WO2023160671A1 - 诱导性成熟肝脏细胞及其制备的方法 - Google Patents

诱导性成熟肝脏细胞及其制备的方法 Download PDF

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WO2023160671A1
WO2023160671A1 PCT/CN2023/078284 CN2023078284W WO2023160671A1 WO 2023160671 A1 WO2023160671 A1 WO 2023160671A1 CN 2023078284 W CN2023078284 W CN 2023078284W WO 2023160671 A1 WO2023160671 A1 WO 2023160671A1
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cells
induced
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liver cells
cell
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丁胜
郝婕
马天骅
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清华大学
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Definitions

  • the invention relates to a composition for preparing and inducing induced mature liver cells, a method for preparing induced mature liver cells, and induced induced mature liver cells.
  • liver cells play an important role in the scientific research and clinical treatment of liver diseases.
  • liver cells can be used to study pathogenesis and screen related drugs through the construction of disease models; transplantation of liver cells can treat chronic liver diseases and alleviate advanced liver failure.
  • transplantation of liver cells can treat chronic liver diseases and alleviate advanced liver failure.
  • the main way to obtain primary liver cells is to separate cells from donor livers.
  • the acquisition of liver cell resources has brought great challenges to scientific research and clinical applications. Here comes a serious hindrance.
  • inducible liver cells The emergence of inducible liver cells holds great promise for solving this bottleneck.
  • Melissa K. Carpenter's research group took the lead in successfully differentiating human embryonic stem cells (embryonic stem cells, ESCs) into inducible liver cells expressing liver marker genes in vitro (Lakshmi Rambhatla, 2003b Generation of Hepatocyte-Like Cells From Human Embryonic Stem Cells. Cell Transplantation, 12, 1-11.).
  • induced pluripotent stem cells induced pluripotent stem cells, iPSCs
  • the method of using induced pluripotent stem cells to induce liver cells was also quickly constructed (Song et al., 2009Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.Cell Res 19,1233-1242.10.1038/cr.2009.107.; Takahashi et al.,2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell 131,861- 872.10.1016/j. cell.2007.11.019.).
  • liver cells In recent years, although the technology of in vitro induction of liver cells has been continuously updated and improved, the induced liver cells obtained so far have only obtained some characteristics of primary liver cells, and these induced liver cells are more similar to fetal liver cells than mature primary liver cells. Liver cells (Baxter et al., 2015 Phenotypic and functional analyzes show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes. J Hepatol 62, 581-589.10.1016/j.jhep.2 014.10.016.).
  • liver cells are relatively three-dimensional, with clear nuclei in the cells, and some cells are dual-nucleated, with small nuclei and relatively low nuclear-cytoplasmic ratios.
  • liver cells induced in the prior art have a polygonal shape unique to liver cells, the cells are relatively adherent and not three-dimensional, and there are basically no clear nuclei in the cells. in primary liver cells.
  • the induced liver cells in the prior art have the ability to secrete albumin, the secretion amount per million cells per 24 hours is about 2-5 ⁇ g, while the secretion ability of high-quality primary liver cells can reach 10 ⁇ g Above (Zhu et al., 2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93-97.10.1038/nature13020.).
  • the ability of CYP450 metabolism and urea synthesis in the prior art to induce liver metabolism is far from that of primary liver cells.
  • the prior art induced liver cells still lack liver polarity; in terms of in vivo reconstitution, when the prior art induced liver cells were transplanted into a mouse model of chronic liver injury, human The time required to source albumin is relatively long, and the highest concentration of albumin detected in blood is only about a few hundred nanograms per milliliter, which indicates that Albumin secretory capacity was low, while in vivo reconstitution of induced hepatocytes was less than 1%.
  • the albumin secretion ability of primary liver cells in mice with chronic liver injury can reach more than 1mg/ml, and the in vivo reconstruction efficiency can reach more than 90% (Zhang et al., 2018 In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell Stem Cell 23, 806-819e804.10.1016/j.stem.2018.10.018.).
  • chimerism-induced liver cells in mice with chronic liver damage mainly chimerized near the hepatic portal vein because they could not migrate (Wang et al., 2019 Human ESC-derived expandable hepatic organoids enable therapeutic liver repopulation and pathophysiological modeling of alcoholic liver injury.
  • the invention aims to establish a method for preparing induced mature liver cells which are close to or not inferior to primary liver cells in terms of morphology, gene expression, cell function and the like by using the composition.
  • the inventors have surprisingly found that it is possible to differentiate induced immature liver cells into induced mature liver cells by using a specific combination of compounds as an additive to the basal medium for inducing induced immature liver cells. Efficient, able to obtain induced mature liver cells similar to primary liver cells. therefore,
  • the present invention provides a first composition comprising: (a) HDAC inhibitor, (b) ERK signaling pathway inhibitor.
  • the first composition further comprises (c) a steroid compound, (d) a Notch signaling pathway inhibitor, and/or (e) a TGF-beta signaling pathway inhibitor.
  • the first composition further comprises: (f) a p38 MAPK inhibitor.
  • the present invention also provides a first kit, (a) HDAC inhibitor, (b) ERK signaling pathway inhibitor, and corresponding instructions for use.
  • the first kit further comprises (c) a steroid compound, (d) a Notch signaling pathway inhibitor, and/or (e) a TGF-beta signaling pathway inhibitor.
  • the first kit further comprises: (f) a p38 MAPK inhibitor.
  • the present invention also provides a first medium, which comprises: (a) HDAC inhibitor, (b) ERK signaling pathway inhibitor, and basal medium.
  • the first culture medium further comprises (c) steroid compounds, (d) Notch signaling pathway inhibitors; and/or (e) TGF-beta signaling pathway inhibitors.
  • the basal medium is selected from media such as DMEM, Knockout DMEM, RPMI 1640, DMEM/F12, HCM and William'E, more preferably, the basal medium is HCM.
  • the first culture medium further comprises: (f) p38 MAPK inhibitor.
  • the present invention also provides a second composition comprising: (a) a cAMP activator, (b) a steroid compound; preferably, the second composition further comprises (c) a ROCK inhibitor And/or (d) TGF-beta signaling pathway inhibitors.
  • the present invention also provides a second kit, which includes: (a) cAMP activator, (b) steroid compound; and corresponding instructions for use; preferably, the second kit further Comprising (c) ROCK inhibitors and/or (d) TGF-beta signaling pathway inhibitors.
  • the present invention also provides a second culture medium, which comprises: (a) cAMP activator, (b) steroid compound, and basal medium; preferably, the basal medium is selected from DMEM, Mediums such as Knockout DMEM, RPMI 1640, DMEM/F12, HCM and William'E, more preferably, the base medium is William'E.
  • the second medium contains 0.05%-20% by volume of serum relative to the total volume of the second medium, preferably 1% by volume.
  • the first kit further comprises (c) ROCK inhibitor and/or (d) TGF-beta signaling pathway inhibitor.
  • the present invention also provides the use of the first composition, the first kit, the first culture medium, the second composition, the second kit, and the second culture medium for preparing induced mature liver cells .
  • the HDAC (histone deacetylase) inhibitor is selected from Mocetinostat (MGCD0103), Abexinostat (PCI-24781), Fimepinostat (CUDC-907), ITSA-1, Vorinostat (SAHA), MS-275 , Panobinostat (LBH589), Quisinostat, FK228, SB939, Trichostatin A (TSA), etc.
  • the HDAC (histone deacetylase) inhibitor is MS-275.
  • the steroid compound is selected from prednisone, methylprednisone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone and the like.
  • the steroid compound is dexamethasone.
  • the Notch signaling pathway inhibitor is selected from IMR-1, RO4929097, Semagacestat (LY450139), Avagacestat (BMS-708163), Dibenzazepine (YO-01027), Crenigacestat (LY3039478), Compound E, DAPT and the like.
  • the Notch signaling pathway inhibitor is Compound E.
  • the ERK signaling pathway inhibitor is selected from SeluMetinib (AZD6244), Trametinib (GSK1120212), PD0325901, U0126-ETOH, U0126, PD184352 (CI-1040) and the like.
  • the ERK signaling pathway inhibitor is PD0325901.
  • the TGF-beta signaling pathway inhibitor is selected from SD-208, LY2109761, BIBF-0775, A83-01, SB431542 and the like.
  • the TGF-beta signaling pathway inhibitor is A83-01.
  • the p38 MAPK inhibitor is selected from SB202190 (FHPI), SB203580, TAK-715, Doramapimod (BIRB 796) and the like.
  • the p38 MAPK inhibitor is SB203580.
  • the cAMP activator is selected from Forskolin, 8-Br-cAMP and the like.
  • the cAMP activator is Forskolin.
  • the ROCK inhibitor is selected from Y-27632, Thiazovivin, GSK429286A, RKI-1447, AT13148 and the like.
  • the ROCK inhibitor is Y-27632.
  • the medium comprises basal medium.
  • the base medium is selected from media such as DMEM, Knockout DMEM, RPMI 1640, DMEM/F12, HCM and William'E.
  • the present invention provides a method for preparing induced mature liver cells, the method comprising the steps of: culturing induced immature liver cells in the medium described herein, thereby preparing the induced immature liver cells mature liver cells.
  • the method comprises step (a) culturing the induced immature liver cells in the first medium described herein.
  • the induced mature liver cells were prepared.
  • the method further comprises culturing the cells obtained in step (a) in the second culture medium described herein, thereby preparing an induced mature liver that is more similar to primary liver cells cell.
  • the induced immature liver cells are prepared from pluripotent stem cells; preferably, in some embodiments, the induced immature liver cells are prepared from pluripotent stem cells by the method described herein .
  • the pluripotent stem cells are selected from embryonic stem cells or induced pluripotent stem cells.
  • the present invention provides an induced mature liver cell having one or more of the following characteristics:
  • the induced mature hepatocytes exhibit typical morphological characteristics of primary hepatic cells, preferably, basically consistent with the primary human-derived hepatic cells;
  • the induced mature liver cells have a significant increase in the transcription or protein expression of one or more liver cell markers selected from the following, or the accessibility of chromosomes: CYP1A2, CYP2B6 , CYP2D6, CYP2C9, CYP2C19, CYP3A4, CYP7A1;
  • said induced mature liver cells have decreased transcription or protein expression of the following markers: AFP;
  • the induced mature hepatocytes have increased or first acquired function of one or more hepatocytes selected from the group consisting of: albumin secretion capacity, urea synthesis capacity, hepatic cell polarity , CYP metabolic enzyme activity, regenerative capacity of mature liver cells, transplant survival rate, in vivo reconstitution rate; and
  • liver cells Compared with primary liver cells, the function of one or more of the induced mature liver cells selected from the following liver cells is close to or not lower than that of primary liver cells: albumin secretion ability, urea synthesis ability, liver Cell polarity, CYP metabolic enzyme activity, regeneration ability of mature liver cells, transplant survival rate, in vivo reconstitution rate;
  • the induced mature liver cells can be prepared according to the methods described herein.
  • induced mature liver cell prepared by the methods described herein.
  • the invention provides an organoid produced from the induced mature liver cells described herein.
  • the invention provides a tissue produced from the induced mature liver cells described herein, preferably the tissue is the liver.
  • the present invention provides an induced mature liver cell disclosed herein in preparation of cells for liver cell transplantation, construction of liver-related disease models, screening and identification of drugs related to liver diseases, screening and identification of drug hepatotoxicity, in vitro Use in constructing liver organs (including 3D printing liver organs).
  • the invention provides an in vitro method of screening for a substance for treating a disease comprising contacting an induced mature liver cell, organoid or tissue disclosed herein with the substance to be screened.
  • the induced immature liver cells described herein can be prepared by conventional methods in the art, including but not limited to (Zhu et al., 2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93- 97.10.1038/nature13020), the induced immature liver cells are also called "f-iHep", “iHep” and the like in the art. Induced immature hepatocytes are also referred to as "induced hepatocytes" in some prior art.
  • the induced immature liver cells can be prepared by the methods described herein.
  • the invention provides a culture comprising the medium described herein and cells produced during the induction process.
  • the culture comprises a medium described herein and induced mature liver cells.
  • the present invention provides a kit comprising HEM-1 medium and HEM-2 medium, wherein the HEM-1 medium comprises ROCK inhibitor, hepatocyte growth factor (HGF), tumor suppressor Prime M (oncostatinM, OSM), steroid compound, Notch signaling pathway inhibitor, TGF-beta signaling pathway inhibitor and basal medium;
  • HGF hepatocyte growth factor
  • OSM tumor suppressor Prime M
  • steroid compound hepatocyte growth factor
  • Notch signaling pathway inhibitor TGF-beta signaling pathway inhibitor
  • basal medium is the HCM culture that removes epidermal growth factor (EGF) base.
  • the HEM-2 medium comprises hepatocyte growth factor (HGF), oncostatin M (oncostatinM, OSM), steroid compounds, Notch signaling pathway inhibitors, TGF-beta signaling pathway inhibitors and basal medium; preferably , the basal medium is HCM medium without epidermal growth factor (EGF).
  • HGF hepatocyte growth factor
  • oncostatinM oncostatinM
  • OSM osteroid compounds
  • Notch signaling pathway inhibitors hepatocyte growth factor
  • TGF-beta signaling pathway inhibitors hepatocyte growth factor (HGF), oncostatinM (oncostatinM, OSM)
  • basal medium is HCM medium without epidermal growth factor (EGF).
  • the ROCK inhibitor is Y-27632
  • the steroid compound is dexamethasone
  • the Notch signaling pathway inhibitor is Compound E
  • TGF-beta signaling pathway inhibitor is A83-01 .
  • the HEM-1 medium and the HEM-2 medium comprise basal medium selected from DMEM, Knockout DMEM, RPMI 1640, DMEM/F12, HCM and William'E; more preferably , in some embodiments, the basal medium is HCM medium.
  • the invention provides a method of expanding induced immature liver cells comprising contacting the induced immature liver cells with a HEM-1 medium or a HEM-2 medium of the invention.
  • the method of expanding induced immature liver cells comprises contacting the induced immature liver cells with HEM-1 medium followed by contacting the induced immature liver cells with HEM-2 medium.
  • the present invention provides a use of a HEM-1 medium and a HEM-2 medium for expanding induced immature liver cells.
  • the induced mature liver cells obtained by the method described herein can maintain key liver cell functions and morphology after long-term (for example: more than 3 months) expansion and culture in vitro.
  • Figure 1 Morphological characteristics of induced mature hepatocytes.
  • Cell 1 induced immature liver cells: induced immature liver cells induced by pluripotent stem cells
  • Cell 2 (5C medium) induced immature liver cells were treated with 5C medium for 15 days, showing Cell morphology
  • Cell 2 (6C medium) Cell morphology of induced immature liver cells treated with 6C medium for 15 days
  • Cell 3 (4C medium) Cell 1 (induced immature liver cells) treated with 6C After medium treatment, the cell morphology presented after being treated with 4C medium for 60 days.
  • Figure 2 Induced mature liver cells highly express a series of marker genes related to liver cell maturation.
  • B) mature liver cells For the expression of genes at the protein level, the protein expressions of maturation-related genes CYP1A2, CYP2C9, CYP2D6, and CYP3A4 in cell 3 (induced mature liver cells) were identified and verified by immunofluorescence.
  • Figure 3 Induced mature hepatocytes have hepatocyte polarity and a series of liver-related functions are also significantly improved.
  • Figure 4 Induced mature liver cells can reestablish migration in FRG of mice with chronic liver injury and alleviate and improve the liver injury and survival rate of mice.
  • HSA human serum albumin
  • the expression of hALB gene in the mouse liver has a significant effect on the expression of hALB gene in the mouse liver for more than 3 months after transplantation.
  • Figure 5 Expansion of Cell 1 (induced immature liver cells) using HEM-1 medium and HEM-2 medium.
  • Figure 6 Component validation and optimization of 4C medium and 6C medium.
  • the term "about” or “approximately” refers to a change of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in quantity, level, value, quantity, frequency, percentage, dimension, size, volume, weight or length.
  • the term "about” or “approximately” refers to ⁇ 15%, ⁇ 10%, ⁇ 9% around a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length , ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% of quantity, level, value, quantity, frequency, percentage, scale, size, amount, weight or length range.
  • the term “substantially/essentially” means about 90%, 91%, compared to a reference amount, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater in quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length.
  • the term “substantially the same” refers to a quantity, level, value, quantity, frequency, A percentage, measure, size, amount, weight, or length range.
  • the term "substantially free” when used to describe a composition such as a cell population or a culture medium means free of a specified substance, for example 95% free, 96% free, 97% free, 98% free A composition that is free, 99% free of the specified substance, or is undetectable as measured by conventional means.
  • a similar meaning applies to the term “absent” when referring to the absence of a particular substance or component of the composition.
  • the term “substantial” refers to an amount, level, value, amount, frequency, percentage, dimension, size, amount, weight or length range that is readily detectable by one or more standard methods.
  • the terms “not-appreciable” and “not appreciable” and equivalents mean an amount, level, value, quantity, frequency, percentage that is not readily detectable or detectable by standard methods , scale, size, volume, weight, or length range. In one embodiment, an event is not substantial if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less.
  • Consisting of means including, but limited to, anything following the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
  • Consisting essentially of is meant to include any of the elements listed after the phrase “consisting essentially of” and is limited to activities or actions specified in the disclosure that do not interfere with or contribute to the listed elements other elements. Thus, the phrase “consisting essentially of” is to indicate that the listed elements are required or mandatory, but that no other elements are optional, and depending on whether they affect the activities or actions of the listed elements and may or may not exist.
  • references to "one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “an embodiment,” “another embodiment,” or “further embodiments” A combination thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.
  • the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment.
  • the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
  • ex vivo generally refers to activities that take place outside a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside the living organism, preferably with minimal changes from natural conditions.
  • "ex vivo" procedures involve living cells or tissues obtained from an organism and cultured in a laboratory apparatus, usually under sterile conditions, and usually for several hours or up to about 24 hours, but including up to 48 or 72 hours, depending on circumstances. In certain embodiments, such tissues or cells can be harvested and frozen, then thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than several days using living cells or tissues are generally considered "in vitro," although in certain embodiments the term is used interchangeably with ex vivo.
  • in vivo generally refers to activities that occur within an organism.
  • pluripotent stem cell refers to a cell that has the ability to form all lineages of an organism or body (ie, an embryonic body).
  • embryonic stem cells are capable of forming from three A type of pluripotent stem cell of cells of the respective germ layers (ectoderm, mesoderm and endoderm).
  • the pluripotent stem cells can be embryonic stem cells or induced pluripotent stem cells.
  • culture means a population of cells grown in culture and optionally passaged.
  • a cell culture can be a primary culture (eg, a culture that has not been subcultured) or can be a passage or subculture (eg, a population of cells that has been subcultured or passaged one or more times).
  • gene expression or “gene transcription” refers to the expression/transcription and and/or relative levels of expression/transcriptional patterns.
  • the mature hepatocytes are induced mature hepatocytes.
  • the immature liver cells are induced immature liver cells.
  • protein expression refers to the relative level of expression and/or expression pattern of a protein in a biological sample (e.g., mature liver cells/immature liver cells or a population of cells comprising mature liver cells/immature liver cells) .
  • a biological sample e.g., mature liver cells/immature liver cells or a population of cells comprising mature liver cells/immature liver cells.
  • the mature hepatocytes are induced mature hepatocytes.
  • the immature liver cells are induced immature liver cells.
  • detecting expression/transcription means determining the amount or presence of an RNA transcript of a gene or its expression product.
  • Methods for detecting gene expression/transcription i.e., gene expression/transcription profiling, including polynucleotide-based hybridization analysis methods, polynucleotide-based sequencing methods, immunohistochemical methods, and proteomics-based methods method.
  • the methods generally detect the expression/transcript product (eg, mRNA) of a gene of interest.
  • PCR-based methods such as reverse transcription PCR (RT-PCR) (Weis et al., TIG8:263-64, 1992), and array-based methods such as microarrays (Schena et al., Science 270:467 -70, 1995).
  • RT-PCR reverse transcription PCR
  • array-based methods such as microarrays (Schena et al., Science 270:467 -70, 1995).
  • detecting protein expression means determining the amount or presence of a protein encoded by a gene. Methods for detecting protein expression, including methods based on Western blot, immunofluorescence, flow cytometry, protein mass spectrometry.
  • “Adherent” refers to the attachment of cells to a vessel, eg, to a sterile plastic (or coated plastic) cell culture dish or flask, in the presence of an appropriate medium. Certain types of cells cannot be maintained or grow in culture unless they adhere to the cell culture vessel. Certain classes of cells (“non-adherent cells”) are maintained and/or proliferated in culture without attachment.
  • Cell culture refers to the maintenance, growth and/or differentiation of cells in an in vitro setting.
  • Cell culture medium refers to nutritional compositions for growing cell cultures.
  • “Culture” or “cell culture” refers to a cultured substance, such as a cell, and/or a medium in which a cultured substance, such as a cell, is present.
  • “Cultivate” refers to the maintenance, propagation (growth) (expansion) and/or differentiation of cells outside a tissue or organism, eg, in sterile plastic (or coated plastic) cell culture dishes or flasks. “Cultivation” may utilize a culture medium as a source of nutrients, hormones, and/or other factors that help to propagate and/or maintain cells.
  • dissociated cells refer to cells that have been substantially separated or purified from other cells or surfaces (eg, the surface of a culture plate).
  • cells can be dissociated from animals or tissues by mechanical or enzymatic methods.
  • cells aggregated in vitro can be dissociated from each other enzymatically or mechanically, for example by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters.
  • adherent cells are dissociated from a culture plate or other surface. Dissociation may thus involve disrupting the interaction of cells with the extracellular matrix (ECM) and substrate (eg, culture surface), or disrupting the ECM between cells.
  • ECM extracellular matrix
  • substrate eg, culture surface
  • the term “enrich” refers to increasing the amount of a specified component in a composition, such as a composition of cells, and "enriched" when used to describe a composition of cells, such as a population of cells, is refers to a population of cells that has a proportionally increased amount of a specified component compared to the proportion of such component in the population of cells prior to enrichment.
  • a composition such as a cell population
  • can be enriched for a target cell type i.e., cells with a specified characteristic
  • target cell type i.e., cells with a specified characteristic
  • Cell populations can be enriched for target cell types by cell selection and sorting methods known in the art.
  • the population of cells is enriched by sorting or selection methods.
  • the method of enriching the target cell population results in the cell population being enriched for the target cell population
  • a cell population is at least about 20% enriched, thereby meaning that the enriched cell population contains proportionally about 20% more target cell types than in the cell population before the cell population was enriched.
  • the method of enriching the target cell population proportionally enriches the cell population by at least about 30+%, 40+%, 50+%, 60+%, 70+%, 80% relative to the target cell population %, 85%, 90%, 95%, 97%, 98%, or 99%, or at least about 98%, or in particular embodiments, about 99%.
  • Genomic stability refers to the ability of cells to faithfully replicate DNA and maintain the integrity of the DNA replication process.
  • the terms “genomically stable cells” and “cells with genomic stability” refer to cells that exhibit a certain frequency of mutations and chromosomal abnormalities such as translocations, aneuploidies, copy number variations, and duplications , said frequency being substantially similar to the frequency of mutations and chromosomal abnormalities relative to normal human cells.
  • “Ingredient” means any compound or other material, whether chemical or biological in origin, that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation.
  • component means any compound or other material, whether chemical or biological in origin, that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation.
  • component means any compound or other material, whether chemical or biological in origin, that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation.
  • component nutrient
  • “ingredient” are used interchangeably.
  • Conventional ingredients for cell culture media may include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like.
  • Other components that promote and/or maintain ex vivo or in vitro cell culture can be selected by those of ordinary skill in the art as needed for the desired effect.
  • Isolate means to separate and collect a composition or material from its natural environment, eg, separation of individual cells or cell cultures from a tissue or body.
  • a cell population or composition is substantially free of cells and materials with which it is associated in nature.
  • isolated or purified or “substantially pure” with respect to a target cell population means at least about 50%, at least about 75%, at least about 85% of the target cells making up the total cell population , at least about 90%, and in particular embodiments, at least about 95% pure cell population.
  • the purity of a cell population or composition can be assessed by appropriate methods well known in the art.
  • a substantially pure population of liver cells refers to at least about 50%, at least about 75%, at least about 85%, at least about 90%, and in particular embodiments, at least about A population of cells that is about 95%, and in certain embodiments, about 98% pure.
  • Passage refers to the act of subdividing and plating cells onto multiple cell culture surfaces or vessels when the cells have proliferated to the desired extent. In some embodiments, “passaging” refers to subdividing, diluting, and plating cells. When cells are transferred from primary culture surfaces or containers Subsequent cultures may be referred to herein as “subcultures” or “first passages” or the like when passaged to subsequent sets of surfaces or containers. Each subdivision and plating into a new culture vessel is considered a passage.
  • Platinum refers to placing one or more cells into a culture vessel such that the cells adhere to and spread on the cell culture vessel.
  • Proliferation refers to the property of a cell dividing into two substantially equivalent cells or a population of cells increasing in number (eg, to replicate).
  • Propagation or “expansion” refers to growing (eg, replicating via cell proliferation) cells of a tissue or outside an organism, eg, in sterile containers, eg, plastic (or coated plastic) cell culture dishes or flasks.
  • iPSC induced pluripotent stem cell
  • Cells may be isolated by disintegration of an appropriate organ or tissue serving as the source of the cells using techniques known to those skilled in the art.
  • a tissue or organ can be mechanically disintegrated and/or treated with digestive enzymes and/or chelating agents that weaken connections between adjacent cells so that the tissue can be dispersed to form a suspension of individual cells without perceptible cell damage .
  • Mechanical disruption can also be accomplished by a variety of methods including, but not limited to, the use of pulverizers, mixers, sieves, homogenizers, pressure cells, or insonators.
  • Substantially purified populations of liver cells can be obtained by extraction from culture sources (eg, by density gradient centrifugation and/or flow cytometry). Purity can be measured by any suitable method.
  • liver cells can be 99%-100% purified by flow cytometry (eg, FACS analysis).
  • flow cytometry eg, FACS analysis
  • a molecule e.g., an antibody, antibody derivative, ligand, or Fc-peptide fusion molecule
  • positively selecting i.e., positively selects
  • Other examples of positive selection methods include methods that preferentially promote the growth of desired cell types in a mixed population of desired and undesired cell types.
  • undesired cells containing such a marker can be removed from desired cells (ie, negative selection).
  • Other negative selection methods include preferential killing or inhibiting the growth of undesired cell types in a mixed population of desired and undesired cell types.
  • Processes for isolation may include magnetic separation, use of antibody-coated magnetic beads, affinity chromatography, cytotoxic agents linked to monoclonal antibodies, or such agents used in conjunction with monoclonal antibodies, such as complement and cytotoxins , and "panning" using antibodies attached to a solid substrate (eg, a plate), or other convenient techniques.
  • Technologies that provide accurate separation include fluorescence-activated cell sorters, which can have varying degrees of complexity, such as multiple color channels, low- and obtuse-angle light-scattering detection channels, and impedance channels.
  • Antibodies can be associated with labels such as magnetic beads that allow for direct isolation, biotin that can be removed using support-bound avidin or streptavidin, or fluorescent light that can be used with fluorescence-activated cell sorters. Dye-conjugated to allow easy isolation of specific cell types. Any technique that is not unduly detrimental to the viability of the induced pluripotent stem cells can be employed.
  • a given culture condition can cause an increase or decrease in the transcription or protein expression of a marker by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000% or more increase or decrease.
  • the transcription of the marker gene can be determined by RT-qPCR (Reverse transcription Quantitative polymerase chain reaction) method.
  • RT-qPCR Reverse transcription Quantitative polymerase chain reaction
  • the transcription of marker genes is detected using the methods disclosed herein.
  • the protein expression of the marker gene can be detected by Western blot, immunofluorescence, flow detected by cytometry method. In some embodiments, the protein expression of the marker gene can be detected by immunofluorescence.
  • the "typical morphological characteristics of primary liver cells” mentioned in this article means that the cells have the unique polygonal shape of primary liver cells, and the cells are relatively three-dimensional, and the nuclei are clear, and some cells are binucleated, and the cell diameter is generally about 15-25 ⁇ M. Preferably about 20 ⁇ M.
  • the "albumin secretion ability” mentioned herein refers to the secretion amount of albumin per million cells per 24 hours, which can be measured by any conventional method in the art.
  • the assay method is an enzyme-linked immunosorbent assay method.
  • the albumin secretory capacity is determined by the methods disclosed herein.
  • urea synthesis capacity refers to the relative amount of urea synthesized per million cells per 24 hours, which can be determined by any conventional method in the art. In some embodiments, the urea synthesis capacity is determined by the methods disclosed herein.
  • the "activities of CYP metabolizing enzymes" described herein refer to the activities of CYP metabolizing enzymes in cells that can be measured by any conventional method in the art.
  • the CYP metabolic enzymes refer to members of the cytochrome P450 enzyme system family, including but not limited to CYP1A2, CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP3A4, and CYP7A1.
  • the activity of the CYP metabolizing enzyme is determined by the methods disclosed herein.
  • liver polarity is mainly reflected in the morphological structure and function of liver cells. From a morphological point of view, the liver cell membrane is divided into three different regions, namely the basement membrane, the lateral membrane, and the bile canalicular region; from a functional point of view, the basement membrane is connected with the hepatic sinusoids and can directly exchange substances, and the lateral membrane mainly uses For the connection of adjacent cells, the bile canaliculus region is mainly used for bile duct secretion (Treyer, 2013). Liver cell polarity can be determined by any method routine in the art.
  • the assay method is a cell staining method
  • the dyes used for cell staining include but are not limited to using CDFDA (5-(and-6)-carboxy-2',7'-dichlorofluorescein ( CDFDA)).
  • CDFDA 5-(and-6)-carboxy-2',7'-dichlorofluorescein
  • the "liver polarity", “cell polarity” or “liver cell polarity” is determined by the methods disclosed herein.
  • Transplantation survival refers to the transplantation of liver cells (such as induced mature liver cells, primary liver cells or induced immature liver cells) to liver injury model.
  • the survival rate of the model organisms after typing the survival rate refers to the ratio of the number of surviving model organisms to the model organisms at the beginning of the experiment after a certain period of time. It can be determined by any method routine in the art.
  • the model organism is a mouse.
  • the mouse model is a FRG mouse model (such as Grompe M, Strom S. Mice with human livers. Gastroenterology. 2013 Dec; 145(6):1209-14.doi:10.1053/j.gastro .2013.09.009. Epub 2013 Sep 13. PMID: 24042096).
  • the survival rate is determined by the methods disclosed herein.
  • the "in vivo reconstitution rate" as described herein refers to the liver reconstitution rate of a model organism after liver cells (such as induced mature liver cells, primary liver cells or induced immature liver cells) are transplanted into a liver injury model, which refers to the percentage of the number of cells derived from transplanted cells to the number of all cells in the tissue, which can be determined by any conventional method in the art.
  • the model organism is a mouse.
  • the assay method includes but not limited to assay by means of immunofluorescence staining and immunohistochemistry.
  • the in vivo reconstitution rate is determined by the methods disclosed herein.
  • the "intrahepatic migration ability" as described herein means that the liver cells transplanted through the hepatic portal vein can migrate in the liver parenchyma region, which can be determined by any conventional method in the art.
  • the assay is immunohistochemistry or immunohistofluorescence.
  • the "intrahepatic migration capacity" is determined by the methods disclosed herein.
  • the "regenerative ability of mature liver cells” mentioned in this article means that after transplantation of mature liver cells, the transplanted cells can migrate in the liver parenchyma; Multiple hepatic clusters are formed; and these hepatic cell clusters can clearly and highly express a series of marker genes unique to mature liver cells.
  • human albumin signal refers to the amount of human albumin expressed by human liver cells (primary liver cells, induced immature liver cells, induced mature liver) transplanted into mice. .
  • the assay method is an enzyme-linked immunosorbent assay.
  • the "human albumin signal” is determined by the methods disclosed herein.
  • the "peak time of human albumin” as described herein refers to the time when the amount of human albumin in mouse blood reaches the minimum limit (300ng/ml) detected by enzyme-linked immunosorbent assay (ELISA) after dilution at a ratio of 1:100.
  • the required time can be determined by any conventional method in the art.
  • the assay method is an enzyme-linked immunosorbent assay.
  • the "peak time of human albumin” is determined by the method disclosed herein.
  • the "cell proliferation rate” as described herein refers to the number of transplanted cells increasing in a mouse within a certain time range, which can be measured by any conventional method in the art.
  • the assay is ELISA, immunohistochemistry, and immunohistofluorescence.
  • the "cell proliferation rate” is determined by the methods disclosed herein.
  • Proliferating cell quality refers to the ability to express key liver cells in cells after proliferation in mice. It can be determined by any method routine in the art. In some embodiments, the assay method is ELISA, immunohistofluorescence, or immunohistochemistry. In some embodiments, the "cell proliferation rate" is determined by the methods disclosed herein.
  • the "source cells” mentioned herein refer to cells that can be used to prepare induced immature liver cells.
  • the source cells include pluripotent stem cells, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), mesenchymal stem cells, fibroblasts, adipose-derived stem cells (ADSC), neurogenic stem cells, blood cells, spinous process cells , intestinal epithelial cells, and other somatic cells other than liver cells.
  • the source cells are pluripotent stem cells (such as: embryonic stem cells, induced pluripotent stem cells). More preferably, in some embodiments, the source cells are embryonic cells or induced pluripotent stem cells.
  • Starting cells suitable for particular embodiments may be derived from essentially any suitable source, heterogeneous or homogeneous with respect to cell type or differentiation state.
  • suitable source cells may be mammalian in origin, for example Such as from rodents, cats, dogs, pigs, goats, sheep, horses, cattle, or primates such as humans.
  • the starting cells are human cells.
  • induced immature liver cell refers to an induced immature liver cell which does not occur naturally.
  • the induced immature liver cells in terms of morphology, although the induced immature liver cells have a polygonal shape characteristic of primary liver cells, compared with primary liver cells, the induced immature liver cells are more adherent under in vitro culture conditions.
  • the walls are not three-dimensional, and there are basically no clear nuclei in the cells. In the cells with partial nuclei, the nucleoplasmic ratio is relatively high.
  • the transcriptional or protein expression of one or more liver cell markers selected from the group consisting of CYP1A2, CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP3A4, CYP7A1 of the immature liver cells is induced in primary liver cells. 1%, 0.1%, 0.01%, 0.001% or less of cells, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the induced mature liver cells have decreased transcription or protein expression of the following marker: AFP relative to the induced immature liver cells.
  • transcriptional or protein expression of the following markers in induced mature liver cells: AFP is 100-fold, 1000-fold, 1000-fold or more that of primary liver cells, or any of the foregoing values as endpoints constitutes a range or any value in it.
  • the induced immature liver cells do not have the cell polarity of primary liver cells.
  • the urea synthesis capacity of the induced immature liver cells is 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of that of the primary liver cells, or any of the foregoing values A range or any value within it as endpoints.
  • the activity of one or more of the following metabolic enzymes of the induced immature liver cells is 10%, 5% of the primary liver cells , 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001% or less, or any of the foregoing values as a range constituted by the endpoints or any value therein.
  • the induced immature liver cells secrete albumin at most about 8 ⁇ g per million cells per 24 hours, more preferably about 7 ⁇ g, 6 ⁇ g, 5 ⁇ g, 4 ⁇ g, 3 ⁇ g, 2 ⁇ g, 1 ⁇ g, 0.5 ⁇ g, 0.2 ⁇ g, 0.1 ⁇ g, 0.05 ⁇ g, 0.02 ⁇ g, 0.01 ⁇ g, 0.001 ⁇ g, 0.0001 ⁇ g or less, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the transplant survival rate of the induced immature liver cells is 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.01% or less than that of primary liver cells, or Any of the foregoing values as a range consisting of endpoints or any value therein.
  • the in vivo reconstitution rate of induced immature liver cells is 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.01% or less, or any of the foregoing values, of primary liver cells A range or any value within it as endpoints.
  • the induced immature liver cells can be prepared from starting cells. In some embodiments, induced immature liver cells can be used to make induced mature liver cells.
  • Induced immature liver cells can be prepared by conventional methods known in the art.
  • the method for preparing the induced immature liver cells is as described in (Zhu et al., 2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93-97.10.1038/nature13020.), The entire contents of which are incorporated herein by reference in their entirety.
  • the induced immature liver cells can be prepared by the methods disclosed in the present invention.
  • the cells produced by conventional methods known in the art for producing induced immature liver cells are induced immature liver cells.
  • induced immature liver cells are also referred to as "f-iHep” or “iHep” in the art.
  • Induced immature hepatocytes are also referred to as “induced hepatocytes” in some prior art.
  • Induced immature liver cells suitable for use in certain embodiments may be derived from essentially any suitable source, heterogeneous or homogeneous with respect to cell type or differentiation state.
  • suitable said induced immature liver cells may be mammalian in origin, such as from rodents, cats, dogs, pigs, goats, Sheep, horses, cattle, or primates such as humans.
  • the induced immature liver cells are human cells.
  • induced mature liver cells refers to induced mature liver cells. It is non-naturally occurring. In some embodiments, induced mature liver cells can be produced by the methods or culture platforms described herein.
  • the mature hepatocytes induce typical morphological characteristics of primary liver cells.
  • the induced mature liver cells have increased transcriptional and/or protein expression of one or more liver cell markers selected from the group consisting of: CYP1A2, CYP2B6, CYP2D6, CYP2C9 relative to the induced immature liver cells , CYP2C19, CYP3A4, CYP7A1; in some embodiments, the transcription or protein expression of one and/or more liver cell markers selected from the group consisting of: CYP1A2, CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP3A4, CYP7A1, 10-fold, 20-fold, 30-fold, 100-fold, 1000-fold or higher than that of induced immature liver cells, or any of the aforementioned values as the range constituted by the endpoint or any value therein.
  • liver cell markers selected from the group consisting of: CYP1A2, CYP2B6, CYP2D6, CYP2C9 relative to the induced immat
  • the induced mature liver cells have transcription or protein expression of one or more liver cell markers selected from: CYP1A2, CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP3A4, CYP7A1, are primary liver cells 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, or 150% of, or Any of the foregoing values as a range consisting of endpoints or any value therein.
  • the induced mature liver cells have decreased transcription or protein expression of the following marker: AFP relative to the induced immature liver cells.
  • the transcriptional and/or protein expression of the following markers of the induced mature liver cells: AFP is 10%, 9%, 8%, 5%, 3%, 1% of the induced immature liver cells , 0.1%, 0.01% or less, or any of the foregoing values as a range constituting the endpoints or any value therein.
  • the transcriptional and/or protein expression of the following markers in mature liver cells is induced: AFP, which is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 160%, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the induced mature liver cells have cell polarity relative to the induced immature liver cells. In some embodiments, the cell polarity of the induced mature liver cells is close to, not lower than, or the same as that of the primary liver cells.
  • the urea synthesis ability of induced mature liver cells is 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.2 times, 3.4 times, 3.6 times, 3.8 times, 4.0 times, 4.2 times times, 4.4 times, 4.6 times, 4.8 times, 5.0 times, 5.2 times, 5.4 times, 5.6 times, 5.8 times, 6.0 times or more, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the urea synthesis ability of induced mature liver cells is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105% of primary liver cells , 110%, 115%, 120%, 130%, 140%, 150%, 160%, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the activity of one or more of the following metabolic enzymes of induced mature liver cells is 10 times, 100 times that of induced immature liver cells times, 1000 times, 5000 times, 10000 times, 50000 times, 100000 times, 500000 times or more, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the activity of one or more of the following metabolic enzymes in induced mature liver cells is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 160%, or any of the preceding values as A range of endpoints or any value within it.
  • the induced mature liver cells secrete at least about 8 ⁇ g of albumin per million cells per 24 hours, more preferably at least about 9 ⁇ g, 10 ⁇ g, 11 ⁇ g, 12 ⁇ g, 13 ⁇ g, 14 ⁇ g, 15 ⁇ g, 16 ⁇ g, 17 ⁇ g, 18 ⁇ g, 19 ⁇ g, 20 ⁇ g, 21 ⁇ g, 22 ⁇ g, 23 ⁇ g, 24 ⁇ g, 25 ⁇ g, 26 ⁇ g or more, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the transplantation survival rate of induced mature liver cells is 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times , 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, 40 times, 41 times, 42 times, 43 times, 44 times times, 45 times, 46 times, 47 times, 48 times, 49 times, 50 times, 51 times, 52 times, 53 times, 54 times, 55 times, 56 times, 57 times, 58 times, 59 times, 60 times, 61 times, 62 times, 63 times, 64 times, 65 times, 66 times, 67 times, 68 times, 69 times, 70 times, 71 times, 72 times, 73 times, 74 times, 75 times,
  • the transplant survival rate of induced mature liver cells is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105% of primary liver cells , 110%, 115%, 120%, 130%, 140%, 150%, 160%, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the in vivo reconstitution rate of induced mature liver cells is 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times , 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, 40 times, 41 times, 42 times, 43 times, 44 times times, 45 times, 46 times, 47 times, 48 times, 49 times, 50 times, 51 times, 52 times, 53 times, 54 times, 55 times, 56 times, 57 times, 58 times, 59 times, 60 times, 61 times, 62 times, 63 times, 64 times, 65 times, 66 times, 67 times, 68 times, 69 times, 70 times, 71 times, 72 times, 73 times, 74 times, 75
  • the in vivo reconstitution rate of induced mature liver cells is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85% of primary liver cells , 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 160%, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • induced mature liver cells according to the present invention may be characterized by one or more of the above characteristics. Such characterizations can be performed using any of the methods described herein or known to those skilled in the art. In some embodiments, such characterizations are performed using the methods described herein. In some embodiments, such characterizations are performed using the methods described in the Examples herein.
  • transcriptome sequencing can also be used to analyze the changes in the transcript levels of induced immature liver cells obtained compared with induced immature liver cells. This could include analysis of the enrichment of mature hepatocyte marker genes in induced immature and induced mature hepatocytes (GSEA analysis). After analysis, it is found that the specific expression of mature liver cell marker genes can be significantly enriched in the induced mature liver cells according to the present invention.
  • Cluster analysis can be used to analyze the similarity of the entire transcriptome level between the initially used induced immature liver cells and the induced mature liver cells according to the present invention and primary liver cells.
  • PCA principal component analysis
  • the primary liver cell marker gene set can be significantly enriched in the induced mature liver cells according to the present invention.
  • the obtained induced mature hepatocytes can be observed using a microscope, whereby the morphology of the induced mature hepatocytes can be judged. It can be found that the morphology of the induced mature liver cells obtained according to the present invention is consistent with that of the primary liver cells, the polarity between the cells is clear and the nucleus-to-cytoplasm ratio is obviously reduced, and some cells show a binuclear morphology.
  • one or more cells can be cultured, dissociated and passaged using the compositions and methods contemplated herein.
  • single cells are cultured, dissociated and passaged using the compositions and methods contemplated herein.
  • a cell population or plurality of cells is cultured, dissociated, and passaged using the compositions and methods contemplated herein.
  • the present invention provides a culture platform that can be used to induce the production of induced mature liver cells, which adopts a specific composition.
  • compositions, kit, medium, or culture of the invention can vary and can be optimized for specific culture conditions, including the specific molecules and combinations used, the type of cells being cultured in the medium, and application.
  • the present invention provides a first composition comprising: (a) HDAC inhibitor, (b) ERK signaling pathway inhibitor.
  • the first composition further comprises (c) a steroid compound, (d) a Notch signaling pathway inhibitor, and/or (e) a TGF-beta signaling pathway inhibitor.
  • the first composition further comprises: (f) a p38 MAPK inhibitor.
  • the present invention provides a first kit comprising: (a) HDAC inhibitor, (b) ERK signaling pathway inhibitor, and corresponding instructions for use.
  • the first kit further comprises (c) steroid compounds, (d) Notch signaling pathway inhibitors, and/or (e) TGF-beta signaling pathway inhibitors.
  • the first kit further comprises: (f) a p38 MAPK inhibitor.
  • the present invention provides a second composition comprising: (a) a cAMP activator, (b) a steroid compound.
  • the second composition further comprises (c) ROCK inhibitor and/or (d) TGF-beta signaling pathway inhibitor.
  • the present invention provides a second kit, (a) cAMP activator, (b) steroid compound, and corresponding instructions for use.
  • the second kit further comprises (c) ROCK inhibitor and/or (d) TGF-beta signaling pathway inhibitor.
  • HDAC (histone deacetylase) inhibitors may be various substances capable of inhibiting HDAC.
  • HDAC inhibitors include, but are not limited to, Mocetinostat (MGCD0103), Abexinostat (PCI-24781), Fimepinostat (CUDC-907), ITSA-1, Vorinostat (SAHA), MS-275, Panobinostat (LBH589), Quisinostat, FK228, SB939, Trichostatin A (TSA).
  • the HDAC inhibitor is MS-275, shown in the formula
  • Steroid compounds refer to various substances derived from steroid compounds with tetracyclic structure.
  • Steroid compounds include, but are not limited to, prednisone, methylprednisone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone.
  • the steroid compound is dexamethasone, as shown in the following formula
  • Notch signaling pathway inhibitors may be various substances capable of inhibiting Notch signaling pathway.
  • Notch signaling pathway inhibitors include, but are not limited to, IMR-1, RO4929097, Semagacestat (LY450139), Avagacestat (BMS-708163), Dibenzazepine (YO-01027), Crenigacestat (LY3039478), Compound E, DAPT.
  • the Notch signaling pathway inhibitor, Compound E is shown in the following formula
  • the ERK signaling pathway inhibitor may be various substances capable of inhibiting the ERK signaling pathway.
  • ERK signaling pathway inhibitors include, but are not limited to, SeluMetinib (AZD6244), Trametinib (GSK1120212), PD0325901, U0126-ETOH, PD184352 (CI-1040).
  • the ERK signaling pathway inhibitor is PD0325901, as shown in the following formula
  • TGF-beta signaling pathway inhibitors may be various substances capable of inhibiting ALK.
  • TGF-beta signaling pathway inhibitors include, but are not limited to, SD-208, LY2109761, BIBF-0775, A83-01, SB431542.
  • the TGF-beta signaling pathway inhibitor is A83-01, as shown in the following formula
  • the p38 MAPK inhibitor may be various substances capable of inhibiting p38 MAPK.
  • p38 MAPK inhibitors include, but are not limited to, SB202190 (FHPI), SB203580, TAK-715, Doramapimod (BIRB 796).
  • the p38 MAPK inhibitor is SB203580, as shown in the following formula
  • the cAMP activator can be various substances capable of activating cAMP.
  • Exemplary cAMP activators include, but are not limited to, Forskolin, 8-Br-cAMP.
  • the cAMP activator is Forskolin, as shown in the following formula
  • cAMP activators can be various substances capable of inhibiting ROCK.
  • exemplary ROCK inhibitors include, but are not limited to, Y-27632, Thiazovivin, GSK429286A, RKI-1447, AT13148.
  • the ROCK inhibitor is Y-27632, as shown in the following formula
  • the HDAC inhibitor is present in a composition, kit, medium or culture of the invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature hepatocytes.
  • the HDAC inhibitor is dosed at 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 ⁇ M or higher or the concentration of any two of the foregoing values in the range of composition exists in the present invention
  • the HDAC inhibitor is present in the first medium or culture of the invention at a concentration of 0.01-1 ⁇ M, preferably 0.1-1 ⁇ M, more preferably 1.0 ⁇ M.
  • the HDAC inhibitor is MS-275, which is present in the first medium or culture of the invention at a concentration of 0.2 ⁇ .
  • the steroid compound is present in the composition, kit, medium or culture of the present invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature liver cells.
  • the steroid compound is 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 ⁇ M, or a range of any two of the foregoing values are present in the first medium or culture of the present invention .
  • the steroid compound is present in the first medium or culture of the present invention at a concentration of 0.1-4.0 ⁇ M, preferably 0.2-2.0 ⁇ M, more preferably 0.1 ⁇ M.
  • the steroid compound is dexamethasone, which is present in the first medium or culture of the invention at a concentration of 0.1 ⁇ .
  • the Notch signaling pathway inhibitor is present in the composition, kit, medium or culture of the present invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature liver cells.
  • the Notch signaling pathway inhibitor is dosed at 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 ⁇ M or higher or any two of the above values is present in the first medium or culture of the invention.
  • the Notch signaling pathway inhibitor is present in the first medium or culture of the present invention at a concentration of 0.1-4.0 ⁇ M, preferably 0.2-2.0 ⁇ M, more preferably 0.1 ⁇ M.
  • the Notch signaling pathway inhibitor is Compound E, which is present in the first medium or culture of the present invention at a concentration of 0.1 ⁇ M.
  • the ERK signaling pathway inhibitor is present in the composition, kit, medium or culture of the present invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature liver cells.
  • the ERK signaling pathway inhibitor is dosed at 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 ⁇ M or higher or the concentration of any two of the foregoing values in the range of composition exists in the second aspect
  • the ERK signaling pathway inhibitor is present in the first medium or culture of the present invention at a concentration of 0.05-5 ⁇ M, preferably 0.1-2 ⁇ M, more preferably 1.0 ⁇ M.
  • the ERK signaling pathway inhibitor is PD0325901, which is present in the first medium or culture of the present invention at a concentration of 1.0 ⁇ M.
  • the TGF-beta signaling pathway inhibitor is present in the composition, kit, medium or culture of the present invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature liver cells.
  • the TGF-beta signaling pathway inhibitor is dosed at 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 ⁇ M or higher or the concentration of any two of the foregoing values in the range of composition
  • the TGF-beta signaling pathway inhibitor is present in the first medium or culture of the present invention at a concentration of 0.05-5 ⁇ M, preferably 0.1-2 ⁇ M, more preferably 0.5 ⁇ M.
  • the TGF-beta signaling pathway inhibitor is A83-01, which is present in the first medium or culture of the present invention at a concentration of 0.5 ⁇ M.
  • the p38 MAPK inhibitor is present in a composition, kit, medium or culture of the invention in an amount or concentration sufficient, alone or in combination with other substances, to induce the production of induced mature hepatocytes.
  • the p38 MAPK inhibitor is dosed at 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.0, 14.0, 16.0 , 18.0, 20.0, 30.0, 40.0, 50.0 ⁇ M or any two of the foregoing values are present in the composition, kit, medium or culture of the present invention.
  • the p38 MAPK inhibitor is present in the first medium or culture of the invention at a concentration of 1.0-50.0 ⁇ M, preferably 2.0-40.0 ⁇ M, more preferably 30.0 ⁇ M.
  • said p38 MAPK inhibitor is SB203580, which is present in the first medium or culture of the invention at a concentration of 30 ⁇ .
  • the HDAC inhibitor is MS-275
  • the steroid compound is dexamethasone
  • the Notch signaling pathway inhibitor is Compound E
  • the ERK signaling pathway inhibitors are PD0325901
  • the TGF-beta signaling pathway inhibitor is A83-01.
  • the concentration of MS-275 in the first medium or culture of the present invention is 0.2 ⁇ M
  • the concentration of dexamethasone in the first medium or culture of the present invention is 0.1 ⁇ M
  • the concentration of Compound E in the first medium or culture of the present invention is 0.1 ⁇ M
  • the concentration of PD0325901 in the first medium or culture of the present invention is 1.0 ⁇ M
  • A83-01 is in the first medium or culture of the present invention
  • the concentration in medium or culture is 0.5 ⁇ M.
  • the first composition further comprises a p38 MAPK inhibitor.
  • the HDAC inhibitor is MS-275
  • the steroid compound is dexamethasone
  • the Notch signaling pathway inhibitor is Compound E
  • the ERK signaling pathway inhibitor is PD0325901
  • the TGF-beta signaling pathway inhibitor is A83-01
  • the p38 MAPK inhibitor is SB203580.
  • the concentration of MS-275 in the first medium or culture of the present invention is 0.2 ⁇ M
  • the concentration of dexamethasone in the first medium or culture of the present invention is 0.1 ⁇ M
  • the concentration of Compound E in the first medium or culture of the present invention is 0.1 ⁇ M
  • the concentration of PD0325901 in the first medium or culture of the present invention is 1.0 ⁇ M
  • A83-01 is in the first medium or culture of the present invention
  • the concentration in the medium or culture is 0.5 ⁇ M
  • the concentration of SB203580 in the first medium or culture of the present invention is 30.0 ⁇ M.
  • the cAMP activator is present in a composition, kit, medium or culture of the invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature liver cells.
  • the cAMP activator is dosed at 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0 ⁇ M or higher or a concentration of any two of the foregoing values exists in the second culture of the present invention.
  • the cAMP activator is present in the second medium or culture of the invention at a concentration of 1.0-50.0 ⁇ M, preferably 10.0-40.0 ⁇ M, more preferably 30.0 ⁇ M.
  • the cAMP activator is Forskolin, which is present in the second medium or culture of the invention at a concentration of 30.0 ⁇ .
  • the ROCK inhibitor is present in a composition, kit, medium or culture of the invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature hepatocytes.
  • the ROCK inhibitor is dosed at 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.0, 14.0, 16.0, A concentration of 18.0, 20.0, 30.0, 40.0, 50.0 ⁇ M or higher or a range consisting of any two of the foregoing values is present in the second medium or culture of the present invention.
  • said ROCK inhibitor is present in the second medium or culture of the invention at a concentration of 1.0-50.0 ⁇ M, preferably 10.0-40.0 ⁇ M, more preferably 10.0 ⁇ M.
  • the ROCK inhibitor is Y-27632, which is present in the second medium or culture of the invention at a concentration of 10.0 ⁇ .
  • the TGF-beta signaling pathway inhibitor is present in the composition, kit, medium or culture of the present invention in an amount or concentration sufficient to induce the production of induced mature liver cells alone or in combination with other substances .
  • the TGF-beta signaling pathway inhibitor is dosed at 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 ⁇ M or higher or the concentration of any two of the foregoing values in the range of
  • the TGF-beta signaling pathway inhibitor is present in the second medium or culture of the present invention at a concentration of 0.05-5.0 ⁇ M, preferably 0.1-2.0 ⁇ M, more preferably 0.5 ⁇ M.
  • the TGF-beta signaling pathway inhibitor is A83-01, which is present in the second medium or culture of the present invention at a concentration of 0.5 ⁇ M.
  • the steroid compound is present in the combinations of the invention in an amount or concentration sufficient alone or in combination with other substances to induce the production of induced mature liver cells substances, kits, media or cultures.
  • the steroid compound is 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 ⁇ M or the concentration of any two of the foregoing values in the range of composition exists in this In the second medium or culture of the invention.
  • the steroid compound is present in the second medium or culture of the present invention at a concentration of 0.1-4.0 ⁇ M, preferably 0.2-2.0 ⁇ M, more preferably 0.1 ⁇ M.
  • the steroid compound is dexamethasone, which is present in the second medium or culture of the invention at a concentration of 0.1 ⁇ .
  • HDAC inhibitors facilitate the generation of induced mature liver cells.
  • the HDAC inhibitor is MS-275.
  • the cAMP activator is Forskolin
  • the ROCK inhibitor is Y-27632
  • the TGF-beta signaling pathway inhibitor is A83-01
  • the The steroid compound is dexamethasone. More preferably, the concentration of Forskolin in the second medium or culture of the present invention is 30.0 ⁇ M, the concentration of Y-27632 in the second medium or culture of the present invention is 10.0 ⁇ M, A83-01 in the present invention
  • the concentration of dexamethasone in the second medium or culture of the present invention is 0.5 ⁇ M, and the concentration of dexamethasone in the second medium or culture of the present invention is 0.1 ⁇ M.
  • the invention provides a first culture medium. In one aspect, the invention provides a second culture medium. In some aspects, the first medium and the second medium described herein can be used to prepare the induced mature liver cells described herein.
  • media of the invention comprise basal media.
  • basal media include, but are not limited to, DMEM, Knockout DMEM, RPMI 1640, DMEM/F12, HCM, and William'E.
  • the basal medium is selected from DMEM, Knockout DMEM, RPMI 1640, DMEM/F12, HCM, and William'E.
  • the basal medium can be purchased commercially.
  • the present invention provides a kit comprising HEM-1 medium and HEM-2 medium, wherein the HEM-1 medium comprises ROCK inhibitor, hepatocyte growth factor (HGF), tumor suppressor Oncostatin M (OSM), steroid compounds, Notch signaling pathway inhibitors, TGF-beta signaling pathway inhibitors and HCM medium without epidermal growth factor (EGF); the HEM-2 medium contains liver cell growth factor (HGF), oncostatin M (oncostatinM, OSM), steroid compounds, Notch signaling pathway inhibitors, TGF-beta signaling pathway inhibitors and removal of epidermal growth factor (EGF) HCM medium.
  • HGF hepatocyte growth factor
  • OSM tumor suppressor Oncostatin M
  • steroid compounds Notch signaling pathway inhibitors
  • TGF-beta signaling pathway inhibitors HCM medium without epidermal growth factor (EGF)
  • HGF liver cell growth factor
  • OSM oncostatinM
  • Notch signaling pathway inhibitors TGF-beta signal
  • the ROCK inhibitor is Y-27632; the steroid compound is dexamethasone; the Notch signaling pathway inhibitor is Compound E; and the TGF-beta signaling pathway inhibitor is A83-01.
  • the ROCK inhibitor is present in HEM-1 at 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ⁇ M, or a range consisting of any of the foregoing values as endpoints or any value therein medium. More preferably, in some embodiments, the ROCK inhibitor is Y-27632 and is present in HEM-1 medium at 10.0 ⁇ .
  • the hepatocyte growth factor takes 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35 ng/ml, or any of the aforementioned values as the endpoints of the range or range thereof Arbitrary value, present in HEM-1 medium or HEM-2 medium. More preferably, in some embodiments, the hepatocyte growth factor is present in HEM-1 medium or HEM-2 medium at 20 ng/ml.
  • Oncostatin M takes 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35 ng/ml, or any of the aforementioned values as the endpoints of the range or range thereof Arbitrary value, present in HEM-1 medium or HEM-2 medium.
  • the Oncostatin M is present in HEM-1 medium or HEM-2 medium at 20 ng/ml.
  • the steroid compound is present in HEM-1 with 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ⁇ M, or any of the aforementioned values as the endpoints of the range or any value therein.
  • culture medium or HEM-2 medium More preferably, in some embodiments, the steroid compound is dexamethasone, and is present in HEM-1 medium or HEM-2 medium at 0.1 ⁇ M.
  • the Notch signaling pathway inhibitor is present in the HEM at 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ⁇ M, or any of the foregoing values as endpoints of a range or any value therein -1 medium or HEM-2 medium. More preferably, in some embodiments, the Notch signaling pathway inhibitor is Compound E, and is present in HEM-1 medium or HEM-2 medium at 0.1 ⁇ M.
  • the TGF-beta signaling pathway inhibitor is present at 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ⁇ M, or any of the aforementioned values as endpoints or any value therein. in HEM-1 medium or HEM-2 medium. More preferably, in some embodiments, the TGF-beta signaling pathway inhibits The formulation was A83-01 and was present at 0.5 ⁇ M in HEM-1 medium or HEM-2 medium.
  • the medium as described herein comprises fetal bovine serum (FBS), and the content of fetal bovine serum can be routinely adjusted according to the knowledge of those skilled in the art.
  • the content of fetal bovine serum in the second medium as described herein is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% of the total volume of the medium , 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0 %, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, or any of the aforementioned values as the range constituted by the endpoints or any value therein.
  • the content of fetal bovine serum in the second culture medium is 0.05%-10% of the total volume of the second culture medium, more preferably 1%.
  • the content of fetal bovine serum in the second culture medium is 0.05%-10% of the total volume of
  • the culture medium of the invention contains cytokines and/or growth factors. In specific embodiments, the culture medium of the invention is substantially free or free of cytokines and/or growth factors. In certain embodiments, the culture medium contains one or more supplements including, but not limited to, serum, extracts, growth factors, hormones, cytokines, and the like. In some embodiments, the medium described herein comprises: penicillin/streptomycin, non-essential amino acids, L-alanyl-L-glutamine or L-glutamine, nicotinamide, N-acetyl-L - Cysteine.
  • Any suitable vessel or cell culture vessel can be used as a support for cell culture in basal media and/or cell culture supplements.
  • a matrix coating on the support is not necessary.
  • coating the surface of a culture vessel with an attachment-promoting matrix e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.
  • an attachment-promoting matrix e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.
  • Suitable matrices for culturing and passaging cells include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, naturally occurring cell line production Mixtures of substrates such as Matrigel TM and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminated monolayers.
  • the present invention provides a method for preparing induced mature liver cells, the method comprising culturing induced immature liver cells in a culture medium, thereby preparing the induced mature liver cells.
  • the medium is the first medium and the second medium described herein.
  • the present invention provides a method for preparing induced mature liver cells, the preparation method comprising contacting the induced immature liver cells with the first composition or the second composition described herein, thereby preparing The induced mature liver cells.
  • induced immature liver cells are prepared by culturing induced immature liver cells using a first composition or a first medium described herein.
  • the culture time of using the first composition and the first medium to culture the induced immature liver cells is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or a range consisting of any of the foregoing any value in it.
  • the culture time is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days, or any of the aforementioned values as the range constituted by the endpoint or wherein any value of .
  • the culture time is 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days, or any of the aforementioned values as the range formed by the endpoint or any value therein .
  • the above-mentioned cells cultured with the first composition or the first medium are further cultured with the second composition or the second medium described herein, so as to obtain induced mature liver cells.
  • the second composition or the second medium is used for culturing, and the culturing time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 60, 90, 120 days, 150 days, 180 days, 270 days, 360 days or longer, or a range constituted by any of the foregoing values as endpoints or any value therein.
  • the culture time is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 60, 90, 120 days, 150 days, or any of the aforementioned values as the range formed by the endpoints or any value therein. More preferably, the culture time is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 60, 90 days, or any of the aforementioned values as the range formed by the endpoint or any value therein .
  • the methods of the present invention comprise culturing and inducing pluripotent stem cells in a culture medium using the methods described herein, thereby producing the induced immature liver cells.
  • induced immature liver cells which can be prepared from a variety of starting cells as described herein.
  • induced immature liver cells can be prepared from pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • the method comprises reprogramming non-pluripotent cells into pluripotent stem cells.
  • the non-pluripotent cells are selected from somatic cells and/or adult stem cells.
  • the reprogramming of non-pluripotent cells into pluripotent stem cells includes expressing one or more recombinant proteins selected from Oct4, Sox2, Klf4, and c-Myc in the non-pluripotent cells. programming factor.
  • the invention provides a method of expanding induced immature liver cells comprising contacting the induced immature liver cells described herein with HEM-1 medium or HEM-2 of the invention.
  • the induced immature liver cells are prepared by the methods described herein.
  • the HEM-1 medium of the present invention is used to culture the induced immature liver cells for 1 day, and then the HEM-2 medium is used for continuous culture for 7 days. More preferably, medium changes (ie medium refreshment) are performed every two days.
  • expansion of induced immature liver cells according to the invention provided herein allows said induced immature liver cells to maintain their differentiated state.
  • the expansion of the induced immature liver cells according to the invention provided by the present invention can make the induced immature liver cells every 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 days, or a range of any of the preceding values as endpoints, or any value therein, doubled.
  • the invention provides a culture comprising a medium as described herein and induced mature liver cells.
  • the medium comprises the first medium, the second medium described herein.
  • the cells contained in the cultures described herein can be a wide variety of cells as described herein.
  • the cells may be starting cells such as induced immature liver cells, starting cells such as pluripotent stem cells (eg embryonic stem cells, induced pluripotent stem cells) for the culture or induction described herein.
  • the cells may be intermediate or final cells cultured or induced as described herein.
  • the intermediate cells may be cells with various developmental potentials that differ from the starting and final cells.
  • the final cells may be induced mature liver cells as described herein.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
  • a culture according to the invention comprises a medium as described herein and pluripotent stem cells.
  • the induced immature liver cells are prepared from pluripotent stem cells.
  • the pluripotent stem cells are embryonic stem cells.
  • the pluripotent stem cells are induced pluripotent stem cells.
  • the induced immature liver cells can be prepared by the methods described herein.
  • the pluripotent stem cells can be obtained by methods known in the art.
  • the induced immature liver cells can be obtained by methods known in the art.
  • the induced mature liver cells according to the present invention can be used in various applications expected in scientific research, industry and clinic. For example, preparation of cells for liver cell transplantation, construction of liver-related disease models, screening and identification of drugs related to liver diseases, screening and identification of drug hepatotoxicity, in vitro construction of liver organs (including 3D printed liver organs) or other potential scientific research, treatment and diagnostic applications.
  • the induced mature liver cells according to the present invention can be used to induce the production of organoids.
  • the organoids can be used for constructing disease models, transplantation therapy or other potential scientific research, treatment and diagnosis applications.
  • the invention provides an organoid produced from the induced mature liver cells described herein.
  • the organoid may include, but is not limited to, liver.
  • the induced mature liver cells according to the present invention can be used to induce tissue (tissue), such as 3D printed liver.
  • tissue such as 3D printed liver.
  • the tissue can be used to prepare cells for liver cell transplantation, construct liver-related disease models, screen and identify drugs related to liver diseases, screen and identify drugs for liver toxicity, construct liver organs in vitro (including 3D printed liver organs) or other potential scientific research , therapeutic and diagnostic applications.
  • the invention provides a tissue produced from the induced mature liver cells described herein. Such tissues include, but are not limited to, the liver.
  • Example 1 Induction and production of inducible mature liver cells
  • D2 medium was removed and D3 medium was added, cultured in an incubator at 37°C, 5% CO 2 for 72 hours to obtain endoderm cells.
  • the D3 medium was removed, and HDM medium was added, and cultured in an incubator at 37° C. with 5% CO 2 for 96 hours, and the medium was changed once during 48 hours of culture, so as to differentiate endoderm cells into hepatic stem cells.
  • the resulting hepatic stem cells were differentiated into Cell 1 by removing the HDM medium, and adding HCM induction medium, and culturing in an incubator at 37°C with 5% CO2 for 10-14 days, during which time every 48 hours The medium was changed once, thereby obtaining Cell 1.
  • Cells 1 obtained above were digested with Accutase TM (STEMCELL) at 37° C. for 3 minutes, and then passaged (passage ratio 1:4) into the culture dish treated with Matrigel.
  • the resulting Cell 1 was cultured and expanded in an incubator at 37 °C, 5% CO2 using Medium 1 and Medium 2 as follows. Culture medium 1 was used for 1 day, and then culture medium 2 was used for 7 days, during which the medium was changed every 48 hours.
  • the HEM medium was removed, and 5C medium or 6C medium was added, and cultured in an incubator at 37° C. with 5% CO 2 for 10 to 15 days, during which the medium was changed every 48 hours, thereby obtaining cell 2.
  • results As shown in FIG. 1 , cells 2 cultured in 5C medium or 6C medium exhibited typical morphological characteristics of primary liver cells. The polarity between cells became clear, and the nucleoplasmic ratio of cells became smaller, and some cells showed binucleation.
  • cell 2 obtained by culturing the 6C medium, remove the 6C medium, and use the following 4C medium to continue culturing for 60 days in an incubator at 37°C and 5% CO 2 , during which the medium was changed every 72 hours, thus obtaining cell3.
  • Cell 3 exhibited the same morphological characteristics as primary liver cells. Compared with cell 1, the polarity between cells 3 became clearer, and the nucleoplasmic ratio of cells became smaller and smaller, and some cells showed binucleation.
  • Cell 1 in Example 1 belongs to the induced immature liver cell described in the present invention.
  • Cell 2 and Cell 3 in the example belong to the induced mature liver cells described in the present invention.
  • Medium 1 in Example 1 belongs to the HEM-1 medium described in the present invention.
  • Medium 2 belongs to the HEM-2 medium described in the present invention.
  • Both the 5C medium and the 6C medium in Example 1 belong to the first medium of the present invention, and the 4C medium belongs to the second medium of the present invention.
  • Embodiment 2 Determination of the expression of liver maturation-related marker genes in induced mature liver cells Express
  • Example 1 Collect Cell 1 (as a control), Cell 2 and Cell 3 in Example 1. And collect the cell 1 (ie cell 2 control (DMSO) and cell 3 control (DMSO)) treated in parallel with DMSO as a control, and then use the Trizol kit (Gibico) to extract total mRNA according to the method in the kit. Then, the expression of liver maturation-related genes in all collected mRNAs was detected by RT-qPCR method (Bio-rad).
  • DMSO cell 2 control
  • DMSO cell 3 control
  • the primers marked with "*” were selected from published literature (Zhu et al., 2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93-97.10.1038/nature13020.).
  • the inventors also detected the expression of maturation-related marker genes by immunofluorescence.
  • the immunofluorescence detection method used was as follows: the cells 1, 2, and 3 were washed three times with DPBS, and then 4% polymer was used to Formaldehyde (sigma, P6148) fixed the cells at room temperature for 30 minutes; removed 4% paraformaldehyde, added 0.1% Triton-100 (0.1 volume % Triton-100 dissolved in PBS) for 30 minutes at room temperature; then removed 0.1% Triton-100, and added 3.5% BSA for 2 hours; dilute the primary antibody (here, anti-albumin antibody (Bethyl, A80-129A), anti-albumin antibody (Bethyl, A80-129A) and HNF4alph antibody (Cell Signaling Technology, 3113s), anti-AFP antibody (Cell Signaling Technology, 4448), anti-CK18 antibody (Abcam, ab82254), anti-CYP1A2 antibody (BIO-RAD, AHP622
  • cells 2 and 3 can express multiple marker genes related to liver maturation.
  • Cell 1 in Example 2 belongs to the induced immature liver cell described in the present invention.
  • Cell 2 and Cell 3 in the example belong to the induced mature liver cells described in the present invention.
  • the inventor further tested the liver polarity and liver-related functions of Cell 1 (as a control), Cell 2 and Cell 3 obtained in Example 1.
  • the experimental method was as follows: Using 20 ⁇ M CDFDA (5-(and-6)- Cell 1, cell 2 and cell 3 were stained with carboxy-2',7'-dichlorofluorescein (sigma, 21884) for 30 minutes, washed 3 times with HBSS (Gibico), and then observed under a fluorescent microscope. In this way, Determine the hepatic polarity of the cells.
  • CYP450 activity detection kit (Luciferin-IPA, V8421 and V9001), according to the method described in the kit, the activities of CYP1A2 and CYP3A4 in the cells were detected.
  • ALB ELISA detection kit (E80-129; Bethyl) and urea detection kit (BioAssay Systems) were used to detect the ability of cells to secrete albumin and synthesize urea according to the method described in the kit.
  • the albumin secretion capacity of cells 2 and 3 reached 10 ⁇ g/1 million cells/24 hours and 20 ⁇ g/1 million cells/24 hours, respectively, approaching or even exceeding the albumin secretion capacity of primary liver cells (Figure 3B) .
  • the urea synthesis ability of cell 2 and cell 3 was also significantly improved, approaching the level reported for primary liver cells (Fig. 3C).
  • Cell 1 in Example 3 belongs to the induced immature liver cell described in the present invention.
  • Cell 2 and Cell 3 in the example belong to the induced mature liver cells described in the present invention.
  • Example 4 Induced mature liver cells in chronic liver injury mouse FRG model The in vivo reconstitution rate, and induced mature liver cells can effectively alleviate and improve the liver of mice Visceral damage and survival
  • the inventor also tested the cell 1', cell 2 and cell 3 obtained in Example 1 in liver injury mouse FRG (such as Grompe M, Strom S. Mice with h ⁇ Man livers. Gastroenterology. 2013 Dec; 145 (6): 1209 -14.doi:10.1053/j.gastro.2013.09.009.Epub 2013 Sep 13.PMID:24042096 described in the in vivo reconstitution rate and the effect on the improvement of the liver damage and the survival rate of the mice.
  • liver injury mouse FRG such as Grompe M, Strom S. Mice with h ⁇ Man livers. Gastroenterology. 2013 Dec; 145 (6): 1209 -14.doi:10.1053/j.gastro.2013.09.009.Epub 2013 Sep 13.PMID:24042096 described in the in vivo reconstitution rate and the effect on the improvement of the liver damage and the survival rate of the mice.
  • nitisinone (Nitisinone, selleck, S5325, CAS No. 104206-65-7) needs to be added to the daily drinking water at a concentration of 16 mg/ml.
  • Ad-uPA Yecuris, 20-0029 virus
  • the injection volume is 5*10 7 pfu of Ad-uPA per gram of mouse body weight - uPA virus.
  • mice received an injection of adult primary liver cells at a dosage of 500,000 cells per mouse. Thirteen mice received cell 1 injections, 21 mice received cell 2 injections, and 12 mice received cell 3 injections, all at 1 million cells per mouse.
  • mice After transplantation, when the mice lost 20% of their body weight, the mice were treated with NTBC for 3-4 days. Afterwards, the state of the mice was monitored, and the days of death of the mice were recorded.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • the inventors also tested the in vivo reconstruction rate of the transplanted mouse liver by means of immunohistofluorescence.
  • the experimental scheme was as follows: After embedding the tissue in paraffin, it was sliced at a thickness of 5 ⁇ m. The sliced samples were dewaxed with xylene for 3 times, 5 minutes each time; then the following reagents were used for gradient rehydration: 100% ethanol, 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, 5 minutes each.
  • the inventor also used the ALB ELISA detection kit (E80-129; Bethyl) to detect the concentration of albumin in the blood of mice on different days after transplantation according to the method described in the kit, thereby determining the peak time of human albumin .
  • Cell 1 in Example 4 belongs to the induced immature liver cells described in the present invention.
  • Cell 2 and Cell 3 in the example belong to the induced mature liver cells described in the present invention.
  • Example 5 uses HEM-1 medium and HEM-2 medium to amplify inducibility cooked liver cells
  • the inventors also tested the use of the HEM medium of the present invention to expand the cell 1 obtained in Example 1.
  • the specific amplification method is as follows: after digesting the cells on day 0, culture the cells for 24 hours using medium 1, which consists of adding 10 ⁇ M Y- 27632 (Selleck, S6390), 20ng/ml hepatocyte growth factor (HGF) (Peprotech, 100-39H), 20ng/ml oncostatin M (OSM) (Peprotech, 300-10), 0.1 ⁇ M dexamethasone (Gibco , D4902), 0.5 ⁇ M A83-01 (Tocris, 2939), 0.1 ⁇ M Compound E (Tocris, 6476/1).
  • the composition of the medium 2 was to add 20 ng/ml liver cells to the HCM medium (Lonza, 3198) in which the EGF factor was removed.
  • Growth factor (HGF) (Peprotech, 100-39H), 20ng/ml oncostatin M (OSM) (Peprotech, 300-10), 0.1 ⁇ M dexamethasone (Gibco, D4902), 0.5 ⁇ M A83-01 (Tocris, 2939), 0.1 ⁇ M Compound E (Tocris, 6476/1).
  • HGF Growth factor
  • OSM oncostatin M
  • 0.1 ⁇ M dexamethasone (Gibco, D4902)
  • 0.5 ⁇ M A83-01 (Tocris, 2939)
  • 0.1 ⁇ M Compound E (Tocris, 6476/1).
  • Medium 2 was used to change the medium every two days during amplification.
  • the mRNA in cell 1 on the 7th day (D7) and the 0th day (D0) was detected by the method described in Example 2. As shown in Figure 5B, the mRNA expression levels of liver cell-related markers were at 0 Day and the 7th day are basically the same.
  • the albumin secretion ability of cell 1 on day 7 (D7) and day 0 (D0) was detected by the method described in Example 3. As shown in FIG. 5D , the albumin secretion ability of the two cells was basically the same.
  • the urea synthesis ability of cell 1 on day 7 (D7) and day 0 (D0) was detected by the method described in Example 3. As shown in FIG. 5E , the urea synthesis ability of the two cells was basically the same.
  • the CYP3A4 activity of cell 1 on day 7 (D7) and day 0 (D0) was detected by the method described in Example 3. As shown in FIG. 5F , the CYP3A4 activity of the two was basically the same.
  • liver-related markers were still significantly expressed.
  • kit Periodic acid–Schiff Sigma-Aldrich
  • kit BODIPY493/503 Life Technologies
  • kit Oil Red O kit ICG (Aladdin) on the 7th day (D7) cells 1 Glycogen synthesis, lipid droplet synthesis, and cell absorption were tested.
  • kit ICG Alddin
  • Cell 1 in Example 5 belongs to the induced immature liver cells described in the present invention.
  • the medium 1 in Example 5 belongs to the HEM-1 medium described in the present invention, and the medium 2 belongs to the HEM-2 medium described in the present invention.
  • the inventor analyzed and verified the key components of the 6C medium in Example 1.
  • the specific method is as follows: the inventors subtracted each component in the 6C medium one by one to construct six kinds of medium. Afterwards, immature induced liver cells, cell 1, were added to these 6 kinds of medium and 6C medium respectively, and the treatment time was 2 weeks. After two weeks, cells were harvested, RNA was extracted, and the effects of each small molecule were assessed using RNA-seq experiments.
  • the removal of HDAC inhibitors and ERK signaling pathway inhibitors greatly affects the differentiation effect of cell 1 to cell 2, mainly reflected in the removal of HDAC inhibitor (MS-275) or ERK from 6C medium alone After the signaling pathway inhibitor (PD0325901), the transcriptome profile of the treated cells is far from that of cell 2, and it is basically impossible to induce the differentiation of immature liver cells into induced liver cells to a certain extent.
  • steroid compounds namely Dex
  • Notch signaling pathway inhibitor Compound E
  • TGF-beta signaling pathway inhibitor A83-01
  • p38 MAPK inhibitor SB203580
  • Cell 1 in Example 6 belongs to the induced immature liver cell described in the present invention.
  • the cell 2 in the example belongs to the induced mature liver cell described in the present invention.
  • the inventor analyzed and verified the key components of the 4C medium in Example 1.
  • the specific method is as follows: the inventors subtracted each component in the 4C medium one by one to construct four kinds of medium. Afterwards, the cells 2 induced by the 4C medium and the 4C medium were respectively added to the 6C medium, and the treatment time was 2 weeks. Two weeks later, the cells were observed under a microscope; RNA was extracted, and RT-qPCR experiments were used to test the expression levels of key genes in mature liver cells to evaluate the effect of each small molecule.
  • cAMP As shown in Figure 6B and Figure 6C, cAMP The removal of activators (Forskolin, FSK) and steroid compounds (dexamethasone, namely Dex) will greatly affect the induction effect in terms of cell morphology and the expression of key genes of mature liver cells, and it is basically impossible to obtain mature induced liver cells .
  • ROCK inhibitor Y-27632
  • TGF-beta signaling pathway inhibitor A83-01
  • Cell 1 in Example 7 belongs to the induced immature liver cell described in the present invention.
  • Cell 2 and Cell 3 in the example belong to the induced mature liver cells described in the present invention.

Abstract

本发明涉及用于制备并诱导产生诱导性成熟肝脏细胞的组合物，制备诱导性成熟肝脏细胞的方法，以及产生的诱导性成熟肝脏细胞。

Description

诱导性成熟肝脏细胞及其制备的方法 技术领域
本发明涉及用于制备并诱导产生诱导性成熟肝脏细胞的组合物，制备诱导性成熟肝脏细胞的方法，以及诱导产生的诱导性成熟肝脏细胞。
背景技术
原代肝脏细胞在肝脏疾病的科学研究和临床治疗中发挥着重要的作用。例如，肝脏细胞可以通过疾病模型的构建用于发病机制的研究和相关药物的筛选；肝脏细胞的移植可以治疗慢性肝脏疾病和缓解晚期肝衰竭等。目前，原代肝脏细胞主要的获取方式是从供肝中分离细胞，但是由于供肝数量有限和原代肝脏细胞在体外较难培养等因素，肝脏细胞资源的获取给科研和临床中的应用带来了严重阻碍。
诱导性肝脏细胞的出现为解决这个瓶颈带来了巨大的希望。在2003年，Melissa K.Carpenter课题组率先在体外成功地将人源胚胎干细胞(embryonic stem cell，ESC)分化为表达肝脏标记基因的诱导性肝脏细胞(Lakshmi Rambhatla,2003b Generation of Hepatocyte-Like Cells From Human Embryonic Stem Cells.Cell Transplantation,12,1-11.)。随着人源诱导性多能干细胞(induced pluripotent stem cell，iPSC)的出现，利用诱导性多能干细胞诱导肝脏细胞的方法也很快被构建出来(Song et al.,2009Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.Cell Res 19,1233-1242.10.1038/cr.2009.107.；Takahashi et al.,2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell 131,861-872.10.1016/j.cell.2007.11.019.)。此外，还有研究通过转分化的方式将成纤维细胞直接诱导为肝脏细胞(Du et al.,2014 Human hepatocytes with drug metabolic function induced from fibroblasts by lineage  reprogramming.Cell Stem Cell 14,394-403.10.1016/j.stem.2014.01.008；Huang et al.,2014 Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.Cell Stem Cell 14,370-384.10.1016/j.stem.2014.01.003.；Zhu et al.,2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature 508,93-97.10.1038/nature13020.)
近年来，虽然体外诱导肝脏细胞的技术在不断更新和完善，但是目前得到的诱导肝脏细胞只获得了原代肝脏细胞的部分特征，这类诱导肝脏细胞更类似于胎肝细胞而不是成熟的原代肝脏细胞(Baxteretal.,2015 Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.J Hepatol 62,581-589.10.1016/j.jhep.2014.10.016.)。
从形态上来看，人源原代肝脏细胞相对比较立体，细胞中呈现出清晰的细胞核，部分细胞呈双核，细胞核比较小且核质比相对较低。而现有技术中诱导的肝脏细胞虽然呈肝脏细胞所特有的多角形，但是细胞相对贴壁而不立体，细胞中基本看不到清晰的细胞核，含有双核的细胞极少，核质比也远大于原代肝脏细胞。
在基因表达方面，现有技术诱导的肝脏细胞中一系列与肝脏代谢相关的重要蛋白酶都没有显著表达，例如CYP3A4，CYP2C9等。
在功能方面，现有技术中诱导的肝脏细胞虽然具有白蛋白分泌能力，但是每24小时每百万个细胞的分泌量在2-5μg左右，而高质量原代肝脏细胞的分泌能力可以达到10μg以上(Zhu etal.,2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature 508,93-97.10.1038/nature13020.)。此外，现有技术中诱导肝脏代谢的CYP450代谢能力和尿素合成的能力也与原代肝脏细胞相差较远。另外，现有技术中的诱导性肝脏细胞仍然缺失肝脏极性；在体内重建方面，当将现有技术中的诱导性肝脏细胞移植到慢性肝脏损伤的小鼠模型体内时，血液中检测到人源白蛋白所需要的时间相对较长，且血液中检测到的白蛋白最高的浓度仅有约几百纳克每毫升，这说明 白蛋白分泌能力较低，同时诱导性肝脏细胞在体内的重建率不到1％。而原代肝脏细胞在慢性肝脏损伤小鼠体内白蛋白的分泌能力可以达到1mg/ml以上，体内重建效率可以达到90％以上(Zhang et al.,2018 In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity.Cell Stem Cell 23,806-819e804.10.1016/j.stem.2018.10.018.)。此外，在慢性肝损小鼠体内嵌合的诱导性肝脏细胞因为不能迁移而主要在肝门静脉附近发生嵌合(Wang et al.,2019Human ESC-derived expandable hepatic organoids enable therapeutic liver repopulation and pathophysiological modeling of alcoholic liver injury.Cell Res 29,1009-1026.10.1038/s41422-019-0242-8.；Yan et al.,2017 Human embryonic stem cell-derived hepatoblasts are an optimal lineage stage for hepatitis C virus infection.Hepatology 66,717-735.10.1002/hep.29134.)；而原代肝脏细胞可以从肝门静脉处迁移至肝实质区域，继而再生增殖。此外相比于原代肝脏细胞，再生后的诱导肝脏细胞与原代肝脏细胞还存在一定程度的差异。上述各项研究结果说明，现有技术中的诱导性肝脏细胞还是一种不成熟的状态，功能与原代肝脏细胞相比仍相差较大。
本发明旨在建立一种利用组合物制备在形态结构，基因表达，细胞功能等方面接近或不低于原代肝脏细胞的诱导性成熟肝脏细胞。
发明内容
该发明内容中提供的本发明实施方式仅意在示例性的并意在提供本文所公开的选择性实施方式的概述。发明内容(其为示例性的和选择性的)并不限制任何权利要求的范围，并非提供在本文中公开或考虑的本发明实施方式的整个范围，且不应解释为限制或约束本公开内容或任何请求保护的本发明实施方式的范围。除非明确地排除，本文公开和考虑的本发明实施方式的其他特征、目的和优势可以与任何其他实施方式组合。
发明人令人惊讶地发现，通过采用特定的化合物组合作为用于诱导诱导性不成熟肝脏细胞的基础培养基的添加剂，可以将诱导性不成熟肝脏细胞分化为诱导性成熟肝脏细胞，该诱导方法有效，能够得到与原代肝脏细胞类似的诱导性成熟肝脏细胞。因此，
在一个方面，本发明提供了一种第一组合物，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂。优选地，在一些实施方式中，所述第一组合物进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂。优选地，在一些实施方式中，所述第一组合物进一步包含：(f)p38 MAPK抑制剂。
在另一方面，本发明还提供了一种第一试剂盒，(a)HDAC抑制剂、(b)ERK信号通路抑制剂、以及相应的使用说明。优选地，在一些实施方式中，所述第一试剂盒进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂。优选地，在一些实施方式中，所述第一试剂盒进一步包含：(f)p38 MAPK抑制剂。
在又一个方面，本发明还提供了一种第一培养基，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂、以及基础培养基。优选地，所述第一培养基进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂；和/或(e)TGF-beta信号通路抑制剂。优选地所述基础培养基选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E等培养基，更优选地，基础培养基是HCM。优选地，在一些实施方式中，所述第一培养基进一步包含：(f)p38 MAPK抑制剂。
在一个方面，本发明还提供了一种第二组合物，其包含：(a)cAMP激活剂、(b)类固醇类化合物；优选地，所述第二组合物进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
在另一方面，本发明还提供了一种第二试剂盒，其包含：(a)cAMP激活剂、(b)类固醇类化合物；以及相应的使用说明；优选地，所述第二试剂盒进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
在又一个方面，本发明还提供了一种第二培养基，其包含：(a)cAMP激活剂、(b)类固醇类化合物、以及基础培养基；优选地所述基础培养基选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E等培养基，更优选地，基础培养基是William’E。优选地，在一些实施方式中，所述第二培养基包含相对于第二培养基总体积的0.05％～20体积％的血清，优选1体积％。优选地，所述第一试剂盒进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
在一方面，本发明还提供了第一组合物、第一试剂盒、第一培养基、第二组合物、第二试剂盒、第二培养基在用于制备诱导性成熟肝脏细胞中的用途。
在一些实施方式中，HDAC(组蛋白去乙酰化酶)抑制剂选自Mocetinostat(MGCD0103)、Abexinostat(PCI-24781)、Fimepinostat(CUDC-907)、ITSA-1、Vorinostat(SAHA)、MS-275、Panobinostat(LBH589)、Quisinostat、FK228、SB939、Trichostatin A(TSA)等。优选地，在一些实施方式中，HDAC(组蛋白去乙酰化酶)抑制剂是MS-275。
在一些实施方式中，类固醇类化合物选自泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、泼尼松龙、氢化可的松、地塞米松等。优选地，在一些实施方式中，类固醇类化合物是地塞米松。
在一些实施方式中，Notch信号通路抑制剂选自IMR-1、RO4929097、Semagacestat(LY450139)、Avagacestat(BMS-708163)、Dibenzazepine(YO-01027)、Crenigacestat(LY3039478)、Compound E、DAPT等。优选地，在一些实施方式中，Notch信号通路抑制剂是Compound E。
在一些实施方式中，ERK信号通路抑制剂选自SeluMetinib(AZD6244)、Trametinib(GSK1120212)、PD0325901、U0126-ETOH、U0126、PD184352(CI-1040)等。优选地，在一些实施方式中，ERK信号通路抑制剂是PD0325901。
在一些实施方式中，TGF-beta信号通路抑制剂选自SD-208、LY2109761、BIBF-0775、A83-01、SB431542等。优选地，在一些实施方式中，TGF-beta信号通路抑制剂是A83-01。
在一些实施方式中，所述p38 MAPK抑制剂选自SB202190(FHPI)、SB203580、TAK-715、Doramapimod(BIRB 796)等。优选地，在一些实施方式中，p38 MAPK抑制剂是SB203580。
在一些实施方式中，所述cAMP激活剂选自Forskolin、8-Br-cAMP等。优选地，在一些实施方式中，cAMP激活剂是Forskolin。
在一些实施方式中，所述ROCK抑制剂选自Y-27632、Thiazovivin、GSK429286A、RKI-1447、AT13148等。优选地，在一些实施方式中，ROCK抑制剂是Y-27632。
在一些实施方式中，所述培养基包含基础培养基。
在一些实施方式中，所述基础培养基选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E等培养基。
在另一方面，本发明提供了一种制备诱导性成熟肝脏细胞的方法，所述方法包括以下步骤：在本文所述的培养基中培养诱导性不成熟肝脏细胞，由此制备所述诱导性成熟肝脏细胞。在一些实施方式中，所述方法包括步骤(a)在本文所述的第一培养基中培养诱导性不成熟肝脏细胞。由此制备所述诱导性成熟肝脏细胞。优选地，在一些实施方式中，所述方法进一步包括在本文所述的第二培养基中培养所述步骤(a)所得的细胞，由此制备与原代肝脏细胞更加类似的诱导性成熟肝脏细胞。
在一些实施方式中，所述诱导性不成熟肝脏细胞由多能干细胞制备得到；优选地，在一些实施方式中，所述诱导性不成熟肝脏细胞通过本文所述的方法由多能干细胞制备得到。优选地，在一些实施方式中，所述多能干细胞选自胚胎干细胞或诱导性多能干细胞。
在一些实施方式中，本发明提供了一种诱导性成熟肝脏细胞，其具有以下一种或多种特征：
(a)诱导性成熟肝脏细胞显示出原代肝脏细胞的典型形态特征，优选地，与原代人源肝脏细胞的形态基本一致；
(b)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞选自以下的一种或多种肝脏细胞标志物的转录或蛋白表达，或染色体的可及性显著增加：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1；(c)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞的以下标志物的转录或蛋白表达减少：AFP；
(d)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞的转录组和染色体的可及性与原代人源肝脏细胞接近或一致；
(e)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞选自以下的一种或多种肝脏细胞的功能增加或首次获得：白蛋白分泌能力、尿素合成能力、肝脏细胞极性、CYP代谢酶活力、成熟肝脏细胞的再生能力，移植存活率、体内重建率；和
(f)相对于原代肝脏细胞，所述诱导性成熟肝脏细胞选自以下的一种或多种肝脏细胞的功能接近或不低于原代肝脏细胞：白蛋白分泌能力、尿素合成能力、肝脏细胞极性、CYP代谢酶活力、成熟肝脏细胞的再生能力，移植存活率、体内重建率；
优选地，在一些实施方式中，所述诱导性成熟肝脏细胞可通过根据本文所述的方法制备。
在一些方面，本文提供了一种诱导性成熟肝脏细胞，其可通过根据本文所述的方法制备。
在另一方面，本发明提供了一种类器官，其产生自本文所述的诱导性成熟肝脏细胞。
在另一方面，本发明提供了一种组织，其产生自本文所述的诱导性成熟肝脏细胞，优选所述组织是肝脏。
在一些方面，本发明提供了一种本文公开的诱导性成熟肝脏细胞在制备用于肝脏细胞移植的细胞、构建肝脏相关疾病模型、肝脏疾病相关药物筛选与鉴定，药物肝脏毒性筛选与鉴定，体外构建肝脏器官(含3D打印肝脏器官)中的用途。
在一些方面，本发明提供了一种筛选用于治疗疾病的物质的体外方法，其包括使本文公开的诱导性成熟肝脏细胞、类器官或组织与待筛选物质接触。
在一些实施方式中，本文所述诱导性不成熟肝脏细胞可由本领域常规方法制备得到，包括但不限于如(Zhu et al.,2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature 508,93-97.10.1038/nature13020)中所描述的方法，所述诱导性不成熟肝脏细胞在本领域中亦被称为“f-iHep”、“iHep”等。在一些现有技术中，诱导性不成熟肝脏细胞也被称为“诱导性肝脏细胞”。优选地，所述诱导性不成熟肝脏细胞可由本文所述方法制备得到。
在另一个方面，本发明提供了一种培养物，其包含本文所述的培养基和诱导过程中产生的细胞。在一些实施方式中，所述培养物包含本文所述的培养基和诱导性成熟肝脏细胞。
在一些方面，本发明提供了一种试剂盒，其包含HEM-1培养基和HEM-2培养基，其中所述HEM-1培养基包含ROCK抑制剂、肝脏细胞生长因子(HGF)、抑瘤素M(oncostatinM,OSM)、类固醇类化合物、Notch信号通路抑制剂、TGF-beta信号通路抑制剂和基础培养基；优选地，所述基础培养基是去除表皮细胞生长因子(EGF)的HCM培养基。所述HEM-2培养基包含肝脏细胞生长因子(HGF)、抑瘤素M(oncostatinM,OSM)、类固醇类化合物、Notch信号通路抑制剂、TGF-beta信号通路抑制剂和基础培养基；优选地，所述基础培养基是去除表皮细胞生长因子(EGF)的HCM培养基。在一些实施方式中，所述ROCK抑制剂是Y-27632；所述类固醇类化合物是地塞米松；所述Notch信号通路抑制剂是Compound E；所述TGF-beta信号通路抑制剂是A83-01。优选地，在一些实施方式中，所述HEM-1培养基和HEM-2培养基包含选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E的基础培养基；更优选地，在一些实施方式中，所述基础培养基是HCM培养基。
在一些方面，本发明提供了一种扩增诱导性不成熟肝脏细胞的方法，其中包括使诱导性不成熟肝脏细胞接触本发明的HEM-1培养基或HEM-2培养基。在一些实施方式中，所述扩增诱导性不成熟肝脏细胞的方法包括使诱导性不成熟肝脏细胞接触HEM-1培养基，之后使诱导性不成熟肝脏细胞接触HEM-2培养基。
在一些方面，本发明提供了一种HEM-1培养基和HEM-2培养基在用于扩增诱导性不成熟肝脏细胞的用途。
通过本文所述的方法获得的诱导性成熟肝脏细胞可以体外长期(例如：3个月以上)扩增培养后，仍然维持关键的肝脏细胞功能和形态。
附图说明
图1：诱导性成熟肝脏细胞的形态特征。细胞1(诱导性不成熟肝脏细胞)：由多能性干细胞诱导得到的诱导性不成熟肝脏细胞；细胞2(5C培养基)：诱导性不成熟肝脏细胞经5C培养基处理15天后，呈现的细胞形态；细胞2(6C培养基)：诱导性不成熟肝脏细胞经6C培养基处理15天后，呈现的细胞形态；细胞3(4C培养基)：细胞1(诱导性不成熟肝脏细胞)经6C培养基处理之后，又经4C培养基处理60天后，呈现的细胞形态。
图2：诱导性成熟肝脏细胞高表达一系列肝脏细胞成熟相关的标记基因。A)成熟肝脏细胞相关基因的mRNA表达水平，利用RT-qPCR的方法对诱导第一阶段的细胞1(诱导性不成熟肝脏肝细胞)，诱导第二阶段的对照组细胞2对照(DMSO)和细胞2(6C培养基)，以及诱导第三阶段的对照组细胞3对照(DMSO)和细胞3(4C培养基)中成熟相关的一系列基因进行mRNA表达水平的鉴定；B)成熟肝脏细胞相关基因的蛋白水平表达，通过免疫荧光的方法，对细胞3(诱导性成熟肝脏细胞)中成熟相关基因CYP1A2、CYP2C9、CYP2D6、CYP3A4的蛋白表达进行鉴定验证。
图3：诱导性成熟肝脏细胞具有肝脏细胞极性且其一系列肝脏相关功能也得到显著提升。A)利用CDF荧光检测细胞1(诱导性不成熟肝脏细胞)，细胞2(6C培养基)以及细胞3(4C培养基)的肝脏极性；B)利用酶联免疫吸附实验鉴定细胞1(诱导性不成熟肝脏细胞)、细胞2(6C培养基)和细胞3(4C培养基)分泌白蛋白的能力；C)诱导肝脏细胞合成尿素能力的鉴定；D)利用化学发光实验鉴定不同诱导细胞中CYP1A2和CYP3A4代谢酶的活力。
图4：诱导性成熟肝脏细胞可以在慢性肝脏损伤小鼠FRG体内重建迁移并缓解和改善小鼠的肝脏损伤情况和存活率。A)原代肝脏细胞，细胞2(6C培养基)，以及细胞3(4C培养基)在移植到FRG小鼠体内后，小鼠血液中人源白蛋白的含量(HSA，human serum albumin)，其中纵坐标为小鼠血液中人源白蛋白的浓度，横坐标为移植后的天数；B)移植3个月后，小鼠肝脏中hALB基因的表达，对移植后存活3个月以上的小鼠的肝脏进行组织免疫荧光染色，其中绿色的区域代表人源ALB阳性的区域，蓝色为DAPI；C)对移植细胞3后存活3个月以上的小鼠的肝脏进行组织免疫荧光染色，其中绿色的为人源ALB，红色的为hCYP3A4，hCYP2C9和FAH基因，蓝色的为DAPI；D)FRG小鼠移植细胞后的存活情况，其中纵坐标代表存活的百分比，横坐标代表移植后的天数；E)移植细胞后FRG小鼠中AST和ALT的情况。
图5：使用HEM-1培养基和HEM-2培养基扩增细胞1(诱导性不成熟肝脏细胞)。A)细胞1(D0)，以及细胞1传代扩增后第3天(D3)和第7天(D7)的细胞形态；B)细胞1(D0)，以及细胞1传代扩增后第1天(D1)第3天(D3)和第7天(D7)中肝脏相关基因的mRNA表达；C)免疫荧光检测扩增后细胞1(D7)中肝脏相关基因的蛋白表达；D)细胞1扩增前(D0)和扩增后(D7)的白蛋白分泌能力；E)细胞1扩增前(D0)和扩增后(D7)的尿素合成能力；F)细胞1扩增前(D0)和扩增后(D7)的CYP3A4的代谢能力；G)细胞1扩增后(D7)的ORO，Bodipy，PAS和ICG的检测。
图6:4C培养基和6C培养基的组分验证和优化。A)对照组细胞1，6C培养基处理2周的细胞2，以及样本组6C培养基逐一减去每一组分后建立培养基处理2周的细胞的转录组；B)基于RT-PCR实验，验证4C培养基中每一组分对诱导效果的影响；C)利用4C培养基对细胞2处理30天后的细胞形态，和利用4C培养基逐一减去每一组分后构建的培养基，对细胞2处理30天后的细胞形态。
具体实施方式
一个或多个本发明实施方式的详细内容在本文的附图、权利要求书和说明书中进行了阐述。除非明确地排除，本文公开和考虑的本发明实施方式的其他特征、目的和优势可以与任何其他实施方式组合。
A.概述
除非另有特别指出，否则本发明的实施将采用在本领域技术内的化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学、细胞生物学、干细胞方案、细胞培养和转基因生物学的常规方法，其中许多在下文描述用于举例说明的目的。这样的技术在文献中充分说明。参见例如Sambrook,et al,Molecular Cloning:A Laboratory Manual(3rd Edition,2001)；Sambrook,et al.,Molecular Cloning:A Laboratory Manual(2nd Edition,1989)；Maniatis et al,Molecular Cloning:A Laboratory Manual(1982)；Ausubel et al.,Current Protocols in Molecular Biology(John Wiley and Sons,updated July 2008)；Short Protocols in Molecular Biology:A CompendiμM of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience；Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985)；Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)；Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991)；Oligonucleotide Synthesis(N.Gait,Ed.,1984)；Nucleic Acid Hybridization(B.Hames&S.Higgins,Eds., 1985)；Transcription and Translation (B.Hames&S.Higgins,Eds.,1984)；Animal Cell Culture (R.Freshney,Ed.,1986)；Perbal,A Practical Guide to Molecular Cloning (1984)；Fire et al.,RNA Interference Technology: From Basic Science to Drug Development (Cambridge University Press,Cambridge,2005)；Schepers,RNA Interference in Practice (Wiley-VCH,2005)；Engelke,RNA Interference (RNAi): The Nuts&Bolts of siRNA Technology (DNA Press,2003)；Gott,RNA Interference,Editing,and Modification: Methods and Protocols (Methods in Molecular Biology；HμMan Press,Totowa,NJ,2004)；Sohail,Gene Silencing by RNA Interference: Technology and Application (CRC,2004)；Clarke and Sanseau,microRNA: Biology,Function&Expression(Nuts&Bolts series；DNA Press,2006)；Immobilized Cells And Enzymes (IRL Press,1986)；the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.)；Gene Transfer Vectors For Mammalian Cells (J.H.Miller and M.P.Calos eds.,1987,Cold Spring Harbor Laboratory)；Harlow and Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998)；Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds.,Academic Press,London,1987)；Handbook Of Experimental Immunology,VolμMes I-IV (D.M.Weir and C.Blackwell,eds.,1986)；Riott,Essential Immunology,6th Edition,(Blackwell Scientific Publications,Oxford,1988)；Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology)(Kurstad Turksen,Ed.,2002)；Embryonic Stem Cell Protocols: VolμMe I: Isolation and Characterization (Methods in Molecular Biology)(Kurstad Turksen,Ed.,2006)；Embryonic Stem Cell Protocols: VolμMe II: Differentiation Models (Methods in Molecular Biology)(Kurstad Turksen,Ed.,2006)；HμMan Embryonic Stem Cell Protocols (Methods in Molecular Biology)(Kursad Turksen Ed.,2006)；Mesenchymal Stem Cells: Methods and  Protocols(Methods in Molecular Biology)(Darwin J.Prockop,Donald G.Phinney,and Bruce A.Bunnell Eds.,2008)；Hematopoietic Stem Cell Protocols(Methods in Molecular Medicine)(Christopher A.Klug,and Craig T.Jordan Eds.,2001)；Hematopoietic Stem Cell Protocols(Methods in Molecular Biology)(Kevin D.Bunting Ed.,2008)Neural Stem Cells:Methods and Protocols(Methods in Molecular Biology)(Leslie P.Weiner Ed.,2008)；Hogan et ah,Methods of Manipulating the Mouse Embyro(2nd Edition,1994)；Nagy et al,Methods of Manipulating the Mouse Embryo(3rd Edition,2002),和The Zebrafish book.A guide for the laboratory use of zebrafish(Danio rerio),4th Ed.,(Univ.of Oregon Press,Eugene,OR,2000)。
本文引用的所有公开文件、专利和专利申请通过引入全文并入本文。
B.定义
除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域普通技术人员通常理解的相同的含义。为了本发明的目的，下文定义了下述术语。
冠词"一个/一种(a/an)"和"该/所述(the)"在本文中用于指一个/一种或超过一个/一种(即至少一个/一种)所述冠词的语法对象。例如，"要素"意指一个/一种要素或超过一个/一种要素。
替代(例如"或")的使用应理解为意指替代方案中任一、两者或其任何组合。
术语"和/或"应理解为意指替代方案中任一或两者。
如本文使用的，术语"约"或"大约”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较，改变多达15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。在一个实施方式中，术语"约"或"大约”是指围绕参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度±15％、±10％、±9％、 ±8％、±7％、±6％、±5％、±4％、±3％、±2％或±1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的，术语"基本上(substantially/essentially)”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较，是约90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。在一个实施方式中，术语"基本上相同”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度大约相同的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的，术语"基本上不含"当用于描述组合物例如细胞群或培养基时，指不含指定物质，例如95％不含、96％不含、97％不含、98％不含、99％不含指定物质的组合物，或如通过常规手段测量是无法检测的。类似含义可应用于术语"不存在"，当指不存在组合物的特定物质或组分时。
如本文使用的，术语"可观的”是指通过一种或多种标准方法可容易地检测到的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。术语"不可观的(not-appreciable)"和"不可观的(notappreciable)"以及等价方式是指通过标准方法无法容易地检测到或无法检测到的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。在一个实施方式中，如果事件以小于5％、4％、3％、2％、1％、0.1％、0.01％、0.001％或更少的次数发生，则它是不可观的。
在本说明书全文，除非上下文另有要求，否则术语"包含"，“包括”、“含有”和“具有”应理解为暗示包括所述步骤或要素或者步骤或要素组，但不排除任何其他步骤或要素或者步骤或要素组。在特定实施方式中，术语"术语"包含"，“包括”、“含有”和“具有”同义使用。
"由……组成"意指包括但限于在短语"由……组成"后的任何。因此，短语"由……组成”是指示所列出的要素是需要的或强制性的，并且没有其他要素是可以存在的。
"基本上由……组成"意指包括在短语"基本上由……组成"后列出的任何要素，并且限于不干扰或贡献于所列出的要素的公开内容中指定的活动或动作的其他要素。因此，短语"基本上由……组成”是指示所列出的要素是需要的或强制性的，但没有其他要素是任选的，并且取决于它们是否影响所列出的要素的活动或动作而可以存在或不存在。
在本说明书全文，提到"一个实施方式"、"实施方式"、"特定实施方式"、"相关实施方式"、"某个实施方式"、"另外的实施方式"或"进一步的实施方式"或其组合意指与所述实施方式结合描述的特定特征、结构或特性被包括在本发明的至少一个实施方式中。因此，前述短语在本说明书全文的各个地方的出现不一定全部指相同实施方式。此外，特定特征、结构或特性可以以任何合适方式在一个或多个实施方式中组合。
术语"离体"一般指在生物体外发生的活动，例如在生物体外的人工环境中在活组织中或活组织上完成的实验或测量，优选伴随天然条件的最低限度改变。在特定实施方式中，"离体"程序涉及从生物体中获得的活细胞或组织，在实验室仪器中培养，通常在无菌条件下，且通常为数小时或高达约24小时，但包括高达48或72小时，取决于环境。在某些实施方式中，可收集且冷冻这样的组织或细胞，随后解冻用于离体处理。使用活细胞或组织的持续长于数天的组织培养实验或程序通常视为"体外"，尽管在某些实施方式中，该术语可与离体互换使用。
术语"体内"一般指在生物体内发生的活动。
如本文使用的，术语"多能干细胞"是指具有形成机体或躯体(即胚体)的所有谱系能力的细胞。例如，胚胎干细胞是能够形成来自三个 胚层(外胚层、中胚层和内胚层)各自的细胞的一类多能干细胞。在一些方面，多能干细胞可以是胚胎干细胞或诱导性多能干细胞。
如在本文中所使用的，"培养物"意指在培养基中生长并且任选传代的细胞群体。细胞培养物可以是原代培养物(例如，尚未被传代的培养物)或者可以是传代或继代培养物(例如，已经传代培养或传代一次或多次的细胞群体)。
如本文使用的，"基因表达"或“基因转录”是指生物样品(例如成熟肝脏细胞/不成熟肝脏细胞或包含成熟肝脏细胞/不成熟肝脏细胞的细胞群)中的基因的表达/转录和/或表达/转录模式的相对水平。在特定实施方式中，成熟肝脏细胞是诱导性成熟肝脏细胞。在特定实施方式中，不成熟肝脏细胞是诱导性不成熟肝脏细胞。
如本文使用的，"蛋白表达"是指生物样品(例如成熟肝脏细胞/不成熟肝脏细胞或包含成熟肝脏细胞/不成熟肝脏细胞的细胞群)中的蛋白的表达和/或表达模式的相对水平。在特定实施方式中，成熟肝脏细胞是诱导性成熟肝脏细胞。在特定实施方式中，不成熟肝脏细胞是诱导性不成熟肝脏细胞。
本文涵盖本领域可用的用于检测表征本发明细胞的基因的表达/转录的任何方法。如本文使用的，术语"检测表达/转录"意指测定基因的RNA转录物或其表达产物的数量或存在。用于检测基因的表达/转录的方法，即基因表达/转录谱分析，包括基于多核苷酸的杂交分析的方法、基于多核苷酸的测序的方法、免疫组织化学方法、和基于蛋白质组学的方法。所述方法一般检测感兴趣的基因的表达/转录产物(例如mRNA)。在一些实施方式中，使用基于PCR的方法例如逆转录PCR(RT-PCR)(Weis等人，TIG8:263-64，1992)，以及基于阵列的方法例如微阵列(Schena等人，Science270:467-70，1995)。
本文涵盖本领域可用的用于检测表征本发明细胞的蛋白表达的任何方法。如本文使用的，“检测蛋白表达”意指测定基因编码的蛋白质的数量或存在。用于检测蛋白表达的方法，包括基于蛋白质印迹法、免疫荧光、流式细胞仪、蛋白质质谱的方法。
"贴壁"是指在适当培养基的存在下，细胞附着至容器，例如细胞附着至无菌塑料(或涂布塑料)细胞培养皿或烧瓶。某些类别的细胞在培养中无法维持或不生长，除非它们贴壁至细胞培养容器。某些类别的细胞("非贴壁细胞")在培养中维持和/或增殖而无需贴壁。
"培养(culture)"或"细胞培养"是指在体外环境中的细胞的维持、生长和/或分化。"细胞培养基"、"培养基"、"培养介质"、"介质"、"补充物"和"培养基补充物”是指培养细胞培养物的营养组合物。
"培养物(culture)"或"细胞培养物"是指被培养的物质如细胞，和/或其中存在有被培养的物质如细胞的培养基。
"培养(cultivate)"是指在组织或机体外，例如在无菌塑料(或经涂覆塑料)细胞培养皿或烧瓶中的细胞的维持、繁殖(生长)(扩增)和/或分化。"培养"可利用培养基作为营养素、激素和/或帮助使细胞繁殖和/或维持细胞的其他因子的来源。
如本文使用的，"解离的"细胞是指已经从其他细胞或表面(例如培养板表面)基本上分开或者纯化的细胞。例如，细胞可通过机械或酶促方法从动物或组织解离。或者，体外聚集的细胞可以被酶促地或机械地彼此解离，例如通过解离成簇、单细胞或单细胞和簇的混合物的悬浮液。在另外一个可替代实施方式中，贴壁细胞从培养板或其他表面解离。解离因此可涉及破坏细胞与细胞外基质(ECM)和基底(例如培养表面)的相互作用，或破坏细胞之间的ECM。
如本文使用的，术语"富集(enrich)"是指增加组合物例如细胞组合物中的指定组分的量，并且当用于描述细胞的组合物例如细胞群时，"富集的"是指与富集前细胞群中的这样的组分的比例相比较，具有按比例增加的量的指定组分的细胞群。例如，组合物例如细胞群可就靶细胞类型(即具有指定特征的细胞)而言进行富集，因此与富集前细胞群中存在的靶细胞的比例相比较，具有增加的比例或百分比的靶细胞类型。细胞群可通过本领域已知的细胞选择和分选方法就靶细胞类型进行富集。在一些实施方式中，细胞群通过分选或选择方法进行富集。在一个特定实施方式中，就靶细胞群进行富集的方法使细胞群就靶细 胞群而言富集至少约20％，从而意指富集的细胞群包含比细胞群被富集前细胞群中按比例多约20％的靶细胞类型。在一个实施方式中，就靶细胞群富集的方法使细胞群就靶细胞群而言按比例富集至少约30+％、40+％、50+％、60+％、70+％、80％、85％、90％、95％、97％、98％或99％，或至少约98％，或在特定实施方式中，约99％。
基因组稳定性是指细胞忠实复制DNA且维持DNA复制过程的完整性的能力。如本文使用的，术语"基因组稳定的细胞"和"具有基因组稳定性的细胞"是指显示出一定频率的突变和染色体异常(例如易位、非整倍体、拷贝数变异和重复)的细胞，所述频率基本上类似于相对于正常人体细胞的突变和染色体异常的频率。
"成分”是指可用于细胞培养基中以维持和/或促进细胞生长和/或分化的任何化合物或其他材料，无论其来源是化学还是生物的。术语"组分"、"营养素"和"成分"可互换使用。用于细胞培养基的常规成分可包括但不限于氨基酸、盐、金属、糖、脂质、核酸、激素、维生素、脂肪酸、蛋白质等等。促进和/或维持离体或体外细胞培养的其他成分可通过本领域普通技术人员根据期望效果的需要而加以选择。
"分离(isolate)"是指使组合物或材料与其天然环境分开且收集，例如个体细胞或细胞培养物与组织或机体的分开。在一个方面，细胞群或组合物基本上不含其在自然界中可与之结合的细胞和材料。就靶细胞群而言，"分离的"或"纯化的"或"基本上纯的"是指就构成总细胞群的靶细胞而言，至少约50％、至少约75％、至少约85％、至少约90％，并且在特定实施方式中，至少约95％纯的细胞群。细胞群或组合物的纯度可通过本领域众所周知的适当方法进行评价。例如，基本上纯的肝脏细胞群是指就构成总细胞群的肝脏细胞而言，至少约50％、至少约75％、至少约85％、至少约90％，并且在特定实施方式中，至少约95％，并且在某些实施方式中，约98％纯的细胞群。
"传代(passage)"是指当细胞已增殖至所需程度时，将细胞细分且铺板到多个细胞培养表面或容器内的动作。在一些实施方式中，"传代"是指将细胞细分、稀释且铺板。当细胞从原代培养表面或容器传 代到后续组的表面或容器时，后续培养在本文中可被称为"继代培养"或"第一次传代"等。每个细分和铺板到新培养容器内的动作被视为一次传代。
"铺板(plating)"是指将一个或多个细胞置于培养容器内，使得细胞贴壁至细胞培养容器且在细胞培养容器上铺展。
"增殖"是指一个细胞分裂成两个基本上等同的细胞或者细胞群数量增加(例如，以进行复制)的性质。
"繁殖"或"扩增"是指使组织或机体外的细胞例如在无菌容器例如塑料(或经涂覆塑料)细胞培养皿或烧瓶中生长(例如经由细胞增殖来复制)。
如在本文中所使用的术语“诱导性多能干细胞”(iPSC)是通过人工方式来源于非多能细胞的多能干细胞类型。
可以通过使用本领域技术人员已知的技术将充当细胞来源的适当的器官或组织分解来分离细胞。例如，可以将组织或器官机械分解和/或用消化酶和/或削弱相邻细胞之间连接的螯合剂处理，从而可以将组织分散以形成个体细胞的悬浮液，而没有可察觉的细胞损坏。可以通过将组织切碎并且用一种或多种酶如胰蛋白酶、糜蛋白酶、胶原酶、弹性蛋白酶、和/或透明质酸酶、DNA酶、链霉蛋白酶、分散酶等处理切碎的组织来完成酶解离。也可以通过多种方法完成机械破坏，包括但不限于使用粉碎机、搅拌机、筛、均化器、压力盒(pressure cell)、或声波器(insonator)。
通过从培养源中提取(例如，通过密度梯度离心和/或流式细胞仪)，可以得到基本上纯化的肝脏细胞群体。纯度可以通过任何适当的方法进行测量。例如，通过流式细胞仪(例如，FACS分析)，可以将肝脏细胞99％-100％纯化。例如，通过使用与肝脏细胞上的标记结合的分子(例如，抗体、抗体衍生物、配体或Fc-肽融合分子)并且从而对结合分子的细胞进行阳性选择(即，阳性选择)，可以将肝脏细胞分离。阳性选择方法的其他实例包括优先促进在希望的和不希望的细胞类型的混合群体中的希望的细胞类型的生长的方法。备选地，通过使 用与在希望的细胞类型上不存在、但是在不希望的细胞类型上存在的标记结合的分子，可以从希望的细胞中移除含有这样的标记的不希望的细胞(即，阴性选择)。其他阴性选择方法包括优先杀伤在希望的和不希望的细胞类型的混合群体中的不希望的细胞类型或抑制其生长。因此，通过使用阴性选择、阳性选择、或它们的组合，可以制备干细胞的富集群体。
用于分离的过程可以包括磁性分离，使用涂布有抗体的磁珠，亲和色谱，与单克隆抗体连接的细胞毒性剂，或与单克隆抗体结合使用的这样的试剂，例如补体和细胞毒素，以及使用附着至固体基质(例如，平板)的抗体的“淘选”，或其他方便的技术。提供准确分离的技术包括荧光活化的细胞分选器，其可以具有不同的复杂程度，例如多个颜色通道、低角度和钝角光散射检测通道、和阻抗通道。抗体可以与标记如允许直接分离的磁珠、可以利用与支持物结合的抗生物素蛋白或链霉抗生物素蛋白移除的生物素、或可以与荧光活化的细胞分选器一起使用的荧光染料缀合，以允许特定细胞类型的容易的分离。可以采用对诱导的多能干细胞的活力不过度地有害的任何技术。
在特定实施方式中，给定的培养条件引起的标志物的转录或蛋白表达的增加或减少的，可以是与适当的对照相比，至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000％或更多的增加或减少。
检测标志基因的转录的方法是本领域已知的。在一些实施方式中，标志基因的转录可由RT-qPCR(Reverse transcription Quantitative polymerase chain reaction)方法测定。优选地，使用本文公开的方法检测标志基因的转录。
检测标志基因的蛋白表达的方法是本领域已知的。在一些实施方式中，标志基因的蛋白表达可以通过蛋白质免疫印迹，免疫荧光，流 式细胞仪方法进行检测。在一些实施方式中，标志基因的蛋白表达可以通过免疫荧光的方法进行检测。
本文所述的“原代肝脏细胞的典型形态特征”是指细胞具有原代肝脏细胞所特有的多角形，且细胞相对立体，同时细胞核清晰，部分细胞呈双核，细胞直径一般约15-25μM，优选约20μM。
本文所述的“白蛋白分泌能力”是指可通过任何本领域常规的方法测定的每24小时每百万个细胞的白蛋白分泌量。在一些实施方式中，测定方法是酶联免疫吸附检测方法。在一些实施方式中，所述白蛋白分泌能力由本文公开的方法测定。
本文所述的“尿素合成能力”是指可通过任何本领域常规的方法测定的每24小时每百万个细胞的合成尿素的相对量。在一些实施方式中，所述尿素合成能力由本文公开的方法测定。
本文所述的“CYP代谢酶的活力”是指可通过任何本领域常规的方法测定的细胞中CYP代谢酶的活力。所述CYP代谢酶是指细胞色素P450酶系统家族成员，其包括但不限于CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1。在一些实施方式中，所述CYP代谢酶的活力由本文公开的方法测定。
本文所述的“肝脏极性”、“细胞极性”或“肝脏细胞极性”主要体现在肝脏细胞的形态结构和功能等方面。从形态上来看，肝脏细胞膜分为3个不同的区域，即基底膜，侧膜和胆小管区；从功能上看，基底膜与肝血窦相连接，可以直接进行物质交换，侧膜主要用于相邻细胞的连接，而胆小管区则主要用于胆管的分泌(Treyer,2013)。肝脏细胞极性可通过任何本领域常规方法进行测定。在一些实施方式中，测定方法是细胞染色方法，优选地，用于细胞染色的染料包括但不限于使用CDFDA(5-(和-6)-羧基-2’,7’-二氯荧光素(CDFDA))。在一些实施方式中，所述“肝脏极性”、“细胞极性”或“肝脏细胞极性”由本文公开的方法测定。
如本文所述的“移植存活率”是指将肝脏细胞(如诱导性成熟肝脏细胞、原代肝脏细胞或诱导性不成熟肝脏细胞)移植到肝脏损伤模 型后的模式生物的存活率，所述存活率是指在一定时间后，存活的模式生物的数量与实验初始时的模式生物的比值。其可通过任何本领域常规方法进行测定。在一些实施方式中，所述模式生物是小鼠。在一些实施方式中，所述小鼠模型是FRG小鼠模型(如Grompe M,Strom S.Mice with human livers.Gastroenterology.2013 Dec；145(6):1209-14.doi:10.1053/j.gastro.2013.09.009.Epub 2013 Sep 13.PMID:24042096中所描述的)。在一些实施方式中，所述存活率由本文公开的方法测定。
如本文所述的“体内重建率”是指将肝脏细胞(如诱导性成熟肝脏细胞、原代肝脏细胞或诱导性不成熟肝脏细胞)移植到肝脏损伤模型后的模式生物的肝脏重建率，其是指移植细胞来源的细胞数占组织中所有细胞数量的百分比，其可通过任何本领域常规方法进行测定。在一些实施方式中，所述模式生物是小鼠。所述测定方法包括但不限于通过免疫荧光染色、免疫组织化学的方式进行测定。在一些实施方式中，所述体内重建率由本文公开的方法测定。
如本文所述的“肝脏内迁移能力”是指通过肝门静脉移植的肝脏细胞可以在肝实质区域迁移，其可通过任何本领域常规方法进行测定。在一些实施方式中，所述测定方法是是免疫组化或者免疫组织荧光。在一些实施方式中，所述“肝脏内迁移能力”由本文公开的方法测定。
文中所述“成熟肝脏细胞的再生能力”是指移植成熟的肝脏细胞后，移植细胞能够在肝实质区迁移；并在肝脏损伤信号的刺激下，主动进入细胞周期，展开增殖，在肝实质区形成多个肝脏再生小团(hepatic cluster)；且这些肝脏细胞小团可以清晰的高表达一系列成熟肝脏细胞所特有的标记基因。
如本文所述的“人源白蛋白信号”是指移植入小鼠体内的人源肝脏细胞(原代肝脏细胞，诱导不成熟肝脏细胞，诱导成熟肝脏)表达人源白蛋白的量。。在一些实施方式中，所述测定方法是酶联免疫吸附。在一些实施方式中，所述“人源白蛋白信号”由本文公开的方法测定。
如本文所述的“人源白蛋白起峰时间”是指按照1:100比例稀释后，小鼠血液中人源白蛋白的量达到酶联免疫吸附实验检测的最低限(300ng/ml)所需要的时间，其可通过任何本领域常规方法进行测定。在一些实施方式中，所述测定方法是酶联免疫吸附。在一些实施方式中，所述“人源白蛋白起峰时间”由本文公开的方法测定。
如本文所述的“细胞增殖速度”是指在一定时间范围内，移植细胞在小鼠体内增长的数量，其可通过任何本领域常规方法进行测定。在一些实施方式中，所述测定方法是酶联免疫吸附实验、免疫组化，和免疫组织荧光实验。在一些实施方式中，所述“细胞增殖速度”由本文公开的方法测定。
如本文所述的“增殖细胞质量”是指在小鼠体内增殖后的细胞中表达关键肝脏细胞的能力。其可通过任何本领域常规方法进行测定。在一些实施方式中，所述测定方法是酶联免疫吸附，免疫组织荧光或者免疫组化。在一些实施方式中，所述“细胞增殖速度”由本文公开的方法测定。
C.细胞
1.原料细胞
本文所述的“原料细胞”是指可以用于制备获得诱导性不成熟肝脏细胞的细胞。所述原料细胞包括多能干细胞胚胎干细胞(ESC)、诱导性多能干细胞(iPSC)、间充质干细胞、成纤维细胞、脂肪源性干细胞(ADSC)、神经源性干细胞、血细胞、棘突细胞、肠上皮细胞和其他非肝脏细胞的体细胞。优选地，在一些实施方式中，所述原料细胞是多能干细胞(如：胚胎干细胞、诱导性多能干细胞)。更优选地，在一些实施方式中，所述原料细胞是胚胎细胞或诱导性多能干细胞。
适用于特定实施方式的原料细胞可来源于基本上任何合适的来源，就细胞类型或分化状态而言可以是异质或同质的。优选地，在一些实施方式中，合适的所述原料细胞在来源方面可以是哺乳动物，例 如来自啮齿类动物、猫、犬、猪、山羊、绵羊、马、牛，或灵长类动物，例如人。在一个实施方式中，所述原料细胞是人细胞。
2.诱导性不成熟肝脏细胞
本文所述的术语“诱导性不成熟肝脏细胞”是指一种经诱导得到的不成熟的肝脏细胞，其是非天然存在的。在一些实施方式中，在形态上，诱导性不成熟肝脏细胞虽然具有原代肝脏细胞所特有的多角形，但是相对于原代肝脏细胞，诱导性不成熟肝脏细胞在体外培养条件下更为贴壁而不立体，细胞中基本没有清晰的细胞核，在部分呈现细胞核的细胞中，其核质比也相对较高。
在一些实施方式中，诱导性不成熟肝脏细胞的选自以下的一种或多种肝脏细胞标志物的转录或蛋白表达：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1，是原代肝脏细胞的1％、0.1％、0.01％、0.001％或更少，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，相对于诱导性不成熟肝脏细胞，诱导性成熟肝脏细胞的以下标志物的转录或蛋白表达减少：AFP。在一些实施方式中，诱导性成熟肝脏细胞的以下标志物的转录或蛋白表达：AFP，是原代肝脏细胞的100倍、1000倍、1000倍或更多，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性不成熟肝脏细胞不具有原代肝脏细胞的细胞极性。
在一些实施方式中，诱导性不成熟肝脏细胞的尿素合成能力是原代肝脏细胞的25％、20％、15％、10％、5％、2％、1％或更低，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性不成熟肝脏细胞的下述一种或多种代谢酶的活性：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1，是原代肝脏细胞的10％、5％、2％、1％、0.1％、0.01％、0.001％、0.0001％或更低，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，所述诱导性不成熟肝脏细胞每24小时每百万个细胞的白蛋白分泌量至多约为8μg，更优选地约7μg、6μg、5μg、4μg、3μg、2μg、1μg、0.5μg、0.2μg、0.1μg、0.05μg、0.02μg、0.01μg、0.001μg、0.0001μg或更少，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性不成熟肝脏细胞的移植存活率是原代肝脏细胞的10％、5％、2％、1％、0.5％、0.2％、0.1％、0.01％或更低、或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性不成熟肝脏细胞的体内重建率是原代肝脏细胞的5％、2％、1％、0.5％、0.2％、0.1％、0.01％或更低、或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性不成熟肝脏细胞可以由原料细胞制备得到。在一些实施方式中，诱导性不成熟肝脏细胞可用于制备诱导性成熟肝脏细胞。
诱导性不成熟肝脏细胞可由本领域已知的常规方法制备得到。在一些实施方式中，诱导性不成熟肝脏细胞的制备方法如(Zhu et al.,2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature 508,93-97.10.1038/nature13020.)中所描述的，其全部内容通过引入全文并入本文。在一些优选的实施方式中，所述诱导性不成熟肝脏细胞可以由本发明公开的方法制备得到。在一些实施方式中，由本领域已知的制备诱导性不成熟肝脏细胞的常规方法制备得到的细胞是诱导性不成熟肝脏细胞。
所述诱导性不成熟肝脏细胞在本领域中亦被称为“f-iHep”、“iHep”。在一些现有技术中，诱导性不成熟肝脏细胞也被称为“诱导性肝脏细胞”。
适用于特定实施方式的诱导性不成熟肝脏细胞可来源于基本上任何合适的来源，就细胞类型或分化状态而言可以是异质或同质的。优选地，在一些实施方式中，合适的所述诱导性不成熟肝脏细胞在来源方面可以是哺乳动物，例如来自啮齿类动物、猫、犬、猪、山羊、 绵羊、马、牛，或灵长类动物，例如人。在一个实施方式中，所述诱导性不成熟肝脏细胞是人细胞。
3.诱导性成熟肝脏细胞
本文所述的术语“诱导性成熟肝脏细胞”是指经诱导得到的成熟的肝脏细胞。其是非天然存在的。在一些实施方式中，诱导性成熟肝脏细胞可由本文所述的方法或培养平台制备得到。
在一些实施方式中，诱导性成熟肝脏细胞原代肝脏细胞的典型形态特征。
在一些实施方式中，相对于诱导性不成熟肝脏细胞，诱导性成熟肝脏细胞的选自以下的一种或多种肝脏细胞标志物的转录和/或蛋白表达增加：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1；在一些实施方式中，诱导性成熟肝脏细胞的选自以下的一种和/或多种肝脏细胞标志物的转录或蛋白表达：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1，是诱导性不成熟肝脏细胞的10倍、20倍、30倍、100倍、1000倍或更高，或者任意前述值作为端点构成的范围或其中的任意值。在一些实施方式中，诱导性成熟肝脏细胞的选自以下的一种或多种肝脏细胞标志物的转录或蛋白表达：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1，是原代肝脏细胞的50％、60％、70％、75％、80％、85％、90％、95％、100％、105％、110％、115％、120％、130％、140％或150％，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，相对于诱导性不成熟肝脏细胞，诱导性成熟肝脏细胞的以下标志物的转录或蛋白表达减少：AFP。在一些实施方式中，诱导性成熟肝脏细胞的以下标志物的转录和/或蛋白表达：AFP，是诱导性不成熟肝脏细胞的10％、9％、8％、5％、3％、1％、0.1％、0.01％或更低，或者任意前述值作为端点构成的范围或其中的任意值。在一些实施方式中，诱导性成熟肝脏细胞的以下标志物的转录和/或蛋白表达：AFP，是原代肝脏细胞的50％、60％、70％、75％、80％、85％、 90％、95％、100％、105％、110％、115％、120％、130％、140％、150％、160％，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，相对于诱导性不成熟肝脏细胞，诱导性成熟肝脏细胞具有细胞极性。在一些实施方式中，诱导性成熟肝脏细胞的细胞极性接近、不低于或与原代肝脏细胞相同。
在一些实施方式中，诱导性成熟肝脏细胞的尿素合成能力是诱导性不成熟肝脏细胞的1.1倍、1.2倍、1.3倍、1.4倍、1.5倍、1.6倍、1.7倍、1.8倍、1.9倍、2.0倍、2.1倍、2.2倍、2.3倍、2.4倍、2.5倍、2.6倍、2.7倍、2.8倍、2.9倍、3.0倍、、3.2倍、3.4倍、3.6倍、3.8倍、4.0倍、4.2倍、4.4倍、4.6倍、4.8倍、5.0倍、5.2倍、5.4倍、5.6倍、5.8倍、6.0倍或更多，或者任意前述值作为端点构成的范围或其中的任意值。在一些实施方式中，诱导性成熟肝脏细胞的尿素合成能力是原代肝脏细胞的50％、60％、70％、75％、80％、85％、90％、95％、100％、105％、110％、115％、120％、130％、140％、150％、160％，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性成熟肝脏细胞的下述一种或多种代谢酶的活性：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1，是诱导性不成熟肝脏细胞的10倍、100倍、1000倍、5000倍、10000倍、50000倍、100000倍、500000倍或更多，或者任意前述值作为端点构成的范围或其中的任意值。在一些实施方式中，诱导性成熟肝脏细胞的下述一种或多种代谢酶的活性：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1，是原代肝脏细胞的50％、60％、70％、75％、80％、85％、90％、95％、100％、105％、110％、115％、120％、130％、140％、150％、160％，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，所述诱导性成熟肝脏细胞每24小时每百万个细胞的白蛋白分泌量至少约为8μg，更优选地至少约9μg、10μg、11μg、12μg、13μg、14μg、15μg、16μg、17μg、18μg、19μg、 20μg、21μg、22μg、23μg、24μg、25μg、26μg或更多，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性成熟肝脏细胞的移植存活率是诱导性不成熟肝脏细胞的2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、11倍、12倍、13倍、14倍、15倍、16倍、17倍、18倍、19倍、20倍、21倍、22倍、23倍、24倍、25倍、26倍、27倍、28倍、29倍、30倍、31倍、32倍、33倍、34倍、35倍、36倍、37倍、38倍、39倍、40倍、41倍、42倍、43倍、44倍、45倍、46倍、47倍、48倍、49倍、50倍、51倍、52倍、53倍、54倍、55倍、56倍、57倍、58倍、59倍、60倍、61倍、62倍、63倍、64倍、65倍、66倍、67倍、68倍、69倍、70倍、71倍、72倍、73倍、74倍、75倍、76倍、77倍、78倍、79倍、80倍、81倍、82倍、83倍、84倍、85倍、86倍、87倍、88倍、89倍、90倍、91倍、92倍、93倍、94倍、95倍、96倍、97倍、98倍、99倍、100倍或更多，或者任意前述值作为端点构成的范围或其中的任意值。在一些实施方式中，诱导性成熟肝脏细胞的移植存活率是原代肝脏细胞的50％、60％、70％、75％、80％、85％、90％、95％、100％、105％、110％、115％、120％、130％、140％、150％、160％，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，诱导性成熟肝脏细胞的体内重建率是诱导性不成熟肝脏细胞的2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、11倍、12倍、13倍、14倍、15倍、16倍、17倍、18倍、19倍、20倍、21倍、22倍、23倍、24倍、25倍、26倍、27倍、28倍、29倍、30倍、31倍、32倍、33倍、34倍、35倍、36倍、37倍、38倍、39倍、40倍、41倍、42倍、43倍、44倍、45倍、46倍、47倍、48倍、49倍、50倍、51倍、52倍、53倍、54倍、55倍、56倍、57倍、58倍、59倍、60倍、61倍、62倍、63倍、64倍、65倍、66倍、67倍、68倍、69倍、70倍、71倍、72倍、73倍、74倍、75倍、76倍、77倍、78倍、79倍、80倍、81倍、82 倍、83倍、84倍、85倍、86倍、87倍、88倍、89倍、90倍、91倍、92倍、93倍、94倍、95倍、96倍、97倍、98倍、99倍、100倍或更多，或者任意前述值作为端点构成的范围或其中的任意值。在一些实施方式中，诱导性成熟肝脏细胞的体内重建率是原代肝脏细胞的10％、20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％、100％、105％、110％、115％、120％、130％、140％、150％、160％，或者任意前述值作为端点构成的范围或其中的任意值。
如前所述，根据本发明的诱导性成熟肝脏细胞可以通过以上特征中的一种或多种进行表征。该等表征可以使用本文所述或者本领域技术人员熟知的任一方法进行。在一些实施方式中，该等表征使用本文所述的方法进行。在一些实施方式中，该等表征使用本文实施例所述的方法进行。
为了进一步探索诱导性成熟肝脏细胞的分子生物学特征，还可以利用转录组测序(RNA-seq)分析与诱导性不成熟肝脏细胞相比获得的诱导性不成熟肝脏细胞的转录水平的变化。这可以包括分析成熟肝脏细胞标志基因在诱导性不成熟肝脏细胞和诱导性成熟肝脏细胞中的富集情况(GSEA分析)。经分析发现成熟肝脏细胞标志基因的特异表达可以在根据本发明的诱导性成熟肝脏细胞中显著富集。
可以通过聚类分析来分析起始使用的诱导性不成熟肝脏细胞和根据本发明的诱导性成熟肝脏细胞与原代肝脏细胞的全转录组水平的相似性。
可以通过全转录组的主成分分析(PCA)，确认根据诱导性成熟肝脏细胞接近于原代肝脏细胞。
可以发现，原代肝脏细胞标志基因集可以在根据本发明的诱导性成熟肝脏细胞中显著富集。
可以使用显微镜对获得的诱导性成熟肝脏细胞进行观察，由此可以判断诱导性成熟肝脏细胞的形态。可以发现，根据本发明获得的诱导性成熟肝脏细胞的形态与原代肝脏细胞一致，细胞之间的极性清晰且细胞核质比明显缩小，部分细胞呈现出双核的形态。
在一些特定实施方式中，可使用本文考虑的组合物和方法进行培养、解离和传代一个或多个细胞。在一些实施方式中，使用本文考虑的组合物和方法进行培养、解离和传代单细胞。在一些实施方式中，使用本文考虑的组合物和方法进行培养、解离和传代细胞群或多个细胞。
D.可用于诱导产生诱导性成熟肝脏细胞的培养平台
细胞建库、疾病建模和细胞疗法等应用已对制备高质量的诱导性成熟肝脏细胞提出了持续增加的需求。本发明提供了可用于诱导产生诱导性成熟肝脏细胞的培养平台，其采用特定的组合物。
本发明的组合物、试剂盒、培养基或培养物中的具体化合物的量可以发生变化，可以根据具体培养条件进行优化，包括所用具体分子和组合、在培养基中培养的细胞的类型，以及具体应用。
在另一个方面，本发明提供了一种第一组合物，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂。优选地，在一些实施方式中，所述第一组合物进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂。优选地，在一些实施方式中，所述第一组合物进一步包含：(f)p38 MAPK抑制剂。
在一个方面，本发明提供了一种第一试剂盒，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂、以及相应的使用说明。优选地，所述第一试剂盒进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂。优选地，在一些实施方式中，所述第一试剂盒进一步包含：(f)p38 MAPK抑制剂。
在另一个方面，本发明提供了一种第二组合物，其包含：(a)cAMP激活剂、(b)类固醇类化合物。优选地，在一些实施方式中，所述第二组合物进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
在一个方面，本发明提供了一种第二试剂盒，(a)cAMP激活剂、(b)类固醇类化合物、以及相应的使用说明。优选地，在一些实施方式 中，所述第二试剂盒进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
1.HDAC抑制剂
HDAC(组蛋白去乙酰化酶，histone deacetylase)抑制剂可以是能够抑制HDAC的各种各样的物质。HDAC抑制剂包括，但不限于，Mocetinostat(MGCD0103)、Abexinostat(PCI-24781)、Fimepinostat(CUDC-907)、ITSA-1、Vorinostat(SAHA)、MS-275、Panobinostat(LBH589)、Quisinostat、FK228、SB939、Trichostatin A(TSA)。优选地，在一些实施方式中，HDAC抑制剂是MS-275，如下式所示
2.类固醇类化合物
类固醇类化合物是指由四环结构的甾族化合物衍生出来的各种各样的物质。类固醇类化合物包括，但不限于，泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、泼尼松龙、氢化可的松、地塞米松。优选地，在一些实施方式中，类固醇类化合物是地塞米松，如下式所示
3.Notch信号通路抑制剂
Notch信号通路抑制剂可以是能够抑制Notch信号通路的各种各样的物质。Notch信号通路抑制剂包括，但不限于，IMR-1、RO4929097、 Semagacestat(LY450139)、Avagacestat(BMS-708163)、Dibenzazepine(YO-01027)、Crenigacestat(LY3039478)、Compound E、DAPT。优选地，在一些实施方式中，Notch信号通路抑制剂、Compound E，如下式所示
4.ERK信号通路抑制剂
ERK信号通路抑制剂可以是能够抑制ERK信号通路的各种各样的物质。ERK信号通路抑制剂包括，但不限于，SeluMetinib(AZD6244)、Trametinib(GSK1120212)、PD0325901、U0126-ETOH、PD184352(CI-1040)。优选地，在一些实施方式中，ERK信号通路抑制剂是PD0325901，如下式所示
5.TGF-beta信号通路抑制剂
TGF-beta信号通路抑制剂可以是能够抑制ALK的各种各样的物质。TGF-beta信号通路抑制剂包括，但不限于，SD-208、LY2109761、BIBF-0775、A83-01、SB431542。优选地，在一些实施方式中，TGF-beta信号通路抑制剂是A83-01，如下式所示
6.p38 MAPK抑制剂
p38 MAPK抑制剂可以是能够抑制p38 MAPK的各种各样的物质。p38 MAPK抑制剂包括，但不限于，SB202190(FHPI)、SB203580、TAK-715、Doramapimod(BIRB 796)。优选地，在一些实施方式中，p38 MAPK抑制剂是SB203580，如下式所示
7.cAMP激活剂
cAMP激活剂可以是能够激活cAMP的各种各样的物质。示例性cAMP激活剂包括，但不限于，Forskolin、8-Br-cAMP。优选地，在一些实施方式中，cAMP激活剂是Forskolin，如下式所示
8.ROCK抑制剂
cAMP激活剂可以是能够抑制ROCK的各种各样的物质。示例性ROCK抑制剂包括，但不限于，Y-27632、Thiazovivin、GSK429286A、RKI-1447、AT13148。优选地，在一些实施方式中，ROCK抑制剂是Y-27632，如下式所示
9.组分的量
第一组合物
在一些实施方式中，HDAC抑制剂以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，HDAC抑制剂以0.01、0.02、0.03、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.4、1.6、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8、8.0、8.2、8.4、8.6、8.8、9.0、9.2、9.4、9.6、9.8、10.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第一培养基或培养物中。优选地，HDAC抑制剂以0.01-1μM，优选0.1-1μM，更优选1.0μM的浓度存在于本发明的第一培养基或培养物中。最优选地，在一个实施方式中，所述HDAC抑制剂是MS-275，其以0.2μM的浓度存在于本发明的第一培养基或培养物中。
在一些实施方式中，类固醇类化合物以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，类固醇类化合物以0.01、0.02、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、2.0、2.2、2.4、2.6、 2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0μM或前述数值中的任意两者组成的范围的浓度存在于本发明的第一培养基或培养物中。优选地，类固醇类化合物以0.1-4.0μM，优选0.2-2.0μM，更优选0.1μM的浓度存在于本发明的第一培养基或培养物中。最优选地，在一个实施方式中，所述类固醇类化合物是地塞米松，其以0.1μM的浓度存在于本发明的第一培养基或培养物中。
在一些实施方式中，Notch信号通路抑制剂以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，Notch信号通路抑制剂以0.01、0.02、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第一培养基或培养物中。优选地，Notch信号通路抑制剂以0.1-4.0μM，优选0.2-2.0μM，更优选0.1μM的浓度存在于本发明的第一培养基或培养物中。最优选地，在一个实施方式中，所述Notch信号通路抑制剂是Compound E，其以0.1μM的浓度存在于本发明的第一培养基或培养物中。
在一些实施方式中，ERK信号通路抑制剂以足以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，ERK信号通路抑制剂以0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.4、1.6、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8、8.0、8.2、8.4、8.6、8.8、9.0、9.2、9.4、9.6、9.8、10.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第一培养基或培养物中。优选地，ERK信号通路抑制剂以0.05-5μM，优选0.1-2μM，更优选1.0μM的浓度存在于本发明的第一培养基或培养物中。最优 选地，在一个实施方式中，所述ERK信号通路抑制剂是PD0325901，其以1.0μM的浓度存在于本发明的第一培养基或培养物中。
在一些实施方式中，TGF-beta信号通路抑制剂以足以单独或与其他物质来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，TGF-beta信号通路抑制剂以0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.4、1.6、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8、8.0、8.2、8.4、8.6、8.8、9.0、9.2、9.4、9.6、9.8、10.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第一培养基或培养物中。优选地，TGF-beta信号通路抑制剂以0.05-5μM，优选0.1-2μM，更优选0.5μM的浓度存在于本发明的第一培养基或培养物中。最优选地，在一个实施方式中，所述TGF-beta信号通路抑制剂是A83-01，其以0.5μM的浓度存在于本发明的第一培养基或培养物中。
在一些实施方式中，p38 MAPK抑制剂以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，p38 MAPK抑制剂以0.01、0.05、0.1、0.5、1.0、2.0、3.0、4.0、5.0、6.0、7.0、7.5、8.0、8.5、9.0、9.5、10.0、12.0、14.0、16.0、18.0、20.0、30.0、40.0、50.0μM或前述数值中的任意两者组成的范围的浓度存在于本发明的组合物、试剂盒、培养基或培养物中。优选地，p38 MAPK抑制剂以1.0-50.0μM，优选2.0-40.0μM，更优选30.0μM的浓度存在于本发明的第一培养基或培养物中。最优选地，在一个实施方式中，所述p38 MAPK抑制剂是SB203580，其以30μM的浓度存在于本发明的第一培养基或培养物中。
优选地，在一些实施方式中，所述第一组合物中，所述HDAC抑制剂是MS-275、所述类固醇类化合物是地塞米松、所述Notch信号通路抑制剂是Compound E、所述ERK信号通路抑制剂是 PD0325901、所述TGF-beta信号通路抑制剂是A83-01。更优选地，在一些实施方式中，MS-275在本发明的第一培养基或培养物中的浓度为0.2μM，地塞米松在本发明的第一培养基或培养物中的浓度为0.1μM，Compound E在本发明的第一培养基或培养物中的浓度为0.1μM，PD0325901在本发明的第一培养基或培养物中的浓度为1.0μM，A83-01在本发明的第一培养基或培养物中的浓度为0.5μM。
优选地，在一些实施方式中，所述第一组合物中进一步包含p38 MAPK抑制剂。优选地，在一些实施方式中，所述HDAC抑制剂是MS-275、所述类固醇类化合物是地塞米松、所述Notch信号通路抑制剂是Compound E、所述ERK信号通路抑制剂是PD0325901、所述TGF-beta信号通路抑制剂是A83-01、所述p38 MAPK抑制剂是SB203580。更优选地，在一些实施方式中，MS-275在本发明的第一培养基或培养物中的浓度为0.2μM，地塞米松在本发明的第一培养基或培养物中的浓度为0.1μM，Compound E在本发明的第一培养基或培养物中的浓度为0.1μM，PD0325901在本发明的第一培养基或培养物中的浓度为1.0μM，A83-01在本发明的第一培养基或培养物中的浓度为0.5μM，SB203580在本发明的第一培养基或培养物中的浓度为30.0μM。
第二组合物
在一些实施方式中，cAMP激活剂以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，cAMP激活剂以0.01、0.05、0.1、0.5、1.0、2.0、3.0、4.0、5.0、6.0、7.0、7.5、8.0、8.5、9.0、9.5、10.0、12.0、14.0、16.0、18.0、20.0、25.0、30.0、35.0、40.0、45.0、50.0、60.0、70.0、80.0、90.0、100.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第二培养基或培养物中。优选地，所述cAMP激活剂以1.0-50.0μM，优选10.0-40.0μM，更优选30.0μM的浓度存在于本发明的第二培养基或培养物中。 最优选地，在一个实施方式中，所述cAMP激活剂是Forskolin，其以30.0μM的浓度存在于本发明的第二培养基或培养物中。
在一些实施方式中，ROCK抑制剂以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，ROCK抑制剂以0.01、0.05、0.1、0.5、1.0、2.0、3.0、4.0、5.0、6.0、7.0、7.5、8.0、8.5、9.0、9.5、10.0、12.0、14.0、16.0、18.0、20.0、30.0、40.0、50.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第二培养基或培养物中。优选地，所述ROCK抑制剂以1.0-50.0μM，优选10.0-40.0μM，更优选10.0μM的浓度存在于本发明的第二培养基或培养物中。最优选地，在一个实施方式中，所述ROCK抑制剂是Y-27632，其以10.0μM的浓度存在于本发明的第二培养基或培养物中。
在一些实施方式中，TGF-beta信号通路抑制剂以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合物、试剂盒、培养基或培养物中。在一些实施方式中，TGF-beta信号通路抑制剂以0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.4、1.6、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8、8.0、8.2、8.4、8.6、8.8、9.0、9.2、9.4、9.6、9.8、10.0μM或更高或前述数值中的任意两者组成的范围的浓度存在于本发明的第二培养基或培养物中。优选地，所述TGF-beta信号通路抑制剂以0.05-5.0μM，优选0.1-2.0μM，更优选0.5μM的浓度存在于本发明的第二培养基或培养物中。最优选地，在一个实施方式中，所述TGF-beta信号通路抑制剂是A83-01，其以0.5μM的浓度存在于本发明的第二培养基或培养物中。
在一些实施方式中，类固醇类化合物以足以单独或与其他物质组合来诱导制备诱导性成熟肝脏细胞的量或浓度存在于本发明的组合 物、试剂盒、培养基或培养物中。在一些实施方式中，类固醇类化合物以0.01、0.02、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0μM或前述数值中的任意两者组成的范围的浓度存在于本发明的第二培养基或培养物中。优选地，类固醇类化合物以0.1-4.0μM，优选0.2-2.0μM，更优选0.1μM的浓度存在于本发明的第二培养基或培养物中。最优选地，在一个实施方式中，所述类固醇类化合物是地塞米松，其以0.1μM的浓度存在于本发明的第二培养基或培养物中。
在一些实施方式中，HDAC抑制剂有助于制备诱导性成熟肝脏细胞。优选地，所述HDAC抑制剂是MS-275。
在一些实施方式中，优选地，所述第二组合物中，所述cAMP激活剂是Forskolin、所述ROCK抑制剂是Y-27632、所述TGF-beta信号通路抑制剂是A83-01、所述类固醇类化合物是地塞米松。更优选地，Forskolin在本发明的第二培养基或培养物中的浓度为30.0μM，Y-27632在本发明的第二培养基或培养物中的浓度为10.0μM，A83-01在本发明的第二培养基或培养物中的浓度为0.5μM，地塞米松在本发明的第二培养基或培养物中的浓度为0.1μM。
本文所述的部分物质的CAS号如下：


本领域技术人员能够理解，上述物质的相应的药学上可接受的盐也能够应用于本发明中，同时也在本发明的保护范围内。
E.培养基
在一个方面，本发明提供了一种第一培养基。在一个方面，本发明提供了一种第二培养基。在一些方面，本文所述的第一培养基和第二培养基可以用于制备本文所述的诱导性成熟肝脏细胞。
在一些实施方式中，本发明的培养基包含基础培养基。示例性基础培养基包括，但不限于，DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E。在一些实施方式中，所述基础培养基选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E。所述基础培养基可以市售购买。
在一些方面，本发明提供了一种试剂盒，其包含HEM-1培养基和HEM-2培养基，其中所述HEM-1培养基包含ROCK抑制剂、肝脏细胞生长因子(HGF)、抑瘤素M(oncostatinM,OSM)、类固醇类化合物、Notch信号通路抑制剂、TGF-beta信号通路抑制剂和去除表皮细胞生长因子(EGF)的HCM培养基；所述HEM-2培养基包含肝脏细胞生长因子(HGF)、抑瘤素M(oncostatinM,OSM)、类固醇类化合物、Notch信号通路抑制剂、TGF-beta信号通路抑制剂和去除表皮细胞生长因子 (EGF)的HCM培养基。优选地，所述ROCK抑制剂是Y-27632；所述类固醇类化合物是地塞米松；所述Notch信号通路抑制剂是Compound E；所述TGF-beta信号通路抑制剂是A83-01。优选地，在一些实施方式中，ROCK抑制剂以0.01、0.05、0.1、0.2、0.4、0.6、0.8、1.0μM，或者任意前述值作为端点构成的范围或其中的任意值，存在于HEM-1培养基中。更优选地，在一些实施方式中，所述ROCK抑制剂是Y-27632，且以10.0μM存在于HEM-1培养基中。优选地，在一些实施方式中，肝脏细胞生长因子以1、2、4、6、8、10、15、20、25、30、35ng/ml，或者任意前述值作为端点构成的范围或其中的任意值，存在于HEM-1培养基或HEM-2培养基中。更优选地，在一些实施方式中，所述肝脏细胞生长因子以20ng/ml存在于HEM-1培养基或HEM-2培养基中。优选地，在一些实施方式中，抑瘤素M以1、2、4、6、8、10、15、20、25、30、35ng/ml，或者任意前述值作为端点构成的范围或其中的任意值，存在于HEM-1培养基或HEM-2培养基中。更优选地，在一些实施方式中，所述抑瘤素M以20ng/ml存在于HEM-1培养基或HEM-2培养基中。优选地，在一些实施方式中，类固醇类化合物以0.01、0.05、0.1、0.2、0.4、0.6、0.8、1.0μM，或者任意前述值作为端点构成的范围或其中的任意值，存在于HEM-1培养基或HEM-2培养基中。更优选地，在一些实施方式中，所述类固醇类化合物是地塞米松，且以0.1μM存在于HEM-1培养基或HEM-2培养基中。优选地，在一些实施方式中，Notch信号通路抑制剂以0.01、0.05、0.1、0.2、0.4、0.6、0.8、1.0μM，或者任意前述值作为端点构成的范围或其中的任意值，存在于HEM-1培养基或HEM-2培养基中。更优选地，在一些实施方式中，所述Notch信号通路抑制剂是Compound E，且以0.1μM存在于HEM-1培养基或HEM-2培养基中。优选地，在一些实施方式中，TGF-beta信号通路抑制剂以0.01、0.05、0.1、0.2、0.4、0.6、0.8、1.0μM，或者任意前述值作为端点构成的范围或其中的任意值，存在于HEM-1培养基或HEM-2培养基中。更优选地，在一些实施方式中，所述TGF-beta信号通路抑 制剂是A83-01，且以0.5μM存在于HEM-1培养基或HEM-2培养基中。
在一些实施方式中，如本文所述的培养基包含胎牛血清(Fetal Bovine Serum,FBS)，胎牛血清的含量可以根据本领域技术人员的知识进行常规调整。优选地，在一些实施方式中，如本文所述的第二培养基的胎牛血清的含量为培养基总体积的0.05％、0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1.0％、2.0％、3.0％、4.0％、5.0％、6.0％、7.0％、8.0％、9.0％、10.0％、11.0％、12.0％、13.0％、14.0％、15.0％、16.0％、17.0％、18.0％、19.0％、20.0％或者任意前述值作为端点构成的范围或其中的任意值。优选地，在一些实施方式中，第二培养基中的胎牛血清的含量为第二培养基总体积的0.05％～10％，更优选地为1％。优选地，在一些实施方式中，所述第一培养基不含有血清。
在特定实施方式中，本发明的培养基含有细胞因子和/或生长因子。在特定实施方式中，本发明的培养基基本上不含或不含细胞因子和/或生长因子。在某些实施方式中，培养基含有一种或多种补充物，包括但不限于血清、提取物、生长因子、激素、细胞因子等等。在一些实施方式中，本文所述的培养基包含：青霉素/链霉素，非必需氨基酸，L-丙氨酰-L-谷氨酰胺或L-谷氨酰胺，烟酰胺，N-乙酰-L-半胱氨酸。
任何合适的容器或细胞培养容器均可用作基础培养基和/或细胞培养补充物中的细胞培养的支持物。在支持物上的基质涂层不是必要的。然而，用贴壁促进基质(例如胶原、纤连蛋白、含RGD多肽、明胶等等)涂布培养容器的表面促进细胞的附着，并且在特定实施方式中，可增强本文公开的培养基和补充物的效应。用于培养和传代细胞的合适基质是本领域已知的，并且包括但不限于玻连蛋白、明胶、层粘连蛋白、纤连蛋白、胶原、弹性蛋白、骨桥蛋白、天然存在的细胞系生产的基质的混合物例如MatrigelTM以及合成或人造表面例如聚胺单层和羧基封端单层。
F.细胞制备方法
在一个方面，本发明提供了一种制备诱导性成熟肝脏细胞的方法，所述方法包括在培养基中培养诱导性不成熟肝脏细胞，由此制备所述诱导性成熟肝脏细胞。在一些实施方式中，所述培养基是本文所述的第一培养基和第二培养基。在一个方面，本发明提供了一种制备诱导性成熟肝脏细胞的方法，所述制备方法包括使诱导性不成熟肝脏细胞与本文所述的第一组合物或第二组合物接触，由此制备所述诱导性成熟肝脏细胞。
在一些实施方式中，使用本文所述的第一组合物或第一培养基培养诱导性不成熟肝脏细胞，由此制备诱导性成熟肝脏细胞。在一些实施方式中，使用所述第一组合物、第一培养基培养诱导性不成熟肝脏细胞的培养时间为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30天，或者任意前述值作为端点构成的范围或其中的任意值。优选地，培养时间为5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20天，或者任意前述值作为端点构成的范围或其中的任意值。更优选地，培养时间为6、7、8、10、11、12、13、14、15、16、17、18、19或20天，或者任意前述值作为端点构成的范围或其中的任意值。优选地，在一些实施方式中，进一步使用本文所述的第二组合物或第二培养基培养上述经第一组合物或第一培养基培养后的细胞，以获得诱导性成熟肝脏细胞。在一些实施方式中，使用所述第二组合物或第二培养基进行培养，培养时间为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、45、60、90、120天、150天、180天、270天、360天或更长，或者任意前述值作为端点构成的范围或其中的任意值。优选地，培养时间为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、 37、38、39、40、45、60、90、120天、150天，或者任意前述值作为端点构成的范围或其中的任意值。更优选地，培养时间为30、31、32、33、34、35、36、37、38、39、40、45、60、90天，或者任意前述值作为端点构成的范围或其中的任意值。
在一些实施方式中，本发明的方法包括使用本文所述的方法在培养基中培养并诱导多能干细胞，由此制备所述的诱导性不成熟肝脏细胞。
用作本发明的方法的起始细胞是诱导性不成熟肝脏细胞，所述诱导性不成熟肝脏细胞可以由如本文所述的各种各样的原料细胞制备得到。例如，可以由多能干细胞例如胚胎干细胞或诱导性多能干细胞制备得到诱导性不成熟肝脏细胞。
在一些实施方式中，所述多能干细胞是胚胎干细胞或诱导性多能干细胞。
在一些实施方式中，所述方法包括将非多能性细胞重编程为多能干细胞。在一些实施方式中，所述非多能性细胞选自体细胞和/或成体干细胞。
在一些实施方式中，所述将非多能性细胞重编程为多能干细胞包括在所述非多能性细胞内表达选自Oct4、Sox2、Klf4和c-Myc中的一种或多种重编程因子。
到目前为止，还没有关于扩增诱导性不成熟肝脏细胞的方法的报道。
在一些方面，本发明提供了一种扩增诱导性不成熟肝脏细胞的方法，其包括使用本发明的HEM-1培养基或HEM-2接触本文所述的诱导性不成熟肝脏细胞。优选地，诱导性不成熟肝脏细胞通过本文所述的方法制备得到。优选地，使用本发明的HEM-1培养基培养诱导性不成熟肝脏细胞1天后，再使用HEM-2培养基连续培养7天。更优选地，每两天进行换液(即更新培养基)。
在一些实施方式中，根据本发明提供的发明扩增诱导性不成熟肝脏细胞可使得所述诱导性不成熟肝脏细胞维持其分化状态。在一些实 施方式中，根据本发明提供的发明扩增诱导性不成熟肝脏细胞可使得所述诱导性不成熟肝脏细胞每7、6、5、4.5、4、3.5、3、2.5、2、1.5或1天，或者任意前述值作为端点构成的范围或其中的任意值，扩增一倍。
G.培养物
在一个方面，本发明提供了一种培养物，其包含本文所述的培养基和诱导性成熟肝脏细胞。在一些实施方式中，所述培养基包含本文所述的第一培养基、第二培养基。
包含在本文所述的培养物中的细胞可以是如本文所述的各种各样的细胞。例如，所述细胞可以是用于本文所述的培养或诱导的起始细胞如诱导性不成熟肝脏细胞、原料细胞如多能干细胞(如：胚胎干细胞、诱导性多能干细胞)。或者，所述细胞可以是本文所述的培养或诱导产生的中间细胞或最终细胞。所述中间细胞可以是具有不同于起始细胞和最终细胞的各种各样发育潜能的细胞。所述最终细胞可以是如本文所述的诱导性成熟肝脏细胞。
在一些实施方式中，所述多能干细胞是胚胎干细胞或诱导性多能干细胞。
在一些实施方式中，根据本发明的培养物包含本文所述的培养基和多能干细胞。
在一些实施方式中，所述诱导性不成熟肝脏细胞由多能干细胞制备获得。在一些实施方式中，所述多能干细胞是胚胎干细胞。在一些实施方式中，所述多能干细胞是诱导性多能干细胞。
优选地，在一些实施方式中，所述诱导性不成熟肝脏细胞可通过本文所述的方法制备。
在一些实施方式中，所述多能干细胞可以由本领域已知的方法获得。在一些实施方式中，所述诱导性不成熟肝脏细胞可以由本领域已知的方法获得。
H.应用
根据本发明的诱导性成熟肝脏细胞可以用于科研、产业和临床上期望的各种各样的用途。例如，制备用于肝脏细胞移植的细胞、构建肝脏相关疾病模型、肝脏疾病相关药物筛选与鉴定，药物肝脏毒性筛选与鉴定，体外构建肝脏器官(含3D打印肝脏器官)或其他潜在科研、治疗和诊断应用。
根据本发明的诱导性成熟肝脏细胞可以用于诱导产生类器官(organoid)。所述类器官可用于构建疾病模型、移植治疗或其他潜在科研、治疗和诊断应用。在一个方面，本发明提供了一种类器官，其产生自本文所述的诱导性成熟肝脏细胞。所述类器官可以是包括但不限于肝脏。
根据本发明的诱导性成熟肝脏细胞可以用于诱导产生组织(tissue)，例如3D打印肝脏。所述组织可用于制备用于肝脏细胞移植的细胞、构建肝脏相关疾病模型、肝脏疾病相关药物筛选与鉴定，药物肝脏毒性筛选与鉴定，体外构建肝脏器官(含3D打印肝脏器官)或其他潜在科研、治疗和诊断应用。在一个方面，本发明提供了一种组织，其产生自本文所述的诱导性成熟肝脏细胞。所述组织包括但不限于肝脏。
本说明书中引用的所有公开文件、专利申请和授权专利以引用的方式并入本文，如同每个个体公开文件、专利申请或授权专利特别且各自指出以引用的方式并入。
尽管本发明已通过举例说明和例子略微详细地进行描述用于明确理解的目的，但对于本领域普通技术人员显而易见的是，根据本发明的教导，可对其作出某些改变和修饰，而不背离所附权利要求的精神或范围。提供下述实施例仅作为例子而不是限制。本领域技术人员容易认识到可改变或修饰以获得基本上相似的结果的各种非关键参数。
实施例
以下实施例作为例子提供，不限制本发明的范围。本领域技术人员容易认识到可改变或修饰以获得基本上相似的结果的各种非关键参数。
以下实施例中使用的培养基组成如表1



实施例1:诱导性成熟肝脏细胞的诱导与产生
在37℃，5％CO2的培养箱中，用mTeSR(STEMCELL，5850)将胚胎干细胞H9(Wicell)培养于被Matrigel(Corning，354248)处理过的细胞培养皿中，待细胞生长至相对于培养孔底面积的密度为90％时准备进行分化。去掉培养皿中原有的培养基，然后用DPBS(Invitrogen,14190367)润洗一次。加入D1培养基，在37℃，5％CO2的培养箱中培养24小时。
去掉D1培养基并加入D2培养基，在37℃，5％CO2的培养箱中培养24小时。
去掉D2培养基并加入D3培养基，在37℃，5％CO2的培养箱中培养72小时，得到内胚层细胞。
去掉D3培养基，并加入HDM培养基，在37℃，5％CO2的培养箱中培养96小时，期间培养48小时的时候进行一次换液，由此将内胚层细胞分化为肝干细胞。
之后，通过以下步骤将得到的肝干细胞分化为细胞1：去掉HDM培养基，并加入HCM诱导培养基，在37℃，5％CO2的培养箱中培养10-14天，期间每48小时进行一次换液，由此得到细胞1。
将上述所得的细胞1用AccutaseTM(STEMCELL)在37℃消化3分钟，之后将其传代(传代比例为1：4)到被Matrigel处理过的培养皿中。使用如下培养基1和培养基2在37℃，5％CO2的培养箱中对得到的细胞1进行培养和扩增。使用培养基1培养1天，之后换用培养基2培养7天，期间每48小时换液一次。
去掉HEM培养基，并加入5C培养基或6C培养基，在37℃，5％CO2的培养箱中培养10～15天，期间每48小时换液一次，由此得到细胞2。
结果如图1所示，经5C培养基或6C培养基培养得到的细胞2呈现出原代肝脏细胞的典型形态特征。细胞之间的极性变得清晰，而且细胞的核质比变得小，部分细胞呈现出了双核。
进一步使用经6C培养基培养得到的细胞2，去掉6C培养基，使用如下4C培养基在37℃，5％CO2的培养箱中继续培养60天，期间每72小时换液一次，由此得到细胞3。
结果如图1所示，细胞3呈现出了与原代肝脏细胞相一致的形态特征。与细胞1相比，细胞3之间的极性变得更加清晰，而且细胞的核质比变得越来越小，部分细胞呈现出了双核。
本实施例1中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。实施例中的细胞2和细胞3属于本发明所述的诱导性成熟肝脏细胞。本实施例1中的培养基1属于本发明所述的HEM-1培养基。培养基2属于本发明所述的HEM-2培养基。本实施例1中的5C培养基和6C培养基均属于本发明所述的第一培养基，4C培养基属于本发明所述的第二培养基。
实施例2:测定诱导性成熟肝脏细胞中肝脏成熟相关标记基因的 表达
收取实施例1中的细胞1(作为对照)、细胞2和细胞3。以及收取经DMSO平行处理的细胞1(即细胞2对照(DMSO)和细胞3对照(DMSO))作为对照，然后使用Trizol试剂盒(Gibico)，按照试剂盒中的方法分别提取总mRNA。之后利用RT-qPCR方法(Bio-rad)检测所有收取的mRNA中的肝脏成熟相关基因的表达。首先按照Axcygen mRNA提取试剂盒(AP-MN-250)提取细胞中的总RNA；之后利用Bio-Rad的iScript cDNA synthesis试剂盒(1708890)将1μg-2μg的mRNA反转为cDNA；然后利用Bio-Rad的SYBR Green Supermix(1708882AP)在Biorad的C1000仪器上展开qRT-PCR实验。实验中所用到的引物如下表(表1)所示。
表1：RT-qPCR引物序列


“*”所标注的引物选自已经发表的文献(Zhu et al.,2014 Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature 508,93-97.10.1038/nature13020.)。
结果如图2A所示。通过RT-qPCR的实验结果可以看出，相对于细胞1，细胞2和细胞3的CYP1A2基因和CYP3A4基因的表达有了近30倍和近100倍的提高，酪氨酸代谢相关的TAT基因的表达也有了近100倍的提升。此外，细胞2和细胞3的胆酸代谢相关基因CYP7A1和白蛋白(ALB)基因的表达增加，细胞2和细胞3的胎肝相关的AFP基因的表达降低。
发明人还通过免疫荧光的方法检测了成熟相关标记基因的表达，所用的免疫荧光检测方法如下：用DPBS将上述细胞1、细胞2和细胞3的润洗三遍，之后利用4％的多聚甲醛(sigma，P6148)将细胞在室温固定30分钟；去掉4％的多聚甲醛，加入0.1％Triton-100(0.1体积％的Triton-100溶于PBS中)室温处理30分钟；之后去掉0.1％的Triton-100，并加入3.5％的BSA处理2小时；用封闭液(含有3重量％的BSA的PBS溶液)稀释一抗抗体(此处即抗白蛋白抗体(Bethyl,A80-129A)、抗HNF4alph抗体(Cell Signaling Technology,3113s)、抗AFP抗体(Cell Signaling Technology,4448)、抗CK18抗体(Abcam,ab82254)、抗CYP1A2抗体(BIO-RAD,AHP622Z)、抗CYP3A4抗体(BIO-RAD,AHP610Z)、抗CYP2D6抗体(Sigma-Aldrich,AV41675)和抗CYP29抗体(BIO-RAD,AHP617Z)，并在4℃条件下孵育过夜，其 中抗白蛋白抗体的稀释比例为1:3000，抗HNF4alpha抗体、抗AFP抗体、抗CK18抗体、抗CYP1A2抗体、抗CYP3A4抗体、抗CYP2D6抗体和抗CYP29抗体稀释比例为1:1000；洗去一抗，用封闭液润洗三遍，每次15分钟；加入二抗(donky anti-rabbit 555/donky anti-goat488(1:1000,invitrogen))，室温孵育2小时；去掉二抗，DPBS润洗三遍，加入DAPI(Roche)对细胞核进行染色，之后利用荧光显微镜(Leica)观察染色的效果。
结果如图2B所示，细胞2和细胞3可以表达多个与肝脏成熟相关的标记基因。
本实施例2中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。实施例中的细胞2和细胞3属于本发明所述的诱导性成熟肝脏细胞。
实施例3对诱导性成熟肝脏细胞的肝脏极性和功能的分析
发明人进一步对实施例1中获得细胞1(作为对照)、细胞2和细胞3的肝脏极性以及肝脏相关功能进行了测试，实验方法如下：利用20μM的CDFDA(5-(和-6)-羧基-2’,7’-二氯荧光素(sigma,21884)对细胞1、细胞2和细胞3染色30分钟后，用HBSS(Gibico)清洗3遍，之后在荧光显微镜下观察。以此，测定细胞的肝脏极性。
利用CYP450活性检测试剂盒(Luciferin-IPA,V8421和V9001)，按照该试剂盒中所述的方法，对细胞中的CYP1A2和CYP3A4活性进行检测。利用ALB ELISA检测试剂盒(E80-129；Bethyl)和尿素检测试剂盒(BioAssay Systems)，按照该试剂盒中所述的方法，检测细胞分泌白蛋白和合成尿素的能力。
结果如图3所示，相比于细胞1，细胞2和细胞3的细胞极性具有显著提升，并与原代肝脏细胞的极性相近(图3A，从图3A中可以看出，细胞2和细胞3中的荧光染料能够在细胞内流动转运，即能够从细胞的一侧运输到细胞的另一侧；而细胞1中的荧光染料则没有表现出流动性，这说明细胞2和细胞3的细胞极性相对于细胞2和细胞 3具有显著改善)。相比于细胞1，细胞2和细胞3的CYP1A2和CYP3A4代谢酶活力具有显著提升(图3D)。细胞2和细胞3的白蛋白分泌能力分别达到了10μg/1百万细胞/24小时以上和20μg/1百万细胞/24小时，接近甚至超过原代肝脏细胞的白蛋白分泌能力(图3B)。相比于细胞1，细胞2和细胞3的尿素合成能力也有了显著提升，接近了已报道的原代肝脏细胞水平(图3C)。
本实施例3中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。实施例中的细胞2和细胞3属于本发明所述的诱导性成熟肝脏细胞。
实施例4诱导性成熟肝脏细胞在慢性肝脏损伤小鼠FRG模型中 的体内重建率，并诱导性成熟肝脏细胞能够有效缓解和改善小鼠的肝 脏损伤情况和存活率
发明人还测试了实施例1中获得的细胞1,、细胞2和细胞3在肝伤小鼠FRG(如Grompe M,Strom S.Mice with hμMan livers.Gastroenterology.2013 Dec；145(6):1209-14.doi:10.1053/j.gastro.2013.09.009.Epub 2013 Sep 13.PMID:24042096中所描述的)中的体内重建率和对该小鼠的肝脏损伤改善情况和存活率的影响。
实验方案如下：该模型小鼠在饲养条件下，需将尼替西农(Nitisinone，selleck，S5325，CAS号为104206-65-7)以16mg/ml的浓度添加到日常的饮用水中。移植前1天，停止在饮用水中添加尼替西农，同时给小鼠注射Ad-uPA(Yecuris，20-0029)病毒，注射量为每克小鼠的体重注射5*107pfu的Ad-uPA病毒。24小时之后，在异氟烷的麻醉下，在腹部左侧切口，脾内注射肝脏细胞。
其中，12只小鼠接受了成人原代肝脏细胞注射，用量为每只小鼠50万细胞。13只小鼠接受了细胞1注射，21只小鼠接受了细胞2注射，12只小鼠接受了细胞3注射，用量均为每只小鼠100万细胞。
移植术后，当小鼠体重下降20％时，小鼠接受3-4天的NTBC治疗。之后，监测小鼠的状态，记录小鼠的死亡天数。
结果如图4D所示。相比于细胞1，细胞2和细胞3移植后小鼠的存活率显著升高，并与成人原代肝脏细胞移植后的存活率基本相同。
此外，通过Cobas的501仪器，并按照仪器说明书测定谷草转氨酶(AST)和谷丙转氨酶(ALT)的血液表达量。
结果如图4E所示。相比于细胞1，细胞2和细胞3移植后小鼠的谷草转氨酶(AST)和谷丙转氨酶(ALT)的血液表达量显著降低。
发明人还通过免疫组织荧光的方法测试了移植后小鼠肝脏的体内重建率，实验方案如下：将组织石蜡包埋后，按照5μm的厚度对其进行切片。切片后的样本用二甲苯脱蜡3次，每次5分钟；之后使用下列试剂进行梯度复水：100％乙醇，100％乙醇，90％乙醇，80％乙醇，70％乙醇，50％乙醇，各5分钟。之后使用去离子水浸泡1分钟；然后用pH＝6.0的柠檬酸钠进行抗原修复，之后浸泡在PBS中备用；在样品周围用ImmunoPen画圈，用3％的BSA-PBS室温孵育封闭20分钟，之后用PBS洗3次；加一抗，其中ALB(Bethyl,A80-229A)，CYP1A2(BIO-RAD,AHP622Z)，CYP3A4(BIO-RAD,AHP610Z)均按照1:1000的比例稀释，将切片放在湿盒内，封好，4度过夜；PBS洗三次，每次5分钟；加二抗驴抗羊488IgG(invitrogen，1:1000)和驴抗兔555IgG(Invitrogen，1:1000)，室温孵育30分钟。PBS洗三次，每次5分钟。DAPI(1:3000)染色10min，之后用中性树脂封片保存，之后镜下观察。
结果如图4B所示，细胞3具有与原代肝脏细胞类似的体内重建率。同时，从图4B中能够看出，移植后的原代肝脏细胞与细胞3均能够均匀分布在肝脏实质中，而非仅仅主要集中在肝门静脉处，由此说明细胞3具有优异的肝脏内迁移能力。从图4C中看出，细胞3增殖形成的再生小团充分表达一系列成熟肝脏细胞的特异表达基因。
发明人还通过ALB ELISA检测试剂盒(E80-129；Bethyl)，按照试剂盒中所述方法，检测移植后不同天数的小鼠血液中的白蛋白浓度，由此测定人源白蛋白起峰时间。
结果如图4A所示，细胞3移植后，小鼠血液中的白蛋白浓度的升高速度与原代肝脏细胞移植后基本相同，人源白蛋白起峰时间与原代肝脏细胞移植后基本相同。综上所述，细胞3移植后，可以迁移到肝实质区，并在肝脏损伤信号的刺激下，迅速自动进入增殖的状态，形成多个肝脏再生小团。通过免疫荧光染色发现，所形成的肝脏再生小团可以充分清晰地高表达一系列成熟肝脏细胞的特异表达基因。上述实验结果之前只在移植人源肝脏细胞的实验样本中观察到，因此也充分说明了本实验诱导的细胞3是一种真正具有肝脏细胞再生能力的诱导肝脏细胞。
本实施例4中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。实施例中的细胞2和细胞3属于本发明所述的诱导性成熟肝脏细胞。
实施例5使用HEM-1培养基和HEM-2培养基扩增诱导性不成 熟肝脏细胞
发明人还测试了使用本发明的HEM培养基扩增实施例1所得的细胞1。具体扩增方法如下：在第0天消化细胞后，使用培养基1培养细胞24小时，所述培养基1的组成为，在去除EGF因子的HCM培养基(Lonza,3198)中加入10μM Y-27632(Selleck，S6390)、20ng/ml肝脏细胞生长因子(HGF)(Peprotech，100-39H)、20ng/ml抑瘤素M(OSM)(Peprotech，300-10)、0.1μM地塞米松(Gibco,D4902)、0.5μM A83-01(Tocris，2939)、0.1μM Compound E(Tocris,6476/1)。
之后，即第1天同一时间，进行换液，将培养基替换为培养基2，所述培养基2的组成为在去除EGF因子的HCM培养基(Lonza,3198)中加入20ng/ml肝脏细胞生长因子(HGF)(Peprotech，100-39H)、20ng/ml抑瘤素M(OSM)(Peprotech，300-10)、0.1μM地塞米松(Gibco, D4902)、0.5μM A83-01(Tocris，2939)、0.1μM Compound E(Tocris,6476/1)。扩增时每两天使用培养基2换液一次。
结果如图5所示，可以看出，此方法能够有效地扩增细胞1。由此扩增得到的第7天的细胞1，通过显微镜观察，其细胞的形态与第0天的细胞基本上相同(图5A)。
通过实施例2中记载的方法对第7天(D7)与第0天(D0)的细胞1中的mRNA进行检测，结果如图5B所示，肝脏细胞相关标志物的mRNA表达水平在第0天和第7天时基本相同。
通过实施例3记载的方法对第7天(D7)与第0天(D0)的细胞1的白蛋白分泌能力进行检测，结果如图5D所示，两者白蛋白分泌能力基本相同。
通过实施例3记载的方法对第7天(D7)与第0天(D0)的细胞1的尿素合成能力进行检测，结果如图5E所示，两者的尿素合成能力基本相同。
通过实施例3记载的方法对第7天(D7)与第0天(D0)的细胞1的CYP3A4活性进行检测，结果如图5F所示，两者的CYP3A4活性基本相同。
通过实施例2记载免疫荧光染色方法，对第7天(D7)细胞1中的一系列肝脏相关标志物的蛋白表达情况进行检测，结果如图5C所示，肝脏相关的标志物仍然显著表达。
通过使用试剂盒Periodic acid–Schiff(Sigma-Aldrich)、试剂盒BODIPY493/503(Life Technologies)以及试剂盒Oil Red O,(Sigma-Aldrich)，试剂盒ICG(Aladdin)对第7天(D7)细胞1合成糖原，合成脂滴，以及细胞吸收能力方面进行检测，结果如图5G所示，细胞1具有肝脏所特有的合成糖原，合成脂滴以及吸收的功能/性质。
本实施例5中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。本实施例5中的培养基1属于本发明所述的HEM-1培养基，培养基2属于本发明所述的HEM-2培养基。
实施例6 6C培养基组分的验证
发明人对实施例1中6C培养基的关键组分进行了分析和验证。具体方法如下：发明人分别逐个减去6C培养基中的每一组分，构建出6种培养基。之后利用这6种培养基和6C培养基分别加入不成熟的诱导肝脏细胞即细胞1，处理时间为2周。两周后，收取细胞，提取RNA，并利用RNA-seq实验评估每个小分子的效果。结合图6A所示，HDAC抑制剂和ERK信号通路抑制剂的去除，大大影响细胞1向细胞2分化的效果，主要体现在从6C培养基中，单独去除HDAC抑制剂(MS-275)或ERK信号通路抑制剂(PD0325901)后，处理后的细胞的转录组图谱与细胞2相比，相差甚远，基本无法实现一定程度的诱导不成熟肝脏细胞朝着诱导型肝脏细胞分化的效果。
此外，从6C培养基中，单独去除类固醇类化合物(地塞米松，即Dex)、Notch信号通路抑制剂(Compound E)、TGF-beta信号通路抑制剂(A83-01)或p38 MAPK抑制剂(SB203580)后，处理后的细胞的转录组图谱与细胞2相比，虽然差距仍然较大，但是可以看出其转录组图谱朝着细胞2的转录组图谱方向发生变化，仍然能够实现一定程度的细胞1向细胞2的分化，即实现一定程度的诱导不成熟肝脏细胞朝着诱导型肝脏细胞分化的效果。
本实施例6中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。实施例中的细胞2属于本发明所述的诱导性成熟肝脏细胞。
实施例7 4C培养基的建立和优化
发明人对实施例1中4C培养基的关键组分进行了分析和验证。具体方法如下：发明人分别逐个减去4C培养基中的每一组分，构建出4种培养基。之后利用这4种培养基和4C培养基分别加入6C培养基诱导得到的细胞2，处理时间为2周。两周后，显微镜下观察细胞；并提取RNA，利用RT-qPCR实验测试成熟肝脏细胞的关键基因的表达水平来评估每个小分子的效果。结合图6B和图6C所示，cAMP 激活剂(Forskolin，FSK)和类固醇类化合物(地塞米松，即Dex)的去除，会在细胞形态和成熟肝脏细胞的关键基因的表达方面大大影响诱导效果，基本无法获得成熟的诱导性肝脏细胞。
此外，ROCK抑制剂(Y-27632)和TGF-beta信号通路抑制剂(A83-01)的去除也在会在不同程度上影响诱导效果，但仍可一定程度上实现成熟的诱导性肝脏细胞。
本实施例7中的细胞1属于本发明所述的诱导性不成熟肝脏细胞。实施例中的细胞2和细胞3属于本发明所述的诱导性成熟肝脏细胞。

Claims (31)

  1. 一种第一组合物，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂；
    优选地，所述第一组合物进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂；
    优选地，所述第一组合物进一步包含(f)p38 MAPK抑制剂。
  2. 一种第一试剂盒，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂、以及相应的使用说明；
    优选地，所述第一试剂盒进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂；
    优选地，所述第一试剂盒进一步包含(f)p38 MAPK抑制剂。
  3. 一种第一培养基，其包含：(a)HDAC抑制剂、(b)ERK信号通路抑制剂、以及基础培养基，优选地所述基础培养基选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E等培养基；
    优选地，所述第一培养基进一步包含(c)类固醇类化合物、(d)Notch信号通路抑制剂、和/或(e)TGF-beta信号通路抑制剂
    优选地，所述第一培养基进一步包含(f)p38 MAPK抑制剂。
  4. 一种第二组合物，其包含：(a)cAMP激活剂、(b)类固醇类化合物；
    优选地，所述第二组合物进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
  5. 一种第二试剂盒，其包含：(a)cAMP激活剂、(b)类固醇类化合物、以及相应的使用说明；
    优选地，所述第二试剂盒进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
  6. 一种第二培养基，其包含：(a)cAMP激活剂、(b)类固醇类化合物、以及基础培养基，
    优选地所述基础培养基选自DMEM、Knockout DMEM、RPMI 1640、DMEM/F12、HCM和William’E等培养基。
    优选地，所述第二培养基进一步包含(c)ROCK抑制剂和/或(d)TGF-beta信号通路抑制剂。
  7. 根据权利要求1-3中任一项所述的组合物、试剂盒或培养基，其中所述HDAC抑制剂选自Mocetinostat(MGCD0103)、Abexinostat(PCI-24781)、Fimepinostat(CUDC-907)、ITSA-1、Vorinostat(SAHA)、MS-275、Panobinostat(LBH589)、Quisinostat、FK228、SB939、Trichostatin A(TSA)等；优选地，所述HDAC抑制剂是MS-275。
  8. 根据权利要求1-6中任一项所述的组合物、试剂盒或培养基，其中所述类固醇类化合物选自泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、泼尼松龙、氢化可的松、地塞米松等；优选地，所述类固醇类化合物是地塞米松。
  9. 根据权利要求1-3中任一项所述的组合物、试剂盒或培养基，其中所述Notch信号通路抑制剂选自IMR-1、RO4929097、Semagacestat(LY450139)、Avagacestat(BMS-708163)、Dibenzazepine(YO-01027)、Crenigacestat(LY3039478)、Compound E、DAPT等；优选地，所述Notch信号通路抑制剂是Compound E。
  10. 根据权利要求1-3中任一项所述的组合物、试剂盒或培养基，其中所述ERK信号通路抑制剂选自SeluMetinib(AZD6244)、Trametinib (GSK1120212)、PD0325901、U0126、U0126-ETOH、PD184352(CI-1040)等；优选地，所述ERK信号通路抑制剂是PD0325901。
  11. 根据权利要求1-6中任一项所述的组合物、试剂盒或培养基，其中所述TGF-beta信号通路抑制剂选自SD-208、LY2109761、BIBF-0775、A83-01、SB431542等；优选地，所述TGF-beta信号通路抑制剂是A83-01。
  12. 根据权利要求1-3中任一项所述的组合物、试剂盒或培养基，其中所述p38 MAPK抑制剂选自SB202190(FHPI)、SB203580、TAK-715、Doramapimod(BIRB 796)等；优选地，所述p38 MAPK抑制剂是SB203580。
  13. 根据权利要求4-6中任一项所述的组合物、试剂盒或培养基，其中所述cAMP激活剂选自Forskolin、8-Br-cAMP等；优选地，所述cAMP激活剂是Forskolin。
  14. 根据权利要求4-6中任一项所述的组合物、试剂盒或培养基，其中所述ROCK抑制剂选自Y-27632、Thiazovivin、GSK429286A、RKI-1447、AT13148等；优选地，所述ROCK抑制剂是Y-27632。
  15. 前述任一项权利要求所述的组合物、试剂盒或培养基在用于制备诱导性成熟肝脏细胞中的用途。
  16. 根据权利要求6所述的第二培养基，其还包含相对于第一培养基总体积的0.05％～20体积％的血清，优选1体积％。
  17. 一种制备诱导性成熟肝脏细胞的方法，所述方法包括以下步骤
    (a)在根据前述任一项权利要求所述的第一培养基中培养诱导性不成熟肝脏细胞；
    由此制备所述诱导性成熟肝脏细胞。
  18. 根据权利要求17所述的方法，其进一步包含以下步骤
    (b)在根据前述任一项权利要求所述的第二培养基中进一步培养步骤(a)所得的细胞；
    由此制备所述诱导性成熟肝脏细胞。
  19. 根据权利要求17-18任一项所述的方法，其中所述诱导性不成熟肝脏细胞由多能干细胞制备得到；优选地所述诱导性不成熟肝脏细胞通过本文所述的方法由多能干细胞制备得到。
  20. 根据权利要求19所述的方法，其中所述多能干细胞选自胚胎干细胞或诱导性多能干细胞。
  21. 一种诱导性成熟肝脏细胞，其具有以下一种或多种特征：
    (a)诱导性成熟肝脏细胞显示出原代肝脏细胞的典型形态特征，优选地与原代人源肝脏细胞的形态基本一致；
    (b)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞选自以下的一种或多种肝脏细胞标志物的转录或蛋白表达，或染色体的可及性显著增加：CYP1A2、CYP2B6、CYP2D6、CYP2C9、CYP2C19、CYP3A4、CYP7A1；
    (c)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞的以下标志物的转录或蛋白表达减少：AFP；
    (d)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞的转录组和染色体的可及性与原代人源肝脏细胞接近或一致；
    (e)相对于诱导性不成熟肝脏细胞，所述诱导性成熟肝脏细胞选自以下的一种或多种肝脏细胞的功能增加或获得：白蛋白分泌能力、 尿素合成能力、肝脏细胞极性、CYP代谢酶活力、成熟肝脏细胞的再生能力、移植存活率、体内重建率；和
    (f)相对于原代肝脏细胞，所述诱导性成熟肝脏细胞选自以下的一种或多种肝脏细胞的功能接近或不低于原代肝脏细胞：白蛋白分泌能力、尿素合成能力、肝脏细胞极性、CYP代谢酶活力、成熟肝脏细胞的再生能力、移植存活率、体内重建率；
    优选地，所述诱导性成熟肝脏细胞可通过根据权利要求17-20中任一项所述的方法制备。
  22. 一种诱导性成熟肝脏细胞，其可通过根据权利要求17-20中任一项所述的方法制备。
  23. 一种类器官，其产生自权利要求21-22中任一项所述的诱导性成熟肝脏细胞。
  24. 一种组织，其产生自权利要求21-22中任一项所述的诱导性成熟肝脏细胞，优选所述组织是肝脏。
  25. 根据权利要求21-22中任一项所述的诱导性成熟肝脏细胞，其在制备用于肝脏细胞移植的细胞、构建肝脏相关疾病模型、肝脏疾病相关药物筛选与鉴定，药物肝脏毒性筛选与鉴定，体外构建肝脏器官(含3D打印肝脏器官)中的用途。
  26. 一种筛选用于治疗疾病的物质的体外方法，其包括使权利要求21-22所述的诱导性成熟肝脏细胞，权利要求23所述的类器官或权利要求24所述的组织与待筛选物质接触。
  27. 一种试剂盒，其包含HEM-1培养基和HEM-2培养基，其中所述HEM-1培养基包含ROCK抑制剂、肝脏细胞生长因子(HGF)、抑瘤素 M(oncostatinM,OSM)、类固醇类化合物、Notch信号通路抑制剂、TGF-beta信号通路抑制剂和基础培养基；所述HEM-2培养基包含肝脏细胞生长因子(HGF)、抑瘤素M(oncostatinM,OSM)、类固醇类化合物、Notch信号通路抑制剂、TGF-beta信号通路抑制剂和基础培养基；
    优选地，所述HEM-1培养基和HEM-2培养基包含基础培养基；更优选地，所述基础培养基是去除表皮细胞生长因子(EGF)的HCM培养基。
  28. 根据权利要求27所述的培养基，其中所述ROCK抑制剂是Y-27632；所述类固醇类化合物是地塞米松；所述Notch信号通路抑制剂是Compound E；所述TGF-beta信号通路抑制剂是A83-01。
  29. 一种扩增诱导性不成熟肝脏细胞的方法，其中包括使诱导性不成熟肝脏细胞接触根据权利要求27或28所述的培养基。
  30. 根据权利要求29所述的方法，其中包括使诱导性不成熟肝脏细胞接触根据权利要求27或28所述的HEM-1培养基，之后使诱导性不成熟肝脏细胞接触根据权利要求27或28所述的HEM-2培养基。
  31. 根据权利要求27或28所述的培养基，其在用于扩增诱导性不成熟肝脏细胞的用途。
PCT/CN2023/078284 2022-02-24 2023-02-24 诱导性成熟肝脏细胞及其制备的方法 WO2023160671A1 (zh)

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