Method for differentiating stem cell-derived hepatocytes by using fasudil and gelatin

Abstract

The present invention relates to: a method for differentiating stem cell-derived hepatocytes by using fasudil and gelatin; and the like. The method for differentiating stem cell-derived hepatocytes, according to the present invention, induces, by means of fasudil, the differentiation of stem cells into endoderm in an early stage of differentiation of stem cells into hepatocytes, inhibits lipid droplet formation through high-viscosity gelatin coating of a culture container, activates mitochondrial functions, up-regulates the expression of endoderm genes, and down-regulates the expression of ectoderm and mesoderm genes, thereby improving the efficiency of differentiation of stem cells into hepatoblasts. In addition, low-viscosity gelatin is added to the medium in a differentiation and maturation (late) stage so as to suppress the accumulation of lipid droplets, mitochondria are activated to improve the efficiency of differentiation of hepatoblasts into hepatocytes, and the function of differentiated hepatocytes is enhanced to improve the efficiency of differentiation of stem cells into hepatocytes. Therefore, hepatocytes obtained using the method are expected to be effectively used as a cell therapeutic agent, a transplant, and the like for treating liver diseases.

Description

Stem cell-derived hepatocyte differentiation method using fasudil and gelatin

[0001] The present invention relates to a method for differentiating stem cells from hepatocytes using fasudil and gelatin.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2024-0085745, filed June 28, 2024, the entire disclosure of which is incorporated herein by reference.

[0003] In general, differentiation refers to the process by which cells in the early stages acquire the characteristics of each tissue. A representative example of this can be seen in the development of animals. In other words, a single cell called a fertilized egg, created when a sperm and an egg unite, must undergo 'differentiation' to develop into various tissue cells such as bone, heart, and skin. Stem cells, which possess this differentiation ability, are cells at the stage before differentiating into the individual cells that make up a tissue. They are cells that can proliferate indefinitely in an undifferentiated state and have the potential to differentiate into cells of various tissues when stimulated by specific differentiation stimuli.

[0004] Among stem cells, mesenchymal stem cells (MSCs) are derived from human fetal and adult tissues, such as bone marrow, umbilical cord matrix, and placenta, as well as various adult tissues. In particular, fetal tissues such as the umbilical cord matrix and placenta are excellent sources of human MSCs due to their unique advantages, including painless collection procedures, rapid self-renewal, and the ability to differentiate into three germ layers. Therefore, human umbilical cord matrix-derived MSCs (hUCM-MSCs) and their differentiated cells have recently been utilized in therapeutic medicine for tissue regeneration to treat various diseases.

[0005] Meanwhile, the liver, accounting for approximately 3% of human body weight, is an organ that performs crucial functions. Hepatocytes, the core cells of the liver, are responsible for the biosynthesis and breakdown of glycogen, proteins, lipids, nucleic acids, and vitamins, as well as the detoxification of toxic substances. However, most patients with liver disease lack this normal liver function due to liver tissue damage, which reduces the regenerative capacity of hepatocytes. Currently, the number of deaths from liver disease in Korea is reported to be as high as 11,000 per 100,000 people. While histocompatible liver tissue transplantation is currently the most effective treatment for liver disease, it suffers from the significant difficulty of finding donors. As an alternative, with the increasing research on stem cells, significant attention is being focused on cell therapy using stem cells. However, the use of differentiated cells from stem cells for therapeutic purposes remains limited.

[0006] One of the major obstacles to using differentiated cells from stem cells is the efficiency of differentiation protocols and the limited phenotype of mature cells. For example, hepatocyte-like cells differentiated from human mesenchymal stem cells using known protocols exhibit characteristics more similar to fetal hepatocytes than adult cells in terms of transcriptomic profile, liver function, and metabolic activity. Therefore, various differentiation methods are being developed to address these challenges, including approaches utilizing genetic modification, microenvironmental manipulation, and the addition of cytokines and growth factors.

[0007] Meanwhile, transcriptome changes are closely related to cell type differentiation when determining stem cell fate. Therefore, sequencing tools such as RNA sequencing have recently been widely used to analyze the transcriptome of differentiated cells worldwide. However, cell fate is not simply determined by changes in transcriptional regulation. Cell differentiation and lineage commitment are influenced by signaling pathways that involve communication between the nucleus and various biological processes, as well as interactions between cytoplasmic macromolecules and organelles. In particular, metabolic changes accompany stem cell differentiation, which are known to play a crucial role in stem cell fate determination. Mitochondria play a pivotal role in cellular metabolism, determining cell fate and function. Furthermore, lipid droplets (LDs), storage organelles central to lipid and energy homeostasis, have been reported to be related to stem cell fate determination. While the organelle status of stem cells is crucial for determining stem cell fate, it remains unknown whether regulation of organelle status in stem cells influences hepatic differentiation.

[0008] Against this backdrop, the present inventors confirmed that the differentiation of hUCM-MSCs into hepatocytes is affected not only by changes in the transcriptome but also by the state of cell organelles, and when fasudil, which induces differentiation into endoderm in the early stage of differentiation of stem cells into hepatocytes, is used in a culture vessel coated with high-viscosity gelatin, the differentiation efficiency of stem cells into hepatocytes can be improved by suppressing the formation of lipid droplets and activating the function of mitochondria, and when low-viscosity gelatin is added to the medium in the differentiation maturation (late) stage, the differentiation efficiency of hepatocytes into hepatocytes and the function of differentiated hepatocytes can be improved by reducing the accumulation of lipid droplets and activating mitochondria, thereby completing the present invention.

[0009] The present inventors conducted extensive research to develop an efficient stem cell-derived hepatocyte differentiation method, and as a result, they confirmed that when fasudil, which induces differentiation into endoderm in the early stage of differentiation, is used in a culture vessel coated with high-viscosity gelatin, the differentiation efficiency of stem cells into hepatocytes can be improved by suppressing the formation of lipid droplets and activating the function of mitochondria, and when low-viscosity gelatin is added to the medium in the differentiation maturation (late) stage, the accumulation of lipid droplets can be reduced and the function of differentiated hepatocytes can be improved by activating mitochondria, thereby completing the present invention.

[0010] Accordingly, the purpose of the present invention is to provide a method for differentiating stem cells into hepatoblasts, which comprises a step of culturing stem cells in a medium containing a ROCK inhibitor in a culture vessel coated with gelatin to induce differentiation into hepatoblasts.

[0011] Another object of the present invention is to provide a composition for culturing hepatocytes, comprising at least one selected from the group consisting of (a) a first composition comprising a ROCK inhibitor as an active ingredient; and (b) a second composition comprising gelatin as an active ingredient, characterized in that the first composition is added to a culture vessel coated with gelatin.

[0012] Another object of the present invention is to provide a hepatocyte culture kit comprising a hepatocyte culture composition according to the present invention and an instruction manual.

[0013]

[0014] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0015] To achieve the above purpose, the present invention provides a method for differentiation of stem cells derived from hepatocytes, comprising a step of culturing stem cells in a culture medium containing a ROCK inhibitor in a gelatin-coated culture vessel to induce differentiation into hepatoblasts.

[0016] In one embodiment of the present invention, the stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord stroma, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, but are not limited thereto.

[0017] In another embodiment of the present invention, the ROCK inhibitor may be at least one selected from the group consisting of Fasudil, Ripasudil, RKI-1447, Y-27632, GSK429286A, and Y-30141, but is not limited thereto.

[0018] In another embodiment of the present invention, the ROCK inhibitor may be included in the medium at a concentration of, but not limited to, 1 to 50 μM.

[0019] In another embodiment of the present invention, the gelatin may be coated on a culture vessel at a concentration of 0.1 to 10%, but is not limited thereto.

[0020] In another embodiment of the present invention, the step of inducing differentiation into hepatoma cells may be performed for 1 to 10 days, but is not limited thereto.

[0021] In another embodiment of the present invention, the step of inducing differentiation into hepatocytes may include, but is not limited to, the following steps:

[0022] (a-1) a step of culturing stem cells in a medium containing a ROCK inhibitor (in a culture vessel coated with high viscosity gelatin) to induce differentiation into hepatic endoderm; and

[0023] (a-2) A step of culturing the hepatic endoderm in a medium containing growth factors, bone morphogenetic proteins, and wnt signal activators to induce differentiation into hepatocytes;

[0024] However, the above step (a-2) is performed in a culture vessel that is not coated with gelatin.

[0025] In another embodiment of the present invention, the method may further include, but is not limited to, a step of culturing hepatocytes in a medium containing gelatin to induce differentiation into hepatocytes.

[0026] In another embodiment of the present invention, the gelatin may be included in the medium at a concentration of, but not limited to, 0.1 mg / ml to 10 mg / ml.

[0027] In another embodiment of the present invention, the step of inducing differentiation into hepatocytes may include, but is not limited to, the following steps:

[0028] (b-1) a step of culturing the hepatocytes in a medium containing oncostatin M; and

[0029] (b-2) A step of adding gelatin to the medium containing the above oncostatin M and further culturing to induce differentiation into hepatocytes.

[0030] In addition, the present invention provides a composition for culturing hepatocytes, comprising at least one selected from the group consisting of (a) a first composition comprising a ROCK inhibitor as an active ingredient; and (b) a second composition comprising gelatin as an active ingredient.

[0031] The present invention provides a composition for culturing hepatocytes, characterized in that the first composition is added to a culture vessel coated with gelatin.

[0032] In one embodiment of the present invention, the culture may induce differentiation of stem cells into hepatocytes, but is not limited thereto.

[0033] In another embodiment of the present invention, the stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord matrix, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, but are not limited thereto.

[0034] In another embodiment of the present invention, the ROCK inhibitor may be any one selected from the group consisting of, but not limited to, Fasudil, Ripasudil, RKI-1447, Y-27632, GSK429286A, and Y-30141.

[0035] In another embodiment of the present invention, the ROCK inhibitor may be included at a concentration of, but not limited to, 1 to 50 μM.

[0036] In another embodiment of the present invention, the gelatin of the second composition may be included in the medium at a concentration of 0.1 mg / ml to 10 mg / ml, but is not limited thereto.

[0037] In another embodiment of the present invention, the second composition may further comprise, but is not limited to, oncostatin M.

[0038] In another embodiment of the present invention, gelatin may be coated on the culture vessel at a concentration of 0.1 to 10%, but is not limited thereto.

[0039] In another embodiment of the present invention, the first composition and the second composition may be used simultaneously, separately, or sequentially for culturing hepatocytes, but are not limited thereto.

[0040] In addition, the present invention provides a hepatocyte culture kit including a composition for hepatocyte culture according to the present invention and an instruction manual.

[0041] In one embodiment of the present invention, the description may describe a method for differentiating stem cells derived from hepatocytes according to the present invention, but is not limited thereto.

[0042] The method for differentiating stem cells into hepatocytes according to the present invention induces differentiation of stem cells into endoderm with a ROCK inhibitor in the initial stage of differentiation of stem cells into hepatocytes, but suppresses the formation of lipid droplets that occur during this process by culturing cells in a culture dish coated with high-viscosity gelatin, activates mitochondrial function, upregulates the expression of endodermal genes, and downregulates the expression of ectoderm and mesodermal genes, thereby improving the differentiation efficiency of stem cells into hepatocytes. In addition, by adding low-viscosity gelatin to the medium in the differentiation maturation (late) stage, the accumulation of lipid droplets is suppressed, mitochondria are activated, thereby improving the differentiation efficiency of hepatocytes into hepatocytes, and the function of differentiated hepatocytes is improved, thereby improving the differentiation efficiency of stem cells into hepatocytes. Therefore, hepatocytes obtained using this method are expected to be useful as cell therapeutic agents for the treatment of liver diseases, transplants, etc.

[0043] Figure 1a shows the results of confirming the change in the expression of endoderm markers (GATA4, SOX17, FOXA2) after treating hUCM-MSCs with 10 μM fasudil.

[0044] Figure 1b shows the results of confirming the change in the expression of endoderm markers (GATA4, SOX17, FOXA2) after treating hUCM-MSCs with fasudil for 72 hours.

[0045] Figure 1c shows the results of tomographic analysis of hUCM-MSCs after pasudil treatment (red: mitochondria, green: lipid droplets; microscopic scale bar = 200 μm, tomographic scale bar = 10 μm).

[0046] Figure 1d shows the results of measuring the number of lipid droplets in hUCM-MSCs after treatment with Pasudil.

[0047] Figure 1e illustrates a protocol for inducing differentiation of hUCM-MSCs into hepatoblasts.

[0048] Figure 1f shows the results of confirming the morphology of the endoderm after treatment with Pasudil (scale bar = 100 μm).

[0049] Figure 1g shows the results of confirming the expression of hepatocyte-related genes (AFP and HNF4A) on day 0, day 3, or day 7 of pasudil treatment.

[0050] Figure 1h shows the results of confirming mitochondrial function in hUCM-MSCs after treatment with Pasudil.

[0051] Figure 2a shows the results of examining mitochondrial function after culturing hUCM-MSCs on 0.1% or 1% gelatin-coated dishes.

[0052] Figure 2b shows the protocol for differentiation of hUCM-MSCs into hepatoblasts using a gelatin-coated dish and a phosphatase inhibitor.

[0053] Figure 2c shows the results of examining the morphology of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish (scale bar = 100 μm).

[0054] Figure 2d shows the results of confirming the expression of endoderm markers (GATA4, SOX17, FOXA2) in hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish on days 0, 3, and 7.

[0055] Figure 2e shows the results of confirming the expression of hepatocyte-related genes (AFP, HNF4A) in hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish on days 0, 3, and 7 of differentiation.

[0056] Figure 2f shows the results of confirming the expression of ectoderm markers (PAX6, SOX1, OTX2) on the third day of differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish.

[0057] Figure 2g shows the results of confirming the expression of mesodermal markers (MIXL2, CDX2) on the third day of differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish.

[0058] Figure 3a shows the results of tomographic analysis on day 3 of differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish, according to the viscosity of the coated gelatin (red: mitochondria, green: lipid droplets; scale bar = 10 μm).

[0059] Figure 3b shows the results of comparing the number of lipid droplets on the third day of differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish according to the viscosity of the coated gelatin.

[0060] Figures 3c and 3d show the results of comparing the mitochondrial function according to the viscosity of the coated gelatin on the third day of differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish. Figure 3c shows the results of OCR measurement, and Figure 3d shows the results of confirming the changes in mitochondrial respiration.

[0061] Figure 3e shows the results of comparing the number of mycochondria copies on the third day of differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish according to the viscosity of the coated gelatin.

[0062] Figure 4a shows a differentiation protocol from hepatocytes to hepatocytes using 0.1% or 1% gelatin.

[0063] Figure 4b shows the results of checking the morphology of cells on the 5th day after differentiation by adding 0.1% or 1% gelatin to hUCM-MSCs differentiated by treating with Fasudil in a gelatin-coated dish (scale bar = 100 μm).

[0064] Figure 4c shows the results of confirming the expression of mature hepatocyte-related genes (ALB, CYP3A4, CYP1A2, HNF1A, and HNF4A) on day 5 after differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin.

[0065] Figure 4d shows the results of Western blotting to confirm the expression of ALB and CYP3A4 proteins on the 5th day after differentiation of hUCM-MSCs treated with fasudil in a gelatin-coated dish and differentiation by adding 0.1% or 1% gelatin.

[0066] Figure 4e shows the results of Western blotting to confirm the expression of ALB and CYP3A4 proteins on day 5 after differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin, as confirmed by densitometric analysis.

[0067] Figure 4f shows the results of confirming the expression of hepatocyte-specific miRNA (miR-122, miR-192) on the 5th day after differentiation of hUCM-MSCs treated with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin.

[0068] Figure 5a shows the results of measuring the amount of human albumin secreted on the 5th day after differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin (undiff: undifferentiated cells).

[0069] Figure 5b shows the results of measuring CYP3A4 activity on the 5th day after differentiation of hUCM-MSCs treated with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin.

[0070] Figure 5c shows the results of confirming lipid droplets on the 5th day after differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin (blue: nuclei, green: lipid droplets; scale bar = 20 μm).

[0071] Figure 5d shows the results of checking the fluorescence intensity of lipid droplets on the 5th day after differentiation of hUCM-MSCs treated with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin.

[0072] Figure 5e shows the results of checking the number of mitochondrial copies on the fifth day after differentiation of hUCM-MSCs differentiated by treating with fasudil in a gelatin-coated dish and adding 0.1% or 1% gelatin.

[0073] Figure 6 schematically illustrates a method for differentiating stem cell-derived hepatocytes according to the present invention.

[0074] The present inventors confirmed that when fasudil, which induces differentiation into endoderm in the early stage of differentiation of stem cells into hepatocytes, was used in a culture dish coated with high-viscosity (1%) gelatin, the formation of lipid droplets that occur during differentiation into endoderm was suppressed and mitochondrial function was activated, thereby improving the differentiation efficiency of stem cells into hepatocytes. In addition, when low-viscosity (0.1%) gelatin was added to the medium at the differentiation maturation (late) stage, the accumulation of lipid droplets was reduced and mitochondria were activated, thereby improving the differentiation efficiency of hepatocytes into hepatocytes and the function of differentiated hepatocytes.

[0075]

[0076] Hereinafter, the present invention will be described in detail.

[0077]

[0078] The present invention provides a method for differentiation of stem cells into hepatocytes, comprising a step of culturing stem cells in a culture medium containing a ROCK inhibitor in a gelatin-coated culture vessel to induce differentiation into hepatoblasts.

[0079] In the present invention, the stem cell may be a mesenchymal stem cell derived from one or more types of tissue selected from the group consisting of umbilical cord stroma, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, and according to one embodiment of the present invention, the stem cell may be a human umbilical cord stroma mesenchymal stem cell, but is not limited thereto.

[0080] In the present invention, the ROCK inhibitor may be at least one selected from the group consisting of Fasudil, Ripasudil, RKI-1447, Y-27632, GSK429286A, and Y-30141, and according to one embodiment of the present invention, it may be Fasudil, but is not limited thereto.

[0081] The above-mentioned Fasudil can not only increase the viability of stem cells, but is also characterized by being effective in differentiation as a small molecule, unlike general cytokines (protein type) used for differentiation.

[0082] The term "gelatin" used in the present invention refers to a derived protein obtained by hydrolyzing collagen, a natural polymer protein that constitutes animal bones, leather, tendons, cartilage, etc. The general composition of gelatin may be composed of about 84 to 90% protein, 8 to 12% water, and 2 to 4% mineral salts. In the present invention, gelatin may have various gel strengths and viscosities. In the present invention, gelatin may be isolated or synthesized from natural substances, and the type of its raw material is not limited, and may be produced by hydrolyzing collagen isolated from leather, bone shell, etc. of mammals or fish.

[0083] In the present invention, the gelatin is 0.1 to 10%, 0.1 to 9%, 0.1 to 8%, 0.1 to 7%, 0.1 to 6%, 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2%, 0.1 to 1%, 0.2 to 1%, 0.3 to 1%, 0.4 to 1%, 0.5 to 1%, 0.6 to 1%, 0.7 to 1%, 0.8 to 1%, 0.9 to 1%, 0.3 to 8%, 0.3 to 7%, 0.3 to 6%, 0.3 to 5%, 0.3 to 4%, 0.3 to 3%, 0.3 to 2%, 0.3 to It can be coated on a culture vessel at a concentration of 1%, 0.5 to 6%, 0.5 to 5%, 0.5 to 4%, 0.5 to 3%, 0.5 to 2%, 0.5 to 1%, 0.7 to 5%, 0.7 to 4%, 0.7 to 3%, 0.7 to 2%, 0.7 to 1%, 0.9 to 5%, 0.9 to 3%, 0.9 to 1%, 1 to 5%, 1 to 3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, and preferably, it can be coated on a culture vessel at a concentration of 1 to 5% (high viscosity), but is not limited thereto.

[0084] In the present invention, the step of inducing differentiation into hepatocytes may be performed for 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 7 days, 5 to 7 days, 6 to 7 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, but is not limited thereto.

[0085] In the present invention, the step of inducing differentiation into hepatocytes may include or consist of the following steps:

[0086] (a-1) a step of culturing stem cells in a medium containing a ROCK inhibitor (in a culture vessel coated with high viscosity gelatin) to induce differentiation into hepatic endoderm; and

[0087] (a-2) A step of culturing the hepatic endoderm in a medium containing growth factors, bone morphogenetic proteins, and wnt signal activators to induce differentiation into hepatocytes;

[0088] However, the above step (a-2) is performed in a culture vessel that is not coated with gelatin.

[0089] According to one embodiment of the present invention, the medium containing the ROCK inhibitor of step (a-1) is a DMEM / F12 medium supplemented with 1 to 20% fetal bovine serum, 0.1 to 5% non-essential amino acids, 0.1 to 5% antibiotic-antimycotic, and 0.05 to 5 mM L-ascorbic acid, wherein the ROCK inhibitor is present at a concentration of 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 2 to 40 μM, 2 to 30 μM, 2 to 20 μM, 2 to 10 μM, 5 to 30 μM, 5 to 20 μM, 5 to 10 μM, 10 to 30 μM, 10 to 20 μM, 1 μM, 2 μM, 3 μM, It may be included in the medium at a concentration of, but is not limited to, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

[0090] According to one embodiment of the present invention, the step (a-1) may be performed in a culture vessel coated with gelatin for 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 4 days, 2 to 3 days, 3 to 5 days, 3 to 4 days, 1 day, 2 days, or 3 days, but is not limited thereto.

[0091] According to one embodiment of the present invention, the medium containing the growth factor, bone morphogenetic protein, and wnt signal activator of step (a-2) may be IMDM (Iscove's Modified Dulbecco's Medium, Gibco) supplemented with 0.01 to 1% polyvinyl alcohol, 1 to 50 mM nicotinamide, 1 to 50 ng / ml hHGF, 0.1 to 5% ITS, and 0.1 to 5% penicillin / streptomycin, but is not limited thereto.

[0092] In the present invention, the "growth factor" may be at least one selected from the group consisting of basic fibroblast growth factor (bFGF, FGF2), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1), and according to one embodiment of the present invention, the growth factor may be FGF2 (bFGF), but is not limited thereto.

[0093] In the present invention, the "bone morphogenic protein" may include any protein belonging to the BMP family, and may be, for example, at least one selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, and BMP10. According to one embodiment of the present invention, the bone morphogenic protein may be BMP4, but is not limited thereto.

[0094] In the present invention, the "wnt signal activator" is CHIR99021, BIO((2'Z,3'E)-6-Bromoindirubin-3'-oxime), BIO-acetoxime((2'Z,3'E)-6-Bromoindirubin-3'-acetoxime), 3F8(5-Ethyl-7,8-dimethoxy-H-pyrrolo[3,-4-c]isoquinoline1,3(2H)-dione), A070722(1-(7-Methoxyquinoiin-4-yl)-3-[6-(tr-ifluoromethyl)pyridin-2-yl]urea), ARA014418(N-[(4-Methoxyphenyl)methyl]-N'-(5-ni-tro-2-thiazolyl)urea), SB216763(3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286(3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), TC-G24 (N-(3-Chioro-4-methylphenyl)-5-(4-ni-trophenyl)-1,3,4-oxadiazol-2-amine), TCS2002 (2-ethyl-5-[3-[4-(methylsulfinyi)ph-enyl]-5-benzofuranyi]-1,3,4-oxadiazole), and It may be at least one selected from the group consisting of TWS119 (3-[[6-(3-Aminophenyl)-7H-pyrrolo[2,-3-d]pyrimidin-4-yl]oxyphenol ditrifluoroacetate), and according to one embodiment of the present invention, the wnt signal activator may be CHIR99021, but is not limited thereto.

[0095] In the present invention, in a medium containing the growth factor, bone morphogenetic protein, and wnt signal activator, the growth factor is present at a concentration of 1 to 50 ng / ml, 1 to 30 ng / ml, 1 to 10 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 5 to 10 ng / ml, 10 to 20 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml;

[0096] The bone morphogenetic protein is present at a concentration of 1 to 50 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 1 to 10 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 10 to 20 ng / ml, 20 to 30 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, or 20 ng / ml; and

[0097] The wnt signal activator may be included at a concentration of, but is not limited to, 1 to 5 μM, 1 to 4 μM, 1 to 3 μM, 1 to 2 μM, 2 to 3 μM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

[0098] According to one embodiment of the invention, the step (a-2) may be performed in a culture vessel without a gelatin coating for, but is not limited to, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 4 days, 2 to 3 days, 3 to 5 days, 3 to 4 days, 4 to 5 days, 1 day, 2 days, 3 days, or 4 days.

[0099] In the present invention, the method may further include a step of culturing hepatocytes in a medium containing gelatin to induce differentiation into hepatocytes, and the step of inducing differentiation into hepatocytes may include or consist of the following steps:

[0100] (b-1) a step of culturing the hepatocytes in a medium containing oncostatin M; and

[0101] (b-2) A step of adding gelatin to the medium containing the above oncostatin M and further culturing to induce differentiation into hepatocytes.

[0102] The term "oncostatin M" in the present invention is a pleiotropic cytokine belonging to the interleukin-6 (IL-6) family, and is known to be quite similar to leukemia inhibitory factor (LIF) in terms of structure and function. It is known to be an important factor in liver development, hematopoiesis, infection, and central nervous system development, and there are also reports that it is associated with bone formation and destruction.

[0103] According to one embodiment of the present invention, the medium containing oncostatin M of the step (b-1) is IMDM supplemented with 0.1 to 5 μM dexamethasone, 0.1 to 5% ITS, 1 to 50 ng / ml hHGF, and 0.1 to 5% penicillin / streptomycin, wherein the oncostatin M may be included in the medium at a concentration of 1 to 50 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 1 to 10 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 10 to 20 ng / ml, 20 to 30 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, or 20 ng / ml, It is not limited to this.

[0104] According to one embodiment of the present invention, the step (b-1) may be performed in a culture vessel without gelatin coating for 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 9 days, 7 to 8 days, 8 to 9 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, but is not limited thereto.

[0105] In the present invention, the gelatin of the step (b-2) is 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 9 mg / ml, 0.1 mg / ml to 8 mg / ml, 0.1 mg / ml to 7 mg / ml, 0.1 mg / ml to 6 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 4 mg / ml, 0.1 mg / ml to 3 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.1 mg / ml to 1 mg / ml, 0.5 mg / ml to 8 mg / ml, 0.5 mg / ml to 6 mg / ml, 0.5 mg / ml to 5 mg / ml, 0.5 mg / ml to 3 mg / ml, 0.5 mg / ml to 1 mg / ml, 1 mg / ml to 8 mg / ml, 1 It can be added (included) to the medium at a concentration of 1 mg / ml to 7 mg / ml, 1 mg / ml to 6 mg / ml, 1 mg / ml to 5 mg / ml, 1 mg / ml to 4 mg / ml, 1 mg / ml to 3 mg / ml, 1 mg / ml to 2 mg / ml, 1 mg / ml to 1.5 mg / ml, or 0.5 mg / ml to 1.5 mg / ml, and preferably, it can be added (included) to the medium at a concentration of 1 mg / ml to 5 mg / ml, but is not limited thereto, and any low viscosity gelatin can be included.

[0106] In the present invention, the gelatin of the step (b-2) is 0.01 to 1%, 0.01 to 0.9%, 0.01 to 0.8%, 0.01 to 0.7%, 0.01 to 0.6%, 0.01 to 0.5%, 0.01 to 0.4%, 0.01 to 0.3%, 0.01 to 0.2%, 0.01 to 0.1%, 0.01 to 0.05%, 0.05 to 0.8%, 0.05 to 0.6%, 0.05 to 0.5%, 0.05 to 0.3%, 0.05 to 0.1%, 0.1 to 0.8%, 0.1 to 0.7%, 0.1 to 0.6%, It can be added (included) to the medium at a concentration of 0.1 to 0.5%, 0.1 to 0.4%, 0.1 to 0.3%, 0.1 to 0.2%, or 0.05 to 0.2%, and preferably can be added (included) to the medium at a concentration of 0.1 to 0.5%, but is not limited thereto, and any low viscosity gelatin can be included.

[0107] According to one embodiment of the present invention, the step (b-2) may be performed in a culture vessel without gelatin coating for 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 5 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 1 day, 2 days, 3 days, 4 days, or 5 days, but is not limited thereto.

[0108] In addition, in the present invention, the method for differentiating stem cells from hepatocytes may include steps generally applicable to those skilled in the art of the present invention, such as a step of pretreating stem cells to make them suitable for culturing, a step of obtaining differentiated hepatocytes, etc.

[0109]

[0110] In addition, the present invention provides a method for differentiating stem cell-derived hepatocytes, comprising the following steps:

[0111] (a) a step of culturing stem cells (in a culture vessel coated with high viscosity gelatin) in a medium containing a ROCK inhibitor to induce differentiation into hepatic endoderm;

[0112] (b) a step of culturing the hepatic endoderm in a medium containing growth factors, bone morphogenetic proteins, and wnt signal activators to induce differentiation into hepatocytes;

[0113] (c) a step of culturing the hepatocytes in a medium containing oncostatin M; and

[0114] (d) A step of adding gelatin to the medium containing the above oncostatin M and further culturing to induce differentiation into hepatocytes.

[0115]

[0116] In addition, the present invention provides a composition for culturing hepatocytes, comprising at least one selected from the group consisting of (a) a first composition comprising a ROCK inhibitor as an active ingredient; and (b) a second composition comprising gelatin as an active ingredient.

[0117] The present invention provides a composition for culturing hepatocytes, characterized in that the first composition is added to a culture vessel coated with gelatin.

[0118] In the present invention, the gelatin in the culture vessel is 0.1 to 10%, 0.1 to 9%, 0.1 to 8%, 0.1 to 7%, 0.1 to 6%, 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2%, 0.1 to 1%, 0.2 to 1%, 0.3 to 1%, 0.4 to 1%, 0.5 to 1%, 0.6 to 1%, 0.7 to 1%, 0.8 to 1%, 0.9 to 1%, 0.3 to 8%, 0.3 to 7%, 0.3 to 6%, 0.3 to 5%, 0.3 to 4%, 0.3 to 3%, 0.3 to 2%, 0.3 to It can be coated at a concentration of, but is not limited to, 1%, 0.5 to 6%, 0.5 to 5%, 0.5 to 4%, 0.5 to 3%, 0.5 to 2%, 0.5 to 1%, 0.7 to 5%, 0.7 to 4%, 0.7 to 3%, 0.7 to 2%, 0.7 to 1%, 0.9 to 5%, 0.9 to 3%, 0.9 to 1%, 1 to 5%, 1 to 3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

[0119] In the present invention, the term "culture" means the proliferation, growth, maintenance and differentiation of cells isolated from a living body, a two-dimensional or three-dimensional aggregate thereof, a tissue or a part of a tissue in vitro. Accordingly, the term "culture" encompasses the entire process of obtaining a target substance under an artificial environment using a starting material (cell, tissue or tissue analog), and "culture composition" includes all of a "proliferation composition", a "growth composition", a "maintenance composition" and a "differentiation-inducing composition". Specifically, in the hepatocyte culture composition, "culture" means inducing differentiation of stem cells into hepatocytes to obtain hepatocytes.

[0120] In the present invention, the culture composition may additionally include a basal media composition for stem cell culture. Various media used in the art for stem cell culture may be used as the basal media, and examples thereof include, but are not limited to, IMDM (Iscove's Modified Dulbecco's Medium), α-MEM (Alpha Modification of Eagle's Medium), F12 (Nutrient Mixture F-12), and DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F12). Specifically, the basal media for stem cell culture used in the present invention may be DMEM / F12 and IMDM media, but are not limited thereto.

[0121] According to one embodiment of the present invention, the first composition may be a DMEM / F12 medium composition supplemented with 1 to 20% fetal bovine serum including a ROCK inhibitor, 0.1 to 5% nonessential amino acids, 0.1 to 5% antibiotic-antimycotic, and 0.05 to 5 mM L-ascorbic acid, and the second composition may be, but is not limited to, IMDM supplemented with 0.1 to 5 μM dexamethasone including gelatin, 0.1 to 5% ITS, 1 to 50 ng / ml hHGF, and 0.1 to 5% penicillin / streptomycin, and the second composition may further include oncostatin M.

[0122] In the present invention, adding the first composition to a culture vessel coated with gelatin means adding the first composition as a medium composition to the culture vessel coated with gelatin, and culture may be performed in a culture vessel coated with gelatin using the first composition as a medium.

[0123] In the present invention, the ROCK inhibitor in the first composition may be included at a concentration of, but is not limited to, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 2 to 40 μM, 2 to 30 μM, 2 to 20 μM, 2 to 10 μM, 5 to 30 μM, 5 to 20 μM, 5 to 10 μM, 10 to 30 μM, 10 to 20 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

[0124] In the present invention, the gelatin of the second composition is 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 9 mg / ml, 0.1 mg / ml to 8 mg / ml, 0.1 mg / ml to 7 mg / ml, 0.1 mg / ml to 6 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 4 mg / ml, 0.1 mg / ml to 3 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.1 mg / ml to 1 mg / ml, 0.5 mg / ml to 8 mg / ml, 0.5 mg / ml to 6 mg / ml, 0.5 mg / ml to 5 mg / ml, 0.5 mg / ml to 3 mg / ml, 0.5 mg / ml to 1 mg / ml, 1 mg / ml to 8 mg / ml, 1 mg / ml to It may be included at a concentration of 7 mg / ml, 1 mg / ml to 6 mg / ml, 1 mg / ml to 5 mg / ml, 1 mg / ml to 4 mg / ml, 1 mg / ml to 3 mg / ml, 1 mg / ml to 2 mg / ml, 1 mg / ml to 1.5 mg / ml, or 0.5 mg / ml to 1.5 mg / ml, and preferably, but not limited to, 1 mg / ml to 5 mg / ml.

[0125] In the present invention, the gelatin of the second composition is 0.01 to 1%, 0.01 to 0.9%, 0.01 to 0.8%, 0.01 to 0.7%, 0.01 to 0.6%, 0.01 to 0.5%, 0.01 to 0.4%, 0.01 to 0.3%, 0.01 to 0.2%, 0.01 to 0.1%, 0.01 to 0.05%, 0.05 to 0.8%, 0.05 to 0.6%, 0.05 to 0.5%, 0.05 to 0.3%, 0.05 to 0.1%, 0.1 to 0.8%, 0.1 to 0.7%, 0.1 to 0.6%, 0.1 to It may be included in a concentration of 0.5%, 0.1 to 0.4%, 0.1 to 0.3%, 0.1 to 0.2%, or 0.05 to 0.2%, preferably, but not limited to, 0.1 to 0.5%.

[0126] In the present invention, the first composition and the second composition can be used simultaneously, separately, or sequentially for culturing hepatocytes, and according to one embodiment of the present invention, in the process of differentiating stem cells into hepatocytes, the first composition can be used in the step of inducing differentiation from stem cells into hepatocytes, preferably the step of inducing differentiation from stem cells into hepatic endoderm, and the second composition can be used in the step of inducing differentiation from hepatocytes into hepatocytes, but is not limited thereto.

[0127]

[0128] In addition, the present invention provides a hepatocyte culture kit including a composition for hepatocyte culture according to the present invention and an instruction manual.

[0129] In the present invention, the above description may describe a method for differentiating stem cells derived from hepatocytes according to the present invention, but is not limited thereto.

[0130] In the present invention, a "kit" refers to a tool that enables differentiation of stem cells into hepatocytes using the method according to the present invention. In addition to the above-mentioned materials, the kit of the present invention may include other components, compositions, solutions, devices, etc. typically required for storage and processing methods thereof. Specifically, each component may be applied at least once without limitation, there is no restriction on the order in which each material is applied, and the application of each material may be performed simultaneously or in microseconds.

[0131] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0132]

[0133] In the present invention, when the term "comprising" is used, unless otherwise specifically stated, it does not exclude other components, but rather means that other components may be included. As used throughout the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0134] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0135]

[0136] [Experimental Method]

[0137] 1. Cell culture

[0138] Human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs) were obtained from the Asan Stem Cell Center (Asan Institute for Life Sciences, Seoul). Stem cells were cultured on 0.1% gelatin-coated cell culture dishes in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS; GenDEPOT, USA), 1% non-essential amino acids (NEAA), 1% antibiotic-antimycotic (Gibco, USA), and 0.2 mM L-ascorbic acid. Cells were manually subcultured every 3–4 days at a 1:3–1:5 dilution.

[0139]

[0140] 2. Quantitative RT-PCR

[0141] Total RNA was extracted using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using the Ultrascript 2.0 cDNA Synthesis Kit (PCR Biosystems, UK), and qRT-PCR was performed using HOT FIREPol EvaGreen qPCR Supermix (SOLIS BIODYNE, Tartu, Estonia) on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA). For analysis, mRNA levels were normalized to GAPDH. Primer sequences are listed in Table 1.

[0142]

[0143]

[0144] RT-qPCR was performed to evaluate the expression of hepatic mature miRNAs (miR-122 and miR-192) in undifferentiated and differentiated cells. Specifically, cDNA was synthesized from total RNA using the miRCURY LNA RT Kit (Qiagen, Germany) according to the manufacturer's instructions. RT-qPCR analysis was performed using the miRCURY LNA SYBR Green PCR Kit (Qiagen, Germany) with miRNA-specific primers purchased from Qiagen. The cycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 s, and annealing and extension at 56°C for 1 min. The cycle threshold was determined using Bio-Rad CFX Maestro software (CFX Maestro, version 1.1; Bio-Rad Laboratories). All experiments were repeated three times, and RNU6B was used as an internal control.

[0145]

[0146] 3. Predicting mitochondrial DNA copy number

[0147] Mitochondrial DNA (mtDNA) copy number was measured using the Absolute Human Mitochondrial DNA Copy Number Quantification qPCR assay kit (ScienCell, CA, USA). Before assessing mtDNA copy number, total DNA was isolated using the QIAamp DNA 117 Mini Kit (Qiagen). Briefly, cycle thresholds for each sample were measured in triplicate using nuclear-specific and mitochondrial-specific probes. The assay was performed according to the manufacturer's instructions.

[0148]

[0149] 4. Detection of secreted human albumin

[0150] Human albumin secreted from differentiated cells was detected using a Human Albumin ELISA kit (Bethyl Laboratories, TX, USA) according to the manufacturer's instructions. Albumin secretion was normalized to culture days and total cell number.

[0151]

[0152] 5. Measurement of CYP3A4 activity in vitro

[0153] Enzyme activity was measured using the P450-Glo CYP3A4 kit (Promega, USA) according to the manufacturer's instructions. Luminescence was measured using a GloMax 96 Microplate Luminometer (Promega). CYP3A4 activity was normalized to the culture date and double-stranded DNA content of each sample.

[0154]

[0155] 6. In vitro liver differentiation

[0156] Hepatic differentiation was performed by modifying some of the previously reported methods (Cells. 2020;9(6), Stem Cell Research & Therapy. 2021;12(1):569). Briefly, stem cells were cultured in cell culture medium at a density of 7000 cells / cm 2 Stem cells were seeded on 0.1% or 1% gelatin-coated dishes. After culturing for 1 day to allow the stem cells to attach to the coated dishes, the cells were pretreated with stem cell culture medium (DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS; GenDEPOT, USA), 1% non-essential amino acids (NEAA), 1% antibiotic-antimycotic (Gibco, USA), and 0.2 mM L-ascorbic acid) and 10 μM fasudil (AdooQ Bioscience, CA, USA) for 3 days. Next, the cells were cultured for 4 days in hepatoblast induction medium consisting of stage 1 basal medium, 10 ng / ml FGF2, 20 ng / ml BMP4, and 3 μM CHIR99021. Finally, differentiated cells were cultured in liver maturation medium consisting of stage 2 basal medium and 20 ng / ml oncostatin M (OSM) for 8 days. After 8 days, the medium was replaced with liver maturation medium consisting of stage 2 basal medium, 20 ng / ml OSM, and 0.1% gelatin (1 mg / ml) or 1% gelatin (10 mg / ml), and cultured for 5 days. The differentiation medium was changed every 2 days.

[0157] The composition of the first-stage basal medium was as follows: IMDM (Iscove's Modified Dulbecco's Medium, Gibco) supplemented with 0.1% polyvinyl alcohol (PVA; Sigma Aldrich), 10 mM nicotinamide (Sigma Aldrich), 20 ng / ml hHGF (human hepatocyte growth factor; Peprotech, USA), 1% ITS (insulin-transferrin-selenium; Gibco), and 1% penicillin / streptomycin (GeneDirx, Taiwan) was used.

[0158] The composition of the 2-step basal medium is as follows: IMDM supplemented with 1 μM dexamethasone, 1% ITS, 20 ng / ml hHGF, and 1% penicillin / streptomycin.

[0159] All growth factors were purchased from Peprotech.

[0160]

[0161] 7. Protein extraction and Western blotting

[0162] For Western blotting, cells were trypsinized, washed with ice-cold PBS, and lysed in RIPA lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 1% Triton X-100) containing protease and phosphatase inhibitor cocktail (Sigma Aldrich). After lysis, the cells were centrifuged at 13,000 rpm for 20 min to remove cell debris. Then, the protein concentration was measured using the Bradford assay. Total cellular protein (15 μg) was separated by 8–15% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to Immobilon PVDF membranes (Immobilon polyvinylidene fluoride membranes; Millipore, MA, USA). The membrane was blocked with 8% bovine serum albumin (BSA; GenDEPOT) in TBST (Tris-buffered saline with Tween 20; 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) and probed with anti-albumin (Abcam, UK) and anti-CYP3A4 (Santa Cruz, USA) primary antibodies. After washing with TBST, the primary antibodies were detected using horseradish peroxidase-conjugated anti-mouse secondary antibodies and an enhanced chemiluminescence detection system (Amersham, UK).

[0163]

[0164] 8. Analysis of organelles in differentiated cells

[0165] Label-free optical diffraction tomography (ODT) using refractive index (RI) tomography was performed on hUCM-MSCs using an ODT microscope (HT-X1; Tomocube Inc., Korea). ODT reconstructed a single hUCM-MSC from 48 overlapping two-dimensional holograms captured at different angles illuminated by a 450 nm light-emitting diode (LED) in a controlled atmosphere of 5% CO2 at 37°C using three-dimensional RI tomography. An HT-X1 microscope with an integrated Mach-Zehnder interferometer was used for three-dimensional RI tomographic reconstruction of cells. LD quantification and volumetric analysis were performed using TomoAnalysis software from TomoCube, and fluorescent staining was used to ensure precision. 250 nM MitoTracker dye (Invitrogen, CA, USA) for mitochondrial labeling and Biotium LipidSpot 488 lipid droplet stain (1:1000 dilution) were used to stain mitochondria and LDs, respectively. Live-cell staining was performed according to the manufacturer's instructions.

[0166] Additionally, cells were observed using a confocal laser scanning microscope, Zeiss LSM 880 (Carl Zeiss, Germany). Specifically, cells were fixed overnight with 4% formaldehyde, washed with PBST (PBS containing Tween 20), permeabilized with 0.5% Triton X-100, and blocked with PBST containing 1% BSA. The samples were then stained with Biotium LipidSpot 488 lipid droplet stain. Nuclei were counterstained with NucBlue fixed cell ready probe reagent (DAPI; Invitrogen) for 10 min, and the fluorescence signals were detected using a Zeiss LSM 880 confocal laser scanning microscope.

[0167]

[0168] 9. Seahorse assay: An experiment to quantify the rate of ATP production.

[0169] To measure the oxygen consumption rate (OCR) in differentiated cells, stem cells were seeded at 7000 cells / cm on 0.1% or 1% gelatin-coated XFe24 cell culture plates (Agilent Technologies, Santa Clara, CA, USA). 2 After seeding, differentiation was induced. Mitochondrial OCR was measured using the XF Cell MitoStress test kit on an XF24 extracellular flux analyzer (Agilent Technologies). OCR values ​​were normalized to the amount of cellular DNA.

[0170]

[0171] 10. Statistical Analysis

[0172] Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, USA). Comparisons of three or more data sets were performed using one-way or two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. Comparisons between two groups were performed using a two-tailed Student's t-test. A P value less than 0.05 was considered statistically significant.

[0173]

[0174] [Example]

[0175] Example 1. Organelle status of differentiated cells affecting the early stage of liver differentiation.

[0176] In a previous study, we investigated the effect of fasudil, a ROCK inhibitor, on the differentiation of hUCM-MSCs into hepatic endoderm, a critical process in the early stages of cell differentiation. The results showed that the ROCK inhibitor not only improved stem cell viability but also promoted the induction of human pluripotent stem cells (hPSCs) into endoderm. In addition, the differentiation efficiency was found to increase when a small molecule (fasudil) was used compared to when only general proteins used for differentiation (e.g., cytokines) were used (Cell Stem Cell. 2009;4(4):348-58). Therefore, to determine the effect of fasudil on the hepatic differentiation of hUCM-MSCs, we first determined the optimal concentration and treatment time of fasudil. As a result, as shown in Fig. 1a and Fig. 1b, unlike the previously reported concentration of fasudil used in hPSCs (Molecular metabolism. 2017;6(7):640-50), endoderm markers (GATA4, SOX17, FOXA2) were significantly increased at a high concentration of 10 μM, and it was effective when treated for 72 hours (P < 0.05). In addition, tomographic analysis was performed to confirm the status of organelles such as LDs (lipid droplets) and mitochondria in differentiated cells, which are known to be important for metabolism and regulation of stem cell fate. As a result, as shown in Fig. 1c, the stem cells changed to a slightly more ovoid shape and the mitochondrial morphology changed. In addition, as shown in Fig. 1c and Fig. 1d, LDs were induced inside the cells, and the number of LDs increased (P < 0.001).

[0177] Next, as shown in Figure 1e, we examined whether the use of fasudil increased the differentiation efficiency of hepatoblasts by evaluating the next stage of hepatic endoderm. As a result, as shown in Figure 1f, the phenotypes of differentiated cells were similar in both the control and fasudil-treated groups. However, as shown in Figure 1g, the expression of hepatocyte-related genes (AFP and HNF4A) was suppressed in the fasudil-treated group (P<0.001). To determine why hepatocyte differentiation efficiency was reduced despite the upregulation of endoderm genes, we performed a mitochondrial function test. Mitochondrial functions, such as adenosine triphosphate (ATP) production, are related to differentiation efficiency and are reduced when excessive LDs are induced. Therefore, we hypothesized that fasudil-induced LDs would affect mitochondrial function, and to confirm this, we examined mitochondrial function in fasudil-treated cells. As expected, as shown in Fig. 1h, mitochondrial function was reduced in cells treated with fasudil on the third day of differentiation (P <0.001).

[0178] From the above results, it was found that fasudil transcriptionally affected the early stage of hepatic differentiation of hUCM-MSCs, but did not affect the subsequent stage of endoderm due to the state of the cell organelles.

[0179]

[0180] Example 2. Effect of gelatin viscosity on hepatoblast induction of hUCM-MSCs.

[0181] Based on the results of Fasudil treatment, we hypothesized that mitochondrial function was correlated with differentiation efficiency. Therefore, the next step was to enhance mitochondrial function through extracellular matrix (ECM) components. Furthermore, as confirmed in the results of this experiment, when hUCM-MSCs were cultured on 0.1% or 1% gelatin-coated dishes, mitochondrial activation levels, such as basal oxidative phosphorylation, maximal oxidative phosphorylation, ATP production, and proton leak, increased (P <0.001), as shown in Figure 2a. Previous studies have shown that increased oxidative phosphorylation levels and ATP production in differentiated cells are required for specific lineage differentiation (Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells. 2006;348(4):1472-8, Glycolytic network restructuring integral to the energetics of embryonic stem cell cardiac differentiation. 2010;48(4):725-34). Therefore, it was expected that gelatin, used as an ECM, could synergistically improve differentiation efficiency together with fasudil.

[0182] To evaluate the effect of gelatin on hepatic differentiation, hUCM-MSCs were cultured on 0.1% or 1% gelatin-coated dishes, as shown in Fig. 2b, and differentiation was performed under these conditions. As shown in Fig. 2c, the phenotypes of differentiated cells were similar in all groups on day 7. Next, the dynamics of gene expression related to hepatic endoderm and hepatoblasts were analyzed on days 0, 3, and 7 of differentiation. As a result, as shown in Fig. 2d and 2e, endoderm-related genes (SOX17, FOXA2) and hepatoblast-related genes (AFP, HNF4A), except for GATA4, were significantly upregulated in the 1% gelatin and fasudil-treated groups compared to the other groups. However, as shown in Fig. 2e, interestingly, as hepatoblast differentiation progressed, hepatoblast markers (AFP and HNF4A) decreased in the 0.1% gelatin and fasudil group, unlike the 1% gelatin and fasudil group (#a and #b, P <0.01).

[0183] To confirm the differences in differentiation efficiency observed between the low-viscosity gelatin groups and the high-viscosity gelatin groups, the initial differentiation fate of the stem cells was examined. In particular, fasudil is known as a promoter for differentiation of cells derived from the ectoderm and mesoderm, including neurons and cardiomyocytes. Therefore, the gene expression of ectoderm (PAX6, SOX1, OTX2) and mesoderm (MIXL2, CDX2) markers was examined in differentiated cells on day 3 of differentiation. As shown in Figure 2f, the expression of ectoderm genes (PAX6 and OTX2) was suppressed in the 1% gelatin and fasudil groups (P<0.05). In addition, as shown in Figure 2g, the mesodermal gene MIXL2 was downregulated in the 1% gelatin and fasudil groups (P<0.01).

[0184] From the above results, it was found that high viscosity gelatin induces endoderm fate by suppressing the expression of ectoderm- and mesoderm-related genes.

[0185] Considering that the fate of stem cells depending on gelatin viscosity may be affected by the state of cell organelles, we next examined the effect of high-viscosity gelatin on the cell organelles of differentiated cells.

[0186]

[0187] Example 3. High viscosity gelatin inhibited the induction of LD and improved mitochondrial function.

[0188]

[0189] To analyze the effects of high-viscosity gelatin on stem cell fate through regulation of organelles, we performed tomography and mitochondrial function analyses. Tomography is known to be able to observe organelles more accurately than confocal laser scanning microscopy. Therefore, we analyzed the LDs and morphology of mitochondria in differentiated cells on day 3 using tomography. As shown in Figures 3a and 3b, the group treated with 1% gelatin and fasudil showed lower LD induction than the group treated with 0.1% gelatin and fasudil (P <0.001), suggesting that high-viscosity gelatin inhibits fasudil-induced LD formation. Furthermore, as shown in Figure 3a, the 0.1% gelatin and fasudil group showed hyperfusion-induced mitochondrial morphology.

[0190] Next, hippocampal analysis was performed to determine how excessive LD induction and hyperfused mitochondria affect cellular mitochondrial function. As shown in Figures 3c and 3d, the 1% gelatin and fasudil groups exhibited the highest overall OCR values ​​(P <0.05). Furthermore, mtDNA copy numbers were measured in all groups. Previous studies have shown that mtDNA levels gradually increase to support differentiation. Therefore, it was expected that more efficient differentiation would lead to higher mtDNA copy numbers. As shown in Figure 3e, in this experiment, the 1% gelatin and fasudil groups exhibited higher mtDNA copy numbers (P <0.001).

[0191] In summary, the improved differentiation efficiency in the 1% gelatin and fasudil groups may be attributed to the downregulation of ectodermal and mesodermal gene expression as well as the modulation of organelle status, including reduced LD production and activated mitochondrial function.

[0192]

[0193] Example 4. Low viscosity gelatin that synergistically improves the efficiency and function of hepatocyte-like cells (HLCs).

[0194] Finally, to determine the effect of gelatin on hepatocyte maturation, hepatoblasts induced using 1% gelatin coating and fasudil were differentiated into HLCs, as shown in Figure 4a. At this stage, the plates were not coated, and gelatin was added to the stage 2 differentiation medium. Specifically, HLCs were induced from hepatoblasts using OSM for 8 days, and maturation was performed for 5 days by adding gelatin at various viscosities of 0% (no gelatin added), 0.1%, or 1%. As shown in Figure 4b, on day 20 of differentiation, the HLC phenotypes were similar in all groups. However, transcriptional analysis results showed that the expression of mature hepatocyte-related genes (ALB, CYP3A4, CYP1A2, HNF1A, and HNF4A) was significantly increased in the low-viscosity (0.1%) gelatin group compared to the other groups (P <0.01), as shown in Figure 4c. Furthermore, as shown in Figures 4d and 4e, the expression of these proteins (ALB (albumin) and CYP3A4) was confirmed through Western blotting. The ALB level in the 0.1% gelatin group was similar to that in the 0% gelatin group, but the expression of CYP3A4 was significantly higher (P <0.01). In addition, the maturation efficiency was evaluated through the expression of hepatocyte-specific miRNAs such as miR-122 and miR-192. As a result, as shown in Figure 4f, hepatocyte-specific miRNAs were significantly upregulated in the presence of 0.1% gelatin.

[0195] Next, considering the key role of hepatocytes in protein synthesis and detoxification, albumin secretion and CYP3A4 activity were measured. Consistent with previous studies, as shown in Figures 5a and 5b, liver function was significantly improved in HLCs supplemented with 0.1% gelatin (P <0.001). Overall, hepatocyte specification and maturation appeared to be more efficient in low-viscosity gelatin, in contrast to hepatoblast differentiation. Therefore, to understand this, we analyzed the status of differentiated organelles, focusing on LDs and mtDNA copy numbers in HLCs on day 20. Confocal image analysis results confirmed that LDs were induced less when 0.1% gelatin was used for hepatocyte maturation (P <0.05), and a significant increase in mtDNA copy number was confirmed in the same group (P <0.001), as shown in Figures 5c and 5d.

[0196] The above results imply that low viscosity gelatin is beneficial for the maturation of HLCs, unlike its role in the early differentiation stage, and emphasize that organelle status plays a pivotal role in determining the efficiency and function of hepatocytes.

[0197]

[0198] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0199] The method for differentiating stem cells into hepatocytes according to the present invention induces differentiation of stem cells into endoderm with a ROCK inhibitor in the initial stage of differentiation of stem cells into hepatocytes, but suppresses the formation of lipid droplets that occur during this process by culturing cells in a culture dish coated with high-viscosity gelatin, activates mitochondrial function, upregulates the expression of endodermal genes, and downregulates the expression of ectoderm and mesodermal genes, thereby improving the differentiation efficiency of stem cells into hepatocytes. In addition, by adding low-viscosity gelatin to the medium in the differentiation maturation (late) stage, the accumulation of lipid droplets is suppressed, mitochondria are activated, thereby improving the differentiation efficiency of hepatocytes into hepatocytes, and the function of differentiated hepatocytes is improved, thereby improving the differentiation efficiency of stem cells into hepatocytes. Therefore, hepatocytes obtained using this method are expected to be useful as cell therapeutic agents for treating liver diseases, transplants, etc., and thus have industrial applicability.

Claims

1. A method for differentiating stem cells derived from hepatocytes, comprising a step of culturing stem cells in a culture medium containing a ROCK inhibitor in a gelatin-coated culture vessel to induce differentiation into hepatoblasts.

2. In paragraph 1, A method for differentiating stem cells derived from hepatocytes, characterized in that the stem cells are mesenchymal stem cells derived from at least one type of tissue selected from the group consisting of umbilical cord stroma, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

3. In paragraph 1, A method for differentiating stem cell-derived hepatocytes, characterized in that the ROCK inhibitor is at least one selected from the group consisting of Fasudil, Ripasudil, RKI-1447, Y-27632, GSK429286A, and Y-30141.

4. In paragraph 1, A method for differentiating stem cell-derived hepatocytes, characterized in that the ROCK inhibitor is included in the medium at a concentration of 1 to 50 μM.

5. In paragraph 1, A method for differentiating stem cell-derived hepatocytes, characterized in that the gelatin is coated on a culture vessel at a concentration of 0.1 to 10%.

6. In paragraph 1, A method for differentiating stem cells into hepatocytes, characterized in that the step of inducing differentiation into hepatocytes is performed for 1 to 10 days.

7. In paragraph 1, A method for differentiating stem cells into hepatocytes, characterized in that the step of inducing differentiation into hepatocytes comprises the following steps: (a-1) a step of culturing stem cells in a medium containing a ROCK inhibitor to induce differentiation into hepatic endoderm; and (a-2) A step of culturing the hepatic endoderm in a medium containing growth factors, bone morphogenetic proteins, and wnt signal activators to induce differentiation into hepatocytes; However, the above step (a-2) is performed in a culture vessel that is not coated with gelatin.

8. In paragraph 1, A method for differentiating hepatocytes derived from stem cells, characterized in that the method further comprises a step of culturing hepatocytes in a medium containing gelatin to induce differentiation into hepatocytes.

9. In paragraph 8, A method for differentiating stem cell-derived hepatocytes, characterized in that the gelatin is included in the medium at a concentration of 0.1 mg / ml to 10 mg / ml.

10. In paragraph 8, A method for differentiating stem cells from hepatocytes, characterized in that the step of inducing differentiation into hepatocytes comprises the following steps: (b-1) a step of culturing the hepatocytes in a medium containing oncostatin M; and (b-2) A step of adding gelatin to the medium containing the above oncostatin M and further culturing to induce differentiation into hepatocytes.

11. A composition for culturing hepatocytes, comprising at least one selected from the group consisting of (a) a first composition comprising a ROCK inhibitor as an active ingredient; and (b) a second composition comprising gelatin as an active ingredient. A composition for culturing hepatocytes, characterized in that the first composition is added to a culture vessel coated with gelatin.

12. In paragraph 11, A composition for culturing hepatocytes, characterized in that the above culturing induces differentiation of stem cells into hepatocytes.

13. In paragraph 12, A composition for culturing hepatocytes, characterized in that the stem cells are mesenchymal stem cells derived from at least one type of tissue selected from the group consisting of umbilical cord stroma, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

14. In paragraph 11, A composition for culturing hepatocytes, characterized in that the ROCK inhibitor is at least one selected from the group consisting of Fasudil, Ripasudil, RKI-1447, Y-27632, GSK429286A, and Y-30141.

15. In paragraph 11, A composition for culturing hepatocytes, characterized in that the ROCK inhibitor is included at a concentration of 1 to 50 μM.

16. In paragraph 11, A composition for culturing hepatocytes, characterized in that the gelatin of the second composition is included at a concentration of 0.1 mg / ml to 10 mg / ml.

17. In paragraph 11, A composition for culturing hepatocytes, characterized in that the second composition further comprises oncostatin M.

18. In paragraph 11, A composition for culturing hepatocytes, characterized in that the culture vessel is coated with gelatin at a concentration of 0.1 to 10%.

19. In paragraph 11, A composition for culturing hepatocytes, characterized in that the first composition and the second composition are used simultaneously, separately, or sequentially for culturing hepatocytes.

20. A hepatocyte culture kit comprising a composition for hepatocyte culture according to any one of claims 11 to 19, and an instruction manual.

21. In paragraph 20, The above description is a kit for culturing hepatocytes, which describes a method for differentiating hepatocytes derived from stem cells according to the first clause.

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