Process for creating a cell population of the hepatic lineage from endoderm cells, and cell composition containing the same

Abstract

To provide a process of making a posterior foregut cell from an endodermal cell, and to provide a population of obtained posterior foregut cells.SOLUTION: A process comprises contacting a posterior foregut cell with a second culture medium, and comprises a second set of additives under conditions that permit differentiation of the posterior foregut cell into a hepatic progenitor cell, the second set of additives comprising or consisting essentially of: an activator of an insulin signaling pathway; an activator of a bone morphogenetic protein (BMP) signaling pathway; an activator of a fibroblast growth factor (FGF) signaling pathway; an activator of the hepatocyte growth factor (HGF) signaling pathway; and an inhibitor of a Wnt signaling pathway.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 676,582, filed May 25, 2018, the entire contents of which are incorporated herein by reference. used.

Background Art

[0002] It has been proven difficult to obtain viable and functional hepatocyte - like cells in high yields, and in particular, to obtain such cells by differentiating them from pluripotent stem cells such as induced pluripotent stem cells. It has also been proven difficult to obtain a homogeneous cell population in a reproducible manner.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Accordingly, there is a long - felt need for cells derived from hepatocyte lineages that exhibit biological activities that enable the metabolism of molecules such as therapeutic agents and / or potential therapeutic agents.

Means for Solving the Problems

[0004] The present disclosure relates to a process for differentiating pluripotent cells into viable and functional hepatocyte - like cells by providing or excluding specific additives during culture. This process does not promote (and in some embodiments, does not allow) the differentiation of pluripotent cells into the mesodermal lineage, and is also a process for differentiating pluripotent cells into the endodermal lineage. This process is for promoting differentiation into the endoderm ​​​​​​​​​, including activation of the Wnt pathway (permitting Nodal expression) and the TGFβ pathway. The endoderm The first shift in the anteroposterior pattern of the endoderm leaflet begins with a combination of Wnt, FGF, and BMP signaling at the posterior end of the embryonic endoderm. Inhibition of the TGFβ pathway, as well as the use of FGF and BM P signaling, in combination with the initial suppression of the Wnt pathway in the anterior endoderm, enables the expression of Hex (required for the development of the liver (and pancreas)). Immediately after the initial suppression of Wnt signaling, the same pathway undergoes activation for liver growth. Continuous signaling, including FGF, BMP, Wnt, and HGF pathways derived from hepatic mesenchyme and endothelial cells, to promote differentiation. For maturation into hepatocyte-like cells, cytokines, glucocorticoids, HGF , and Wnt are useful. Cytokines such as OSM induce morphological maturation into polarized epithelium. In a first aspect, the present disclosure provides a process for creating posterior foregut cells from endoderm cells. This process involves contacting the endoderm cells with a first culture medium that does not contain insulin and including a first additive set that permits differentiation of the endoderm cells into posterior foregut cells. The first additive set does not contain insulin and includes an activator of the bone morphogenetic protein (BMP) signaling pathway; an activator of the fibroblast growth factor (FGF)

[0005] signaling pathway; an inhibitor of the Wnt signaling pathway; and an inhibitor of the transforming growth factor β (TGFβ) signaling pathway, or consists essentially of them . In certain embodiments, the first culture medium contains serum. In another embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4 . In a first aspect, the present disclosure provides a process for creating posterior foregut cells from endoderm cells. This process involves contacting the endoderm cells with a first culture medium that does not contain insulin and including a first additive set that permits differentiation of the endoderm cells into posterior foregut cells. The first additive set does not contain insulin and includes an activator of the bone morphogenetic protein (BMP) signaling pathway; an activator of the fibroblast growth factor (FGF) signaling pathway; an inhibitor of the Wnt signaling pathway; and an inhibitor of the transforming growth factor β (TGFβ) signaling pathway, or consists essentially of them . In certain embodiments, the first culture medium contains serum. In another embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4 In another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist. In a further embodiment, inhibition of the Wnt signaling pathway is The factor is capable of inhibiting the biological activity of porcupine, e.g., IWP2. In yet another embodiment, the inhibitor of the TGFβ signaling pathway is ALK4, ALK 5, or ALK7, e.g., inhibiting the biological activity of A83-01. In one embodiment, the endoderm cells can be inhibited by expressing SOX17, GATA4, FO expresses at least one of XA2, CXCR4, or EOMES, and / or , which do not substantially express c-Kit. The authors concluded that "c-Kit is a viable cell line that is not viable" if less than 3% of the cells are positive for the c-Kit marker. Therefore, cells derived from the posterior foregut cells cannot express them qualitatively. Due to their endodermal origin, they also fail to substantially express c-Kit. , posterior foregut cells express SOX2, FOXA1, FOXA2, HNF4a, AFP, or The present disclosure relates to the process described herein. A population of posterior foregut cells obtainable or obtained thereby is also provided.

[0006] In a second aspect, the present disclosure provides a process for generating hepatic progenitor cells from hindgut and foregut cells. This process involves the differentiation of posterior foregut cells into hepatic progenitor cells under conditions that permit differentiation. contacting the posterior foregut cells with a second culture medium comprising a second set of additives; The two additive sets are: activators of the insulin signaling pathway; bone morphogenetic proteins (BMP) Signal transduction pathway activator; fibroblast growth factor (FGF) signal transduction Pathway activator; hepatocyte growth factor (HGF) signal transduction pathway activator; and, W nt signal transduction pathway activator, or consisting essentially of them. In one embodiment form, the second culture medium contains serum. In another embodiment, insulin signal transduction The activator of the pathway is an insulin receptor agonist, for example, insulin. In another example In the form, the activator of the BMP signal transduction pathway is a BMP receptor agonist, for example , BMP4. In a further embodiment, the activator of the FGF signal transduction pathway is F GF receptor agonist, for example, basic FGF. In yet another embodiment, HGF The activator of signal transduction is an HGF receptor agonist, for example, HGF. Further In another embodiment, the activator of the Wnt signal transduction pathway is GSK3, for example, CHI The biological activity of R99021 can be inhibited. In one embodiment, posterior foregut cells Express at least one of SOX2, FOXA1, FOXA2, HNF4a, AFP, or albumin . In another embodiment, hepatocyte progenitor cells are α-fetoprotein Protein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, or at least one of HNF4a Express at least one. The present disclosure also provides a population of hepatocyte progenitor cells obtainable or obtained by the processes described herein.

[0007] According to a third aspect, the present disclosure provides a process for creating hepatocyte-like cells from hepatic progenitor cells Provided. This process includes (i) contacting hepatocyte lineage cells with a third culture medium containing a third additive set under conditions for obtaining hepatocyte progenitor cells, (ii) contacting hepatocyte lineage cells with a fourth culture medium containing a fourth additive set under conditions for obtaining immature hepatocyte-like cells, and (iii) contacting immature hepatocyte-like cells with a fifth culture medium containing no cytokine and containing a fifth additive set under conditions for obtaining mature hepatocyte-like cells. The third additive set includes an activator of the insulin signaling pathway, an activator of the bone morphogenetic protein (BMP) signaling pathway, an activator of the fibroblast growth factor (FGF) signaling pathway, an activator of the hepatocyte growth factor (HGF) signaling pathway, an activator of the Wnt signaling pathway, an inhibitor of the transforming growth factor β (TGFβ) signaling pathway, a cytokine, and a glucocorticoid, or consists essentially of them. The fourth additive set includes a cytokine and a glucocorticoid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist cells, and (ii) contacting hepatocyte lineage cells with a fourth culture medium containing a fourth additive set under conditions for obtaining immature hepatocyte-like cells, and (iii) contacting immature hepatocyte-like cells with a fifth culture medium containing no cytokine and containing a fifth additive set under conditions for obtaining mature hepatocyte-like cells. The third additive set includes an activator of the insulin signaling pathway, an activator of the bone morphogenetic protein (BMP) signaling pathway, an activator of the fibroblast growth factor (FGF) signaling pathway, an activator of the hepatocyte growth factor (HGF) signaling pathway, an activator of the Wnt signaling pathway, an inhibitor of the transforming growth factor β (TGFβ signaling pathway, a cytokine, and a glucocorticoid, or consists essentially of them. The fourth additive set includes a cytokine and a glucocorticoid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist cells with a fifth culture medium containing no cytokine and containing a fifth additive set under conditions for obtaining mature hepatocyte-like cells. The third additive set includes an activator of the insulin signaling pathway, an activator of the bone morphogenetic protein (BMP) signaling pathway, an activator of the fibroblast growth factor (FGF) signaling pathway, an activator of the hepatocyte growth factor (HGF) signaling pathway, an activator of the Wnt signaling pathway, an inhibitor of the transforming growth factor β (TGFβ signaling pathway, a cytokine, and a glucocorticoid, or consists essentially of them. The fourth additive set includes a cytokine and a glucocorticoid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist tor, an activator of the bone morphogenetic protein (BMP) signaling pathway, an activator of the fibroblast growth factor (FGF) signaling pathway, an activator of the hepatocyte growth factor (HGF) signaling pathway, an activator of the Wnt signaling pathway, an inhibitor of the transforming growth factor β (TGFβ signaling pathway, a cytokine, and a glucocorticoid, or consists essentially of them. The fourth additive set includes a cytokine and a glucocorticoid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist signaling pathway, an activator of the Wnt signaling pathway, an inhibitor of the transforming growth factor β (TGFβ ) signaling pathway, a cytokine, and a glucocorticoid, or consists essentially of them. The fourth additive set includes a cytokine and a glucocorticoid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist signaling pathway, a cytokine, and a glucocorticoid, or consists essentially of them. The fourth additive set includes a cytokine and a glucocorticoid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist oid, or consists essentially of them. The fifth additive set contains no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist no cytokine and contains a glucocorticoid, or consists essentially of it. In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist In certain embodiments, the fourth, fifth, and / or sixth culture media contain serum. In another embodiment, the activator of the insulin signaling pathway is an insulin receptor agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist agonist, such as insulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist ulin. In a further embodiment, the activator of the BMP signaling pathway is a BMP receptor agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist agonist, such as BMP4. In yet another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist agonist, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist ist, such as an HGF receptor agonist The factor is, for example, HGF. In yet another embodiment, activation of the Wnt signaling pathway The factor can inhibit the biological activity of GSK3, for example, CHIR99021 . In yet another embodiment, the inhibitor of the TGFβ signaling pathway is at least one of ALK4, ALK 5, or ALK7, for example, can inhibit the biological activity of A83-01 . In another embodiment, the cytokine is oncostatin M (OSM) . In another embodiment, the glucocorticoid is dexamethasone. In yet another embodiment , the hepatic progenitor cells express at least one of α-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, or HNF4a. In yet another embodiment , the immature hepatocyte-like cells and / or mature hepatocyte-like cells express α-fetoprotein (AFP), albumin (ALB), ASGR1, HNF4a, or at least one of SOX9 . In one embodiment, the mature hepatocyte-like cells have detectable Cyp 3A4 activity and express detectable levels of albumin and / or urea . The present disclosure also provides a population of hepatocyte-like cells obtainable or obtained by the processes described herein .

[0008] According to a fourth aspect, the present disclosure provides a process for creating hepatic progenitor cells from endoderm cells . The process comprises (a) performing the process described herein to obtain posterior foregut cells or providing a population of posterior foregut cells described herein; and, ( b) subjecting posterior foregut cells to the processes described herein to obtain hepatic progenitor cells, comprising, or consisting essentially of, the same. The present disclosure also provides a population of hepatic progenitor cells obtainable by, or obtained by, the processes described herein.

[0009] According to a fifth aspect, the present disclosure provides a process for creating hepatocyte-like cells from hepatic progenitor cells. The process comprises: (a) performing the processes described herein to obtain hepatic progenitor cells, or providing a population of hepatic progenitor cells described herein; and (b) subjecting the hepatic progenitor cells to the processes described herein to obtain hepatocyte-like cells, comprising, or consisting essentially of, the same. The present disclosure also provides a population of hepatocyte-like cells obtainable by, or obtained by, the processes described herein.

[0010] According to a sixth aspect, the present disclosure provides a process for creating hepatocyte-like cells from endoderm cells. The process comprises: (a) optionally performing the processes described herein to obtain posterior foregut cells, or optionally providing a population of posterior foregut cells described herein; (b) subjecting the posterior foregut cells to the processes described herein to obtain hepatic progenitor cells, or providing a population of hepatic progenitor cells described herein; and (c) subjecting the hepatic progenitor cells to the processes described herein to obtain hepatocyte-like cells, comprising, or consisting essentially of, the same. The present disclosure also provides a population of hepatocyte-like cells obtainable by, or obtained by, the processes described herein.

[0011] According to a seventh aspect, the present disclosure provides a process for creating encapsulated liver tissue. . This process involves: (a) providing a population of hepatocyte-like cells as described herein; (b) combining hepatocytes, mesenchymal cells, and optional endothelial cells in suspension and culturing to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm; and (c) at least partially covering at least one of said liver organoids with a first biocompatible cross-linked polymer. In certain embodiments, endodermal cells and hepatocyte-like cells are combined at a ratio of 1:0.2 to 7 prior to culturing. In another embodiment, hepatocytes and endothelial cells are combined at a ratio of 1:0.2 to 1 prior to culturing. In yet another embodiment, at least one of hepatocytes, endodermal cells, and endothelial cells is obtained by differentiating pluripotent cells such as pluripotent stem cells. In certain embodiments, the endothelial cells are endothelial progenitor cells. In a further embodiment, this process further involves substantially covering at least one liver organoid with a first biocompatible cross-linked polymer, for example, where the cross-linked polymer comprises poly(ethylene glycol) glycol (PEG). In another embodiment, this process further involves covering at least a portion of the first biocompatible cross-linked polymer with a second biocompatible cross-linked polymer and, in some embodiments, substantially covering it. In certain embodiments, at least a portion of the first biocompatible cross-linked polymer and / or the second biocompatible cross-linked polymer is biodegradable. In yet another embodiment, the second biocompatible cross-linked polymer comprises poly(ethylene) glycol (PEG). This disclosure pertains to what is described herein. In suspension, combine hepatocytes, mesenchymal cells, and optional endothelial cells and culture to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In suspension, combine hepatocytes, mesenchymal cells, and optional endothelial cells and culture to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In suspension, combine hepatocytes, mesenchymal cells, and optional endothelial cells and culture to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In suspension, combine hepatocytes, mesenchymal cells, and optional endothelial cells and culture to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In suspension, combine hepatocytes, mesenchymal cells, and optional endothelial cells and culture to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In suspension, combine hepatocytes, mesenchymal cells, and optional endothelial cells and culture to obtain at least one liver organoid comprising: (i) a cell core at least partially covered with hepatocyte-like cells and / or cholangiocyte epithelial cells and containing mesenchymal cells and optional endothelial cells, (ii) a spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In certain embodiments, endodermal cells and hepatocyte-like cells are combined at a ratio of 1:0.2 to 7 prior to culturing. In another embodiment, hepatocytes and endothelial cells are combined at a ratio of 1:0.2 to 1 prior to culturing. In yet another embodiment, at least one of hepatocytes, endodermal cells, and endothelial cells is obtained by differentiating pluripotent cells such as pluripotent stem cells. In certain embodiments, endodermal cells and hepatocyte-like cells are combined at a ratio of 1:0.2 to 7 prior to culturing. In certain embodiments, the endothelial cells are endothelial progenitor cells. In a further embodiment, this process further involves substantially covering at least one liver organoid with a first biocompatible cross-linked polymer, for example, where the cross-linked polymer comprises poly(ethylene glycol) glycol (PEG). In another embodiment, this process further involves covering at least a portion of the first biocompatible cross-linked polymer with a second biocompatible cross-linked polymer and, in some embodiments, substantially covering it. In another embodiment, this process further involves covering at least a portion of the first biocompatible cross-linked polymer with a second biocompatible cross-linked polymer and, in some embodiments, substantially covering it. In another embodiment, this process further involves covering at least a portion of the first biocompatible cross-linked polymer with a second biocompatible cross-linked polymer and, in some embodiments, substantially covering it. In certain embodiments, at least a portion of the first biocompatible cross-linked polymer and / or the second biocompatible cross-linked polymer is biodegradable. In yet another embodiment, the second biocompatible cross-linked polymer comprises poly(ethylene) glycol (PEG). This disclosure pertains to what is described herein. Also provided are encapsulated liver tissues obtainable by, or obtained by, the processes described below.

[0012] According to an eighth aspect, the present disclosure provides an additive set and a medium containing the same. . In certain embodiments, the present disclosure provides the first additive set described herein, and a first culture medium that contains the first additive set and does not contain an activator of the insulin signaling pathway. In certain embodiments, the first culture medium further contains endoderm cells and / or foregut posterior cells. In another embodiment, the present disclosure provides the second additive set described herein, and a second culture medium that contains the second additive set. In certain embodiments, the second culture medium contains foregut posterior cells and / or hepatic progenitor cells. In yet another embodiment, the present disclosure provides the third additive set described herein, and a third culture medium that contains the third additive set. In still another embodiment, the present disclosure provides the fourth additive set described herein, and a fourth culture medium that contains the fourth additive set. In yet another embodiment, the present disclosure provides the fifth additive set described herein, and a fifth culture medium that contains the fifth additive set and does not contain cytokines. The present disclosure also provides a kit for generating foregut posterior cells, hepatic progenitor cells, or hepatocyte-like cells. The kit contains at least one additive set described herein and / or at least one medium described herein; and instructions for generating foregut posterior cells, hepatic progenitor cells, or hepatocyte-like cells (e.g., for performing the processes described herein). In some embodiments, the kit contains endoderm cells, foregut posterior cells, or hepatocyte-like cells. It further includes foregut cells and / or hepatic progenitor cells.

[0013] The gist of the present invention is as described above. Next, preferred embodiments thereof will be described with reference to the attached drawings for illustrative purposes, without being limited thereto.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Cells, and a process for creating a composition containing the same According to the present invention, there is provided a process for differentiating endoderm cells into cells of a competent hepatocyte lineage (e.g., posterior foregut cells, liver progenitor cells, and / or hepatocytes). Cells of the hepatocyte lineage can be cells that can differentiate into hepatocytes, or cells that are hepatocytes. In some embodiments, the process of the present disclosure is such that they are a large number of cells of the hepatocyte lineage, and / or It is advantageous because it enables the production of cells that are biologically more potent.

[0016] In certain embodiments, this process can be used to generate various cell populations from endodermal cells. As used herein, the term “endodermal cells” refers to cells having the characteristics of cells derived from the endoderm. As is known in the art of developmental biology, the endoderm is the innermost of the three main germ layers. Endodermal cells are generally flat and play a role in generating most of the cells of the gastrointestinal tract, respiratory tract, liver, pancreas, endocrine organs, and urinary organs. Endodermal cells can be identified by those skilled in the art using various techniques known in the art. For example, endodermal cells can be identified by determining the presence or absence, and expression levels, of at least one of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments, endodermal cells express at least two of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In yet another embodiment, endodermal cells can be identified by detecting and optionally measuring the expression of at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells can be identified by detecting and optionally measuring the expression of at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. In still another embodiment, endodermal cells express at least three of the following genes: SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or any combination thereof, or the polypeptides encoded thereby. A and / or at least 4 of EOMES, or any combination thereof expression can be detected, and optionally measured and identified. In yet another embodiment endoderm cells have the following genes (or polypeptides related thereto): SOX 17, GATA4, FOXA2, CXCR4, and / or EOMES expression detected and optionally measured and identified. In some embodiments, endoderm cells are caused to express, and the following genes: SOX2, SOX17, GATA4, FOXA2, CXCR4, and / or EOMES, or the expression level of the polypeptides encoded thereby is compared to the expression level of the same gene / polypeptide in (undifferentiated) stem cells and identified. In certain embodiments, endoderm cells have strong expression of SOX17, GATA4, FOXA2, CXCR4 compared to the corresponding levels in undifferentiated pluripotent (stem) cells and / or the EOMES gene, or the polypeptides encoded thereby.

[0017] Endoderm cells can be of any origin, particularly those that can be derived from mammals and in some embodiments, can be derived from humans.

[0018] Endoderm cells can be obtained from pluripotent cells (e.g., embryonic stem cells or induced pluripotent stem cells) that have differentiated into endoderm cells. In some embodiments, endoderm cells can be obtained by differentiating induced pluripotent stem cells (i PSCs). Pluripotent (stem) cells can be of any origin, particularly those that can be derived from mammals and in some embodiments, can be derived from humans. In some embodiments of differentiating pluripotent (stem) cells into endoderm cells, the pluripotent (stem) cells Compounds capable of activating the Nodal / Activin signaling pathway, such as contacting with Nodal / Activin receptor agonists such as Activin A can be done. In some further embodiments, the pluripotent (stem) cells are Wnt signaling activators of the pathway, such as Wnt receptor agonists, or compounds capable of inhibiting the biological activity of GSK3 such as CHIR99021, can also be contacted. .

[0019] Before differentiating into endodermal cells, pluripotent (stem) cells are exposed to one or more activators of the APELA / ELABELA signaling pathway, such as agonists of the APELA / ELABELA receptor, such as APELA / ELABELA polypeptide or its self-replication, and / or functional fragments for inducing, optimizing, and maintaining pluripotency (such as those described in U.S. Patent No. 9,309,314, etc.).

[0020] The present disclosure provides a first process for creating posterior foregut cells from endodermal cells. This process involves contacting one or more endodermal cells with a first culture medium containing a first set of additives under conditions that permit differentiation of the endodermal cells into posterior foregut cells. The first process does not involve contacting the cultured cells with an activator of the insulin signaling pathway, such as insulin. As used in the present disclosure, "posterior foregut cells" refers to cells having the biological characteristics of cells of the posterior foregut. As is known in the field of developmental biology, the posterior foregut is the region of the endoderm that will subsequently form the liver. Thus, the posterior foregut cells have the biological characteristics of cells of the posterior foregut. foregut refers to the region of the endoderm that will subsequently form the liver. Thus, the posterior foregut The cells can further differentiate into the liver, pancreas, stomach, and a part of the small intestine. Those skilled in the art can identify the posterior foregut cells using various techniques known in the art . For example, the posterior foregut cells can be identified by determining the presence or absence, and the expression level of at least one of any combination of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or albumin, or the polypeptides encoded thereby . In certain embodiments, the posterior foregut cells express at least two of any combination of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP , and / or albumin, or the polypeptides encoded thereby . In yet another embodiment, the posterior foregut cells express at least three of any combination of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP , and / or albumin, or the polypeptides encoded thereby . In still another embodiment, the posterior foregut cells express at least four of any combination of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP , and / or albumin, or the polypeptides encoded thereby . In still another embodiment, the posterior foregut cells express at least five of any combination of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP , and / or albumin, or the polypeptides encoded thereby . In still another embodiment, the posterior foregut cells express at least one of any combination of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP , and / or albumin, or the polypeptides encoded thereby . In still another embodiment, the posterior foregut cells express all of the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or albumin, or the polypeptides encoded thereby . In still another embodiment In the form, the posterior foregut cells are the following genes (or the corresponding polypeptides): S OX2, FOXA1, FOXA2, HNF4a, AFP, and / or the expression of albumin can be detected, and optionally measured and identified. In some embodiments, expressed in posterior foregut cells, and the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or albumin, or the polypeptide encoded by them The expression level of can be identified by comparing with the expression level of the same gene / polypeptide in (undifferentiated) stem cells or endoderm cells. In certain embodiments, The posterior foregut cells strongly express SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or the albumin gene, or the polypeptide encoded by them, compared to the corresponding levels in pluripotent (stem) cells or endoderm cells. In further embodiments, The posterior foregut cells express the SOX2 gene, or the polypeptide encoded by it, at a high level compared to the corresponding level in endoderm cells. In further embodiments, the posterior fore gut cells strongly express the FOXA1 gene, or the polypeptide encoded by it, compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the FOXA2 gene, or the polypeptide encoded by it, strongly compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the HNF4a gene, or the polypeptide encoded by it, strongly compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the corresponding level in endoderm cells compared to the level of the same gene / polypeptide in (undifferentiated) stem cells or endoderm cells. In certain embodiments, the posterior foregut cells strongly express SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or the albumin gene, or the polypeptide encoded by them, compared to the corresponding levels in pluripotent (stem) cells or endoderm cells. In further embodiments, The posterior foregut cells express the SOX2 gene, or the polypeptide encoded by it, at a high level compared to the corresponding level in endoderm cells. In further embodiments, the posterior fore gut cells strongly express the FOXA1 gene, or the polypeptide encoded by it, compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the FOXA2 gene, or the polypeptide encoded by it, strongly compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the HNF4a gene, or the polypeptide encoded by it, strongly compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the corresponding level in endoderm cells compared to the level of the same gene / polypeptide in (undifferentiated) stem cells or endoderm cells. In certain embodiments, the posterior foregut cells strongly express the FOXA2 gene, or the polypeptide encoded by it, compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the HNF4a gene, or the polypeptide encoded by it, strongly compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the corresponding level in endoderm cells compared to the level of the same gene / polypeptide in (undifferentiated) stem cells or endoderm cells. In certain embodiments, the posterior foregut cells strongly express the HNF4a gene, or the polypeptide encoded by it, compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the corresponding level in endoderm cells compared to the corresponding level in endoderm cells. In further embodiments, the posterior foregut cells express the corresponding level in endoderm cells Compared with the loop, it strongly expresses the AFP gene or the polypeptide encoded thereby. In a further embodiment, the posterior foregut cells strongly express the A LB gene or the albumin polypeptide encoded thereby, as compared to the corresponding level in endoderm cells.

[0021] The posterior foregut cells can be of any origin, and in particular can be derived from mammals and, in some embodiments, can be derived from humans.

[0022] The first culture medium used in the first process can be serum-free (e.g., not supplemented with serum ). In another embodiment, the first culture medium used in the first process can contain serum, and the serum can be KnockOut Serum Replacem ent (trademark) (ThermoFisher Scientific). In certain embodiments the first culture medium contains about 0.1 to about 5% (v / v) serum. In yet another embodiment the first culture medium contains at least about 0.1, 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 % or more serum. In another embodiment, the first culture medium contains about 5, 4.5, 4, 3.5 , 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0 .3, 0.2% or less serum. In still another embodiment, the first culture medium contains about 0. 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5% and about 5, 4.5, 4, 3.5, 3, 2 .5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or Alternatively, it contains serum between 0% and 0.2%. In certain embodiments, the first culture medium contains about 1% blood serum.

[0023] The first culture medium contains a first set of additives, which consists of, or consists essentially of, activators of the bone morphogenetic protein (BMP) signaling pathway; activators of the fibroblast growth factor (FGF) signaling pathway; inhibitors of the Wnt signaling pathway; and inhibitors of the transforming growth factor β (TGFβ) signaling pathway. The first set of additives does not contain activators of the insulin signaling pathway such as insulin. As used in the context of the present disclosure, the expression "the first culture medium consists essentially of the first set of additives" refers to a first culture medium that contains additional additives that are non-essential in the differentiation of endoderm cells into posterior foregut cells, but that can promote differentiation. Examples of these additional additives include, but are not limited to, retinoic acid, vitamins, and minerals. (TGFβ) signaling pathway. The first set of additives does not contain activators of the insulin signaling pathway such as insulin. As used in the context of the present disclosure, the expression "the first culture medium consists essentially of the first set of additives" refers to a first culture medium that contains additional additives that are non-essential in the differentiation of endoderm cells into posterior foregut cells, but that can promote differentiation. Examples of these additional additives include, but are not limited to, retinoic acid, vitamins, and minerals.

[0024] The first culture medium contains an activator of the bone morphogenetic protein (BMP) signaling pathway. During development, activators of the BMP signaling pathway are normally provided from the cardiac mesoderm and promote the differentiation of endoderm cells into posterior foregut cells. As used in the context of the present disclosure, "activator of the BMP signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of BMP to its cognate receptors (e.g., BMPR1 and / or BMPR2). The signal transduction BMP receptors occur via the SMAD and MAP kinase pathways and effect the transcription of BMP target genes. This compound ​​​​​​​​​​​​, an agonist of a BMP receptor (specific for BMPR1 or BMPR2 or binding to both receptors and capable of activating), an activator of a polypeptide known to activate the BMP signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the BMP signaling pathway. Known BMPs include, but are not limited to, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. In certain embodiments, the activator is DM3189. In another embodiment, the activator is BMP4 (which may be provided in recombinant or purified form). BMP4 is a member of the transforming growth factor-β (TGF-β) family and binds to two different types of serine-threonine kinase receptors known as BMPR1 and BMPR2. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of less than at least about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or less ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is the first... , and a polypeptide activator known to activate the BMP signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the BMP signaling pathway. Known BMPs include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15, but are not limited thereto. In one embodiment, the activator is DM3189. In another embodiment, the activator is BMP4 (which may be provided in recombinant or purified form). BMP4 is a member of the transforming growth factor-β (TGF-β) family and binds to two different types of serine-threonine kinase receptors known as BMPR1 and BMPR2. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of less than at least about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or less ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is the first... , and binds to two different types of serine-threonine kinase receptors known as BMPR1 and BMPR2. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of less than at least about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or less ng / mL. , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, , 25, 26, 27, 28, 29, or more ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 can be provided in the first culture medium at a concentration of less than at least about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or less ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is the first... , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, , 11, or less ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is the first... In the first culture medium, it can be provided at a concentration between about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and about 30, 29 , 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 ng / mL. In some specific embodiments, BMP4 can be provided in the first culture medium at a concentration of about 20 ng / mL.

[0025] The first culture medium also contains an activator of the fibroblast growth factor (FGF) signaling pathway. During the growth process, the activator of the FGF signaling pathway is usually provided from the cardiac mesoderm and promotes the differentiation of endodermal cells into posterior foregut cells. As used in the context of this disclosure, the "activator of the FGF signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of FGF to its cognate receptors (e.g., FGFR1, FGFR2, FGFR3, and / or FGFR4). This compound can be an agonist of the FGF receptor (specific for FGFR1, FGFR2, FGFR3, and / or FGFR4 or capable of binding to and activating multiple receptors), an activator of a polypeptide known to activate the FGF signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the FGF signaling pathway. Known FGFs include FGF1, FGF2, FGF3, FGF4, FGF5 , FGF6, FGF7, FGF8a, FGF8b, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, F GF18, FGF19, FGF20, FGF21, FGF22, FGF23, FGF24, and FGF25. GF18, FGF19, FGF20, FGF21, FGF22, FGF23, FGF24, or FGF25.​​​​​ There are GF18, FGF20, FGF21, FGF22, and FGF23, but not limited to these. In certain embodiments, the activating factor is basic FGF or FGF2 (which can be provided in recombinant form or purified form). FGF2 binds to two different types of receptors known as FGFR2 (also known as CD332) and FGFR3. In embodiments where basic FGF is provided as an activator of the FGF signaling pathway, basic FGF can be provided in the first culture medium at a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than n g / mL. In embodiments where basic FGF is provided as an activator of the FGF signaling pathway, basic FGF can be provided in the first culture medium at a concentration of less than at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than ng / mL. In embodiments where basic FGF is provided as an activator of the FGF signaling pathway, basic FGF can be provided in the first culture medium at a concentration between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, or 19 and about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 2 ng / mL. In some specific embodiments, basic FGF can be provided in the first culture medium at a concentration of about 5 ng / mL. The first culture medium further contains an inhibitor of the Wnt signaling pathway. The Wnt signal transduction pathway is also inhibited. In some embodiments, the first culture medium can be a basal medium such as DMEM / F12, RPMI 1640, or McCoy's 5A, supplemented with fetal bovine serum, penicillin, streptomycin, and glutamine.

[0026] The first culture medium further contains an inhibitor of the Wnt signaling pathway. The Wnt signal The presence of an inhibitor of the signaling pathway, in combination with an inhibitor of the TGFβ signaling pathway, advantageously acts on the expression of the HEX and PROX1 genes encoding polypeptides necessary for liver development. As used in the context of the present disclosure, an "inhibitor of the Wnt signaling pathway" refers to a compound that can inhibit the signaling pathway associated with the binding of a Wnt protein ligand to its cognate Frizzled receptor (e.g., FZD1 , FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 , or FZD10). The family of Frizzled receptors are G protein-coupled receptor proteins. This compound can be an antagonist of the Frizzled receptor that is specific for any one of (FZD1, FZD2, FZD3, FZD4, FZD 5, FZD6, FZD7, FZD8, FZD9, or FZD10), or binds to and can inhibit multiple receptors, a known inhibitor of a polypeptide that activates the Wnt signaling pathway, and / or a known activator of a polypeptide that inhibits the Wnt signaling pathway. Known Wnt proteins include, but are not limited to, WNT1 , WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, , WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B , WNT10A, WNT10B, WNT11, and WNT16. In certain embodiments, the inhibitor can inhibit the biological activity of one or more Frizzled receptors. In another embodiment, the inhibitor can inhibit the biological activity of the Porcupine protein. For example, the Porcupine protein ,... ... ... ... ... In certain embodiments, the inhibitor can inhibit the biological activity of one or more Frizzled receptors. In another embodiment, the inhibitor can inhibit the biological activity of the Porcupine protein. For example, the Porcupine protein ... ... An inhibitor that can inhibit the biological activity of a substance can be IWP2. When using IWP2 in an embodiment as an inhibitor of the Wnt signaling pathway, IWP2 is in the first culture medium and can be provided at a concentration of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0. 8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6. 5, 7, 7.5, 8, 8.5, 9, 9.5 μM, or a concentration exceeding that. When using IWP2 in an embodiment as an inhibitor of the Wnt signaling pathway, IWP2 is in the first culture medium and can be provided at a concentration of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6. 5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0. 8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 μM, or a concentration less than that. When using IWP2 as an inhibitor of the Wnt signaling pathway, IWP2 is in the first culture medium and can be provided at a concentration of about 0.1, 0.2, 0.3, 0.4 , 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4 , 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5 and about 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0. 4, 0.3, or 0.2 μM. When using IWP2 as an inhibitor of the Wnt signaling pathway, IWP2 can be provided to the first culture medium at a concentration of about 4 μM.

[0027] The first culture medium further contains an inhibitor of the transforming growth factor β (TGFβ) signaling pathway. The presence of the inhibitor of the TGFβ signaling pathway, in combination with an inhibitor of the Wnt signaling pathway, favorably acts on the expression of the HEX and PROX1 genes encoding polypeptides necessary for liver development. As used in the context of the present disclosure, an "inhibitor of the TGFβ signaling pathway" refers to a compound capable of inhibiting the signaling pathway associated with the binding of TGFβ to its cognate receptor. The family of TGFβ receptors mediates signal transduction via SMAD proteins. This compound can be an antagonist of the TGFβ receptor, an inhibitor of a polypeptide known to activate the TGFβ signaling pathway, and / or an activator of a polypeptide known to inhibit the TGFβ signaling pathway. Known TGFβ proteins include, but are not limited to, TGFB1, TGFB2, TGFB3, and TGFB4. In certain embodiments, the inhibitor can inhibit at least one biological activity of the ALK4, ALK5, or ALK7 polypeptide. In some embodiments, the inhibitor can inhibit the biological activities of the ALK4, ALK5, and ALK7 polypeptides. For example, an inhibitor capable of inhibiting the biological activities of the ALK4, ALK5, and ALK7 polypeptides can be A83-01. Alternatively, or in combination, SB431542, and / or LY364947 can be used as the inhibitor. In embodiments where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 is present in the first culture medium at least at 0.1, 0.2, 0.3, 0. Furthermore, the presence of the inhibitor of the TGFβ signaling pathway, in combination with an inhibitor of the Wnt signaling pathway, favorably acts on the expression of the HEX and PROX1 genes encoding polypeptides necessary for liver development. Furthermore, the presence of the inhibitor of the TGFβ signaling pathway, in combination with an inhibitor of the Wnt signaling pathway, favorably acts on the expression of the HEX and PROX1 genes encoding polypeptides necessary for liver development. As used in the context of the present disclosure, an "inhibitor of the TGFβ signaling pathway" refers to a compound capable of inhibiting the signaling pathway associated with the binding of TGFβ to its cognate receptor. As used in the context of the present disclosure, an "inhibitor of the TGFβ signaling pathway" refers to a compound capable of inhibiting the signaling pathway associated with the binding of TGFβ to its cognate receptor. As used in the context of the present disclosure, an "inhibitor of the TGFβ signaling pathway" refers to a compound capable of inhibiting the signaling pathway associated with the binding of TGFβ to its cognate receptor. The family of TGFβ receptors mediates signal transduction via SMAD proteins. The family of TGFβ receptors mediates signal transduction via SMAD proteins. This compound can be an antagonist of the TGFβ receptor, an inhibitor of a polypeptide known to activate the TGFβ signaling pathway, and / or an activator of a polypeptide known to inhibit the TGFβ signaling pathway. Known TGFβ proteins include, but are not limited to, TGFB1, TGFB2, TGFB3, and TGFB4. Known TGFβ proteins include, but are not limited to, TGFB1, TGFB2, TGFB3, and TGFB4. In certain embodiments, the inhibitor can inhibit at least one biological activity of the ALK4, ALK5, or ALK7 polypeptide. In certain embodiments, the inhibitor can inhibit at least one biological activity of the ALK4, ALK5, or ALK7 polypeptide. In some embodiments, the inhibitor can inhibit the biological activities of the ALK4, ALK5, and ALK7 polypeptides. In some embodiments, the inhibitor can inhibit the biological activities of the ALK4, ALK5, and ALK7 polypeptides. For example, an inhibitor capable of inhibiting the biological activities of the ALK4, ALK5, and ALK7 polypeptides can be A83-01. For example, an inhibitor capable of inhibiting the biological activities of the ALK4, ALK5, and ALK7 polypeptides can be A83-01. Alternatively, or in combination, SB431542, and / or LY364947 can be used as the inhibitor. In embodiments where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 is present in the first culture medium at least at 0.1, 0.2, 0.3, 0. 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5 μM, or can be provided at a concentration exceeding that. In an embodiment where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 is in the first culture medium, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0. 4, 0.3, 0.2 μM, or at a concentration less than that. A83 -01 is used as an inhibitor of the TGFβ signaling pathway in an embodiment, A83- 01 is in the first culture medium at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, 1.9, 2, 2.5, 3, 3.5, 4, or between 4.5 and approximately 5, 4.5, 4, 3 .5, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1 .2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 μM can be provided. In an embodiment where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 can be provided to the first culture medium at a concentration of approximately 1 μM.

[0028] The first culture medium is contacted with endoderm cells and posterior foregut cells for at least 1 day or more until differentiation occurs. If it is intended to contact the first culture medium with the cultured cells for 2 days or more, the medium can be exchanged daily. In some embodiments of the process of the present disclosure In one embodiment, the first culture medium is cultured for at least 1, 2, 3, 4, or more days. In another embodiment, the first culture medium is allowed to contact the cultured cells for 5, 4, 3, 2, or 3 days. In yet another embodiment, the first The culture medium is then incubated for at least 1, 2, 3, 4 or more days, and then incubated for 5, 4, The cultured cells are allowed to remain in contact for 3, 2, or fewer days. The first culture medium is then left in contact with the cultured cells for about 1 to 5 days.

[0029] The first culture medium containing endoderm cells was used to differentiate the endoderm cells into posterior foregut cells. Thus, the present disclosure relates to posterior foregut cells obtained by the processes described herein. In the population of posterior foregut cells of the present disclosure, the majority of the cells are from the posterior foregut cells, and in some embodiments, may include some endodermal cells. This can be done.

[0030] The present disclosure relates to a method for producing hepatic progenitor cells (also referred to herein as hepatoblasts) from posterior foregut cells. This process provides a second process for the release of one or more of the posterior foregut cells into the posterior The first culture containing a second set of additives was incubated under conditions that permitted differentiation of the posterior foregut cells from the posterior foregut cells. The posterior foregut cells used in the second process are contacted with a culture medium of It can be obtained by running process 1.

[0031] As used herein, "hepatic progenitor cells" or "hepatoblasts" refer to cells derived from bile duct cells and hepatocytes. It refers to a bipotential progenitor cell that can differentiate into either one of two types of cells. Using various techniques known in the art, hepatic progenitor cells can be identified. For example, hepatic progenitor cells can be identified by determining the presence or absence, and expression levels, of the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX gene, and / or at least one of HNF4a, or any combination thereof, and / or the polypeptides they encode. In certain embodiments, hepatic progenitor cells express at least one of the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, or HNF4a, or any combination thereof, or the polypeptides they encode. In yet another embodiment, hepatic progenitor cells express at least one of the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, or HNF4a, or the polypeptides they encode. In still another embodiment, hepatic progenitor cells express at least one of the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, or HNF4a, or the polypeptides they encode. In yet another embodiment, hepatic progenitor cells express at least two of any combination of the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, and / or HNF4a, or the polypeptides they encode. In still another embodiment, hepatic progenitor cells express at least Fetal protein (AFP), albumin (ALB), cytokeratin 7 (CK7), cyto keratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX , and / or at least three of any combination of HNF4a, or a polypeptide encoded by them is expressed. In yet another embodiment, the hepatic progenitor cells are the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7( CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpC AM, HHEX, and / or at least four of any combination of HNF4a , or a polypeptide encoded by them is expressed. In yet another embodiment, the hepatic progenitor cells express at least five of the following genes: alpha-fetoprotein (AFP), albumin (ALB), cyto keratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PR OX1, EpCAM, HHEX, and / or any combination of HNF4a within. In yet another embodiment, the hepatic progenitor cells express the following genes: alpha- fetal protein (AFP), albumin (ALB), cytokeratin 7 (CK7), sa cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHE X, and / or at least six or more polypeptides encoded by any combination of HNF4a . In yet another embodiment, the hepatic progenitor cells are the following genes: alpha-fetal protein (AFP), albumin (ALB), cytokeratin 7 (CK7), sa cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX . In yet another embodiment, the hepatic progenitor cells express the following genes: alpha-fetal , and / or, at least seven or more polypeptides encoded by any combination of HNF4a are expressed. In yet another embodiment, the hepatic progenitor cells are the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cyto keratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, , and / or, at least eight or more polypeptides encoded by any combination of HNF4a are expressed. In yet another embodiment, the hepatic progenitor cells are the following genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cyto keratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, and / or, at least nine or more polypeptides encoded by any combination of HNF4a are expressed. In yet another embodiment, the hepatic progenitor cells are the following genes (or the polypeptides encoded by them): alpha-fetoprotein (AFP), albumin (ALB ), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX 1, PROX1, EpCAM, HHEX, and / or, HNF4a are expressed. In some embodiments, expression is induced in the hepatic progenitor cells, and the following genes: alpha-fetoprotein ( AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 ( CK19), SOX9, PDX1, PROX1, and / or, HNF4a, or the expression levels of the polypeptides encoded by them can be identified by comparison with the expression levels of the same genes / polypeptides in posterior foregut cells. In certain embodiments, the hepatic progenitor cells express substantially the same amount of albumin as posterior foregut cells. In certain embodiments, the hepatic progenitor cells , and / or, HNF4a, or the polypeptides encoded by them , and the expression levels of the polypeptides encoded by them can be identified by comparison with the expression levels of the same genes / polypeptides in posterior foregut cells. In certain embodiments, the hepatic progenitor cells express substantially the same amount of albumin as posterior foregut cells. In certain embodiments, the hepatic progenitor cells express substantially the same amount of albumin as posterior foregut cells. In certain embodiments, the hepatic progenitor The cells express substantially the same amount of AFP as the posterior foregut cells. In certain embodiments, the hepatic progenitor cells express the CK19 gene more strongly than the posterior foregut cells. In certain embodiments, the hepatic progenitor cells express the CK7 gene more strongly than the posterior foregut cells. In certain embodiments, the hepatic progenitor cells express the PDX1 gene more strongly than the posterior foregut cells. In certain embodiments, the hepatic progenitor cells express the SOX9 gene more strongly than the posterior foregut cells. In certain embodiments, the hepatic progenitor cells express the PROX1 gene more strongly than the posterior foregut cells. In certain embodiments, the hepatic progenitor cells express the HHEX gene, but at a lower level than the posterior foregut cells. In certain embodiments, the hepatic progenitor cells, compared to undifferentiated pluripotent cells (such as iPSCs), express TRA-1-60, and / or substantially do not express the Nanog gene, or express these genes at very low levels and express.

[0032] The hepatic progenitor cells can be of any origin, and in particular can be derived from mammals, and in some embodiments can be derived from humans.

[0033] The second culture medium used in the second process can be serum-free (e.g., not supplemented with serum ). In another embodiment, the second culture medium used in the second process can contain serum, and the serum can be KnockOut Serum Replacem ent (trademark) (ThermoFisher Scientific). In certain embodiments, the second culture medium contains about 0.1 to about 5% (v / v) serum. In yet another embodiment the second culture medium contains at least about 0.1, 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 contains more than %. In another embodiment, the second culture medium is about 5, 4.5, 4, 3.5 , 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0 .3, contains serum of 0.2% or less. In yet another embodiment, the second culture medium is about 0. 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5% and about 5, 4.5, 4, 3.5, 3, 2 .5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or contains serum between 0.2%. In one embodiment, the second culture medium contains about 2% of the blood serum.

[0034] The second culture medium contains a second set of additives, the set including activators of the insulin signaling pathway, activators of the bone morphogenetic protein (BMP) signaling pathway, activators of the fibroblast growth factor (FGF) signaling pathway, activators of the hepatocyte growth factor (H GF) signaling pathway, and activators of the Wnt signaling pathway, or consisting essentially of them. The expression "the second culture medium is consisting essentially of the second set of additives" as used in the context of the present disclosure refers to a second culture medium that contains additional additives that are non-essential for the differentiation of posterior foregut cells into hepatic progenitor cells but can promote differentiation . These additional additives include, for example, but are not limited to, B27 supplement, retinoic acid, vitamins, and minerals. The second culture medium also contains an activator of the insulin signaling pathway. In the context of the present disclosure refers to a second culture medium that contains additional additives that are non-essential for the differentiation of posterior foregut cells into hepatic progenitor cells but can promote differentiation . These additional additives include, for example, B27 supplement, retinoic acid, vitamins, and minerals, but are not limited thereto.

[0035] The second culture medium also contains an activator of the insulin signaling pathway. In the context of the present disclosure The "activators of the insulin signaling pathway" used herein are insulin and its cognate receptors. Activating signaling pathways associated with binding to receptors (tyrosine kinase receptors) This compound is a compound that can bind to the insulin receptor (insulin, IGF- agonists of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II), which activate the insulin signaling pathway. activators of polypeptides known to inhibit the insulin signaling pathway and / or The inhibitor may be any of the polypeptides known to inhibit the activity of The activating agent is insulin (which may be provided in recombinant or purified form). In embodiments where insulin is used as an inhibitor of the insulin signaling pathway, The sulin is added to the second culture medium at least about 1, 5, 10, 15, 20, 25, 30, 3 5, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and Insulin can be provided in amounts greater than ng / mL. In an embodiment where insulin is used as an inhibitor of 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 3 Available in concentrations of 0, 25, 20, 15, 10, 5, or less than sub-ng / mL Insulin can be used as an inhibitor of the insulin signaling pathway. In certain embodiments, insulin is present in the second culture medium at a concentration of about 1, 5, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, Or, 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, It can be provided at a concentration between 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin can be provided in a second culture medium at a concentration of about 10 mg / ml. In yet another embodiment, insulin is provided in the form of a B27 supplement, an HBM / HCM Bulletkit™, and / or in the form of a primary hepatocyte (PHH) supplement.

[0036] The second culture medium contains an activator of the bone morphogenetic protein (BMP) signaling pathway. During the growth process, the activator of the BMP signaling pathway is usually provided from the cardiac mesoderm and promotes the differentiation of endodermal cells into posterior foregut cells. As used in the context of the present disclosure, the "activator of the BMP signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of BMP to its cognate receptors (e.g., BMPR1 and / or BMPR2). The signaling BMP receptors occur via the SMAD and MAP kinase pathways and perform the transcription of BMP target genes. This compound can be an agonist of the BMP receptor (specific for BMPR1 or BMPR2 or binding to and activating both receptors), an activator of a polypeptide known to activate the BMP signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the BMP signaling pathway. Known BMPs include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7 BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. ​​​​However, it is not limited to these. In one embodiment, the activating factor is DM3189. In another embodiment, the activating factor is BMP4 (provided in recombinant form, or in purified form ). BMP4 is a member of the transforming growth factor-β (TGF-β) family and binds to two different types of serine-threonine kinase receptors known as BMPR1 and BMPR2. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway BMP4 can be provided in the second culture medium at a concentration of at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway BMP4 can be provided in the second culture medium at a concentration of less than at least about 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or less ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is in the first 2 culture medium and can be provided at a concentration between about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and about 30, 29 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 ng / mL. In some specific embodiments, BMP4 can be provided in the second culture medium at a concentration of about 20 ng / mL . In a further embodiment, BMP4 is used in both the first and second additive sets . . In some specific embodiments, BMP4 can be provided in the second culture medium at a concentration of about 20 ng / mL . In a further embodiment, BMP4 is used in both the first and second additive sets It can be provided as an activator in a square.

[0037] The second culture medium also contains an activator of the fibroblast growth factor (FGF) signaling pathway. During the growth process, the activator of the FGF signaling pathway is usually provided from the cardiac mesoderm and promotes the differentiation of endodermal cells into posterior foregut cells. As used in the context of the present disclosure, the "activator of the FGF signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of FGF to its cognate receptors (e.g., FGFR1, FGFR2, FGFR3, and / or FGFR4). This compound can be an agonist of the FGF receptor (which is specific for FGFR1, FGFR2, FGFR3, and / or FGFR4 or can bind to and activate multiple receptors), an activator of a polypeptide known to activate the FGF signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the FGF signaling pathway. Known FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8a, FGF8b, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, and FGF23. In certain embodiments, the activator is basic FGF or FGF2 (which can be provided in recombinant or purified form). FGF2 binds to two different types of receptors known as FGFR2 (also known as CD332) and FGFR3. Specific for FGFR1, FGFR2, FGFR3, and / or FGFR4, or Can bind to and activate multiple receptors) an agonist of the FGF receptor, An activator of a polypeptide known to activate the FGF signaling pathway, and / or Or, an inhibitor of a polypeptide known to inhibit the FGF signaling pathway, either Can be either. Known FGFs include FGF1, FGF2, FGF3, FGF4, FGF5 , FGF6, FGF7, FGF8a, FGF8b, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, F GF18, FGF20, FGF21, FGF22, and FGF23, but are not limited to these. In certain embodiments, the activator is basic FGF or FGF2 (which can be provided in recombinant form or purified form). FGF2 binds to two different types of receptors known as FGFR2 (also known as C D332) and FGFR3. Binds to two different types of receptors known as FGFR2 (also known as CD332) and FGFR3. In embodiments that provide basic FGF as an activator of the FGF signaling pathway, The active FGF is present in the second culture medium at a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 n is 0, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more Basic FGF can be provided at a concentration of 1000 mg / mL. In an embodiment in which basic FGF is provided as a activating factor, the second culture medium comprises at least Approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, May be provided in concentrations of 6, 5, 4, 3, 2, or less than ng / mL. In an embodiment in which basic FGF is provided as an activator of the FGF signaling pathway, The basic FGF is added to the second culture medium at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ,11, 12, 13, 14, 15, 16, 17, 18, or ,19 and about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 In certain embodiments, the base may be provided at a concentration of between 1 ng / mL and 2 ng / mL. Active FGF may be provided in the second culture medium at a concentration of about 10 ng / mL. Basic FGF acts as an activator in both the first and second additive sets. This can be provided.

[0038] The second culture medium also contains an activator of the hepatocyte growth factor (HGF) signaling pathway. During development, activators of the HGF signaling pathway mediate the differentiation of endoderm cells into hepatic progenitor cells. As used in the context of this disclosure, an "activator of the HGF signaling pathway" refers to an activator of the HGF signaling pathway that activates HGF. Signal transduction pathways associated with the binding of GFs to their cognate receptors (e.g., c-Met) Refers to a compound that can be activated. This compound is an agonist of the HGF receptor , an activator of a polypeptide known to activate the HGF signaling pathway, and / or or an inhibitor of a polypeptide known to inhibit the HGF signaling pathway . In certain embodiments, the activator is HGF (which may be provided in recombinant or purified form). In embodiments where HGF is provided as an activator of the HGF signaling pathway , HGF can be provided in the second culture medium at a concentration of at least about 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or greater ng / mL. In embodiments where HGF is provided as an activator of the HGF signaling pathway , HGF can be provided in the second culture medium at a concentration of less than at least about 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 2 9, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16 , 15, 14, 13, 12, 11, or less than ng / mL. In embodiments where HGF is provided as an activator of the HGF signaling pathway , HGF can be provided in the second culture medium at a concentration of about 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, , 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 and about 40, 3 , 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 , 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 and about 40, 3 , 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 , 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12. Or it can be provided at a concentration between 11 ng / mL. In some specific embodiments HGF can be provided to the second culture medium at a concentration of about 20 ng / mL.

[0039] The second culture medium further contains an activator of the Wnt signaling pathway. In some embodiments it is important to activate the Wnt signaling pathway in posterior foregut cells only when inhibition has already occurred (e.g., as shown in the first process). As used in the context of this disclosure, the "activator of the Wnt signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of a Wnt protein ligand to its cognate Frizzled receptor (e.g., FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10). The family of Frizzled receptors are G protein-coupled receptor proteins. This compound can be an agonist of the Frizzled receptor (specific for any one of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, or FZD10, or capable of binding to and activating multiple receptors), an activator of a polypeptide known to activate the Wnt signaling pathway, and / or it can be an inhibitor of a polypeptide known to inhibit the Wnt signaling pathway, either specific for one of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, or FZD10, or capable of binding to and activating multiple receptors). Known Wnt proteins include WNT1, WNT2, WNT2B, WNT3 WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, W NT11, etc. WNT11, etc. WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, W There are NT11 and WNT16, but not limited to these. In certain embodiments, the activator is Wnt3a, SB-216763, and / or LY2090314. In certain embodiments, the activator is capable of inhibiting the biological activity of the GSK3 protein. For example, the activator that can inhibit the biological activity of the GSK3 protein can be CHIR99021. In embodiments where CHIR99021 is used as an activator of the Wnt signaling pathway, CHIR99021 can be provided in the second culture medium at a concentration of at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5 .5, 6, 6.5, 7, 7.5 μM, or a concentration exceeding that. . In embodiments where CHIR99021 is used as an activator of the Wnt signaling pathway , CHIR99021 can be provided in the second culture medium at a concentration less than 8, 7.5, 7, 6.5, 6, 5.5 , 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or less than μM. In embodiments where CHIR99021 is used as an activator of the Wnt signaling pathway, CHIR99021 can be provided in the second culture medium at a concentration between about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5 and about 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3. 5, 3, 2.5, 2, 1.5, or 1 μM. In embodiments where CHIR99021 is used as an inhibitor of the Wnt signaling pathway, CHIR99021 can be provided in the second culture medium at a concentration of about 3 μM.

[0040] The second culture medium is brought into contact with the posterior foregut cells and the hepatic progenitor cells for at least one day or more in order to enable differentiation. If it is intended to bring the second culture medium into contact with the cultured cells for two days or more, the medium can be exchanged daily. In some embodiments of the process of the present disclosure, the second culture medium is brought into contact with the cultured cells for at least 1, 2, 3, 4, or more days. In another embodiment, the second culture medium is brought into contact with the cultured cells for 5, 4, 3, 2, or fewer days. In yet another embodiment, the second culture medium is brought into contact with the cultured cells for at least 1, 2, 3, 4, or more days and then for 5, 4, 3, 2, or fewer days. In still another embodiment, the second culture medium is brought into contact with the cultured cells for about 1 day to 5 days. Using a second culture medium containing posterior foregut cells can differentiate the posterior foregut cells into hepatic progenitor cells. Thus, the present disclosure provides a population of hepatic progenitor cells obtained by the process described herein. In the population of hepatic progenitor cells of the present disclosure, most of the cells are considered to be hepatic progenitor cells, and in some embodiments, can include some hepatic progenitor cells. In one embodiment, the population of hepatic progenitor cells obtained by the second process comprises hepatic progenitor cells (e.g., which can be identified by determining the expression of CK19 or EpCAM) at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,

[0041] 96, 97, 98, or 99%. The present disclosure provides a third process for creating hepatocyte-like cells from hepatic progenitor cells.

[0042] ​​​​​​​This process enables the differentiation of hepatocyte progenitor cells into hepatocytes under conditions that allow such differentiation, and one or more hepatocyte progenitor cells are contacted with a third culture medium containing a third set of additives (which promotes the differentiation of hepatocyte progenitor cells into cells of the hepatocyte lineage), followed by contact with a fourth culture medium containing a fourth set of additives (which promotes the differentiation of cells of the hepatocyte lineage into immature cells), and then contact with a fifth culture medium containing a fifth set of additives (which promotes the differentiation of immature hepatocytes into mature hepatocytes). The hepatocyte progenitor cells used in the third process can be obtained by performing the first process and / or the second process as described herein. As used herein, the term "hepatocyte-like cells" generally refers to cells of the hepatocyte lineage, immature hepatocyte-like cells, and mature hepatocyte-like cells. Cells of the hepatocyte lineage cannot differentiate into cholangiocytes but can differentiate into hepatocytes. In some embodiments, hepatocyte-like cells (particularly, mature hepatocyte-like cells) can reproduce liver-specific functions such as the production of certain proteins (such as albumin, coagulation factors, alpha-1-antitrypsin, etc.), the detoxification of ammonia to urea, the metabolism of drugs, the storage of glycogen, the conjugation of bilirubin, and the synthesis of bile. One of ordinary skill in the art can identify hepatocyte-like cells using various techniques known in the art. For example, hepatocyte-like cells can be identified by determining the presence or absence, and the expression levels, of at least one of the following genes: alpha-fetoprotein (AFP), albumin (ALB), ASGR1, ASGPR, HNF4a, or SOX9, or any combination thereof, or the polypeptides encoded by them.

[0043] can be identified. In certain embodiments, the hepatocyte-like cells express the following genes: alpha-fetoprotein (AFP), albumin (ALB), ASGR1 (ASGPR), HNF4 alpha, and / or at least one of SOX9, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments, the hepatocyte -like cells express at least two of the following genes: alpha-fetoprotein (AFP), albumin (ALB), AS GR1 (ASGPR), HNF4alpha, and / or SOX9, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments, the hepatocyte -like cells express at least three of the following genes: alpha-fetoprotein (AFP ), albumin (ALB), ASGR1 (ASGPR), HNF4alpha, and / or SOX9, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments, the hepatocyte -like cells express at least four of the following genes: alpha-fetoprotein (AFP ), albumin (ALB), ASGR1 (ASGPR), HNF4alpha, and / or SOX9, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments the hepatocyte-like cells express the following genes: alpha-fetoprotein (AFP ), albumin (ALB), ASGR1 (ASGPR), HNF4alpha, and / or SOX9, or any combination thereof, or the polypeptides encoded thereby. In yet another embodiment, the hepatocyte-like cells express the following genes : alpha-fetoprotein (AFP), albumin (ALB), ASGR1 (ASGPR) , HNF4alpha, and / or at least one of SOX9, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments the hepatocyte-like cells express at least four of the following genes: alpha-fetoprotein (AFP), albumin (ALB ), ASGR1 (ASGPR), HNF4alpha, and / or SOX9, or any combination thereof, or the polypeptides encoded thereby. In certain embodiments the hepatocyte-like cells express the following genes: alpha-fetoprotein (AFP), albumin (AL B), ASGR1 (ASGPR), HNF4alpha, and / or SOX9, or the polypeptides encoded thereby. In yet another embodiment, the hepatocyte-like cells express the following genes : alpha-fetoprotein (AFP), albumin (ALB), ASGR1 (AS GPR), HNF4alpha, and / or at least one of SOX9, or any combination thereof of them detect the expression of any combination, or the polypeptides they encode, and can be identified by optionally measuring. In yet another embodiment, the expression is induced in hepatocyte-like cells, and the following genes: alpha-fetoprotein (AFP), albumin ( ALB), ASGR1, HNF4a, and / or at least one of SOX9, or detect the expression of one or more polypeptides encoded by any combination thereof, and can be identified by optionally measuring. In some embodiments, the expression is induced in hepatocyte-like cells, and the following genes: alpha-fetoprotein (AFP), albumin (ALB), ASGR1, HNF4a, and / or SOX9, or the expression levels of the polypeptides they encode, are compared with the expression levels of the same genes / polypeptides in hepatocytes (e.g., fetal hepatocytes, etc.) for identification. In certain embodiments, the hepatocyte-like cells strongly express the SOX9 gene, or the polypeptide it encodes, compared to the corresponding levels in fetal hepatocytes. In certain embodiments, the hepatocyte-like cells express HNF4a, AFP, ALB, and the ASGPR gene at substantially the same level compared to fetal hepatocytes. Mature hepatocyte-like cells can have a detectable level of CyP3A4, such as at least 10000 units per million cells, for example. In yet another embodiment, mature hepatocyte-like cells can have stronger CyP3A4 activity than immature hepatocyte-like cells. Mature hepatocyte-like cells can have, for example, at least about 5, 6, 7, 8, 9, 10, 11, 12 μg / L / 10 / 24 hours, or or or or or 6 or It is possible to produce detectable levels of albumin exceeding this. The mature hepatocyte-like cells can, for example, produce detectable levels of albumin of at least about 10, 100, or 1000 μg / L / 10 6 / 24 hours, or detectable levels of albumin exceeding this.

[0044] The hepatocyte-like cells can be of any origin, particularly those that can be derived from mammals and, in some embodiments, can be derived from humans.

[0045] The third, fourth, and fifth culture media used in the third process can be serum-free (e.g., without supplemented serum). In another embodiment, the third, fourth, and fifth culture media used in the third process can contain serum, and the serum can be Knock Out Serum Replacement (trademark) (ThermoFisherSc ientific). In certain embodiments, the third, fourth, and fifth culture media contain about 0.1 to about 5% (v / v) serum. In yet another embodiment, the third, fourth, and fifth culture media contain at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5% or more serum. In another embodiment, the third, fourth, and fifth culture media contain about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2% or less serum. In still another embodiment, the third, fourth, and fifth culture media contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5% and about 5 , 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6 , 0.5, 0.4, 0.3, or serum between 0.2%. In certain embodiments, The third culture medium contains about 2% serum. In another embodiment, the third culture medium is about 1% serum. In another embodiment, the fourth culture medium contains about 1% serum. In yet another embodiment, the fifth culture medium contains about 1% serum.

[0046] The third culture medium contains a third set of additives, which set includes activators of the insulin signaling pathway, activators of the bone morphogenetic protein (BMP) signaling pathway, activators of the fibroblast growth factor (FGF) signaling pathway, activators of the hepatocyte growth factor (H GF) signaling pathway, activators of the Wnt signaling pathway, inhibitors of the TGFβ signaling pathway, cytokines, and glucocorticoids, or consists essentially of them. The expression "the third culture medium consists essentially of the third set of additives" as used in the context of the present disclosure refers to a third culture medium that contains additional additives that are not essential for the differentiation of hepatocyte progenitor cells into hepatocyte-like cells, but can promote differentiation. These additional additives include, for example, but are not limited to, B27 supplement, primary hepatocyte supplement ( PHH), HBM / HCM Bulletkit™ retinoic acid, insulin, vi tamins, and minerals.

[0047] The third culture medium also includes an activator of the insulin signaling pathway. As used in the context of the present disclosure, "activator of the insulin signaling pathway" refers to insulin and its cognate receptor Activating a signal transduction pathway related to binding to a receptor (tyrosine kinase receptor) refers to a compound capable of this. This compound is an agonist of an insulin receptor (insulin, IGF- I, or IGF-II), an activator of a polypeptide known to activate the insulin signal transduction pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signal transduction pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium either of the inhibitors of polypeptides known to inhibit the insulin signal transduction pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less ng / mL. In embodiments where insulin is used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin is in the third culture medium used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, 15, 20, 25, 30, used as an activator of the insulin signal transduction pathway, insulin can be provided in the third culture medium at at least about 1, 5, 10, and can be provided at a concentration of about 10 mg / ml. In yet another embodiment, the ins ulin is provided in the form of a B27 supplement, HBM / HCM Bulletkit™, and / or in the form of a primary hepatocyte (PHH) supplement.

[0048] The third culture medium contains an activator of the bone morphogenetic protein (BMP) signaling pathway. During the growth process, the activator of the BMP signaling pathway is usually provided from the cardiac mesoderm, and promotes the differentiation of endodermal cells into posterior foregut cells. As used in the context of the present disclosure, the "activator of the BMP signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of BMP to its cognate receptors (e.g., BMPR1 and / or BMPR2). The signaling BMP receptors occur via the SMAD and MAP kinase pathways and effect the transcription of BMP target genes. This compound can be an agonist of the BMP receptor (specific for BMPR1 or BMPR2 or binding to and capable of activating both receptors), an activator of a polypeptide known to activate the BMP signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the BMP signaling pathway. Known BMPs include, but are not limited to, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. In one embodiment, the activator is DM3189. In another embodiment, the activator is BMP4 (provided in recombinant or purified form). is possible). BMP4 is a member of the transforming growth factor-β (TGF-β) family and binds to two different types of serine-threonine kinase receptors known as BMPR1 and BMPR2 . In embodiments where BMP4 is provided as an activator of the BMP signaling pathway , BMP4 can be provided in the third culture medium at a concentration of at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or greater than ng / mL . In embodiments where BMP4 is provided as an activator of the BMP signaling pathway , BMP4 can be provided in the third culture medium at a concentration of less than at least about 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or less than ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway , BMP4 is in the third culture medium, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and about 30, 29 , 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 ng / mL can be provided at a concentration between . In some particular embodiments, BMP4 can be provided in the third culture medium at a concentration of about 20 ng / mL . In further embodiments, BMP4 can be provided as an activator in both the first, second, and third additive sets t.

[0049] The third culture medium also contains an activator of the fibroblast growth factor (FGF) signaling pathway During development, activators of the FGF signaling pathway are normally provided by the cardiac mesoderm. and promotes differentiation of endoderm cells into posterior foregut cells. An "activator of the FGF signaling pathway" refers to an activator of the FGF and its cognate receptor (e.g., F GFR1, FGFR2, FGFR3, and / or FGFR4) This refers to a compound that can activate the signal transduction pathway. specific for FGFR1, FGFR2, FGFR3, and / or FGFR4, or FGF receptor agonists (capable of binding to and activating multiple receptors); Activators of polypeptides known to activate the FGF signaling pathway, and / or or an inhibitor of a polypeptide known to inhibit the FGF signaling pathway. Known FGFs include FGF1, FGF2, FGF3, FGF4, and FGF5. , FGF6, FGF7, FGF8a, FGF8b, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, F There are FGF18, FGF20, FGF21, FGF22, and FGF23. In one embodiment, the activator is basic FGF or FGF2 (recombinant FGF2 is a cytoplasmic fibroblast growth factor (FGFR2) which is a fibroblast growth factor (FGF) receptor. D332) and FGFR3. In embodiments that provide basic FGF as an activator of the FGF signaling pathway, The active FGF is present in the first culture medium at a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, or n greater than or equal to that It can be provided at a concentration of g / mL. In embodiments where basic FGF is provided as an activator of the FGF signaling pathway, basic FGF is present in the first culture medium at at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than ng / mL. In embodiments where basic FGF is provided as an activator of the FGF signaling pathway, basic FGF can be provided in the first culture medium at a concentration less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, or 19 and between about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 , 2 ng / mL. In some specific embodiments, basic FGF can be provided to the third culture medium at a concentration of about 10 ng / mL. In further embodiments , basic FGF can be provided as an activator in both the second and third additive sets. The third culture medium also contains an activator of the hepatocyte growth factor (HGF) signaling pathway. During the growth process, the activator of the HGF signaling pathway promotes the differentiation of endodermal cells into hepatocyte lineage cells. As used in the context of the present disclosure, an "activator of the HGF signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of HGF to its cognate receptor (e.g., c-Met). This compound is an agonist of the HGF receptor, an activator of a polypeptide known to activate the HGF signaling pathway,

[0050] The third culture medium also contains an activator of the hepatocyte growth factor (HGF) signaling pathway. During the growth process, the activator of the HGF signaling pathway promotes the differentiation of endodermal cells into hepatocyte lineage cells. As used in the context of the present disclosure, an "activator of the HGF signaling pathway" refers to a compound that can activate the signaling pathway associated with the binding of HGF to its cognate receptor (e.g., c-Met). This compound is an agonist of the HGF receptor, an activator of a polypeptide known to activate the HGF signaling pathway, refers to a compound that can activate the signaling pathway associated with the binding of HGF to its cognate receptor (e.g., c-Met). This compound is an agonist of the HGF receptor, an activator of a polypeptide known to activate the HGF signaling pathway, The signaling pathway can be a compound that can activate the signaling pathway associated with the binding of HGF to its cognate receptor (e.g., c-Met). This compound is an agonist of the HGF receptor, an activator of a polypeptide known to activate the HGF signaling pathway, an activator of a polypeptide known to activate the HGF signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the HGF signaling pathway It may be any of them. In certain embodiments, the activator is HGF (in recombinant form, or provided in purified form). When HGF is provided as an activator of the HGF signaling pathway in an embodiment, HGF can be provided in the third culture medium at a concentration of at least about 10, 11, 1 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or greater than ng / mL. When HGF is provided as an activator of the HG F signaling pathway in an embodiment, HGF can be provided in the third culture medium at a concentration of less than at least about 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 3 0, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17 , 16, 15, 14, 13, 12, 11, or less than ng / mL can be provided. When HGF is provided as an activator of the HGF signaling pathway in an embodiment, HGF can be provided in the third culture medium at about 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 and about 4 0, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 ng / mL. In some specific embodiments, HGF can be provided in the third culture medium at a concentration of about 20 ng / mL. HGF can be an activating factor in the second and third additive sets.

[0051] The third culture medium further contains an activating factor of the Wnt signaling pathway. Related to the present disclosure The "activating factor of the Wnt signaling pathway" used herein refers to a compound that can activate the signaling pathway related to the binding of a Wnt protein ligand and its homologous Frizzle receptor (e.g., FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10). The Frizzl ed receptor family is a G protein-coupled receptor protein. This compound can be an agonist of the Frizzled receptor (specific to any one of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, or FZD10, or can bind to and inhibit multiple receptors), an activating factor of a polypeptide known to activate the Wnt t signaling pathway, and / or an inhibitory factor of a polypeptide known to inhibit the Wnt signaling pathway. Known Wnt proteins include, but are not limited to, WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WN T11, and WNT16. In certain embodiments, the activating factor is Wnt3a, SB-216763, and / or LY2090314. In certain embodiments, the activating factor inhibits the biological activity of the GSK3 protein and can be one of them. In certain embodiments, the activating factor inhibits the biological activity of the GSK3 protein It is possible. For example, an activator that can inhibit the biological activity of GSK3 protein can be CHIR99021. In an embodiment where CHIR99021 is used as an activator of the Wnt signaling pathway, CHIR99021 can be provided in the third culture medium at a concentration of at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5. 5, 6, 6.5, 7, 7.5 μM, or a concentration exceeding that. In an embodiment where CHIR99021 is used as an activator of the Wnt signaling pathway CHIR99021 can be provided in the third culture medium at a concentration less than 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or less than 1 μM. In an embodiment where CHIR99021 is used as an activator of the Wnt signaling pathway, CHIR99021 can be provided in the third culture medium at a concentration between about 0 .5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 and about 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5 , 3, 2.5, 2, 1.5, or 1 μM. In an embodiment where CHIR99021 is used as an inhibitor of the Wnt signaling pathway, CH IR99021 can be provided in the third culture medium at a concentration of about 3 μM. In certain embodiments , CHIR99021 can be an activator in the second and third additive sets. The third culture medium further contains an inhibitor of the transforming growth factor β (TGFβ) signaling pathway. The presence of an inhibitor of the TGFβ signaling pathway inhibits the Wnt signaling pathway.

[0052] When combined with a harmful factor, it favorably acts on the expression of HEX and PROX1 genes that encode polypeptides necessary for liver development. The "inhibitor of the TGFβ signaling pathway" used in the context of the present disclosure refers to a compound that can inhibit the signaling pathway related to the binding of TGFβ and its cognate receptor. The family of TGFβ receptors mediates signal transduction via SMAD proteins. This compound can be an antagonist of the TGFβ receptor, an inhibitor of a polypeptide known to activate the TGFβ signaling pathway, and / or an activator of a polypeptide known to inhibit the TGFβ signaling pathway. Known TGFβ proteins include, but are not limited to, TGFB1, TGFB2, TGFB3, and TGFB4. In certain embodiments, the inhibitor can inhibit at least one biological activity of the ALK4, ALK5, or ALK7 polypeptide. In some embodiments, the inhibitor can inhibit the biological activities of the ALK4, ALK5, and ALK7 polypeptides. For example, an inhibitor that can inhibit the biological activities of the ALK4, ALK5, and ALK7 polypeptides can be A83-01. Alternatively, or in combination, SB431542 and / or LY364947 can be used as the inhibitor. In embodiments where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 is present in the third culture medium at at least 0.1, 0.2, 0.3, 0.4 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1 .5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5 μM, or ​​ or can be provided at a concentration exceeding that. In an embodiment where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 can be provided in the third culture medium at 5 , 4.5, 4, 3.5, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1 .4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 .3, 0.2 μM, or at a concentration less than that. In an embodiment where A83- 01 is used as an inhibitor of the TGFβ signaling pathway, A83-0 1 can be provided in the third culture medium at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1. 8, 1.9, 2, 2.5, 3, 3.5, 4, or 4.5 and between about 5, 4.5, 4, 3. 5, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1. 2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 μ M. In an embodiment where A83-01 is used as an inhibitor of the TGFβ signaling pathway, A83-01 can be provided in the second culture medium at a concentration of about 1 μM. In some embodiments, A83-01 can be an inhibitor in the first and third additive sets. The third culture medium also includes cytokines such as, for example, oncostatin M (OSM). In an embodiment where oncostatin M is used as a cytokine, oncostatin M is in the third culture medium at at least 10, 11, 12, 13, 14, 15, 16, 17, 18

[0053] ​​​, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 ng / ml, or it can be present at a concentration equal to or higher than that. In embodiments where oncostatin M is used as a cytokine, oncostatin M is in the third culture medium at 30, 29, 28 , 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 ng / ml, or at a concentration less than or even lower than that. In embodiments where oncostatin M is used as a cytokine, oncostatin M is in the third culture medium at about 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 ng / ml. In certain embodiments, oncostatin M is present in the third culture medium at a concentration of about 20 ng / ml. The third culture medium further contains a glucocorticoid such as dexamethasone, for example. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is in the third culture medium at at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 μM, or it can be present at a concentration equal to or higher than that. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is in the third culture medium at 15, 14, 1 3, 12, 11, 10, 9, 8, 7, 6 μM, or at a concentration less than or even lower than that. In embodiments where dexamethasone is used as a glucocorticoid,

[0054] ​ Dexamethasone can be present in the third culture medium at a concentration between about 5, 6, 7, 8, 9, 10, 11, 12, 13 , or 14 and about 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 μ M. In certain embodiments, dexamethasone is present in the third culture medium at a concentration of about 1 0 μM.

[0055] The third culture medium is contacted with hepatocyte progenitor cells and cells of the hepatocyte lineage for at least 1 day or more to enable differentiation. If it is intended to contact the third culture medium with the cultured cells for 2 days or more, the medium can be changed daily. In some embodiments of the process of the present disclosure, the third culture medium is contacted with the cultured cells for at least 1, 2, 3, 4, or more days. In another embodiment, the third culture medium is contacted with the cultured cells for 5, 4, 3, 2, or less days. In yet another embodiment, the third culture medium is contacted with the cultured cells for at least 1, 2, 3, 4, or more days, and then for 5, 4, 3, 2, or less days. In still another embodiment, the third culture medium is contacted with the cultured cells for about 1 to 5 days.

[0056] Using a third culture medium containing posterior foregut cells can differentiate hepatocyte progenitor cells into cells of the hepatocyte lineage. Thus, the present disclosure provides a population of cells of the hepatocyte lineage obtained by the process described herein. In the population of cells of the hepatocyte lineage of the present disclosure, most of the cells are considered to be cells of the hepatocyte lineage, and in some embodiments, can include some hepatocyte progenitor cells and / or endodermal cells.

[0057] The fourth culture medium contains a fourth additive set, and the set contains activators of the insulin signaling pathway, cytokines, and glucocorticoids, or consists essentially of them. The expression "the fourth culture medium consists essentially of the fourth additive set" used in the context of the present disclosure refers to a fourth culture medium that contains additional additives that are non-essential for the differentiation of hepatocyte lineage immature hepatocyte-like cells but can promote differentiation. Examples of these additional additives include, but are not limited to, B27 supplement, primary hepatocyte supplement (PHH), HBM / HCM Bulletkit (trademark) retinoic acid, insulin, vitamins, and minerals. The fourth culture medium also contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin and its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin is present in the fourth culture medium at least at about 1, 5, 10, 15, 20, 25, 30,

[0058] ​​​​​​​​​​​​​​​​35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more ng / mL can be provided. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin can be provided in the fourth culture medium at a concentration of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than ng / mL. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin can be provided in the fourth culture medium at a concentration between about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin can be provided in the fourth culture medium at a concentration of about 10 mg / ml. In yet another embodiment, insulin is provided in the form of B27 supplement, HBM / HCM Bulletkit™, and / or primary hepatocyte (PHH) supplement. Alternatively, it can be provided at a concentration above that. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin can be provided in the fourth culture medium at a concentration of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than ng / mL. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin can be provided in the fourth culture medium at a concentration of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than ng / mL. 0, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 、30, 25, 20, 15, 10, 5, or less than ng / mL. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin can be provided in the fourth culture medium at a concentration of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than ng / mL. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin can be provided in the fourth culture medium at a concentration of about 1, 5, 10, 15, 20 、25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 and about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin can be provided in the fourth culture medium at a concentration of about 10 mg / ml. In yet another embodiment, insulin is provided in the form of B27 supplement, HBM / HCM Bulletkit™, and / or primary hepatocyte (PHH) supplement. The fourth culture medium includes cytokines such as, for example, oncostatin M (OSM). In embodiments where oncostatin M is used as a cytokine, oncostatin M is

[0059] The fourth culture medium includes cytokines such as, for example, oncostatin M (OSM). In embodiments where oncostatin M is used as a cytokine, oncostatin M is in the fourth culture medium at at least 10, 11, 12, 13, 14, 15, 16, 17, 18 、19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 ng / ml, or more. Alternatively, it can be present at a concentration above that. In embodiments where oncostatin M is used as a cytokine, oncostatin M is present in the fourth culture medium at a concentration of 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 ng / ml, or at a concentration less than or even lower than that. In embodiments where oncostatin M is used as a cytokine, oncostatin M is present in the fourth culture medium at a concentration of about 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 ng / ml. In certain embodiments, oncostatin M is present in the fourth culture medium at a concentration of about 20 ng / ml. The fourth culture medium further contains a glucocorticoid such as dexamethasone, for example. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fourth culture medium at a concentration of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 μM, or at a concentration above that. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fourth culture medium at a concentration of 15, 14, 1 3, 12, 11, 10, 9, 8, 7, 6 μM, or at a concentration less than or even lower than that. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fourth culture medium at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13

[0060] ​ or be present at a concentration between 14 and about 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 μ M. In certain embodiments, dexamethasone is present in the fourth culture medium at a concentration of about 1 0 μM.

[0061] The fourth culture medium is contacted with cells of the hepatocyte lineage and immature hepatocyte-like cells for at least 1 day or more to allow for differentiation. If it is intended to contact the fourth culture medium with the cultured cells for 2 days or more, the medium can be changed daily. In some embodiments of the processes of the present disclosure, the fourth culture medium is contacted with the cultured cells for at least 1, 2, 3, 4, or or more days. In another embodiment, the fourth culture medium is contacted with the cultured cells for 5, 4, 3, 2, or less days. Further in another embodiment, the fourth culture medium is contacted with the cultured cells for at least 1, 2, 3, 4, or more days and then for 5, 4, 3, 2, or less days. In yet another embodiment, the fourth culture medium is contacted with the cultured cells for about 1 to 5 days and then left in contact. Using a fourth culture medium containing posterior foregut cells can differentiate cells of the hepatocyte lineage into immature hepatocyte-like cells. Accordingly, the present disclosure provides a population of immature hepatocyte-like cells obtained by the processes described herein. In the population of immature hepatocyte-like cells of the present disclosure most of the cells are thought to be immature hepatocyte-like cells, and in some embodiments can include some cells of the hepatocyte lineage, hepatic progenitor cells, and / or endodermal cells.

[0062] Using a fourth culture medium containing posterior foregut cells can differentiate cells of the hepatocyte lineage into immature hepatocyte-like cells. Thus, the present disclosure provides a population of immature hepatocyte-like cells obtained by the processes described herein. In the population of immature hepatocyte-like cells of the present disclosure most of the cells are believed to be immature hepatocyte-like cells, and in some embodiments can include some cells of the hepatocyte lineage, hepatic progenitor cells, and / or endodermal cells. In some embodiments, it can include some cells of the hepatocyte lineage, hepatic progenitor cells, and / or endodermal cells. It can be possible.

[0063] The fifth culture medium contains a fifth additive set, which contains an activator of the insulin signaling pathway and a glucocorticoid, or consists essentially of them. The expression "the fifth culture medium consists essentially of the fifth additive set" as used in the context of the present disclosure refers to a fifth culture medium that contains additional additives that are non-essential for the differentiation of immature hepatocyte-like cells into mature hepatocyte-like cells, but can promote differentiation. These additional additives include, for example, but are not limited to, B27 supplement, primary hepatocyte supplement, retinoic acid, insulin, vitamins, HBM / HCM Bulletkit™, and melatonin. The fifth culture medium also contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" as used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin to its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, the insulin is present in the fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more. The fifth culture medium contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" as used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin to its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, the insulin is present in the fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more. The fifth culture medium contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" as used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin to its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, the insulin is present in the fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30,

[0064] 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more. The fifth culture medium contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" as used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin to its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, the insulin is present in the fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more. The fifth culture medium contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" as used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin to its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, the insulin is present in the fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more. The fifth culture medium contains an activator of the insulin signaling pathway. The "activator of the insulin signaling pathway" as used in the context of the present disclosure refers to a compound that can activate the signal transduction pathway associated with the binding of insulin to its cognate receptor (tyrosine kinase receptor). This compound can be an agonist of the insulin receptor (insulin, IGF-I, or IGF-II), an activator of a polypeptide known to activate the insulin signaling pathway, and / or an inhibitor of a polypeptide known to inhibit the insulin signaling pathway. In certain embodiments, the activator is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an activator of the insulin signaling pathway, the insulin is present in the fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more. or can be provided at ng / mL above that. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin is present in the fifth culture medium at a concentration of less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than ng / mL. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin is present in the fifth culture medium at a concentration of about 10 0, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 , 30, 25, 20, 15, 10, 5, or less than ng / mL. In embodiments where insulin is used as an activator of the insulin signaling pathway, insulin is present in the fifth culture medium at a concentration between about 1, 5, 10, 15, 20 and 90, or 95 and between about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In some specific embodiments, insulin can be provided in the fifth culture medium at a concentration of about 10 mg / ml. In yet another embodiment, insulin is provided in the form of B27 supplement, HBM / HCM Bulletkit™, and / or in the form of primary hepatocyte (PHH) supplement. The fifth culture medium further contains a glucocorticoid such as dexamethasone, for example. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone can be present in the fifth culture medium at a concentration of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 μM, or above that. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fifth culture medium at a concentration of 15, 14, 1 In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fifth culture medium at a concentration of 15, 14, 1 In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fifth culture medium at a concentration of 15, 14, 1 In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fifth culture medium at a concentration of 15, 14, 1

[0065] or above that. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fifth culture medium at a concentration of 15, 14, 1 or above that. or above that. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is present in the fifth culture medium at a concentration of 15, 14, 1 Present at a concentration of 3, 12, 11, 10, 9, 8, 7, 6 μM, or less than that and not even reaching that concentration This can be done. In embodiments where dexamethasone is used as a glucocorticoid, dexamethasone is in the fifth culture medium at about 5, 6, 7, 8, 9, 10, 11, 12, 13 , or between 14 and about 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 μ M. In certain embodiments, dexamethasone is present in the fifth culture medium at a concentration of about 1 0 μM. In certain embodiments, dexamethasone is present in the fifth culture medium at a concentration of about 10 μM.

[0066] The fifth culture medium is contacted with immature hepatocyte-like cells and mature hepatocyte-like cells for at least 1 day or more to enable differentiation. If it is intended to contact the fifth culture medium with the cultured cells for 2 days or more, the medium can be exchanged daily. In some embodiments of the process of the present disclosure, the fifth culture medium is contacted with the cultured cells for at least 1, 2, 3, 4, or more days. In another embodiment, the fifth culture medium is contacted with the cultured cells for 5, 4, 3, 2, or less days. Further in another embodiment, the fifth culture medium is contacted with the cultured cells for at least 1, 2, 3, 4, or more days and then for 5, 4, 3, 2, or less days. Still in another embodiment, the fifth culture medium is contacted with the cultured cells for about 1 day to 5 days . Using the fifth culture medium containing posterior foregut cells can differentiate immature hepatocyte-like cells into mature hepatocyte-like cells. Therefore, the present disclosure provides a process described herein .

[0067] cells. Thus, the present disclosure provides a process described herein for differentiating immature hepatocyte-like cells into mature hepatocyte-like cells using the fifth culture medium containing posterior foregut cells. Therefore, the present disclosure provides a process described herein ​To provide a population of obtained mature hepatocyte-like cells. In the population of mature hepatocyte-like cells of the present disclosure, most of the cells are considered to be mature hepatocyte-like cells, and in some embodiments , it can include some immature hepatocyte-like cells, cells of the hepatocyte lineage, hepatic progenitor cells, and / or endodermal cells.

[0068] Since the medium described herein can promote the formation of cholangiocytes, there is no particular need to include EGF.

[0069] The present disclosure provides a combination of the first, second, and / or third processes as disclosed herein. For example, the first process can be combined with the second process to create hepatic progenitor cells from endodermal cells. In another example, the second process can be combined with the third process to create hepatocyte-like cells from posterior foregut cells. In a further example, the first, second, and third processes can be combined to create hepatocyte-like cells from endodermal cells. The processes described herein can generate numerous hepatocyte-like cells that have strong biological activities (e.g., high Cyp3A4 activity, high albumin expression levels, and / or high urea production levels) and / or can metabolize therapeutic agents (or potential therapeutic agents). This particular embodiment is particularly useful in creating hepatocyte-like cells intended to be incorporated into encapsulated liver tissue, as described below.

[0070] The present disclosure also provides components for kits for creating posterior foregut cells, hepatic progenitor cells, and / or hepatocyte-like cells. Generally, the kit includes at least one as described herein. ​​​​​​​​​​​​​An additive set, or at least one culture medium described herein, any cells, and including instructions for carrying out the processes described herein. A kit for producing posterior foregut cells can include, for example, a first additive set, or a first culture medium, any endoderm cells, and instructions for carrying out the first process. A kit for producing hepatic progenitor cells can include, for example, a second additive set, or a second culture medium, any posterior foregut cells, and instructions for carrying out the second process. A kit for producing hepatocyte-like cells can include, for example, a third additive set or a third culture medium, a fourth additive set or a fourth culture medium, a fifth additive set or a fifth culture medium, any hepatic progenitor cells, cells of the

[0071] Encapsulated liver tissue Encapsulated liver tissue includes at least one (and in some embodiments, a plurality) of liver organoids at least partially coated with a biocompatible crosslinked polymer. As used in connection with the present disclosure, the term "liver organoid" refers to a mixture of cultured hepatocytes, mesenchymal cells, and endothelial cells, wherein the hepatocytes are obtained using the processes described herein. In some embodiments, the liver organoid includes a mixture of cultured hepatocytes, mesenchymal cells, and endothelial cells. Liver organoids generally have a spherical morphology and their surface can be In an embodiment, during the culture, it is produced and constructed from an extracellular matrix, hepatocytes, mesenchymal cells, and any optional endothelial cells. The liver organoids can be obtained by culturing the cells in suspension. In some embodiments, in particular, before culturing / differentiating the encapsulated liver tissue, the surface of the liver organoids is at least partially coated (substantially coated in some embodiments) with hepatocytes, such as hepatocytes and / or cholangiocytes. In another embodiment, the hepatocytes are dispersed throughout the cell core (but not necessarily uniformly). The organoids present in the encapsulated liver tissue are at least partially coated (substantially coated in some embodiments) with a first biocompatible crosslinked polymer.

[0072] The liver organoids before encapsulation do not contain exogenous extracellular matrix. The liver organoids are substantially composed of cultured hepatocytes, mesenchymal cells, and any optional endothelial cells. Furthermore, the liver organoids (either encapsulated in the first biocompatible polymer or not) exhibit liver function. For example, the liver organoids synthesize albumin and clotting factors, exhibit CyP3A4 activity, detoxify ammonia to urea, and are capable of metabolizing liver-specific drugs (i.e., tacrolimus or rifampicin).

[0073] The liver organoids of the present disclosure have a substantially spherical morphology and have a relative diameter in the micrometer range (e.g., its diameter is less than 1 mm). In certain embodiments, the liver organoids, before their encapsulation, are at least about 50, 60, 70, 80, 9 0, 100, 110, 120, 130, 140, 150, 160, 170, 180, 19 0, 200, 210, 220, 230, 240, 250, 260, 270, 280, 29 0, 300, 310, 320, 330, 340, 350, 360, 370, 380, 39 0, 400, 410, 420, 430, 440, 450, 460, 470, 480, and has a relative diameter of 490 μm. In yet another embodiment, the liver organoid before encapsulation is about 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or has a relative diameter of 60 μm or less . In another embodiment, the liver organoid, prior to encapsulation, is at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 280, 290, 300, 310, 320, 330, 340, 350, 360, 370 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 480, or 490 μm and about 500, 490, 480, 470, 460, 450 440, 430, 420, 410, 400, 390, 380, 370, 360, 350 340, 330, 320, 310, 300, 290, 280, 270, 260, 250 240, 230, 220, 210, 200, 190, 180, 170, 160, 150 and have a relative diameter with 140, 130, 120, 110, 100, 90, 80, 70, or 60 μm or less. In some embodiments, the liver organoid, prior to its encapsulation, has at least about 100, 110, 120, 130, 140, 150, 160, 170, 180 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290 μm and about 300, 290, 280, 270, 260, 250, 240 230, 220, 210, 200, 190, 180, 170, 160, 150, 140 130, 120, 110, 100, 90, 80, 70, or 60 μm or less. In yet another embodiment, the liver organoid prior to encapsulation is at least about 100 μm and has a relative diameter of about 300 μm or less. For example, prior to encapsulation the liver organoid is at least about 150, 160, 170, 180, or 190 μm and has a relative diameter smaller than 200, 190, 180, 170, or 160 μm In still further embodiments, the liver organoid prior to encapsulation is at least about 150 μm and has a relative diameter of about 200 μm or less. Depending on the size of the liver organoid the cells contained therein are increasingly exposed to various nutrients and the biological fluids / cells in contact with the encapsulated liver tissue In some embodiments, this eliminates the need for blood vessel formation of the liver organoid in the host's vascular system (e.g., the vasculature of the host in which the encapsulated liver tissue is transplanted), enabling it to survive in vivo and maintain a biologically active state

[0074] The hepatocytes of the liver organoid can be dispersed throughout the organoid. In some embodiments​​ In this state, some of them can be located on the surface of the cell core of the liver organoid. The hepatocytes of the liver organoid can be, for example, cells derived from embryonic endoderm, posterior foregut cells, cells of the hepatocyte lineage, or hepatocyte progenitor cells, or hepatocyte-like cells. The hepatocytes of the liver organoid can be hepatocyte-like cells and / or cholangiocyte cells. The hepatocytes of the liver organoid can be derived from a single cell type (e.g., embryonic endoderm cells, posterior foregut cells , cells of the hepatocyte lineage, hepatocyte-like cells, or cholangiocyte cells), or from a mixture of cell types (e.g., a mixture of at least two of the following cell types: embryonic endoderm cells, posterior foregut cells, cells of the hepatocyte lineage , hepatocyte-like cells, and / or cholangiocyte cells). During in vitro cell culture of the liver organoid, or when the liver organoid is transplanted in vivo, the hepatocyte type(s) can change or the hepatocytes can differentiate. For example, the hepatocytes of the liver organoid can differentiate (from embryonic endoderm, posterior foregut, or cells of the hepatocyte lineage into hepatocyte-like cells or cholangiocyte cells) while co-culturing with mesenchymal cells and any epithelial cells, or when transplanted in vivo. To determine whether hepatocyte-like cells are present in the liver organoid, the activity of cytochrome P450 family 3 subfamily A member 4 (CyP3A4) can be determined by means known in the art. To determine the presence or absence of hepatocyte-like cells in the liver organoid, the synthesis / production of albumin, clotting factors, and urea, as well as the activity of CyP3A4 can also be monitored. For embryonic endoderm in the liver organoid ​​​​​Alternatively, to determine the presence or absence of posterior foregut cells, the expression of SOX17, FOXA2, CXCR4 , and GATA4 can be determined by means known in the art.

[0075] The mesenchymal cells of the liver organoids can be, for example, mesenchymal stem / progenitor cells, adipocytes, myocytes, hepatic stellate cells, myofibroblasts, and / or fibroblasts of different origins (bone marrow (including blood), umbilical cord, or adipose tissue). The mesenchymal cells of the liver organoids can be of a single cell type (e.g., mesenchymal stem / progenitor cells, adipocytes, myocytes, or fibroblasts ), or can be derived from a mixture of cell types (e.g., a mixture of at least two of the following cell types: mesenchymal stem / progenitor cells, adipocytes, myocytes, hepatic stellate cells, myofibroblasts, and / or fibroblasts). The type of mesenchymal cells of the liver organoids can differentiate (from mesenchymal stem / progenitor cells to fibroblasts, adipocytes, or myocytes) during co-culture with hepatocytes and any endothelial cells, or upon transplantation in vivo. Mesenchymal stem / progenitor cells are known to express, among other genes, α-smooth muscle actin (α SMA), fibronectin, CD90, and CD73. To determine the position or presence of mesenchymal cells in liver organoids, the expression of genes or proteins that are, among others, specific to the mesenchymal lineage or associated with the mesenchymal lineage can be determined. The endothelial cells of the liver organoids, when present, can be, for example, endothelial progenitor cells of various origins, and / or endothelial cells. The endothelial cells of the liver organoids can be of a single cell type. Among a number of genes, mesenchymal stem / progenitor cells are known to express, in particular, α-smooth muscle actin (α SMA), fibronectin, CD90, and CD73. To determine the position or presence of mesenchymal cells in liver organoids, the expression of genes or proteins that are, among others, specific to the mesenchymal lineage or associated with the mesenchymal lineage can be determined. Among others, genes or proteins that are specific to the mesenchymal lineage or associated with the mesenchymal lineage can be determined. The endothelial cells of the liver organoids, when present, can be, for example, endothelial progenitor cells of various origins,

[0076] and / or endothelial cells. The endothelial cells of the liver organoids can be of a single cell type. Derived from a cell type (e.g., endothelial progenitor cells, or endothelial cells), or a mixture of cell types (e.g., a mixture of endothelial progenitor cells and endothelial cells). The endothelial cell type of the liver organoid can differentiate (from endothelial progenitor cells to endothelial cells) during in vitro co - culture of endoderm cells and mesenchymal cells, or upon in vivo transplantation. In some embodiments, the endothelial cells of the liver organoid can organize into capillaries or capillary - like structures where endothelial cells line the inner surface of the lumen (which can be assumed to be partial).

[0077] As described above, the cell core of the liver organoid is composed of hepatocytes, mesenchymal cells, and any endothelial cells, and in some embodiments, consists of an extracellular matrix produced and constructed by cells during culture. The cell core of the liver organoid has substantially few necrotic cells / apoptotic cells (e.g., the cell core of the liver organoid has no necrotic regions when examined histologically), because nutrients can diffuse from the medium in which the liver organoid is cultured throughout the cell core, thereby delivering nutrients to the cells within the cell core, and furthermore, because the metabolic waste products of the cells in the cell core can diffuse outside the liver organoid. The liver organoid itself (before encapsulation) does not contain exogenous extracellular matrix or synthetic polymer materials (e.g., they are not present). In some embodiments, hepatocytes can be present on the surface of the cell core. In another embodiment, hepatocytes can produce and construct extracellular matrix materials (e.g., collagen and fibronectin) in combination with the cells of the cell core, and in addition (e.g., collagen, and fibronectin). Thus, in some embodiments, the basement membrane material can also be produced and constructed.

[0078] As described above, hepatocytes can coat at least partially the surface of the cell core of the liver organoid. In the context of the present disclosure, the expression "hepatocytes coat at least partially the surface of the cell core" means that hepatocytes occupy at least about 10%, 20%, 30%, or 40% of the surface of the cell core. In some embodiments, hepatocytes substantially coat the surface of the cell core. In the context of the present disclosure, the expression "hepatocytes substantially coat the surface of the cell core" means that hepatocytes occupy most of the surface of the cell core, for example, at least about 50%, 60%, 70%, 80%, 9 0%, 95%, 99% of the surface of the cell core. In certain embodiments, hepatocytes completely coat the surface of the cell core (e.g., coat more than 99% of the surface of the cell core with hepatocytes). In certain embodiments, the liver organoids of the present disclosure, before being encapsulated with the first crosslinked biocompatible polymer, have a higher proportion of mesenchymal cells (and, if present, endothelial cells) than hepatocyte-like cells and / or cholangiocyte epithelial cells, when compared to those found in the mammalian liver. However, after being encapsulated with the first crosslinked biocompatible polymer, the liver organoids of the present disclosure have a higher proportion of hepatocytes than mesenchymal cells (and, if present, endothelial cells).

[0079] It is known that about 90% of the mammalian liver is composed of hepatocytes. Thus, in some embodiments of the present disclosure, the proportion of hepatocytes in the liver organoid is about 90%, about 85%, about 80%, or about 75% (when compared to the total number of cells in the liver organoid). less than.​​​​​​

[0080] Liver organoids can be created from cells of different origins. In certain embodiments at least one of hepatocytes, mesenchymal cells, or endothelial cells is from a mammal, such as a human In another embodiment, at least two of hepatocytes, mesenchymal cells, or endothelial cells are from a mammal, such as a human. In yet another embodiment, hepatocytes, mesenchymal cells, and endothelial cells are all from a mammal, such as a human. Within the liver organoid, cells from different origins can be combined. For example, mesenchymal cells and endothelial cells can be from mouse or pig origin while hepatocytes are from human origin . Such combinations are not exhaustive and one of ordinary skill in the art can envision additional combinations that are suitable in the context of the present disclosure .

[0081] The cells of the liver organoid can be from different sources. For example, the cells of the liver organoid can be from primary cell cultures, established cell lines, or differentiated stem cells . Within the liver organoid, cells from different sources can be combined . For example, hepatocytes can be from primary cell cultures, mesenchymal cells can be from established cell lines, and endothelial cells can be from differentiated cell lines . Alternatively, within the liver organoid, cells from the same source (e.g., differentiated stem cells ) can be combined. This embodiment is particularly useful because cells can be obtained from a single cell source (e.g , stem cells) to create encapsulated liver tissue . In certain embodiments, the cells of the liver organoid are hepatocytes, mesenchymal cells, and optionally any inner derived from a single population of stem cells that have differentiated into epithelial cells. The population of stem cells can be derived from embryonic stem cells or induced pluripotent stem cells. In certain embodiments, the cells of the liver organoid are derived from a single population of pluripotent stem cells that have differentiated into hepatocytes, mesenchymal cells, and optionally endothelial cells.

[0082] A polymer (also known as a polymer matrix) that can be used in encapsulated liver tissue forms a hydrogel around the liver organoid(s). As is well known in the art, a hydrogel refers to a hydrophilic polymer chain in which water is the dispersion medium. Hydrogels can be obtained from natural or synthetic polymer networks. In the context of the present disclosure, encapsulation in a hydrogel prevents the encapsulated liver organoids from leaking out of the polymer, so that the cells of the liver organoids are excluded or inhibited from causing an immune reaction or tumor in the recipient's body during transplantation. In certain embodiments, the liver organoids are each individually encapsulated, and the encapsulated liver organoids are further included in a polymer matrix in another embodiment. In yet another embodiment, the liver organoids are incorporated into the polymer matrix in a manner that encapsulates them.

[0083] In the context of the present disclosure, a polymer is considered to be "biocompatible" if it does not exhibit toxicity when introduced into a subject (e.g., a human). In the context of the present disclosure, a biocompatible polymer preferably does not exhibit toxicity to the cells of the liver organoid or when transplanted in vivo into a subject (e.g., a human). Hepatotoxicity can be, for example, the percentage of apoptotic death of hepatocyte-like cells (e.g., an increase in apoptosis indicates hepatotoxicity), ​ Transaminase levels (e.g., an increase in transaminase levels indicates hepatotoxicity), hypertrophy of hepatocyte-like cells (e.g., an increase in hypertrophy indicates hepatotoxicity), microvesicular degeneration in hepatocyte-like cells (e.g., an increase in degeneration indicates hepatotoxicity), the rate of bile duct cell death (e.g., an increase in the mortality rate of bile duct cells indicates hepatotoxicity), γ-glutamyl transpeptidase (GGT) levels (e.g., an increase in GGT levels indicates hepatotoxicity) can be determined and measured. As biocompatible polymers, carbohydrates (glycosaminoglycans such as hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, cellulose, etc., and / or their derivatives), proteins (collagen, elastin, fibrin, albumin, poly(amino acids), glycoproteins, antibodies, and / or their derivatives), and / or synthetic polymers (e.g., those based on poly(ethylene glycol) (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), and / or poly(vinyl alcohol) (PVA)) are available, but not limited to these. The biocompatible polymer can be a single polymer or a mixture of different polymers (e.g., those described in US2012 / 0142069). Examples of biocompatible polymers include poly(ethylene) glycol, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), fibrin, polysaccharide materials (such as chitosan, proteoglycan, or glycosaminoglycan (GAG)), alginate, collagen, thiolated ), etc. ), the rate of bile duct cell death (e.g., an increase in the mortality rate of bile duct cells indicates hepatotoxicity), γ-glutamyl transpeptidase (GGT) levels (e.g., an increase in GGT levels indicates hepatotoxicity) can be determined and measured. As biocompatible polymers, carbohydrates (glycosaminoglycans such as hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, cellulose, etc., and / or their derivatives), proteins (collagen, elastin, fibrin, albumin, poly(amino acids), glycoproteins, antibodies, and / or their derivatives), and / or synthetic polymers (e.g., those based on poly(ethylene glycol) (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), and / or poly(vinyl alcohol) (PVA)) are available, but not limited to these. The biocompatible polymer can be a single polymer or a mixture of different polymers (e.g., those described in US2012 / 0142069). Examples of biocompatible polymers include poly(ethylene) glycol, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), fibrin, polysaccharide materials (such as chitosan, proteoglycan, or glycosaminoglycan (GAG)), alginate, collagen, thiolated ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. ), etc. There are heparin and their mixtures, but not limited thereto. In some embodiments , the biocompatible polymer can be linear or branched, and optionally can incorporate peptides (e.g., RGD), growth factors, integrins, or drugs. .

[0084] In some embodiments, the polymer is a "low immunogenic polymer" that does not induce an immune response in the recipient or induces only a minimal immune response (i.e., as a result, this polymer will not undergo degradation, modification, or loss of function). This low immunogenic polymer blocks one or more antigenic determinants of cells and can suppress or even block the immune response to such antigenic determinants when introduced into allogeneic subjects.

[0085] The polymer present in the encapsulated liver tissue of the present disclosure is preferably crosslinkable, for example, can be crosslinked. The polymer can be crosslinked by heat, chemically (e.g., using one or more peptides such as VPMS, RGD), or by using pH or light (e.g., photopolymerization using UV light). In some embodiments, crosslinking can be performed after dispersing the liver organoid (regardless of the presence or absence of encapsulation by the polymer matrix) within the polymer matrix.

[0086] The polymer of the present disclosure can be wholly or partially biodegradable (e.g., easily hydrolyzed by the metabolism of the living body), or can show wholly or partially resistance to biodegradation (e.g., hydrolysis resistance when subjected to the metabolism . Examples of biocompatible and biodegradable polymers include, but are not limited to, poly(ethylene-glycol)-(maleimide) (PEG-Mal) 8-arms. Examples of biocompatible and biodegradation-resistant polymers include, but are not limited to, poly(ethylene-glycol)-vinyl sulfone (PEG-VS).

[0087] The encapsulated liver tissue comprises a first biocompatible crosslinked polymer that at least partially (in some cases, substantially) coats the liver organoid. The first biocompatible polymer physically contacts the cells of the liver organoid. In the context of the present disclosure, the expression "liver organoid(s) at least partially coated with a first biocompatible crosslinked polymer" refers to at least about 10%, 20%, 30%, or 40% of the surface of the liver organoid being occupied by the first biocompatible crosslinked polymer. In some embodiments, the first biocompatible crosslinked polymer substantially coats the surface of the liver organoid(s). In the context of the present disclosure, the expression "liver organoid(s) substantially coated with a first biocompatible crosslinked polymer" refers to the majority of the surface of the liver organoid being occupied by the first biocompatible crosslinked polymer, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% of the surface of the organoid being occupied by the first biocompatible crosslinked polymer. In certain embodiments, the first biocompatible crosslinked polymer completely coats the surface of the liver organoid (e.g., coating more than 99% of the surface of the liver organoid with the first biocompatible crosslinked polymer).

[0088] In some embodiments, the encapsulated liver tissue comprises at least a portion of the first biocompatible crosslinked polymer also includes a second biocompatible crosslinked polymer that at least partially (in some cases, substantially) covers it is possible. The second biocompatible polymer is in physical contact with the first biocompatible crosslinked polymer and, in some embodiments, is in physical contact with the cells of the liver organoid. In the context of the present disclosure the expression "a first biocompatible crosslinked polymer at least partially covered by a second biocompatible crosslinked polymer" refers to at least about 10%, 20%, 30%, or 40% of the surface of the first biocompatible crosslinked polymer being occupied by the second biocompatible crosslinked polymer In some embodiments, the second biocompatible crosslinked polymer substantially covers the surface of the first biocompatible crosslinked polymer. In the context of the present disclosure, the expression "a first biocompatible crosslinked polymer substantially covered by a second biocompatible crosslinked polymer" refers to the second biocompatible crosslinked polymer occupying a majority of the surface of the first biocompatible crosslinked polymer and, for example, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% of the surface of the first biocompatible crosslinked polymer being occupied by the second biocompatible crosslinked polymer In certain embodiments, the second biocompatible crosslinked polymer completely covers the surface of the first biocompatible crosslinked polymer (e.g., covering more than 99% of the surface of the first biocompatible crosslinked polymer with the second biocompatible crosslinked polymer) In yet another embodiment, the second biocompatible crosslinked polymer forms a matrix in which liver organoids (at least partially covered with the first biocompatible crosslinked polymer) are dispersed In such embodiments, the liver organoids (at least partially covered with the first biocompatible crosslinked polymer) In one embodiment, the second biocompatible crosslinked polymer completely covers the surface of the first biocompatible crosslinked polymer (e.g., covering more than 99% of the surface of the first biocompatible crosslinked polymer with the second biocompatible crosslinked polymer) In yet another embodiment, the second biocompatible crosslinked polymer forms a matrix in which liver organoids (at least partially covered with the first biocompatible crosslinked polymer) are dispersed In such embodiments, the liver organoids (at least partially covered with the first biocompatible crosslinked polymer) also includes a second biocompatible crosslinked polymer that at least partially (in some cases, substantially) covers it is possible. The second biocompatible polymer is in physical contact with the first biocompatible crosslinked polymer and, in some embodiments, is in physical contact with the cells of the liver organoid. In the context of the present disclosure The (thing) can have a second biocompatible crosslinked matrix present around it, and alternatively, can be physically contacted with another liver organoid (at least partially coated with a first biocompatible crosslinked polymer). The encapsulated liver tissue can include a further biocompatible crosslinked polymer that coats the second biocompatible crosslinked polymer.

[0089] The first biocompatible crosslinked polymer and the second biocompatible crosslinked polymer can be the same or alternatively, different. In certain embodiments, the first biocompatible crosslinked polymer is at least partially (in some embodiments, completely) a biodegradable polymer. Combined or alternatively, the second biocompatible crosslinked polymer is at least partially (in some embodiments, completely) resistant to biodegradation. In yet another embodiment, the first biocompatible crosslinked polymer is a biodegradable polymer and the second biocompatible crosslinked polymer is resistant to biodegradation. In such embodiments, the first biocompatible crosslinked polymer can have a higher biodegradability (e.g., lower resistance to biodegradation) compared to the second biocompatible crosslinked polymer.

[0090] In some embodiments, the first biocompatible crosslinked polymer includes a plurality of liver organoids. In such embodiments, the encapsulated liver tissue can include at least about 50, 60 2 per cm , 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 35 0, 400, 450, or 500 liver organoids. In yet another embodiment, the encapsulated liver tissue can include a maximum of about 500, 450, 400, 3 2 per cm 50, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, or can include 50 liver organoids. In yet another embodiment, the encapsulated liver tissue is cm 2 per and contains about 50, 60, 70, 80, 90, 100, 125, 1 50, 175, 200, 250, 300, 350, 400, or 450 and about 500 , 450, 400, 350, 300, 250, 200, 175, 150, 125, 100 , 90, 80, 70, or a number of liver organoids between 60 and contains. In yet another embodiment the encapsulated liver tissue is cm 2 per and contains about 50 - 500 liver organoids . In another embodiment, the encapsulated liver tissue is cm 3 per and contains at least about 250, 300, 3 50, 400, 450, 500, 550, 600, 650, 700, 750, 800, 8 50, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500 , 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300 , 2400, or 2500 liver organoids. In yet another embodiment, the encapsulated liver tissue is cm 3 per and contains a maximum of about 2500, 2400, 2300, 2200, 21 00, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 13 00, 1200, 1100, 1000, 950, 900, 850, 800, 750, 70 0, 650, 600, 550, 500, 450, 400, 350, 300, or 25 0 liver organoids. In yet another embodiment, the encapsulated liver tissue is cm 3 per and contains about 250, 300, 350, 400, 450, 500, 550, 600, 650, 70 0, 750, 800, 850, 900, 950, 1000, 1100, 1200, 130 0, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 210 0, 2200, 2300, or 2400 and approximately 2500, 2400, 2300, 22 00, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 14 00, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 7 50, 700, 650, 600, 550, 500, 450, 400, 350, or 3 00 and contains a number of liver organoids between. In yet another embodiment, the encapsulated liver tissue is cm 3 per and contains approximately 250 to 2500 liver organoids.

[0091] In certain embodiments, when the encapsulated liver tissue is cultured or transplanted in vivo, hepatocytes , mesenchymal cells, and genes and proteins associated with any endothelial cells can be expressed . In further embodiments, the encapsulated liver tissue (in vitro or in vivo) can produce albumin, synthesize urea from ammonia, exhibit CyP3A4 activity , and / or metabolize drugs (such as tacrolimus and / or rifampicin). In some embodiments, the encapsulated liver tissue can produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg of albumin per gram of liver organoids within the tissue . In another embodiment, the encapsulated liver tissue can express genes and proteins associated with hepatocytes, mesenchymal cells, and any endothelial cells, produce albumin, in one or more freeze - thaw cycles. . cells, and genes and proteins associated with any endothelial cells, albumin Producing min, synthesizing urea from ammonia, and exhibiting CyP3A4 activity , and / or metabolizing drugs (such as tacrolimus and / or rifampicin, which are known to undergo metabolism in the liver) can be performed. In some embodiments, the encapsulated liver tissue after freezing, per gram of liver organoids in the tissue, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 2 0 mg of albumin can be produced.

[0092] Process for creating encapsulated liver tissue In the process for creating encapsulated liver tissue, first, it is necessary to create liver organoid(s), and then it is necessary to encapsulate this (these) liver organoid(s) at least partially with a first biocompatible crosslinking polymer (optionally further encapsulating with a second biocompatible crosslinking polymer and additional biocompatible crosslinking polymers).

[0093] Liver organoids can be created by co - culturing hepatocytes, mesenchymal cells, and optional endothelial cells (all as described above) under conditions necessary to obtain liver organoids having (i) a cell core containing hepatocytes, mesenchymal cells, and optional endothelial cells, (ii) a substantially spherical morphology, and (iii) a relative diameter of about 50 to about 500 μm. In some embodiments these conditions include suspension culture of cells (e.g., ultra - low attachment conditions) to promote the formation of liver organoids.

[0094] Hepatocytes to be included in the encapsulated liver tissue can be of different origins (e.g., mammalian) and sources (primary cells) provided they are subjected to at least one of the processes described herein. It can be obtained from (cell culture, cell line, differentiated stem cells). Hepatocytes are embryonic endoderm cells, hindgut foregut cells, cells of the hepatocyte lineage, hepatocyte-like cells, and / or different types such as cholangiocyte epithelial cells can be derived from. Hepatocytes derived from a single organoid can be of the same or different origin, the same or different sources, and the same or different types. .

[0095] Mesenchymal cells included in the encapsulated liver tissue can be obtained from different origins (e.g., mammals) and sources ( primary cell culture, cell line, differentiated stem cells). Mesenchymal cells can be derived from different types such as mesenchymal stem cells, adipocytes, myocytes, or fibroblasts. Mesenchymal cells derived from a single organoid can be of the same or different origin, the same or different sources, and the same or different types. In certain embodiments, mesenchymal stem cells / progenitor cells are used. In yet another embodiment, mesenchymal stem cells / progenitor cells are obtained from differentiated stem cells (such as pluripotent stem cells). In yet another embodiment, mesenchymal stem cells / progenitor cells are obtained from differentiated pluripotent stem cells (e.g., by culturing pluripotent stem cells in uncoated plastic in DMEM high in KnockOut Serum Replacement). Mesenchymal cells can be used fresh or cryopreserved until used for forming liver organoids.

[0096] Endothelial cells, if present, included in the encapsulated liver tissue can be obtained from different origins (e.g., mammals) and sources (primary cell culture, cell line, differentiated stem cells). Endothelial cells can be derived from different types such as endothelial progenitor cells and endothelial cells. In an embodiment, endothelial progenitor cells are used. Endothelial cells derived from a single organoid can be from the same or different origins, the same or different sources, and the same or different types. In yet another embodiment, the endothelial progenitor cells are obtained from differentiating stem cells (such as pluripotent stem cells). In yet another embodiment, the endothelial progenitor cells are obtained from differentiating pluripotent stem cells (for example, obtained by culturing pluripotent stem cells in combination with BMP4, bFGF, and / or VEGF, CHIR 99021, and / or Activin A). The endothelial cells can be used fresh or cryopreserved until used to form liver organoids.

[0097] In one embodiment, liver organoids are prepared from a single population of pluripotent stem cells. The pluripotent stem cells can be induced using methods known in the art, such as viral transduction (for example, using a Sendai virus system), or using a synthetic mRNA approach. The population of pluripotent stem cells can be obtained from more than one colony of induced pluripotent stem cells (iPSCs). In an embodiment where liver organoids are prepared from the same population of pluripotent stem cells, the iPSC population is split into at least two (and in some embodiments, at least three) subpopulations, each of which is subjected to different culture conditions for generating hepatocytes and mesenchymal cells (and in some embodiments, endothelial cells). Once each of the different cells is obtained, these cells are mixed and suspension-cultured to generate liver organoids. To control the size of the liver organoids, 100 - 100

[0098] Once each of the different cells is obtained, these cells are mixed and suspension-cultured to generate liver organoids. To control the size of the liver organoids, 100~100 Ultra-low adhesion conditions (e.g., suspension) using microcavities having a diameter of 0 μm allow cells to be cultured. In some embodiments, 2 per cm, there are multiple microcavities having a diameter and depth of about 500 μm. In some embodiments, once the initial liver organoids are formed, such liver organoids can be suspension-cultured (for growth purposes) in a bioreactor. In certain embodiments, hepatocytes and mesenchymal cells are mixed prior to culturing at a ratio of 1 endodermal cell to 0.1 - 0.7 mesenchymal cells. In yet another embodiment, when endothelial cells are present, endothelial cells and endodermal cells are mixed prior to culturing at a ratio of 1 endodermal cell to 0.2 - 1 endothelial cells. In yet another embodiment, the pre-culture ratio of hepatocytes, mesenchymal cells, and endothelial cells is 1:0.2:0.7. If preferentially growing a part, those that preferentially differentiate will appear, so it is understood that the ratio between different cells can change during culturing. It is also understood that other ratios can be used to obtain the liver organoids described herein. During the process of creating liver organoids, a physical scaffold or an exogenous matrix material (other than tissue culture vessels) is not necessary. Liver organoids can be used directly to create encapsulated liver tissue. In certain embodiments, liver organoids can be cryopreserved until they are introduced into the encapsulated liver tissue. Polymers that can be used in encapsulated liver tissue form a hydrogel around the liver organoid(s). As is known in the art, a hydrogel is a material in which water is dispersed throughout. and forms a continuous phase.

[0099]

[0100] ​​​​​​ Refers to a hydrophilic polymer chain that is a dispersion medium. Hydrogels can be obtained from natural or synthetic polymer networks. In the context of the present disclosure, encapsulation within the hydrogel prevents the embedded liver organoids from leaking from the polymer, so that the cells of the liver organoids eliminate or suppress the risks of immune reactions or tumors in the recipient's body during transplantation.

[0101] In the context of the present disclosure, a polymer is considered "biocompatible" if it does not exhibit toxicity to the cells of the liver organoids or does not exhibit toxicity when introduced into a subject (e.g., a human). In the context of the present disclosure, a biocompatible polymer preferably does not exhibit toxicity to the cells of the liver organoids when transplanted in vivo into a subject (e.g., a human). Hepatotoxicity can be determined and measured, for example, by the rate of apoptotic death of hepatocyte-like cells (e.g., an increase in apoptosis indicates hepatotoxicity), transaminase levels (e.g., an increase in transaminase levels indicates hepatotoxicity), hypertrophy of hepatocyte-like cells (e.g., an increase in hypertrophy indicates hepatotoxicity), microvesicular degeneration in hepatocyte-like cells (e.g., an increase in degeneration indicates hepatotoxicity), the rate of death of bile duct cells (e.g., an increase in the mortality rate of bile duct cells indicates hepatotoxicity), γ-glutamyl transpeptidase (GGT) levels (e.g., an increase in GGT levels indicates hepatotoxicity). As a biocompatible polymer, carbohydrates (hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, sulfate, heparan sulfate, alginic acid, chitosan, heparin, agarose, dextran, Glycosaminoglycans such as cellulose, and / or their derivatives), proteins (collagen, elastin, fibrin, albumin, poly(amino acids), glycoproteins , antibodies, and / or their derivatives), and / or synthetic polymers (e.g., poly (ethylene glycol)(PEG), poly(hydroxyethyl methacrylate)(PHE MA), and / or those based on poly(vinyl alcohol)(PVA)), but are not limited to these. The biocompatible polymer can be a single polymer, or a mixture of different polymers (e.g., those described in US2012 / 0142069). Examples of biocompatible polymers include poly(ethylene) glycol, polylactic acid (PLA ), polyglycolic acid (PGA), polycaprolactone (PCL), fibrin, polysaccharide materials (such as chitosan, proteoglycan, or glycosaminoglycan (GAG)), alginate, collagen, thiolated heparin, and mixtures thereof, but are not limited to these. In some embodiments, the biocompatible polymer can be linear, branched chain, and optionally can incorporate peptides (e.g., RGD), growth factors, integrins , or drugs.

[0102] In some embodiments, the polymer is a "low immunogenic polymer" that does not induce an immune response in the recipient, or induces only a minimal immune response. This low immunogenic polymer blocks one or more antigenic determinants of the cell, and can suppress or even prevent an immune response against such antigenic determinants when introduced into an allogeneic subject.

[0103] ​​​​​ The polymer present in the encapsulated liver tissue of the present disclosure is preferably crosslinkable, for example it can be crosslinked. The polymer can be crosslinked thermally, chemically (e.g., using any one or more of VPMS, RGD peptides), or using pH or light (e.g., photopolymerization using UV light).

[0104] The polymer of the present disclosure can be made biodegradable (e.g., easily hydrolyzed by the metabolism of the living body) or can be made entirely or partially resistant to biodegradation (e.g., hydrolysis resistance when subjected to the metabolism of the living body). Examples of biocompatible and biodegradable polymers include, but are not limited to, poly(ethylene - glycol)-(maleimide)(PEG - Mal) 8 - arm. Examples of biocompatible and biodegradation - resistant polymers include, but are not limited to, poly(ethylene - glycol)-vinyl sulfone (PEG - VS).

[0105] When obtaining liver organoids, the liver organoids are contacted with a first biocompatible crosslinkable polymer to at least partially (in some embodiments, substantially) coat the liver organoids. The polymer can be used at various concentrations. In certain embodiments the concentration of the polymer upon contact with the liver organoids is between about 1% - 15% (weight / volume). In certain embodiments the concentration of the polymer upon contact with the liver organoids is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% 13%, or 14%. In yet another embodiment, upon contact with the liver organoids The concentration of the polymer at that time is about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8 %, 7%, 6%, 5%, 4%, 3%, or 2% or less. When the liver organoid contacts the first polymer, the first polymer is crosslinked (using any of heat, chemistry, or pH or light). The crosslinking of the first biocompatible polymer is achieved by generating additional bonds (and, in some embodiments, additional covalent bonds) between different molecules of the polymer and / or within the same molecule of the polymer. In some embodiments, the crosslinking of the first biocompatible polymer generates additional bonds (and, in some embodiments, additional covalent bonds) between the polymer molecule and the surface of the liver organoid. In some embodiments, the first

[0106] polymer is at least partially biodegradable. In some embodiments, the liver organoid (at least partially) coated or encapsulated with the first biocompatible crosslinked polymer is contacted with a second biocompatible crosslinkable polymer to at least partially (substantially in some embodiments) coat the encapsulated liver tissue. When the encapsulated liver organoid contacts the second polymer, the latter crosslinks (crosslinks using any of heat, chemistry, or pH or light). The crosslinking of the second biocompatible polymer is achieved by generating additional bonds (and, in some embodiments, additional covalent bonds) between different molecules of the polymer and / or within Between the surface of the nodule, additional bonds (and, in some embodiments, additional covalent bonds) are generated. In some embodiments, the second polymer is at least partially resistant to biodegradation.

[0107] In some embodiments, this process involves encapsulating a liver organoid (a first biocompatible cross- linked polymer / partially coated with a second biocompatible cross-linked polymer) contacting the encapsulated liver organoid with an additional biocompatible cross-linkable polymer to coat it. When the liver organoid contacts the additional polymer, the latter cross-links (either thermally, chemically, or using any of pH or light). The cross-linking of the additional biocompatible polymer is achieved by generating additional bonds (and, in some embodiments, additional covalent bonds) between different molecules of the polymer and / or within the same molecule of the polymer. In some embodiments, the cross-linking of the additional biocompatible polymer results in additional bonds (and, in some embodiments, additional covalent bonds) between the polymer molecule and the second biocompatible cross-linked polymer, and, in some embodiments, between the polymer molecule and the first biocompatible cross-linked polymer and / or the surface of the liver organoid.

[0108] This process can be designed such that a plurality of monodisperse liver organoids are included within the first biocompatible cross-linked polymer. For example, hepatic progenitor cells, endothelial progenitor cells, and mesenchymal progenitor cells are obtained from differentiable single iPSCs. These cells are mixed and then suspension-cultured to form liver organoids. In some embodiments, cells of the hepatocyte has differentiated into hepatocyte-like cells (before introducing liver organoids into the encapsulated liver tissue) , and this hepatocyte-like cell substantially covers a cell core formed by mesenchymal and endothelial progenitor cells . In a further embodiment, the liver organoid has a substantially spherical morphology and a relative diameter of about 150 μm. Next, a cross-linking agent (UV light shown in the examples) can be used to encapsulate the liver organoid with a first biocompatible cross-linkable matrix . This encapsulated liver tissue can be included in a regenerative medicine and used as transplantable liver tissue (for example, having a size of 5 mm to 10 cm) . Alternatively, the liver organoid can be designed for a multi-well plate and used in pharmaceutical development to determine the metabolism or hepatotoxicity of screened compounds .

[0109] This process can be designed to provide a plurality of liver organoids individually (at least partially) coated with a first biocompatible cross-linking polymer , and then this plurality of liver organoids is incorporated into a matrix consisting of a second biocompatible cross-linking polymer . In such an embodiment, first, a plurality of liver organoids individually (at least partially) coated with a first biocompatible cross-linking polymer are formed, and then this plurality of liver organoids is contacted with a second biocompatible cross-linkable polymer that cross-links .

[0110] This process can also be designed to provide a plurality of individual (for example, monodisperse) liver organoids coated with a first compatible cross-linking polymer and any second compatible cross-linking polymer . In such an embodiment, the encapsulated liver tissue is at least per cm 2 ​​​​It can also include about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250 , 300, 350, 400, 450, or 500 liver organoids. In yet another embodiment, the encapsulated liver tissue is, per cm , at most about 500, 45 2 0, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90 , 80, 70, 60, or 50 liver organoids. In still another embodiment, the encapsulated liver tissue is, per cm , between about 50, 60, 70, 80, 90, 10 2 0, 125, 150, 175, 200, 250, 300, 350, 400, or 45 0 and about 500, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, or 60 liver organoids. In still another embodiment, the encapsulated liver tissue is, per cm , between about 50 - 500 liver organ 2 oids. In another embodiment, the encapsulated liver tissue is, per cm , at least about 250, 3 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 , 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200 , 2300, 2400, or 2500 liver organoids. In yet another embodiment, the encapsulated liver tissue is, per cm , at most about 2500, 2400, 2300, 22 00, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 14 3 00, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 7 00, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 7 00, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 7 50, 700, 650, 600, 550, 500, 450, 400, 350, 300, or comprises 250 liver organoids. In yet another embodiment, the encapsulated liver tissue is per 3 comprises approximately 250, 300, 350, 400, 450, 500, 550, 600, 6 50, 700, 750, 800, 850, 900, 950, 1000, 1100, 120 0, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 200 0, 2100, 2200, 2300, or 2400 and approximately 2500, 2400, 23 00, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 15 00, 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, or 300 liver organoids. In yet another embodiment, the encapsulated liver tissue is 3 per approximately 250 - 2500 liver organoids.

[0111] In certain embodiments, the encapsulated liver tissue can be used directly in the treatment methods and screening described herein, or can be cryopreserved to extend its shelf life.

[0112] Therapeutic Use of Encapsulated Liver Tissue The encapsulated liver tissue described herein can be used as a medicament. Since the encapsulated liver tissue described herein exhibits some of the biological functions of the liver, it can be used in vivo or ex vivo to restore or improve the liver function of a subject in need thereof. Liver function can be determined and evaluated, for example, by measuring the synthesis of albumin and coagulation factors (e.g., fibrinogen, pro thrombin, factors V, VII, VIII, IX, X, XI, XIII, as well as protein C, protein S, and antithrombin). On the other hand, an increase in the synthesis of albumin and / or coagulation factors indicates the recovery or improvement of liver function. Liver function can also be evaluated by measuring the international normalized ratio, or INR (e.g., a decrease in INR indicates the recovery or improvement of liver function). Liver function can also be evaluated by measuring the detoxification of ammonia to urea (e.g., a decrease in ammonia level and / or an increase in urea level indicates the recovery or improvement of liver function).

[0113] In such embodiments, the encapsulated liver tissue is intended to be contacted with the biological fluid of the subject being treated. In such embodiments, the encapsulated liver can even release the synthesized proteins and metabolites (albumin, coagulation factors, and / or urea) required by the subject into the biological fluid and absorb the toxic substances (such as ammonia, unconjugated bilirubin, cholesterol, tyrosine, etc.) to be metabolized from the biological fluid. The encapsulated liver tissue can be used to restore the enzyme function that is deficient or reduced in congenital abnormalities of liver metabolism.

[0114] For the purpose of restoring or improving liver function, encapsulated liver tissue can be transplanted in vivo into a subject whose liver function has decreased to almost nil or nil. Thus, the encapsulated liver tissue can be transplanted into the peritoneal cavity, for example, to contact ascites. Alternatively, the encapsulated ​​​​​​​​​​​​​​​The liver tissue can be transplanted into the recipient's liver so as to contact the liver fluid. Further In another example, the encapsulated liver tissue can be transplanted subcutaneously or intramuscularly so as to contact the lymph or blood.

[0115] Alternatively, encapsulated liver tissue can be used as a cell component of an ex vivo detoxification device (e.g., an extracorporeal device) to restore or improve liver function. In such an embodiment proteins, and metabolites (albumin, clotting factors, and / or urea) are supplied, and the blood and / or ascites of the subject to be treated are contacted ex vivo with the encapsulated liver tissue to absorb or metabolize potentially toxic substances (such as ammonia, unconjugated bilirubin, cholesterol, tyrosine, etc.).

[0116] The encapsulated liver tissue can be used in various subjects, including mammals, particularly humans, who are expected to benefit from the restoration or improvement of liver function. The cells of the encapsulated liver tissue can be autologous, allogeneic, or xenogeneic with respect to the subject intended to be treated. However, since the encapsulated liver tissue can be designed to prevent physical contact with the cells of the intended recipient (particularly, immune cells), there is no need to use autologous cells or immunosuppressive agents to prevent immunological recognition and reaction by the intended recipient. This can be achieved, for example, by using an encapsulated liver tissue containing only one biocompatible cross-linked polymer, or by using an encapsulated liver tissue containing both a first biocompatible cross-linked polymer and a second biocompatible cross-linked polymer, and / or by using a low immunogenic polymer. and / or by using a low immunogenic polymer. recipient, there is no need to use autologous cells or immunosuppressive agents to prevent immunological recognition and reaction by the intended recipient. This can be achieved, for example, by using an encapsulated liver tissue containing only one biocompatible cross-linked polymer, or by using an encapsulated liver tissue containing both a first biocompatible cross-linked polymer and a second biocompatible cross-linked polymer, and / or by using a low immunogenic polymer. polymer, and / or by using a low immunogenic polymer. polymer, and / or by using a low immunogenic polymer. This can be achieved, for example, by using an encapsulated liver tissue containing only one biocompatible cross-linked polymer, or by using an encapsulated liver tissue containing both a first biocompatible cross-linked polymer and a second biocompatible cross-linked polymer, and / or by using a low immunogenic polymer.

[0117] In some embodiments, the encapsulated liver tissue can be manipulated using surgery, e.g., laparoscopic procedures, and designed to be introduced into a subject. Further, since the liver tissue is encapsulated in a biocompatible (and in some embodiments, low immunogenicity) polymer, the encapsulated liver tissue can be removed from the subject when liver function has recovered or when the encapsulated liver tissue can no longer improve liver function. The encapsulated liver tissue can be used for the treatment of liver failure. Liver failure occurs when most of the liver undergoes damage that exceeds repair, and such a liver no longer functions. Initial symptoms of liver failure include nausea, loss of appetite, fatigue, and diarrhea. As the symptoms progress, symptoms such as jaundice, bleeding, abdominal distension, confusion, and disorientation (known as hepatic encephalopathy), drowsiness, and coma can also be observed. Liver failure can be acute, chronic, or chronic with acute exacerbation. The most common causes of chronic liver failure are non-alcoholic steatohepatitis, hepatitis B, hepatitis C, long-term alcohol intake, cirrhosis, hemochromatosis,

[0118] and malnutrition. In chronic liver failure, hepatocyte transplantation is most often performed via the portal circulation. However, in cases where chronic liver failure develops following cirrhosis, when the small pores of the sinusoids disappear (capillarization), the injected cells injected via the portal circulation cannot reach the liver parenchyma, and the uptake into the liver lobules is hindered. This prevents the maturation and function of the transplanted cells and results in complications such as sinusoidal and portal vein thrombosis. The encapsulated liver tissue described herein does not require portal vein injection or immunosuppression, so it can benefit hundreds of thousands of liver failure patients. patients. The most common causes of chronic liver failure are non-alcoholic steatohepatitis, hepatitis B, hepatitis C, long-term alcohol intake, cirrhosis, hemochromatosis, and malnutrition. In chronic liver failure, hepatocyte transplantation is most often performed via the portal circulation. However, in cases where chronic liver failure develops following cirrhosis, when the small pores of the sinusoids disappear (capillarization), the injected cells injected via the portal circulation cannot reach the liver parenchyma, and the uptake into the liver lobules is hindered. This prevents the maturation and function of the transplanted cells and results in complications such as sinusoidal and portal vein thrombosis. The encapsulated liver tissue described herein does not require portal vein injection or immunosuppression, so it can benefit hundreds of thousands of liver failure patients. failure patients. Patients with cirrhosis, and patients with chronic (or acutely deteriorating chronic) liver failure, who are unsuitable for transplantation can also be treated to prevent or suppress severe complications (such as hepatic encephalopathy, coagulation disorders, etc.) and improve the survival rate. It is considered possible.

[0119] The encapsulated liver tissue described in this specification can also be used for the treatment of acute liver failure. The most common causes of acute liver failure are reactions to prescription drugs and herbal medicines derived from plants, or their excessive intake, viral infections (including hepatitis A, hepatitis B, and hepatitis C), as well as the ingestion of toxic wild mushrooms, autoimmune hepatitis, or Wilson's disease. Acute liver failure develops suddenly, and the time to onset may be less than 48 hours, so prevention is difficult. Furthermore, in acute liver failure, liver function is impaired to such an extent that it becomes necessary to transplant fully mature functional hepatocytes. In some embodiments, the encapsulated liver tissue can be used for the treatment or alleviation of the symptoms of acute liver failure. The encapsulated liver tissue can be transplanted into a subject in need thereof, or used in an external (ex vivo) detoxification device (extracorporeal liver function assistance, bioartificial liver device, or liver dialysis) for treating the blood of a subject in need thereof. Depending on the number of liver organoids in the encapsulated liver tissue and the severity of the condition, one or more encapsulated liver tissues can be used to treat the subject. The encapsulated liver tissue(s) can be used simultaneously or sequentially. When using the encapsulated liver tissue to treat or alleviate the symptoms of liver failure, allogeneic cells can be used for the subject to be treated. tissue can be used for the treatment or alleviation of the symptoms of acute liver failure. The encapsulated liver tissue can be transplanted into a subject in need thereof, or used in an external (ex vivo) detoxification device (extracorporeal liver function assistance, bioartificial liver device, or liver dialysis) for treating the blood of a subject in need thereof. Depending on the number of liver organoids in the encapsulated liver tissue and the severity of the condition, one or more encapsulated liver tissues can be used to treat the subject. The encapsulated liver tissue(s) can be used simultaneously or sequentially. When using the encapsulated liver tissue to treat or alleviate the symptoms of liver failure, allogeneic cells can be used for the subject to be treated. liver dialysis) for treating the blood of a subject in need thereof. Depending on the number of liver organoids in the encapsulated liver tissue and the severity of the condition, one or more encapsulated liver tissues can be used to treat the subject. The encapsulated liver tissue(s) can be used simultaneously or sequentially. When using the encapsulated liver tissue to treat or alleviate the symptoms of liver failure, allogeneic cells can be used for the subject to be treated. tissue(s) can be used simultaneously or sequentially. When using the encapsulated liver tissue to treat or alleviate the symptoms of liver failure, allogeneic cells can be used for the subject to be treated. The encapsulated liver tissue(s) can be used simultaneously or sequentially. When using the encapsulated liver tissue to treat or alleviate the symptoms of liver failure, allogeneic cells can be used for the subject to be treated. When using the encapsulated liver tissue to treat or alleviate the symptoms of liver failure, allogeneic cells can be used for the subject to be treated. It is possible to use allogeneic cells for the subject to be treated.

[0120] The encapsulated liver tissue is used for a monogenic congenital abnormality of liver metabolism (for example, Crigler-Najjar syndrome syndromes, familial hypercholesterolemia, urea cycle disorders, N-acetylglutamate synthase deficiency, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, citrullinemia, argininosuccinate lyase deficiency, arginase deficiency, etc. , type I hypertyrosinemia, etc.) can also be used to treat or alleviate the symptoms. In this embodiment, the encapsulated liver tissue imparts the lacking metabolic function, alleviates the symptoms, prevents or suppresses complications, and / or suppresses or eliminates the need for lifelong treatment or dietary restrictions.

[0121] The encapsulated liver tissue can be designed as an implantable product (e.g., an encapsulated liver tissue sheet) for treating acute and chronic liver failure without requiring immunosuppression. In such an 2 embodiment, the implantable tissue sheet contains about thousands of liver organoids per cm . In some embodiments, the encapsulated liver tissue sheet can be placed in a container (e.g., a custom-made permeable bag, etc.) that facilitates manipulation and fixation to the desired implantation site. In another embodiment, for ease of manipulation, the implantable tissue sheet has a thickness of at least 1 mm, and in some further embodiments, a width of at least 5 mm to 10 cm.

[0122] Liver metabolism, and methods and kits for screening liver toxicity The encapsulated liver tissue described herein retains at least some liver functions Investigating the metabolism of agents (e.g., drug candidates) by the human body to streamline drug discovery and development The encapsulated liver tissue described herein can be used as an in vitro model. It can also be used to determine the presence or absence of hepatotoxicity due to the substance. The vast majority of (suspect) therapeutic drugs (approved or in development) are in some form or another In some embodiments, the agent (e.g., a putative therapeutic agent) is metabolized by cells in the liver. If hepatotoxicity (e.g., drug-induced hepatotoxicity) is present, the encapsulation methods described herein may be used. The cultured liver tissue can be used to determine hepatotoxicity. Drugs, toxins, and other substances that have been reported to cause liver damage include: There are more than 900 medicinal herbs, and drugs are the main cause of fulminant hepatic failure in 20-40% of all cases. Approximately 75% of idiosyncratic drug reactions result in liver transplantation or death. Drug-induced liver injury is the most common reason for the withdrawal of approved drugs. Early determination of the hepatotoxicity profile of drugs (e.g., serotonin-dependent inhibitors) can streamline new drug discovery and development. This is useful.

[0123] The encapsulated liver tissue described herein does indeed exhibit at least some liver functions and therefore acts Hepatic metabolism of substances (e.g. chemical agents, biological agents, natural drug preparations or mixtures) and / or This method can be used in vitro to determine hepatotoxicity or hepatotoxicity. The present invention can be used to determine the hepatic metabolism of a drug or combination of drugs.

[0124] To do so, at least one hepatic organoid of encapsulated liver tissue is An agent is provided under conditions sufficient to act on at least one (and in some embodiments, two or three) cell types. The test substance, or a combination of agents, is subjected to contact with the encapsulated liver tissue. The test mixture comprises the agent and the encapsulated liver tissue. Next, at least one (and in some embodiments, at least two or three) cell types of at least one liver organoid of the encapsulated liver tissue are determined, or at least one agent-related liver metabolite of the agent in the test mixture is determined. As used in connection with the present disclosure, the expression "agent-related metabolite" refers to a metabolite that can be formed by hydrolysis of the agent being tested.

[0125] Alternatively, or in combination, at least one (and in some embodiments, at least two or three) cell types of at least one liver organoid of the encapsulated tissue are determined, or at least one liver parameter in the test mixture is determined. Liver parameters that can be determined include albumin production, urea production, ATP production, glutathione production, cytochrome P450 (CYP) metabolic activity, liver-specific genes or proteins (e.g., the expression of CYP enzymes (CyP2C9, CyP3A4, CyP1A1, CyP1A2, CyP2B6, and / or CyP2D6), the response to liver toxins, cell death (e.g., as determined by measurement of lactate dehydrogenase or transaminase in the test mixture), apoptosis of cells, necrosis of cells, metabolic activity of cells (e.g., live / dead assay, caspase 3 / 7 assay, MTT assay, or a test based on WST-1), mitochondrial The liver function and / or bile acid production, among others, but not limited thereto. At least one (Or a plurality of) liver parameters are obtained, and the corresponding control liver parameters are compared with those liver parameters. In certain embodiments, the control liver parameters are obtained in the absence of the screened agent (or a combination of the screened agents) or can be obtained in the presence of a medium that dissolves the screened agent (or a combination of the screened agents). The determination step can be performed on all or part of the cells of the encapsulated liver tissue. In certain embodiments, the determination step is performed on the hepatocyte-like cells and / or cholangiocyte epithelial cells of the encapsulated liver tissue. This method also includes a comparison to determine whether the liver organoids of the encapsulated liver tissue metabolize the agent and / or whether the agent is hepatotoxic to the cells of the liver organoids of the encapsulated liver tissue. To do so, a comparison is made between the agent-related liver metabolites to be measured and the control agent-related liver metabolites. For example, the control agent-related metabolite can be the agent itself in an intact (e.g., not hydrolyzed) form. When the presence of agent-related metabolites different from the control agent-related metabolites is determined, then the metabolism of the agent in hepatocytes is determined. A comparison can also be made between the measured liver parameters and the control liver parameters. For example, the control liver parameters can be obtained in the absence of the agent. The control liver parameters and the liver parameters

[0126] ​​​​​​​​​​​​ Once it is determined that there is a difference, it is then determined whether the agent exhibits hepatotoxicity. .

[0127] In certain embodiments, this method is used to determine whether a screened agent (or a combination of screened agents) exhibits hepatotoxicity. In such embodiments, the screened agent (or combination of screened agents) is contacted to determine whether toxicity is induced in at least one cell (e.g., a hepatocyte or a cholangiocyte) of the liver organoids of the encapsulated liver tissue. Toxicity can be determined and measured, for example, by cell death (e.g., measurement of lactate dehydrogenase or transaminase in the test mixture), cell metabolic performance (e.g., live / dead assay, caspase 3 / 7 assay, MTT assay, or a WST-1 based test), mitochondrial function (e.g., a decrease in mitochondrial function indicates hepatotoxicity), regulation of the activity of one or more enzymes of the cytochrome P450 system (e.g., CYP2E1, etc.) (e.g., an increase in the activity of the cytochrome P450 system enzyme(s) indicates hepatotoxicity), and / or regulation of bile acid production (e.g., an increase in bile acid production indicates hepatotoxicity). This method can include comparing the toxicity results of the screened agent to the toxicity results of a control agent (known not to induce hepatotoxicity or known to induce hepatotoxicity).

[0128] This method involves contacting a screened agent (or multiple screened agents) to encapsulated liver tissue obtained using liver organoids with different metabolic activities. ​ This can also include causing. For example, in order to implement specific metabolic functions at different levels, liver organoids can be created using cells from different origins and sources (thereby obtaining the diversity observed among individuals in the general population). For example, the obtained encapsulated liver tissues with different metabolic activities can be tested for the screened agents while comparing each of them and all of them to each other. This can be done in different wells of a single plate. In certain embodiments, the liver organoids can be derived from different genders, ethnicities, and / or genotypes. Testing the screened agents against such different genders, ethnicities, and / or genotypes enables determination of metabolic differences or determination of whether hepatotoxicity is present in all or only some of the gender, ethnicity, and / or genotype. In certain embodiments, among multiple liver organoids, the mesenchymal components and / or endothelial components of the liver organoids can be made the same, but the hepatocyte-like cells and cholangiocyte epithelial cells are derived from different genders, ethnicities, and / or genotypes. As an example, each different encapsulated liver tissue can be placed in different wells (optionally as multiple replicate specimens), and the same screened agent can be contacted with each different encapsulated liver tissue. In some embodiments, the encapsulated liver tissue used in this screening method does not contain a second biocompatible crosslinking polymer or a further biocompatible crosslinking polymer. Instead, it contains the liver organoids described herein and the first biocompatible crosslinking polymer.

[0129] ​ It becomes qualitative.

[0130] In this screening method, individually encapsulated liver organoids, or liver organoids encapsulated in a matrix containing a plurality of liver organoids can be used. In the latter case, the encapsulated liver tissue is located at the bottom of the well, whereby the addition of the screened agent and the washing of the encapsulated liver tissue before the determination step are very convenient.

[0131] The present disclosure also provides a kit for determining liver metabolism or hepatotoxicity. This kit includes the encapsulated liver tissue described in this specification and an instruction manual for implementing the method described in this specification. In some embodiments, this kit further includes a tissue culture support, which can optionally include at least one well. In further embodiments, the encapsulated liver tissue can be placed at the bottom of at least one well and, if necessary, attached to the surface of the well (covalently or non-covalently). This kit can also include reagents for performing measurements of liver metabolism or hepatotoxicity (e.g., live / dead assay, caspase 3 / 7 assay, MTT assay, WST -1 assay, and / or measurement of LDH).

[0132] By referring to the following examples, the present invention can be easily understood. However, the same examples are described for illustrative purposes of the present invention and do not limit the scope of the present invention.

Examples

[0133] Production and characterization of hepatocyte-like cells ​​​Hepatocyte-like cells (HLCs) were obtained using two different protocols: the protocol described herein (referred to as Protocol B), and the standard protocol described in PCT / CA2017 / 051404 (referred to as Protocol A). Then, the HLCs were compared.

[0134] Differentiation protocol (Protocol B) iPSC preparation (days -3 to 0). Three days before starting differentiation, single-cell passage was performed using TrypLE. The iPSCs were placed on plates coated with laminin and cultured in Essential 8 Flex medium. This medium was supplemented with RevitaCell™ (ThermoFisher Scientif ic) for the first 24 hours only. The medium was changed daily. Details of definitive endoderm (days 1 to 2). The cells were washed with DMEM / F-12 medium. Next, the cells were

[0135] cultured in RPMI / B27 without insulin, containing 1% knockout serum replacement (KOSR), supplemented with 100 ng / ml Activin A, and 3 μM CHIR99021. The cells were cultured at 37 °C in an atmosphere of O2 / 5% CO2 for 2 days.

[0136] The medium was changed daily.

[0137] Involvement of definitive endoderm (intraembryonic endoderm, days 3 to 5). The cells were cultured in RPMI / B27 without insulin, containing 1% knockout serum replacement and supplemented with 100 ng / ml Activin A. The cells were cultured at 37 °C in an atmosphere of O2 / 5% CO2 for 3 days. The medium was changed daily.

[0137] Posterior foregut (days 6 to 10). Without insulin, containing 1% knockout serum replacement , 20 ng / ml BMP4, 5 ng / ml bFGF, 4 μM IWP2, and 1 μ M A83-01 were supplemented, and cells were cultured in RPMI / B27. At 37 °C, in an atmosphere of O2 / 5% CO2, cells were cultured for 5 days. The medium was changed daily.

[0138] Details of the liver (bipotent progenitor cells, days 11 - 15). Insulin, 2% knockout serum replacement were included, and cells were cultured in RPMI / B27 supplemented with 20 ng / ml BMP4, 10 ng / ml bFGF, 20 ng / ml HGF, and 3 μM CHIR99021. At 37 °C, in an atmosphere of O2 / 5% CO2, cells were cultured for 5 days. The medium was changed daily.

[0139] Liver maturation 1 (immature hepatocyte-like cells, days 16 - 20:). 1% knockout serum replacement was included, and cells were cultured in HBM / HCM medium (containing EGF, Lonza) supplemented with 20 ng / ml HGF, 3 μM CHIR99021, 20 ng / ml BMP 4, 10 ng / ml bFGF, 20 ng / ml OSM, 10 μM dexamethasone, and 1 μM A83-01. Cells were cultured at 37 °C in an atmosphere of O2 / 5% CO2 for 5 days. The medium was changed daily. Equivalent results were obtained using RPMI / B27 containing insulin, 2% knockout serum replacement instead of HBM / HCM medium (data not shown).

[0140] Liver maturation 2 (immature hepatocyte-like cells, days 21 - 25). 1% knockout serum replacement was included, and cells were cultured in HBM / HCM medium (not containing EGF, Lonza) supplemented with 20 ng / ml OSM, 10 μM dexamethasone. At 37 °C, in an atmosphere of O2 / 5% C O2 / 5% CO2, cells were cultured for 5 days. The medium was changed daily.​​​​ The cells were cultured with O2 for 5 days. The medium was changed daily. Instead of the HBM / HCM medium, equivalent results were obtained (data not shown) using William’s E medium supplemented with 1% knockout serum replacement and Primary Hepatocyte Maintenance Supplement (trademark) (ThermoFisher Scientific).

[0141] Liver maturation 3 (mature hepatocyte-like cells, days 25 - 30). The cells were cultured in HBM / HCM medium (containing EGF, Lonza) supplemented with 1% knockout serum replacement and 10 μM dexamethasone. The cells were cultured for 5 days at 37 °C in an atmosphere of O2 / 5% CO2. The medium was changed every other day. Instead of the HBM / HCM medium, equivalent results were obtained (data not shown) using William’s E medium supplemented with 1% knockout serum replacement and Primary Hepatocyte Maintenance Supplement (trademark) (ThermoFisher Scientific).

[0142] Table 1. Details of two protocols for obtaining hepatocyte-like cells compared in this example.

Table 1-1

Table 1-2

Table 1-3

[0143] ​​​​​​​​Cell microscopy. Phase contrast microscopy (EVOS FL Cell Imaging Sys tem, Thermo Fisher Scientific) was used to observe the live cells at the end of the differentiation pro cess and study their morphology.

[0144] Cell counting. Cells were harvested from the culture plates using TrypLE, and then counted using an automated cell counter, Countess II FL Automated Cell Counter , Thermo Fisher Scientific.

[0145] Immunofluorescence. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 5 minutes at room temperature. Nonspecific sites were blocked by incubating the cells with a 3% blocking serum solution (corresponding to the antibody) for 30 minutes at room temperature. The fixed and permeabilized cells were then incubated with the primary antibody solution (the antibody was diluted to 2% with PBS-BSA) for 1 hour at room temperature. The cells were incubated with the secondary labeled antibody solution (fluorescent) for 30 minutes at room temperature while protecting from light. During the last 15 minutes of incubation with the secondary labeled antibody, a dye (Pureblue e nuclei staining, BioRad) was added to stain the nuclei. Next the cells were fixed with an anti-fading reagent (ProLong Gold). Fluorescence was analyzed the day after this procedure. The following antibodies were used: anti-human SOX17 from ABCAM diluted 1:100 , anti-human FOXA2 from ABCAM diluted 1:100; anti-human CXCR4 from ABCAM diluted 1 :100; anti-human AFP from DAKO diluted 1:100; anti-human albumin (AL ) from DAKO diluted 1:100; The cells were fixed with an anti-fading reagent (ProLong Gold). The next day, fluorescence was analyzed. The following antibodies were used: anti-human SOX17 from ABCAM diluted 1:100, anti-human FOXA2 from ABCAM diluted 1:100; anti-human CXCR4 from ABCAM diluted 1:100; anti-human AFP from DAKO diluted 1:100; anti-human albumin (AL ), anti-human albumin (AL ) from DAKO diluted 1:100; :100; anti-human AFP from DAKO diluted 1:100; anti-human albumin (AL B) Dilution 1:100; Anti-human CK19 from Abcam diluted 1:100, and anti-human CK7 from Abcam diluted 1:200.

[0146] FACS analysis. 0.5 - 1×10 6 cells were dispensed into each assay tube . The cells were stained with 100 μL of the fluorescent dye-conjugated primary antibody solution (membrane antigen) for 20 minutes at room temperature while protecting from light. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were permeabilized with 1% Triton X-100. 100 μL of the fluorescent dye-conjugated antibody solution (intracellular antigen) was used to stain the cells, and then the cells were incubated in the dark at room temperature for 20 minutes. The cells were resuspended in 0.5 mL of PBS-BSA 1%, kept at 4°C, and then analyzed. The following antibodies were used for FACS: Per-CP -Cy 5.5 anti-human SOX17 (BD Bioscience), APC anti-human CD1 84 (CXCR4) (BD Bioscience), PE anti-human FOXA2 (BD B ioscience), PE anti-human EpCAM (BD Bioscience), APC anti-human albumin (R&D Systems), FITC anti-human TRA1-60 (BD Bios cience), Alexa647 anti-human Nanog (BD Bioscience), APC anti-human Brachyury (Bio-Techne), and PerCP-Cy5 .5 anti-human c-Kit (CD117) (BD Bioscience).

[0147] Real-time RT-PCR. Total RNA was extracted from cultured cells or organoids (Rneasy Plus Mini Kit) to be used as a template for synthesizing single-stranded cDNA. Kit, Qiagen). Reverse transcription was performed to obtain cDNA. After preparing the PCR reaction mixture, it was loaded onto a plate. The plate was sealed, centrifuged, and then loaded onto the instrument. Standard TaqMan qPCR reaction conditions were used. To calculate the relative quantification of gene expression, the data were analyzed using the comparative CT (ΔΔCT) method. The following TaqMan gene expression assays (obtained from Thermo Fisher scientific) were used: Hs1053049_S1 SOX2 Taqman gene expression assay, Hs00751752_S1 SOX17 Taqman gene expression assay, Hs00171403_M1 GATA4 Taqman gene expression assay, Hs002230853_M1 HNF4A Taqman gene expression assay, Hs00173490_M1 AFP Taqman gene expression assay, Hs00609411_M1 albumin Taqman gene expression assay, Hs99999905_M1 GAPDH Taqman gene expression assay, Hs04187555_m1 FOXA1 Taqman gene expression assay, Hs00242160 ml HHEX Taqman gene expression assay, Hs00236830 ml PDX1 Taqman gene expression assay, Hs00232764 ml FOXA2 Taq expression assay, Hs01005019_m1 ASGR1 Taqman gene expression assay, Hs00173490 AFP Taqman gene expression assay, Hs00607978 s1 CXCR4 Taqman gene expression assay, Hs00761767_s1 KRT19 Taqman gene expression assay, Hs00559840_m1 KRT7 Taqman gene expression assay After preparation, it was loaded onto a plate. The plate was sealed, centrifuged, and then loaded onto the instrument Standard TaqMan qPCR reaction conditions were used. To calculate the relative quantification of gene expression, the data were analyzed using the comparative CT (ΔΔCT) method using the comparative CT (ΔΔCT) method. The following TaqMan gene expression assays (obtained from Thermo Fisher scientific ) were used: Hs1053049_S1 SOX2 Taqman gene expression assay , Hs00751752_S1 SOX17 Taqman gene expression assay, Hs0 0171403_M1 GATA4 Taqman gene expression assay, Hs00223 0853_M1 HNF4A Taqman gene expression assay, Hs00173490 _M1 AFP Taqman gene expression assay, Hs00609411_M1 albumin Taqman gene expression assay, Hs99999905_M1 GAPDH Taqman gene expression assay, Hs04187555_m1 FOXA1 Taqm an gene expression assay, Hs00242160 ml HHEX Taqman gene expression assay, Hs00236830 ml PDX1 Taqman gene expression assay assay, Hs00232764 ml FOXA2 Taq expression assay, Hs010050 19_m1 ASGR1 Taqman gene expression assay, Hs00173490 A FP Taqman gene expression assay, Hs00607978 s1 CXCR4 T aqman gene expression assay, Hs00761767_s1 KRT19 Taqma n gene expression assay, Hs00559840_m1 KRT7 Taqman gene expression assay Current assay and Hs00944626_m1 TAT Taqman gene expression assay assay.

[0148] Cyp 3A4 activity. Promega's "P450-Glo™ Assay" was used according to the manufacturer's instructions to evaluate Cyp3A4 activity. urea synthesis. Gentaur's "Quantichrom urea assay kit" was used according to the manufacturer's instructions to measure urea synthesis.

[0149] albumin production. Abcam's "Albumin human ELISA kit" was used according to the manufacturer's instructions to evaluate albumin production. was used according to the manufacturer's instructions to measure urea synthesis.

[0150] Albumin production. Abcam's "Albumin human ELISA kit" was used according to the manufacturer's instructions to evaluate albumin production.

[0151] Mitochondrial respiration analysis. Using a Seahorse Bioscience XF96 analyzer (Seahorse Bioscience Inc.), in a 96-well plate at 37 °C, according to the manufacturer's instructions with some modifications, a mitochondrial stress test was performed. Briefly, cells were seeded at 1 × 10 cells / well 5 and pre-treated 24 hours before the assay with different doses of acetaminophen (APAP-2, 4, 8 mM), and amiodarone (AMIO-2, 4, 8, 19 μM). On the test day, the growth medium was removed, washed twice, and then exchanged with XF assay medium (non-buffered DM EM, d5030 Sigma, 25 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate, pH 7.4), and the plate was incubated at 37 °C for 1 hour in a CO 2-free incubator. Appropriate to the hydration cartridge sensor, was incubated. Loaded mitochondrial modulators to achieve the final concentration in each well: oligomycin (2 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (2 μM), and rotenone / antimycin A (both 1 μM). Next, as described in the manufacturer's protocol, the levels of basal respiration, ATP production, proton leak, maximal respiration, and non-mitochondrial respiration were analyzed from the OCR values. Table 2. Abbreviations used.

[0152]

[0153] When the endoderm induction process of hiPSCs was carried out over 5 days, a homogeneous monolayer of cells expressing the specific endoderm markers SOX17, FOXA2, GATA4, CXCR4, and EOMES was obtained (Figure 1). The homogeneity of the population was confirmed by flow cytometry analysis and it was found that more than 80% of the cells were triple positive for SOX17, FOXA2, and CXCR4 and that the cells did not express c-Kit (Figure 2). Immunostaining revealed that most cells were positive for SOX17, FOXA2, and CXCR4, which are markers of definitive endoderm (lower panel of Figure 3). Similar results were obtained when human embryonic stem cells (hESCs, data not shown) were differentiated instead of iPS cells.

[0154] After inducing endoderm, the cells were treated for 5 days to induce differentiation into posterior foregut. At that stage, signals such as FGF-2 and BMP4 that normally emerge from cardiac mesoderm were observed. ​​​​​​​​In addition, the Wnt / β-catenin and TGFβ signaling pathways were inhibited (using WP2 and A83-01 respectively), enabling the expression of Hex and Proxl. As shown in Figure 4, the cells enhanced the expression of the foregut-specific markers FOXA2, SOX2, FOXA1, HNF4A, AFP, and albumin.

[0155] Subsequently, the FGF-2 and BMP4 signals were maintained, HGF was added, and the Wnt pathway promoting liver growth was activated (using CHIR99021) to induce liver specificity (polygonal liver bud cells) for 5 days. These cells showed the expression of the liver-specific markers AFP, albumin, CK19, CK7, and EpCAM (Figure 5). It was also confirmed that the iPSC-derived liver progenitor cell population did not contain undifferentiated cells (Figure 6). RT-qPCR showed the expression of characteristic liver bud cell / liver cell markers such as albumin, AFP, AFP, CK19, CK7, PDX1, SOX9, PROX1, HNF4α, and HHEX (Figure 7). As shown in Figure 8, the liver progenitor cells showed a significant increase in cell yield compared to endoderm cells or undifferentiated iPSCs.

[0156] To further clarify the involvement in the liver, TGFβ signaling was inhibited (using A83-01 to avoid cholangiocytes), and the Wnt pathway was activated (using CHIR99021). FGF-2, BMP4, HGF, OSM, and dexamethasone were included. At the final stage of differentiation, OSM was removed (since hematopoiesis does not occur in the liver after birth), and dexamethasone was maintained.

[0157] During the differentiation process, the cell population gradually acquired the typical morphology of hepatocyte-like cells with a large ratio of cytoplasm to nucleus, numerous vacuoles and vesicles, and prominent nucleoli. Some cells were found to be binuclear (Figure 9A). These cells also showed the expression of AFP, albumin, and CK19 (Figure 9B). Immunofluorescence showed albumin expression, increased expression, and decreased expression of AFP and CK19 compared with the hepatoblast stage (Figure 9B and data not shown). Flow cytometry analysis showed that the majority of cells (98.5%) were positive for albumin (Figure 10). RT-qPCR analysis showed that the expression of specific liver genes such as albumin, AFP, HNF4a, ASGR1, and SOX9 was similar between HLC and FPHH (Figure 11). Figure 12 compares HLC obtained by protocol B with primary human hepatocytes HepG2, undifferentiated iPSCs, DE cells, or PFG cells. These results revealed that HLC-B and FPH

[0158] H showed similar CyP3A4 activity (Figure 12A) and urea production (Figure 12C). HLC-B cells produced albumin at levels equal to or lower than those of adult hepatocytes (Figure 12B). HLC obtained by protocol B achieved significant differentiation compared with HLC obtained by protocol A, which showed high expression of liver markers (Figure 13), significantly greater CyP 3a4 activity (Figure 14A), albumin production (Figure 14B), and cell yield (Figure 14C), indicating its significant importance.

[0159]

[0160] ​​​​Metabolic function of hepatocyte-like cells (obtained using Protocol B), mitochondrial respiratory capacity Power, and respiration related to ATP, under basal conditions, and after increasing the doses of drugs that are specifically metabolized in the liver, acetaminophen (APAP ), and amiodarone (AMIO), were evaluated (Figure 15). The results shown in Example 15 indicate that upon contact with the drugs, the HLCs obtained with Protocol B regulate their respiration and are thus metabolically active.

[0161] Although the present invention has been described in relation to its specific embodiments, it should be understood that the claims are not limited by the preferred embodiments described in the examples and should be given the broadest interpretation consistent with the entire specification. ​​​

Claims

**Claim 1** A process for generating hepatic progenitor cells from posterior foregut cells, the process comprising contacting the posterior foregut cells with a second culture medium and comprising a second set of additives under conditions that permit the differentiation of the posterior foregut cells into the hepatic progenitor cells, the second set of additives comprising: ● Insulin as an activator of the insulin signaling pathway, ● BMP4 as an activator of the bone morphogenetic protein (BMP) signaling pathway, ● Basic fibroblast growth factor as an activator of the fibroblast growth factor (FGF) signaling pathway, ● Hepatocyte growth factor as an activator of the hepatocyte growth factor (HGF) signaling pathway, and ● CHIR99021 as an inhibitor of the Wnt signaling pathway, the process. **Claim 2** The process according to claim 1, wherein the second culture medium comprises serum. **Claim 3** The process according to claim 1 or 2, wherein the posterior foregut cells express at least one of SOX2, FOXA1, FOXA2, HNF4a, AFP, or albumin. **Claim 4** The process according to claim 1 or 2, wherein the hepatic progenitor cells express at least one of alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, HHEX, HNF4a, or epithelial cell adhesion molecule (EpCAM).

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