CN114317398A

CN114317398A - Hepatic progenitor cell population co-labeled by Gli1 and EpCAM genes and application thereof

Info

Publication number: CN114317398A
Application number: CN202011034452.3A
Authority: CN
Inventors: 赵允; 高栋; 彭甲银
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2022-04-12
Anticipated expiration: 2040-09-27
Also published as: CN114317398B

Abstract

The invention provides a hepatic progenitor cell population jointly marked by Gli1 and EpCAM genes and application thereof. The invention discloses a novel isolated cell population, which is a Gli1+ cell population, preferably an EpCAM +/Gli1+ cell population. The cells have characteristics of liver stem/progenitor cells, have functions of differentiation/transdifferentiation to form liver cells, liver injury repair effects, and can form liver organoids. The invention also provides a method for sorting the cells, a method for preparing liver organoids by using the cells and the like. The invention provides a new support for liver injury repair mechanism and a new way for research and clinical application of liver regeneration.

Description

Hepatic progenitor cell population co-labeled by Gli1 and EpCAM genes and application thereof

Technical Field

The invention belongs to the field of cell biology; more specifically, the invention relates to a hepatic progenitor cell population co-labeled with Gli1 and EpCAM genes and uses thereof.

Background

The liver, the largest parenchymal organ in the body, and the largest digestive gland in the body, is in the central position in the process of maintaining the metabolism of the body, and has strong detoxification capability and regeneration capability. When the liver is damaged by virus, medicine, alcohol and other pathogenic factors, the residual liver tissue can be regenerated and restored to the original volume and weight, and finally the reconstruction of the liver tissue structure and the restoration of the liver function are achieved. Clinically common chronic and persistent liver injury has obvious damage to liver function. Research shows that the cell types involved in the liver injury repair process are closely related to the degree of liver injury. When the liver is slightly injured, proliferation of liver parenchymal cells occurs mainly, and injury repair is performed. When the liver is seriously damaged and the regeneration of the liver cells is obstructed, the necrosis of the liver cells is increased, the liver cells cannot be repaired only by the regeneration of the liver cells, and the immaterial liver cells are often needed to participate. In this case, the liver tissue will initiate processes including hepatic stem/progenitor differentiation, cholangiocellular transdifferentiation, and hepatic cell dedifferentiation to repair the damaged liver (fig. 1).

However, to date, little is known about the molecular mechanisms of how the damaged liver initiates this process. In recent years, research shows that the liver cell transplantation can be used as an effective alternative treatment means, help partial liver failure patients to pass through a dangerous period to wait for liver transplantation, and even can directly obtain satisfactory curative effect in the liver cell transplantation of partial liver failure patients, thereby providing a new choice for treating the liver failure. Although hepatocytes have strong damage repair ability in vivo, long-term culture of mature hepatocytes in vitro has been a worldwide problem, which also imposes restrictions on clinical treatment of related liver damage diseases.

Therefore, the molecular mechanism of liver injury repair process and the long-term culture of hepatocytes in vitro and further artificial intervention on hepatocyte proliferation based on the molecular mechanism are still one of the key points of current research in life science.

Disclosure of Invention

The invention aims to provide a hepatic progenitor cell population jointly marked by Gli1 and EpCAM genes and application thereof.

In a first aspect of the invention, there is provided the use of a cell or cell culture for: preparing a composition that transdifferentiates to form hepatocytes, preparing a composition for performing liver injury repair, or preparing a liver organoid; wherein the cell is a Gli1+ cell and comprises the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids.

In a preferred embodiment, the composition comprises a culture.

In another preferred embodiment, the cells are Gli1+ and EpCAM + cells.

In another preferred embodiment, the cell further comprises a feature selected from the group consisting of: have epithelial (like) cell characteristics, or express an epithelial cell marker; having mesenchymal (like) cell characteristics, or expressing mesenchymal cell markers; can form organoid with the characteristics of the cells of the bile duct (like) or express bile duct cell markers.

In another preferred embodiment, the cholangiocellular marker comprises a marker selected from the group consisting of: KRT19, SOX 9.

In another preferred embodiment, the epithelial cell markers comprise markers selected from the group consisting of: EpCAM, KRT7 and KRT 19.

In another preferred embodiment, the stromal cell marker comprises a marker selected from the group consisting of: PDGFR α and PDGFR β.

In another preferred embodiment, said cells comprise passaged cells.

In another preferred embodiment, the passaged cells include passage 1-30, more specifically 2, 3, 5, 8, 10, 12, 15, 18, 20, and 25.

In another aspect of the invention, there is provided an isolated cell or cell culture, said cell being a Gli1+ cell and comprising the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids.

In a preferred embodiment, the cells are Gli1+ and EpCAM + cells.

In another preferred example, the Gli1+ cells are isolated by a method comprising: gli1+ cells were sorted from liver tissue isolates using binding molecules (e.g., antibodies) or capture molecules that specifically bind or capture Gli 1.

In another preferred embodiment, said Gli1+ and EpCAM + cells are isolated by a method comprising: gli1+ and EpCAM + cells were sorted from liver tissue isolates using binding molecules (e.g., antibodies) or capture molecules that specifically bind or capture Gli1 and EpCAM.

In another preferred embodiment, the isolated liver tissue comprises: liver tissue or products resulting from the processing of the tissue, more specifically cell suspensions.

In another aspect of the invention, there is provided a method of isolating or enriching a cell or cell culture, said cell being a Gli1+ cell and comprising the following features: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids; the method comprises the following steps: gli1+ cells were sorted from liver tissue isolates using binding molecules (e.g., antibodies) or capture molecules that specifically bind or capture Gli 1.

In a preferred embodiment, the cells are Gli1+ and EpCAM + cells, and the method comprises: gli1+ and EpCAM + cells were sorted from liver tissue isolates using binding molecules (e.g., antibodies) or capture molecules that specifically bind or capture Gli1 and EpCAM.

In another preferred embodiment, the method of sorting cells includes (but is not limited to): flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion.

In another aspect of the present invention, there is provided a method of preparing a liver organoid, comprising: culturing a cell or cell culture as described in any one of the preceding paragraphs, allowing growth and differentiation, thereby obtaining a liver organoid.

In a preferred embodiment, the method comprises:

(1) providing a cell or cell culture as defined in any of the preceding claims, or obtaining a cell or cell culture using a method as defined in any of the preceding claims;

(2) growing the cells or cell cultures of (1); preferably, the generation is carried out for 1-5 times (such as 2, 3, 4 times);

(3) and (3) carrying out differentiation culture on the culture obtained in the step (2), thereby obtaining the liver organoid.

In another preferred example, (2) the medium for growth culture contains Advance DMEM/F12 medium to which HEPES, GlutaMAX-1, primocin, B27, N-acetyl cysteine, Nicotinamide, A83-01, Y27632, EGF, FGF10, FGF2, R-Spondin, Noggin is added.

In another preferred example, (3) the medium for the differentiation culture contains an Advance DMEM/F12 medium to which EGF, DAPT, HEPES, GlutaMAX-1, primocin, B27, Y27632, DAPT, BMP7, HGF, and oncostatin M are added.

In another preferred embodiment, the medium in which the growth culture is carried out comprises: an Advance DMEM/F12 culture solution, and: 10mM HEPES, 2nM GlutaMAX-1, 500 XPrimycin, 1 XB 27, 1.56mM N-Acetylcysteine, 10mM Nicotinamide, 0.5. mu. M A83-01, 10. mu. M Y27632, 50ng/mL EGF, 10ng/mL FGF10, 1ng/mL FGF2, R-Spondin (10%) and Noggin (10%); the respective content formulas can be varied up and down within the range of 50% (preferably within the range of 40%, more preferably within the range of 30%, such as within the ranges of 20%, 15%, 10%, 5%).

In another preferred embodiment, the medium for differentiation culture contains: in an Advance DMEM/F12 culture medium, and 50ng/mL EGF, 10. mu.M DAPT, 10mM HEPES, 2mM GlutaMAX-1, 500 XPrimycin, 1 XB 27, 10. mu. M Y27632, 10. mu.M DAPT, 25ng/mL BMP7, 25ng/mL HGF and 20ng/mL oncostatin M; the respective content formulas can be varied up and down within the range of 50% (preferably within the range of 40%, more preferably within the range of 30%, such as within the ranges of 20%, 15%, 10%, 5%).

In another preferred embodiment, the organoids further comprise organoids selected from the group consisting of: passaged organoids, continuously cultured organoids, cryo-preserved and/or resuscitated organoids.

In another preferred embodiment, the organoids are passaged for 1-30 passages, more specifically for 2, 3, 5, 8, 10, 12, 15, 18, 20, and 25 passages.

In another preferred embodiment, the culturing comprises: three-dimensional (3D) culture, or two-dimensional (2D) culture; preferably three-dimensional (3D) culture.

In another preferred embodiment, the method is an in vitro method.

In another preferred embodiment, the method is one that does not have a direct purpose of disease diagnosis or treatment (non-diagnostic or non-therapeutic).

In another aspect of the invention there is provided an artificially created liver organoid obtained from the growth and differentiation of a cell or cell culture as defined in any one of the preceding; or, it is prepared by any one of the methods for preparing liver organoids.

In another aspect of the invention there is provided a kit for isolating or enriching a cell or cell culture as defined in any one of the preceding claims, comprising: gli1 binding or capture molecule, and EpCAM binding or capture molecule.

In another aspect of the present invention, there is provided a kit for preparing a liver organoid, comprising: gli1 binding or capture molecule, EpCAM binding or capture molecule, and an agent for organoid culture.

In another aspect of the present invention, there is provided a kit for preparing a liver organoid, comprising: a cell or cell culture as described in any of the preceding, and an agent for organoid culture.

In a preferred embodiment, the agent for organoid culture comprises: a medium for performing growth culture comprising: an advanced DMEM/F12 culture medium to which HEPES, GlutaMAX-1, primocin, B27, N-acetyl cysteine, Nicotinamide, A83-01, Y27632, EGF, FGF10, FGF2, R-Spondin, Noggin are added.

In another preferred example, the medium for differentiation culture comprises an Advance DMEM/F12 medium to which EGF, DAPT, HEPES, GlutaMAX-1, primocin, B27, Y27632, DAPT, BMP7, HGF, and oncostatin M are added.

In another aspect of the present invention, there is provided the use of Gli1 for screening liver stem/progenitor cells, or for preparing a composition for screening liver stem/progenitor cells.

In another aspect of the invention there is provided the use of a combination of Gli1 and EpCAM for screening for liver stem/progenitor cells, or for the preparation of a composition for screening for liver stem/progenitor cells.

In another aspect of the present invention, there is provided a method of screening for a substance (potential substance) that promotes hepatocyte regeneration or promotes liver repair, the method comprising:

(1) treating an isolate of liver tissue with a candidate substance; and

(2) and (3) detecting the cell surface molecular characteristics of the liver tissue isolate, wherein if the Gli1+ cells are statistically increased (for example, the percentage of Gli1+ cells in the whole isolate cell population is increased by more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, and the like), the candidate substance is a substance (potential substance) for promoting the regeneration of the liver cells or promoting the liver repair.

In another preferred embodiment, in detecting the cell surface molecular property of the isolate, further comprising detecting EpCAM; if the Gli1+ EpCAM + cells are statistically increased (e.g., the proportion of Gli1+ EpCAM + cells is increased by more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, etc., in the whole isolate cell population), the candidate substance is a substance (potential substance) for promoting hepatocyte regeneration or liver repair.

In another preferred example, step (1) includes: adding a candidate substance to the isolate in the test group; and/or.

In another preferred example, the step (2) includes: detecting the amount of Gli1+ cells or Gli1+ EpCAM + cells in the isolate; and comparing to a control, wherein said control is an isolate to which said candidate substance is not added; if Gli1+ cells or Gli1+ EpCAM + cells in the test group are statistically increased, the candidate substance is a substance that promotes hepatocyte regeneration or promotes liver repair.

In another preferred embodiment, the candidate substance includes (but is not limited to): regulatory molecules or constructs thereof (e.g., gene editing reagents, overexpression vectors, recombinant viral or non-viral constructs for recombinant engineering (e.g., overexpression), etc.), small chemical molecules (e.g., specific agonists, enhancers), interactive molecules, etc., designed to target hepatocytes or Gli1 or its encoded gene therein.

In another preferred example, the method further comprises: the obtained potential substance is subjected to further cell experiments and/or animal experiments to further select and determine a substance useful for promoting hepatocyte regeneration or promoting liver repair from the candidate substances.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, liver injury repair process.

FIG. 2, schematic diagram of Hh signal transduction pathway.

FIG. 3, expression of Gli1 in different liver cells;

a and B: alb and HNF4a are markers specific for mature hepatocytes, respectively. qPCR results found that Alb and HNF4a were mainly expressed in liver cells;

c and D: CK19 is a marker specifically expressed by bile duct cells, and EpCAM is a marker specifically expressed by bile duct cells and stem/progenitor cells; the results of qPCR found that CK19 and EpCAM were mainly expressed in bile duct cells;

e: the qPCR results found that Gli1 was mainly expressed in bile duct cells.

FIG. 4, Gli1 is expressed in normal liver predominantly in cells surrounding the bile duct and partially co-expressed with EpCAM, PDGFRa and a-SMA genes;

a.8-10 week old Gli1-LacZ mice liver frozen section, x-gal staining. Blue is X-gal staining. Red is nuclear fast red dyeing;

b.8-10 week old Gli1-LacZ mice, livers were cryosectioned and co-stained with β -gal antibody and cholangiocellular markers (KRT1 and OPN), hepatocyte markers (Alb and GS), immunocyte markers (F4/80), endothelial marker (VE-CAD) antibody, respectively. Red is antibody staining of different cell markers, and green is staining of beta-gal antibody;

c.8-10 week old Gli1-LacZ mice, liver was cryosectioned and co-stained with β -gal antibody and mesenchymal cell marker (PDGFRa and a-SMA) antibody, respectively. Red is antibody staining of different cell markers, and green is staining of beta-gal antibody;

8-10 week old Gli1-LacZ mice, livers were cryosectioned and co-stained with X-gal and hepatic progenitor marker (EpCAM) antibodies, respectively. Green staining for EpCAM antibody and blue staining for X-gal.

FIG. 5, Gli1+ cells were predominantly distributed around bile ducts and co-localized with EpCAM + progenitor cells;

gli1+ cell lineage tracing pattern map. Gli1-CreER^T2(ii) a Injecting TM to induce Cre enzyme expression after Ai9 mice are born for 6 weeks, taking livers after one week as frozen sections, and carrying out immunofluorescence staining;

B. immunofluorescence staining is carried out by using mesenchymal cell markers (PDGFRa and a-SMA) and a hepatic progenitor cell marker (EpCAM), the positioning of Gli1+ cells, the mesenchymal cell markers and the hepatic progenitor cell markers is detected, and the result shows that Gli1 can be partially co-positioned with the mesenchymal cell markers and the hepatic progenitor cell markers;

C. the localization of Gli1+ cells and bile duct cells is detected by immunofluorescence staining with bile duct cell markers (KRT19 and SOX9), and the result shows that Gli1+ cells are distributed around the bile duct cells and cannot be co-localized with KRT19 and SOX 9;

D. immunofluorescent staining was performed with hepatocyte markers (HNF4a and Alb), immunocyte markers (F4/80), endothelial marker (VE-CAD), and it was found that Gli1+ cells could not co-localize with hepatocyte markers, immunocyte markers and endothelial markers.

Figure 6, Gli1 positive cells increased after liver injury;

a.8-10 weeks old Gli1-LacZ mice are respectively subjected to partial liver resection (PH) for 24h and 48h, then the liver is taken for frozen section, and x-gal staining is carried out;

b.8-10 week old Gli1-LacZ mice according to CCl₄Oil 1:3 ratio using CCl₄Intraperitoneal injection is carried out at 2mL/Kg, 1 time is carried out every 3 days for 28 days, only the same amount of Oil is injected into a control group, and the liver is taken to be frozen section for carrying out x-gal staining;

C-E.8-10 week old Gli1-LacZ mice were fed with normal feed and special feed containing 0.1% DDC, CDE and MCD, respectively, and after feeding for 3-4 weeks, livers were frozen and sectioned for x-gal staining. After DDC is fed for 2 weeks, epithelial hyperplasia of small bile ducts can be seen in the area of the confluence, the number of immature bile ducts is increased, and inflammatory cells are gathered around the immature bile ducts.

FIG. 7, Gli1+ cells can transdifferentiate to form hepatocytes;

A. lineage tracing the shift in the properties of Gli1+ cells in the liver of DDC liver-injured mice. Gli1-CreER^T2(ii) a Injecting TM to induce Cre enzyme expression after Ai9 mice are born for 6 weeks, after TM treatment for 2 weeks, feeding common feed and special feed containing 0.1% DDC in groups, taking livers after feeding for 4 weeks, freezing and slicing, and carrying out immunofluorescence staining;

B. fluorescence observation of the whole embedded liver tissue, and existence of tdtomato positive epithelial-like cells can be observed after liver injury;

C. liver sections are cut, and immunofluorescence staining can find that tdtomato positive hepatocyte-like cells exist;

immunofluorescence staining was performed with bile duct marker CK19, hepatic stem/progenitor marker molecule EpCAM, hepatic cell marker HNF4a and tdtomato, respectively. As a result, tdtomato was found to label a part of hepatocytes.

FIG. 8, Single cell transcriptome analysis of EpCAM and Gli 1-labeled cell lineages;

A. from Gli1-CreERt 2; sorting populations of EpCAM +/Gli1-, EpCAM-/Gli1+ and EpCAM +/Gli1+ cells in the liver of Ai9 mice for single cell RNA sequencing; e + G-, EpCAM +/Gli 1-; E-G +, EpCAM-/Gli1 +; e + G +, EpCAM +/Gli1 +;

b and c. isolated 527 single cells t-random adjacent insert (tSNE) plots;

D. violin plots drawn by scRNA-seq data show the expression of EpCAM and tdTomato. E + G-, EpCAM +/Gli 1-; E-G +, EpCAM-/Gli1 +; e + G +, EpCAM +/Gli1 +; h, hepatocytes;

E. gene differential heatmap shows the differential genes of 4 different clusters of scRNA-seq data;

F. violin plots show the selection of the expression of specific lineage-associated genes from the scra-seq data;

GO annotation analysis of epcam +/Gli1+ cells;

H. the BioCarta gene set of GSEA was used to analyze the signaling pathway significantly expressed in EpCAM +/Gli1+ cells.

FIG. 9, establishment of 3D organoid long-term culture system of Gli1+ cells;

gli1+ cell in vitro 3D organoid culture pattern diagram;

gli1+ cells were cultured in matrigel in 3D to form organoids. The growth status of the organoids in culture is shown for 1 day, 3 days, 6 days, and 9 days. Meanwhile, the results show that liver organoids generated by culturing the Gli1+ cell subset can be stably passaged.

FIG. 10, EpCAM +/Gli1+ cell 3D organoid culture;

A. in vitro 3D organoid culture profile;

EpCAM +/Gli1-, EpCAM +/Gli1+, EpCAM-/Gli1+ and EpCAM-/Gli 1-cell flow sorting;

C. the ability of the four cell types to form organoids;

comparison of organoids formed by EpCAM +/Gli 1-and EpCAM +/Gli1+ at 0, 2, 4, 6, 8 and 10 days of culture.

E. Statistics of the efficiency and number of organoids formed by the four cell types;

FIG. 11, the organoids formed by EpCAM +/Gli1+ express bile duct cell markers;

A. fluorescence observation of the whole embedded and cultured organoid;

B. the organoids were immunofluorescent stained with cholangiocyte-specific markers (KRT19, SOX9), progenitor cell marker (EpCAM) and cell proliferation marker (Ki 67).

FIG. 12, the organoids formed by EpCAM +/Gli1+ can differentiate into functional hepatocytes in vitro;

qpcr analyzed the expression of mature hepatocyte markers (HNF4a and Alb) and cholangiocellular markers (KRT19, EpCAM) in organoids in the presence of growth medium and differentiation medium, respectively;

B. hepatocyte markers HNF4a and Alb) immunofluorescent staining of differentiated organoids;

pas and LDL uptake staining.

FIG. 13, biological function of EpCAM +/Gli1+ cells in liver injury repair.

FIG. 14, establishment of a human hepatocyte 3D organoid long-term culture system;

A. after the human liver tissue block is mechanically sheared and digested into single cells by collagenase, the human liver tissue block is cultured in matrigel in a 3D way to form organoids. The growth states of the organoids in culture for 1 day, 3 days, 6 days, and 9 days are shown; the liver organoids can be stably passaged for a long time, and can still be stably proliferated after 25 generations of culture;

b-c. cultured organoids can express specific molecules of cholangiocytes (CK19) and hepatic stem/progenitor markers (EpCAM).

FIG. 15 shows that the Gli inhibitor GANT-61 inhibits the growth of 3D organoids of human liver cells;

A-A ", human liver cell 3D organoid without any treatment;

B-D ", cultured human liver cell 3D organoids treated with inhibitors of the Hh signaling pathway, SANT-1(10nM), GANT-61(10uM) and agonist SAG (100nM), and observed on

days

0, 1 and 2, respectively, to show that only GANT-61 inhibits the growth of liver cell 3D organoids.

Detailed Description

The present inventors have conducted intensive studies in an effort to study the regeneration of hepatocytes and have revealed a novel isolated cell population, which is Gli1+ cell population, preferably Gli1+ and EpCAM + (EpCAM +/Gli1+) cell population. The cells have characteristics of liver stem/progenitor cells, have the functions of differentiation/transdifferentiation to form liver cells, have the effect of repairing liver injury, and can form liver organoids. The invention also provides a method for sorting the cells, a method for preparing liver organoids by using the cells and the like. The invention provides a new theoretical support for the liver injury repair mechanism and a new way for the research and clinical application of liver regeneration.

Term(s) for

As used herein, the term "isolated" refers to a substance that is separated from its original environment (e.g., the natural body or environment). If living cells are not isolated and purified when present in vivo, but if the same cells are separated from other materials that are present, the cells are isolated or purified, e.g., from their native state/original environment.

As used herein, "isolated cell (culture)" includes "isolated cell line (culture)".

As used herein, "enriching" a cell (population) refers to increasing the content of desired cells of interest/cells of interest contained in a mixed cell population, and generally refers to increasing the frequency of "cells of interest" in the total cells. Thus, an enriched cell population refers to a cell population that has a higher frequency of cells of interest as a result of the enrichment step.

As used herein, unless otherwise specified, the "cell of interest/target cell" is a cell of interest in the present invention having the intended function; more specifically, the term "target cell" refers to a Gli1+ cell population, or to an EpCAM +/Gli1+ cell population.

As used herein, the term "Gli 1+ (Gli1 positive)" or "EpCAM + (EpCAM positive)" means that the cells express Gli1 or EpCAM, and are bound or captured by their specific binding molecules (e.g., antibodies) or capture molecules, thereby isolating and enriching the positive cells (population).

As used herein, "cell population" includes pre-enrichment and post-enrichment cell populations, and when subjected to an enrichment step, the cell populations may be post-enrichment. Each cell population can be used directly in the next step, and can be partially or fully frozen for long or short term storage and for subsequent steps. Meanwhile, cells of a cell population may be individually suspended to obtain a single cell suspension.

As used herein, "marker" is used interchangeably with "marker". In the present invention, unless otherwise stated, the "marker" is a molecule of interest (e.g., a protein surface molecule) that is used to determine a particular cell (population) or property thereof. For example, in the present invention, Gli1 is used as a marker to obtain Gli1+ cells; or obtaining Gli1+ and EpCAM + cells by using Gli1 and EpCAM as markers.

New isolated cell (population)

As one of the most important morphogens in the development process of multicellular animals, the evolutionarily highly conserved Hedgehog (Hh) signal plays an important role in controlling the proliferation and differentiation of cells, embryogenesis, morphogenesis, tissue organ development and the like. In mammalian organisms, the classical Hh signaling pathway can be briefly summarized as Hh/Ptch/Smo/Sufu/Gli signaling axis. It is composed mainly of extracellular Hh ligand, 12 transmembrane protein receptor molecules patched (ptch) on the surface of cell membrane, 7 transmembrane proteins smoothened (smo), intracellular reverse regulatory factor Sufu protein, and nuclear transcription factor Gli family protein (fig. 2). Meanwhile, researches show that various non-classical Hh signal paths exist besides the classical Hh signal switching path. Wherein mainly include: signal transduction that is dependent on Hh ligand and receptor Ptch function, but not on Smo; smo-independent, but Gli-dependent signal transduction, and the like. Among the pathways, Gli family proteins are key transcription factors located at the extreme ends of Hh signaling pathways, and upregulation of their activity is one of the most important markers of Hh signaling pathway activation. Hh signaling pathway activation, for whatever reason, will transduce to Gli, and ultimately translate into modulation of Gli activity. Gli family protein members include Gli1, Gli2, and Gli 3. These 3 Gli proteins all contain highly conserved DNA binding regions. Gli1 differs structurally from Gli2, Gli3 in that Gli1 does not have an N-terminal inhibition region. Although the 3 proteins are structurally similar, there are major differences in their functions. Gli1 mainly exists in the form of a transcription activator, has positive feedback regulation with an Hh signal transduction pathway, and plays an important role in amplifying and maintaining an Hh pathway signal; gli2 and Gli3 exist in the form of transcription factors directly responding to Hh ligand, and have both activating and inhibiting functions, wherein Gli2 is mainly activating and Gli3 is mainly inhibiting. At present, several main antagonists aiming at the Hh signal pathway, such as Cyclopamine, SANT1/2/3/4, GDC-0449 and the like, mainly play a role by regulating the activity of Smo protein in the Hh signal pathway or signals upstream of the Smo protein, and have no obvious effect on the activation of Smo downstream molecules in the Hh pathway or the loss-of-function mutation of Hh signal pathway inhibitors; the Gli inhibitor, GANT61, mainly inhibits the binding of Gli family proteins to DNA, thereby exerting an inhibitory function on the pathway. From the viewpoint of biological functions, the Hh signaling pathway is a signaling pathway having functions of regulating cell proliferation, apoptosis, migration, and differentiation. At present, the specific biological functions and molecular mechanisms in the liver injury repair process are still unclear.

The inventor aims to research the molecular mechanism and the biological function regulated and controlled in the liver injury repair process. In previous studies, little is known about the molecular mechanism and biological function of the transcription factor Gli1 involved in liver injury repair. In the research, the inventor finds that Gli1 can be used as a marker gene of liver stem/progenitor cells and is possibly involved in the liver injury repair process through a non-classical Hh signal path. Meanwhile, the mechanism and the function of the Gli1+ cells in liver injury repair are taken as research focuses, and the biological functions of various liver injury models, genetically modified animals, somatic cell tracking and 3D-type Gli1+ cells in the liver injury repair process are integrated and utilized; meanwhile, on the basis, the technology of in vitro culture of the Hh signal organ and the like, and the methods of molecular biology, cell biology, information biology, chemical biology and the like are researched for further research. By carrying out in vivo lineage tracing, the cell attribute conversion of the Gli1+ cells in the damage repair process is identified, and the Gli1 is clear⁺Hepatic stem/progenitor cell characteristics of the cells; normal EpCAM +/Gli1+ cells were isolated and enriched using lineage tracing labeling technique, and stem/progenitor cell characteristics of EpCAM +/Gli1+ cells were revealed by single cell sequencing; the effects of pathway agonists and inhibitors on cultured human organoids are elucidated by using 3D organoid culture techniques, Fah-deficient animal models, in combination with corresponding human normal and liver disease clinical samples.

Based on the new findings of the present inventors, there is provided an isolated cell (population) or cell culture, said cell being a Gli1+ cell; the cells have the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids.

Based on the inventors' novel findings, there is also provided an isolated cell (population) or cell culture, said cell being an EpCAM +/Gli1+ cell; the cells have the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids.

The amino acid sequence of Gli1, a transcription factor from the Gli family, can be found in GenBank accession No. 2735 (human) or GenBank accession No. 14632 (mouse). It is to be understood that the present invention also encompasses Gli1 homologues, i.e. such proteins that are of different species origin but functionally identical.

The EpCAM is an epithelial adhesion protein and its amino acid sequence can be found in GenBank accession No. 4072 (human) or GenBank accession No. 17075 (mouse). It is to be understood that the present invention also encompasses Gli1 homologues, i.e. such proteins that are of different species origin but functionally identical.

In a preferred form of the invention, the cell further has one or more characteristics including one or more selected from the group consisting of: have epithelial (like) cell characteristics, or express an epithelial cell marker; having mesenchymal (like) cell characteristics, or expressing mesenchymal cell markers; has the characteristics of bile duct (like) cells, or expresses bile duct cell markers.

In a preferred form of the invention, the cells according to the invention are isolated from hepatocytes or are passaged cells isolated from hepatocytes.

According to the research results of the inventor, the cell of the invention can be expanded, cultured, passaged and preserved after being separated from the natural environment (such as organism), and the good biological activity of the cell can be maintained. Therefore, the cells of the present invention can be cultured and applied in a large scale or industrially.

Method for isolating cell(s)

The present inventors have discovered a population of progenitor cells co-labeled with the Gli1 gene or the Gli1/EpCAM gene that are involved in the liver repair process immediately after the onset of chronic liver disease. Genetic evidence of the Gli1+ cells or EpCAM +/Gli1+ cells for liver repair provides new insights for cell and molecular mechanisms of liver diseases and regeneration, and helps for research on human liver regeneration.

According to the new findings of the present inventors, the present invention also provides a method for isolating or enriching a cell or cell culture, said cell being a Gli1+ cell, preferably it is an EpCAM +/Gli1+ cell. The cells have the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids; the method comprises the following steps: hepatocytes are provided and Gli1+ cells are sorted using a binding molecule (e.g., an antibody) or capture molecule that specifically binds to Gli 1.

The methods of the invention can be used to enrich (or isolate) at least one target cell of interest. The methods of the invention comprise a series of culturing and selection steps that can be used separately, in combination, sequentially, repeatedly, or periodically.

Thus, methods for isolating or enriching cells with specific identifiable signals (e.g., EpCAM + and/or Gli1+) are known to those of skill in the art and a variety of methods for isolating or enriching cells can be used in the present invention. In the examples of the present invention, specific cell sorting methods are provided, however, it is to be understood that, after the cell surface molecules of interest have been known in the present invention, cell sorting is not limited to those specifically listed in the examples, and other methods are also applicable. Some cell sorting methods commonly used in the art include, but are not limited to, the following:

(a) immunomagnetic bead cell sorting, which is a technique for separating target cells from a cell population by magnetic beads; firstly, the magnetic beads are combined with specific cell surface proteins on target cells through antibodies, biotin or avidin; then the sample is placed in a magnetic field, and the cells marked by the magnetic beads are adsorbed under the action of the magnetic field; the unlabeled cells are retained in the supernatant, thereby physically separating the cells of interest from the non-cells of interest in the sample; because of its rapidity and simplicity of operation, immunomagnetic bead cell sorting is one of the most common methods used by researchers to isolate specific cell subsets of high purity.

(b) Flow cytometric sorting, a method of separating heterogeneous cell populations by flow cytometry and fluorescent probes; when the single cell suspension after fluorescent staining or marking is put into a sample tube, the single cell suspension is pressed into a flow chamber under high pressure; the flowing chamber is filled with sheath liquid, and cells are arranged into a single row and are sprayed out from a nozzle of the flowing chamber at a certain speed under the wrapping and pushing of the sheath liquid; an ultrahigh frequency piezoelectric crystal is arranged on a nozzle of the flow chamber, and the ultrahigh frequency piezoelectric crystal vibrates after being charged, so that the sprayed liquid flow is broken into uniform liquid drops, and cells to be detected are dispersed in the liquid drops; the liquid drops are charged with different positive and negative charges, when the liquid drops flow through a deflection plate with several kilovolts, the liquid drops are deflected under the action of a high-voltage electric field and fall into respective collection containers, and the liquid drops which are not charged fall into a middle waste liquid container, so that the separation of cells is realized; it is particularly useful for sorting of single cells, sorting based on intracellular markers, sorting based on expression levels of cell surface markers, sorting when complex cell types are separated by multiple markers and purity requirements are high.

(c) Microfluidic cell sorting, which performs single-cell separation by manipulating fluids at the microscopic level, microfluidic technology is often built on microchips, also commonly referred to as "lab-on-a-chip"; microfluidic technology has great advantages when the volumes of sample and reagents used are small; furthermore, lab-on-a-chip is portable and can be used almost anywhere, and is therefore particularly suitable as a tool for field diagnostics. Several different microfluidic cell sorting methods include: ultrasonic sorting, double water phase system, bionic micro-fluidic sorting, affinity sorting, deterministic lateral shift, electrophoretic sorting, field flow classification, gravity and sedimentation, magnetophoresis, microfiltration, and light sorting.

(d) An adhesion method which can separate a cell of interest from a heterogeneous cell population based on the unique adhesion ability of different types of cells; the ability to selectively promote or inhibit cell adhesion by selection of appropriate growth factors and cell culture plates allows for the separation of adhered cells from suspension, and the effective use of this method is further based on the observation of the adhesion properties of the cell populations isolated in the present invention.

Different types of identifiable signals may be selected depending on the sorting method. For example, the cell population is enriched by flow cytometric sorting to form an enriched cell population comprising at least one cell of interest, the identifiable signal employed preferably being a fluorescent dye, which is interpreted according to the wavelength of excitation light or emission light specific to the fluorescent dye.

Binding or capture molecules for isolating or enriching specific cells are readily prepared or obtained by one skilled in the art. More commonly used binding molecules are for example but not limited to: antibodies, ligands, which may be free or immobilized on a solid support.

In addition to being isolated or enriched from hepatocytes, the cells of the invention may also be artificially cultured/scale-cultured in vitro. After the first generation cells are artificially isolated from the natural environment, large scale propagation may be performed by appropriate conditions, which may employ cell culture methods known in the art. Preferably, reference may be made to the method in the examples of the invention, but the invention is not limited to this preferred method.

In addition, the present invention also includes precursor cells (cell cultures) which, after certain transformation/transdifferentiation/artificial conditions, are capable of forming Gli1+ cells as in the present invention, or EpCAM +/Gli1+ cells.

The cells obtained by the method can be frozen, revived, passaged and maintained and cultured for a long time.

Liver organoids and preparation thereof

The invention also provides an artificially created liver organoid obtained by growing and differentiating a cell or cell culture according to any of the preceding aspects of the invention; or, it is prepared by any of the methods for preparing liver organoids according to the present invention.

The culture of the liver organoid of the present invention comprises subjecting the aforementioned cell or cell culture to growth culture and differentiation culture, thereby obtaining the liver organoid. More specifically, the method comprises the following steps: (1) providing said cell or cell culture; (2) growing the cells or cell cultures of (1); preferably, the generation is carried out for 1-5 times (such as 2, 3, 4 times); (3) and (3) carrying out differentiation culture on the culture obtained in the step (2), thereby obtaining the liver organoid.

Several growth and differentiation culture media or culture methods known in the art may be used in the present invention. In a preferred embodiment of the present invention, the medium for growth culture contains: an advanced DMEM/F12 culture medium to which HEPES, GlutaMAX-1, primocin, B27, N-acetyl cysteine, Nicotinamide, A83-01, Y27632, EGF, FGF10, FGF2, R-Spondin, Noggin are added. The research results of the inventor find that the addition of the components is beneficial to maintaining good cell state, keeping good activity and being beneficial to obtaining organoid.

In a preferred embodiment of the present invention, the medium for differentiation culture contains an Advance DMEM/F12 medium to which EGF, DAPT, HEPES, GlutaMAX-1, primocin, B27, Y27632, DAPT, BMP7, HGF, and oncostatin M are added. The research results of the inventor find that the addition of the components is beneficial to the specific differentiation of cells and the obtainment of organoids.

In the present invention, the organoids also include, but are not limited to: subcultured organoids, continuously cultured organoids, cryo-preserved and/or resuscitated organoids, and the like. The subcultured organoids can include subcultured 1-100 generations, more specifically 2, 3, 5, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 and 90 generations.

The culture method and the culture medium of the present invention can be used for culturing in a two-dimensional (2D) or three-dimensional (3D) culture system. In a preferred embodiment of the present invention, the culture is performed under three-dimensional (3D) conditions. 3D culture is a culture method for growing cells in the form of multicellular aggregates in vitro, called a morphology with a three-dimensional structure (organoid), and compared with 2D culture (adherent culture), 3D culture can be closer to corresponding physiological characteristics in tissues/organs in vivo by simulating the interaction among a three-dimensional cell network, cells and matrixes and cells.

The organoid obtained by the method of the invention can be frozen, revived, subcultured and maintained and cultured for a long time.

Drug screening

After knowing the specific properties of the cells co-labeled with Gli1 and EpCAM genes and their close correlation with hepatocyte regeneration/liver repair, substances (potential substances) that promote hepatocyte regeneration or promote liver repair can be screened based on this feature. From said substances, drugs can be found which are really useful for promoting hepatocyte regeneration or promoting liver repair.

Accordingly, the present invention provides a method of screening for a substance (potential substance) that promotes hepatocyte regeneration or promotes liver repair, the method comprising: (1) treating an isolate of liver tissue with a candidate substance; and (2) detecting the cell surface molecular characteristics of the isolate, wherein if the Gli1+ cells are statistically increased, the candidate substance is a substance (potential substance) that promotes hepatocyte regeneration or promotes liver repair. In a more preferred form, the method further comprises detecting EpCAM; if the Gli1+ EpCAM + cells are statistically increased, the candidate substance is a substance (potential substance) that promotes hepatocyte regeneration or promotes liver repair.

In a preferred embodiment of the present invention, a Control group (Control) may be provided in order to more easily observe the change in the amount of Gli1+ cells or Gli1+ EpCAM + cells during screening, and the Control group may be a isolate to which the candidate substance is not added.

As a preferred embodiment of the present invention, the method further comprises: further cellular and/or animal tests and/or clinical tests are performed on the obtained potential substances to further select and identify substances that are truly useful for promoting hepatocyte regeneration or promoting liver repair.

Potential substances which can promote the regeneration of liver cells or promote the repair of liver and are obtained by the screening method of the invention can form a screening library, so that people can further screen medicines finally.

Reagent kit

Based on the new findings of the present inventors, the present invention also provides a kit for isolating or enriching the cells or cell cultures described in the present invention, comprising: gli1 binding or capture molecule, and EpCAM binding or capture molecule.

Based on the new findings of the present inventors, the present invention also provides a kit for preparing a liver organoid, comprising: gli1 binding or capture molecule, EpCAM binding or capture molecule, and an agent for organoid culture. The organoid culture reagents may include, but are not limited to, reagents selected from the group consisting of: matrigel gel, growth medium, differentiation medium.

In a preferred embodiment of the invention, the kit may further comprise a reagent for hydrolyzing/disintegrating cells, such as collagenase, lytic enzyme; reagents for cell suspension; a medium for cell culture, and the like.

For the convenience of the skilled person, the kit of the present invention further comprises instructions for isolating or enriching the cells or cell cultures of the present invention or preparing the liver organoid. For example, the aforementioned method of the present invention can be described in the specification.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Experimental materials and methods

1. Animal experiments

Transgenic mice and strains used in this experiment were as follows: gli1-lacZ (Jackson lab, 008211, C57BL/6), Gli1-CreERt2(Jackson lab, 007913, C57BL/6), B6.Cg-Gt (ROSA) 26Sor^tm9(CAG ^{-tdTomato)Hze/J}(Ai9) (Jackson lab, 007909, C57BL/6), HNF4 α -DreERt2, NR1, and Ai66 mice were obtained from Zhou and researchers laboratories (institute for biochemistry and cell, China academy of sciences, 129x1/SvJ), Fah and researchers laboratories (national institute for biochemistry and cell, China), Japan, and Japan, and New, Umbers, and New, Japan, and New, Japanese, and New, Japanese, Utility, and New, Japan, New^-/-rag2^-/-Il2rg^-/-(FRG) mice (obtained from Xin Wang, C57BL/6J and 129S6/SvEvtac mixed background). For genealogical tracing experiments Tamoxifen (Sigma, TAM) was diluted with corn oil (Sigma) to a concentration of 20 mg/ml. In order to induce Cre and Dre enzymes to enter the nucleus, mice of 6-8 weeks old were injected intraperitoneally with TAM at an amount of 200ug per gram of body weight for 3 consecutive days. All lineage tracing experiments included at least 5 mice. Animal experiments were performed according to the guidelines of the animal ethics committee. Animals were housed in SPF facilities.

2. Liver injury model construction

Mouse acute and chronic liver injury models are the main models for studying the liver injury repair mechanism and function. The inventor adopts 2/3 liver resection model (PH) to prepare acute liver injury model. The method comprises the following specific steps: after the abdominal opening is performed, the roots of the left and right leaves are ligated, and the left and right leaves are cut off; then ligating the middle lobe approximately at the gallbladder-up position and approximately 2-3mm from the superior vena cava, and cutting the middle lobe and gallbladder; the abdomen was sutured and placed on a hot table to incubate until the mice were awake. Surgery was performed in SPF grade animal room operating room to avoid infection. The method is a mouse liver partial excision model internationally recognized at present. By using CCl₄Injecting and DDC and CDE feed to prepare a chronic liver injury model. CCl₄Establishing an induced chronic liver injury model: in the experiment, according to CCl₄Oil 1:3 ratio using CCl₄2mL/Kg for intraperitoneal injection, 3 timesThe injection was performed 1 time a day for 28 days, and the control group was injected with an equal amount of Oil only. Establishment of a chronic liver injury model based on DDC and CDE feeds: the mice aged 8 weeks are divided into two groups, and are fed with common feed and special feed containing 0.1% DDC respectively, and the DDC chronic injury mice are obtained and tested after 2 weeks of feeding. In the experiment, blood and liver samples of different liver injury model mice are respectively taken, and liver weight and body weight are recorded simultaneously for detection of liver injury indexes alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) and H&E, dyeing with sirius red, and finally determining that the liver injury model is successfully constructed.

3. Immunofluorescence and galactosidase (LacZ) staining

Livers were removed under sterile conditions and fixed with 4% paraformaldehyde at 4 ℃ for 1h, washed 3 times with PBS after fixation, then dehydrated in 30% sucrose at 4 ℃ overnight, and the next day tissues were embedded with OCT. The tissue was cut into 10 μm sections with an ice cutter, and when stained, the sections were air-dried at room temperature for 10 minutes. The dried sections were washed with PBS and then blocked with 1% BSA diluted in PBST (0.1% Triton X-100) for 30 min at room temperature. Diluting primary antibody with blocking solution, and incubating overnight at 4 ℃; PBST washing is carried out for three times, 10 minutes each time; diluting the secondary antibody with a confining liquid, and incubating for 60 minutes at room temperature in a dark place; PBST washing for three times; then dyeing with DAPI, washing with deionized water twice; mounting, air drying overnight, and storing at-20 deg.C. The antibody information used is as in the following table (table 1).

TABLE 1 antibody information

To perform X-gal staining of the sections, the sections were first washed with Wash buffer (PBS containing 2mM MgCl. sub.MgCl. sub.₂0.02% NP-40, 0.01% sodium deoxycholate, pH 7.4) 3 times for 5 minutes each. The staining solution was then stained with X-gal (Wash buffer contains 5mM K)₃Fe(CN)₆，5mM K₄Fe(CN)₆And 1mg/ml X-gal) were stained at 37 ℃ for 12 hours in the dark. After staining, the cells were washed 3 times with PBS for 5 minutes each. The nucleus is fixed red and stained, and the photographing and the observation are carried out under the mirror.

4. Cell isolation and 3D organoid culture

The reverse collagenase perfusion method of the inferior vena cava cannula is adopted for liver cell separation: firstly, the perfusion needle is reversely inserted into the inferior vena cava and then fixed, and the portal vein is opened to be used as an outflow tract. Then perfusing with perfusion liquid containing EGTA and then Ca²⁺The collagenase is fully perfused. After the perfusion is finished, the cells are filtered and cleaned, and then the prepared hepatocyte suspension is obtained by adopting density gradient centrifugation. In the invention, the method of jointly treating collagenase and clastic enzyme is adopted to separate bile duct cells: collecting Gli1-creERt2 with the age of 6-8 weeks; the liver of Ai9 and Gli1-lacZ mice was first cut to 0.5mm³Then treating the small blocks by 0.125mg/mL collagenase and lyase together, obtaining the bile duct structure by centrifugation, and picking out the bile duct with intact morphology by a stereomicroscope for subsequent experiments. After separating to obtain the liver cells and the bile duct cells, further extracting RNA of the liver cells and the bile duct cells, detecting the expression level of the Gli1 gene by using qPCR, or obtaining positive cells by sorting to culture a 3D organoid.

The inventors sorted EpCAM + and Gli1+ (tdTomato + or CUG +) cells by flow with EpCAM-APC (eBioscience, Clone G8.8, 1:50) antibody. The isolated positive cells were mixed with Matrigel gel (BD), the cell suspension was mixed according to the ratio of Matrigel to growth medium 5:3, and then 80. mu.l of each pack was seeded in a 24-well suspension plate. The formulation of the growth medium (EM) was as follows: the Advance DMEM/F12 medium was supplemented with 10mM HEPES (Gibco, USA), 2nM GlutaMAX-1(Gibco, USA), 500 XPrimocin (InvivoGen, USA), 1 XB 27(Gibco, USA), 1.56mM N-Acetylcysteine (Sigma, Germany), 10mM Nicotinamide (Sigma, Germany), 0.5. mu. M A83-01(Tocris, USA), 10. mu. M Y27632, 50ng/mL EGF (Peprotech, USA), 10ng/mL FGF10(Peprotech, USA), 1ng/mL FGF2(Peprotech, USA), homemade R-Spondin (10%) and Noggin (10%) conditioned medium. The culture medium is changed every 3 days in the culture process, and the subculture is carried out after continuous culture for 10 days, wherein the subculture steps are as follows: sucking culture solution, blowing and beating organoid and Matrigel glue for several times, transferring to a 15ml centrifuge tube with a pointed bottom, centrifuging at 1500rpm for 5 minutes, sucking supernatant, adding 1ml of TrypLE Express, digesting at 37 ℃ for 8 minutes, repeatedly blowing and beating by a pipette until a single cell suspension is formed, and then adding 1ml of culture solution containing serum for neutralization. Cells were passaged at 1:10 by centrifugation at 1500rpm for 5 minutes, and then seeded in 24-well plates in the same manner. To induce differentiation of cells into hepatocytes, single cell-derived organoids were maintained in EM for 3 days after passage, followed by replacement of Differentiation Medium (DM) and further culture for 10 days, and then the characteristics and functions of cells were verified by immunofluorescence, PAS, and LDL uptake experiments. The DM medium formulation is as follows: the Advance DMEM/F12 medium was supplemented with 50ng/mL EGF, 10. mu.M DAPT, 10mM HEPES (Gibco, USA), 2mM GlutaMAX-1(Gibco, USA), 500 XPrimocin (InvivoGen, USA), 1 XB 27, 10. mu. M Y27632, 10. mu.M DAPT (Selleck), 25ng/mL BMP7(Peprotech), 25ng/mL HGF (Peprotech) and 20ng/mL Oncostatin (Oncostatin) M.

5. Real-time quantitative PCR

Total RNA was extracted from organoids and isolated cells using TRIzol (Invitrogen). OD of all samples_260/280Ratio of>1.8 and<2.1. the ReverTra Ace qPCR RT master Mix with gDNA Remover kit (Invitrogen) kit reverse transcribes total RNA (500ng) from organoids and cells into cDNA. The real-time quantitative PCR was performed by ABI Fast 7500 fluorescent quantitative PCR, and the reaction system was 25. mu.L, where SYBR Green (Takara) was 12.5. mu.L, template cDNA was 2. mu.L, and primers were 1. mu.M. The reaction program was 95 ℃ for 10min, 94 ℃ for 15s, 60 ℃ for 30s, 72 ℃ for 30s, 40 cycles. Internal control was performed with GAPDH and each experiment was repeated at least three times. The primers used were as follows:

EpCAM：Fw-TGTGGACATAGCTGATGTGGCTTAC(SEQ ID NO:1)；

Rv-CACCCTCAGGTCCATGCTCTTA(SEQ ID NO:2)；

Krt19：Fw-GTCCTACAGATTGACAATGC(SEQ ID NO:3)；

Rv-CACGCTCTGGATCTGTGACA(SEQ ID NO:4)；

HNF4α：Fw-AGCTCGAGGCTCCGTAGTGTTT(SEQ ID NO:5)；

Rv-GAAAATGTGCAGGTGTTGACCA(SEQ ID NO:6)；

Alb：Fw-GCTGAGACCTTCACCTTCCA(SEQ ID NO:7)；

Rv-TCTTCAGTTGCTCCGCTGTA(SEQ ID NO:8)；

GAPDH：Fw-AGGTCGGTGTGAACGGATTTG(SEQ ID NO:9)；

Rv-TGTAGACCATGTAGTTGAGGTCA(SEQ ID NO:10)。

6. transplantation experiments

FRG mice were maintained on drinking water with 7.5 mg/L2- (2-nitro-4-trifluoromethylbenzoyl) -1,3 cyclohexanedione (NTBC). FRG male mice 8 to 12 weeks old were used for transplantation experiments. Organoid cells for transplantation were obtained from three different Gli1-creERt 2; ai9 mice, cultured in vitro for at least 2 months. After organoid passage, it was kept in EM for 3 days, followed by replacement of Differentiation Medium (DM) and further culture for 10 days. Then digested into single cells with TrypLE Express, and 1X 10 cells were added⁶Single cell suspensions from individual organoids were injected into the spleen of FRG mice. The mice were transplanted and then given NTBC medication in drinking water for 4 days. The normal drinking water was then changed and the health and body weight of the mice were measured every 1 day. Mice were deprived of liver for experimental use 1 month or 3 months after transplantation. Non-transplanted littermates served as negative controls.

7. Single cell banking and sequencing

This experiment used the Smart-seq2 method to construct scRNA-seq libraries. The specific operation steps are as follows: after UV irradiation of the 96-well plate, 2ul of cell lysis buffer (diluted to 0.2% (vol/vol) with Triton X-100, 190ul of 0.2% Triton X was added to 10ul of RNase inhibitor, followed by 1ul of oligo-dT primer (10uM) and 1ul of dNTP mix. Sealing, and storing at 4 deg.C. FACS was plated, centrifuged at 700g for 1min at 4 ℃ and placed on ice. The lysis was performed by incubation for 3 minutes at 72 ℃ in a PCR instrument. Next, the present inventors reverse transcribed single-cell RNA using SuperScript II reverse transcriptase in the presence of oligonucleotide primer (TSO). The products of the reverse transcription were then preamplified using a Kapa Ready Mix (Kapa Biosystems). The amplified products were purified using VAHTS DNA Clean Beads and quality controlled using QIxcel (to confirm correct product size) and Qubit (to determine quantity). Next, a single cell Library was generated using the TruePrep DNA Library Prep Kit V2 for Illumina Library construction Kit by Vazyme. Each single cell library was encoded by PCR using Index primers. The encoded single cells were pooled together and sequenced in groups of 80 cells.

Example 1 expression analysis of Gli1 in Normal liver

The regeneration of liver involves a relatively complex mechanism, and after extensive research and experiments, the inventors focus on the molecular mechanism and biological function of Gli1 in liver injury repair. First, the present inventors examined the expression profile of Gli1 in normal liver in order to provide experimental basis for further studies. The specific experimental procedures and results are as follows:

1. gli1 is mainly expressed in liver luminal cells

The liver is mainly composed of hepatocytes and cholangiocytes. To investigate the expression profile of Gli1 in liver cells, the inventors isolated hepatocytes and cholangiocytes from normal mouse liver tissue. The reverse collagenase perfusion method of the inferior vena cava cannula is adopted for liver cell separation: firstly, the perfusion needle is reversely inserted into the inferior vena cava and then fixed, and the portal vein is opened to be used as an outflow tract. Then perfusing with perfusion liquid containing EGTA and then Ca²⁺The collagenase is fully perfused. After the perfusion is finished, the cells are filtered and cleaned, and then the prepared hepatocyte suspension is obtained by adopting density gradient centrifugation. The method for separating bile duct cells by adopting collagenase and lyase for co-treatment comprises the following steps: firstly, the liver tissue is cut into 0.5mm³Then treating the small blocks by 0.125mg/mL collagenase and lyase together, obtaining the bile duct structure by centrifugation, and picking out the bile duct with intact morphology by a stereomicroscope for subsequent experiments. After separating and obtaining the liver cells and the bile duct cells, further extracting RNA of the liver cells and the bile duct cells respectively, and detecting the expression level of the Gli1 gene by qPCR.

As shown in fig. 3A-B, Alb and HNF4a are markers specific to mature hepatocytes, respectively, and Alb and HNF4a are mainly expressed in liver cells; as shown in fig. 3C to D, CK19 is a marker specifically expressed in cholangiocytes, EpCAM is a marker specifically expressed in cholangiocytes and stem/progenitor cells, and CK19 and EpCAM are mainly expressed in cholangiocytes. The above results indicate that the isolated cells are relatively specific.

As in fig. 3E, Gli1 is predominantly expressed in cholangiocytes.

2.Gli1 is expressed predominantly in cells surrounding the bile duct of the liver and is partially co-expressed with EpCAM, PDGFRa and a-SMA genes

In order to observe the expression distribution of the Gli1 gene in the liver more intuitively, a Gli1-LacZ transgenic mouse was used as a study object, and a normal liver injury adult mouse was used as a study object. The livers were removed under sterile conditions and frozen sections were X-gal stained. In order to eliminate the possibility of false positive results caused by X-gal staining, the beta-gal antibody is adopted to carry out immunofluorescence staining, the results are repeated, and the immunofluorescence co-staining is carried out with various liver cell markers (including markers of various different types of cells such as bile duct cells, liver cells, interstitial cells, liver stem cells, liver progenitor cells and the like), so as to identify the cell type expressing the Gli1 gene.

The results showed that Gli1+ cells were found around bile ducts by X-gal staining and by immunofluorescence staining (FIG. 4).

3. In vivo cell lineage tracing confirms distribution of Gli1+ cells

The cell lineage tracing is to mark specific cells in the organism by various ways and carry out in vivo tracing observation on cell activities including proliferation, differentiation, migration and the like of progeny cells of the cells, and is an effective research method for detecting the cell transdifferentiation process of specific types of cells in development, diseases and regeneration. Based on the above experimental results, to further determine the cell type expressing Gli1 gene, Gli1-CreER was used^T2The mouse is crossed with a Cre reporter gene Ai9 mouse to obtain Gli1-creER^T2(ii) a Ai9 double-transgenic mouse, injecting Tamoxifen (TM), taking liver tissue as frozen section for immunofluorescence staining, detecting tdTomato marking condition of Gli1+ cells in mouse liver, and mixing with various liver cell markers (including bile duct cell, liver cell, interstitial cell, liver stem cell and liver progenitor cell)Markers of these multiple different types of cells) were separately immunohistochemically co-stained to further clarify the distribution of Gli1+ cells in the liver.

Immunofluorescent staining results showed that Gli1+ cells could be labeled by tdTomato. The experimental results also showed that Gli1+ cells were distributed around the bile duct and co-localized with the hepatic progenitor marker EpCAM (fig. 5).

Example 2 expression analysis of Gli1 after liver injury and transdifferentiation of lineage-traced Gli1+ cells in liver injury repair

1. Expression analysis of Gli1 after liver injury

Mouse acute and chronic liver injury models are the main models for studying the liver injury repair mechanism and function. An acute liver injury model was prepared using the 2/3 hepatectomy model (PH). Reference is made in the experiments primarily to the method published by Mitchell et al on Nat Protoc in 2008. The method comprises the following specific steps: after the abdominal opening is performed, the roots of the left and right leaves are ligated, and the left and right leaves are cut off; then ligating the middle lobe approximately at the gallbladder-up position and approximately 2-3mm from the superior vena cava, and cutting the middle lobe and gallbladder; the abdomen was sutured and placed on a hot table to incubate until the mice were awake. Surgery was performed in SPF grade animal room operating room to avoid infection. The method is a mouse liver partial excision model internationally recognized at present. By using CCl₄And (3) preparing a chronic liver injury model by injecting DDC, CDE and MCD feeds. CCl₄Establishing an induced chronic liver injury model: in the experiment, according to CCl₄Oil 1:3 ratio using CCl₄2mL/Kg were injected intraperitoneally 1 time every 3 days for 28 days, and the control group was injected with an equal amount of Oil. Establishment of a chronic liver injury model based on DDC, CDE and MCD feeds: the mice aged 8 weeks are divided into two groups, and fed with common feed and special feed containing 0.1% DDC, CDE and MCD, respectively, and after feeding for 3-4 weeks, the mice with chronic injury are obtained and tested. The Gli1 gene was observed in partial liver resection (PH), CCl by the above model₄Gli1 activation in the liver of mice model acute/chronic liver injury by injection, DDC, CDE and MCD feed.

The experimental results show that Gli1 is mainly expressed around bile ducts of normal liver,acute liver injury (PH) did not cause significant change in Gli1, whereas chronic liver injury (CCl)₄Injection, DDC, CDE, and MCD models) can cause a significant increase in Gli1 levels and an increase in Gli1+ cells (fig. 6).

The above experimental results indicate that the slow liver injury repair process requires the regulation of Gli1 expression and the involvement of Gli1+ cells.

2. Lineage tracing confirms transdifferentiation of Gli1+ cells in liver injury repair

Based on the above experimental results, to further observe whether Gli1+ cells in mouse liver could differentiate into mature hepatocytes. The inventor firstly puts Gli1-CreER of C57BL/6 strain^T2The mice are hybridized with Ai9 mice to obtain Gli1-CreER^T2(ii) a Ai9 double transgenic mice. On this basis, stable genetic labeling of Gli1+ cells in the liver (i.e. labeled tdTomato) was achieved by injection of tamoxifen (tm). Subsequently, the Gli1-CreER that had been genetically labeled in this Gli1+ cell was used^T2(ii) a Ai9 mice a DDC liver injury model was prepared to track whether Gli1+ cells have the potential to transdifferentiate into hepatocytes and differentiate into cholangiocytes.

The results showed that the number of Gli1+ cells around the bile duct increased and Gli1+ cells could transdifferentiate into hepatocytes (fig. 7). The above experimental results show that Gli1+ cells have important roles in liver injury repair process and the ability of Gli1+ cells to differentiate and transform cell attributes.

Example 3 Single cell sequencing analysis of the characteristics of Gli1+ cells

The above experimental results demonstrate that Gli1 and EpCAM can be co-localized and Gli1+ cells can be transdifferentiated in vivo to form hepatocytes, and in order to verify whether EpCAM + and Gli1+ cells are the same cell and have characteristics of hepatic stem/progenitor cells, three cell subsets, EpCAM-/Gli1+, EpCAM +/Gli 1-and EpCAM +/Gli1+ in mouse livers were separately isolated and pooled by the method of Smart-seq2, followed by single cell sequencing and hepatocyte control, and characteristics of different types of cells were observed at the single cell level. A total of 527 cells (144 EpCAM-/Gli1+ cells, 148 EpCAM +/Gli 1-cells, 202 EpCAM +/Gli1+ cells, 33 hepatocytes) were analyzed by single cell sequencing, and Unsupervised clustering (Unsupervised clustering) analysis found that these cells could be divided into four distinct populations of cells, which were clearly identified as EpCAM +/Gli1-, EpCAM-/Gli1+, EpCAM +/Gli1+ and hepatocytes, respectively, and these cells exhibited different biological characteristics. As a result, it was found that EpCAM was mainly expressed in EpCAM +/Gli 1-cells and EpCAM +/Gli1+ cells, whereas tdTomato was mainly expressed in EpCAM-/Gli1+ cells and EpCAM +/Gli1+ cells. The above results are consistent with the results of flow sorting, providing evidence for high quality of sequencing data. Then, the first 200 genes of each cell population were detected by cluster analysis, and representative differential genes of each cell population were detected by violin plots. Hepatocytes specifically express hepatocyte marker genes such as Alb and CYP7a 1. EpCAM +/Gli 1-cells specifically express KRT18, KRT7 and KRT19, which are known markers of biliary cells. EpCAM-/Gli1+ highly expressed extracellular matrix (ECM) genes (Col1A1, Col1A2, Col3A1 and DCN) and mesenchymal cell related marker genes (PDGFR α and PDGFR β). Interestingly, it was found that EpCAM +/Gli1+ cells were intermediate between EpCAM + Gli 1-and EpCAM-Gli1+ cells, co-expressing both EpCAM + Gli 1-and EpCAM-Gli1+ cell-specific genes. EpCAM +/Gli1+ cells expressed marker genes for epithelial cells and mesenchymal cells, suggesting that EpCAM +/Gli1+ cells may be facultative and function as both cells. To study the biological function of EpCAM +/Gli1+ cells, GO analysis was performed on differentially expressed genes in EpCAM +/Gli1+ cells. This analysis revealed several significantly enriched biological processes associated with tissue morphogenesis, injury response and stem cell differentiation. The function of the EpCAM +/Gli1+ cell subset is characterized by using GSEA (gene set evolution analysis), and the result shows that the EpCAM +/Gli1+ cells are enriched in the ways of tissue morphogenesis, injury reaction, stem cell differentiation and the like compared with other three cells. The EpCAM +/Gli1+ cell population co-expressed the epithelial cell markers (EpCAM, KRT7 and KRT19) and the mesenchymal cell markers (PDGFR α and PDGFR β) to support the progenitor signature of this population of cells, suggesting that EpCAM +/Gli1+ cells are the source of Gli1+ hepatocytes after liver injury (fig. 8).

Example 4 isolation and 3D organoid culture of Gli1+ cells characterization of Gli1+ cells

The above experimental results demonstrate that Gli1+ cells can differentiate/transdifferentiate into hepatocytes. Therefore, it is assumed that Gli1+ cells are likely to be liver stem cells or progenitor cells, and thus play an important role in the repair process after liver injury. 3D organoid culture is a latest cell in vitro culture technology, can well simulate the microenvironment of cells in vivo, and is widely applied to the research of characteristics of stem cells or progenitor cells. Based on this, the characteristics of Gli1+ cells will be further determined using 3D organoid culture techniques. Using Gli1-CreER^T2(ii) a Ai9 transgenic mice, after TM induction, liver is taken to separate and obtain liver cell suspension, the cells are divided into two cell subsets of Gli1+ and Gli 1-by flow sorting, then 5,000 cells are respectively taken and mixed with Matrigel in equal proportion by culture solution, 3D organoid culture is carried out, and the organoid forming capability of the two cell subsets of Gli1+ and Gli 1-is observed. Experimental results show that the Gli1+ cells can form organoids through 3D culture, the formed organoids can be subcultured continuously, and meanwhile, the Gli1+ organoid cells can be subjected to cryopreservation and resuscitation. Whereas Gli 1-cells were unable to form organoids by the same culture method (FIG. 9). Based on this, Gli1+ cells were confirmed to have hepatic stem/progenitor cell characteristics.

EpCAM is a molecule specifically expressed by hepatic stem/progenitor cells. Liver EpCAM + cells are able to form organoids in adult mice by 3D culture, whereas EpCAM-cells are unable to form organoids in culture. In combination with the results found in the above experiments: the ability of Gli1 and EpCAM to co-localize and Gli1+ cells to form organoids in vitro suggests that different subpopulations of liver progenitors may be present and that they play an important role in the repair of liver damage. On the basis of the separation of the hepatocytes and the cholangiocytes, the cells of the mouse liver are further divided into a plurality of cell subsets such as EpCAM +/Gli1-, EpCAM +/Gli1+, EpCAM-/Gli1+ and EpCAM-/Gli 1-through flow cytometry sorting, and are respectively cultured in a 3D organoid, and the capacity of forming organoids in vitro of the cells of each subset is detected.

The experimental results show that the EpCAM +/Gli 1-and EpCAM +/Gli1+ cells can form organoids through 3D culture, and the organoid forming efficiency and the same day size of the EpCAM +/Gli1+ cells are obviously higher than those of the EpCAM +/Gli 1-cells. Whereas EpCAM-/Gli1+ and EpCAM-/Gli 1-were not able to form organoids by the same culture method. In addition, the organoids formed by EpCAM + Gli 1-and EpCAM + Gli1+ cells were morphologically similar to organoids previously cultured from cholangiocytes (FIG. 10).

Example 5 organoids formed by EpCAM +/Gli1+ express biliary cell markers and can differentiate into functional hepatocytes in vitro

1. Organoid expression bile duct cell marker formed by EpCAM +/Gli1+

Whereas EpCAM + Gli 1-and EpCAM + Gli1+ cells formed organoids that were morphologically similar to organoids previously cultured from cholangiocytes. The inventor realizes that the organoids formed by EpCAM + Gli 1-and EpCAM + Gli1+ cells may express markers of corresponding bile duct cells, and shows that the EpCAM + Gli1+ organoid specifically expresses tdtomato while the EpCAM + Gli 1-organoid does not express tdtomato through staining, thereby proving that the cultured organoid is better in specificity. By immunofluorescent staining of organoids with cholangiocyte-specific markers (KRT19, SOX9) and cell proliferation marker (Ki67), it was found that both cell-cultured organoids highly expressed cholangiocyte markers and had high proliferation potency (fig. 11).

2. The organoid formed by EpCAM +/Gli1+ can be differentiated into functional liver cells in vitro

To further study the in vitro differentiation capacity of the organoids formed by EpCAM +/Gli1+, the passaged cells were first cultured in growth medium for 3 days, then replaced with differentiation medium for 10 days, and the expression of mature hepatocyte markers (HNF4a and Alb) and cholangiocellular markers (KRT19, EpCAM) in the organoids in the presence of growth medium and differentiation medium, respectively, was first analyzed by qPCR.

The results show that KRT19, EpCAM are predominantly expressed in organoids in the presence of growth medium, whereas hepatocyte-specific markers (HNF4a and Alb) are predominantly expressed in organoids in the presence of differentiation medium. The differentiated organoids were then immunofluorescent stained by using hepatocyte-specific markers (HNF4a and Alb). High expression of hepatocyte-specific markers was also found in organoids in the presence of differentiation medium. Glycogen storage (PAS staining) and LDL uptake are important methods to verify that mature hepatocytes have liver function. The organoids induced by differentiation medium were found to have normal liver function by staining with PAS and staining with LDL uptake (fig. 12).

These experiments established that EpCAM +/Gli1+ cells have the ability to form liver mature cells in vitro.

Example 6 biological function of Gli1+ cells in liver injury repair

The hepatic stem/progenitor cell characteristics of Gli1+ cells in the liver were confirmed by the above experiments. However, the specific biological function of Gli1 in the repair of liver injury is still not well understood. To investigate whether Gli1+ cells in vivo could enhance liver injury repair and promote hepatocyte regeneration, equal numbers of in vitro cultured EpCAM +/Gli 1-and EpCAM +/Gli1+ organoid cells were transplanted into model mice (Fah knockout mice) examined for liver regeneration ability, respectively, and the efficiency of proliferation of both cells in the liver and the ability to restore liver function in Fah-deficient mice were analyzed at different time points, respectively. The ability to restore liver function was tested with FAH antibodies 1 month and 3 months after transplantation of EpCAM +/Gli 1-and EpCAM +/Gli1+ organoid cells, respectively.

As a result, sporadic FAH-positive cells were observed at 1 month after transplantation, and many FAH-positive cells were observed at 3 months after transplantation, but there was no significant difference between the transplanted EpCAM +/Gli 1-and EpCAM +/Gli1+ organoid cells (fig. 13).

Thus, the efficiency of the organoids formed by EpCAM +/Gli1+ was higher, and at the same time the organoids were relatively large, in addition to the other phenotypes of EpCAM +/Gli 1-and EpCAM +/Gli1+ organoids being identical.

Example 7 3D organoid culture of human liver cells and Effect of Hh Signal pathway agonists and inhibitors on cultured organoids

In earlier work of the inventor, EpCAM + cells are separated by flow cytometry on the basis of obtaining a human liver sample, and a 3D organoid culture system of the human liver cells is successfully established. Liver organoid cells can proliferate rapidly (fig. 14) and can be cultured continuously for more than 25 passages, while liver organoid cells can be cryopreserved and revived.

To determine the regulatory effect of the Hh signaling pathway on cultured human liver cells 3D organoids, the inventors treated cultured 3D organoids with inhibitors of the Hh signaling pathway, SANT-1 (targeting Smo), GANT-61 (targeting Gli), and agonist of Smo, SAG, respectively.

As a result, it was found that only the inhibitor of Gli, GANT-61, was able to inhibit the growth of human 3D organoids (FIG. 15). On one hand, the result suggests that the non-classical Hh pathway represented by Gli1 and Gli1+ cells may play more important roles in the liver injury repair process, and also lays a solid foundation for further screening related small-molecule lead compounds by using 3D organs and performing artificial intervention on cell proliferation.

Summary and discussion

The liver is one of the largest and most important metabolic organs and has a very high regeneration potential after injury. Hepatocytes are the main functional cell types for physiological functions of the liver. Therefore, the cellular source of new hepatocytes has been the focus of research on regeneration after liver injury. The regenerative capacity of the liver depends mainly on the self-proliferation capacity of the two epithelia, hepatocytes and cholangiocytes. Recent studies have shown that in severe liver injury, hepatocytes and cholangiocytes may themselves be mutually facultative liver stem/progenitor cells (LPC) and participate in the repair of damaged liver. However, the role of non-epithelial cells including mesenchymal cells and immune cells in liver regeneration is still not completely understood. When hepatocytes and cholangiocytes are severely damaged, LPC may promote liver regeneration by generating new hepatocytes. Indeed, the identification of LPC-specific markers would greatly facilitate the development of the field of liver regeneration. Previous studies suggest that the mesenchymal marker Foxl1 can be recognized as a marker of facultative LPC and can generate hepatocytes and biliary cells after liver injury. However, it is emphasized that the Foxl1 gene is not expressed prior to liver injury and therefore cannot be used for pre-labelling cells produced during liver regeneration. In addition, a variety of surface markers have been used to isolate bile duct cell subsets with liver regeneration capability, including EpCAM, TROP2, CD133, Lgr5, CD44, and Thy-1. However, these results were based on a single selectable marker or did not completely exclude other non-progenitor cell populations. In the present invention, the inventors have discovered a novel Gli1+ mesenchymal-like cell population that contributes to the formation of new hepatocytes in chronic injury. The inventors found that Gli1+ cells in the liver were a heterogeneous population including PDGFRa + stromal cells and a very small population of EpCAM + cells. By scRNA-seq sequencing, the inventors found that EpCAM +/Gli1+ cells appear to be in a double phenotypic state, co-expressing epithelial markers and mesenchymal markers. At the same time, the data of the present inventors also show that Gli1 and EpCAM mark a specific cell population present in healthy liver, which is expandable in vitro to epithelial organoids and differentiable into functional hepatocytes both in vivo and in vitro. Thus, Gli1 and EpCAM can be used as markers for screening hepatic progenitors. Based on mouse studies, the inventors studied the effects of various inhibitors and agonists of the Hh pathway on human liver organoids using cultured EpCAM + human liver organoids, and showed that only Gli inhibitors could inhibit the formation, growth, and proliferation of human liver organoids. Therefore, the expression of Gli1 in cultured human organoids can be confirmed, and the progenitor cell population co-expressed by Gli1 and EpCAM is separated and cultured and amplified in vitro, which provides a new way for human liver regeneration. In summary, the genetic evidence of EpCAM +/Gli1+ cells for liver repair provides new insights into the cellular and molecular mechanisms of liver disease and regeneration.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. Use of a cell or cell culture for: preparing a composition that transdifferentiates to form hepatocytes, preparing a composition for performing liver injury repair, or preparing a liver organoid;

wherein the cell is a Gli1+ cell and comprises the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids.

2. The use of claim 1, wherein the cells are Gli1+ and EpCAM + cells.

3. The use of claim 1 or 2, wherein the cell further comprises a characteristic selected from the group consisting of:

having epithelial cell characteristics, or expressing an epithelial cell marker;

having mesenchymal cell characteristics, or expressing mesenchymal cell markers;

can form organoid with the characteristics of inter-biliary duct cells or express bile duct cell markers.

4. The use of claim 3, wherein the cholangiocellular marker comprises a marker selected from the group consisting of: KRT19, SOX 9;

the epithelial cell markers include markers selected from the group consisting of: EpCAM, KRT7 and KRT 19; or

The stromal cell marker comprises a marker selected from the group consisting of: PDGFR α and PDGFR β.

5. The use according to any one of claims 1 to 4, wherein the cells comprise passaged cells.

6. An isolated cell or cell culture, said cell being a Gli1+ cell and comprising the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids.

7. The cell or cell culture of claim 6, wherein the cell is a Gli1+ and EpCAM + cell.

8. The cell or cell culture of claim 7, wherein the cell further comprises a characteristic selected from the group consisting of:

9. The cell or cell culture of any one of claims 6 to 8, wherein said Gli1+ cells are isolated by a method comprising: sorting Gli1+ cells from liver tissue isolates using binding or capture molecules that specifically bind or capture Gli 1; or

The Gli1+ and EpCAM + cells were isolated by a method comprising: gli1+ and EpCAM + cells were sorted from liver tissue isolates using binding or capture molecules that specifically bind or capture Gli1 and EpCAM.

10. A method of isolating or enriching a cell or cell culture, said cell being a Gli1+ cell and comprising the following characteristics: (i) has hepatic stem/progenitor cell characteristics, (ii) has the function of differentiating/transdifferentiating to form hepatocytes, (iii) has a liver injury repair effect, and/or (iv) is capable of forming liver organoids; the method comprises the following steps: gli1+ cells were sorted from liver tissue isolates using binding or capture molecules that specifically bind or capture Gli 1.

11. The method of claim 10, wherein the cells are Gli1+ and EpCAM + cells, the method comprising: gli1+ and EpCAM + cells were sorted from liver tissue isolates using binding or capture molecules that specifically bind or capture Gli1 and EpCAM.

12. The method of claim 10 or 11, wherein the method of sorting cells comprises: flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion.

13. A method of preparing a liver organoid, comprising: culturing the cell or cell culture of any one of claims 6 to 9 to grow and differentiate to obtain a liver organoid.

14. The method of claim 13, wherein the method comprises:

(1) providing a cell or cell culture according to any one of claims 6 to 9, or a cell or cell culture obtained by a method according to any one of claims 1 to 5;

(2) growing the cells or cell cultures of (1); preferably, passage is carried out for 1-5 times;

15. The method according to claim 14, wherein in (2), the medium for growth culture contains an advanced DMEM/F12 medium to which HEPES, GlutaMAX-1, primocin, B27, N-acetyl cysteine, Nicotinamide, a83-01, Y27632, EGF, FGF10, FGF2, R-Spondin, Noggin; or

(3) The medium for differentiation culture contained advanced DMEM/F12 medium, to which EGF, DAPT, HEPES, GlutaMAX-1, primocin, B27, Y27632, DAPT, BMP7, HGF, and oncostatin M were added.

16. The method of claim 13, wherein said organoids further comprise organoids selected from the group consisting of: passaged organoids, continuously cultured organoids, cryo-preserved and/or resuscitated organoids.

17. The method of claim 13, wherein the culturing comprises: three-dimensional culture or two-dimensional culture; preferably three-dimensional cultivation.

18. An artificially created liver organoid obtained by growing and differentiating the cell or cell culture of any one of claims 6 to 9; or, it is prepared by the method for preparing liver organoid according to any one of claims 13 to 17.

19. A kit for isolating or enriching a cell or cell culture according to any one of claims 6 to 9, comprising: gli1 binding or capture molecule, and EpCAM binding or capture molecule.

20. A kit for preparing a liver organoid, comprising: gli1 binding or capture molecule, EpCAM binding or capture molecule, and an agent for organoid culture; or

The method comprises the following steps: the cell or cell culture of any of claims 6 to 9, and a reagent for organoid culture.

21. The kit of claim 20, wherein the reagents for organoid culture comprise:

a medium for performing growth culture comprising: an advanced DMEM/F12 culture solution, wherein HEPES, GlutaMAX-1, primocin, B27, N-acetyl cysteine, Nicotinamide, A83-01, Y27632, EGF, FGF10, FGF2, R-Spondin and Noggin are added; and/or

A culture medium for differentiation culture which comprises an Advance DMEM/F12 culture medium supplemented with EGF, DAPT, HEPES, GlutaMAX-1, primocin, B27, Y27632, DAPT, BMP7, HGF, and oncostatin M.

Use of Gli1 for screening liver stem/progenitor cells, or for preparing a composition for screening liver stem/progenitor cells.

Use of a combination of Gli1 and EpCAM for screening liver stem/progenitor cells, or for the preparation of a composition for screening liver stem/progenitor cells.

24. A method of screening for an agent that promotes hepatocyte regeneration or promotes liver repair, the method comprising:

(1) treating an isolate of liver tissue with a candidate substance; and

(2) and detecting the cell surface molecular characteristics of the liver tissue isolate, wherein if the Gli1+ cells are increased statistically, the candidate substance is a substance for promoting hepatocyte regeneration or promoting liver repair.

25. The method of claim 24, wherein in detecting the cell surface molecular property of the isolate, further comprising detecting EpCAM; if the Gli1+ EpCAM + cells are statistically increased, the candidate substance is a substance that promotes hepatocyte regeneration or promotes liver repair.

26. The method of claim 24 or 25, wherein step (1) comprises: adding a candidate substance to the isolate in the test group; and/or the presence of a gas in the gas,

the step (2) comprises the following steps: detecting the amount of Gli1+ cells or Gli1+ EpCAM + cells in the isolate; and comparing to a control, wherein said control is an isolate to which said candidate substance is not added; if Gli1+ cells or Gli1+ EpCAM + cells in the test group are statistically increased, the candidate substance is a substance that promotes hepatocyte regeneration or promotes liver repair.