Method for inducing hepatic stem cells or hepatic progenitor cells

Abstract

The present invention provides, inter alia: a method for inducing hepatic stem cells or hepatic progenitor cells from non-hepatic stem cells or non-hepatic progenitor cells; a method for producing hepatic stem cells or hepatic progenitor cells; and a method for producing stem cells or bile duct epithelial cells.

Description

Method for inducing hepatic stem cells or hepatic progenitor cells

[0001] The present invention relates to a method for inducing non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells, a method for producing hepatic stem cells or hepatic progenitor cells, and a method for producing hepatocytes or bile duct epithelial cells.

[0002] Techniques for producing hepatocytes by introducing a certain set of genes as reprogramming factors into human fibroblasts are already known. For example, a method for producing induced hepatocytes from non-hepatocytes using reprogramming factors such as CEBPA, HNF4A, FOXA3, GATA4, and HNF1A is known (Patent Document 1).

[0003] Other known methods include reprogramming mouse embryonic fibroblasts into hepatic stem cells by introducing Hnf1β and Foxa3 (Non-Patent Document 1), reprogramming human fibroblasts into mature hepatocytes by expressing FOXA3, HNF1A, and HNF4A (Non-Patent Document 2), and producing induced human hepatocytes by overexpressing HNF1A, HNF4A, and HNF6 together with maturation factors ATF5, PROX1, and CEBPA (Non-Patent Document 3).

[0004] However, the hepatocytes induced in Patent Document 1 and Non-Patent Documents 2 and 3 had poor proliferation ability and could not differentiate into bile duct epithelial cells. Furthermore, Non-Patent Document 1 provides data on mouse cells, not human cells.

[0005] Special Publication No. 2015-527084

[0006] Bing Yu et al., Cell Stem Cell, 13(3):328-40, 2013Pengyu Huang et al., Cell Stem Cell, 14(3): 370-384, 2014Yuanyuan Du et al., Cell Stem Cell, 14(3), 394-403, 2014

[0007] In one embodiment, an objective of the present invention is to provide a method for inducing hepatic stem cells or hepatic progenitor cells or a method for producing hepatic stem cells or hepatic progenitor cells.

[0008] The present inventors have found that non-hepatic stem cells or non-hepatic progenitor cells can be induced to become hepatic stem cells or hepatic progenitor cells by using a specific combination of reprogramming factors.

[0009] (1) A method for inducing non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells, comprising introducing the following combination into non-hepatic stem cells or non-hepatic progenitor cells: a combination comprising an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and a Wnt signaling activator. (2) A method for producing hepatic stem cells or hepatic progenitor cells, comprising introducing the following combination into non-hepatic stem cells or non-hepatic progenitor cells: a combination comprising an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and a Wnt signaling activator. (3) The method according to (1) or (2), wherein the combination consists of an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and a Wnt signaling activator. (4) The method according to any one of (1) to (3), wherein HNF1 is HNF1A. (5) The method according to any one of (1) to (4), wherein FOXA is FOXA3. (6) The method according to any one of (1) to (5), wherein the Wnt signal activator is CHIR99021 or BIO (6-bromoindirubin-3-oxime). (7) The method according to (6), wherein the Wnt signal activator is CHIR99021. (8) The method according to any one of (1) to (7), wherein the non-hepatic stem cells or non-hepatic progenitor cells are vascular endothelial cells or blood-derived cells. (9) The method according to (8), wherein the vascular endothelial cells are derived from umbilical vein, peripheral blood, or umbilical cord blood. (10) The method according to (8), wherein the blood-derived cells are peripheral blood T cells or umbilical cord blood T cells. (11) The method according to any one of (1) to (10), wherein the combination further comprises an HNF6 gene or HNF6 protein. (12) A method for producing hepatocytes or bile duct epithelial cells, comprising: (a) inducing hepatic stem cells or hepatic progenitor cells by the method according to any one of (1) and (3) to (11), and (b) differentiating the induced hepatic stem cells or hepatic progenitor cells into hepatocytes or bile duct epithelial cells. (13) The following combination, or a kit or composition comprising said combination: a combination comprising an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and a Wnt signal activator.(14) The combination according to (13), or a kit or composition comprising the combination, wherein the combination further comprises an HNF6 gene or an HNF6 protein. (15) The combination according to (13) or (14), or a kit or composition comprising the combination, for use in the method according to any one of (1) to (11).

[0010] The present invention provides a method for inducing non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells, a method for producing hepatic stem cells or hepatic progenitor cells, and a method for producing hepatocytes or bile duct epithelial cells.

[0011] Figure 1 shows the results of albumin (ALB) immunostaining (A) and colony counting (B) of colonies formed on days 9 and 16 after transfection of human iHepPC inducers (FOXA3, HNF1A, and HNF6) into HUVECs and addition of the Wnt activator CHIR99021 or BIO to the culture medium. Cell DNA was stained with DAPI (blue). Figure 2 shows the results of albumin (ALB) immunostaining (A) and ALB gene expression analysis (B) of colonies formed on days 9, 16, 23, and 44 after transfection of human iHepPC inducers (FOXA3, HNF1A, and HNF6) into HUVECs and addition of the Wnt inhibitor IWP2 to the culture medium. The group without Wnt signal inhibitors is designated as Control. Cell DNA was stained with DAPI (blue). Figure 3 shows the results of albumin (ALB) and E-cadherin (E-CAD) expression analysis in three groups: Group 1, in which human iHepPC inducing factors (FOXA3, HNF1A, and HNF6) were introduced into HUVECs; Group 2, in which partial human iHepPC inducing factors (FOXA3 and HNF1A) were introduced; and Group 3, in which a Wnt signaling activator (CHIR99021: 3 μM) was added to the culture medium after partial human iHepPC inducing factors (FOXA3 and HNF1A) were introduced. Cell DNA was stained with DAPI (blue).

[0012] In one embodiment, the present invention relates to a method for inducing non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells, comprising introducing into the non-hepatic stem cells or non-hepatic progenitor cells a combination comprising the following: an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and a Wnt signal activator.

[0013] In one embodiment, the present invention relates to a method for producing hepatic stem cells or hepatic progenitor cells, comprising introducing into non-hepatic stem cells or non-hepatic progenitor cells a combination of the following: an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and a Wnt signal activator, thereby inducing the non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells.

[0014] As used herein, "non-hepatic stem cells" and "non-hepatic progenitor cells" refer to cells other than "hepatic stem cells" and "hepatic progenitor cells" induced or produced by the methods described herein. Examples of "non-hepatic stem cells" and "non-hepatic progenitor cells" include fibroblasts, endothelial cells, blood cells, umbilical cord blood cells, bone marrow cells, keratinocytes, hepatocytes, bile duct epithelial cells, myofibroblasts, neural cells, and epithelial cells. Examples of "non-hepatic stem cells" and "non-hepatic progenitor cells" include vascular endothelial cells or blood-derived cells. Vascular endothelial cells include those derived from umbilical vein, peripheral blood, or umbilical cord blood, and blood-derived cells include peripheral blood cells or umbilical cord blood cells (e.g., peripheral blood T cells or umbilical cord blood T cells). Examples of "non-hepatic stem cells" and "non-hepatic progenitor cells" used herein include cells derived from mammals such as mice, rats, rabbits, cats, dogs, monkeys, and humans. In one embodiment, the "non-hepatic stem cells" and "non-hepatic progenitor cells" are cells of human origin.

[0015] As used herein, "non-hepatic stem cells" and "non-hepatic progenitor cells" can be derived not only from fetuses but also from adults. Therefore, the methods described herein enable liver function tests and drug screening using adult-derived cells, such as peripheral blood-derived hepatic stem cells or hepatic progenitor cells.

[0016] As used herein, "hepatic stem cells" or "hepatic progenitor cells" refer to cells that have the ability to differentiate into both hepatocytes and bile duct epithelial cells, as well as the ability to self-proliferate or self-renew. Markers of hepatocytes and hepatic progenitor cells include, for example, albumin and E-cadherin.

[0017] The combinations described herein may be combinations of reprogramming factors. As used herein, "reprogramming" refers to the process of changing the differentiation state of a cell to a differentiated state different from that of the cell or to an undifferentiated state. As used herein, reprogramming may refer to the induction of the above-mentioned "non-hepatic stem cells" and "non-hepatic progenitor cells" into "hepatic stem cells" and "hepatic progenitor cells." The methods described herein enable the induction of "hepatic stem cells" and "hepatic progenitor cells" from "non-hepatic stem cells" and "non-hepatic progenitor cells" without first passing through pluripotent stem cells. Factors (reprogramming factors) used for such reprogramming include combinations containing or consisting of the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and a Wnt signal activator. Specific combinations include any of the following combinations 1) to 4), such as combination 1) or 2). 1) A combination of the HNF1 gene, the FOXA gene, and a Wnt signaling activator. 2) A combination of the HNF1 protein, the FOXA protein, and a Wnt signaling activator. 3) A combination of the HNF1 gene, the FOXA protein, and a Wnt signaling activator. 4) A combination of the HNF1 protein, the FOXA gene, and a Wnt signaling activator.

[0018] Hepatocyte Nuclear Factor 1 (HNF1) is a homeodomain protein with two isoforms, HNF1A and HNF1B.

[0019] FOXA is a hepatocyte nuclear factor (transcription factor) required for the earliest stage of liver tissue formation, and includes FOXA1, FOXA2, and FOXA3. These FOXA transcription factors share over 90% amino acid homology in the common forkhead / winged helix domain, suggesting that they have functional complementarity with each other.

[0020] The combinations described herein may contain other factors as long as they do not inhibit the effects of the methods described herein, such as induction into hepatic stem cells or hepatic progenitor cells or production of hepatic stem cells or hepatic progenitor cells. Such factors include, for example, MYC. MYC family genes are known as transcription factors that function by binding to nuclear DNA, and in humans, they include c-MYC, L-MYC, and N-MYC.

[0021] The amino acid sequences of the above reprogramming factors and the nucleotide sequences of the genes encoding these factors are shown in Table 1.

[0022] Genes encoding these factors or portions thereof can be cloned with reference to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) or the like, or can be obtained from addgene or the like.

[0023] Herein, the gene encoding HNF1A is referred to as the "HNF1A gene," and the gene encoding HNF6 is referred to as the "HNF6 gene." Genes encoding other factors can also be designated in the same manner as above.

[0024] The reprogramming factors (genes or proteins) used herein are not limited to nucleic acid sequences having the nucleotide sequences represented by the SEQ ID NOs in Table 1 or amino acid sequences having the amino acid sequences represented by the SEQ ID NOs in Table 1. The following mutant forms can also be used as long as they have the function of a reprogramming factor in the methods described herein: (a) A protein consisting of an amino acid sequence in which one or several (e.g., 10 or less, 5 or less, 4 or less, 3 or less, or 2) amino acids have been deleted, substituted, or added in the amino acid sequence shown in Table 1 (the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, or 8), and which has the function of a reprogramming factor. (b) A protein consisting of an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more (e.g., 90% or more) sequence identity to the amino acid sequence shown in Table 1 (the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, or 8), and which has the function of a reprogramming factor. (c) A nucleic acid encoding the protein of (a) above. (d) A nucleic acid encoding the protein of (b) above. (e) A nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in Table 1 (amino acid sequence shown in SEQ ID NO: 1, 3, 5, or 7) and encodes a protein that functions as a reprogramming factor.

[0025] As used herein, "stringent conditions" may refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, it is expected that DNA and / or RNA with higher homology can be obtained more efficiently as the temperature is increased. However, several factors are thought to affect the stringency of hybridization, including temperature, DNA and / or RNA concentration, DNA and / or RNA length, ionic strength, time, and salt concentration, and one skilled in the art can achieve similar stringency by appropriately selecting these factors.

[0026] Whether a particular protein functions as a reprogramming factor can be tested, for example, as described in the Examples herein. For example, a combination described herein containing a particular protein can be introduced into non-hepatic stem cells or non-hepatic progenitor cells, followed by culturing for a predetermined period of time, and the function can be examined by determining whether the expression of markers of hepatic stem cells or hepatic progenitor cells, such as albumin, α-fetoprotein, and E-cadherin, is increased in the cells.

[0027] As used herein, the type of Wnt signal activator is not limited, and examples include proteins belonging to the Wnt family (e.g., Wnt1), Wnt receptors, Wnt receptor agonists, and glycogen synthase kinase-3 (GSK-3) inhibitors, such as CHIR99021 (CAS No. 252917-06-9), BIO (CAS No. 6-bromoindirubin-3-oxime) (667463-62-9), IQ-1 (CAS No. 331001-62-8), and BML284 (CAS No. 853220-52-7). The Wnt signal activator may be, for example, CHIR99021 or BIO. The concentration of the Wnt signal activator is not limited, and may be, for example, 0.1 μM or more, 0.2 μM or more, 0.5 μM or more, or 1 μM or more, or 50 μM or less, 30 μM or less, 10 μM or less, or 5 μM or less; for example, the concentration of the Wnt signal activator may be about 1 μM or 3 μM.

[0028] The combinations described herein may be, but are not limited to, the following combinations of (1) to (5), for example, combinations of (2) or (3): (1) a combination of (a) the HNF1A gene or HNF1A protein, (b) the FOXA3 gene or FOXA3 protein, and (c) CHIR99021 or BIO, (2) a combination of (a) the HNF1A gene, (b) the FOXA3 gene, and (c) CHIR99021, (3) a combination of (a) the HNF1A protein, (b) the FOXA3 protein, and (c) CHIR99021, (4) a combination of (a) the HNF1A gene, (b) the FOXA3 gene, and (c) BIO, (5) a combination of (a) the HNF1A protein, (b) the FOXA3 protein, and (c) BIO. The combinations may comprise, consist of, or consist essentially of the above factors.

[0029] The combinations described herein may further include other factors, for example, the HNF6 gene or HNF6 protein. The combinations described herein may be, but are not limited to, the following combinations (1) to (5), for example, the combination of (2) or (3): (1) a combination of (a) the HNF1A gene or HNF1A protein, (b) the FOXA3 gene or FOXA3 protein, (c) CHIR99021 or BIO, and (d) the HNF6 gene or HNF6 protein, (2) a combination of (a) the HNF1A gene, (b) the FOXA3 gene, (c) CHIR99021, and (d) the HNF6 gene, (3) a combination of (a) the HNF1A protein, (b) the FOXA3 protein, (c) CHIR99021, and (d) the HNF6 protein, (4) a combination of (a) the HNF1A gene, (b) the FOXA3 gene, (c) BIO, and (d) the HNF6 gene, (5) A combination of (a) HNF1A protein, (b) FOXA3 protein, (c) BIO, and (d) HNF6 protein, which may comprise, consist of, or consist essentially of the factors.

[0030] Generally, small molecular weight compounds are easier to handle than genes or proteins. Therefore, when the Wnt signaling activator is a small molecular weight compound, reprogramming can be performed more easily by replacing the gene or protein used as the reprogramming factor with the Wnt signaling activator.

[0031] Furthermore, the use of a Wnt signal activator can improve the efficiency of induction into hepatic stem cells or hepatic progenitor cells in the method for inducing hepatic stem cells or hepatic progenitor cells or the method for producing hepatic stem cells or hepatic progenitor cells. Thus, the methods described herein can have high induction efficiency into hepatic stem cells or hepatic progenitor cells.

[0032] The method for introducing the combinations described herein into cells is not limited. For example, if the reprogramming factor is a nucleic acid, the nucleic acid may be incorporated into the same or different vectors, or if the reprogramming factor is a protein, a nucleic acid encoding the protein may be incorporated into the same or different vectors, and then the recombinant vectors may be introduced into cells. The vector may be an expression vector in which the reprogramming factor and / or other DNA sequence is operably linked to one or more expression control sequences. Examples of expression control sequences include promoters, enhancers, and transcription termination regions.

[0033] The genes encoding the reprogramming factors may be contained in a single vector, or each gene may be contained in a separate vector. Alternatively, some genes may be contained in one vector and the remaining genes may be contained in another vector.

[0034] Expression vectors that can be used herein include plasmids, bacteriophages, and viral vectors derived from retroviruses, vaccinia viruses, adenoviruses, lentiviruses, adeno-associated viruses, Sendai viruses, etc. The method for introducing the vector into non-hepatic stem cells and non-hepatic progenitor cells is not particularly limited, and examples include lipofection, electroporation, and calcium phosphate methods. When the vector is a viral vector, a method of infecting cells with the viral vector can also be used. The dose of the vector can be adjusted appropriately.

[0035] Alternatively, if the reprogramming factor is a protein, it can be directly introduced into cells. Reprogramming factors can be produced by genetic engineering (see, for example, "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)). The resulting polypeptide can be introduced into cells by linking it to a membrane-permeable peptide or by using a cationic lipid. Reagents for intracellular introduction are also commercially available (e.g., PULSin (PPU), Prote-IN (HYG), BioPORTER Protein Delivery Reagent (GTS)).

[0036] Furthermore, when the reprogramming factor is a low molecular weight compound that penetrates into cells upon contact with the cells, the reprogramming factor may be introduced into the cells simply by contacting the reprogramming factor with the cells. If the introduction efficiency into the cells is low, measures may be taken to increase the introduction efficiency of the reprogramming factor into the cells, for example, by linking the low molecular weight compound to a membrane-permeable peptide or the like.

[0037] When the reprogramming factor is a gene, the gene may be, for example, DNA or RNA (e.g., mRNA). The combination described herein may be introduced into cells by directly introducing nucleic acids such as mRNA, or by introducing them into cells using lipid nanoparticles (LNPs), for example. In one embodiment, the combination described herein is contained in a lipid nanoparticle (LNP). In one embodiment, the present invention relates to an LNP comprising the combination described herein.

[0038] The method for introducing reprogramming factors may be one or more of the above methods in combination.

[0039] The methods for inducing hepatic stem cells / progenitor cells from non-hepatic stem cells and non-hepatic progenitor cells described herein, and the methods for producing hepatic stem cells / progenitor cells from non-hepatic stem cells and non-hepatic progenitor cells described herein, may be performed in vitro or in vivo. When performed in vitro, the non-hepatic stem cells and progenitor cells may be cultured for a predetermined period after introducing a combination described herein. The culture is performed using a medium used for culturing animal cells, and then, by repeated passage as necessary, the cells can be reprogrammed into hepatic stem cells or hepatic progenitor cells.

[0040] The methods described herein can be performed in vivo, for example, by introducing the combinations described herein into non-hepatic stem cells or non-hepatic progenitor cells from animal skin tissue, blood vessels, or the peritoneal cavity. When an expression vector is used to introduce the reprogramming factors, an organ-specific promoter, such as a liver-specific promoter, may be introduced into the vector to make the expression of the reprogramming factors tissue-specific. After introduction of the reprogramming factors, the presence or absence of cells reprogrammed into hepatic stem cells or hepatic progenitor cells in the animal tissue or in cells recovered from the animal tissue may be confirmed as needed. Whether the obtained cells have been reprogrammed into hepatic stem cells or hepatic progenitor cells can be confirmed by determining whether the expression of markers expressed in hepatic stem cells or hepatic progenitor cells is increased. Examples of markers include albumin, alpha-fetoprotein, and E-cadherin.

[0041] In one embodiment, the present invention relates to a method for producing hepatocytes or bile duct epithelial cells, comprising the steps of (a) inducing hepatic stem cells or hepatic progenitor cells by the methods described herein, and (b) differentiating the induced hepatic stem cells or hepatic progenitor cells into hepatocytes or bile duct epithelial cells.

[0042] Differentiation of hepatic stem cells or hepatic progenitor cells into hepatocytes or bile duct epithelial cells can be carried out by conventional methods. For example, differentiation into hepatocytes can be induced by forming aggregates (aggregates) of hepatic stem cells or hepatic progenitor cells in three-dimensional suspension culture, and differentiation into bile duct epithelial cells can be induced by forming bile duct-like organoids by embedding hepatic stem cells or hepatic progenitor cells in Matrigel and culturing them in three dimensions. Culture conditions (temperature, period, medium, etc.) can be in accordance with conventional methods, for example, the method described in WO20198 / 082874 can be followed.

[0043] In one embodiment, the present invention relates to a combination described herein or a kit or composition comprising the combination, for use in a method for inducing non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells or a method for producing hepatic stem cells or hepatic progenitor cells described herein. The kit may include, for example, one or more of an instruction manual, a reagent, and a buffer solution. In this embodiment, the HNF1A gene and the FOXA3 gene that may be included in the combination may be included in a vector.

[0044] In one embodiment, the present invention relates to a pharmaceutical composition comprising cells, such as hepatic stem cells or hepatic progenitor cells, obtained by the method for inducing hepatic stem cells or hepatic progenitor cells, the method for producing hepatic stem cells or hepatic progenitor cells, or the method for producing hepatocytes or bile duct epithelial cells described herein. In one embodiment, the present invention relates to a method for producing a pharmaceutical composition, comprising the steps of obtaining cells, such as hepatic stem cells or hepatic progenitor cells, by the method for inducing hepatic stem cells or hepatic progenitor cells, the method for producing hepatic stem cells or hepatic progenitor cells, or the method for producing hepatocytes or bile duct epithelial cells described herein, and producing a pharmaceutical composition comprising the obtained cells. The pharmaceutical compositions described herein can be used for the treatment and / or prevention of diseases, such as liver disease or liver damage. The dosage form, dosage, frequency, number of administrations, and route of administration of the pharmaceutical composition can be selected depending on the age, sex, weight, symptoms, and route of administration of the subject.

[0045] The pharmaceutical compositions described herein may contain, in addition to the combinations described herein, pharmaceutically acceptable carriers (excipients, fillers, binders, lubricants, etc.) and / or known additives (buffers, isotonicity agents, chelating agents, colorants, preservatives, fragrances, flavoring agents, sweeteners, etc.). For example, lipid nanoparticles (LNPs) can be included as carriers to facilitate gene delivery.

[0046] Example 1: Highly efficient generation of human induced hepatic progenitor cells using a Wnt signaling activator A retroviral vector (gene expression vector) containing human iHepPC-inducing factors (FOXA3, HNF1A, HNF6) was introduced into human umbilical vein endothelial cells (HUVECs (Takara)) by retroviral infection. Cell culture, retrovirus production, and retroviral infection of cells were performed according to Hiroki Inada et al., Nature Communications, Vol. 11, Article number: 5292, 2020. Six hours after retroviral infection, CHIR99021 (Tocris), a Wnt signaling activator, was added to the cells. 3 μM BIO (6-bromoindirubin-3-oxime) (Calbiochem) or 1 μM BIO (6-bromoindirubin-3-oxime) (Calbiochem) was added to the culture medium, and the colonies formed on days 9 and 16 were immunostained for albumin (ALB) and counted. The group without Wnt signaling activators was designated as Control. Cell DNA was stained with DAPI (blue). Immunostaining was performed according to Hiroki Inada et al. (supra). Colony counts were performed by counting colonies observed under a microscope or in photographs taken with a microscope.

[0047] The results are shown in Figure 1. Figure 1A shows the results of immunostaining, and Figure 1B shows the results of colony counting. Figure 1 demonstrates that adding the Wnt signal activators CHIR99021 or BIO to human iHepPC inducers (FOXA3, HNF1A, and HNF6) increased the number of cells expressing albumin, a hepatic progenitor cell marker, and shortened the time until albumin-expressing cells appeared, thereby improving the efficiency of induction of human hepatic progenitor cells.

[0048] Example 2: Reduction in Human iHepPC Colony Formation Frequency by Addition of Wnt Signaling Inhibitor to Culture Medium HUVECs were transfected with a retroviral vector (gene expression vector) containing human iHepPC-inducing factors (FOXA3, HNF1A, and HNF6). Cell culture, retrovirus production, and retroviral infection were performed as described in Example 1. Six hours after retroviral infection, a Wnt signaling inhibitor (IWP2 (Nacalai Tesque): 0.5 μg / ml) was added to the culture medium. Colonies formed on days 9, 16, 23, and 44 were immunostained for albumin (ALB) (A) and analyzed for ALB gene expression (B). The group without Wnt signaling inhibitor was designated as Control. Cell DNA was stained with DAPI (blue). Immunostaining was performed as described in Example 1, and gene expression analysis was performed according to Hiroki Inada et al. (supra).

[0049] The results are shown in Figure 2. Figure 2 shows that adding the Wnt signal inhibitor IWP2 to human iHepPC inducers (FOXA3, HNF1A, and HNF6) reduced the number of cells expressing albumin, a hepatic progenitor cell marker, and therefore reduced the efficiency of induction into human hepatic progenitor cells.

[0050] Example 3: Substitution of HNF6 with a Wnt Signaling Activator We investigated the induction of human iHepPCs in three groups: Group 1, in which human iHepPC inducing factors (FOXA3, HNF1A, HNF6) were introduced into HUVECs; Group 2, in which a portion of the human iHepPC inducing factors (FOXA3, HNF1A) was introduced; and Group 3, in which a Wnt signaling activator (CHIR99021: 3 μM) was added to the culture medium 6 hours after the introduction of a portion of the human iHepPC inducing factors (FOXA3, HNF1A). After the introduction of the iHepPC inducing factors, cells were cultured for one month, and then immunostaining was performed. Immunostaining was performed as described in Example 1.

[0051] The results are shown in Figure 3. Human iHepPCs positive for albumin (ALB) and E-cadherin (E-CAD) were induced by immunohistochemistry only in groups 1 and 3, whereas no human iHepPCs positive for ALB or E-CAD were induced in group 2. This indicates that FOXA3 and HNF1A alone are insufficient for the induction of human iHepPCs, that HNF6 is required, and that HNF6 can be replaced by a Wnt signaling activator.

Claims

1. A method for inducing non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells, comprising introducing into non-hepatic stem cells or non-hepatic progenitor cells the following combination: a combination comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and a Wnt signal activator.

2. A method for producing hepatic stem cells or hepatic progenitor cells, comprising introducing the following combination into non-hepatic stem cells or non-hepatic progenitor cells: a combination comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and a Wnt signal activator, to induce the non-hepatic stem cells or non-hepatic progenitor cells into hepatic stem cells or hepatic progenitor cells.

3. The method of claim 1, wherein the combination consists of the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and a Wnt signal activator.

4. The method of claim 1, wherein HNF1 is HNF1A.

5. The method of claim 1, wherein FOXA is FOXA3.

6. The method according to claim 1, wherein the Wnt signal activator is CHIR99021 or BIO (6-bromoindirubin-3-oxime).

7. The method according to claim 6, wherein the Wnt signal activator is CHIR99021.

8. The method of claim 1, wherein the combination further comprises an HNF6 gene or HNF6 protein.

9. A method for producing hepatocytes or bile duct epithelial cells, comprising: (a) a step of inducing hepatic stem cells or hepatic progenitor cells by the method according to claim 1; and (b) a step of differentiating the induced hepatic stem cells or hepatic progenitor cells into hepatocytes or bile duct epithelial cells.

10. A combination for use in the method according to claim 1 or 2, comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and a Wnt signal activator.

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