KR20170019601A - Method for differentiation of hepatocyte - Google Patents

Abstract

The present invention relates to a differentiation method of highly functional hepatocytes, which comprises the following steps: treating stem cells with activin A (AA) to obtain endoderm cells which are differentiated from the stem cells; treating the endoderm cells with retinoic acid (RA) to obtain hepatoblasts which are differentiated from the endoderm cells, and treating hepatoblasts with nicotinamide (NA) to proliferate the hepatoblasts; and treating the proliferated hepatoblasts with hepatocyte growth factor (HGF) to obtain hepatocytes differentiated from the hepatoblasts.

Description

Methods for differentiation of hepatocytes < RTI ID = 0.0 >

The present invention relates to a method of differentiating high-function hepatocytes and a high-function hepatocyte differentiated therefrom.

Cell-based treatment of irreversible liver loss using human embryonic stem cells (hESCs) and human-derived pluripotent stem cells derived hepatocyte-like cells (HLCs) There is a possibility of development. In addition, stem cell-derived HLCs are used for in vitro drug screening and toxicity studies. Thus, the induction of HLCs from hESCs has been studied as an unlimited source of cells for clinical and experimental studies. As a result, hepatocyte differentiation efficiency using hESCs was greatly increased. However, most of the HLCs produced by the various differentiation methods naturally differentiate into multiple cell lines at the final stage of hepatocyte differentiation without a separate purification process. In addition, the phenotype of most HLCs is more similar to fetal hepatocytes than mature adult hepatocytes. Phase I and II enzyme activities, important functions of mature hepatocytes, are more active in HLCs than in human primary hepatocytes (hPHs) . In addition, phase I and II drug metabolism and hepatocellular tubular structure by hepatic biliary transport are rapidly lost in 2D cultured HLCs. When 3D hepatocytes in the body are used for in vitro experiments, 3D culture method is needed because 2D metabolism loses phase I and II drug metabolism, one of hepatocyte functionalities.

Among the existing research results, hepatocytes differentiated from human embryonic stem cells have been cultured as 3D-shaped spheres, but functional comparison with hPHs has not been performed or functions of drug metabolizing enzymes have been remarkably deteriorated [Non-Patent Document 1] . These key differences between HLCs and hPHs result in limited use of HLCs derived from stem cells as a renewable source of functional mature hepatocytes for in vivo and experimental use.

Studies have been conducted to further mature HLCs derived from stem cells, but the in vivo developmental pathway regulating hepatocyte maturation is not known and is not being performed properly.

The CYP enzyme activity of the hepatocytes derived from the stem cells as reported so far is very low compared to normal hepatocytes.

Therefore, there is a need to develop a method of differentiating hepatocytes that exhibits drug metabolism activity similar to in vivo hepatocytes.

Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells, Stem Cells Dev. 2014 Jan 15; 23 (2): 124-31

Accordingly, the present inventors have made efforts to solve the above-mentioned problems. As a result, they have succeeded in differentiating hepatocytes and hepatocytes, which are endogenous mesenchymal cells, from hepatocytes by sequential differentiation, Thereby completing the present invention.

It is an object of the present invention to provide a method of differentiating high-function hepatocytes that exhibits drug metabolism activity similar to that of hepatocytes in vivo.

Another object of the present invention is to provide a highly functional hepatocyte differentiated by the differentiation method.

As means for solving the above problems,

(a) treating stem cells with AA (activin A) to obtain differentiated endoderm cells from the stem cells;

(b) treating the obtained endoderm cells with RA (retinoic acid) to obtain hepatocytes differentiated from the endoderm cells and treating the obtained hepatocytes with NA (nicotinamide) to grow hepatocytes; And

(c) treating hepatocyte growth factor (HGF) with the proliferated hepaticoblasts to obtain hepatocytes differentiated from the hepaticoblasts

The present invention provides a method of differentiating high-function hepatocytes.

As another means for solving the above problems, the present invention provides a high-function hepatocyte differentiated by the above method.

As another means for solving the above problems, the present invention provides a therapeutic composition for the recovery of liver function necessary for the treatment of acute, chronic or genetic liver damage including high-functioning cells differentiated by the above method do.

Since the differentiation method according to the present invention can be utilized as an analysis and evaluation tool very similar to the human tissue in the development stage of the new drug, the present technology development can be utilized not only for identifying the cause of the disease and for discovering new drug targets, It is expected that technology will be available in most stages of preclinical field from the early stage of new drug development, such as secondary pharmacological efficacy research, safety research, and metabolism research.

To assess the toxicity of differentiated hepatocytes from human omnipotent stem cell lines (human embryonic stem cells and derived pluripotent stem cells) with various genetic backgrounds, it is possible to predict and evaluate the individual differences in drug efficacy and toxicity through drug metabolism It is expected to be possible.

Therefore, when the development of the differentiation factor or the 3-D culture technique for increasing the activity of the essential enzymes related to these drug metabolism and delivery is facilitated, the development of the cell line functionally equivalent to the human hepatocyte is facilitated, This will greatly contribute to the credibility of the development of new drugs, including evaluation.

FIG. 1 is a schematic diagram showing the differentiation of undifferentiated human embryonic stem cells into pluripotent human embryonic stem cells in the absence of nutritious cells through various steps into functional hepatocytes. Step-specific differentiation markers (undifferentiated: OCT4, complete endoderm: Sox17, Cells: AFP, immature hepatocytes: ALB, mature hepatocytes (ALB, ASGPR1) and inducers for induction of differentiation (undifferentiated: mTeSR1 medium, complete endoderm: Activin A, CHIR99021, Sodium butyrate, abundant bacterium: BMP2, FGF4, Oncostatin M, Dexamethesone) were induced by 22 days in total for the differentiation of retinoic acid, B27, hepatocyte proliferation: Nicotinamide, Ascorbic acid, bFGF, B27, immature hepatocytes: HGF and mature hepatocytes.
FIG. 2 shows gene expression by differentiation stages.
FIG. 3 is a photograph showing the step of proliferating hepatocytes after hepatocytes were obtained through RA after differentiation into endoderm cells.
FIG. 4 is a graph showing the survival of cells before stepwise sphere formation (left) and the size of spheres formed by stepwise culturing (right) in order to derive an appropriate step for hepatocyte structure formation (right).
FIG. 5 shows the degree of differentiation of the finally differentiated mature hepatocytes by 2D adherent culture and 3D suspension culture by cell sorting (FACS) with ALB, a mature hepatocyte marker.
FIG. 6 is a graph showing the degree of differentiation of the final differentiated mature hepatocytes by 2D adherent culture and 3D floating culture by cell sorting (FACS) with ASGPR, a mature hepatocellular surface marker.
FIG. 7 is a photograph showing the characteristics of hepatocytes differentiated by 2D attachment culture and 3D floating culture through PAS staining and ICG uptake for confirming LDL uptake and glycogen accumulation.
FIG. 8 shows the characteristics of hepatocytes differentiated by 2D attachment culture and 3D suspension culture through the albumin and urea secretion ability.
FIG. 9 is a photograph showing TEM images of the cell organelles of the differentiated hepatocytes.
10 shows the gene expression of a nuclear receptor involved in CYP enzyme expression in differentiated hepatocytes.
Fig. 11 shows gene expression of drug metabolizing enzyme group 2 in differentiated hepatocytes.
FIG. 12 shows gene expression of drug metabolizing group 1 enzyme in differentiated hepatocytes.
FIG. 13 shows the results of treatment of three different drugs PB, AP, and RIF in differentiated liver cells for 2 days in order to confirm the drug metabolizing enzymatic activity of hepatocytes.
FIG. 14 is a photograph showing the change in hepatocyte characteristics caused by a drug when the drug is treated on the differentiated hepatocytes by fluorescence staining of hepatocyte markers such as albumin (ALB) and AFP.
FIG. 15 is a photograph showing the degree of accumulation of glycocene in order to confirm the change of the hepatocyte characteristics by the drug when the drug is treated on the differentiated hepatocytes.

Hereinafter, the configuration of the present invention will be described in more detail.

The present invention relates to a method of differentiating high-function hepatocytes which exhibits drug metabolism activity similar to that of hepatocytes in vivo.

In the stem cell differentiation induction method of the present invention, the stem cell is a cell derived from a mammal, preferably pluripotent stem cells or an origin cell of the cell, capable of differentiating into hepatocytes in an in vitro culture . ≪ / RTI > The mammal in the present invention is not particularly limited and may be selected from the group consisting of rodentia, lagomorpha, primates, carnivora, perissodactyla and artiodactyla Lt; RTI ID = 0.0 > mammal. ≪ / RTI > In one embodiment, the mammal may be a mouse, a rabbit, a cow, a pig, a monkey, a person, and may be a human, but specifically a mammalian cell having the ability to differentiate into hepatocytes in vitro, .

The term "pluripotent stem cells" refers to cells capable of almost enduring or prolonged cell proliferation maintained in an undifferentiated state by in vitro culture, and capable of proliferating cells of all lineages of trichomes (ectoderm, mesoderm, ≪ / RTI > cells with the ability to differentiate into < RTI ID = 0.0 > At present, pluripotent stem cells are embryonic stem cells (hereinafter abbreviated as "ES cells"), embryonic stem cells (hereinafter referred to as "embryonic stem cells") isolated from early embryos derived from mammals such as mouse, monkey, stage embryonic germ cells (abbreviated as "EG cells") and multipotent adult progenitor cells (hereinafter referred to as "MAPC") isolated from the adult bone marrow ).

In one embodiment, in the method of inducing differentiation of stem cells of the present invention, the pluripotent stem cells (iPSC), which is a pluripotent stem cell produced through the reprogramming of ES cells and various somatic cells, , EG cells, or MAPC, preferably ES cells, more preferably human ES cells.

Specific examples of mouse ES cells include EB3 cells, E4 cells, D3 cells, CCE cells, R1 cells, 129 SV cells, J1 cells, and the like. In addition, standard protocols for the production, transfer, and preservation of ES cells, EG cells, and MAPC have already been established, and known protocols (Matsui et al, Cell 70: 841, 1992; Shamblott et al., Proc. Natl. These pluripotent stem cells can be easily used with reference to US Pat. No. 6,060,622; International Patent Publication No. 01/11011), which is incorporated herein by reference in its entirety, to Jiang et al., Nature 418: 41 have.

In addition, the cells usable in the present invention are not limited to the above three kinds, and all the pluripotent stem cells derived from an embryo of a mammal, an adult tissue such as fetus, umbilical cord blood, adult organs or bone marrow, . As a specific example, stem cells (Sharda & Zahner, International Patent Publication No. 02/051980) obtained by treating medicinal agents such as 5-azacytidine (hereinafter abbreviated as "AZC" (Abuljadayel, Curr. Med. Res. Opin. 19: 355, 2003) or adult stem cells derived from adult cells (Li et al, Nature Med ., Advance online publication). In this case, the ES cell-like trait refers to a cell having a surface (antigen) marker specific to an ES cell, an ES cell specific gene, a teratoma forming function, or a chimeric mouse capable , And the like.

In addition, even if a cell that does not have a trait similar to an ES cell, or a cell that is not an pluripotent stem cell, is a cell capable of differentiating into a cell having a hepatocyte-like trait in at least an in vitro culture, Can be used.

The term "feeder" or "feeder cell" is a term used to describe a type of cell that co-cultures with other types of cells to provide an environment in which a second type of cell can grow.

The present invention

(a) treating stem cells with AA (activin A) to obtain differentiated endoderm cells from the stem cells;

(b) treating the obtained endoderm cells with RA (retinoic acid) to obtain hepatocytes differentiated from the endoderm cells and treating the obtained hepatocytes with NA (nicotinamide) to grow hepatocytes; And

(c) treating hepatocyte growth factor (HGF) with the proliferated hepaticoblasts to obtain hepatocytes differentiated from the hepaticoblasts

The present invention provides a method of differentiating high-function hepatocytes.

The step (b) may further include the step of culturing the endoderm cells obtained in step (a) by adding AA (activin A), sodium butyrate and fetal bovine serum (FBS) before the step (b).

The step (b) may further include pre-culturing the endoderm cells obtained in step (a) by adding bone morphogenetic protein 2 (BMP2) and fibroblast growth factor 4 (FGF4).

When the hepatic stem cells of step (b) are proliferated, basic fibroblast growth factor (bFGF) and ascorbic acid may be further treated.

The step (c) may further include the step of monoclonalizing the hepatocytes by treating trypsin with the hepaticoblasts obtained in step (b).

It is more preferable that the step (c) is performed through 3D floating culture.

And further comprising the step of treating the hepatocytes obtained through the step (c) with OSM (oncostatin M) and DEX (dexamethasone) to mature the hepatocytes.

The hyperfunctional hepatocytes prepared through these steps showed drug metabolism activity similar to hepatocyte in vivo, and showed cytochrome P450 (CYP) enzyme activity in drug metabolism.

The present invention also relates to a therapeutic composition comprising hepatocytes differentiated from stem cells as provided above by induction of differentiation by the method of the present invention, preferably hepatic cells necessary for the treatment of acute, chronic or genetic liver damage A therapeutic composition for recovery, more preferably a composition for treating genetic liver damage.

The therapeutic composition of the present invention may be prepared into a suitable preparation including an acceptable carrier depending on the administration mode. Formulations suitable for the mode of administration are known and may include formulations that facilitate passage, typically through the membrane.

In addition, the therapeutic composition of the present invention can be used in the form of a general pharmaceutical preparation. The parenteral preparation may be in the form of a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion or a lyophilized preparation, or a tablet, troch, capsule, elixir, suspension, syrup or wafer in the case of oral administration. May be formulated in unit dosage ampoules or multiple doses.

In addition, the therapeutic composition of the present invention may be administered together with a pharmaceutically acceptable carrier. For oral administration, for example, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring matters or fragrances may be used. In the case of injections, buffering agents, preservatives, An isotonic agent, an isotonic agent, a stabilizer, etc. may be used in combination. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used.

In addition, the method of treating acute, chronic or genetic liver damage using the therapeutic composition of the present invention may include administration through a common route in which a predetermined substance is introduced into the patient in an appropriate manner. Such administration methods include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration. However, upon oral administration, the cells may be digested, so that the oral composition is preferably formulated to coat the active agent or protect it from being decomposed from above.

In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell. Preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular or drip injectable. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (for example, oleic acid), an alcohol (for example, ethanol, benzyl alcohol, propylene glycol or glycerin) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor Preservatives (for example, mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.). Preferably, a method of treating acute, chronic or genetic liver damage with the therapeutic composition of the present invention comprises administering a therapeutically effective amount of the therapeutic composition of the present invention. The pharmaceutically effective amount may be appropriately selected depending on the kind of the disease, the age, body weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, administration frequency, And can be readily determined by those skilled in the art depending on the factors.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

< Example  1> undifferentiated human Embryonic stem  Cell line and primary cultured hepatocyte culture method

Undifferentiated human embryonic stem cell (hESC) cell lines were grown in DMEM / F12 medium (Invitrogen Life Technologies, USA) containing 4 ng / ml bFGF, 1% nonessential amino acids and 100 mM beta mercaptoethanol (10 μg / ml mitomycin-C) mouse embryonic fibroblasts (MEFs) using mitotically-inactivated cells. The cells were cultured under standard conditions (37 ° C, 5% CO ₂ and saturated humidity) and the medium was changed daily. When hESCs and hiPSC (human induced pluripotent stem cells) colonies are proliferated, all cells of the hESC and hiPSC cell lines are treated with type IV collagenase and then shredded and inoculated into new feeder cells every 5-6 days . BGOl hESCs were used to establish the protocol, and other hESCs and hiPSCs were used to validate the hepatocyte differentiation protocol.

Human primary hepatocytes (hPH) were purchased from BD Biosciences Discovery Labware. Human hepatocytes cultured in 24 well plates coated with type 1 collagen were cultured and maintained for at least 24 hours before the hepatocyte culture medium was treated.

Two human liver cell lines (HepG2 and Huh7) were obtained from the Korean Cell Line Bank. The cells were inoculated into DMEM medium containing 10% FBS and 1% penicillin / streptomycin and cultured at 37 ° C and 5% CO ₂ . When the cell density reached a degree of saturation of 70-80%, subculture was performed. The medium was replaced every 3 days.

< Example  2> Differentiation of Undifferentiated Human Embryonic Stem Cells into Hepatocytes

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a method for differentiating human embryonic stem cells into hepatocytes.

To initiate hepatocyte differentiation, hESCs and hiPSCs were dispersed into single cells for 3 min using TrypLE select (Invitrogen) and harvested by centrifugation at 1000 rpm for 5 min. Matrigel-coated culture dishes were cultured for one day after inoculation with mTeSR1 medium containing a protein kinase inhibitor Y-27632 (ROCK inhibitor). Y-27632 (ROCK inhibitor) -free mTeSR1 medium for 2 days, and hepatocyte differentiation was performed. Human embryonic stem cells were identified by the expression of OCT4, an undifferentiated marker, through qPCR (Fig. 2).

Stage 1 ) To endoderm cells  Differentiation / 2D adherent culture

Endoderm cells can form epithelial cells of the internal organs, including the digestive tract, liver and lungs. The step of differentiating pluripotent stem cells into endoderm cells is necessary for the differentiation of hepatocytes in vitro. In the present invention, hESCs and hiPSCs were first differentiated into endoderm cells for 3 days. Differentiation of hESCs and hiPSCs for the first 1.5 days was initiated in RPMI medium containing 2 μM CHIR99021 (GSK-3 inhibitor) and 100 ng / ㎖ AA (activin A). For the next 1.5 days, the cells were further differentiated in RPMI medium containing 100 ng / ml AA, 0.5-1 mM sodium butyrate and 0.2% FBS in the same culture medium. Induction of endoderm cells was confirmed by measuring the expression level of SOX17, a marker of endoderm cells through qPCR (Fig. 2).

Step 2 ) Hepatic  Differentiation / 2D Adhesion culture

The endoderm cells obtained in step 1 were further cultured for 8 days and differentiated into hepaticoblasts. First, the cells were inoculated into RPMI medium containing B27 adjuvant (Gibco) and cultured for 2 days with 20 ng / ml BMP2 (bone morphogenetic protein 2) and 30 ng / ml FGF4 (fibroblast growth factor 4) . Differentiation into hepatocytes was induced by inoculating the cells into DMEM medium containing B27 adjuvant and treating with 2 μM RA (retinoic acid) for 2 days. The hepatocytes were inoculated into DMEM medium containing B27 adjuvant and cultured for 4 days with 10 mM NA (nicotinamide), 1 ng / ml bFGF, and 100 μM ascorbic acid to grow hepatocytes. The induction of hepatic stem cells was assayed by measuring the expression of HNF4α and AFP (FIG. 2), and proliferation of hepatic stem cells was assessed using the EdU incorporation assay kit (FIG. 3).

Step 3 ) Differentiation into hepatocytes / 2D adherent culture or 3D floating culture

In order to induce final maturation of hepatocytes, the hepatocytes obtained in step 2 are treated with TrypLE select to be single-celled, and inoculated into a culture container coated with type 1 collagen to perform 2D adhesion culture, or inoculated into 3D In order to perform suspension culture, an adhesion inhibition culture container was inoculated. Both were inoculated on ITS medium, treated with 20 ng / ml HGF, and cultured for 4 days to differentiate. The cells were inoculated into ITS medium containing 10 ng / ml of OSM (oncostatin M) and 10 [mu] M of DEX (dexamethasone) and cultured for 4 days for further maturation. Expression was confirmed using AAT and ALB as markers of the final differentiated cells (Fig. 2).

< Example  3> Selection of differentiation stage for spherical formation

The survival rate of 10, ㎕ of cultured cells suspended in single cells, I, II, and III, and 10 ㎕ of trypan blue reagent were analyzed using a cell counter. It was confirmed that the survival rate of the cells was about 80 ~ 90% when they were separated into single cells in step I and step II, but fell below 40% in step III. Thereafter, the spleroids formed in step I and the spheroids formed in step II were cultured in the same manner until the last stage of differentiation, and then five photographic images of independent positions were taken at the same magnification (X40) to compare the sizes of the spheroids As a result, the cells were separated into single cells in step II, and it was confirmed that the culture was successful when the spheroids were formed (FIG. 4).

< Example  4> Flow cell  Analysis of differentiated hepatocytes Marker  Protein expression

TrypLE select was added to 2D cultured HLCs and 3D cultured hepatocyte spheroids to separate them into single cells, and fixed with 4% PFA for 20 minutes at 4 ° C. The cells were washed with FACS washing buffer sold by BD, treated with ALB and ASGPR 1 antibody, respectively, known as mature hepatocyte markers as primary antibodies, and reacted at 4 ° C for 30 minutes. After 3 washing steps, the secondary antibody was reacted at 4 ° C for 30 minutes. The washed cells were then analyzed using a BD-FACS Calibur Flow Cytometer (FACS Calibur, USA). Three independent experiments were performed for each marker. Experimental results were analyzed using Flowjo software. In the case of ALB, when the degree of differentiation was confirmed by the cell separator (FACS), the differentiated cells attached to the culture container (2D) showed 92.84% differentiation efficiency and 91% By confirming the differentiation efficiency, it was confirmed that the two groups showed similar differentiation efficiency (FIG. 5). In the case of ASGPR1, when the degree of differentiation was confirmed by the cell separator (FACS), the differentiated cells attached to the culture container (2D) showed 40.2% differentiation efficiency and 50.85% By confirming the differentiation efficiency, it was confirmed that the expression in the differentiated hepatocytes was enhanced by spherical formation (Fig. 6).

<Example 5> Characterization of differentiated hepatocytes

LDL absorption analysis

In order to analyze the position of 2D-cultured HLCs cells expressing hepatocyte markers and the LDL absorbed in 3D-cultured spleoids, analysis was performed according to the user manual using Dil-Ac-LDL staining kit (Biomedical Technologies, USA) Respectively. Fluorescence expressed in absorbed LDL was photographed with a fluorescence microscope (Carl Zeiss Jena, Germany). It was confirmed that LDL absorption was well performed both in 2D cultured HLCs cells and in 3D-cultured spoloids (Fig. 7).

PAS (Periodic Acid- Schiff ) dyeing

Cells derived from 2D cultured HLCs and 3D cultured hepatocyte spheroids were fixed with a solution containing 4% formalin and 95% ethanol and reacted with PAS solution for 5 minutes at room temperature. After several washes with distilled water, the cells were treated with Schiff's reagent for 15 minutes, washed in running water for 5 minutes, and then stained with hematoxylin for 90 seconds at room temperature. Intracellular glycogen particles (purple) were detected with an optical microscope (Carl Zeiss). In both 2D cultured HLCs cells and 3D cultured spoloids, glycogen accumulation was well established (FIG. 7).

ICG ( Indocyanin  green) dyeing

1 mg / ml ICG was added to the medium of cells derived from 2D cultured HLCs and 3D cultured hepatocyte spheroids and reacted for 1 hour under standard culture conditions. ICG absorption was observed with an optical microscope. Both ICG-HLCs cells and 3D-cultured spoloids showed good ICG uptake (FIG. 7).

ALB  And Urea  Measure

The ALB contained in the cell culture supernatant was measured using an albumin ELISA kit (Bethyl laboratories, Inc., USA). Approximately, an antibody coating solution was added to a 96-well plate and coated for 1 hour, followed by addition of a primary antibody to ALB and reaction at room temperature for 1 hour. Subsequently, 50 [mu] l of CM (conditioned medium-media of the same amount of cells cultured for 48 hours) was dispensed into each well and left at room temperature for 1 hour. ALB bound to the antibody was detected using secondary antibody (Santa Cruz Biotechnology, USA) coupled with HRP (horseradish peroxidase). After reacting at room temperature for 1 to 2 hours, TMB (tetramethylbenzidine) substrate was added to the plate for reaction, and 0.5 M sulfuric acid was added to terminate the reaction. Absorbance was measured at 405 nm. Each sample was tested in triplicate using at least three independent samples.

Urea was also reacted with the solution using a kit according to the user manual and then measured by absorbance. Each sample was tested in triplicate using at least three independent samples. It was confirmed that the hepatocyte differentiated from HepG2 and Huh7, which are commonly known liver cancer cell lines, have high albumin and urea secretion ability. 3D cultured spoloids showed lower albumin and urea secretion than primary cultured human hepatocytes, but were significantly higher than 2D cultured HLCs (Fig. 8).

TEM (Transmission electron microscopy) analysis

Cells derived from 2D cultured HLCs and 3D cultured hepatocyte spheroids were added to 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde-2% PFA and fixed at 4 DEG C for 2 hours. After washing with 0.1 M phosphate buffer, 0.1 M phosphate buffer containing 1% OsO ₄ (osmium tetroxide) was added to the cells and post-fixed in the dark for 1 hour. Post-fixed cells were dehydrated, propylene was added and formatted in an Epon812 mixture (EMS, USA). The block was cut with a Ultracut UCT ultramicrotome (Leica, Germany) into flakes of 60 nm thickness. Uranyl acetate and lead citrate, and the flakes were analyzed by H-7600 TEM (80 kV). Both 2D cultured HLCs and 3D cultured hepatocyte spoloids were able to observe the bile canalicular junction characteristic of hepatocytes (yellow arrow head) and to confirm the presence of intracellular accumulated glycogen (yellow arrow). Round, large nuclei (N) and MV (microvilli) characteristic of hepatocytes were also confirmed. The ruler represents 2 [mu] m (Fig. 9).

< Example  6> Enzymes related to drug metabolism in differentiated hepatocytes Nuclear receptor  Confirm gene expression

Total RNA was obtained from cells that had been repeatedly exposed to biological debris using 2D cultured HLCs, cells derived from 3D cultured hepatocyte spheroids and TRIzol reagent, and a reverse transcription procedure using a reverse transcription system (Promega Corp., USA) Respectively. PCR amplification conditions were 94 ° C for 5 minutes, 35 cycles (94 ° C for 30 seconds, 50-57 ° C for 30 seconds and 72 ° C for 30 seconds) and 72 ° C for 10 minutes.

qPCR analysis was performed using SYBR Green PCR Master Mix (Applied Biosystems, USA). The PCR reactions consisted of 12.5 SY of SYBR Green PCR Master Mix, 25 포함 containing 0.8 10 10 mM primer, 10.4 증 distilled water and 0.5 주 template cDNA, respectively, and amplification was confirmed under conditions appropriate for each primer. The relative expression levels of each gene were normalized using &lt; RTI ID = 0.0 &gt; ss-actin. &Lt; / RTI &gt;

The sequences of the primers used are as follows (Table 1).

gene Forward (5'3 ') Reverse direction (5'3 ') Product size
(bp) CYP1A2
CYP2B6
CYP2C9
CYP2D6-v1
CYP2D6-v2
CYP3A4
CAR
PXR
PPARα
GSTA2
GSTA4
GSTM4
GAPDH CCAGTCTGTTCCCTTCTCGG
AGCTTCATGACCGAGCCAAA
ATTTGTGTGGGAGAAGCCCT
GTTCCCAAGGGGTGTTCCTG
CCCAAGGACGCCCCTTTC
AGCCTGGTGCTCCTCTATCT
GACCTGCCTGTCTTCCGTTC
CCACTGGGAGTGCAGGGGC
TCGGCGAGGATAGTTCTGGA
CCTTCTTTCAGTGGGAGGGA
CCGAGTGGACTCCAGAAAGC
TGTACACAAGGGTGGCTGTC
TTCAGTGGTGGACCTGACCT GCTGGCTCATCCTTGACAGT
CAGGATTGAAGGCGTCTGGT
AAAGAGAGCTGCAGGGACTG
GGCTTTGTCCAAGAGACCGT
GCTGGGATATGCAGGAGGAC
CCCTTATGGTAGGACAAAAT
GATTTCCACAGCTGCTCCCT
TGGCAGCCGGAAATTCTTGA
TGGTGAAAGCGTGTCCGTGA
GCCATGGTAGCAGTCTCCTG
GGCACTTGTTGGAACAGCAG
GAAAGGAACGAGGAGGCAGG
CACCACCCTGTTGCTGTAGC 200
216
224
200
205
116
72
101
108
140
200
144
256

In the CAR, PPARα, and PXR nuclear receptors involved in the expression of drug metabolizing enzymes of hepatocytes, 3D culture spoloids were found to be lower than that of the primary cultured human hepatocytes but higher than 2D culture HLCs (FIG. 10).

The GSTA2, GSTA4, and GSTM4 enzymes of the hepatocyte-mediated metabolism group 2 were found to have the highest expression of 3D culture spoloid (FIG. 11).

In the case of CYP2D6-v1, the expression of 3D-cultured spleroid was similar to that of primary cultured human hepatocyte. The expression of CYP2D6-v2, CYP3A4, CYP2B6 and CYP2C9 was higher than that of primary cultured human hepatocyte Expression was increased in 3D cultured spolide than in 2D cultured HLCs. In the case of CYP1A2, it was confirmed that the expression of 3D cultured spoloid was the highest (Fig. 12).

< Example  7 > cytochrome P450 of differentiated hepatocytes ( CYP ) Enzyme drug metabolism activity analysis

Cell activity of CYP3A4, CYP2C19, CYP1A2 and CYP2D6 was confirmed using cell-based CYP enzyme activity assay. Approximately, treatment with PB (10, 25, 50, 100 and 200 ㎛), AP (10, 25, 50, 100 and 200 ㎛) and RIF (0.5, 1 and 5 ㎛) Respectively. Luciferin matrix (luciferin-IPA for CYP3A4, luciferin-H EGE for CYP2C19, luciferin-ME for CYP1A2 and P450-Glo for CYP2D6) was added to 2D cultured HLCs and 3D cultured hepatocyte spheroids in a 24 well culture vessel, And reacted for 1 hour. At the end of the reaction, 50 [mu] l of each reagent was dispensed into a 96 well opaque white luminometer plate at room temperature. Next, 50 占 퐇 of luciferin detection reagent was added to each well, and the reaction was carried out in a dark place for 40 to 60 minutes to induce a fluorescence reaction. The resulting fluorescence levels were measured. After measuring the fluorescence level, the protein concentration per well was measured, and the fluorescence value per protein concentration was calculated to confirm various CYP activities. It was confirmed that the drug metabolizing enzyme activity of the 3D culture spoloid was highest or appeared to be similar to that of the primary cultured human hepatocyte (Fig. 13). * Indicates p <0.05 and this value is statistically significant as compared to the control group in which each drug group was not treated.

< Example  8> Identification of differentiated hepatocyte characteristics due to drug treatment

For immunofluorescence staining, PB, AP, and RIF, which exhibited the highest drug metabolizing enzyme activity, were selected and exposed to 2D culture HLCs for 48 hours. PBS containing 4% PFA (paraformaldehyde) . Fixed 2D cultured hepatocytes were blocked and punctured by treatment with 0.1% BSA / PBS containing 0.3% Triton X-100 / PBS and 10% serum (using PBS without 0.1% BSA for ALB antibody) , ALB, and AFP were diluted and treated and reacted overnight at 4 ° C. Homologous mouse IgG or normal donkey serum was used as a negative control, but no fluorescence was detected in the negative control. After washing three times with PBS, a secondary antibody diluted with 1: 400 conjugated with Alexa flour 488 and Alexa flour 594 fluorescent material was added and reacted at room temperature for 1 hour and 30 minutes. Next, the nuclei of the cells were stained using 1 ㎍ / ml DAPI (4 ', 6-diamidino-2-phenylindole). It was also confirmed that the expression of hepatocyte markers was maintained at a high level even in the treatment with various drugs (PB, AP, RIF) (FIG. 14). The ruler represents 100 μm.

In order to confirm the accumulation of glycogen, red stained PAS staining was performed to confirm that hepatocyte characteristics were maintained regardless of treatment with various drugs (PB, AP, RIF) (FIG. 15). The ruler represents 100 μm.

< Example  9> Hepatocyte  Identify the proliferative phase

EdU staining

To confirm hepatocyte proliferation during hepatocyte differentiation, 10 μM EdU (5-ethynyl-2'-deoxyuridine) was added to the culture medium group treated with the basic culture solution group and NA, AsA and bFGF for 4 hours to reproduce DNA The cells were incubated with EdU. Cells exposed to EdU were washed three times with PBS and fixed in PBS containing 4% PFA (paraformaldehyde) for 20 minutes. Then, staining was performed according to the manual of the EdU incorporation assay kit (invitrogen), and ALB, a hepatocyte marker, was stained with the above immunofluorescent staining method. Fluorescence photographs of EdU were taken with a fluorescence microscope (Carl Zeiss Jena, Germany). As a result, it was observed that the number of cells was increased in the group treated with NA, and that ALB and EDU fluorescence stained more proliferation in the group treated with NA (FIG. 3). The number of proliferated cells was counted and it was confirmed that a large number of cell proliferation occurred in the group treated with NA (FIG. 3). The ruler represents 100 μm.

Claims (10)

(a) treating stem cells with AA (activin A) to obtain differentiated endoderm cells from the stem cells;
(b) treating the obtained endoderm cells with RA (retinoic acid) to obtain hepatocytes differentiated from the endoderm cells and treating the obtained hepatocytes with NA (nicotinamide) to grow hepatocytes; And
(c) treating hepatocyte growth factor (HGF) with the proliferated hepaticoblasts to obtain hepatocytes differentiated from the hepaticoblasts
&Lt; / RTI &gt;
The method according to claim 1,
Further comprising the step of culturing the endoderm cells obtained in step (a) by adding AA (activin A), sodium butyrate and fetal bovine serum (FBS) before the step (b).
The method according to claim 1,
Further comprising the step of pre-culturing the endoderm cells obtained in step (a) by adding bone morphogenetic protein 2 (BMP2) and fibroblast growth factor 4 (FGF4) before the step (b).
The method according to claim 1,
The method according to any one of claims 1 to 3, wherein the growth of the hepatic stem cells of step (b) is further treated with bFGF (basic fibroblast growth factor) and ascorbic acid.
5. The method of claim 4,
Further comprising the step of monoclonalizing the hepatocyte by treating trypsin with the hepaticoblast obtained in step (b) before the step (c).
The method according to claim 1,
Wherein step (c) is performed through 3D suspension culture.
The method according to claim 1,
Further comprising the step of treating the hepatocytes obtained through the step (c) with OSM (oncostatin M) and DEX (dexamethasone) to mature hepatocytes.
A high-function hepatocyte differentiated by the method of claim 1.
9. The method of claim 8,
Hepatocyte showing cytochrome P450 (CYP) enzyme activity.
A therapeutic composition for the recovery of hepatic function required for the treatment of acute, chronic or genetic liver impairment comprising high function cells differentiated by the method of claim 1.

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