduce the innate human liver insulin response in vitro. In some embodiments, these HLOs can be further engineered to exhibit a fatty liver phenotype. Assays for evaluating insulin resistance in the human liver by comparing normal and fatty liver organoid functions are further described. term

[0023] The following detailed description refers to the accompanying drawings, which form part of it. In the drawings, unless otherwise indicated in the context, similar symbols typically identify similar components. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. The aspects of this disclosure generally described herein and shown in the drawings may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0024] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as they would be generally understood by those skilled in the art when this disclosure is read in light of it. For the purposes of this disclosure, the following terms are defined below:

[0025] This disclosure uses positive language to describe many embodiments. This disclosure also includes embodiments in which subject matter such as substances or materials, method steps and conditions, protocols or procedures are completely or partially excluded.

[0026] The articles "a" and "an" are used herein to refer to one or more (e.g., at least one) grammatical objects of the article. For example, "element" means one or more elements.

[0027] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by approximately 10% relative to the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length being referenced.

[0028] Throughout this specification, unless otherwise required by context, the words “comprise,” “comprises,” and “comprise” shall be interpreted as meaning to include the described step or element or group of steps or elements, but not to exclude any other step or element or group of steps or elements. “Consisting of” means to include everything that follows the phrase “consisting of.” Therefore, the phrase “consisting of” indicates that the enumerated elements are required or mandatory, and no other elements can be present. “Consisting essentially of” means to include all elements enumerated before this phrase, and other elements are limited to those that do not interfere with or contribute to the activity or action expressed in this disclosure with respect to the enumerated elements. Therefore, the phrase "essentially consists of" indicates that the enumerated elements are required or mandatory, while the other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0029] As used herein, the terms “individual,” “subject,” or “patient” have their general and ordinary meanings as understood in light of this specification and mean human or non-human mammals, e.g., dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates, or birds, e.g., chickens, and other vertebrates or invertebrates. The term “mammal” is used in its ordinary biological sense. This includes, specifically, primates including monkeys (chimpanzees, apes, and primates) and humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0030] As used herein, the terms “effective dose” or “effective amount” have their general and ordinary meanings as understood in light of the specification and refer to the amount of the described composition or compound that produces an observable effect. The actual dose levels of the active ingredient in the active composition of the subject currently disclosed may be varied to administer an amount of the active composition or compound that is effective in achieving a desired response for a particular subject and / or use. The selected dose level will depend on a variety of factors, including but not limited to the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, if a minimum dose is administered and there is no dose-limiting toxicity, the dose is increased to the minimum effective dose. This specification is intended to evaluate the determination and adjustment of effective doses, as well as when and how such adjustments should be made.

[0031] As used herein, the terms “function” and “functional” have their obvious and ordinary meanings as understood in light of this specification, and refer to biological, enzymatic, or therapeutic functions.

[0032] As used herein, the term “inhibit” has its general and ordinary meaning as understood in light of this specification and can mean a reduction or prevention of biological activity. The reduction may be by an amount that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or about that, at least that, at least about that, less than or about that, or about less than or about that, or within the range defined by any two of the aforementioned values. As used herein, the term “delay” has its general and ordinary meaning as understood in light of this specification and can mean a delay, postponement, or delay of a biological event to a later time than would otherwise be expected. The delay may be approximately 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a percentage that is approximately that, at least that, at least approximately that, less than or equal to that, or approximately less than or equal to that, or within the range defined by any two of the aforementioned values. The terms inhibition and delay do not necessarily imply 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0033] As used herein, the term “isolated” has the general and ordinary meaning as understood in light of the specification and means a substance and / or entity that (1) when it was first produced (in nature and / or in a laboratory environment) it was separated from at least some of the components to which it was associated, and / or (2) when it was produced, prepared and / or manufactured by human hands it was separated from at least some of the components to which it was associated. An isolated substance and / or entity may be separated from 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or equal to 100%, about the aforementioned values, at least the aforementioned values, at least about the aforementioned values, less than or equal to the aforementioned values, or less than or equal to the aforementioned values ​​(or a range including and / or spanning the aforementioned values). In some embodiments, the isolated agent is approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, substantially 100%, or equivalent to 100% pure, approximately the aforementioned values, at least the aforementioned values, at least approximately the aforementioned values, less than or equal to the aforementioned values, or less than or equal to the aforementioned values ​​(or a range including and / or spanning the aforementioned values). As used herein, “isolated” substance can be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multicellular organism or tissue.

[0034] As used herein, “in vivo” is given its general and ordinary meaning as understood in light herein, and refers to the implementation of the method in living organisms, typically animals, mammals including humans, and plants, as opposed to tissue extracts or dead organisms.

[0035] As used herein, “exvivo” is given its general and ordinary meaning as understood in light of the specification and refers to the execution of a method in vitro with little alteration of natural conditions.

[0036] As used herein, “in vitro” is given its general and ordinary meaning as understood in light of the specification and refers to the execution of the method outside of biological conditions, for example, in a petri dish or test tube.

[0037] As used herein, the terms “nucleic acid” or “nucleic acid molecule” have their general and ordinary meanings as understood herein, and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that occur naturally in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have changes in the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications may include, for example, substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or functionalization of the sugar as an ether or ester. Furthermore, the entire sugar moiety may be replaced with a sterically and electronically similar structure such as aza sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilidetes, or phosphoramidates. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. “Oligonocyte” can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.Nucleic acids (one or more) may be contained in nucleic acid vectors or nucleic acid constructs (e.g., plasmids, viruses, retroviruses, lentiviruses, bacteriophages, cosmids, fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can be used for amplification and / or expression of nucleic acids (one or more) in various biological systems. Typically, vectors or constructs may also contain elements such as promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0038] A nucleic acid or nucleic acid molecule may contain one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences may be adjacent to each other within the same nucleic acid or nucleic acid molecule, or, for example, extra nucleic acids between linker, repeat, or restriction enzyme sites, or any other sequence of any length within the range defined by a base length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300, or any two of the aforementioned lengths. As used herein, the term “downstream” with respect to nucleic acids has its general and ordinary meaning as understood in light of the specification, and, in the case of a double-stranded nucleic acid, refers to the sequence following the 3' end of the sequence preceding the sequence on the strand containing the coding sequence (sense strand). As used herein, the term “upstream” with respect to nucleic acids has its general and ordinary meaning as understood in light of the specification, and, in the case of a double-stranded nucleic acid, refers to the sequence preceding the 5' end of the sequence following the sequence on the strand containing the coding sequence (sense strand). As used herein, the term “grouping” with respect to nucleic acids has its general and ordinary meaning as understood in light of this specification and refers to two or more sequences that occur in close proximity to any other sequence that is, about, at least, at least about, less than, or about less than, any length within the range defined by any two of the aforementioned lengths, but generally does not occur between sequences encoding functional or catalytic polypeptides, proteins, or protein domains.

[0039] The nucleic acids described herein include nucleic acid bases. Primary, standard, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0040] As used herein, the terms “peptide,” “polypeptide,” and “protein” have their general and ordinary meanings as understood herein and refer to macromolecules composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structural, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, though not always, produced biologically by ribosome complexes using nucleic acid templates, but chemosynthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations can be performed, such as substitution, deletion, shortening, addition, replication, or fusion of two or more peptides, polypeptides, and proteins. The fusion of two or more peptides, polypeptides, or proteins can be conjugated adjacent to each other within the same molecule, or, for example, by an extra amino acid between linkers, repeats, epitopes, or tags, or by a base length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300, or any length within the range defined by any two of the aforementioned lengths, or about that, at least that, at least about that, less than or about that. The term “downstream” in relation to polypeptides as used herein has its general and ordinary meaning as understood in light of the specification and refers to the sequence following the C-terminus of the preceding sequence. As used herein, the term “upstream” in relation to polypeptides has its general and ordinary meaning as understood in light of the specification, and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0041] The term “purity” used herein for any given substance, compound, or material has its general and ordinary meaning as understood in light of the specifications and refers to the actual amount of the substance, compound, or material relative to the expected amount. For example, a substance, compound, or material is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, a substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasmas, pyrogens, bacterial endotoxins, and exogenous infectious agents. Purity can be measured using techniques such as electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-Vis spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric analysis, or titration, or any combination thereof.

[0042] The term “yield” for any given substance, compound, or material used herein has its general and ordinary meaning as understood in light of the specifications and refers to the actual total amount of the substance, compound, or material relative to the expected abundance. For example, the yield of a substance, compound, or material may be 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total, or about that, at least that, at least about that, less than or about that, or about less than or about that, including all decimals in between. The yield may be affected by the efficiency of the reaction or process, undesirable side reactions, decomposition, the quality of the input substances, compounds, or materials, or the loss of the desired substance, compound, or material at any step of the manufacturing process.

[0043] As used herein, the term “insulin” has its general and ordinary meaning as understood herein and refers to the major metabolic hormone that regulates glucose uptake in the body’s cells. Insulin typically interacts with transmembrane insulin receptor (INSR) proteins found on the cell surface. Binding of insulin to the receptor induces signaling pathways that affect gluconeogenesis in hepatocytes, glucose uptake in cells such as muscle and adipocytes, downregulation of gluconeogenesis, and upregulation of lipid biosynthesis, among others. In some embodiments, methods involving contact with or treatment with insulin may also be carried out using insulin, insulin aspart, insulin glulisine, insulin lispro, insulin isophane, insulin degludec, insulin detemir, insulin zinc, or insulin glargine, among others, insulin derivatives or mimics thereof. In addition, vanadium, biguanides, metformin, phenformin, buformin, thiazolidinedione, rosiglitazone, pioglitazone, troglitazone, trimidone, sulfonylurea, tolbutamide, acetohexamide, trazamide, chlorpropamide, glipizide, glibenclamide, glimepiride, gliclazide, glyclopyramide, glikidone, meglitinide, repaglinide, nateglinide, α-glucosidase inhibitors, miglitol, acarbose, voglibose, incretins, glucagon-like peptide 1, glucagon-like peptide agonists, exenatide, liraglutide, taspoglutide, lixisenatide, semaglutide Other compounds or compositions that affect glucose metabolism and / or regulation, insulin sensitivity, or are used in the treatment of diabetes, including but not limited to dulaglutide, gastric inhibitory peptides, dipeptidyl peptidase-4 inhibitors, vildagliptin, sitagliptin, saxagliptin, linagliptin, alogliptin, septagliptin, teneligliptin, gemigliptin, pramulintide, dapagliflozin, canapagliflozin, empagliflozin, or remogliflozin, may also be used as substitutes or in combination with insulin or its derivatives or mimics listed herein.

[0044] As used herein, the term “insulin-responsive” has its general and ordinary meaning as understood herein and refers to cells, tissues, or organoids that produce insulin, interact with insulin, or respond to insulin. Insulin-responsive cells, tissues, or organoids include, but are not limited to, pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes, or any tissue or organoid containing one or more of these cells. Dysfunction of the insulin response of these cells or tissues (insulin resistance or hypersensitivity) can cause or be the cause in patients a number of diseases or disorders, including, but not limited to, diabetes mellitus, hyperinsulinemia, weight gain, hypertension, hyperglycemia, dyslipidemia, or inflammatory diseases such as non-alcoholic fatty liver disease (NALFD). In some embodiments, any one of the methods described herein using insulin-responsive cells, tissues, or organoids is applicable to the cells, or their tissues or organoids listed herein. Furthermore, in other embodiments, any one of the stem cells or stem cell compositions described herein can be differentiated into any one of the cells, tissues, or organoids listed herein.

[0045] As used herein, the term “insulin resistance” has its general and ordinary meaning as understood herein and refers to the phenomenon of decreased cellular sensitivity to insulin. This applies to individuals in whom insulin can be endogenously produced by the pancreas or exogenously administered for therapeutic purposes. Excessive blood glucose and / or elevated blood insulin levels (which may occur due to the pancreas's response to high blood glucose levels) can reduce cellular responsiveness to normal levels of insulin release. While this can particularly lead to prediabetes and type 2 diabetes, insulin resistance is associated with other diseases and disorders such as metabolic syndrome, fatty liver disease, steatohepatitis, obesity, cardiovascular disease, polycystic ovary syndrome, hyperglycemia, hyperinsulinemia, or dyslipidemia. Furthermore, although insulin resistance is not generally the primary cause of type 1 diabetes, individuals with type 1 diabetes can also develop insulin resistance.

[0046] As used herein, the term “insulin resistance reporter” has the general and ordinary meaning as understood herein and refers to a biologically relevant construct that responds to changes in insulin levels, insulin-dependent pathway activity, or any target or pathway affected by insulin activity, and exhibits a detectable effect in accordance with such response. Those skilled in the art will understand that the term may apply to nucleic acid constructs that may or may not act directly as reporters, but are translated into proteins that perform the function of a reporter, or are directly applied to proteins. Illustrative reporters commonly used in the art and as used herein include reporters that emit light that can be readily captured by conventional microscopes and other devices. However, other reporters known in the art, such as chemical reporters that induce chemical reactions in response to stimuli (e.g., blue-white screening) or selective marker reporters (e.g., antibiotic resistance), may similarly be used in combination with or as substitutes for other reporters. As disclosed herein, one method for generating an insulin resistance reporter is to functionally link the expression of one or more reporters to the expression of proteins involved in insulin-related pathways. In this case, regulation of the expression of proteins involved in insulin-related pathways (upward or downward regulation of expression) also leads to the same regulation of the reporter.

[0047] It should be understood that the term “insulin resistance reporter” as used herein is not limited to reporters that detect only the phenomenon of insulin resistance. Rather, “insulin resistance reporter” is intended to encompass any embodiment of a reporter construct that can exhibit a detectable effect in response to any modulation of insulin activity, expression, quantity, or function (including insulin sensitivity). Some non-limiting examples of applications of insulin resistance reporters disclosed herein include modeling insulin activity under physiological, normal conditions, modeling insulin resistance, modeling insulin hypersensitivity, and post-transplant monitoring of subjects. The term “insulin response reporter” is used herein interchangeably with “insulin resistance reporter.”

[0048] As used herein, the term “insulin-dependent gene” has its general and ordinary meaning as understood herein and refers to any gene (and the proteins expressed by each) involved in insulin-related pathways. This gene may be involved in the insulin signaling cascade, and when insulin binds to the insulin receptor, it triggers the activation or inhibition of the activity of certain metabolic enzymes, such as PCK1, which may also be accompanied by post-translational modifications of proteins (such as phosphorylation of AKT) or changes in the expression of regulatory genes. Since both gluconeogenesis and lipid biosynthesis are affected by intracellular insulin signaling, genes and proteins that regulate or perform these two metabolic processes fall into the category of insulin-dependent genes. It should be understood that by operably linking any one insulin-dependent gene to an insulin resistance reporter, it becomes possible to measure the insulin-dependent gene and detect any changes in the abundance or function of the insulin-dependent gene, or its absence, in response to insulin stimulation. Those skilled in the art will be expected to understand the intended results of using a particular insulin-dependent gene. For example, proteins involved in gluconeogenesis are expected to show decreased expression in response to insulin, while proteins involved in lipid biosynthesis are expected to show increased expression in response to insulin.

[0049] As used herein, the term “bisistronic element” has its general and ordinary meaning as understood herein and refers to a gene sequence that, based on a coding sequence adjacent to the bisistronic element, results in the expression of two distinct proteins rather than the formation of a fusion of two proteins. Two major bisistronic elements are commonly known: self-cleaving peptides and internal ribosome entry sites (IRESs). Self-cleaving peptides utilize specific amino acid sequences to result in ribosome skipping of peptide bond formation between two amino acids in the sequence. This results in two proteins that are not bound after translation. Well-known self-cleaving peptides include T2A, P2A, E2A, and F2A sequences. IRESs contain RNA sequences that form a secondary structure sufficient to recruit a ribosome without the usual 5' cap. Both types of bisistronic elements arise from the same mRNA and are therefore useful when the expression of two or more distinct proteins is desired and the relative levels of the two or more distinct proteins are to be maintained at generally equal levels. As disclosed herein, a bicistronic element can be placed between an insulin-dependent gene and an insulin resistance reporter to induce equivalent expression of both components, enabling inferences about the level of an insulin-dependent gene by measuring the level of an insulin resistance reporter. Furthermore, a bicistronic element can be placed between two or more reporter genes to enable multiple detection methods (e.g., fluorescence and luminescence).

[0050] As used herein, “pharmaceutically acceptable” means a carrier, excipient, and / or stabilizer that has its obvious and ordinary meaning as understood in light of this specification and is non-toxic to cells or mammals to which cells or mammals are exposed at the doses and concentrations used, or has an acceptable level of toxicity. As used herein, “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” are intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic and absorption retardant, etc., that has their obvious and ordinary meaning as understood in light of this specification and is suitable for administration to human, cat, dog, or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are approved by federal, state, or other regulatory authorities for use in animals, including humans and non-human mammals such as cats and dogs, or are listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The terms diluent, excipient, and / or “carrier” may refer to the diluent, adjuvant, excipient, or vehicle through which the pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin. Water, physiological saline, and aqueous solutions of dextrose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers may also contain one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming inhibitors such as sodium; nonionic surfactants such as TWEEN® and polyethylene glycol (PEG); and PLURONICS®. The composition may also contain small amounts of wetting agents, fillers, emulsifiers, or pH buffers as needed. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulations must be suitable for the administration method.

[0051] Cryotropes are cell composition additives used to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryotropes include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene, glycol, or hydroxyethyl starch. Cryotropes may be used as part of a cryopreservation medium that also contains other components such as nutrients to enhance cell viability after thawing (e.g., albumin, serum, bovine serum, fetal bovine serum [FCS]). In these cryopreservation media, at least one antifreeze agent may be found in concentrations of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or at least that amount, or at least that amount, or at least that amount, or at any percentage within the range defined by any two of the aforementioned numbers.

[0052] Additional excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may include, but are not limited to, serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof, as residues or contaminants from the manufacturing process. The amount of excipients may be found in the composition in any weight percentage within the range defined by 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or about that, at least that, at least about that, less than or about that, or about less than or about that, or any weight percentage within the range defined by any two of the numbers above.

[0053] The term "pharmaceutically acceptable salt" has its plain and ordinary meaning as understood in light of this specification and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including but not limited to analgesics, therapeutic agents, and other materials. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of inorganic bases suitable for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such a class of organic bases may include, but is not limited to, mono, di, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono, di, or trihydroxyalkylamines, including mono-, di-, and triethanolamine; amino acids, including glycine, arginine, and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; and trihydroxymethylaminoethane.

[0054] The appropriate formulation will vary depending on the chosen route of administration. The formulations and techniques for administering the compounds described herein are known to those skilled in the art. Multiple techniques for administering the compounds exist in the art, including, but are not limited to, enteral, oral, rectal, topical, sublingual, oral, intraocular, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intra-arterial, intra-intravenous), intra-portal, intra-articular, intradermal, peritoneal, intrathecal, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. Pharmaceutical compositions will generally be formulated to suit a specific intended route of administration.

[0055] As used herein, “carrier” means a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or organs of the body, in its plain and ordinary sense as understood in light of this specification.

[0056] As used herein, “diluent” has its obvious and ordinary meaning as understood in light of the specification and refers to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It may also be a liquid for dissolving a drug administered by injection, ingestion, or inhalation. Common forms of diluents in the art are buffered aqueous solutions, such as phosphate-buffered saline which mimics the composition of human blood, but are not limited thereto.

[0057] As used herein, the terms “w / w%” or “weight / weight%” have their general and ordinary meanings as understood herein, and refer to a percentage expressed in relation to the weight of an ingredient or drug relative to the total weight of the composition multiplied by 100. As used herein, the terms “v / v%” or “volume / volume%” have their general and ordinary meanings as understood herein, and refer to a percentage expressed in relation to the liquid volume of a compound, substance, ingredient or drug relative to the total liquid volume of the composition multiplied by 100.

[0058] stem cells As used herein, the term “totipotent stem cell” (also known as omnipotent stem cell) refers to a stem cell capable of differentiating into embryonic and extraembryonic cell types. Such cells can construct a complete and viable organism. These cells are produced from the fusion of an egg and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0059] As used herein, the term “embryonic stem cells (ESCs)” is also commonly abbreviated as ES cells and, as used herein, refers to pluripotent cells derived from the inner cell mass of a blastocyst, which is an early embryo, having its obvious and ordinary meaning as understood in light of this specification. For the purposes of this disclosure, the term “ESCs” may be used more broadly to encompass embryonic germ cells.

[0060] As used herein, the term “pluripotent stem cells (PSCs)” has its obvious and ordinary meaning as understood herein and encompasses any cell that can differentiate into any of the body’s nearly all cell types, namely any cell that can differentiate into any of the three germ layers (embryonic epithelium), including the endoderm (endoderm, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), and ectoderm (epidermal tissue and nervous system). PSCs may be descendants of inner cell mass cells of a preimplantation blastocyst, or may be obtained by induction of non-pluripotent stem cells, such as adult somatic cells, by forcing the expression of specific genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0061] As used herein, the term “induced pluripotent stem cells (iPSCs)” has its obvious and common meaning as understood in light of this specification, and is commonly abbreviated as iPS cells. It refers to a type of pluripotent stem cell artificially induced from normally non-pluripotent cells, such as adult somatic cells, by inducing the “forced” expression of specific genes, while hiPSC refers to human iPSCs. In some methods known in the art, iPSCs can be induced by transfection of non-pluripotent cells, such as adult fibroblasts, with specific stem cell-related genes. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcription factors Oct-3 / 4 (POU5F1) and Sox2, but other genes may also improve the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically and are typically isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include first-generation iPSCs, second-generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, retroviral systems are used to transform human fibroblasts into pluripotent stem cells using four critical genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, lentiviral systems are used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4(POU5F1), certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, E-cadherin, or any combination thereof.

[0062] As used herein, the term “progenitor cell” has its obvious and ordinary meaning as understood in light of the specification and encompasses any cell that can be used in the methods described herein, through which one or more progenitor cells acquire the ability to regenerate themselves or to differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or capable of becoming pluripotent. In some embodiments, progenitor cells are subjected to treatment with an extrinsic factor (e.g., a growth factor) to acquire pluripotency. In some embodiments, progenitor cells may be totipotent (or omnipotent) stem cells, pluripotent stem cells (inducible or uninducible), polypotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells may be derived from an embryo, infant, child, or adult. In some embodiments, progenitor cells may be somatic cells subjected to treatment such that pluripotency is conferred via genetic engineering or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblastic stem cells (EpiSCs).

[0063] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from totipotent cells of an early mammalian embryo and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, which is an early stage embryo. Methods for inducing embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESCs) are used in the exemplary embodiments described herein, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0064] Additional stem cells that can be used in embodiments of this disclosure include, but are not limited to, those listed in databases obtained from the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC cell bank at the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, California), Cellartis AB (Goteborg, Sweden), ES Cell International Pte Ltd (Singapore), Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database, which is maintained by Princeton University and the University of Pennsylvania. Examples of embryonic stem cells that can be used in embodiments of this disclosure include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (13), TE04 (14), TE06 (16), UC01 (HSF1), UC06 (HSF6), WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), and WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0065] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term “directed differentiation” describes the process by which less specialized cells become a specific specialized target cell type. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or alter the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0066] In some embodiments, adenoviruses can be used to transport the four required genes, resulting in embryonic stem cell-virtually identical iPSCs. Since adenoviruses do not combine their own genes with the target host, the risk of tumor formation is eliminated. In some embodiments, non-viral-based techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system, albeit with very low efficiency. In other embodiments, iPSCs are generated using direct protein delivery, thus eliminating the need for viruses or gene modification. In some embodiments, mouse iPSC generation is possible using similar methodologies. Repeated treatment of cells with specific proteins delivered to cells via polyarginine anchors has been sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0067] As used herein, the term “feeder cell” has its general and ordinary meaning as understood herein and refers to cells that support the proliferation of pluripotent stem cells by secreting growth factors into the culture medium or displaying them on the cell surface, etc. Feeder cells are generally adherent cells and may cease to proliferate. For example, feeder cells may cease to proliferate by irradiation (e.g., gamma rays), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., formaldehyde or glutaraldehyde). However, feeder cells do not necessarily cease to proliferate. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or heterogeneous to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human precutaneous fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal myocytes, human fetal fibroblasts, or human adult Fallopian tube epithelial cells. In some embodiments, a conditioned medium prepared from the feeder cells is used instead of, or in combination with, the feeder cell co-culture. In some embodiments, the feeder cells are not used during the proliferation of target stem cells.

[0068] gene editing Any of the cells disclosed herein, such as stem cells, pluripotent stem cells, iPSCs, ESCs, endoderm cells, foregut endoderm cells, anterior foregut cells (or anterior foregut spheroids), or organoids (including, but not limited to, liver organoids), can be genetically modified to express an insulin resistance reporter. In some embodiments, iPSCs or ESCs are genetically modified before differentiation into endoderm cells, anterior foregut spheroids, or organoids, or any combination thereof. In some embodiments, iPSCs are first differentiated into endoderm cells before genetic modification. In some embodiments, endoderm cells are genetically modified before differentiation into anterior foregut spheroids or organoids, or both. In some embodiments, endoderm cells are first differentiated into anterior foregut spheroids before genetic modification. In some embodiments, anterior foregut spheroids are genetically modified before differentiation into organoids. In some embodiments, anterior foregut spheroids are differentiated into organoids before genetic modification. In some embodiments, organoids are genetically modified.

[0069] Cells disclosed herein can be modified with insulin resistance reporters using methods commonly known in the art. In some embodiments, cells are genetically modified using CRISPR nucleases, TALENs, zinc finger nucleases, meganucleases, or megaTALs. In some embodiments, cells can be genetically modified using non-homologous end joining or homology-directed repair approaches. In some embodiments, cells are genetically modified using CRISPR nucleases. In some embodiments, cells are genetically modified using homology approaches. In some embodiments, cells are genetically modified using homology approaches with CRISPR nucleases. In some embodiments, the CRISPR nuclease is Cas9, Cpf1, Cas12a, Cas12b, Cas13a, Cas13b, Cas13c, Cas13d, or Cas14a. In some embodiments, the CRISPR nuclease is Cas9.

[0070] In some embodiments, cells are genetically modified with an insulin resistance reporter. In some embodiments, the insulin resistance reporter is a gluconeogenesis reporter. In some embodiments, the insulin resistance reporter is a lipid biosynthesis reporter. In some embodiments, the insulin resistance reporter comprises one or more nucleic acid sequences encoding a reporter protein. In some embodiments, the reporter protein is a fluorescent protein. In some embodiments, the insulin resistance reporter comprises one or more nucleic acid sequences encoding a fluorescent protein. In some embodiments, the fluorescent protein is mScarlet. In some embodiments, mScarlet is encoded by the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, mScarlet comprises the peptide sequence of SEQ ID NO: 15. In some embodiments, the reporter protein is a luminescent protein. In some embodiments, the insulin resistance reporter comprises one or more nucleic acid sequences encoding a luminescent protein. In some embodiments, the luminescent protein is luciferase. In some embodiments, the luciferase is encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the luciferase comprises the peptide sequence of SEQ ID NO: 17. In some embodiments, the insulin resistance reporter comprises one or more nucleic acid sequences encoding a resistance marker. In some embodiments, the resistance marker is a neomycin resistance marker (neoR). In some embodiments, the neomycin resistance marker is encoded by the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the neomycin resistance marker includes the peptide sequence of SEQ ID NO: 18. In some embodiments, the insulin resistance reporter includes one or more bisistronic elements. In some embodiments, the bisistronic elements are self-cleaving peptides or IRESs. In some embodiments, the insulin resistance reporter includes one or more nucleic acid sequences encoding self-cleaving peptides.In some embodiments, one or more self-cleaving peptides are P2A, T2A, E2A, or F2A, or any combination thereof. In some embodiments, the P2A self-cleaving peptide comprises the nucleic acid sequence of SEQ ID NO: 7 and the peptide sequence of SEQ ID NO: 14. In some embodiments, the T2A self-cleaving peptide comprises the nucleic acid sequence of SEQ ID NO: 9 and the peptide sequence of SEQ ID NO: 16. In some embodiments, the insulin resistance reporter comprises one or more nucleic acid sequences homologous to an endogenous gene. In some embodiments, the endogenous gene is a gene involved in gluconeogenesis or lipid biosynthesis. In some embodiments, the gene is PCK1. In some embodiments, the gene is selected from the group consisting of PCK1, G6PC, G6PC2, G6PC3, FOXO1, CREB1, GSK3A, GSK3B, MTOR, SREBP1C, ACC, ACLY, FASN, and GCK. In some embodiments, the insulin resistance reporter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the insulin resistance reporter is inserted into an endogenous gene in a cell such that the expression of the endogenous gene results in the expression of a fluorescent protein, a luminescent protein, a resistance marker, or any combination thereof.

[0071] In some embodiments, cells are genetically modified with an insulin resistance reporter. In some embodiments, the insulin resistance reporter is operably linked to an insulin-dependent gene in the cell. In some embodiments, the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. In some embodiments, the insulin-dependent gene is any gene (or resulting protein) that is regulated in some way in response to insulin signaling. In some embodiments, the insulin-dependent gene is PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, CREB 1、The insulin-dependent gene is selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC. In some embodiments, the insulin-dependent gene is PCK1. In some embodiments, the expression of the insulin-dependent gene results in the expression of an insulin resistance reporter. In some embodiments, the insulin-dependent gene and the insulin resistance reporter are separated by a bisistronic element. In some embodiments, the bisistronic element is a self-cleaving peptide or IRES. In some embodiments, the self-cleaving peptide is selected from P2A, T2A, E2A, or F2A, or any combination thereof. In some embodiments, the P2A self-cleaving peptide includes the nucleic acid sequence of SEQ ID NO: 7 and the peptide sequence of SEQ ID NO: 14. In some embodiments, the T2A self-cleaving peptide includes the nucleic acid sequence of SEQ ID NO: 9 and the peptide sequence of SEQ ID NO: 16. In some embodiments, the insulin resistance reporter is integrated into the locus of the insulin-dependent gene. In some embodiments, the insulin resistance reporter is integrated into the locus of the insulin-dependent gene using a CRISPR nuclease. In some embodiments, the CRISPR nuclease is Cas9, Cpf1, Cas12a, Cas12b, Cas13a, Cas13b, Cas13c, Cas13d, or Cas14a. In some embodiments, the CRISPR nuclease is Cas9. In some embodiments, the insulin resistance reporter is incorporated into the locus of an insulin-dependent gene using homology-directed repair. Exemplary regions of homology to PCK1 for the incorporation of the insulin resistance reporter by homology-directed repair are provided as SEQ ID NOs. 6 and 13. In some embodiments, the insulin resistance reporter comprises two or more reporter genes, the two or more reporter genes being separated by one or more bicistronic elements.

[0072] In some embodiments, iPSCs, endoderm cells, anterior foregut spheroids, or organoids are genetically modified or edited according to methods known in the art. For example, gene editing using CRISPR nucleases such as Cas9 is described in PCT Publications WO2013 / 176772, WO2014 / 093595, WO2014 / 093622, WO2014 / 093655, WO2014 / 093712, WO2014 / 093661, and WO2014 / 204. This is discussed in Nos. 728, WO2014 / 204729, WO2015 / 071474, WO2016 / 115326, WO2016 / 141224, WO2017 / 023803, and WO2017 / 070633, each of which is expressly incorporated herein in whole by reference. Insulin resistance reporter

[0073] Insulin resistance reporters are disclosed herein. These reporters are embodied as nucleic acid constructs and resulting expressed proteins used to visualize, measure, or quantify systems associated with gluconeogenesis, lipid biosynthesis, or other pathways related to insulin activity in biological cells. Generally, the insulin resistance reporters disclosed herein function by the expression of one or more reporter proteins that occur in conjunction with the expression of proteins involved in the insulin pathway, and changes in the expression of insulin-related proteins also apply to the expression of one or more reporter proteins.

[0074] An insulin resistance reporter is provided herein, comprising one or more reporter genes adjacent to the 5' homologous region and 3' homologous region associated with an insulin-dependent gene. The 5' homologous region and 3' homologous region are used for homology-oriented gene editing of one or more reporter genes at the locus of the insulin-dependent gene. In some embodiments, the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. In some embodiments, the insulin-dependent gene is PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, CREB 1、The insulin-dependent gene is selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC. In some embodiments, the insulin-dependent gene is PCK1. In some embodiments, at least one of one or more reporter genes and 5' homologous regions associated with the insulin-dependent gene is separated by a bicistronic element. In some embodiments, the insulin resistance reporter is intended to be inserted on the 3' side of the insulin-dependent gene. In some embodiments, at least one of one or more reporter genes and 3' homologous regions associated with the insulin-dependent gene is separated by a bicistronic element. In some embodiments, the insulin resistance reporter is intended to be inserted on the 5' side of the insulin-dependent gene. In some embodiments, the bicistronic element is a self-cleaving peptide or IRES. In some embodiments, the self-cleaving peptide is a P2A, T2A, E2A, or F2A self-cleaving peptide. In some embodiments, one or more reporter genes include a gene encoding a fluorescent protein or a gene encoding a luminescent protein, or both. In some embodiments, the fluorescent protein includes mScarlet, or the luminescent protein includes luciferase. However, any other fluorescent protein and any other luminescent protein commonly known in the art may be used. In some embodiments, mScarlet is encoded by the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, mScarlet comprises the peptide sequence of SEQ ID NO: 15. In some embodiments, luciferase is encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, luciferase comprises the peptide sequence of SEQ ID NO: 17. In some embodiments, one or more reporter genes further comprise resistance markers, such as neomycin resistance markers. In some embodiments, the neomycin resistance marker is encoded by the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the neomycin resistance marker comprises the peptide sequence of SEQ ID NO: 18.In some embodiments, the insulin resistance reporter comprises two or more reporter genes, which are separated by one or more bicistronic elements. In some embodiments, the one or more bicistronic elements comprises one or more self-cleaving peptides or IRESs. In some embodiments, the one or more self-cleaving peptides comprises P2A, T2A, E2A, or F2A self-cleaving peptides. The separation of two or more reporter genes by one or more bicistronic elements allows for the expression of multiple distinct (i.e., unfused) reporter proteins. In some embodiments, the 5' homologous region associated with the insulin-dependent gene comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 6. In some embodiments, the 3' homologous region associated with the insulin-dependent gene includes a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 13. In some embodiments, the insulin resistance reporter includes a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. Cells, tissues, or organoids, including liver organoids, can be genetically modified to express any of these insulin resistance reporters.

[0075] Cell, tissue, and organoid compositions The methods disclosed herein allow for the direct use or differentiation of pluripotent stem cells (PSCs) into downstream cell types. In some embodiments, PSCs are differentiated into embryonic endoderm cells. In some embodiments, PSCs are differentiated into anterior foregut cells. In some embodiments, PSCs are differentiated into insulin-responsive cells, tissues, or organoids. In some embodiments, the insulin-responsive cells, tissues, or organoids are or include pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes. In some embodiments, the insulin-responsive cells, tissues, or organoids are liver organoids. In some embodiments, PSCs are differentiated into liver organoids. In some embodiments, PSCs are differentiated into pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes, or their tissues or organoids, by methods known in the Art.

[0076] Insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter are disclosed herein. In some embodiments, the insulin resistance reporter is one of the insulin resistance reporters disclosed herein. In some embodiments, the insulin resistance reporter is operably linked to an insulin-dependent gene of an insulin-responsive cell, tissue, or organoid. In some embodiments, the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. In some embodiments, the insulin-dependent gene is PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, CREB 1、The insulin-dependent gene is selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC. In some embodiments, the insulin-dependent gene is PCK1. In some embodiments, expression of the insulin-dependent gene results in expression of an insulin resistance reporter. In some embodiments, the insulin-dependent gene and the insulin resistance reporter are separated by a bicistronic element. In some embodiments, the insulin resistance reporter is located at the 3' end of the insulin-dependent gene so that the insulin resistance reporter and the insulin-dependent gene are separated by the bicistronic element. In some embodiments, the insulin resistance reporter is located at the 5' end of the insulin-dependent gene so that the insulin resistance reporter and the insulin-dependent gene are separated by the bicistronic element. In some embodiments, the bicistronic element is a self-cleaving peptide or IRES. In some embodiments, the self-cleaving peptide is a P2A, T2A, E2A, or F2A self-cleaving peptide. In some embodiments, the insulin resistance reporter is incorporated into the locus of the insulin-dependent gene using a CRISPR nuclease. In some embodiments, the CRISPR nuclease is Cas9. However, an insulin resistance reporter can be incorporated into the locus of an insulin-dependent gene using any other method of gene editing. In some embodiments, the insulin resistance reporter comprises one or more reporter genes. In some embodiments, one or more reporter genes comprises a gene encoding a fluorescent protein or a gene encoding a luminescent protein, or both. In some embodiments, the fluorescent protein comprises mScarlet, or the luminescent protein comprises luciferase. However, any other fluorescent protein and any other luminescent protein commonly known in the art can be used. In some embodiments, mScarlet is encoded by the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, mScarlet comprises the peptide sequence of SEQ ID NO: 15.In some embodiments, one or more reporter genes further comprise a resistance marker. In some embodiments, the luciferase is encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the luciferase comprises the peptide sequence of SEQ ID NO: 17. In some embodiments, one or more reporter genes further comprise a resistance marker. In some embodiments, the resistance marker is a neomycin resistance marker. In some embodiments, the neomycin resistance marker is encoded by the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the neomycin resistance marker comprises the peptide sequence of SEQ ID NO: 18. In some embodiments, the insulin resistance reporter comprises two or more reporter genes, and the two or more reporter genes are separated by one or more bicistronic elements. In some embodiments, one or more bicistronic elements comprise one or more self-cleaving peptides or IRESs. In some embodiments, one or more self-cleaving peptides comprise P2A, T2A, E2A, or F2A self-cleaving peptides. In some embodiments, the insulin resistance reporter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the insulin-responsive cells, tissues, or organoids are stem cells, induced pluripotent stem cells, embryonic stem cells, endoderm cells, or foregut cells. In some embodiments, the insulin-responsive cells, tissues, or organoids are mammalian or human insulin-responsive cells, tissues, or organoids. In some embodiments, the insulin-responsive cells, tissues, or organoids are derived from pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells. In some embodiments, insulin-responsive cells, tissues, or organoids are derived from cells from subjects who have or are at risk of developing diseases or disorders associated with insulin dysfunction.In some embodiments, insulin dysfunction may include insulin resistance or insulin hypersensitivity. In some embodiments, diseases or disorders associated with insulin dysfunction include diabetes mellitus, metabolic syndrome, fatty liver disease, steatohepatitis, obesity, cardiovascular disease, polycystic ovary syndrome, hyperglycemia, hyperinsulinemia, dyslipidemia, or any combination thereof.

[0077] Methods for generating liver organoids have been studied, for example, in PCT Publications WO2018 / 085615, WO2018 / 085622, WO2018 / 085623, WO2018 / 191673, WO2018 / 226267, WO2019 / 126626, WO2020 / 023245, and WO2020 / 069285, each of which is expressly incorporated herein in whole by reference. Any known liver organoid composition or method for producing the same is applicable to the human liver organoids (HLOs) described herein.

[0078] Liver organoids containing insulin resistance reporters are also disclosed herein. In some embodiments, the insulin resistance reporter is one of the insulin resistance reporters disclosed herein. In some embodiments, the insulin resistance reporter is operably linked to an insulin-dependent gene. In some embodiments, the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. In some embodiments, the insulin-dependent gene is PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, CREB 1、The insulin-dependent gene is selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC. In some embodiments, the insulin-dependent gene is PCK1. In some embodiments, expression of the insulin-dependent gene results in expression of an insulin resistance reporter. In some embodiments, the insulin-dependent gene and the insulin resistance reporter are separated by a bicistronic element. In some embodiments, the insulin resistance reporter is located at the 3' end of the insulin-dependent gene so that the insulin resistance reporter and the insulin-dependent gene are separated by the bicistronic element. In some embodiments, the insulin resistance reporter is located at the 5' end of the insulin-dependent gene so that the insulin resistance reporter and the insulin-dependent gene are separated by the bicistronic element. In some embodiments, the bicistronic element is a self-cleaving peptide or IRES. In some embodiments, the self-cleaving peptide is a P2A, T2A, E2A, or F2A self-cleaving peptide. In some embodiments, the insulin resistance reporter is incorporated into the locus of the insulin-dependent gene using a CRISPR nuclease. In some embodiments, the CRISPR nuclease is Cas9. However, an insulin resistance reporter can be incorporated into the locus of an insulin-dependent gene using any other method of gene editing. In some embodiments, the insulin resistance reporter comprises one or more reporter genes. In some embodiments, one or more reporter genes comprises a gene encoding a fluorescent protein or a gene encoding a luminescent protein, or both. In some embodiments, the fluorescent protein comprises mScarlet, or the luminescent protein comprises luciferase. However, any other fluorescent protein and any other luminescent protein commonly known in the art can be used. In some embodiments, mScarlet is encoded by the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, mScarlet comprises the peptide sequence of SEQ ID NO: 15.In some embodiments, one or more reporter genes further comprise a resistance marker. In some embodiments, the luciferase is encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the luciferase comprises the peptide sequence of SEQ ID NO: 17. In some embodiments, one or more reporter genes further comprise a resistance marker. In some embodiments, the resistance marker is a neomycin resistance marker. In some embodiments, the neomycin resistance marker is encoded by the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the neomycin resistance marker comprises the peptide sequence of SEQ ID NO: 18. In some embodiments, the insulin resistance reporter comprises two or more reporter genes, and the two or more reporter genes are separated by one or more bicistronic elements. In some embodiments, one or more bicistronic elements comprise one or more self-cleaving peptides or IRESs. In some embodiments, one or more self-cleaving peptides comprise P2A, T2A, E2A, or F2A self-cleaving peptides. In some embodiments, the insulin resistance reporter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the liver organoid is a fatty liver organoid or a fatty liver organoid. In some embodiments, the fatty liver organoid or fatty liver organoid contains a large number of lipid droplets compared to a normal liver organoid. In some embodiments, the fatty liver organoid or fatty liver organoid is produced by contacting the liver organoid with a fatty acid. In some embodiments, the fatty acid includes oleic acid, linoleic acid, palmitic acid, or stearic acid, or any combination thereof. In some embodiments, fatty liver organoids or fatty liver organoids exhibit insulin resistance and / or type 2 diabetes phenotypes.In some embodiments, insulin resistance includes, compared to normal liver organoids, decreased AKT phosphorylation, reduced suppression of PCK1, CREB1, or FOXO1 expression in response to insulin, or reduced suppression of gluconeogenesis in response to insulin, or any combination thereof. In some embodiments, fatty liver organoids or fatty liver organoids exhibit, compared to normal liver organoids, more lipid droplets, increased DGAT1 / 2 expression, or increased expression and / or secretion of pro-inflammatory cytokines, or any combination thereof. In some embodiments, pro-inflammatory cytokines include TNFα, TGFb, IL6, IL8, or IL1b, or any combination thereof. In some embodiments, liver organoids are mammalian or human liver organoids. In some embodiments, liver organoids are derived from pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells. In some embodiments, liver organoids are derived from cells from subjects who have or are at risk of developing diseases or disorders associated with insulin dysfunction. In some embodiments, insulin dysfunction may include insulin resistance or insulin hypersensitivity. In some embodiments, diseases or disorders associated with insulin dysfunction include diabetes mellitus, metabolic syndrome, fatty liver disease, steatohepatitis, obesity, cardiovascular disease, polycystic ovary syndrome, hyperglycemia, hyperinsulinemia, dyslipidemia, or any combination thereof.

[0079] In some embodiments, insulin-responsive cells, tissues, or organoids are treated with a compound or composition for inducing a hyperlipidemia phenotype. In some embodiments, the compound or composition comprises one or more fatty acids. In some embodiments, the one or more fatty acids comprise oleic acid, linoleic acid, palmitic acid, or any combination thereof. In some embodiments, the insulin-responsive cells, tissues, or organoids are liver organoids. In some embodiments, liver organoids treated with a compound or composition for inducing a hyperlipidemia phenotype develop a fatty liver phenotype. In some embodiments, liver organoids having a fatty liver phenotype are fatty liver organoids. In some embodiments, fatty liver organoids resemble liver tissue exhibiting non-alcoholic fatty liver disease (NAFLD). In some embodiments, fatty liver organoids resemble liver tissue exhibiting steatohepatitis (i.e., fatty liver organoids). In some embodiments, insulin-responsive cells, tissues, or organoids exhibit insulin resistance after treatment with a compound or composition used to induce a hyperlipidemia phenotype. Methods for generating fatty liver organoids and / or steatohepatitis organoids using fatty acids are discussed in PCT Publication WO2018 / 085622, which is expressly incorporated herein by reference in its entirety. In some embodiments, the hyperlipidemia phenotype is completely or partially reversed by treating insulin-responsive cells, tissues, or organoids with one or more (e.g., at least one, two, or three) of obeticholic acid, pioglitazone, or metformin, or any combination thereof. The use of obeticholic acid for the treatment of fatty liver disease is discussed in PCT Publication WO2018 / 085623, which is expressly incorporated herein by reference in its entirety.

[0080] Also disclosed herein are stem cells containing an insulin resistance reporter. In some embodiments, the insulin resistance reporter is one of the insulin resistance reporters disclosed herein. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the stem cells are iPSCs. In some embodiments, the stem cells are derived from human subjects. In some embodiments, the stem cells include one or more nucleic acid sequences encoding a reporter protein and one or more nucleic acid sequences encoding self-cleaving peptides that isolate each of the one or more nucleic acid sequences encoding the reporter protein. In some embodiments, the insulin resistance reporter comprises one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the sequence encoding the reporter protein, and one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the sequence encoding a self-cleaving peptide that separates each of the sequences encoding the reporter protein. In some embodiments, the reporter protein is a fluorescent protein. In some embodiments, the fluorescent protein is mScarlet or any other fluorescent protein known in the art. In some embodiments, mScarlet is encoded by the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, mScarlet comprises the peptide sequence of SEQ ID NO: 15. In some embodiments, the reporter protein is a luminescent protein. In some embodiments, the luminescent protein is luciferase or any other luminescent protein known in the art. In some embodiments, the luciferase is encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the luciferase comprises the peptide sequence of SEQ ID NO: 17. In some embodiments, the reporter protein further comprises a resistance marker. In some embodiments, the resistance marker is a neomycin resistance marker.In some embodiments, the neomycin resistance marker is encoded by the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the neomycin resistance marker includes the peptide sequence of SEQ ID NO: 18. In some embodiments, the insulin resistance reporter further includes one or more nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence associated with an insulin-dependent gene. In some embodiments, the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene. In some embodiments, the insulin-dependent gene is PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, or CREB. 1、The insulin-dependent reporter is selected from the group consisting of TFE3, SREBP1C, FASN, ACLY, and ACC. In some embodiments, the insulin-dependent gene is PCK1. In some embodiments, one or more nucleic acid sequences homologous to the sequence associated with the insulin-dependent gene are adjacent to the sequence encoding the reporter protein and the sequence encoding the self-cleaving peptide, and the sequence homologous to the sequence associated with the insulin-dependent gene acts as a homologous region for recombination into the liver organoid genome. In some embodiments, the insulin resistance reporter is incorporated into the stem cell genome using a CRISPR nuclease. In some embodiments, the CRISPR nuclease is Cas9. However, the insulin resistance reporter can be incorporated into the stem cell genome using any other method of gene editing known in the art. In some embodiments, the sequence associated with the insulin-dependent gene includes a 5' homologous region and a 3' homologous region associated with the insulin-dependent gene. The 5' homologous region and the 3' homologous region enable the incorporation of the insulin resistance reporter into the locus of the insulin-dependent gene by homology-directed repair. In some embodiments, the 5' homologous region associated with the insulin-dependent gene includes a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 6, and / or the 3' homologous region associated with the insulin-dependent gene includes a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 13. In some embodiments, the insulin resistance reporter includes a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4.

[0081] Embryonic endoderm cells differentiated from any of the stem cells disclosed herein are also disclosed herein. Anterior foregut cells differentiated from any of the stem cells disclosed herein are also disclosed herein. Insulin-responsive cells, tissues, or organoids differentiated from any of the stem cells disclosed herein are also disclosed herein. In some embodiments, the insulin-responsive cells, tissues, or organoids include pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes. In some embodiments, the insulin-responsive cells, tissues, or organoids are liver organoids. In some embodiments, the liver organoids are fatty liver organoids after treatment with one or more fatty acids. In some embodiments, the one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. Pancreatic cells, brain cells, adipocytes, muscle cells, or hepatocytes differentiated from any of the stem cells disclosed herein are also disclosed herein. In some embodiments, any of the cells differentiated from any of the stem cells disclosed herein contains any one of the insulin resistance reporters disclosed herein. In some embodiments, any of the cells disclosed herein exhibit insulin dysfunction. In some embodiments, any of the cells disclosed herein exhibit insulin resistance.

[0082] In some embodiments, any of the cells disclosed herein can be cryopreserved for later use. The cells can be cryopreserved according to methods commonly known in the art.

[0083] Screening method, method of use as an indicator, and pharmaceutical composition An in vitro method for screening candidate compounds for the treatment of diseases or disorders associated with insulin insufficiency is disclosed herein. In some embodiments, the method comprises contacting a liver organoid containing an insulin resistance reporter, or an insulin-responsive cell, tissue, or organoid containing an insulin resistance reporter, with a candidate compound, and observing improvement in the liver organoid or insulin-responsive cell, tissue, or organoid for diseases or disorders associated with insulin insufficiency. In some embodiments, diseases or disorders associated with insulin insufficiency include diabetes mellitus, metabolic syndrome, fatty liver disease, steatohepatitis, obesity, cardiovascular disease, polycystic ovary syndrome, hyperglycemia, hyperinsulinemia, dyslipidemia, or any combination thereof. In some embodiments, the liver organoid containing the insulin resistance reporter is any one of the liver organoids disclosed herein. In some embodiments, the insulin-responsive cell, tissue, or organoid containing the insulin resistance reporter is any one of the insulin-responsive cell, tissue, or organoids disclosed herein. In some embodiments, the liver organoid containing the insulin resistance reporter is a fatty liver organoid or a fatty liver organoid. In some embodiments, observing improvement in insulin dysfunction-related disease or impairment in the liver organoid includes observing an increase in AKT phosphorylation, an increase in the suppression of insulin-responsive PCK1, CREB1, or FOXO1 expression, or an increase in insulin-responsive gluconeogenesis suppression, or any combination thereof, compared to before the contact step. In some embodiments, observing improvement in insulin dysfunction-related disease or impairment in the liver organoid includes observing a decrease in the number of lipid droplets, a decrease in DGAT1 / 2 expression, or a decrease in the expression and / or secretion of pro-inflammatory cytokines, or any combination thereof, compared to before the contact step.In some embodiments, the insulin resistance reporter is located at the 3' end of the insulin-dependent gene in a liver organoid or insulin-responsive cell, tissue, or organoid, so that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element. In some embodiments, the insulin resistance reporter is located at the 5' end of the insulin-dependent gene in a liver organoid or insulin-responsive cell, tissue, or organoid, so that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element.

[0084] Also disclosed herein are in vitro methods for evaluating insulin resistance in insulin-responsive cells, tissues, or organoids, including insulin resistance reporters. In some embodiments, the insulin resistance reporter is any one of the insulin resistance reporters disclosed herein. In some embodiments, the method includes quantifying the baseline expression level of one or more reporter proteins of the insulin resistance reporter; contacting insulin-responsive cells, tissues, or organoids with insulin or its derivatives or mimics; quantifying the expression level of one or more reporter proteins after treatment; and determining that the insulin-responsive cells, tissues, or organoids exhibit insulin resistance based on the change or absence of the expression level of one or more reporter proteins. In some embodiments, the method further includes contacting insulin-responsive cells, tissues, or organoids with one or more of obeticholic acid (OCA), pioglitazone, or metformin, or any combination thereof. In some embodiments, the method further comprises contacting insulin-responsive cells, tissues, or organoids with one or more fatty acids before quantifying baseline expression levels. In some embodiments, the method further comprises contacting insulin-responsive cells, tissues, or organoids with one or more fatty acids after quantifying baseline expression levels and before contacting the insulin-responsive cells, tissues, or organoids with insulin or its derivatives or mimetic. In some embodiments, insulin-responsive cells, tissues, or organoids are any one of the insulin-responsive cells, tissues, or organoids disclosed herein. In some embodiments, one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. In some embodiments, insulin-responsive cells, tissues, or organoids are derived from human subjects requiring treatment for insulin resistance. In some embodiments, insulin-responsive cells, tissues, or organoids are liver organoids, and insulin resistance is hepatic insulin resistance.In some embodiments, hepatic insulin resistance is caused by non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some embodiments, the insulin resistance reporter is located 3' to the insulin-dependent gene in a liver organoid or insulin-responsive cell, tissue, or organoid, so that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element. In some embodiments, the insulin resistance reporter is located 5' to the insulin-dependent gene in a liver organoid or insulin-responsive cell, tissue, or organoid, so that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element.

[0085] Also disclosed herein are in vitro methods for screening compounds or compositions for treating insulin resistance. These methods include contacting insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter with one or more fatty acids; quantifying the baseline expression level of one or more reporter proteins of the insulin resistance reporter; contacting insulin-responsive cells, tissues, or organoids with a compound or composition; quantifying the expression level of one or more reporter proteins after treatment; and determining whether the compound or composition can treat insulin resistance based on the change or absence of the expression level of one or more reporter proteins. In some embodiments, the insulin-responsive cells, tissues, or organoids are any one of the insulin-responsive cells, tissues, or organoids disclosed herein. In some embodiments, the insulin resistance reporter is any one of the insulin resistance reporters disclosed herein. In some embodiments, the one or more fatty acids include oleic acid, linoleic acid, palmitic acid, or any combination thereof. In some embodiments, insulin-responsive cells, tissues, or organoids are derived from human subjects requiring treatment for insulin resistance. In some embodiments, the insulin-responsive cells, tissues, or organoids are liver organoids, and the insulin resistance is hepatic insulin resistance. In some embodiments, hepatic insulin resistance is caused by non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some embodiments, the insulin resistance reporter is located 3' to the insulin-dependent gene of the liver organoid or insulin-responsive cells, tissues, or organoids, so that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element.In some embodiments, the insulin resistance reporter is located 5' to the insulin-dependent gene in a liver organoid or an insulin-responsive cell, tissue, or organoid, so that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element.

[0086] Also disclosed herein are compounds or compositions identified by any of the screening methods disclosed herein, which have been found to have an effect on treating, improving, or preventing diseases or disorders associated with insulin resistance or insulin dysfunction.

[0087] Some embodiments described herein relate to pharmaceutical compositions comprising, essentially, or consisting of, an effective amount of any one of the insulin-responsive cells, tissues, or organoids described herein, and a pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary applications. In some embodiments, insulin-responsive cells, tissues, or organoids (such as liver organoids) containing an insulin resistance reporter can be used as a detection device to measure the level of circulating insulin in a subject.

[0088] Methods for monitoring the insulin response in a subject are also disclosed herein. In some embodiments, the method includes transplanting a liver organoid containing an insulin resistance reporter, or insulin-responsive cells, tissues, or organoids containing an insulin resistance reporter, into a subject, and monitoring the expression of the insulin resistance reporter in the liver organoid or insulin-responsive cells, tissues, or organoids. In some embodiments, the liver organoid containing an insulin resistance reporter is any one of the liver organoids disclosed herein. In some embodiments, the insulin-responsive cells, tissues, or organoids is any one of the insulin-responsive cells, tissues, or organoids disclosed herein. In some embodiments, the subject has or is at risk of developing a disease or disorder associated with insulin dysfunction. In some embodiments, insulin dysfunction may include insulin resistance or insulin hypersensitivity. In some embodiments, the subject does not have or is at risk of developing a disease or disorder associated with insulin dysfunction, and normal physiological activity of insulin is monitored. [Examples]

[0089] Example 1. Human liver organoids (HLOs) can be used as a model for the insulin response. Liver organoids, such as those derived from human cells, can be produced according to the methods described herein and or otherwise known in the art. For example, exemplary methods for producing liver organoids from pluripotent stem cells are described in PCT Publications WO2018 / 085615, WO2018 / 191673, WO2018 / 226267, WO2019 / 126626, WO2020 / 023245, and WO2020 / 069285, each of which is expressly incorporated herein in whole by reference.

[0090] A schematic embodiment of liver organoid generation is shown in Figure 1A. Briefly, pluripotent stem cells (egiPSCs or ESCs) are first differentiated into endoderm cells by culturing with activin A and BMP4, and then the endoderm cells are cultured in foregut induction medium containing FGF pathway activators (e.g., FGF4) and Wnt pathway activators (which may be in the form of spheroids, or GSK3 inhibitors (e.g., CHIR99021) for generating foregut cells). If necessary, these foregut cells can be cryopreserved for later use. The foregut cells are embedded in a basement membrane matrix (e.g., Matrigel) and cultured in differentiation medium containing FGF2, VEGF, EGF, CHIR99021, and a TGF-β inhibitor to generate liver organoids. The obtained liver organoids have a uniform morphology (Figure 1B), closely resemble liver tissue, and express liver-specific markers such as albumin (ALB) and hepatocyte nuclear factor 4 (HNF4), as well as the epithelial cell marker E-cadherin (Figure 1C).

[0091] Single-cell RNA sequencing of constituent cells of liver organoids (Figure 2A) revealed two distinct populations: 1) parenchymal cells (approximately 82.4%) expressing characteristic hepatocyte markers such as ALB, APOE, RBP4, and TDO2, and 2) non-parenchymal cells (approximately 17.5%) expressing characteristic markers of hepatic stellate cells, biliary cells, and cholangiocarcinoma cells (COL1A1, PDGFRA, ACTA2, BMP4, WNT6) (Figure 2B). These pluripotent stem cell-derived liver organoids have the potential to emulate the behavior of human liver tissue.

[0092] HLOs represent genes and pathways involved in the insulin response. Figure 3A shows a schematic diagram of the insulin response in the liver. Profiling of single-cell RNA sequencing of HLOs revealed the expression of the insulin receptor (INSR) and insulin receptor substrates 1 and 2 (IRS1 / 2). INSR and IRS2 were concentrated in the hepatocyte population of multicomponent HLOs, but IRS1 was also expressed in the astrocytic cell population.

[0093] HLOs exhibited insulin-stimulated responses to AKT phosphorylation, gluconeogenesis, and lipid biosynthesis in vitro. Insulin responsiveness of HLOs was analyzed using Western blotting and qPCR. Prepared HLOs were cultured under insulin starvation for 24 hours before being exposed to insulin.

[0094] We analyzed the phosphorylation of AKT, which is activated downstream of insulin signaling. HLO was treated with 0 ng / mL, 10 ng / mL, and 100 ng / mL insulin for 20 minutes, and then the proteins were extracted for Western blotting. Insulin induces AKT phosphorylation in HLO (Figure 4A).

[0095] To analyze the insulin responsiveness of gluconeogenesis and lipid biosynthesis regulatory genes in HLO, HLO was treated with 100 ng / mL insulin for 8 hours, and then RNA was extracted for qPCR. In insulin-treated HLO, gluconeogenesis regulatory gene expression (FOXO1, CREB) was reduced. 1、 PCK1 was suppressed by insulin stimulation (Figure 4B). Conversely, the expression of lipid biosynthesis regulatory genes (SREBP, FASN, ACLY, ACC) was induced by insulin stimulation (Figure 4C).

[0096] Example 2. Insulin response reporters can be established in iPSCs and downstream cells. To visualize and quantify HLO insulin responsiveness, iPSCs were gene-edited using the CRISPR / Cas9 system, and a reporter construct containing the mScarlet (fluorescence) and luciferase (luminescence) genes was inserted downstream of the gluconeogenesis regulatory gene PCK1, at the 3' end of exon 10 (Figure 5A). Figure 5A also shows a schematic of the reporter function. When gluconeogenesis is enhanced by exposure to glucagon or cAMP, mScarlet and luciferase are expressed along with increased PCK1 expression. When gluconeogenesis is suppressed by exposure to insulin, the expression of mScarlet and luciferase also decreases, correlated with the downregulation of PCK1. An exemplary reporter construct is represented as the nucleic acid sequence of SEQ ID NO: 4 and the peptide sequence of SEQ ID NO: 5. However, it is conceivable that other reporter constructs, such as those using alternative reporters, may be used. Furthermore, alternative insulin pathway regulatory genes used instead of PCK1 are shown in Figure 3C. Exemplary sgRNAs for orienting Cas9 to the 3' side of PCK1 are provided as Sequence IDs 1-3.

[0097] Example 3. Insulin responsiveness can be observed using the PCK1 reporter HLO. Human iPSCs were gene-edited to insert a fluorescent and luminescent reporter construct into the 3' end of PCK1. The insertion of the construct at the desired location was verified by locus amplification (Figure 5B), and normal morphology was observed (Figure 5C). These gene-edited iPSCs were subsequently differentiated into HLOs. To analyze insulin responsiveness, HLOs were treated with 100 μM cAMP for 24 hours. Subsequently, 100 ng / mL insulin was applied for 3 hours, and fluorescence and luciferase activity were measured. Following cAMP treatment, PCK1-mScarlet fluorescence and luciferase activity increased in response to gluconeogenesis (Figures 6A-B). In addition, after cAMP treatment, treatment with 100 ng / mL insulin for 3 hours resulted in a decrease in PCK1-mScarlet fluorescence and PCK1 luciferase activity in response to insulin stimulation. Figures 6C-D show the detection of PCK1-luciferase luminescence measured by in vitro imaging. HLOs derived from iPSCs that were not gene-edited with the reporter construct did not show luciferase signaling under any of the conditions (cAMP, insulin).

[0098] Using a reporter HLO, we tested various parameters of the insulin response.

[0099] The effect of insulin concentration was investigated. PCK1-luciferase HLOs were depleted of insulin for 24 hours, followed by treatment with 0 nM, 10 nM, 100 nM, or 1000 nM insulin for 1 hour for luciferase imaging. PCK1 luciferase activity decreased in response to insulin stimulation (Figure 6E).

[0100] The effect of insulin treatment time was tested. Insulin-depleted HLOs were treated with 100 nM insulin for 1, 2, or 3 hours (without an insulin control). PCK1 luciferase activity decreased after 1 hour of treatment (Figure 6F).

[0101] The effects of cAMP treatment were investigated. Insulin-depleted HLO cells were treated with 100 μM cAMP for 24 hours, followed by luciferase imaging. Under alternative conditions, cAMP-treated HLO cells were treated with 100 nM insulin for 3 hours. PCK1-luciferase activity increased with cAMP treatment and was inhibited by insulin stimulation (Figure 6G).

[0102] Example 4. A human liver organoid (sHLO) model of fatty liver disease exhibits symptoms of type 2 diabetes. Fatty acid treatment induced a steatohepatitis phenotype in HLOs. Exposure of HLOs to 300 μM oleic acid for 72 hours induced lipid accumulation (Figure 7A). Lipid imaging and NMR-based analysis showed numerous lipid droplets in sHLOs (Figures 7B-C). DGAT1 / 2, which catalyzes the formation of triglycerides from diacylglycerol and acyl-CoA, was increased in sHLOs compared to normal HLOs (Figure 7D). Expression and secretion of pro-inflammatory cytokines were also increased in sHLOs (Figure 7E).

[0103] sHLO showed excessive gluconeogenesis through a certain level of PCK1 activation, a characteristic of type 2 diabetes. A significant increase in PCK1 was observed in sHLO induced by excessive fat accumulation, and this was accompanied by increased glucose production (Figure 7F-H).

[0104] Insulin responsiveness in sHLO was investigated. To detect insulin responsiveness, insulin depletion was performed for 24 hours in both sHLO and HLO control groups, followed by insulin stimulation.

[0105] Insulin signal analysis was performed by Western blotting, using 100 nM insulin for 20 minutes. AKT phosphorylation was inhibited in sHLO (Figure 7I). The insulin responsiveness of PCK1, CREB1, and FOXO1 was not suppressed in sHLO (Figures 7J-K). These results indicate that PCK1 and other gluconeogenic genes do not respond to insulin in sHLO. Furthermore, glucose production was not suppressed after insulin stimulation in sHLO (Figure 7L).

[0106] Example 5. Screening of insulin resistance and fatty liver improvement drugs using fatty liver organoids. Since fatty liver HLO was confirmed to exhibit insulin resistance, it was applied to the screening of drugs that improve NAFLD and insulin resistance. The fatty liver phenotype was induced by treating HLO with fatty acids for 6 days. Then, candidate drugs were exposed to sHLO for 48 hours.

[0107] Oveticolic acid (OCA) treatment reduced fat accumulation and the expression of inflammatory genes (TNFa, NFKB1, NFKB2) (Figures 8A-B). Conversely, metformin (MET) treatment showed improvement in fat accumulation in HLOs, but the improvement in inflammatory responses was limited. HLOs treated with pioglitazone (PIO) showed limited improvement in fat accumulation and inflammatory responses.

[0108] To investigate the improvement of insulin responsiveness in fatty liver HLOs by treatment with candidate drugs, sHLOs were incubated under insulin depletion conditions for 24 hours and then exposed to insulin.

[0109] To analyze insulin-responsive gluconeogenesis, HLOs were treated with 100 ng / mL insulin for 3 hours, and then luciferase activity was measured. Based on the PCK1-luciferase assay, OCA-treated fatty liver HLOs showed improved insulin responsiveness (Figure 8C).

[0110] To analyze the expression of genes involved in the insulin response, sHLO was treated with 100 ng / mL for 8 hours, and then RNA was extracted. OCA-treated sHLO improved insulin responsiveness and resulted in suppression of gluconeogenesis regulatory genes and upregulation of lipid biosynthesis regulatory genes (Figure 8D).

[0111] In summary, INSR and IRS2 are expressed in the liver population of multicomponent liver organoids, suggesting that the HLO liver population responds to insulin signaling and mimics the human insulin response. Human liver organoids exhibited hepatic insulin resistance when they accumulated fat and showed a fatty liver phenotype. PCK1 was continuously activated and gluconeogenesis was promoted in fatty liver organoids. OCA treatment improved fat accumulation, inflammatory response, and gluconeogenesis / lipid biosynthesis in fatty liver HLOs. This suggests that drugs beneficial to fat accumulation may also have the potential to improve hepatic insulin responsiveness. The human liver organoid models disclosed herein provide an opportunity to investigate hepatic insulin resistance and fat metabolism distinct from the metabolism of other peripheral tissues found in vivo.

[0112] In at least some of the embodiments described above, one or more elements used in the embodiment may be interchangeably used in another embodiment unless such substitution is not technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter as defined by the appended claims.

[0113] With regard to substantially all use of plural and / or singular terms herein, those skilled in the art can paraphrase from plural to singular and / or singular to plural as appropriate to the context and / or use. For clarity, various singular / plural substitutions can be clearly described herein.

[0114] Those skilled in the art will understand that the terms used herein and especially in the appended claims (e.g., the text of the appended claims) are generally intended to be "open" terms (for example, the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," and the term "include" should be interpreted as "including but not limited to," etc.). Those skilled in the art will further understand that where there is an intended specific number of claim details to be introduced, such intent is explicitly detailed in this claim, and where no such detail is present, no such intent exists. For example, for the sake of understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce claim details. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim detail by the indefinite article "a" or "an" limits any particular claim containing the claim detail thus introduced to only one embodiment containing such detail (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), even if the same claim contains the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an"), and the same applies to the use of the definite article used to introduce a claim detail. In addition, if the specific number of claim details to be introduced is explicitly detailed, such details should be interpreted as meaning at least the number detailed (for example, the mere detail "two details" without other modifying phrases means at least two details or two or more details). Furthermore, where conventions similar to "at least one of A, B, and C, etc." are used, such constructions are usually intended to have meanings that a person skilled in the art would understand (for example, "a system having at least one of A, B, and C" includes, but is not limited to, A only, B only, C only, A and B together, A and C together, B and C together, and / or a system having A, B, and C together, etc.).Where conventions similar to “at least one of A, B, or C, etc.” are used, such constructions are usually intended to have a meaning that a person skilled in the art would understand (for example, “a system having at least one of A, B, or C” includes, but is not limited to, A only, B only, C only, A and B together, A and C together, B and C together, and / or a system having A, B, and C together, etc.). A person skilled in the art will further understand that any substantially any disjunct word and / or disjunct phrase presenting two or more alternative terms in the specification, claims, or drawings should be understood to be intended to include the possibility of including one of the terms, either or both of the terms. For example, the phrase “A or B” would be understood to include the possibilities of “A” or “B” or “A and B”.

[0115] In addition, if any feature or aspect of the present disclosure is described by the Markush Group, a person skilled in the art will recognize that the present disclosure may also be described by any individual member or subgroup of a member of the Markush Group.

[0116] For all purposes, including the standpoint of documenting the subject matter in a manner that can be understood by those skilled in the art, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. It is readily apparent that any scope enumerated may be adequately described and made possible to decompose the same scope into at least two, three, four, five, ten, etc., subscopes. As a non-limiting example, each scope considered herein may be readily decomposed into a lower third, a middle third, an upper third, etc. As can be understood by those skilled in the art, all terms such as “maximum,” “at least,” “greater than,” and “less than” include the numbers detailed and refer to scopes that may later be decomposed into subscopes as considered herein. Finally, as can be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0117] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be obvious to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit, and the true scope and spirit are set forth in the following claims.

[0118] All references cited herein, including but not limited to published and unpublished applications, patents, and documents, are incorporated herein by reference in their entirety and become part of this Specification. To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained in this Specification, this Specification is intended to take precedence over and / or supersede such conflicting material.

Claims

1. A liver organoid comprising an insulin resistance reporter, wherein the insulin resistance reporter is operably linked to an insulin-dependent gene of the liver organoid, and the expression of the insulin-dependent gene results in the expression of the insulin resistance reporter.

2. The liver organoid according to claim 1, wherein the liver organoid is a fatty liver organoid or a fatty liver organoid.

3. The liver organoid according to claim 1, comprising one or more reporter genes adjacent to a 5' homologous region and a 3' homologous region associated with an insulin-dependent gene, which is an insulin resistance reporter.

4. The liver organoid according to claim 1, wherein the insulin-dependent gene is a gluconeogenesis gene or a lipid biosynthesis gene.

5. The liver organoid according to claim 4, wherein the insulin-dependent gene is selected from the group consisting of PCK1, G6PC, G6PC2, G6PC3, GSK3A, GSK3B, MTOR, GCK, FOXO1, CREB1, TFE3, SREBP1C, FASN, ACLY, and ACC.

6. The liver organoid according to claim 5, wherein the insulin-dependent gene is PCK1.

7. The liver organoid according to claim 3, wherein at least one of one or more reporter genes and a 5' homologous region associated with an insulin-dependent gene are separated by a bisistronic element.

8. The liver organoid according to claim 7, wherein the bisistronic element is a self-cleaving peptide or IRES.

9. The liver organoid according to claim 8, wherein the self-cleaving peptide is a P2A, T2A, E2A, or F2A self-cleaving peptide.

10. The liver organoid according to claim 3, wherein one or more reporter genes include a gene encoding a fluorescent protein or a gene encoding a luminescent protein, or both.

11. The liver organoid according to claim 10, wherein the fluorescent protein comprises mScarlet, or the luminescent protein comprises luciferase.

12. The liver organoid according to claim 3, wherein one or more reporter genes further comprise antibiotic resistance markers.

13. The liver organoid according to claim 1, wherein the insulin resistance reporter comprises two or more reporter genes, and the two or more reporter genes are separated by one or more bicistronic elements.

14. The liver organoid according to claim 13, wherein the one or more bisistronic elements comprise one or more self-cleaving peptides or IRESs.

15. The liver organoid according to claim 14, wherein the one or more self-cleaving peptides include P2A, T2A, E2A, or F2A self-cleaving peptides.

16. The liver organoid according to claim 3, wherein the 5' homologous region associated with the insulin-dependent gene comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 6, and / or the 3' homologous region associated with the insulin-dependent gene comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO:

13.

17. The liver organoid according to claim 1, wherein the insulin resistance reporter comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO:

4.

18. An in vitro method for screening candidate compounds for the treatment of diseases or disorders associated with insulin insufficiency, A step of contacting a liver organoid containing an insulin resistance reporter with the candidate compound, An in vitro method comprising the step of observing improvement in the liver organoid of the disease or disorder associated with insulin dysfunction.

19. The method according to claim 18, wherein the diseases or disorders associated with insulin dysfunction include diabetes mellitus, metabolic syndrome, fatty liver disease, steatohepatitis, obesity, cardiovascular disease, polycystic ovary syndrome, hyperglycemia, hyperinsulinemia, dyslipidemia, or any combination thereof.

20. The method according to claim 18, wherein the liver organoid containing the insulin resistance reporter is the liver organoid according to any one of claims 1 to 17.

21. The method according to claim 20, wherein the liver organoid containing the insulin resistance reporter is a fatty liver organoid or a fatty liver organoid.

22. The method according to claim 18, wherein observing improvement of the disease or disorder associated with insulin dysfunction in the liver organoids includes observing an increase in AKT phosphorylation, an increase in the suppression of insulin-responsive PCK1, CREB1, or FOXO1 expression, or an increase in insulin-responsive gluconeogenesis suppression, or any combination thereof, compared to before the step of contacting the candidate compound with the liver organoids.

23. The method according to claim 22, wherein observing improvement of the disease or disorder associated with insulin dysfunction in the liver organoid comprises observing a decrease in the number of lipid droplets, a decrease in the expression of DGAT1 or DGAT2, or a decrease in the expression and / or secretion of pro-inflammatory cytokines, or any combination thereof, compared to before the step of contacting the candidate compound with the liver organoid.

24. A composition for use in monitoring the insulin response in a subject, wherein the composition is Includes liver organoids containing an insulin resistance reporter, The insulin resistance reporter is operably linked to an insulin-dependent gene of the liver organoid, The monitoring involves monitoring the expression of the insulin resistance reporter in the liver organoid. composition.

25. The composition according to claim 24, wherein the liver organoid containing the insulin resistance reporter is the liver organoid according to any one of claims 1 to 17.

26. The composition according to claim 24, wherein the subject has or is at risk of developing a disease or disorder associated with insulin dysfunction.

27. The liver organoid according to claim 1, wherein the insulin resistance reporter is located on the 3' side of the insulin-dependent gene such that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistron element.

28. The composition according to claim 24, wherein the insulin resistance reporter is located on the 3' side of the insulin-dependent gene such that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element.

29. The liver organoid according to claim 1, wherein the insulin resistance reporter is located on the 5' side of the insulin-dependent gene such that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistron element.

30. The composition according to claim 24, wherein the insulin resistance reporter is located on the 5' side of the insulin-dependent gene such that the insulin resistance reporter and the insulin-dependent gene are separated by a bisistronic element.

31. The liver organoid according to claim 1, wherein the liver organoid is generated from pluripotent stem cells.

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