KR102015562B1 - Composition for inducing differentiation of hepatocyte-like cells having polarity and differentiation method of hepatocyte-like cells using the same - Google Patents

Abstract

The present invention relates to a composition for inducing differentiation into hepatocyte-like cells having polarity and a method for differentiation into hepatocyte-like cells using the composition. Specifically, when the compound represented by Formula 1 according to the present invention is treated during the process of differentiating hepatocytes, the differentiated hepatocyte-like cells are similar in shape to hepatocytes, have polarity by inhibiting EMT, and the hepatocyte marker proteins and genes. By increasing expression and exhibiting functional properties of the liver, the composition comprising the compound represented by the formula (1) according to the present invention can be usefully used as a composition for differentiating hepatocyte-like cells from hepatocytes.

Description

COMPOSITION FOR INDUCING DIFFERENTIATION OF HEPATOCYTE-LIKE CELLS HAVING POLARITY AND DIFFERENTIATION METHOD OF HEPATOCYTE-LIKE CELLS USING THE SAME}

The present invention relates to a composition for inducing differentiation into hepatocyte-like cells having polarity and a method for differentiation into hepatocyte-like cells using the composition.

The liver produces and secretes bile; Storage of glycogen, vitamins and minerals; It is an essential organ that contributes to maintaining the body's environment through various functions including the synthesis of proteins. In addition, the main function of the liver is metabolism of xenobiotic. Therefore, the liver is the most important organ to be considered in the development of new drugs, and liver toxicity is one of the major factors that the drug withdraws from the market.

Several in vitro models are used at preclinical stages for hepatotoxicity testing, among which hepatocyte-like cells (HLC) derived from pluripotent stem cells are considered as a model of potential drug screening and have. However, such cells have limited drug metabolic functions compared to primary hepatocytes and lose polarity during cell culture. Therefore, there is a need for the development of new methods that can induce differentiation into HLC with correct cell polarity and drug metabolic function.

In this regard, Korean Patent Laid-Open No. 10-2017-0087756 discloses a system for co-culturing various cells constituting liver tissue by simulating the structure and environment of hepatic lobule, which is a unit structure of the liver, and using it for evaluating liver toxicity and A method for evaluating hepatotoxicity using the same is disclosed.

Epithelial-mesenchymal transition (ETM), on the other hand, is a series of processes in which epithelial cells lose their properties and acquire the properties of mesenchymal cells. Epithelial cells undergoing EMT lose their unique characteristics such as polarity, cell-cell adhesion and cytoskeletal tissue. In addition, EMT is also involved in the differentiation of hepatocytes. It is known from previous studies that hepatocyte differentiation occurs due to signaling mechanisms associated with EMT. In addition, EMT and its transdifferentiation phenomenon, mesenchymal-epithelial transition (MET), are important for liver development and phenotype formation of hepatocytes.

Furthermore, the Aurora-A kinase protein is one of the Aurora mitotic serine / threonine kinase family and is important for cell division. It is also known to be often overexpressed in cancer cells. Aurora-A kinase protein is an important factor included in EMT, and recent studies have reported that Aurora-A kinase protein inhibitors can treat cancer cells.

Accordingly, the present inventors have been trying to produce hepatocyte-like cells showing polarity, and the hepatocyte-like cells obtained by treating phthalazione pyrazole (PP) in the process of differentiating hepatocytes have different shapes from hepatocytes. The present invention was completed by confirming that it is similar, has polarity by inhibiting EMT, increases expression of hepatocyte marker proteins and genes, and exhibits functional properties of the liver.

Republic of Korea Patent Publication No. 10-2017-0087756

It is an object of the present invention to provide a composition for differentiation of hepatoblasts into hepatocyte-like cells comprising a specific compound.

Another object of the present invention is to provide a method for differentiating hepatocyte-like cells from hepatocytes by treating a specific compound, and providing hepatocyte-like cells differentiated by the above methods.

Still another object of the present invention is to provide a method for screening liver toxicity using hepatocyte-like cells differentiated by the method according to the present invention.

In order to achieve the above object, the present invention provides a composition for differentiation into hepatocyte-like cells (HLC) from hepatoblast (hepatoblast) comprising a compound represented by the formula (1) as an active ingredient:

[Formula 1]

.

.

The present invention also provides a method for differentiating hepatocyte-like cells in hepatocytes, the method comprising treating the hepatocytes with a compound represented by Formula 1 below:

[Formula 1]

.

.

The present invention also provides hepatocyte-like cells differentiated by the above method.

Furthermore, the present invention provides a method for screening liver toxicity using the hepatocyte-like cells.

When the compound represented by Formula 1 according to the present invention is treated during the process of differentiating hepatocytes, differentiated hepatocyte-like cells are similar in shape to hepatocytes, have polarity by inhibiting EMT, and increase expression of hepatocyte marker proteins and genes. And, by showing the functional properties of the liver, the composition comprising the compound represented by the formula (1) according to the present invention can be usefully used as a composition for differentiating hepatocyte-like cells from hepatocytes.

1 is a schematic diagram showing a method for producing hepatocyte-like cells (HLC) from embryonic stem cells.
Figure 2 is a photograph of the results of microscopic observation of the form of differentiated HLC by adding an Aurora kinase inhibitor to the medium.
3 is a photograph of the results of observing the morphology of HLC differentiated according to the concentration of added phthalazinone pyrazole (PP) under a microscope.
Figure 4 is a graph confirming the cytotoxicity by the Aurora kinase inhibitor (ES-HLC: HLC differentiated from CHA-hES15 cells, iPS-HLC: HLC differentiated from iPS cells derived from human normal fibroblasts, Huh7: control) .
5 is a photograph showing the results of confirming the change in the expression of protein related to polarity in HLC differentiated by PP treatment by immunocytochemical analysis.
6 is a result of Western blot confirming the expression change of the protein related to polarity in HLC differentiated by treatment with PP (A) and a graph showing the expression level of E-cadherin and N-cadherin protein from the result (B) )to be.
7 is a graph showing the results of mRNA expression change of E-cadherin and N- cadherin in differentiated HLC by treating Aurora kinase inhibitors other than PP.
8 is a graph showing the mRNA expression levels of hepatocyte markers and drug metabolism enzymes in differentiated HLC by treating PP.
9 is a photograph showing the results of confirming the change in expression of hepatocyte markers and drug metabolism enzymes in HLC differentiated by PP treatment by immunocytochemical analysis.
10 shows Ac-LDL uptake and glycogen synthesis changes (A) in HLC differentiated by PP treatment; Change in secretion of bile ducts (B); Change in secretion of albumin (C); And the result of confirming the production change (D) of urea.
FIG. 11 shows Ki67, BrdU and DAPI staining (A), the number of cells positive for BrdU dye in the staining results (B), and nuclei showing positive for DAPI staining. This is the result shown through (B).
Figure 12: Changes in the expression of Aurora-A kinase protein (A) in HLC differentiated by PP treatment; Inhibition of phosphorylation of AKT protein (B); And AKT protein phosphorylation results in the expression change (C) of the lower signaling mechanism protein.
13 is a graph showing the results of a change in the expression of Aurora-A kinase, AKT1, snail and HNF4α gene in PP-treated HLC.
14 is a schematic diagram illustrating a signal transmission mechanism in HLC by treatment with PP.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for differentiation of hepatocyte-like cells (HLC) from hepatoblasts (hepatoblast) comprising a compound represented by the formula (1) as an active ingredient:

[Formula 1]

.

.

The compound represented by Formula 1 may be phthalazinone pyrazole. The "phthalazinone pyrazole (PP)" compound is 4-[(5-methyl-1H-pyrazol-3-yl) amino] -2H-phenyl-1-phthalazinone (4-[( 5-methyl-1H-pyrazol-3-yl) amino] -2H-phenyl-1-phthalazinone) and has the formula C ₁₈ H ₁₅ N ₅ O. Phtharazinone pyrazole compounds are known as inhibitors of Aurora-A kinase. In particular, Aurora-A kinase is overexpressed in various tumor cells such that the compound can be used as an anticancer agent.

The compound represented by Chemical Formula 1 may be used in addition to a medium for hepatocyte culture known in the art. At this time, the addition amount can be appropriately selected by a person skilled in the art, specifically, 0.1 to 20 μM, 0.1 to 10 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.5 to 20 μM, 0.5 to 10 μM, 0.5 to 5 μM, It may be a concentration of 0.5 to 3 μM.

The composition for differentiation according to the present invention may further include one or more components usable for differentiating hepatoblasts into hepatocyte-like cells. Specifically, the component is any one selected from the group consisting of hepatocyte growth factor (HGF), fibroblast growth factor (FGF), oncostatin M (OSM) and dexamethasone (dexamethasone) There may be more than one. At this time, the content of the added component can be appropriately selected by those skilled in the art.

As used herein, the term “hepatoblast” refers to progenitor cells of hepatocytes. The hepatocytes may be derived from embryonic stem cells. The embryonic stem cells may be totipotent cells capable of differentiating into all kinds of cells or pluripotent cells capable of differentiating into various kinds of cells. Embryonic stem cells can be differentiated into hepatic blasts via endoderm cells, and methods for obtaining hepatic blasts from embryonic stem cells are well known in the art.

As used herein, the term "hepatocyte-like cell (HLC)" refers to a cell differentiated to have almost the same characteristics as hepatocytes. The hepatocyte-like cells may be hepatocytes differentiated in vitro from embryonic stem cells, which may be used substantially in the same sense as hepatocytes prepared in vitro.

In one embodiment of the present invention, the present inventors treated hepatic stem cells obtained from embryonic stem cells with various kinds of Aurora-A kinase inhibitors to differentiate hepatocyte-like cells, thereby treating PP with Aurora-A kinase inhibitors to differentiate them. It was confirmed that the hepatocyte-like cells were shown to have a uniform form of HLC (see FIG. 2).

Thus, the expression of the proteins related to polarity in the HLC, such as occlusin, P-gp and OATP2 protein is increased (see Fig. 5), the expression of E-cadherin protein is increased and the expression of N-cadherin protein at the same time It was confirmed that EMT is suppressed by decreasing (see FIG. 6).

In addition, as a result of confirming the expression change of hepatocyte markers in the HLC, expression of hepatocyte marker genes such as ALB, TDO2 and AAT or CYP enzyme genes such as CYP1A1, CYP2C9, CYP2C19 and CYP3A4 increased. In addition, the expression of hepatocyte markers and metabolic enzyme proteins also increased in PP treated HLC, consistent with the results of expression changes in genes (see FIG. 9).

In addition, as a result of confirming the liver function characteristics of the HLC, Ac-LDL absorption and glycogen synthesis and albumin secretion were increased in the HLC treated with PP, but there was no effect on the secretion of bile ducts or the production of urea (Fig. 10).

Therefore, the compound represented by the formula (1) can be usefully used as a composition for differentiating from hepatic stem cells to hepatocyte-like cells exhibiting polarity.

The present invention also provides a method for differentiating hepatocyte-like cells in hepatocytes, the method comprising treating the hepatocytes with a compound represented by Formula 1 below:

[Formula 1]

.

.

The compound represented by Chemical Formula 1 may have the characteristics as described above. For example, the compound represented by Chemical Formula 1 may be a phthalazinone pyrazole compound known as Aurora-A kinase. The compound may be treated at concentrations of 0.1 to 20 μM, 0.1 to 10 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.5 to 20 μM, 0.5 to 10 μM, 0.5 to 5 μM, 0.5 to 3 μM.

Liver blast cells and hepatocyte-like cells in the method according to the invention may have the characteristics as described above. In one example, the hepatic stem cells may be derived from embryonic stem cells, and the obtaining of liver stem cells from embryonic stem cells may be performed by any method well known in the art. Hepatocyte-like cells obtained by the above method may exhibit polarity.

In the method, the differentiation may be performed for 3 to 20 days, 3 to 15 days, 3 to 10 days, 5 to 20 days, 5 to 15 days, or 5 to 10 days, and is represented by Formula 1 during the differentiation period. The compound can be treated. The compound represented by Formula 1 may be treated between 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days or 2 to 3 days to start differentiation. Can be.

In one embodiment of the present invention, the inventors of the present invention showed that the hepatocyte-like cells obtained by treating the hepatocytes with PP exhibited a homogeneous form of HLC (see FIG. 2), and increased the expression of proteins related to polarity (see FIG. 5). ), EMT was inhibited (see FIG. 6), the expression of genes and proteins related to hepatocyte markers and drug metabolism enzymes was increased (see FIGS. 8 and 9), and it was confirmed that they exhibit liver function characteristics (see FIG. 10).

Therefore, the compound represented by the formula (1) from the above can be usefully used for differentiating hepatocyte-like cells from hepatocytes.

The present invention also provides hepatocyte-like cells differentiated by the above method.

The hepatocyte-like cells can be differentiated in the same manner as described above. For example, the hepatocyte-like cells may be obtained by treating the hepatic blast cells with the compound represented by Formula 1 according to the present invention. Compound represented by Formula 1 is 0.1 to 20 μM, 0.1 to 10 μM, 0.1 to 5 μM, 0.1 to 3 μM, 0.5 to 20 μM, 0.5 to 10 μM, 0.5 to 5 μM, 0.5 to 3 μM Can be processed. Meanwhile, the differentiation may be performed for 3 to 20 days, 3 to 15 days, 3 to 10 days, 5 to 20 days, 5 to 15 days, or 5 to 10 days, and is represented by Formula 1 during the differentiation period. The compound can be treated. The compound represented by Formula 1 may be treated between 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days or 2 to 3 days to start differentiation. Can be.

Hepatocyte-like cells differentiated by this method can exhibit polarity.

In one embodiment of the present invention, the inventors of the present invention showed that the hepatocyte-like cells obtained by treating the hepatocytes with PP exhibited a homogeneous form of HLC (see FIG. 2), and increased the expression of proteins related to polarity (see FIG. 5). ), EMT was inhibited (see FIG. 6), the expression of genes and proteins related to hepatocyte markers and drug metabolism enzymes was increased (see FIGS. 8 and 9), and it was confirmed that they exhibit liver function characteristics (see FIG. 10).

Thus, the method can be used to differentiate hepatocyte-like cells.

Furthermore, the present invention provides a method for screening liver toxicity using the hepatocyte-like cells.

In the method for screening liver toxicity according to the present invention, hepatocyte-like cells may have the characteristics as described above. For example, the hepatocyte-like cells may be obtained by treating the hepatic blast cells with the compound represented by Formula 1 according to the present invention. The compound represented by Chemical Formula 1 may be treated in the process of differentiating hepatocyte-like cells from hepatocytes.

The method according to the invention may comprise the step of treating the test substance to the hepatocyte-like cells differentiated by the method of the present invention. The test substance may be a substance to be checked for hepatotoxicity. In addition, the method may determine whether the test substance is hepatotoxic by comparing the hepatocyte-like cells treated with the test substance with the control cells. The method for screening liver toxicity using the hepatocyte-like cells can be carried out using any method well known in the art.

In one embodiment of the present invention, the inventors of the present invention showed that the hepatocyte-like cells obtained by treating the hepatocytes with PP exhibited a homogeneous form of HLC (see FIG. 2), and increased the expression of proteins related to polarity (see FIG. 5). ), EMT was inhibited (see FIG. 6), the expression of genes and proteins related to hepatocyte markers and drug metabolism enzymes was increased (see FIGS. 8 and 9), and it was confirmed that they exhibit liver function characteristics (see FIG. 10).

Therefore, hepatocyte-like cells according to the present invention can be usefully used for screening hepatotoxicity.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example  Aurora Kinase  Confirmation of Morphological Changes in Hepatocyte-like Cells by Addition of Inhibitors

To determine if epithelial-mesenchymal transition (ETM) is inhibited by aurora kinase inhibitors that affect cell polarity in the differentiation of hepatocyte-like cells (HLC) The same experiment was performed (FIG. 1).

First, CHA-hES15 cells (cha hospital, Korea), which are human embryonic stem cells, are grown on mitomycin C (Sigma, USA) on the feeder layer of mouse embryonic fibroblasts. ) Was added and cultured. At this time, 4 ng / ㎖ basic fibroblast growth factor (bFGF, R & D Systems, USA) and 1.2 mg / ㎖ sodium bicarbonate, 20% knockout serum replacement (Knockout serum replacement, Invitrogen, USA), 1% GlutaMax (Invitrogen, USA), 1% non-essential amino acid (Invitrogen, USA), 0.1 mM β-mercaptoethanol (β-mercaptoethanol, Sigma, USA) DMEM / F12 culture medium (Invitrogen, USA) containing 100 U / mL penicillin (Gibco, USA) and 100 μg / mL streptomycin (Gibco, USA) was used.

Cultured cells are dispensed into matrigel-coated plates (Corning Life Science, USA) before starting to differentiate and cultured using mTeSR1 culture medium (Stem Cell Technologies, Canada) containing 4 ng / ml of bFGF. Ready. Next, in order to induce definitive endoderm, the medium of prepared hESC cells was prepared using 0.5 mg / ml bovine serum albumin (BSA, Sigma-Aldrich, USA), 100 ng / ml activin A ( Activin A, Peprotech, USA), replaced with RPMI-1640 culture medium (Lonza, Switzerland) containing 0.5 mM sodium butyrate (Sigma, USA) and 1x B27 supplement (B27 supplement, Gibco, USA) Incubated for After incubation, the culture medium was replaced with a medium prepared so that the concentration of sodium butyrate contained in the RPMI-1640 culture medium was 0.1 mM and further cultured for 4 days. Next, to induce hepatoblasts, definitive endoderm-derived cells were treated with 0.5 mg / ml BSA, 10 ng / ml hepatocyte growth factor (HGF, Peprotech, USA) and 10 ng /. Cultures were carried out for 5 days using RPMI-1640 culture medium containing ㎖ fibroblast growth factor 4 (fibroblast growth factor 4, FGF4, Peprotech, USA). After incubation, hepatic blast cells were treated with 10 ng / ml HGF, 10 ng / ml FGF4, 10 ng / ml Oncostatin M (OSM, Peprotech, USA) and 0.1 μM of dexamethasone (dexamethasone, Sigma-). Differentiation of HLC was induced by culturing for 7 days using hepatocyte culture medium (Aldrich, USA) (hepatocyte culture medium, HCM, Lonza, USA). All cells were incubated at 37 ° C. and 5% CO ₂ conditions and culture medium was changed daily.

On the other hand, as an Aurora-A kinase inhibitor, phthalazinone pyrazole (PP), Aurora A inhibitor I (Sigma, USA), MLN8237 (alisertib), and MK-5108 and Aurora-B kinase inhibitors Hesperadin, barasertib and GSK1070916 were prepared by diluting them in DMSO (dimethylsulfoxide). At this time, the Aurora kinase inhibitor was diluted to a known IC ₅₀ concentration. The diluted Aurora kinase inhibitors were added two days after the start of incubation of the liver blasts with HLC. At this time, cells added with only DMSO were used as a control. 2 shows a photomicrograph of the differentiated HLC at the end of the final culture.

As shown in FIG. 2, during the differentiation into HLC, PP-added cells were denser than those of other Aurora kinase inhibitors, and more frequent diploids and clear nuclei were observed in the cells. In addition, cells added with other Aurora kinase inhibitors except PP showed inconsistent morphological changes with differentiated HLC, but cells with PP showed more uniform HLC morphology (FIG. 2).

From this, it can be seen that PP can be used to differentiate mature HLC in hepatocytes.

Example  2. added PP  According to concentration Of HLC  Confirmation of shape change

In the process of differentiating hepatocytes into HLC, an experiment was performed to determine the shape of HLC according to the concentration of PP. Specifically, the experiment was carried out under the same conditions and methods as in Example 1, except that 0.1, 1 or 10 μM of PP was added during the addition of the Aurora Kinase Inhibitor.

As a result, as shown in Fig. 3, the cells added with PP changed to a form similar to that of hepatocytes, and in particular, when the 1 or 10 μM concentration of PP was added, the shape of the cells changed very similarly to the hepatocytes ( 3).

Example  3. Aurora Kinase  Confirmation of Cytotoxicity by Inhibitors

In general, Aurora kinase inhibitors are being developed as anticancer agents because they induce cell death. Therefore, a cytotoxicity assay was performed to confirm whether the Aurora kinase inhibitor used for differentiation of HLC is toxic to HLC.

Experiments were performed using cells in which iPS cells derived from CHA-hES15 cells or human dermal fibroblasts were differentiated by HLC under the same conditions and methods as in Example 1. At this time, Huh7 cells, a hepatocellular carcinoma used as a control, were added with 10% fetal bovine serum (Lonza, USA), 100 U / mL penicillin (Lonza, USA) and 100 μg / mL streptomycin (Lonza, USA). Prepared by incubation using DMEM culture medium (Invitrogen, USA). All cells were incubated at 37 ° C. and 5% CO ₂ conditions.

Meanwhile, cytotoxicity analysis was performed according to the manufacturer's protocol using Cell Counting Kit-8 (Cell Counting Kit-8, CCK-8, Dojindo, USA). Specifically, the prepared cells were dispensed into 48-well plates and incubated overnight. After incubation, Aurora kinase inhibitor was added and the cells were further incubated for 5 days. At this time, 0, 1, 10 or 100 μM of PP as an aurora kinase inhibitor; Aurora A inhibitor I at 0, 5, 50 or 500 nM; MLN8237 at 0, 1, 10 or 100 μM; 0, 0.5, 5 or 50 μM MK-5108; 0, 0.1, 1 or 10 μM of hesperadin; 0, 0.1, 1 or 10 μM barassultip; Or 0, 5, 50 or 500 μM of GSK1070916. After 5 days of culture, the cell culture plate to which the CCK-8 solution was added was reacted at 37 ° C. for 1 hour, and then absorbance was measured using a microplate reader at a wavelength of 450 nm.

As a result, as shown in FIG. 4, unlike Huh7 cells, ES-HLC or iPC-HPC differentiated from iPS cells derived from CHA-hES15 or human dermal fibroblasts were cytotoxic by aurora kinase inhibitor. Not shown (FIG. 4). From this, it was confirmed that the morphological change in HLC differentiated by PP was not due to toxicity.

Example  4. PP  Having polarity by addition Of HLC  Identify differentiation induction

4-1. PP  Confirmation of Increased Expression of Polarity-Related Proteins by Addition-1

In order to confirm that HLC cells prepared by the addition of PP exhibited the polarity exhibited by hepatocytes by EMT inhibition, the expression of polarity-related proteins was confirmed using immunocytochemistry analysis.

Specifically, HLC was prepared by differentiating under the same conditions and methods as in Example 1, except that 1 or 10 μM of PP was added in the course of adding an Aurora Kinase Inhibitor. At this time, cells added with DMSO were used as a control. Prepared cells were fixed with 4% paraformaldehyde and washed three times with Dulbecco's phosphate-buffered saline (DPBS) solution. The washed cells were added with DPBS to which 0.1% of Triton X-100 was added to make them permeable. DPBS was washed and reacted for 1 hour by adding blocking buffer containing 5% goat serum to the cells. The primary antibodies include E-cadherin (Cell Signaling Technology, USA), CK8 + 18 (Abcam, UK), Occludin (occludin, Thermo, USA), CK19 (Abcam, UK), P-gp (Abcam, UK) and OATP2 (Abcam, UK) proteins, respectively, were added and reacted overnight at 4 ° C. After completion of the reaction, the cells were washed with DPBS and reacted for 1 hour by adding a secondary antibody. After the reaction, the cells were washed with DPBS and the nuclei were stained with DAPI (4'6-diamidino-2-phenylindole). 5 shows the results of the stained cells under the microscope.

As shown in FIG. 5, the expressions of the proteins related to polarity, occlusin, P-gp and OATP2 proteins, increased in the cell membrane of HLC differentiated by the addition of PP. On the other hand, the expression level of the liver cytokeratin CK8 and CK18 protein was similar to the control group, the expression of cholangiocyte marker CK19 protein was reduced compared to the control group. In addition, E-cadherin protein, an epithelial cell surface marker, was increased in cells to which PP was added (FIG. 5).

4-2. PP  Confirmation of Increased Expression of Polarity-Related Proteins by Addition-2

Western blot was performed using HLC differentiated by the addition of PP to confirm the change in the expression of protein related to polarity by the addition of PP.

First, HLC was obtained under the same conditions and methods as in Example 4-1, wherein cells added with DMSO were used as a control. Cells were lysed by adding RIPA buffer (Invitrogen, USA) containing protein and phosphatase inhibitor to the obtained cells. Cell lysates were centrifuged to obtain supernatant and the amount of protein quantified. 20 or 40 μg of protein was separated using SDS-PAGE electrophoresis and transferred to the membrane. The protein-transferred membrane was blocked at room temperature for 1 hour, and as primary antibodies, E-cadherin, occlusin, ZO-1 (Abcam, UK), N-cadherin (BD Biosciences, USA), and bimentin ( Abcam, UK) and β-actin (Santa Cruz Biotechnology, USA) were added and reacted overnight at 4 ° C. After the reaction, the membrane was washed with TBS-T buffer containing 0.1% Tween 20, HRP (horseradish peroxidase) -bound secondary antibody was added and reacted at room temperature. After 1 hour, the membranes were washed with TBS-T buffer and visualized with enhanced chemiluminescence (Ether Fisher Scientific, USA) reagent. The results of visualizing the protein detected on the membrane are shown in FIG. 6A, and the results of graphing the expression levels of E-cadherin and N-cadherin in the above results are shown in FIG. 6B.

As shown in FIG. 6A, the expression levels of E-cadherin and epithelial cell surface marker protein were increased in the cell group to which PP was added. On the other hand, the expression levels of mesenchymal cell markers N-cadherin and non-mentin protein were markedly decreased in the cell group to which PP was added (FIGS. 6A and 6B). In other words, it has been reported that the conversion of E-cadherin to N-cadherin is a typical feature of EMT. HLC differentiated by adding PP by increasing E-cadherin protein expression and decreasing N-cadherin protein expression Was found to inhibit EMT and differentiate polarized HLC.

4-3. PP  Aurora other than Kinase  By adding inhibitor EMT  Suppress Confirmation

In order to confirm whether EMT is inhibited in differentiated HLC even when Aurora kinase inhibitors other than PP were added, mRNA expression changes of E-cadherin and N-cadherin were confirmed as follows.

First, an Aurora Kinase Inhibitor was added under the same conditions and methods as described in Example 4-2, except that 50 nM Aurora A Inhibitor I, 100 μM MLN8237 or 50 μM MK-5108 was added to the cells. Cells were obtained. TRIzol (Invitrogen, USA) was added to the obtained cells and total RNA was obtained according to the manufacturer's protocol. The obtained RNA was quantified and analyzed for purity using a nanodrop system (Thermo Fishcer Scientific, USA), and then synthesized into cDNA using SuperScript II Reverse Transcriptase (Invitrogen, USA). Synthesized cDNA was used as a template and qPCR was performed using a StepOnePlus real-time PCR system (Applied Biosystem, USA) and Power SYBR Green PCR Master Mix (Applied Biosystem, USA). The expression level of all mRNAs was normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA using the ΔΔCt method. As a result, as a result of confirming the expression levels of E-cadherin and N-cadherin mRNA is shown in Figure 7 the graph.

As shown in FIG. 7, unlike PP, E-cadherin mRNA expression was reduced or maintained in cells to which Aurora A inhibitor I, Aurora kinase inhibitors of MLN8237 and MK-5108 were added, and N-cadherin mRNA expression. Increased (FIG. 7). In other words, it was confirmed that the Aurora kinase inhibitor other than PP did not inhibit EMT during the differentiation of HLC.

Example  5. PP  Hepatocytes by Addition Of marker  Confirmation of expression change

5-1. PP  Hepatocytes by Addition Marker  Confirmation of gene expression change

In order to confirm the influence of PP on the maturation of HLC into hepatocytes, mRNA expression levels of hepatocellular markers and drug metabolism enzymes were confirmed by qPCR method.

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained, and qPCR was carried out in the same manner as described in Example 4-3 using the HLC. At this time, qPCR is prepared using albumin ( ALB ); Tryptophan-2,3-dioxygenase (TDO2); Alpha-1 antitrypsin ( AAT ); Genes included in cytochrome P450 (CYP) including CYP1A1 , CYP2C9 , CYP2C19 and CYP3A4 ; Phase II enzyme and drug transporter genes including UGT1A1 , UGT1A6 , multidrug-resistance protein 2 ( MRP2 ) and multidrug resistance protein 1 ( MDR1 ); And the transcription level of the PXR (pregnane X receptor) gene.

As a result, as shown in Figure 8, mRNA expression level of hepatocyte markers ALB, TDO2 and AAT significantly increased in HLC with PP. In addition, the expression of mRNA encoding CYP enzymes including CYP1A1 , CYP2C9 , CYP2C19 and CYP3A4 also increased in these cells. Furthermore, the expression levels of UGT1A1 , UGT1A6 , MRP2 and MDR1 mRNAs increased concentration-dependently of the added PP. However, the expression levels of ALB , TDO2 , AAT , CYP2C9 and CYP2C19 mRNA were decreased when 10 μM of PP was added as compared to when 1 μM of PP was added (FIG. 8). From the above results, it was found that the addition of 1 μM PP was more effective in the maturation of HLC.

5-2. Of PP  Hepatocytes by Addition Of marker  Confirmation of expression change

To confirm the effect of PP on the maturation of HLC into hepatocytes, the expression levels of hepatocyte marker proteins were confirmed using immunocytochemical analysis. Experiments include ALB (DAKO, USA), PXR (Abcam, UK), HNF4A (hepatocyte nuclear factor 4 alpha) (Abcam, UK), CYP1A1 (Abcam, UK), UGT1A1 (R & D system, USA) and MRP2 as primary antibodies. (cell signaling technology, USA) The same conditions and methods as in Example 4-1 were conducted except that the antibody against the protein was used. As a result, the results of taking pictures of the stained cells are shown in FIG.

As shown in FIG. 9, the expression of hepatocyte markers and drug metabolism enzymes was increased in cells to which PP was added (FIG. 9).

Example  6. Of PP  By addition Liver function  Promotion confirmation

The following experiment was conducted to determine how the hepatic function of HLC changes with PP added during the maturation of hepatocytes.

6-1. Ac -LDL uptake and glycogen synthesis changes

First, for the analysis of acetylated-low-density lipoprotein (Ac-LDL) uptake, HLC differentiated by adding 1 or 10 μM of PP, as described in Example 4-1. After final differentiation of HLC, cells were treated with 10 μg / ml of 1,1′-Dioctadecyl-3,3,3 ′, 3′-tetramethylindo-carbocyanine-labeled (1,1′-dioctadecyl- 3,3,3 ', 3'-tetramethylindo-carbocyanine-labeled) Ac-LDL was added. It was further incubated at 37 ° C. for 5 hours and then the cells were washed with PBS. Washed cells were observed using a fluorescence microscope, the results are shown in Figure 10A.

On the other hand, glycogen synthesis was confirmed by PAS (periodic acid-schiff) staining. Specifically, as described in Example 4-1, 4% paraformaldehyde was added to HLC differentiated by adding 1 or 10 μM of PP and fixed for 30 minutes. Fixed cells were washed and permeabilized and stained according to the manufacturer's protocol using the PAS staining kit. The result of photographing the stained cells is shown in Figure 10A.

As shown in Figure 10A, by adding PP during the differentiation of HLC, glycogen synthesis increased with LDL uptake and scale (Figure 10A).

6-2. Change in albumin secretion

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained. The amount of albumin secreted in the culture medium of differentiated HLC was measured using the human albumin ELISA quantification kit (Bethly Laboratory, USA) and a model 680 microplate reader (Bio-Rad, USA). ELISA was performed according to the kit manufacturer's protocol and the resulting data was normalized to the amount of total protein. As a result, the result of confirming the secretion amount of albumin is shown in Figure 10C.

As shown in FIG. 10C, the amount of albumin was significantly increased in cells added with 1 μM PP, but decreased in cells added with 10 μM PP (FIG. 10C).

6-3. Confirmation of changes in secretion of bile ducts

The function of bile canalicular was confirmed using CDFDA (5-carboxy-2,7-dichlorofluorescein diacetate). In polarized hepatocytes, CDFDA is hydrolyzed to fluorescent carboxy-dichlorofluorescein (CDF), which is secreted into the bile duct through the MRP2 protein. Therefore, it was confirmed that the secretion change of the bile duct in the HLC added PP using this.

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained. 10 mM of CDFDA (Life Technologies, USA) was added to differentiated HLC and reacted at 37 ° C. for 15 minutes. Cells were washed with HCM culture medium and observed using a fluorescence microscope. On the other hand, in order to suppress the protein associated with multidrug resistance before adding CDFDA, cells were reacted for 1 hour at 37 ° C. by adding 2 mM of probenecid (probenecid, Sigma-Aldrich, USA). The photograph of the cells observed with the fluorescence microscope is shown in Figure 10B.

As shown in FIG. 10B, inhibition of bile by the MRP2 inhibitor, probeneside, delayed the efflux of CDF, a functional feature of polarized hepatocytes. However, the function of bile ducts was not significantly different between HLC with or without PP (FIG. 10B).

6-4. Urea  Confirmation of generation change

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained. The amount of urea contained in the cultured HLC culture was obtained using a human urea ELISA quantification kit (BioAssay Systems, USA) and a model 680 micro plate reader (Bio-Rad, USA). ELISA quantification was performed according to the kit manufacturer's protocol and the resulting data was normalized to the amount of total protein.

As a result, as shown in Figure 10C, the production of urea was reduced in HLC differentiated with the addition of PP, but no significant change was seen (Figure 10C).

6-5. Cell proliferation confirmation

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained. On the other hand, the number of proliferated cells was confirmed using the BrdU (bromodeoxyuridine, Molecular Probe, USA) analysis. Specifically, 10 μM of BrdU labeled solution was added to the HLC and reacted for 30 hours at 37 ° C. and 5% CO ₂ conditions. After the reaction, cells were stained using anti-BrdU antibody (Invitrogen, USA) and analyzed using a high-content analysis system (Micro XLS, Molecular Devices) and MetaXpress.

As a result, as shown in FIGS. 11A and 11B, the number of cells positive for Ki67 and BrdU in HLC with PP increased, and as shown in FIG. 11C, DAPI for the same area in HLC with PP As the number of nuclei positive for the dye was increased, it was found that PP promoted the proliferative capacity of HLC (FIG. 11).

Therefore, from the above results, it was confirmed that the addition of PP promotes the functional maturation and proliferation of differentiated HLC.

Example  7. On PP  The differentiation mechanism of HPC

7-1. On PP  Aurora-A by Kinase  Confirmation of protein expression inhibition

PP has been known as an inhibitor of Aurora-A kinase protein. Therefore, it was confirmed by the following method whether PP inhibits EMP and matures HLC by inhibiting Aurora-A kinase protein as described above.

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained. The experiments were conducted with the same conditions and methods as described in Example 4-2, except that Aurora-A (Cell Signaling Technology, USA) and Phospho-Aurora-A (Cell Signaling Technology, USA) antibodies were used as primary antibodies. Western blot was performed. As a result, as shown in FIG. 12A, the expression of Aurora-A kinase protein was increased in HLC differentiated by adding PP, but phosphorylation was not significantly reduced. In addition, the ratio of phosphorylated Aurora-A kinase protein to the amount of total Aurora-A kinase protein was reduced in cells with PP addition in a concentration dependent manner (FIG. 12A). Therefore, it can be seen that the PP developed as an inhibitor of the Aurora-A kinase protein does not inhibit the expression of the Aurora-A kinase protein during HLC maturation.

7-2. On PP  by AKT  Check for changes in signaling mechanisms

Inhibitors of Aurora-A kinase protein induce inhibition of AKT signaling mechanisms involved in the induction of EMT in various cancer cell lines. Therefore, the activation of AKT and GSK-3β was confirmed by Western blot to confirm whether the AKT signaling mechanism is involved in PP-induced EMT inhibition.

First, as described in Example 4-1, HLC differentiated by adding 1 or 10 μM of PP was obtained, and western blot was performed using the obtained cells. Specifically, Western blot is a primary antibody, Cell Signaling Technology (AKT), Phospho-AKT (Cell Signaling Technology, USA), GSK-3β (Santa Cruz biochenology, USA), Phospho-GSK-3β (Santa). Example 4, except that Cruz biochenology (US), snail (Snail, Santa Cruz biochenology, USA), HNF4α (Hepatocyte nuclear nuclear factor 4α, Abcam, UK) and GAPDH (Thermo Fisher Scientific, USA) antibodies were used The same conditions and methods as described in -2 were performed. The results of confirming the activity change of the AKT and GSK-3β protein are shown in Figure 12B and 12C.

As shown in Figure 12B, phosphorylation of AKT was significantly inhibited in HLC with PP addition (Figure 12B). On the other hand, activation of GSK-3β protein is induced by this inactivation of AKT protein, which inhibits snail protein expression. In addition, inhibition of the expression of snail protein showed a result contrary to the increased expression of HNF4α protein (FIG. 12C).

7-3. PP  By addition mRNA  Confirmation of expression change

In the above example, the expression of Aurora kinase protein and change in AKT signaling mechanism were confirmed in HLC obtained by adding PP. Therefore, it was confirmed by using a real-time PCR method whether such a change in expression of the protein is also expressed in the expression of mRNA. The experiment was performed in the same manner as described in Example 4-3, except for using a known primer capable of detecting the sequence of the Aurora-A kinase , AKT1 , snail or HNF4α gene. As a result, as a result of confirming the expression change of the gene, a graph is shown in FIG.

As shown in FIG. 13, the expression of Aurora-A kinase mRNA was increased in HLC differentiated with PP, whereas the expression of AKT and snail mRNA was decreased. This was identical to the change in expression of the protein. On the other hand, HNF4α gene increased in HLC differentiated by the addition of 1 μM PP, but decreased in HLC differentiated by the addition of 10 μM PP (Fig. 13).

From the above, it was found that PP added to HLC differentiation inhibits EMT by inhibiting AKT signaling mechanism by upregulating HNF4α rather than directly inhibiting Aurora-A kinase protein.

Claims (11)

Composition for differentiation from hepatoblasts (hepatocyte-like cells, HLC) comprising a compound represented by the formula (1) as an active ingredient:
[Formula 1]

.
According to claim 1, wherein the compound represented by the formula (1) is contained in a concentration of 0.1 to 20 μM, composition for differentiation from hepatic stem cells to hepatocyte-like cells.
The method of claim 1, wherein the composition is from the group consisting of hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and oncostatin M (OSM) and dexamethasone (dexamethasone) A composition for differentiation from hepatic stem cells to hepatocyte-like cells, further comprising any one or more selected
A method of differentiating hepatocyte-like cells in hepatocytes comprising treating the hepatocytes in vitro with a compound represented by Formula 1 below:
[Formula 1]

.
The method of claim 4, wherein the compound represented by Chemical Formula 1 is treated at a concentration of 0.1 to 20 μM.
The method of claim 4, wherein the compound represented by Formula 1 is treated between 1 and 5 days of differentiation.
The method of claim 4, wherein said differentiation is performed for 3 to 20 days.
The method of claim 4, wherein said hepatoblasts are derived from embryonic stem cells.
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