Method for producing hepatocyte-like cells and use thereof

Abstract

To provide a method for producing hepatocyte-like cells and the like.SOLUTION: A method for producing hepatocyte-like cells comprises a step of differentiating hepatoblasts into hepatocyte-like cells, the step involving irradiation of the hepatoblasts with green LED.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing hepatocyte-like cells and its application technology, etc. In particular, the present disclosure relates to a method for producing hepatocyte-like cells and its application technology, etc., which includes irradiating hepatoblasts with green LED light in a step of differentiating hepatoblasts into hepatocyte-like cells. [Background technology]

[0002] Liver transplantation is a common treatment for severe liver disease, but in Japan in particular, there is a shortage of donors, so cell therapy (cell transplantation) is becoming increasingly important as an alternative to liver transplantation.

[0003] To date, studies have been conducted on transplanting mesenchymal stem cells into liver injury model mice, but issues remain, such as insufficient differentiation into hepatocytes. In addition, in recent years, induction of differentiation from stem cells into hepatocyte-like cells has been carried out worldwide, but it has been reported that the function of the differentiated hepatocyte-like cells is inferior to that of actual liver cells, and efforts have been made to develop methods for improving the function of hepatocyte-like cells by adding substances or genetic manipulation, etc. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7078939 [Non-patent literature]

[0005] [Non-Patent Document 1] Surg Today 2021 Mar;51(3):340-349. [Non-patent document 2] J Hepatobiliary Pancreat Sci. 2021 Sep;28(9):705-715. [Non-patent document 3] Hepatol Res 2018 Oct;48(11):926-936. Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present disclosure is to provide a method for producing hepatocyte-like cells, etc. [Means for solving the problem]

[0007] The present inventors discovered that irradiating hepatoblasts with a green LED during the process of differentiating hepatoblasts into hepatocyte-like cells improves the function of the hepatocyte-like cells, and have made further improvements.

[0008] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. A method for producing hepatocyte-like cells, comprising a step of differentiating hepatoblasts into hepatocyte-like cells, The method includes irradiating the hepatoblasts with a green LED in this step. Section 2. Item 1. The method according to item 1, wherein the peak wavelength of the green LED is 500 to 570 nm. Section 3. The green LED irradiation conditions are as follows: Daily cumulative illuminance is 0.3 to 0.9 J / cm 2 , Energy density: 8~40W / m 2 , The exposure time per day is 1 to 10 minutes, and / or Item 3. The method according to Item 1 or 2, wherein the number of days of irradiation is between 5 and 15. Section 4. Item 4. The method according to any one of Items 1 to 3, further comprising the step of differentiating stem cells into hepatoblasts. Section 5. moreover, Differentiating the stem cells into endoderm cells; and Item 4. The method according to any one of Items 1 to 3, which comprises the step of differentiating endoderm cells into hepatoblasts. Section 6. Item 6. Hepatocyte-like cells produced by the method according to any one of Items 1 to 5. Section 7. Compared to hepatocyte-like cells differentiated from hepatoblasts without green LED irradiation, Hepatocyte-like cells that satisfy at least one of the following (A) to (C): (A) The expression level of the alpha-1 antitrypsin (AAT) gene is 1.1 times or more. (B) The expression level of the ornithine transcarbamylase (OTC) gene is 1.3 times or more. (C) The ammonium ion concentration in the cell culture supernatant 24 hours after the addition of ammonium chloride is 0.8 times or less. Section 8. A preparation comprising the hepatocyte-like cells according to Item 6 or 7. [Effects of the Invention]

[0009] A method for producing hepatocyte-like cells with improved function is provided. Also provided are hepatocyte-like cells with improved function and preparations containing the cells. [Brief explanation of the drawings]

[0010] [Figure 1] A schematic diagram of the green LED irradiation protocol is shown. [Figure 2] Microscopic photographs of the cells and the results of cell viability measurements are shown. [Figure 3] The results of measuring the expression levels of albumin (ALB) and α-1 antitrypsin (AAT), which are hepatocyte maturation markers, are shown. [Figure 4] The results of measuring the expression levels of ornithine transcarbamylase (OTC) and carbamyl phosphate synthase 1 (CPS1), which are urea cycle genes involved in ammonia metabolism, are shown. [Figure 5] 1 shows the results of measuring ammonia metabolism and CYP3A4 activity. [Figure 6] The results of measuring ROS (reactive oxygen species) activity are shown. [Figure 7]The results of measuring the expression level of PGC1α and the results of Ca 2+ and ATP assays are shown. [Figure 8] The results of measuring the expression level of OPN3, a photoreceptor, are shown. DETAILED DESCRIPTION OF THE INVENTION

[0011] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses a method for producing hepatocyte-like cells, which includes a step of differentiating hepatoblasts into hepatocyte-like cells. In this specification, this production method may be referred to as the "production method of the present disclosure."

[0012] As used herein, the term "hepatoblast" refers to a precursor of liver cells (hepatocytes). Hepatoblasts are also called hepatoblasts or liver progenitor cells.

[0013] The hepatoblasts may be, for example, primary cultured cells, subcultured cells, or established cell lines.

[0014] The species from which hepatoblasts are derived are preferably mammals, for example. More specifically, examples include humans; rodents such as mice, rats, guinea pigs, and hamsters; dogs, cats, rabbits, cows, monkeys, and pigs. Of these, humans are preferred.

[0015] Hepatoblasts may also be cells differentiated from stem cells, for example. Examples of stem cells include mesenchymal stem cells; induced pluripotent stem cells such as ES cells and iPS cells; and the like. Among these, mesenchymal stem cells are preferred.

[0016] Examples of tissues from which mesenchymal stem cells can be derived include adipose tissue, bone marrow, umbilical cord blood, etc. Among these, adipose tissue-derived mesenchymal stem cells are minimally invasive and can be collected in large quantities, making them the most suitable for clinical use.

[0017] The method for differentiating hepatoblasts into hepatocyte-like cells is not particularly limited, but examples include a method in which hepatocyte growth factor (HGF), oncostatin M, dexamethasone, etc. are added to a culture medium for hepatoblasts and cultured. These can be used alone or in combination of two or more. Among these, it is preferable to add hepatocyte growth factor, oncostatin M, and dexamethasone. The concentration at the time of addition can be appropriately selected depending on the substance to be added. For example, when hepatocyte growth factor is added, the concentration can be about 5 to 80 ng / mL, preferably about 10 to 60 ng / mL. For example, when oncostatin M is added, the concentration can be about 1 to 20 ng / mL, preferably about 5 to 15 ng / mL. For example, when dexamethasone is added, the concentration is 1 x 10 -8 ~1×10 -4 It can be about 1×10 -7 ~1×10 -5 It is about M size. The timing of adding the above-mentioned substances can be appropriately selected depending on the substance to be added. For example, the substance may be added at the start of culture, after the start of culture (e.g., one day after the start of culture), or in two or more divided doses during the culture period.

[0018] The culture medium for hepatoblasts is not particularly limited as long as it allows hepatoblasts to be cultured, and known media can be used, such as minimal essential medium containing non-essential amino acids, DMEM / F-12, etc.

[0019] Hepatoblasts may be cultured either in two-dimensional culture or three-dimensional culture. Three-dimensional culture methods are not particularly limited, but include, for example, methods using scaffolds, gels, etc. More specifically, three-dimensional culture can be performed by the method described in Patent Document 1 or Non-Patent Document 2 (methods using biocompatible polymer blocks).

[0020] The number of days for culturing when differentiating hepatoblasts into hepatocyte-like cells can be, for example, about 5 to 15 days, preferably about 7 to 12 days, and particularly preferably 10 days.

[0021] The culture temperature when differentiating hepatoblasts into hepatocyte-like cells is not particularly limited as long as it allows hepatoblasts to be cultured, and can be, for example, about 30 to 40°C.

[0022] Furthermore, in the step of differentiating hepatoblasts into hepatocyte-like cells, an additive, insulin-transferrin-selenium (ITS), can be further added. The concentration of the additive when added is not particularly limited, but can be, for example, about 0.5 to 3% by mass. The above substances may be added at the start of culture, or after the start of culture (for example, one day after the start of culture), or may be added in two or more divided doses during the culture period.

[0023] Furthermore, in the step of differentiating hepatoblasts into hepatocyte-like cells, bovine serum albumin can also be added. The concentration at which it is added is not particularly limited, but can be, for example, about 0.1 to 3 mg / mL. The above substances may be added at the start of culture, or after the start of culture (for example, one day after the start of culture), or may be added in two or more divided doses during the culture period.

[0024] Furthermore, in the step of differentiating hepatoblasts into hepatocyte-like cells, human umbilical vein endothelial cells (HUVECs) can be further added. The concentration at which they are added is not particularly limited, but for example, the ratio of the number of hepatoblasts to the number of HUVECs (hepatoblasts:HUVECs) can be about 1:0.1 to 1:10. The cells may be added at the start of culture, after the start of culture (for example, one day after the start of culture), or in two or more divided doses during the culture period.

[0025] The production method of the present disclosure may further include a step of differentiating stem cells into hepatoblasts. The method of differentiating stem cells into hepatoblasts is not particularly limited, and examples include a method of differentiating stem cells into endoderm cells and then differentiating the endoderm cells into hepatoblasts. In other words, the production method of the present disclosure may further include a step of differentiating stem cells into endoderm cells and a step of differentiating the endoderm cells into hepatoblasts. In other words, the manufacturing method of the present disclosure includes: Differentiating the stem cells into hepatoblasts; and Differentiating hepatoblasts into hepatocyte-like cells, The step of differentiating the hepatoblasts into hepatocyte-like cells may include irradiating the hepatoblasts with a green LED; differentiating the stem cells into endoderm cells; differentiating endoderm cells into hepatoblasts; and Differentiating hepatoblasts into hepatocyte-like cells, The step of differentiating hepatoblasts into hepatocyte-like cells may include irradiating the hepatoblasts with a green LED.

[0026] The above description can be applied to the stem cells used.

[0027] The method for differentiating stem cells into endoderm cells (more specifically, definitive endoderm cells) is not particularly limited, and examples include a method of culturing stem cells by adding a glycogen synthase kinase 3 inhibitor, activin A, a Wnt (Wnt family member) protein (more specifically, Wnt3a, etc.), a PI3 kinase inhibitor, or the like to a culture medium for stem cells. These can be used alone or in combination of two or more. Among these, it is preferable to add a glycogen synthase kinase 3 inhibitor. The concentration at the time of addition can be appropriately selected depending on the substance to be added. For example, when a glycogen synthase kinase 3 inhibitor is added, the concentration can be about 0.5 to 10 μmol / L, and preferably about 1 to 5 μmol / L. The timing of adding the above-mentioned substances can be appropriately selected depending on the substance to be added. For example, the substance may be added at the start of culture, after the start of culture (e.g., one day after the start of culture), or in two or more divided doses during the culture period.

[0028] The culture medium for stem cells is not particularly limited as long as it can culture stem cells, and any known medium can be used. The culture medium for stem cells may be the same as or different from the culture medium for hepatoblasts. Examples include minimal essential medium containing non-essential amino acids, DMEM / F-12, etc.

[0029] Stem cell culture may be either two-dimensional or three-dimensional. Three-dimensional culture methods are not particularly limited, but examples include methods using scaffolds, gels, etc. More specifically, three-dimensional culture can be performed using the method described in Patent Document 1 or Non-Patent Document 2 (a method using a biocompatible polymer block).

[0030] The number of days for culturing when differentiating stem cells into endoderm cells can be, for example, about 2 to 8 days, preferably about 3 to 7 days, and particularly preferably 5 days.

[0031] The culture temperature when differentiating stem cells into endoderm cells is not particularly limited as long as the stem cells can be cultured, and can be, for example, about 30 to 40°C.

[0032] Furthermore, in the step of differentiating stem cells into endoderm cells, an insulin-transferrin-sodium selenite additive can also be added. The concentration of the additive when added is not particularly limited, but can be, for example, about 0.5 to 3% by mass. The above substances may be added at the start of culture, or after the start of culture (for example, one day after the start of culture), or may be added in two or more divided doses during the culture period.

[0033] Furthermore, in the step of differentiating stem cells into endoderm cells, bovine serum albumin can also be added. The concentration at which it is added is not particularly limited, but can be, for example, about 0.1 to 3 mg / mL. The above substances may be added at the start of culture, or after the start of culture (for example, one day after the start of culture), or may be added in two or more divided doses during the culture period.

[0034] The method for differentiating endoderm cells into hepatoblasts is not particularly limited, but examples include a method in which bone morphogenetic protein (BMP, more specifically, BMP-2, etc.), fibroblast growth factor (FGF, more specifically, FGF-4, etc.), DMSO, etc. are added to a culture medium for endoderm cells and cultured. These can be used alone or in combination of two or more. Among these, it is preferable to add BMP-2 and FGF-4. The concentration at the time of addition can be appropriately selected depending on the substance to be added. For example, when BMP-2 is added, the concentration can be about 5 to 50 ng / mL, preferably about 10 to 30 ng / mL. For example, when FGF-4 is added, the concentration can be about 10 to 60 ng / mL, and preferably about 20 to 50 ng / mL. The timing of adding the above-mentioned substances can be appropriately selected depending on the substance to be added. For example, the substance may be added at the start of culture, after the start of culture (e.g., one day after the start of culture), or in two or more divided doses during the culture period.

[0035] The culture medium for endodermal cells is not particularly limited as long as it can culture endodermal cells, and any known medium can be used. The culture medium for endodermal cells may be the same as or different from the culture medium for stem cells. Examples include minimal essential medium containing non-essential amino acids, DMEM / F-12, etc.

[0036] Furthermore, endoderm cells may be cultured in two-dimensional culture or three-dimensional culture. Three-dimensional culture methods are not particularly limited, but examples include methods using scaffolds, gels, etc. More specifically, three-dimensional culture can be performed by the method described in Patent Document 1 or Non-Patent Document 2 (methods using biocompatible polymer blocks).

[0037] The number of days for culturing when differentiating endoderm cells into hepatoblasts can be, for example, about 2 to 8 days, preferably about 3 to 7 days, and particularly preferably 5 days.

[0038] The culture temperature when differentiating endoderm cells into hepatoblasts is not particularly limited as long as it allows stem cells to be cultured, and can be, for example, about 30 to 40°C.

[0039] Furthermore, in the step of differentiating endoderm cells into hepatoblasts, an insulin-transferrin-sodium selenite additive can also be added. The concentration of the additive when added is not particularly limited, but can be, for example, about 0.5 to 3% by mass. The above substances may be added at the start of culture, or after the start of culture (for example, one day after the start of culture), or may be added in two or more divided doses during the culture period.

[0040] Furthermore, in the step of differentiating endoderm cells into hepatoblasts, bovine serum albumin can also be added. The concentration at which it is added is not particularly limited, but can be, for example, about 0.1 to 3 mg / mL. The above substances may be added at the start of culture, or after the start of culture (for example, one day after the start of culture), or may be added in two or more divided doses during the culture period.

[0041] The manufacturing method of the present disclosure includes irradiating hepatoblasts with green LED light in the step of differentiating hepatoblasts into hepatocyte-like cells.

[0042] Furthermore, when the production method of the present disclosure includes a step of differentiating stem cells into hepatoblasts, the step may involve irradiation with a green LED. Furthermore, when the manufacturing method of the present disclosure includes a step of differentiating stem cells into endoderm cells and a step of differentiating endoderm cells into hepatoblasts, a green LED may be irradiated during the step of differentiating stem cells into endoderm cells and / or the step of differentiating endoderm cells into hepatoblasts.

[0043] The peak wavelength of the irradiated green LED can be, for example, about 500 to 570 nm, preferably about 510 to 560 nm, more preferably about 510 to 550 nm, and particularly preferably 520 nm.

[0044] The daily cumulative illuminance of a green LED is, for example, 0.3 to 0.9 J / cm 2 Preferably, it is 0.4 to 0.8 J / cm 2 and more preferably 0.5 to 0.7 J / cm 2 That's about it.

[0045] The energy density is, for example, 8 to 40 W / m 2 Preferably, it is 10 to 30 W / m 2 That's about it.

[0046] The irradiation time can be, for example, about 1 to 10 minutes per day, preferably about 2 to 8 minutes, and particularly preferably 5 minutes.

[0047] The number of days for irradiation can be, for example, about 5 to 15 days, preferably about 7 to 12 days, and particularly preferably 10 days. For example, in the step of differentiating hepatoblasts into hepatocyte-like cells, green LED irradiation may be performed every day, every other day, or every third day, with daily irradiation being preferred.

[0048] The irradiation distance (the distance between the cells and the light source) can be, for example, about 1 to 100 mm, and is preferably about 5 to 50 mm.

[0049] The production method of the present disclosure may include a step of recovering hepatocyte-like cells. The recovery method is not particularly limited, and known methods, conditions, etc. can be used.

[0050] Without wishing to be bound by theory, it is speculated that according to the production method of the present disclosure, as shown in the examples below, irradiation with green LED light induces an increase in mitochondrial function, such as an increase in intracellular ATP concentration, Ca ion concentration, and / or ROS activity, via a direct pathway, thereby increasing the expression of the AAT gene and / or OTC gene, thereby obtaining hepatocyte-like cells with increased hepatocyte function (e.g., ammonia metabolic ability, CYP3A4 activity, etc.). Furthermore, as shown in the examples below, it is speculated that an increase in the expression of the PGC1α gene, which is involved in mitochondrial function, via the photoreceptor OPN3 induces an increase in mitochondrial function via OPN3 as an indirect pathway, thereby obtaining hepatocyte-like cells with increased hepatocyte function (e.g., ammonia metabolic ability, CYP3A4 activity, etc.).

[0051] The present disclosure also encompasses hepatocyte-like cells produced by the production method of the present disclosure. In this specification, such cells may be referred to as "hepatocyte-like cells of the present disclosure."

[0052] Furthermore, "hepatocyte-like cells of the present disclosure" also include hepatocyte-like cells that satisfy at least one of (A) to (C) compared to hepatocyte-like cells differentiated from hepatoblasts without being irradiated with a green LED. (A) The expression level of the alpha-1 antitrypsin (AAT) gene is 1.1 times or more. (B) The expression level of the ornithine transcarbamylase (OTC) gene is 1.3 times or more. (C) The ammonium ion concentration in the cell culture supernatant 24 hours after the addition of ammonium chloride is 0.8 times or less. The hepatocyte-like cells can be produced by the production method of the present disclosure.

[0053] AAT is a marker of hepatocyte maturation. In this specification, the expression level of the AAT gene is evaluated by measuring the expression level of AAT mRNA by quantitative RT-PCR. More specifically, the expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (mRNA) is used as an internal control, and the expression level is evaluated using the relative expression level (the ratio of the expression level of the AAT gene to the expression level of the GAPDH gene) based on the internal control.

[0054] The hepatocyte-like cells of the present disclosure preferably have an AAT gene expression level 1.1 times or more, preferably 1.15 times or more, and more preferably 1.2 times or more, higher than that of hepatocyte-like cells differentiated from hepatoblasts without green LED irradiation.

[0055] OTC is a urea cycle gene involved in ammonia metabolism. In this specification, the expression level of the OTC gene is evaluated by measuring the expression level of OTC mRNA by quantitative RT-PCR. More specifically, the expression level of the GAPDH gene (mRNA) is used as an internal control, and the expression level is evaluated using the relative expression level (the ratio of the expression level of the OTC gene to the expression level of the GAPDH gene) based on the internal control.

[0056] The hepatocyte-like cells of the present disclosure preferably have an OTC gene expression level 1.3 times or more, preferably 1.5 times or more, and more preferably 1.8 times or more, higher than that of hepatocyte-like cells differentiated from hepatoblasts without being irradiated with a green LED.

[0057] The ammonium ion concentration in the cell culture supernatant 24 hours after the addition of ammonium chloride is measured by the method described in the Examples below. Specifically, NH4Cl diluted to a standard of 300 μmol / L with Hank's balanced salt solution is added to a culture plate of hepatocyte-like cells, and the ammonium ion concentration in the culture supernatant after 24 hours of culture at 37°C is measured.

[0058] The hepatocyte-like cells of the present disclosure preferably have an ammonium ion concentration in the cell culture supernatant 24 hours after addition of ammonium chloride of 0.8 or less, preferably 0.75 or less, and more preferably 0.7 or less, compared to hepatocyte-like cells differentiated from hepatoblasts without irradiation with a green LED.

[0059] The hepatocyte-like cells of the present disclosure preferably satisfy at least one of the above-mentioned (A) to (C), more preferably satisfy any two (more specifically, (A) and (B), (A) and (C), or (B) and (C)), and particularly preferably satisfy all of (A) to (C).

[0060] In addition to the above (A) to (C), the hepatocyte-like cells of the present disclosure may have (D) CYP3A4 activity that is 1.1 times or more, 1.15 times or more, or 1.2 times or more, compared to hepatocyte-like cells differentiated from hepatoblasts without being irradiated with a green LED.

[0061] In this specification, CYP3A4 activity is evaluated by the method described in the Examples below, specifically, by a luminescence method using a luminescent substrate. More specifically, it is evaluated using relative activity normalized to the total number of viable cells. CYP3A4 activity can be measured using, for example, Promega's P450-Glo™ CYP3A4 Assay with Luciferin-IPA kit.

[0062] In addition to the above (A) to (C), the hepatocyte-like cells of the present disclosure may have (E) an expression level of peroxisome proliferator-activated receptor gamma coactivator (PGC) 1α that is 1.3 times or more, 1.4 times or more, or 1.5 times or more, compared to hepatocyte-like cells differentiated from hepatoblasts without being irradiated with a green LED.

[0063] In this specification, the expression level of PGC1α is evaluated by measuring the expression level of PGC1α mRNA by quantitative RT-PCR. More specifically, the expression level of GAPDH gene (mRNA) is used as an internal control, and the expression level is evaluated using the relative expression level (the ratio of the expression level of PGC1α gene to the expression level of GAPDH gene) based on the internal control.

[0064] In addition to the above (A) to (C), the hepatocyte-like cells of the present disclosure may also have (F) an intracellular reactive oxygen content that is 1.5 times or more, or even 2 times or more, or even 3 times or more, compared to hepatocyte-like cells differentiated from hepatoblasts without being irradiated with a green LED.

[0065] In this specification, the amount of intracellular reactive oxygen species is evaluated by the method described in the Examples below, specifically, by a fluorescence method using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). For example, an ROS assay kit from Dojindo Laboratories can be used.

[0066] In addition to the above (A) to (C), the hepatocyte-like cells of the present disclosure may have (G) an intracellular ATP concentration that is 1.3 times or more, 1.5 times or more, or even 2 times or more, compared to hepatocyte-like cells differentiated from hepatoblasts without being irradiated with a green LED.

[0067] In this specification, the intracellular ATP concentration is evaluated by the method described in the Examples below, specifically, by a luminescence method using a luminescent substrate, for example, an adenosine triphosphate (ATP) assay kit manufactured by Fujifilm Corporation.

[0068] In addition to the above (A) to (C), the hepatocyte-like cells of the present disclosure have the following characteristics: (H) intracellular Ca 2+ The concentration may be 1.05 times or more, 1.1 times or more, or 1.2 times or more.

[0069] As used herein, intracellular Ca 2+ The concentration is evaluated by the method described in the Examples below, specifically, by a colorimetric method using a chromogenic substrate. 2+ A detection assay kit can be used.

[0070] The present disclosure also encompasses a preparation comprising the hepatocyte-like cells of the present disclosure. In this specification, such a preparation may be referred to as a "preparation of the present disclosure."

[0071] The content of the hepatocyte-like cells of the present disclosure contained in the formulation of the present disclosure is not particularly limited and can be set appropriately up to a maximum of 100% by mass.

[0072] The formulations of the present disclosure include the hepatocyte-like cells of the present disclosure and may further include other optional ingredients.

[0073] The formulations of the present disclosure can be prepared by conventional methods by combining the hepatocyte-like cells of the present disclosure with other components as needed. Examples of such other components include pharmacologically or food hygienically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, surfactants, antioxidants, preservatives, coating agents, colorants, flavorings, pH adjusters, gastric mucosa protectants, and other pharmaceutical agents and / or other ingredients or materials that can be used as foods. These components can be used alone or in combination of two or more.

[0074] The formulation of the present disclosure contains hepatocyte-like cells with improved function, and can therefore be used for cell transplantation into patients with liver diseases, such as metabolic liver diseases such as urea cycle disorders (more specifically, OTC deficiency and citrulline deficiency).

[0075] Furthermore, since the formulation of the present disclosure contains hepatocyte-like cells with improved function, it can be used for pharmacokinetic evaluation and / or toxicity evaluation in drug discovery.

[0076] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0077] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0078] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0079] Cell culture Primary human hepatocytes (PHH) were purchased from Thermo Scientific Inc. (Waltham, MA, USA) and cultured in hepatocyte medium (Thermo Scientific Inc.) according to the manufacturer's protocol. PHH were used within 3 days of seeding.

[0080] Generation of hepatocyte-like cells (HLC) STEMPRO human adipose tissue-derived mesenchymal stem cells (ADSCs) were purchased from Life Technologies (Tokyo, Japan). ADSCs were used in experiments at passages 2 to 6. HLCs were prepared according to the protocol described in Non-Patent Document 1. Briefly, ADSCs (2 × 10 per well) were cultured in a 2-well culture medium. 6Cells (number of cells) were seeded onto 6-well flat-bottom collagen-coated plates (Nunclon Sphera Microplates, Thermo Scientific Inc.) and incubated with serum-free Dulbecco's modified Eagle's medium (DMEM / F-12) supplemented with F-12 for 24 hours. A three-step differentiation protocol was then performed. In the first step (DE differentiation), cells were cultured for 24 hours in DMEM / F-12 supplemented with 0.5 mg / mL bovine serum albumin fraction V (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 2 μmol / L Chir99021 (glycogen synthase kinase 3 inhibitor; Selleckchem, Tokyo, Japan) to induce definitive endoderm differentiation. The next day, 1% insulin-transferrin-selenium (ITS; Sigma-Aldrich) was added to the medium and cultured at 37°C for 5 days. In the second stage (hepatoblast differentiation), the medium was changed to minimal essential medium containing non-essential amino acids (Thermo Scientific Inc.) containing 0.5 mg / mL BSA, 1% ITS, 20 ng / mL bone morphogenic protein 2 (PeproTech, Inc., NJ, USA), and 30 ng / mL fibroblast growth factor 4 (PeproTech, Inc.), and cultured for 5 days at 37°C. In the third stage (hepatocyte differentiation), the cells were treated with 20 ng / mL hepatocyte growth factor (PeproTech) for 5 days, and then further treated with 20 ng / mL hepatocyte growth factor, 10 ng / mL oncostatin M (PeproTech), and 1 × 10 -6 The cells were cultured in M ​​dexamethasone (Sigma-Aldrich) at 37°C for 5 days.

[0081] Green LED (GLED) illumination A GLED with a wavelength of 520 nm and maximum light intensity was produced using an LED irradiation device (3LH-256 / 3LH-75DPS, Nippon Medical and Chemical Instruments Co., Ltd., Osaka). A photoradiometer (Light Analyzer LA-105, Nippon Medical and Chemical Instruments Co., Ltd.) was used to measure the light intensity. The cell culture plate was placed on the LED irradiation device, and the energy density was adjusted to 21 W / m. 2 In the third stage of HLC production, that is, from the 11th day of differentiation, GLED irradiation was performed at room temperature for 5 minutes every day (for a total of 10 days), with an accumulated irradiance of 0.63 J / cm per day. 2 The GLED irradiation protocol is shown in Figure 1. Control cells were treated in the same way except for not being irradiated with GLED. Sufficient ventilation was provided to prevent changes in the medium temperature during treatment. The experiment was repeated at least three times under the same conditions using purchased ADSCs.

[0082] Cell viability Live and dead nucleated cells were counted in GLED-treated and untreated HLCs on day 21 using a LIVE / DEAD Cell Imaging Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Fluorescent signals were measured using a fluorescence microscope (Keyence Corporation). Cell numbers were counted using Image J software (ver. 1.53, National Institutes of Health, Bethesda, MD, USA).

[0083] RT-PCR of HLCs Total RNA was prepared from each HLC sample using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and cDNA was synthesized using a reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The following TaqMan assays (assay identification numbers and primers) were used: α-1 antitrypsin (AAT) (Hs00165475_m1), albumin (ALB) (Hs00910225_m1), ornithine transcarbamylase (OTC) (Hs00166892_m1), carbamyl phosphate synthase 1 (CPS1) (Hs00157048_m1), OPN3 (Hs00173892_m1), and peroxisome proliferator-activated receptor gamma coactivator (PGC) 1α (Hs00173304_m1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs02786624_g1) was used as an internal control for stable expression levels for normalization. All primers were purchased from Thermo Fisher Scientific. Data were collected from 2 -ΔΔCt The relative expression level was calculated as a ratio to GAPDH expression. The results were presented as the fold change in relative mRNA expression of the group compared to the control group.

[0084] Western blotting Protein extraction was performed using RIPA buffer (Thermo Fisher Scientific, Inc.) containing a protease inhibitor cocktail (Sigma-Aldrich) and a PhosSTOP phosphatase inhibitor cocktail (Roche, Tokyo, Japan). Total protein concentration was assessed using a BCA kit (Thermo Fisher Scientific, Inc.). Equal amounts of extracted protein were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). To assess protein abundance, blots were blocked with 5% skim milk for 1 hour at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-OPN3 (1:1,000; SAB2700986; Sigma-Aldrich) and anti-β-actin (1:1,000; cat. no. 4970, Cell Signaling Technology, Inc., MA, USA). The blots were then incubated with anti-rabbit IgG, HRP-conjugated (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) as the secondary antibody for 1 h at room temperature. Proteins were detected by chemiluminescence (GE, Little Chalfont, Buckinghamshire, UK).

[0085] CYP3A4 activity measurement CYP3A4 enzyme activity was assessed using the P450-Glo™ CYP3A4 Assay with Luciferin-IPA (Promega Corporation, WI, USA) according to the manufacturer's instructions. Luminescence was measured using a microplate reader (SpectraMax i3; Molecular Devices, LLC). Luminescence measurements were normalized to the total number of viable cells.

[0086] Ammonia metabolism assay Ammonia metabolism was assessed by measuring the change in ammonium ion concentration in the cell culture supernatant 24 hours after the addition of ammonium chloride (NH4Cl). Briefly, after washing the plate twice with Hank's balanced salt solution (HBSS, Fujifilm Wako Pure Chemical Corporation), NH4Cl (Fujifilm Wako Pure Chemical Corporation) diluted to a standard concentration of 300 μmol / L in HBSS was added to the culture plate. The plates were placed adjacent to each other in an incubator and cultured at 37°C for 24 hours. The supernatant was collected, and the ammonium ion concentration in the supernatant (24 hours after the addition of NH4Cl) was measured using an ammonia assay kit (Cell Biolabs). The negative control was a culture plate containing only the standard ammonium chloride solution. As a positive control, primary hepatocytes were cultured in the same manner as described above.

[0087] Immunofluorescence staining HLCs were immobilized in iPGell (Geno staff, Tokyo, Japan) and fixed with 4% paraformaldehyde according to the manufacturer's protocol. Frozen, gelatinized cells were excised, mounted on slides, and incubated with anti-OPN3 primary antibody (Abcam, ab140901) overnight at 4°C. Subsequently, the slides were incubated with a fluorescently labeled secondary antibody (Thermo Fisher Scientific, A11008) and then with DAPI (Thermo Fisher Scientific, P306931). Slides were observed under a fluorescence microscope (Keyence Corporation, Itasca, IL, USA).

[0088] Ca 2+ and ATP assay Intracellular Ca in GLED-treated HLCs 2+ The concentration is Ca 2+Measurements were performed using a detection assay kit (Abcam, Cambridge, UK). Briefly, cells were homogenized and centrifuged at 20,000 × g for 5 minutes at 4°C, and the supernatant was collected. The samples were mixed with a color reagent and incubated at room temperature for 10 minutes according to the manufacturer's protocol. The absorbance of each sample was measured at 575 nm using a microplate reader. The readings for each cell lysate sample were 1 × 10 6 Normalized to HLC. To assess the amount of ATP in GLED-treated and non-treated HLCs, the Ca 2+ Cells were homogenized and centrifuged in the same manner as in the assay, and an adenosine triphosphate (ATP) assay kit (Fujifilm Wako Pure Chemical Industries, Ltd.) was used according to the manufacturer's protocol. The measured values ​​for each cell lysate sample were 1 × 10 6 Normalized to HLC.

[0089] Detection of reactive oxygen species (ROS) The formation of intracellular reactive oxygen species was measured using a ROS assay kit (Dojindo Laboratories, Kumamoto, Japan). Briefly, HLCs at day 21 of differentiation (10 days after GLED irradiation) were seeded into 6-well dishes and incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) for 30 minutes in the dark at 37°C. After washing twice with HBSS, the cells were incubated with 100 μmol / L hydrogen peroxide for 30 minutes according to the manufacturer's protocol. Fluorescent signals were measured and analyzed using a FACSVerse cytometer and FACSiute software (Becton Dickinson). Cells were imaged using a fluorescence microscope (Keyence Corporation), and cellular fluorescence levels were measured using the Cell Magic Wand plugin in Image J software.

[0090] statistical analysis All data are presented as mean ± standard deviation. Statistical analysis and graphing were performed using GraphPad Prism v7.0 (GraphPad Software, Inc.) and Image J software. Comparisons between two groups were analyzed using the Mann-Whitney test. A P value of less than 0.05 (two-tailed) was considered statistically significant.

[0091] Microscopic photographs of the cells and the results of measuring cell viability are shown in Figure 2. As shown in Figure 2, it was confirmed that GLED irradiation did not cause any morphological changes, and cell viability was similar regardless of whether irradiation was performed or not.

[0092] The results of measuring the expression levels of ALB and AAT, which are hepatocyte maturation markers, are shown in Figure 3. As shown in Figure 3, it was confirmed that AAT expression increased 1.2-fold with GLED irradiation. A tendency for ALB expression to increase was also confirmed. Furthermore, when a similar evaluation was performed using red LEDs (Non-Patent Document 3), which have been reported to promote hepatocyte proliferation, instead of GLEDs, no effect of promoting differentiation into HLCs was confirmed with red LEDs. Furthermore, when GLED irradiation was performed in the first or second stage of HLC production instead of the third stage, no effect of promoting differentiation into HLC was confirmed.

[0093] The results of measuring the expression levels of OTC and CPS1, which are urea cycle genes involved in ammonia metabolism, are shown in Figure 4. As shown in Figure 4, it was confirmed that the expression of OTC increased approximately two-fold due to GLED irradiation. An increased tendency for expression of CPS1 was also confirmed.

[0094] The results of measuring ammonia metabolism and CYP3A4 activity are shown in Figure 5. As shown in Figure 5, in the GLED-irradiated group, ammonia metabolism improved by approximately 25% compared to the non-GLED-irradiated group (ammonium ion concentration decreased by approximately 0.7 times), and CYP3A4 activity also increased by approximately 1.2 times. These findings indicate that GLED irradiation improves hepatocellular function.

[0095] The results of measuring ROS activity are shown in Figure 6. As shown in Figure 6, it was confirmed that intracellular ROS activity increased approximately three-fold due to GLED irradiation.

[0096] To evaluate mitochondrial function, we measured the expression level of PGC1α. 2+ ATP assay was performed. The results are shown in Figure 7. As shown in Figure 7, it was confirmed that the expression of PGC1α increased by approximately 1.5 times due to GLED irradiation. In addition, the ATP concentration increased by approximately 2 times and the Ca 2+ It was confirmed that the concentration was significantly increased by approximately 1.2 times with GLED irradiation. These results indicate that GLED irradiation improves mitochondrial function.

[0097] The results of measuring the expression level of the photoreceptor OPN3 are shown in Figure 8. As shown in Figure 8, it was confirmed that the expression of OPN3 was significantly increased by GLED irradiation.

Claims

1. A method for producing hepatocyte-like cells, comprising a step of differentiating hepatoblasts into hepatocyte-like cells, The method includes irradiating the hepatoblasts with a green LED in the step.

2. 2. The method of claim 1, wherein the green LED has a peak wavelength of 500 to 570 nm.

3. The green LED irradiation conditions are as follows: Daily cumulative illuminance is 0.3 to 0.9 J / cm ２ , Energy density: 8 to 40 W / m ２ , Irradiation time per day is 1 to 10 minutes, and / or 3. The method according to claim 1, wherein the irradiation period is 5 to 15 days.

4. The method according to claim 1 or 2, further comprising the step of differentiating the stem cells into hepatoblasts.

5. moreover, Differentiating the stem cells into endoderm cells; and The method according to claim 1 or 2, comprising the step of differentiating endoderm cells into hepatoblasts.

6. Hepatocyte-like cells produced by the method of claim 1.

7. Compared with hepatocyte-like cells differentiated from hepatoblasts without green LED irradiation, Hepatocyte-like cells that satisfy at least one of the following (A) to (C): (A) The expression level of the alpha-1 antitrypsin (AAT) gene is 1.1 times or more. (B) The expression level of the ornithine transcarbamylase (OTC) gene is 1.3 times or more. (C) The ammonium ion concentration in the cell culture supernatant 24 hours after the addition of ammonium chloride is 0.8 times or less.

8. A preparation comprising the hepatocyte-like cells of claim 6 or 7.

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