Process for making cell populations of hepatic lineage from endodermal cells, and cellular compositions comprising the same

Abstract

To provide: processes for differentiating an endodermal cell into a posterior foregut cell, a posterior foregut cell into a hepatic progenitor cell, and / or a hepatic progenitor cell into a hepatocyte-like cell; and obtained cell populations.SOLUTION: In some embodiments, the process can be conducted in the absence of serum. The hepatocyte-like cell population obtained from this process have a detectable CyP3A4 activity and / or express a detectable level of albumin and / or of urea. The process can be designed to increase cellular yield.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a priority application of U.S. Provisional Application No. 62 / 676,582, filed May 25, 2018. The entire contents of that application are hereby incorporated by reference. Use. [Background technology]

[0002] Obtaining viable and functional hepatocyte-like cells in high yields, particularly Such cells can be obtained by differentiation from pluripotent stem cells such as induced pluripotent stem cells. Obtaining homogeneous cell populations in a reproducible manner has proven difficult. This has also proven difficult. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, it is particularly important to minimize the metabolism of molecules such as therapeutic agents and / or potential therapeutic agents, if possible. There is a strong demand for cells derived from hepatic cell lines that exhibit biological activity. [Means for solving the problem]

[0004] The present disclosure provides methods for culturing pluripotent cells by providing or excluding specific additives during culture. and the process of differentiation into viable and functional hepatocyte-like cells. This process commits pluripotent cells (or the resulting differentiated cells) to the mesodermal lineage. The pluripotent cells are internalized without being encouraged, and in some embodiments, not allowed, to differentiate. This process is also responsible for differentiation into the germ layer lineage. , the Wnt pathway (which allows for Nodal expression), and activation of the TGFβ pathway. The initial transition of the anterior-posterior pattern of the definitive endoderm is initiated by Wnt, FGF, and BMP at the posterior end of the definitive endoderm. It begins with a combination of signal transduction: inhibition of the TGFβ pathway, and FGF and BM The early suppression of the Wnt pathway in the anterior endoderm, coupled with the use of HexP signaling, is (which is necessary for liver (and pancreas) development). Shortly after the initial inhibition, the same pathways for liver growth are activated. Continuous pathways involving FGF, BMP, Wnt, and HGF pathways derived from hepatic mesenchymal and endothelial cells Maturation into hepatocyte-like cells requires cytokines, glucocorticoids, and HGF. Cytokines such as OSM promote the morphological transformation of polarized epithelia. Induce maturation.

[0005] In a first aspect, the present disclosure provides a process for generating posterior foregut cells from endoderm cells. This process involves contacting the endoderm cells with a first culture medium that does not contain insulin. and the first additive set under conditions that allow differentiation of endoderm cells into posterior foregut cells. The first additive set does not include insulin and is a bone-forming Activator of the bone marrow morphogenetic protein (BMP) signaling pathway; fibroblast growth factor (FG F) activators of signaling pathways; inhibitors of the Wnt signaling pathway; and Transforming Growth Factor β (TGFβ) signaling pathway inhibitors, including or consisting essentially of In one embodiment, the first culture medium comprises serum. In another embodiment, the first culture medium comprises BM Activators of the P signaling pathway are BMP receptor agonists, e.g., BMP4 In another embodiment, the activator of the FGF signaling pathway is an FGF receptor agonist. In a further embodiment, the inhibitor is a cytotoxic agent, such as basic FGF. 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 express SOX17, GATA4, FO expressing at least one of XA2, CXCR4, or EOMES; and / or , which do not substantially express c-Kit. The authors conclude that "c-Kit is not a viable target for the experimental treatment" when less than 3% of the cells are positive for the c-Kit marker. Therefore, cells derived from the posterior foregut cells cannot express these Due to their endodermal origin, they are also unable to substantially express c-Kit. , posterior foregut cells express SOX2, FOXA1, FOXA2, HNF4a, AFP, or The present disclosure relates to the process described herein. Also provided is a population of posterior foregut cells obtainable or obtained thereby.

[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 allow differentiation. contacting the posterior foregut cells with a second culture medium containing a second set of additives; The two additive sets are: activators of the insulin signaling pathway; bone morphogenetic proteins Activators of the (BMP) signaling pathway; fibroblast growth factor (FGF) signaling activators of the hepatocyte growth factor (HGF) signaling pathway; and W In some embodiments, the activator of the nt signaling pathway comprises or consists essentially of an activator of the nt signaling pathway. In one embodiment, the second culture medium comprises serum. Activators of the pathway are insulin receptor agonists, e.g., insulin. In embodiments, the activator of the BMP signaling pathway is a BMP receptor agonist, e.g. In a further embodiment, the activator of the FGF signaling pathway is F In yet another embodiment, the GF receptor agonist is a basic FGF. The signal transduction activator is an HGF receptor agonist, for example, HGF. In another embodiment, the activator of the Wnt signaling pathway is GSK3, e.g., CHI In one embodiment, the biological activity of R99021 can be inhibited in posterior foregut cells. SOX2, FOXA1, FOXA2, HNF4a, AFP, or albumin In another embodiment, the hepatocyte progenitor cells express at least one of alpha-fetal protein. Protein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, or HNF4a The present disclosure also relates to a method for producing a compound obtainable by the process described herein. The present invention provides a population of hepatocyte progenitor cells obtained or isolated therefrom.

[0007] According to a third aspect, the present disclosure provides a process for generating hepatocyte-like cells from hepatic progenitor cells. This process provides a method for (i) culturing hepatic progenitor cells under conditions to obtain cells of hepatic lineage. (ii) contacting the cells with a third culture medium containing a third set of additives; Hepatocyte lineage cells were cultured in a fourth culture medium containing a fourth set of additives under conditions to obtain cells. and (iii) contacting the immature hepatocytes with the ground under conditions to obtain mature hepatocyte-like cells. The cells were cultured with a fifth culture medium containing a fifth set of additives and no cytokines. The third set of additives involves contacting insulin with activators of the insulin signaling pathway. Activator of bone morphogenetic protein (BMP) signaling pathway, fibroblast growth factor activator of the fibronectin (FGF) signaling pathway, and hepatocyte growth factor (HGF) signaling pathway activator of the Wnt signaling pathway, transforming growth factor β (TGFβ ) inhibitors of signal transduction pathways, including cytokines and glucocorticoids, or The fourth set of additives consists essentially of cytokines and glucocorticoids. A fifth set of additives comprises or consists essentially of cytokines. In some embodiments, the composition is free of and comprises or consists essentially of a glucocorticoid. In another embodiment, the fourth, fifth, and / or sixth culture medium comprises serum. Activators of the insulin signaling pathway include insulin receptor agonists, e.g., insulin In a further embodiment, the activator of the BMP signaling pathway is BM In yet another embodiment, the agonist is a P receptor agonist, such as BMP4. Activators of the FGF pathway are FGF receptor agonists, such as basic FGF. In yet another embodiment, the activator of the HGF signaling pathway is an HGF receptor agonist. In yet another embodiment, the activation of the Wnt signaling pathway is The agent is capable of inhibiting the biological activity of GSK3, e.g., CHIR99021. 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 another embodiment, the cytokine is oncostatin M (OSM). In another embodiment, the glucocorticoid is dexamethasone. In an embodiment, the hepatic progenitor cells are derived from alpha-fetoprotein (AFP), albumin (ALB), Cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, In yet another embodiment, the cells express at least one of PROX1 or HNF4a. In this study, immature hepatocyte-like cells and / or mature hepatocyte-like cells express α-fetoprotein (AFP), albumin (ALB), ASGR1, HNF4a, or SOX9 In one embodiment, the mature hepatocyte-like cells express at least one detectable Cyp have 3A4 activity and express detectable levels of albumin and / or urea. The present disclosure relates to a method for producing a compound obtainable by or obtained by the process described herein. A population of hepatocyte-like cells is also provided.

[0008] According to a fourth aspect, the present disclosure provides a process for generating hepatic progenitor cells from endoderm cells. This process provides a method for preparing a posterior foregut by (a) carrying out the process described herein. Obtaining cells or providing a population of posterior foregut cells as described herein; and b) subjecting the posterior foregut cells to the process described herein to obtain hepatic progenitor cells; The present disclosure relates to the process described herein. Also provided is a population of hepatic progenitor cells obtainable or obtained thereby.

[0009] According to a fifth aspect, the present disclosure provides a process for generating hepatocyte-like cells from hepatic progenitor cells. The process provides a method for producing a liver progenitor by (a) carrying out the process described herein. obtaining cells or providing a population of hepatic progenitor cells as described herein; and (b ) subjecting the hepatic progenitor cells to the process described herein to obtain hepatocyte-like cells. The present disclosure provides a method for producing a cellulose acetate solution comprising the steps of: Also provided are populations of hepatocyte-like cells obtainable or obtained by the method.

[0010] According to a sixth aspect, the present disclosure provides a process for producing hepatocyte-like cells from endoderm cells. The process provides a method for: (a) optionally carrying out a process described herein; Obtaining posterior foregut cells or optionally providing a population of posterior foregut cells as described herein. (b) subjecting the posterior foregut cells to the process described herein to obtain hepatic progenitor cells. or providing a population of hepatic progenitor cells as described herein; and (c) hepatic progenitor cells. to the process described herein to obtain hepatocyte-like cells; or The present disclosure relates to a method for producing a medicament for the manufacture of ... Also provided are populations of hepatocyte-like cells that have been or have been obtained.

[0011] According to a seventh aspect, the present disclosure provides a process for producing encapsulated liver tissue. This process (a) provides a population of hepatocyte-like cells as described herein; (b) provides a suspension of the hepatocyte-like cells; Hepatocytes, mesenchymal cells, and optional endothelial cells are combined and cultured in suspension. and (i) at least partially covered with hepatocyte-like cells and / or bile duct epithelial cells. (ii) a cell core containing mesenchymal cells and optionally endothelial cells; (iii) a spherical morphology; and (iii) at least one liver organoid comprising a relative diameter of about 50 to about 500 μm; and (c) subjecting at least one of the liver organoids to a first biocompatible culture medium. In one embodiment, the method comprises at least partially covering the endodermal cells with a cross-linked polymer. The hepatocyte-like cells are combined at a ratio of 1:0.2 to 7 before culturing. Hepatocytes and endothelial cells are combined at a ratio of 1:0.2 to 1:1 before culturing. In terms of morphology, at least one of hepatocytes, endodermal cells, and endothelial cells is a pluripotent stem cell. In one embodiment, the endothelial cells are endothelial progenitor cells. In a further embodiment, this process comprises: For example, the cross-linked polymer may comprise poly(ethylene glycol) glycol (PEG). In another embodiment, this process comprises substantially covering the surface of the tissue with a biocompatible cross-linked polymer. The second biocompatible crosslinked polymer is a crosslinked polymer that is at least partially crosslinked with the first biocompatible crosslinked polymer. This further includes covering, and in some embodiments substantially covering, the portion. In this embodiment, the first biocompatible crosslinked polymer and / or the second biocompatible crosslinked polymer In yet another embodiment, the second biocompatible crosslink is at least partially biodegradable. The polymer comprises poly(ethylene) glycol (PEG). Also provided is encapsulated liver tissue obtainable or obtained by the process.

[0012] According to an eighth aspect, the present disclosure provides an additive set and a culture medium containing the same. In some embodiments, the present disclosure provides a first set of additives as described herein, as well as a second set of additives. a first additive set and no activator of the insulin signaling pathway; In one embodiment, the first culture medium is provided for endodermal cells and / or In another embodiment, the present disclosure provides a method for producing a medicament for the treatment of a medicament comprising administering to a subject the method of the present invention a method for producing a medicament for the treatment of ... and a second culture medium comprising the second set of additives. In some embodiments, the second culture medium comprises posterior foregut cells and / or hepatic progenitor cells. In yet another embodiment, the present disclosure provides a third additive set described herein, and In yet another embodiment, a third culture medium is provided that includes a third set of additives. The illustration shows the fourth additive set described herein, as well as a second additive set comprising the fourth additive set. In yet another embodiment, the present disclosure provides a culture medium according to any one of the fifth to fifth embodiments described herein. A fifth set of supplements was added to the medium, as well as a fifth set of supplements that did not contain cytokines. The present disclosure provides a culture medium for producing posterior foregut cells, hepatic progenitor cells, or hepatocyte-like cells. Also provided are kits for producing the pharmaceutical composition of the present invention. The kits include at least one additive cell described herein. and / or at least one medium described herein; and posterior foregut cells, for generating hepatic progenitor cells or hepatocyte-like cells (e.g., using the processes described herein) In some embodiments, the kit comprises a kit for the production of endoderm cells, endodermal cells, It further comprises foregut cells and / or hepatic progenitor cells.

[0013] Having outlined the gist of the present invention above, reference should now be made to the accompanying drawings, which are given for illustrative purposes only. A preferred embodiment thereof will now be described. [Brief explanation of the drawings]

[0014] [Figure 1] A and B show the expression of endoderm-specific genes. (A) Upregulation of endoderm-specific genes (FOXA2, SOX17, CXCR4, EOMES, GATA4) in iPSC-derived endoderm cells (DE - dark gray bars) compared to undifferentiated iPSCs (iPSC - light gray bars) as measured by RT-qPCR. Results are shown as the log fold change for the various genes tested. Data are means ± standard deviation. N=6 for DE, N=3 for iPSCs. *p<0.05**p<0.01. (B) Time course analysis of endoderm-specific gene (EOMES, FOXA2, SOX17) expression during iPSC differentiation to endoderm using RT-qPCR. Results are shown as the log fold change for the various genes tested (identified on the X-axis). Data are means ± standard deviation. N=3 for all time points. **p<0,01***p<0,001****p<0,0001. [Figure 2] Figure 2 shows representative flow cytometry analysis of iPSC-derived endoderm cells for FoxA2, ​​Cxcr4, Sox17, Brachyury, and c-Kit markers. More than 85% of cells are triple positive for FoxA2, ​​Cxcr4, and Sox17; 90% of cells are positive for brachyury; and less than 1% of cells are positive for c-Kit, indicating the absence of mesodermal cells. Data are mean ± standard deviation. n=4. [Figure 3] Figure 3 shows representative immunofluorescence analysis of endoderm markers Sox17, FoxA2, ​​and Cxcr4 in iPSC-derived endoderm cells (lower panel) and undifferentiated iPSCs (upper panel). The inset shows nuclear (DAPI) staining (scale bar 200 μm). [Figure 4] Figure 1 shows increased expression of posterior foregut-specific genes in iPSC-derived ventral posterior foregut cells, which give rise to hepatic progenitor cells. Results are shown as fold change in mRNA expression of these genes (FOXA2, SOX2, FOXA1, HNF4α, AFP, and albumin (ALB)) in iPSC-derived endoderm cells (DE - dark gray bars) and iPSC-derived posterior foregut cells (PFG - light gray bars). Data are mean ± standard deviation. DE: n=3, PFG: n=6. *p<0.05**p<0.01***p<0.001. [Figure 5] A to D show the expression of liver-specific markers (A AFP, B albumin, C CK19 and CK7, D EpCAM) in iPSC-derived hepatic progenitor cells as measured by immunofluorescence (scale bar 200 μM). [Figure 6] Representative flow cytometry analysis of iPSC-derived hepatic progenitor cells (HB - gray bars) compared to undifferentiated iPSCs (iPSC - white bars) for the pluripotency markers TRA1-60 and Nanog. Data are mean ± standard deviation. n=3. [Figure 7] Expression of hepatoblast- and hepatocyte-specific genes (albumin (ALB), AFP, CK19, CK7, PDX1, SOX9, PROX1, HNF4α, HHEX) in iPSC-derived hepatic progenitor cells (HB - black bars) compared to iPSC-derived posterior foregut cells (PFG - light gray bars) as determined by RT-qPCR. Results are shown as log fold changes for the various genes tested (identified on the x-axis). Data are mean ± standard deviation. n=8 for HB, n=3 for PFG. **p<0.01. [Figure 8] Figure 8 shows the time course of cell proliferation during iPSC differentiation into hepatic progenitor cells, demonstrating a significant increase in cell yield. Data are mean ± standard deviation. n = 6 for undifferentiated iPSCs (iPSCs), n = 3 for iPSC-derived endoderm cells (DE), and n = 6 for iPSC-derived hepatic progenitor cells (HB). **p<0.01. [Figure 9A]1 illustrates the characteristics of iPSC-derived hepatocyte-like cells. Typical aspects of iPSC-derived hepatocyte-like cells (HLCs) at day 28 as measured by light microscopy (scale bars, 1,000 μm in the upper panel and 200 μm in the lower panel). [Figure 9B] Figure 1 illustrates the characteristics of iPSC-derived hepatocyte-like cells. Expression of liver-specific markers (B1 AFP, B2 albumin, B3 and B4 CK19) in iPSC-derived hepatocyte-like cells determined by immunofluorescence (scale bars: 200 μm for upper and lower left panels, 100 μm for lower right panel). [Figure 10] A and B provide (A) a typical flow cytometry result of albumin-expressing iPSC-derived hepatocyte-like cells (HLCs), showing the uniformity of albumin expression (98.5% of gated cells), and (B) related analysis. Data are mean ± standard deviation. n=4. [Figure 11] Figure 1 shows the expression of liver-specific genes (HNF4α, AFP, albumin (ALB), SOX9, ASGPR) in iPSC-derived hepatocyte-like cells (HLC - dark gray bars) compared to freshly isolated fetal hepatocytes (FPHH - light gray bars) as determined by RT-qPCR. Results are shown as the log fold change of the various genes tested (identified on the X-axis). Data are mean ± standard deviation. n=6 for FPHH, N=10 for HLC. **p<0.01; ns=not significant. [Figure 12]Figures A-C show the liver-specific functions of primary human hepatocytes (PHH), human hepatoma cell line (HepG2), undifferentiated iPSCs (iPSCs), iPSC-derived endoderm cells (DE), iPSC-derived ventral posterior foregut cells (PFG), iPSC-derived hepatic progenitor cells (HB), and iPSC-derived hepatocyte-like cells (HLC). (A) Comparison of CyP3A4 activity. Results are shown as activity (RLU / 1 x 10 cells) according to the conditions tested. Data are means ± standard deviation. PHH: n = 10, HepG2 and iPSC: n = 3, HLC: n = 6. *p < 0.05. (B) Comparison of albumin synthesis. Data are means ± standard deviation. iPSC, DE, PFG, and HB: n = 3, HLC: n = 6, PHH: n = 10. **p < 0.01. (C) Comparison of urea. Data are means ± standard deviations. HepG2: n = 3, HLC: n = 6, PHH: n = 10. [Figure 13] Figure 13 provides the expression of liver-specific genes (HNF4α, AFP, albumin (ALB), ASGR1, TAT) in iPSC-derived hepatocyte-like cells (HLC-B, grey bars) compared to iPSC-derived hepatocyte-like cells obtained by standard differentiation protocol (HLC-A, black bars) as determined by RT-qPCR. Results are shown as log fold change for the various genes tested (identified on the X-axis). Data are mean ± standard deviation. n=8 for HLC-A, n=4 for HLC-B. *p<0.05***p<0.001*** *p<0.0001. [Figure 14]Figures A–C compare the characteristics of iPSC-derived hepatocyte-like cells (HLC-A, black bars) and iPSC-derived hepatocyte-like cells (HLC-B, gray bars). (A) Comparison of CyP3A4 activity. Results are shown as activity (RLU / 1 × 10 cells) depending on the condition tested. Data are means ± standard deviation. N = 4 for HLC-A and N = 6 for HLC-B. **p<0.01. (B) Comparison of albumin synthesis. Data are means (μg / 1 × 10 cells / 24 h) ± standard deviation. N = 4 for HLC-A and N = 6 for HLC-B. **p<0.01. (C) Cell yield at the end of differentiation: The novel differentiation protocol (light gray bars) significantly increases cell numbers compared to the amount of undifferentiated iPSCs at the beginning of the process (white bars), whereas the standard differentiation protocol (black bars) reduces cell yields. Data are means ± standard deviation. HLC-A: n = 3, HLC-B: n = 4. *p<0.05. [Figure 15] Oxygen consumption rate (OCR) was measured in Seahorses to assess key parameters of mitochondrial function in iPSC-derived hepatocyte-like cells (HLCs) at baseline (light grey bars) and after different doses of amiodarone (2, 4, 8, 16 μM - dark grey bars) and acetaminophen (2, 4, 8 mM - black bars). Data are means ± standard deviation. n=6. *p<0.05**p<0.01***p<0.001****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0015] Processes for producing cells and compositions containing same According to the present invention, endoderm cells can be isolated from cells of a competent hepatocyte lineage (e.g., posterior foregut cells, hepatocytes, and / or hepatocytes). The cells may be cells that can differentiate into hepatocytes or cells that are hepatocytes. The process may, in some embodiments, involve the production of large numbers of cells of the hepatocyte lineage, and / or This is advantageous as it allows for the production of biologically more potent cells.

[0016] In one embodiment, this process is used to generate diverse cell populations from endoderm cells. As used in this disclosure, "endodermal cells" refer to cells that have the characteristics of cells derived from the endoderm. As is known in the field of embryology, the endoderm is the most abundant of the three primary germ layers. The inner layer of the endoderm. The cells of the endoderm are generally flat and are found in the gastrointestinal tract, respiratory tract, and It is responsible for the development of most of the cells in the liver, pancreas, endocrine system, and urinary system. Endodermal cells may be identified by one of skill in the art using a variety of techniques known in the art. For example, endoderm cells express the following genes: SOX17, GATA4, FOXA2, and CXCRA , and / or EOMES, or any combination thereof or the presence or absence, and expression level, of the polypeptides they encode. In certain embodiments, the endoderm cells are identified by determining the following genetic sequence: Child: SOX17, GATA4, FOXA2, CXCRA, and / or EOMES At least two of them, or any combination thereof, or In yet another embodiment, the endoderm cells express the following gene: SOX 17, GATA4, FOXA2, CXCRA, and / or EOMES or any combination thereof, or the polypeptides they encode. Expression of the gene can be detected, and optionally measured, to allow identification. In morphology, endoderm cells express the following genes: SOX17, GATA4, FOXA2, CXCR At least four of A and / or EOMES, or any combination thereof Expression of the gene can be detected, and optionally measured, to identify the gene. In this state, endoderm cells express the following genes (or their related polypeptides): SOX 17, GATA4, FOXA2, CXCRA, and / or EOMES expression was detected. , and optionally, can be measured to identify the endoderm cells. and the following genes: SOX2, SOX17, GATA4, FOXA2, Expression of CXCRA and / or EOMES or the polypeptides they encode The current levels are compared to the levels of expression of the same gene / polypeptide in (undifferentiated) stem cells. In certain embodiments, endoderm cells can be identified as undifferentiated pluripotent (stem) cells. SOX17, GATA4, FOXA2, and CXCR4 compared with their corresponding levels in cells and / or strongly expressing EOMES genes or the polypeptides they encode. Appear.

[0017] The endoderm cells can be of any origin, and in particular can be derived from mammals. and, in some embodiments, may be of human origin.

[0018] Endodermal cells are pluripotent cells (e.g., embryonic stem cells or pluripotent stem cells) that have differentiated into endodermal cells. In some embodiments, endoderm cells can be obtained from induced pluripotent stem cells (i.e., induced pluripotent stem cells). Pluripotent (stem) cells can be obtained by differentiation of PSCs. , may be of mammalian origin, and in some embodiments, may be of human origin. In some embodiments where pluripotent (stem) cells are differentiated into endoderm cells, the pluripotent (stem) cells are Compounds capable of activating the Nodal / Activin signaling pathway, e.g. , and contacting the Nodal / Activin receptor with a Nodal / Activin receptor agonist, such as Activin A. In some further embodiments, the pluripotent (stem) cells are capable of expressing Wnt signaling. Activators of the pathway, e.g., Wnt receptor agonists, or the biological activity of GSK3 The compound may also be contacted with a compound capable of inhibiting .

[0019] Pluripotent (stem) cells express APELA / ELABELA signaling before differentiating into endoderm cells. one or more activators of the ATP signal transduction pathway, e.g., APELA / ELABELA polypeptides Agonists of the APELA / ELABELA receptor, such as, or its self-renewal and / or or functional fragments for inducing, optimizing, and maintaining pluripotency (U.S. Pat. No. 6,449,399; U ...). The compound can be contacted with a soluble polymer (such as those described in US Pat. No. 9,309,314).

[0020] The present disclosure provides the first process for generating posterior foregut cells from endodermal cells. This process allows the differentiation of one or more endoderm cells into posterior foregut cells. with a first culture medium containing a first set of additives under conditions that The process of 1 involves culturing cells to induce the expression of insulin, e.g., insulin, in the insulin signaling pathway. As used herein, "posterior foregut cells" does not include contacting the cells with an activator. These cells have biological characteristics of the cells of the foregut. The foregut is the region of the endoderm that will eventually form the liver. These cells can further differentiate into parts of the liver, pancreas, stomach, and small intestine. If so, various techniques known in the art can be used to identify posterior foregut cells. For example, posterior foregut cells express the following genes: SOX2, FOXA1, FOXA2, At least one of any combination of HNF4a, AFP, and / or albumin The presence or absence of amino acids or the polypeptides they encode, as well as expression levels In certain embodiments, the posterior foregut cells can be identified by determining the following: Genes: SOX2, FOXA1, FOXA2, HNF4a, AFP in any combination At least two of these, and / or albumin, or the polypeptides they encode In yet another embodiment, the posterior foregut cells express the following genes: SOX2, At least 3 of any combination of FOXA1, FOXA2, HNF4a, and AFP and / or express albumin or the polypeptides they encode. In yet another embodiment, the posterior foregut cells express the following genes: SOX2, FOXA1, FOX At least four of any combination of A2, HNF4a, and AFP; and / or Expressing albumin or the polypeptides they encode. In the posterior foregut cells, the following genes were expressed: SOX2, FOXA1, FOXA2, HNF4a, At least five of any combination of AFP and / or albumin, or In yet another embodiment, the posterior foregut cells express the polypeptides they encode. The following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or express albumin or the polypeptides they encode. In morphology, posterior foregut cells express the following genes (or their corresponding polypeptides): Expression of OX2, FOXA1, FOXA2, HNF4a, AFP, and / or albumin The phenomenon can be detected and optionally measured to identify it. Expression in posterior foregut cells and the following genes: SOX2, FOXA1, FOXA2, HNF4a, AFP, and / or albumin, or the polypeptides they encode The expression level of the peptide was compared with that of the same gene / polypeptide in (undifferentiated) stem cells or endoderm cells. The level of expression of the peptide can be compared to identify the target gene. Foregut cells have a higher S level compared to corresponding levels in pluripotent (stem) cells or endodermal cells. OX2, FOXA1, FOXA2, HNF4a, AFP, and / or albumin genes In a further embodiment, the vectors express the polypeptides encoded by them. Posterior foregut cells express high levels of the SOX2 gene compared with corresponding levels in endoderm cells. In a further embodiment, the gene expresses a polypeptide that encodes the gene. Enterocytes express less FOXA1 gene or its equivalent compared to the corresponding levels in endoderm cells. In a further embodiment, the posterior foregut cells strongly express the polypeptide encoded by The FOXA2 gene or its encoding In a further embodiment, the posterior foregut cells strongly express the polypeptide. the HNF4a gene or its encoded polypeptide, compared to corresponding levels In a further embodiment, posterior foregut cells strongly express α-glucan at levels comparable to those in endoderm cells. The AFP gene or the polypeptide it encodes is more strongly expressed than in normal mice. In a further embodiment, posterior foregut cells exhibit a higher A compared to corresponding levels in endoderm cells. It strongly expresses the LB gene or the albumin polypeptide it encodes.

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

[0022] The first culture medium used in the first process may be serum-free (e.g., not supplemented with serum). In another embodiment, the first culture medium used in the first process can be The serum may be used in combination with KnockOut Serum Replacer. ent™ (ThermoFisherScientific). In one embodiment, the first culture medium contains about 0.1 to about 5% (v / v) serum. In embodiments, the first culture medium has a pH of at least about 0.1, 0.2, 0.3, 0.4, 0.5, or 10% by weight of the first culture medium. ,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5 In another embodiment, the first culture medium comprises about 5, 4.5, 4, 3.5, or more percent serum. ,3,2.5,2,1.5,1,0.9,0.8,0.7,0.6,0.5,0.4,0 .3, containing 0.2% or less serum. In yet 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 approximately 5, 4.5, 4, 3.5, 3, 2 0.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or In one embodiment, the first culture medium contains between about 1% serum and 0.2% serum. Includes Qing.

[0023] The first culture medium includes a first set of additives, the first set including bone morphogenetic proteins. Activator of bone marrow protein (BMP) signaling pathway; fibroblast growth factor (FGF) signaling activators of the Wnt signaling pathway; inhibitors of the Wnt signaling pathway; and transforming growth factor β (TGFβ) signaling pathway inhibitors. One additive set contains activators of the insulin signaling pathway, such as insulin. As used in the context of this disclosure, the phrase "first culture medium" refers to a culture medium that is essentially made up of a first set of additives. "Consists of" means that the cells do not contain any additional additives that are not essential for differentiation of endoderm cells into posterior foregut cells. These additional additives are referred to as the first culture medium, which contains the additional additives but is still capable of promoting differentiation. Examples include, but are not limited to, retinoic acid, vitamins, and minerals. I can't.

[0024] The first culture medium contains activators of the bone morphogenetic protein (BMP) signaling pathway. During development, activators of the BMP signaling pathway are normally expressed in the cardiac mesoderm. and promotes differentiation of endoderm cells into posterior foregut cells. "Activators of the BMP signaling pathway" as used herein refer to the interaction between BMPs and their cognate receptors (e.g., Activates signaling pathways associated with binding to BMPR1 and / or BMPR2 Signaling BMP receptors are compounds that can act as receptors for SMADs and MAs. This compound is involved in the transcription of BMP target genes via the P kinase pathway. , (specific for BMPR1 or BMPR2, or binds to both receptors) agonists of BMP receptors, which can activate BMP signaling pathways activators of polypeptides known to activate the tract and / or BMP signaling The inhibitor can be any of the polypeptides known to inhibit the transduction pathway. MPs include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7 BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. In one embodiment, the activator is DM3189, but is not limited to these. In another embodiment, the activator is BMP4 (provided in recombinant or purified form). BMP4 is a member of the transforming growth factor-β (TGF-β) family. It is a protein that expresses two different types of serine- Binds to leonine kinase receptors, making BMP4 an activator of the BMP signaling pathway In the embodiments provided herein, the BMP4 is present 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, Provided at concentrations of 25, 26, 27, 28, 29, or greater ng / mL In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is added to the first culture medium at least about 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, BMP4 can be provided at concentrations of less than 11 or less than ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, 1 culture medium, approximately 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, Can be provided at concentrations between 15, 14, 13, 12, or 11 ng / mL In certain embodiments, BMP4 is added to the first culture medium at a concentration of about 20 ng / mL. It can be provided.

[0025] The first culture medium also contains activators 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. "Activators of the FGF signaling pathway" refer to FGFs and their cognate receptors (e.g., FGFs). GFR1, FGFR2, FGFR3, and / or FGFR4) This refers to a compound that can activate the signal transduction pathway (FGF). 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 any 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 soluble form of FGFR2 (also known as CGF-1). D332) and FGFR3, two different types of receptors. 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 n 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 embodiments where basic FGF is provided as a activating factor, the first culture medium contains at least Approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, It can be provided in concentrations of 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, The basic FGF is added to the first culture medium at a concentration of 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 first culture medium at a concentration of about 5 ng / mL.

[0026] The first culture medium further comprises an inhibitor of the Wnt signaling pathway. The presence of inhibitors of the signaling pathway in combination with inhibitors of the TGFβ signaling pathway In this study, the expression of HEX and PROX1 genes, which encode polypeptides required for liver development, was As used in the context of this disclosure, "inhibitor of the Wnt signaling pathway" The interaction between Wnt protein ligands and their cognate Frizzle receptors (e.g., FZD1 , FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 or FZD10) and can inhibit signal transduction pathways associated with binding. The Frizzled receptor family is a G protein-coupled receptor This compound is a protein (FZD1, FZD2, FZD3, FZD4, FZD 5. Specific for either FZD6, FZD7, FZD8, FZD9, or FZD10 or capable of binding to and inhibiting multiple receptors) Frizz Antagonists of the led receptor, polynucleotides known to activate the Wnt signaling pathway, Peptide inhibitors and / or peptides known to inhibit the Wnt signaling pathway Known Wnt proteins include WNT1 and WNT2. , WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B , WNT10A, WNT10B, WNT11, and WNT16, but are not limited to these. In certain embodiments, the inhibitor inhibits the biology of one or more Frizzled receptors. In another embodiment, the inhibitor is a porcupine protein. It can inhibit the biological activity of proteins, such as porcupine protein. The inhibitor capable of inhibiting the biological activity of the protein may be IWP2. In embodiments where IWP2 is used as an inhibitor of the Wnt signaling pathway, IWP2 is added to the first culture medium. In the nutrient medium, 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. May be provided at concentrations of 5, 7, 7.5, 8, 8.5, 9, 9.5 μM or greater. In an embodiment in which IWP2 is used as an inhibitor of the Wnt signaling pathway, IWP2 was cultured in the first medium at 10, 9.5, 9, 8.5, 8, 7.5, 7, and 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 less IWP2 can be used as an inhibitor of the Wnt signaling pathway. In some embodiments, IWP2 is present in the first culture medium at a concentration of about 0.1, 0.2, 0.3, 0.4, or 1.0% by weight. ,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, Approximately 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. IWP2 can be provided at a concentration of between 4, 0.3, or 0.2 μM. In embodiments where IWP2 is used as an inhibitor of the nt signaling pathway, IWP2 is administered at a concentration of about 4 μM. The first culture medium may be provided at a concentration of

[0027] The first culture medium further contains an inhibitor of the transforming growth factor β (TGFβ) signaling pathway. The presence of inhibitors of the TGFβ signaling pathway inhibits the Wnt signaling pathway. In combination with the toxic factor, HEX and HEX encode polypeptides required for liver development. As used in the context of this disclosure, "TGFβ synthase" has a beneficial effect on the expression of the PROX1 gene. "Inhibitors of signal transduction pathways" refers to inhibitors of signal transduction pathways associated with the binding of TGF-β to its cognate receptor. The term refers to compounds that can inhibit the TGFβ receptor family. mediates signal transduction through SMAD proteins. This compound inhibits TGFβ receptors. antagonists of the TGFβ signaling pathway, polypeptides known to activate the TGFβ signaling pathway inhibitors of TGFβ and / or polypeptides known to inhibit the TGFβ signaling pathway Known TGFβ proteins include TGFB1, Examples include, but are not limited to, TGFB2, TGFB3, and TGFB4. In some embodiments, the inhibitor inhibits at least one of an ALK4, ALK5, or ALK7 polypeptide. In some embodiments, the inhibitor can inhibit at least one biological activity of: It is possible to inhibit the biological activity of ALK4, ALK5, and ALK7 polypeptides. For example, by inhibiting the biological activity of ALK4, ALK5, and ALK7 polypeptides. The inhibitor that can be used may be A83-01. In combination, SB431542 and / or LY364947 are used as inhibitors. A83-01 is used as an inhibitor of the TGFβ signaling pathway. In this state, A83-01 is added to the first culture medium 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, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5μM, A83-01 can be provided at a concentration higher than that of TGFβ signaling. In embodiments for use as an inhibitor of a transduction pathway, A83-01 comprises, in a 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. It can be provided at concentrations of 4, 0.3, 0.2 μM or less. A83 In embodiments where A83-01 is used as an inhibitor of the TGFβ signaling pathway, 01 is the first culture medium, 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 0.8, 1.9, 2, 2.5, 3, 3.5, 4, or 4.5 and 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 A83-01 can be provided at a concentration between 1 μM and 2 μM. In an embodiment where A83-01 is used as an inhibitor of It may be provided in the culture medium.

[0028] The first culture medium is used to culture endoderm cells and posterior cells for at least one day until differentiation occurs. The first culture medium is kept in contact with the cultured cells for two or more days. Where illustrated, the medium can be changed daily. In some embodiments, the first culture medium is maintained 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 may be left for at least 1, 2, 3, 4, or more days, and then 5, 4, The cultured cells are left in contact for 3, 2, or fewer days. The first culture medium is then allowed to come into contact with the cultured cells for about 1 to 5 days.

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

[0030] The present disclosure provides 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 posterior foregut cells into the posterior foregut. The first culture containing a second set of additives was grown under conditions that allowed differentiation of the posterior foregut cells from the posterior foregut cells. The posterior foregut cells used in the second process are contacted with the second culture medium. It can be obtained by running process 1.

[0031] As used herein, "hepatic progenitor cells" or "hepatoblasts" refers to cells derived from bile duct cells and hepatocytes. Those skilled in the art will understand that the term "progenitor cells" refers to bipotent progenitor cells that can differentiate into either Various techniques known in the art can be used to identify hepatic progenitor cells. For example, Hepatic progenitor cells express the following genes: alpha-fetoprotein (AFP), albumin (ALB), Cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, At least one of PROX1, EpCAM, HHEX gene, and / or HNF4a One or any combination thereof and / or the polypeptides they encode. The presence or absence of a peptide, as well as the expression level, can be determined to identify it. In certain embodiments, the hepatic progenitor cells express the following genes: alpha-fetoprotein (AFP), Albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19) , SOX9, PDX1, PROX1, EpCAM, HHEX, or HNF4a At least one or any combination thereof or the programs they code for In yet another embodiment, the hepatic progenitor cells express the following gene: α-fetal Antigen protein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin Keratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, or at least one of HNF4a, HNF5a, and HNF6a, or the polypeptides they encode. In yet another embodiment, the hepatic progenitor cells express the following gene: alpha-fetoprotein. (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, and / or , HNF4a, or at least two of any combination thereof In yet another embodiment, the hepatic progenitor cells express the following gene: α-fetal Antigen protein (AFP), albumin (ALB), cytokeratin 7 (CK7), Tokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX and / or HNF4a in any combination of at least three of them In yet another embodiment, the hepatic progenitor cells express a polypeptide encoded by Genes: alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 7 ( CK7), cytokeratin 19 (CK19), SOX9, PDX1, PROX1, EpC At least four of any combination of AM, HHEX, and / or HNF4a or express the polypeptides they encode. Progenitor cells express the following genes: alpha-fetoprotein (AFP), albumin (ALB), and cytoplasmic reticulocytes (CRC). Cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PR Any combination of OX1, EpCAM, HHEX, and / or HNF4a In yet another embodiment, the hepatic progenitor cells express at least five of the following genes: α- Fetal protein (AFP), albumin (ALB), cytokeratin 7 (CK7), Itokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHE At least six or more genes encoding any combination of X and / or HNF4a In yet another embodiment, the hepatic progenitor cells express the following gene: α-fetal Antigen protein (AFP), albumin (ALB), cytokeratin 7 (CK7), Tokeratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX and / or any combination of HNF4a and at least seven or more polypeptides encoded by the In yet another embodiment, the hepatic progenitor cells express the following gene: α-fetal Antigen protein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin Keratin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, and / or at least eight or more polynucleotides encoded by any combination of HNF4a. In yet another embodiment, the hepatic progenitor cells express the following gene: α-fetal peptide. Protein (AFP), albumin (ALB), cytokeratin 7 (CK7), cytokeratin Latin 19 (CK19), SOX9, PDX1, PROX1, EpCAM, HHEX, and and / or at least nine or more polypeptides encoded by any combination of HNF4a and HNF5a. In yet another embodiment, the hepatic progenitor cells express the following genes (or Polypeptides encoded by: alpha-fetoprotein (AFP), albumin (ALB), ), cytokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX 1, PROX1, EpCAM, HHEX, and / or HNF4a. In embodiments, hepatic progenitor cells are induced to express and express 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 these genes were compared with those of the same genes / polypeptides in posterior foregut cells. In one embodiment, hepatic progenitor cells can be identified by comparing the expression levels of the hepatic progenitor cells with the expression levels of the hepatic progenitor cells. In one embodiment, hepatic progenitor cells express substantially the same amount of albumin as posterior foregut cells. In one embodiment, the hepatic progenitor cells express substantially the same amount of AFP as the posterior foregut cells. express the CK19 gene more strongly than posterior foregut cells. In one embodiment, hepatic progenitor cells express the CK7 gene more strongly than posterior foregut cells. In one embodiment, hepatic progenitor cells express the PDX1 gene more strongly than posterior foregut cells. In one embodiment, hepatic progenitor cells express the SOX9 gene more strongly than posterior foregut cells. , which express the PROX1 gene more strongly than posterior foregut cells. In one embodiment, hepatic progenitor cells express the HHEX gene, but more weakly than posterior foregut cells. The cells are characterized by the presence of TRA-1-60 and / or TRA-1-60 compared to undifferentiated pluripotent cells (e.g., iPSCs). do not substantially express Nanog genes or express these genes at very low levels. To manifest.

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

[0033] The second culture medium used in the second process may be serum-free (e.g., not supplemented with serum). In another embodiment, the second culture medium used in the second process can be The serum may be used in combination with KnockOut Serum Replacer. ent™ (ThermoFisherScientific). In one embodiment, the second culture medium contains about 0.1 to about 5% (v / v) serum. In embodiments, the second culture medium is 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 In another embodiment, the second culture medium contains about 5, 4.5, 4, 3.5, or more percent serum. ,3,2.5,2,1.5,1,0.9,0.8,0.7,0.6,0.5,0.4,0 .3, containing 0.2% or less serum. In yet another embodiment, the second 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 approximately 5, 4.5, 4, 3.5, 3, 2 0.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or In one embodiment, the second culture medium contains between about 2% serum and 0.2% serum. Includes Qing.

[0034] The second culture medium contains a second set of additives, which is an insulin signaling agent. activator of the bone morphogenetic protein (BMP) signaling pathway Activator of fibroblast growth factor (FGF) signaling pathway, hepatocyte growth factor (HGF) activators of the GFAP signaling pathway and the Wnt signaling pathway As used in the context of this disclosure, the expression "second culture medium" refers to a medium that comprises, or consists essentially of, , essentially consisting of a second set of additives, is responsible for the differentiation of posterior foregut cells into hepatic progenitor cells. A second culture medium that contains additional additives that are not essential for differentiation but can promote differentiation. These additional additives include, for example, B27 supplements, retinoic acid, These include, but are not limited to, vitamins and minerals.

[0035] The second culture medium also contains an activator of the insulin signaling pathway. The term "activator of the insulin signaling pathway" used refers to insulin and its cognate receptors. Activation of signal transduction pathways associated with binding to receptors (tyrosine kinase receptors) This compound is a compound that can bind to insulin receptors (insulin, IGF- IGF-I or IGF-II agonists, activating the insulin signaling pathway activators of polypeptides known to inhibit the insulin signaling pathway and / or In some embodiments, the inhibitor may be any of the polypeptides known to inhibit activity. The activating agent is insulin (which can be provided in recombinant or purified form). In embodiments where insulin is used as an inhibitor of the insulin signaling pathway, The sulin may be added to the second culture medium at a concentration of 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 at levels above 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 some embodiments, insulin is present in the second culture medium at a concentration of about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, 300, 450, 500, 600, 700, 800, 900, 1000, 150 , 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, Available in concentrations between 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 In certain embodiments, insulin can be provided in the second culture medium at a concentration of about In yet another embodiment, insulin can be provided at a concentration of 10 mg / ml. , in the form of B27 supplements, HBM / HCM Bulletkit™, and / or Alternatively, it may be provided in the form of a primary hepatocyte (PHH) supplement.

[0036] The second culture medium contains activators of the bone morphogenetic protein (BMP) signaling pathway. During development, activators of the BMP signaling pathway are normally expressed in the cardiac mesoderm. and promotes differentiation of endoderm cells into posterior foregut cells. "Activators of the BMP signaling pathway" as used herein refer to the interaction between BMPs and their cognate receptors (e.g., Activates signaling pathways associated with binding to BMPR1 and / or BMPR2 Signaling BMP receptors are compounds that can act as receptors for SMADs and MAs. This compound is involved in the transcription of BMP target genes via the P kinase pathway. , (specific for BMPR1 or BMPR2, or binds to both receptors) agonists of BMP receptors, which can activate BMP signaling pathways activators of polypeptides known to activate the tract and / or BMP signaling The inhibitor can be any of the polypeptides known to inhibit the transduction pathway. MPs include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7 BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. In one embodiment, the activator is DM3189, but is not limited to these. In another embodiment, the activator is BMP4 (provided in recombinant or purified form). BMP4 is a member of the transforming growth factor-β (TGF-β) family. It is a protein that expresses two different types of serine- Binds to leonine kinase receptors, making BMP4 an activator of the BMP signaling pathway In the embodiments provided herein, the BMP4 is present 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, Provided at concentrations of 25, 26, 27, 28, 29, or greater ng / mL In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is added to the second culture medium for at least about 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, BMP4 can be provided at concentrations of less than 11 or less than ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, 2 culture medium, approximately 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, Can be provided at concentrations between 15, 14, 13, 12, or 11 ng / mL In certain embodiments, BMP4 is added to the second culture medium at a concentration of about 20 ng / mL. In a further embodiment, BMP4 may be present in both the first and second sets of additives. It can serve as an activator in the method.

[0037] The second culture medium also contains activators 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. "Activators of the FGF signaling pathway" refer to FGFs and their cognate receptors (e.g., FGFs). GFR1, FGFR2, FGFR3, and / or FGFR4) This refers to a compound that can activate the signal transduction pathway (FGF). 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 any 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 soluble form of FGFR2 (also known as CGF-1). D332) and FGFR3, two different types of receptors. In embodiments that provide basic FGF as an activator of the FGF signaling pathway, The FGF is present in the second culture medium at a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 n 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 embodiments where basic FGF is provided as a activating factor, the second culture medium may contain at least Approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, It can be provided in concentrations of 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, In the second culture medium, basic FGF is added at a concentration of 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. In this study, basic FGF was used as an activator in both the first and second additive sets. It 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 promotes differentiation. The signaling pathways associated with the binding of GFs to their cognate receptors (e.g., c-Met) This compound is an agonist of the HGF receptor. activators of polypeptides known to activate the HGF signaling pathway, and / or Alternatively, any of the inhibitors of polypeptides known to inhibit the HGF signaling pathway. In one embodiment, the activator may be HGF (recombinant or purified form). HGF is presented as an activator of the HGF signaling pathway. In a provided embodiment, HGF is present 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, Alternatively, HGF can be provided at a concentration of ng / mL or higher. In embodiments where HGF is provided as an activator of the nucleoside signaling pathway, the second culture medium may comprise: 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 ng / mL The present invention provides HGF as an activator of the HGF signaling pathway. In the second culture medium, HGF is added 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 9, 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, It can be provided at a concentration between 12 or 11 ng / mL. In one embodiment, HGF can be provided in the second culture medium at a concentration of about 20 ng / mL.

[0039] The second culture medium further comprises an activator of the Wnt signaling pathway. In this state, only if you have already inhibited it (e.g., as shown in the first process) It is important to activate the Wnt signaling pathway in the foregut cells. The term "activator of the Wnt signaling pathway" used herein refers to a Wnt protein ligand and , and their cognate Frizzle receptors (e.g., FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10) Frizzle refers to compounds that can activate related signaling pathways. The ed receptor family is a G protein-coupled receptor protein. , (FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, or FZD10, or multiple receptors agonists of Frizzled receptors, W activators of polypeptides known to activate the nt signaling pathway, and / or Any of the polypeptide inhibitors known to inhibit the Wnt signaling pathway Known Wnt proteins include WNT1, WNT2, WNT2B, and WNT3. , WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B , WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, W Examples of the active compounds include, but are not limited to, NT11 and WNT16. The activating factors are Wnt3a, SB-216763, and / or LY2090314. In one embodiment, the activator inhibits the biological activity of the GSK3 protein. For example, activators that can inhibit the biological activity of GSK3 protein can be The offspring can be designated CHIR99021. CHIR99021 can be used to express Wnt signaling. In embodiments where CHIR99021 is used as an activator of a transduction pathway, In the medium, at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5 It can be provided at concentrations of 0.5, 6, 6.5, 7, 7.5 μM or greater. Embodiments in which CHIR99021 is used as an activator of the Wnt signaling pathway In the second culture medium, CHIR99021 was used at 8, 7.5, 7, 6.5, 6, and 5.5 , 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or less μM CHIR99021 can be provided at concentrations that are not sufficient to inhibit the Wnt signaling pathway. In embodiments used as an activator, CHIR99021 is added to the second culture medium at a concentration of 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. It can be provided at a concentration of between 5, 3, 2.5, 2, 1.5, or 1 μM. In embodiments where HIR99021 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 then added to the posterior foregut cells for at least one day to allow differentiation. The second culture medium is allowed to contact with the cultured cells for 2 days or more. If it is intended that the medium be changed daily, the medium may be changed daily. In some embodiments, the second culture medium comprises at least 1, 2, 3, 4, or more In another embodiment, the second culture medium is allowed to remain in contact with the cultured cells for 5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 The cultured cells are left in contact for 3, 2, or fewer days. Then, the second culture medium is incubated for at least 1, 2, 3, 4, or more days, and The cells are left in contact with the cultured cells for 1, 5, 4, 3, 2, or fewer days. In this embodiment, the second culture medium is left in contact with the cultured cells for about 1 to 5 days.

[0041] The second culture medium containing posterior foregut cells differentiates the posterior foregut cells into hepatic progenitor cells. Therefore, the present disclosure provides a method for producing hepatic progenitor cells obtained by the processes described herein. In the population of hepatic progenitor cells of the present disclosure, a majority of the cells are hepatic progenitor cells. and, in some embodiments, may include some hepatic progenitor cells. In one embodiment, the population of hepatic progenitor cells obtained in the second process is hepatic progenitor cells (e.g., For example, the expression of CK19 or EpCAM can be determined to identify the target cell. At least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, Contains 96, 97, 98, or 99%.

[0042] The present disclosure provides a third process for generating hepatocyte-like cells from hepatic progenitor cells. This process involves the production of one or more hepatic progenitor cells under conditions that allow differentiation of the hepatic progenitor cells into hepatocytes. The hepatic progenitor cells were cultured in a medium containing a third set of additives (which differentiates hepatic progenitor cells into cells of the hepatocyte lineage). The cells are then contacted with a third culture medium containing a fourth set of additives (promoting the growth of cells of a hepatocyte lineage). The cells are then contacted with a fourth culture medium (which promotes differentiation of the cells into immature cells), followed by a fifth additive. contacting the cells with a fifth culture medium containing acetone (which promotes differentiation of immature hepatocytes into mature hepatocytes); The hepatic progenitor cells used in the third process are the same as those used in the first process, as described herein. , and / or can be obtained by performing a second process.

[0043] As used in this disclosure, "hepatocyte-like cells" generally refers to cells of the hepatocyte lineage, immature hepatocyte-like cells, Hepatocyte-lineage cells differentiate into bile duct cells and mature hepatocyte-like cells. In some embodiments, hepatocyte-like cells are not capable of differentiation into hepatocytes, but are capable of differentiation into hepatocytes. The cells (especially mature hepatocyte-like cells) express certain proteins (albumin, clotting factors, alpha -1-antitrypsin, etc.), detoxification of ammonia to urea, and drug metabolism It can reproduce liver-specific functions such as glycogen storage, bilirubin conjugation, and bile synthesis. Those skilled in the art can identify hepatocyte-like cells using a variety of techniques known in the art. For example, hepatocyte-like cells express the following gene: α-fetal protein Protein (AFP), albumin (ALB), ASGR1, ASGPR, HNF4a, and SOX9 or any combination thereof, or The presence or absence and expression level of the polypeptides encoded by them are determined, In certain embodiments, the hepatocyte-like cells can be identified by expressing the following gene: α-fetal Antigen protein (AFP), albumin (ALB), ASGR1 (ASGPR), HNF4 a, and / or SOX9, or any combination thereof In certain embodiments, hepatocytes are capable of expressing the polypeptides they encode. The α-fetoprotein (AFP), albumin (ALB), and AS genes were expressed in the α-fetoprotein (AFP), α-fetoprotein (AFP), and albumin (ALB), respectively. At least two of GR1 (ASGPR), HNF4a, and / or SOX9, or or any combination thereof, or any vector capable of expressing the polypeptides they encode. In certain embodiments, the hepatocyte-like cells express the following gene: alpha-fetoprotein (AFP) ), albumin (ALB), ASGR1 (ASGPR), HNF4a, and / or S At least three of the OX9, or any combination of them, or In certain embodiments, the hepatocyte-like cells express a polypeptide encoding the following gene: : alpha-fetoprotein (AFP), albumin (ALB), ASGR1 (ASGPR) , HNF4a, and / or SOX9, or any of them. In certain embodiments, the polypeptides encoded by the vectors are expressed in combination with the vectors. The hepatocyte-like cells express the following genes: alpha-fetoprotein (AFP), albumin (AL), and B), ASGR1 (ASGPR), HNF4a, and / or SOX9, or In yet another embodiment, the hepatocyte-like cells express a polypeptide encoded by Genes: alpha-fetoprotein (AFP), albumin (ALB), ASGR1 (AS GPR), HNF4a, and / or SOX9, or at least one of them detecting expression of any combination thereof or the polypeptides they encode, and In yet another embodiment, hepatocyte-like cells are identified. The cells were then transfected with the following genes: alpha-fetoprotein (AFP), albumin ( ALB), ASGR1, HNF4a, and / or SOX9, or and detecting the expression of one or more polypeptides encoded by any combination thereof. In some embodiments, hepatocyte-like cells can be identified by optionally measuring the amount of hepatocyte-like cells. The cells were allowed to express and express the following genes: alpha-fetoprotein (AFP), albumin (ALB), ASGR1, HNF4a, and / or SOX9, or The level of expression of the polypeptide to be encoded is compared with that of the same gene in hepatocytes (e.g., fetal hepatocytes). The level of expression of the protein / polypeptide can be compared to identify the protein / polypeptide. In this state, hepatocyte-like cells express a significantly higher level of the SOX9 gene, compared to the corresponding levels in fetal liver cells. Alternatively, the polypeptide it encodes may be expressed strongly. The cells expressed HNF4a, AFP, ALB, and IL-1 at virtually the same levels as fetal liver cells. and express the ASGPR gene. The mature hepatocyte-like cells are, for example, with detectable levels of CyP3A4, such as a relative activity of 0 units / million cells In yet another embodiment, mature hepatocyte-like cells are more potent than immature hepatocyte-like cells. Mature hepatocyte-like cells can have stronger CyP3A4 activity than mature hepatocyte-like cells, for example, All were approximately 5, 6, 7, 8, 9, 10, 11, and 12 μg / L / 10 6 / 24 hours or more Mature hepatocyte-like cells can produce detectable levels of albumin exceeding that of normal hepatocytes. , e.g., at least about 10, 100, or 1000 μg / L / 10 6 / 24 hours, Alternatively, detectable levels of albumin can be produced.

[0044] The hepatocyte-like cells can be of any origin, and in particular can be derived from mammals. and, in some embodiments, may be of human origin.

[0045] The third, fourth, and fifth culture media used in the third process are serum-free (e.g., serum-free). In another embodiment, the third process may be The third, fourth, and fifth culture media may contain serum, and the serum may be used in combination with Knockout. Out Serum Replacement™ (ThermoFisherSc In one embodiment, the third, fourth, and fifth culture media may be In yet another embodiment, the third, fourth, and fifth serums each contain about 0.1 to about 5% (v / v) of serum. and the fifth culture medium is 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 In another embodiment, the third, fourth, and fifth culture media contain serum. 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, In yet another embodiment, the third, fourth, and and the fifth 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 approximately 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% serum. The third culture medium comprises about 2% serum. In another embodiment, the third culture medium comprises about 1% In another embodiment, the fourth culture medium comprises about 1% serum. In an embodiment, the fifth culture medium comprises about 1% serum.

[0046] The third culture medium contains a third set of additives, which is an insulin signaling agent. activator of the bone morphogenetic protein (BMP) signaling pathway Activator of fibroblast growth factor (FGF) signaling pathway, hepatocyte growth factor (HGF) activator of the Wnt signaling pathway, TGFβ inhibitors of signal transduction pathways, including cytokines and glucocorticoids; or As used in the context of this disclosure, the expression "third culture medium" refers to a medium that essentially consists of the first The term "consisting of a set of three additives" refers to the use of additives that are not essential for the differentiation of hepatocyte progenitor cells into hepatocyte-like cells. This refers to a third culture medium that contains additional additives necessary but is still able to promote differentiation. These further additives include, for example, B27 supplement, primary hepatocyte supplement ( PHH), HBM / HCM Bulletkit™ Retinoic Acid, Insulin, Examples of suitable nutrients include, but are not limited to, vitamins and minerals.

[0047] The third culture medium also contains an activator of the insulin signaling pathway. The term "activator of the insulin signaling pathway" used refers to insulin and its cognate receptors. Activation of signal transduction pathways associated with binding to receptors (tyrosine kinase receptors) This compound is a compound that can bind to insulin receptors (insulin, IGF- IGF-I or IGF-II agonists, activating the insulin signaling pathway activators of polypeptides known to inhibit the insulin signaling pathway and / or In some embodiments, the inhibitor may be any of the polypeptides known to inhibit activity. The activating agent 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 added to the third culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 450, 500, 600, 700, 800, 900, 1000, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, Insulin may be provided at or above ng / mL. In embodiments where insulin is used as a tract activator, the insulin is added to the third 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 Insulin can be used as an activator of the insulin signaling pathway. In the embodiment used, insulin is added to the third culture medium at a concentration of about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 23 , 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, Concentrations between 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 In certain embodiments, insulin may be provided in the third culture medium. In yet another embodiment, insulin may be provided at a concentration of about 10 mg / ml. Phosphorus is available in the form of B27 supplements, HBM / HCM Bulletkit™, and and / or in the form of a primary hepatocyte (PHH) supplement.

[0048] The third culture medium contains activators of the bone morphogenetic protein (BMP) signaling pathway. During development, activators of the BMP signaling pathway are normally expressed in the cardiac mesoderm. and promotes differentiation of endoderm cells into posterior foregut cells. "Activators of the BMP signaling pathway" as used herein refer to the interaction between BMPs and their cognate receptors (e.g., Activates signaling pathways associated with binding to BMPR1 and / or BMPR2 Signaling BMP receptors are compounds that can act as receptors for SMADs and MAs. This compound is involved in the transcription of BMP target genes via the P kinase pathway. , (specific for BMPR1 or BMPR2, or binds to both receptors) agonists of BMP receptors, which can activate BMP signaling pathways activators of polypeptides known to activate the tract and / or BMP signaling The inhibitor can be any of the polypeptides known to inhibit the transduction pathway. MPs include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7 BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. In one embodiment, the activator is DM3189, but is not limited to these. In another embodiment, the activator is BMP4 (provided in recombinant or purified form). BMP4 is a member of the transforming growth factor-β (TGF-β) family. It is a protein that expresses two different types of serine- Binds to leonine kinase receptors, making BMP4 an activator of the BMP signaling pathway In the embodiments provided above, BMP4 is present 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, Provided at concentrations of 25, 26, 27, 28, 29, or greater ng / mL In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, BMP4 is added to the third culture medium for at least about 30, 29, 28, 27, 26, 25 , 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, BMP4 can be provided at concentrations of less than 11 or less than ng / mL. In embodiments where BMP4 is provided as an activator of the BMP signaling pathway, 3 culture medium, approximately 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, Can be provided at concentrations between 15, 14, 13, 12, or 11 ng / mL In certain embodiments, BMP4 is added to the third culture medium at a concentration of about 20 ng / mL. In a further embodiment, BMP4 may be provided as a first, second, and third additive set. The activator may be provided in both the IL-1 and IL-2 receptors.

[0049] The third culture medium also contains activators 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. "Activators of the FGF signaling pathway" refer to FGFs and their cognate receptors (e.g., FGFs). GFR1, FGFR2, FGFR3, and / or FGFR4) This refers to a compound that can activate the signal transduction pathway (FGF). 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 any 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 soluble form of FGFR2 (also known as CGF-1). D332) and FGFR3, two different types of receptors. 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 n 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 embodiments where basic FGF is provided as a activating factor, the first culture medium contains at least Approximately 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, It can be provided in concentrations of 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, The basic FGF is added to the first culture medium at a concentration of 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 third culture medium at a concentration of about 10 ng / mL. In this study, basic FGF was used as an activator in both the second and third additive sets. It can be provided.

[0050] The third culture medium also contains an activator of the hepatocyte growth factor (HGF) signaling pathway. During development, activators of the HGF signaling pathway induce differentiation of endoderm cells into cells of the hepatocyte lineage. As used in the context of this disclosure, "activator of the HGF signaling pathway" refers to a signal transduction associated with the binding of HGF to its cognate receptor (e.g., c-Met) This refers to a compound that can activate the HGF receptor 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. In certain embodiments, the activator may be HGF (recombinant or HGF is an activator of the HGF signaling pathway. In the embodiments provided herein, HGF is present in a 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, 30, 31, 32, 33, 34, 35, 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, HGF can be provided at a concentration of 39 or more ng / mL. In embodiments where HGF is provided as an activator of the F signaling pathway, HGF is added to the third culture medium. 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 HGF can be provided as an activator of the HGF signaling pathway. In some embodiments, HGF is present in the third 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, , 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, It can be provided at a concentration of between 13, 12, or 11 ng / mL. In this embodiment, HGF may be provided in the third culture medium at a concentration of about 20 ng / mL. HGF can be the activator in the second and third sets of additives.

[0051] The third culture medium further comprises an activator of the Wnt signaling pathway. The term "activator of the Wnt signaling pathway" used herein refers to a Wnt protein ligand and , and their cognate Frizzle receptors (e.g., FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10) Frizzle refers to compounds that can activate related signaling pathways. The ed receptor family is a G protein-coupled receptor protein. , (FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, or FZD10, or multiple receptors agonists of Frizzled receptors, Wn activators of polypeptides known to activate the t signal transduction pathway, and / or , any of the polypeptide inhibitors known to inhibit the Wnt signaling pathway. Known Wnt proteins include WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WN These include, but are not limited to, T11 and WNT16. The factors are Wnt3a, SB-216763, and / or LY2090314. In one embodiment, the activator is capable of inhibiting the biological activity of the GSK3 protein. For example, an activator capable of inhibiting the biological activity of the GSK3 protein CHIR99021 can be used to inhibit Wnt signaling. In embodiments where CHIR99021 is used as an activator of the delivery pathway, On the ground, at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5. It may be provided at a concentration of 5, 6, 6.5, 7, 7.5 μM or greater. In embodiments where CHIR99021 is used as an activator of the Wnt signaling pathway, CHIR99021 was cultured in the third medium at 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or less μM CHIR99021 can be provided at a concentration that does not inhibit the activity of the Wnt signaling pathway. In embodiments where CHIR99021 is used as an activating agent, the CHIR99021 is present in a third culture medium at a concentration of 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 It can be provided at a concentration of between 3, 2.5, 2, 1.5, or 1 μM. In embodiments where IR99021 is used as an inhibitor of the Wnt signaling pathway, HIR99021 may be provided in the third culture medium at a concentration of about 3 μM. CHIR99021 may be an activator in the second and third additive sets.

[0052] The third culture medium further contains an inhibitor of the transforming growth factor β (TGFβ) signaling pathway. The presence of inhibitors of the TGFβ signaling pathway inhibits the Wnt signaling pathway. In combination with the toxic factor, HEX and HEX encode polypeptides required for liver development. As used in the context of this disclosure, "TGFβ synthase" has a beneficial effect on the expression of the PROX1 gene. "Inhibitors of signal transduction pathways" refers to inhibitors of signal transduction pathways associated with the binding of TGF-β to its cognate receptor. The term refers to compounds that can inhibit the TGFβ receptor family. mediates signal transduction through SMAD proteins. This compound inhibits TGFβ receptors. antagonists of the TGFβ signaling pathway, polypeptides known to activate the TGFβ signaling pathway inhibitors of TGFβ and / or polypeptides known to inhibit the TGFβ signaling pathway Known TGFβ proteins include TGFB1, Examples include, but are not limited to, TGFB2, TGFB3, and TGFB4. In some embodiments, the inhibitor inhibits at least one of an ALK4, ALK5, or ALK7 polypeptide. In some embodiments, the inhibitor can inhibit at least one biological activity of: It is possible to inhibit the biological activity of ALK4, ALK5, and ALK7 polypeptides. For example, by inhibiting the biological activity of ALK4, ALK5, and ALK7 polypeptides. The inhibitor that can be used may be A83-01. In combination, SB431542 and / or LY364947 can be used as inhibitors. An embodiment in which A83-01 is used as an inhibitor of the TGFβ signaling pathway In the third culture medium, A83-01 is 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 0.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5 μM, or A83-01 can be provided at a concentration of 100 or more. In an embodiment where A83-01 is used as an inhibitor of the delivery pathway, the A83-01 is added to a third culture medium at a concentration of 5 ,4.5,4,3.5,3,2.5,2,1.9,1.8,1.7,1.6,1.5,1 0.4, 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 less. In embodiments where A83-01 is used as an inhibitor of the TGFβ signaling pathway, 1 is the third culture medium, 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 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, 0.2μ A83-01 can be administered at a concentration of between 100 and 150 mg / mL. In embodiments used as an inhibitor, A83-01 is added to the second culture medium at a concentration of about 1 μM. In some embodiments, A83-01 may be provided in the first and third additive sets. This can be an inhibitor in the

[0053] The third culture medium also contains a cytokine, such as, for example, oncostatin M (OSM). In embodiments where oncostatin M is used as the cytokine, oncostatin M is In the third culture medium, at least 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29ng / ml, Oncostatin M can be present at concentrations greater than or equal to 100 mg / kg of cytokines. In embodiments where oncostatin M is used as a third culture medium, , 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, Present at concentrations of 14, 13, 12, 11 ng / ml or less In embodiments where oncostatin M is used as a cytokine, Chin M is the third culture medium, and the 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, Approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, Present at concentrations between 17, 16, 15, 14, 13, 12, or 11 ng / ml In certain embodiments, oncostatin M is administered at a concentration of about 20 ng / ml. 3 culture medium.

[0054] The third culture medium further comprises a glucocorticoid, such as, for example, dexamethasone. In embodiments where dexamethasone is used as the glucocorticoid, dexamethasone is In culture medium of 3, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 μM, Dexamethasone may be present in concentrations greater than or equal to 100 mg / kg of glucocorticoids. In embodiments where dexamethasone is used as a stimulant, the third culture medium may contain 15, 14, 1 Present at concentrations of 3, 12, 11, 10, 9, 8, 7, 6 μM or less In embodiments where dexamethasone is used as the glucocorticoid, Dexamethasone was added to the third culture medium at approximately 5, 6, 7, 8, 9, 10, 11, 12, 13 , or , 14 and , approximately 15, 14, 13, 12, 11, 10, 9, 8, 7, or , 6 μ In certain embodiments, dexamethasone can be present at a concentration between about 1 M. 0 μM in the third culture medium.

[0055] The third culture medium is used to culture the hepatic progenitor cells for at least one day to allow differentiation. and contacting the hepatocyte lineage cells with the third culture medium. If it is intended that the culture medium be changed daily, the process of the present disclosure In some embodiments, the third culture medium comprises at least 1, 2, 3, 4, or more In another embodiment, the third culture medium is allowed to remain in contact with the cultured cells for the number of days above. The cells may be left in contact with the culture for 4, 3, 2, or fewer days. In some embodiments, the third culture medium is maintained for at least 1, 2, 3, 4, or more days. and leave in contact with the cultured cells for 5, 4, 3, 2, or fewer days. In another embodiment, the third culture medium is allowed to contact the cultured cells for about 1 to 5 days. .

[0056] The use of a third culture medium containing posterior foregut cells differentiates hepatic progenitor cells into cells of the hepatocyte lineage. Thus, the present disclosure provides methods for producing hepatocytes obtained by the processes described herein. In the population of cells of the hepatocyte lineage of the present disclosure, the majority of the cells are A portion of the cells are believed to be cells of the hepatocyte lineage, and in some embodiments, It may include hepatic progenitor cells and / or endodermal cells.

[0057] The fourth culture medium contains a fourth set of additives, which is an insulin signaling inhibitor. activators of the steroid signaling pathway, cytokines, and glucocorticoids, The expression "fourth culture medium" as used in the context of this disclosure essentially consists of a fourth "Comprising a set of additives" means that the additives are non-essential for differentiation of hepatocyte lineage cells into immature hepatocyte-like cells. This refers to a fourth culture medium that contains additional additives but is still able to promote differentiation. Further additives include, for example, B27 supplement, primary hepatocyte supplement (P HH), HBM / HCM Bulletkit™ Retinoic Acid, Insulin, Vitamin Examples of suitable nutrient sources include, but are not limited to, amines and minerals.

[0058] The fourth culture medium also contains an activator of the insulin signaling pathway. The term "activator of the insulin signaling pathway" used refers to insulin and its cognate receptors. Activation of signal transduction pathways associated with binding to receptors (tyrosine kinase receptors) This compound is a compound that can bind to insulin receptors (insulin, IGF- IGF-I or IGF-II agonists, activating the insulin signaling pathway activators of polypeptides known to inhibit the insulin signaling pathway and / or In some embodiments, the inhibitor may be any of the polypeptides known to inhibit activity. The activating agent 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 added to the fourth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, Insulin may be provided at or above ng / mL. In an embodiment for use as a tract activator, insulin is added to the fourth 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 Insulin can be used as an activator of the insulin signaling pathway. In the embodiment used, insulin is added to the fourth culture medium at a concentration of about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 210, 220, 230, 240, 250, 260, 27 , 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, Concentrations between 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 In certain embodiments, insulin may be provided in the fourth culture medium. In yet another embodiment, insulin may be provided at a concentration of about 10 mg / ml. Phosphorus is available in the form of B27 supplements, HBM / HCM Bulletkit™, and and / or in the form of a primary hepatocyte (PHH) supplement.

[0059] The fourth culture medium also contains a cytokine, such as, for example, oncostatin M (OSM). In embodiments where oncostatin M is used as the cytokine, oncostatin M is In the fourth culture medium, at least 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29ng / ml, Oncostatin M can be present at concentrations greater than or equal to 100 mg / kg of cytokines. In embodiments where oncostatin M is used as a fourth culture medium, the fourth culture medium may contain 30, 29, 28 , 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, Present at concentrations of 14, 13, 12, 11 ng / ml or less In embodiments where oncostatin M is used as a cytokine, Chin M is the fourth culture medium, and the 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, Approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, Present at concentrations between 17, 16, 15, 14, 13, 12, or 11 ng / ml In certain embodiments, oncostatin M is administered at a concentration of about 20 ng / ml. 4 culture medium.

[0060] The fourth culture medium further comprises a glucocorticoid, such as, for example, dexamethasone. In embodiments where dexamethasone is used as the glucocorticoid, dexamethasone is In culture medium at 4, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 μM, Dexamethasone may be present in concentrations greater than or equal to 100 mg / kg of glucocorticoids. In embodiments where dexamethasone is used as a marker, the fourth culture medium may contain 15, 14, 1 Present at concentrations of 3, 12, 11, 10, 9, 8, 7, 6 μM or less In embodiments where dexamethasone is used as the glucocorticoid, Dexamethasone was added to the fourth culture medium at approximately 5, 6, 7, 8, 9, 10, 11, 12, 13 , or , 14 and , approximately 15, 14, 13, 12, 11, 10, 9, 8, 7, or , 6 μ In certain embodiments, dexamethasone can be present at a concentration between about 1 M. It is present in the fourth culture medium at a concentration of 0 μM.

[0061] The fourth culture medium is used to culture the hepatocyte lineage cells for at least one day to allow differentiation. The fourth culture medium was left in contact with the cultured cells and immature hepatocyte-like cells for 2 days. If longer contact is intended, the medium can be changed daily. In some embodiments of the process, the fourth culture medium is at least 1, 2, 3, 4, or In another embodiment, the fourth culture medium is left in contact with the cultured cells for a period of 10 days or more. is allowed to contact the cultured cells for 5, 4, 3, 2, or fewer days. In another embodiment, the fourth culture medium comprises at least 1, 2, 3, 4, or more days, and in contact with cultured cells for 5, 4, 3, 2, or fewer days. In yet another embodiment, the fourth culture medium is contacted with the cultured cells for about 1 to 5 days. Leave it there.

[0062] Using a fourth culture medium containing posterior foregut cells, hepatocyte lineage cells were cultured to immature hepatocyte-like cells. Thus, the present disclosure provides methods for the differentiation of cells into the cells described herein. The present disclosure provides a population of immature hepatocyte-like cells obtained by the method. The majority of the cells are believed to be immature hepatocyte-like cells, and in some embodiments In some cases, the cells may contain some cells of the hepatocyte lineage, hepatic progenitor cells, and / or endodermal cells. can.

[0063] The fifth culture medium contains a fifth set of additives, which is an insulin signaling inhibitor. activators of the steroid signaling pathway and glucocorticoids, or As used in the context of this disclosure, the expression "fifth culture medium" refers to a culture medium that is essentially made up of a fifth set of additives. "Consisting of" means that the differentiation of immature hepatocyte-like cells into mature hepatocyte-like cells is not essential. This refers to a fifth culture medium that contains additives that can promote differentiation. Examples of additives include B27 supplements, primary hepatocyte supplements, and retinoin. Acids, insulin, vitamins, HBM / HCM Bulletkit™, and These include, but are not limited to, neral.

[0064] The fifth culture medium also contains an activator of the insulin signaling pathway. The term "activator of the insulin signaling pathway" used refers to insulin and its cognate receptors. Activation of signal transduction pathways associated with binding to receptors (tyrosine kinase receptors) This compound is a compound that can bind to insulin receptors (insulin, IGF- IGF-I or IGF-II agonists, activating the insulin signaling pathway activators of polypeptides known to inhibit the insulin signaling pathway and / or In some embodiments, the inhibitor may be any of the polypeptides known to inhibit activity. The activating agent 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 added in a fifth culture medium at a concentration of at least about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, Insulin may be provided at or above ng / mL. In an embodiment for use as a tract activator, insulin is added to 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 Insulin can be used as an activator of the insulin signaling pathway. In the embodiment used, insulin is added in a fifth 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, Concentrations between 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 In certain embodiments, insulin may be provided in the fifth culture medium. In yet another embodiment, insulin may be provided at a concentration of about 10 mg / ml. Phosphorus is available in the form of B27 supplements, HBM / HCM Bulletkit™, and and / or in the form of a primary hepatocyte (PHH) supplement.

[0065] The fifth culture medium further comprises a glucocorticoid, such as, for example, dexamethasone. In embodiments where dexamethasone is used as the glucocorticoid, dexamethasone is In culture medium at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 μM, Dexamethasone may be present in concentrations greater than or equal to 100 mg / kg of glucocorticoids. In embodiments where dexamethasone is used as a 5th culture medium, the 5th culture medium is used at 15, 14, 1 Present at concentrations of 3, 12, 11, 10, 9, 8, 7, 6 μM or less In embodiments where dexamethasone is used as the glucocorticoid, Dexamethasone was added to the fifth culture medium at approximately 5, 6, 7, 8, 9, 10, 11, 12, and 13 , or , 14 and , approximately 15, 14, 13, 12, 11, 10, 9, 8, 7, or , 6 μ In certain embodiments, dexamethasone can be present at a concentration between about 1 M. In certain embodiments, dexamethasone is present in the fifth culture medium at a concentration of about 0 μM. Present in the fifth culture medium at a concentration of 10 μM.

[0066] The fifth culture medium is used to culture immature hepatocytes for at least one day to allow differentiation. The fifth culture medium was left in contact with the cultured cells for 2 days. If longer contact is intended, the medium can be changed daily. In some embodiments of the process, the fifth culture medium comprises at least 1, 2, 3, 4, or In another embodiment, the fifth culture medium is left in contact with the cultured cells for a period of 10 days or more. is allowed to contact the cultured cells for 5, 4, 3, 2, or fewer days. In another embodiment, the fifth culture medium comprises at least 1, 2, 3, 4, or more days, and in contact with cultured cells for 5, 4, 3, 2, or fewer days. In yet another embodiment, the fifth culture medium is contacted with the cultured cells for about 1 to 5 days. Leave it there.

[0067] The fifth culture medium containing posterior foregut cells allowed the differentiation of immature hepatocyte-like cells into mature hepatocyte-like cells. Thus, the present disclosure provides methods for the differentiation of cells into the cells described herein. The present disclosure provides a population of mature hepatocyte-like cells obtained by the method. The majority of the cells are believed to be mature hepatocyte-like cells, and in some embodiments , some immature hepatocyte-like cells, cells of the hepatocyte lineage, hepatic progenitor cells, and / or endodermal cells. It may contain cells.

[0068] The medium described herein may contain EGF, which can promote the formation of cholangiocytes. There is no particular need.

[0069] The present disclosure provides a method for producing a first, second, and / or third process as disclosed herein. For example, a first process can be combined with a second process to provide In another example, the second process can be performed using a third In combination with the above process, hepatocyte-like cells can be generated from posterior foregut cells. In another example, the first, second, and third processes are combined to generate hepatocytes from endoderm cells. The process described herein can produce cytoplasmic cells with potent biological activity. (e.g., high Cyp3A4 activity, high albumin expression levels, and / or high urea production levels) and / or the ability to metabolize therapeutic agents (or potential therapeutic agents). This specific This embodiment, as described below, is intended to incorporate hepatocyte-like cells into encapsulated liver tissue. It is particularly useful in producing cells.

[0070] The present disclosure provides kits for producing posterior foregut cells, hepatic progenitor cells, and / or hepatocyte-like cells. Generally, the kits include at least one of the components described herein. A set of additives or at least one culture medium described herein, any cells, etc. and instructions for carrying out the processes described herein. The kit for extracting the β-glucan may include, for example, a first set of additives, or a first culture medium, any The method may include the steps of: (a) providing a method for producing a liver cell comprising the steps of: The kit for producing progenitor cells may include, for example, a second set of additives or a second culture medium. The second process may include the culture medium, optional posterior foregut cells, and instructions for carrying out the process. The kit for producing hepatocyte-like cells can be, for example, a third additive set or a third culture medium, a fourth set of additives, or a fourth culture medium, a fifth set of additives, or a fifth culture medium, any hepatic progenitor cells, cells of the hepatocyte lineage, or immature hepatocyte-like cells; , as well as instructions for carrying out the third process.

[0071] encapsulated liver tissue The encapsulated liver tissue is a liver organelle at least partially coated with a biocompatible crosslinked polymer. The term "antibody" as used in the context of this disclosure includes at least one (and in some embodiments, multiple) The term "liver organoid" used herein refers to a mixture of cultured hepatocytes, mesenchymal cells, and endothelial cells. The term refers to hepatocytes obtained using the processes described herein. In this embodiment, the liver organoids are a mixture of cultured hepatocytes, mesenchymal cells, and endothelial cells. Hepatic organoids generally have a spherical shape and an irregular surface. The relative diameter of liver organoids is approximately 50 to 500 μm. The cytoplasmic core is composed of hepatocytes, mesenchymal cells, and optional endothelial cells, and in part In this embodiment, during the culture, the extracellular matrix, hepatocytes, mesenchymal cells, and any endogenous Liver organoids are produced and constructed from epithelial cells. Liver organoids can be obtained by cell suspension culture. In some embodiments, particularly prior to the culture / differentiation of the encapsulated liver tissue, hepatocytes, e.g. , hepatocytes, and / or bile duct epithelial cells, etc., to cover the surface of the liver organoids. In another embodiment, the coating partially covers (and in some embodiments substantially covers) both the surface and the substrate. Hepatocytes are dispersed (but not necessarily uniformly) throughout the cell core. The organoids present in the liver tissue are at least partially crosslinked with a first biocompatible crosslinked polymer. The coating is thoroughly coated (and in some embodiments, substantially coated).

[0072] Before encapsulation, liver organoids do not contain any exogenous extracellular matrix. The ganoids consist essentially of cultured hepatocytes, mesenchymal cells, and optionally endothelial cells. Furthermore, liver organoids (encapsulated in a first biocompatible polymer, or non-encapsulated) exhibit liver function, e.g., liver organoids synthesizes urea and clotting factors, exhibits CyP3A4 activity, and converts ammonia into urea and the detoxification of liver-specific drugs (i.e., tacrolimus, can undergo rifampicin) metabolism.

[0073] The liver organoids of the present disclosure have a substantially spherical morphology and micrometers. The diameter of the sphere may be in the range of 1 / 2 to 1 / 4 of a millimeter (e.g., the diameter may be less than 1 mm). In this study, liver organoids were cultured for at least approximately 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2500, 3000, 45 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 In yet another embodiment, the liver organoids have a relative diameter of 490 μm. Before encapsulation, the values ​​were approximately 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, having a relative diameter of 120, 110, 100, 90, 80, 70, or 60 μm or less In another embodiment, the liver organoids are cultured for at least about 50, 60, or 70 days prior to encapsulation. 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 approximately 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 60 μm or less In some embodiments, the liver organoids have a relative diameter between 100 and 150 mm, and the organoids have a relative diameter between 100 and 150 mm. 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 approximately 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 between 60 μm or less In yet another embodiment, the liver organoids before encapsulation have at least For example, before encapsulation, the diameter of the encapsulated particles is about 100 μm and the relative diameter is about 300 μm or less. liver organoids are at least about 150, 160, 170, 180, or 190 μm and smaller than 200, 190, 180, 170, or 160 μm In yet a further embodiment, the liver organoids before encapsulation have a relative diameter of at least The liver organ has a diameter of at least about 150 μm and a relative diameter of no more than about 200 μm. The size of the id allows the cells contained therein to have access to various nutrients and encapsulated liver tissue. In some embodiments, this increases the exposure of the host to biological fluids / cells that Blood vessels of liver organoids in the vasculature (e.g., the vasculature of a host into which the encapsulated liver tissue has been transplanted) and remain viable and biologically active in vivo, eliminating the need for cell formation. You will be able to do this.

[0074] Hepatocytes in liver organoids can be dispersed throughout the organoid and, 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 organoids may be derived from, for example, definitive endoderm, posterior foregut cells, hepatocyte lineage cells, Alternatively, the cells may be derived from hepatic progenitor cells or hepatocyte-like cells. The hepatocytes of the luganoids can be hepatocyte-like cells and / or bile duct epithelial cells. Hepatocytes in liver organoids are composed of only a single cell type (e.g., definitive endoderm cells, posterior foregut cells) cells of the hepatocyte lineage, hepatocyte-like cells, or bile duct epithelial cells) or derived from cells A mixture of cell types (e.g., the following cell types: definitive endoderm cells, posterior foregut cells, cells of the hepatocyte lineage) , hepatocyte-like cells, and / or bile duct epithelial cells) During in vitro cell culture of liver organoids or Transplantation of the hepatocytes in vivo has been shown to alter the hepatocyte cell type(s) or differentiate hepatocytes. For example, hepatocytes in liver organoids can be differentiated into mesenchymal cells and any endogenous cells. During co-culture with epithelial cells or upon in vivo transplantation, the definitive endoderm, posterior foregut, or differentiation of cells of the hepatocyte lineage into hepatocyte-like cells or bile duct epithelial cells To determine whether hepatocyte-like cells exist in liver organoids, The activity of CyP450 family 3 subfamily A member 4 (CyP3A4) was measured using the The presence of hepatocyte-like cells in liver organoids can be determined by any means known in the art. To determine the presence or absence of albumin, clotting factors, and urea synthesis / production, The activity of CyP3A4 can also be monitored. To determine the presence or absence of SOX17, FOXA2, and CXCR4 in the posterior foregut cells, GATA4 expression can be determined by means known in the art.

[0075] Mesenchymal cells of liver organoids can be derived from different sources, e.g., bone marrow (including blood), umbilical cord, or mesenchymal stem cells / progenitor cells, adipocytes, myocytes, hepatic stellate cells, myofibroblasts (adipose tissue) The mesenchymal cells of liver organoids can be cells and / or fibroblasts. one cell type (e.g., mesenchymal stem / progenitor cells, adipocytes, myocytes, or fibroblasts) ) or a mixture of cell types (e.g., the following cell types: mesenchymal stem cells / progenitor cells At least one of the following cells: alveoli, adipocytes, myocytes, hepatic stellate cells, myofibroblasts, and / or fibroblasts The mesenchymal cell type of liver organoids can be derived from hepatocytes (or a mixture of the two). , and during co-culture with any endothelial cells or when transplanted in vivo (mesenchymal stem cells) cells / progenitor cells into fibroblasts, adipocytes, or myocytes Mesenchymal stem / progenitor cells express, among other genes, α-smooth muscle actin (α They are known to express SMA, fibronectin, CD90, and CD73. To determine the location or presence of mesenchymal cells in liver organoids, among other things, In particular, genes or proteins specific to or associated with the mesenchymal lineage Protein expression can be determined.

[0076] The endothelial cells of liver organoids, if present, are derived from endothelial progenitor cells of various origins, e.g. and / or endothelial cells. The endothelial cells of liver organoids can be single cells. 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 id is formed during in vitro co-culture of endodermal and mesenchymal cells or When transplanted in vivo, they can differentiate (from endothelial progenitor cells to endothelial cells). In some embodiments, the endothelial cells of the liver organoids are endothelial cells lining the inner surface of the lumen (partially Capillaries or capillary-like shapes can be organized (which can be targeted).

[0077] As mentioned above, the cellular core of liver organoids consists of hepatocytes, mesenchymal cells, and any endogenous cells. In some embodiments, the cells are produced and cultured. The cellular core of liver organoids consists of necrotic cells / anti- inflammatory cells and an organized extracellular matrix. Substantially fewer apoptotic cells (e.g., the cell cores of liver organoids are histologically The reason for this is that the culture medium for liver organoids is Nutrients can diffuse throughout the cell core, providing nutrients to the cells within the cell core. Furthermore, metabolic waste products of the cells in the cell core can be delivered to the outside of the liver organoid. The liver organoids themselves (before encapsulation) can be easily dispersed by exogenous cells. Does not contain extracellular matrix or synthetic polymeric materials (e.g., if they are absent) In some embodiments, the hepatocytes can be present on the surface of the cell core. In embodiments, the hepatocytes, in combination with the cells of the cell core, form extracellular matrix material. (e.g., collagen and fibronectin) and Thus, in some embodiments, basement membrane material can also be produced and constructed.

[0078] As described above, hepatocytes at least partially cover the surface of the cell core of the liver organoid. In the context of the present disclosure, "hepatocytes can be coated with a small amount of the surface of the cell core." The phrase "partially cover at least about 10%, 20%, or 30% of the surface of the cell core" means at least about 10%, 20%, or 30% of the surface of the cell core. In some embodiments, the hepatocytes comprise 30% or 40% of the total hepatocytes. In the context of the present disclosure, "hepatocytes" refers to cells that are formed by the cell core. The phrase "substantially cover the surface" means that the hepatocytes occupy the majority of the surface of the cell core. For example, 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%, In some embodiments, the hepatocytes comprise 0%, 95%, or 99% of the surface of the cell core. completely coated (e.g., covering more than 99% of the surface of the cell core with hepatocytes).

[0079] In some embodiments, the liver organoids of the present disclosure are coated with a first crosslinked biocompatible polymer. Prior to encapsulation, hepatocyte-like cells and / or The bile ducts contain a greater proportion of mesenchymal cells (and endothelial cells, if present) than bile duct epithelial cells. However, after encapsulation with the first cross-linked biocompatible polymer, the liver organoids of the present disclosure The endothelial cells contain a greater proportion of hepatocytes than mesenchymal cells (and endothelial cells, if present). It is known that about 90% of the mammalian liver is composed of hepatocytes. Therefore, in some embodiments of the present disclosure, the ratio of hepatocytes in liver organoids is Approximately 90%, 85%, 80%, or 75% of the total cell count in the ganoid is less than.

[0080] Liver organoids can be generated from cells of different origins. At least one of the cells is a hepatocyte, a mesenchymal cell, or an endothelial cell. In another embodiment, at least two of the hepatocytes, mesenchymal cells, or endothelial cells are derived from In yet another embodiment, the cells are derived from hepatocytes, mesenchymal cells, and mammals, such as humans. The endothelial cells are all derived from mammals, such as humans. Cells from different sources can be combined, for example mesenchymal and endothelial cells. are derived from murine or porcine origin, while hepatocytes are derived from human origin. These combinations are not exhaustive and those skilled in the art will appreciate that the present invention Further combinations are conceivable that are appropriate in the context of the disclosure.

[0081] The cells of the liver organoids can be derived from different sources. Ganoid cells are derived from primary cell cultures, established cell lines, or differentiated stem cells. Within liver organoids, cells from different sources can be combined. For example, hepatocytes can be derived from primary cell cultures, and mesenchymal cells can be derived from established The endothelial cells can be derived from differentiated cell lines, and the endothelial cells can be derived from differentiated cell lines. Alternatively, liver organoids can be cultured from the same source (e.g., differentiated stem cells). ) can also be combined. In this embodiment, cells from a single cell source (e.g. This is particularly useful because cells can be obtained from stem cells to generate encapsulated liver tissue. In certain embodiments, the cells of the liver organoids include hepatocytes, mesenchymal cells, and any endogenous cells. They are derived from a single stem cell population that differentiates into epithelial cells. The stem cell population can be embryonic stem cells or human stem cells. In certain embodiments, the cells of liver organoids can be derived from pluripotent stem cells. The results show that a single pluripotent stem cell population differentiates into hepatocytes, mesenchymal cells, and any endothelial cell type. It originates from

[0082] Polymers that can be used in encapsulated liver tissue (also known as polymer matrices) are As is well known in the art, hydrogels are formed around the id(s). Gels are hydrophilic polymer chains in which water is the dispersion medium. Hydrogels are naturally occurring or can be obtained from a synthetic polymer network. The gel encapsulation prevents the embedded liver organoids from leaking out of the polymer. Therefore, the cells of liver organoids will not cause immune reactions or tumors in the recipient's body upon transplantation. In some embodiments, the liver organoids are The organoids are then individually encapsulated and the encapsulated liver organoids are then, in another embodiment, placed in a polymer matrix. In yet another embodiment, the liver organoids are further included in a cell culture medium that encapsulates them. The polymer matrix is ​​then incorporated into the cell.

[0083] In the context of the present disclosure, a polymer is one that is toxic when introduced into a subject (e.g., a human). In the context of this disclosure, biocompatible The polymer is not toxic to the cells of the liver organoids or to the cells of the subject (e.g., It is preferable that the compound does not exhibit toxicity when transplanted in vivo into a living organism (e.g., a human). Hepatotoxicity can occur, for example, when the compound is transplanted into the liver. the rate of apoptotic death of cell-like cells (e.g., increased apoptosis indicates hepatotoxicity); Transaminase levels (e.g., elevated transaminase levels indicate liver toxicity) hypertrophy of hepatocyte-like cells (e.g., increased hypertrophy indicates hepatotoxicity); Microvacuolar degeneration in the liver (e.g., increased degeneration indicates hepatotoxicity), the rate of bile duct cell death (e.g., For example, increased mortality of bile duct cells indicates hepatotoxicity), γ-glutamyl transpeptidase Determine and measure guanylate glutathione (GGT) levels (e.g., elevated GGT levels indicate liver toxicity) Biocompatible polymers include carbohydrates (hyaluronic acid (HA), Droitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, alginic acid, chitosan glycosaminoglycans such as heparin, agarose, dextran, and cellulose; / or their derivatives), proteins (collagen, elastin, fibrin, alginate, (proteins, poly(amino acids), glycoproteins, antibodies, and / or their derivatives), and and / or synthetic polymers (e.g., poly(ethylene glycol) (PEG), poly(hydroxybenzoates) hydroxyethyl methacrylate) (PHEMA) and / or poly(vinyl alcohol) Biocompatible polymers include, but are not limited to, those based on polyvinyl alcohol (PVA). , a single polymer or a mixture of different polymers (e.g., US2012 / 0142 069). Examples of biocompatible polymers include poly( Ethylene glycol, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone Lactone (PCL), fibrin, polysaccharide materials (chitosan, proteoglycan, or glycosaminoglycans (GAGs), alginate, collagen, thiolated In some embodiments, the present invention includes, but is not limited to, heparin and mixtures thereof. The biocompatible polymer can be linear, branched, and optionally, peptide. can incorporate nucleotides (e.g., RGD), growth factors, integrins, or drugs. .

[0084] In some embodiments, the polymer is a "low immunogenic polymer" and is non-immunogenic in the recipient. elicits no or only a minimal immune response (i.e., (So ​​the polymer will not undergo degradation, alteration, or loss of function.) The cytotoxic polymer blocks one or more antigenic determinants on the cell and binds such antigenic determinants to the cell. When introduced into an allogeneic subject, it suppresses or blocks the immune response to that antigenic determinant. You can even stop it.

[0085] The polymer present in the encapsulated liver tissue of the present disclosure is preferably crosslinkable, e.g. For example, the polymer can be crosslinked by thermal or chemical (e.g., VPMS, RGD, etc.). which one or more peptides are used), or the use of pH or light (e.g., UV light) In some embodiments, crosslinking can be achieved by photopolymerization using liver organophosphates. The endothelial cells (with or without encapsulation in a polymer matrix) were then subjected to a polymer matrix This can be done after dispersing the mixture in the liquid.

[0086] The polymers of the present disclosure may be wholly or partially biodegradable (e.g., biodegradable by the metabolism of the living body). readily hydrolyzed) or wholly or partly resistant to biodegradation. It can be used to show properties (e.g., resistance to hydrolysis when metabolized by the living body). An example of a biocompatible and biodegradable polymer is poly(ethylene glycol)-(methylcellulose). Examples include, but are not limited to, PEG-Mal) 8-arm. An example of a biodegradation-resistant polymer is poly(ethylene glycol)-vinyl sulfone. (PEG-VS), but is not limited to this.

[0087] Encapsulated liver tissue can at least partially (and in some cases, substantially) transform liver organoids. The first biocompatible crosslinked polymer is a polymer that is capable of crosslinking the liver. In the context of the present disclosure, the expression "first biocompatible" refers to a method for producing a first biocompatible organoid. "Liver organoid(s) at least partially coated with a synthetic cross-linked polymer" covers at least about 10%, 20%, 30%, or 40% of the surface of the liver organoid. , refers to the first biocompatible crosslinked polymer. In some embodiments, the first biocompatible crosslinked polymer The compatible cross-linked polymer substantially coats the surface of the liver organoid(s). In this context, the expression "a liver substantially coated with a first biocompatible crosslinked polymer" is used. The organoid(s) are then cultured by first covering most of the surface of the liver organoid with a biocompatible crosslinker. This refers to the polymer occupying, e.g., at least about 50% of the surface of the organoid. , 60%, 70%, 80%, 90%, 95%, 99%, the first biocompatible cross-linked polymer In one embodiment, the first biocompatible crosslinked polymer is present on the surface of the liver organoid. Completely cover the surface (e.g., more than 99% of the surface of the liver organoids) with the first biocompatible material. coated with a cross-linked polymer).

[0088] In some embodiments, the encapsulated liver tissue comprises at least in part a first biocompatible crosslinked polymer. It may also include a second biocompatible crosslinked polymer that partially (and in some cases substantially) covers the The second biocompatible polymer is in physical contact with the first biocompatible crosslinked polymer. In some embodiments, the method is in physical contact with the cells of the liver organoids. In the context of the present invention, the expression "at least partially coated with a second biocompatible crosslinked polymer" is used. The term "first biocompatible crosslinked polymer" refers to a crosslinked polymer having a surface area of ​​at least one of the first biocompatible crosslinked polymers. At least about 10%, 20%, 30%, or 40% of the crosslinked polymer is a second biocompatible crosslinked polymer. In some embodiments, the second biocompatible crosslinked polymer is a crosslinked polymer of the first biocompatible polymer. In the context of this disclosure, the expression "second "First biocompatible crosslinked polymer substantially covered with a biocompatible crosslinked polymer" The second biocompatible crosslinked polymer occupies most of the surface of the first biocompatible crosslinked polymer. For example, at least about 50% of the surface of the first biocompatible crosslinked polymer %, 60%, 70%, 80%, 90%, 95%, 99% of the second biocompatible cross-linked polymer In one embodiment, the second biocompatible crosslinked polymer is a crosslinked polymer of the first biocompatible crosslinked polymer. Completely cover the surface of the crosslinked polymer (e.g., 9 mm of the surface of the first biocompatible crosslinked polymer). In yet another embodiment, more than 9% of the surface is coated with a second biocompatible crosslinked polymer. The biocompatible cross-linked polymer is the first biocompatible cross-linked polymer that is less effective in growing liver organoids. Such an embodiment forms a matrix in which the particles (at least partially coated) are interspersed. In this example, liver organoids (at least partially coated with a first biocompatible crosslinked polymer) were cultured. The grafted material may have a second biocompatible crosslinked matrix surrounding it, or Alternatively, another liver organoid (at least partially coated with a first biocompatible cross-linked polymer) may be cultured. The encapsulated liver tissue can be physically contacted with a second biocompatible crosslinker. A further biocompatible cross-linked polymer may be included coating the polymer.

[0089] The first biocompatible cross-linked polymer and the second biocompatible cross-linked polymer may be the same or In one embodiment, the first biocompatible cross-linked polymer The mer is a polymer that is at least partially (and in some embodiments, completely) biodegradable. In combination, the second biocompatible crosslinked polymer may be at least In yet another embodiment, the The first biocompatible crosslinked polymer is a biodegradable polymer, and the second biocompatible crosslinked polymer is a In such an embodiment, the first biocompatible crosslinked polymer is resistant to biodegradation. The polymer is more biodegradable (e.g., biodegradable) than the second biocompatible crosslinked polymer. (reduce resistance to)

[0090] In some embodiments, the first biocompatible crosslinked polymer comprises a plurality of liver organoids. In such an embodiment, the encapsulated liver tissue is 2 At least about 50 or 60 per , 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 35 The culture medium may contain 0, 400, 450, or 500 liver organoids. In an embodiment, the encapsulated liver tissue is 2 Hit, maximum about 500, 450, 400, 3 50, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, In yet another embodiment, the method comprises the steps of: Encapsulated liver tissue is cm 2 Per, approximately 50, 60, 70, 80, 90, 100, 125, 1 50, 175, 200, 250, 300, 350, 400, or 450 and approximately 500 , 450, 400, 350, 300, 250, 200, 175, 150, 125, 100 , 90, 80, 70, or 60 liver organoids. In an embodiment, the encapsulated liver tissue is 2 Contains approximately 50-500 liver organoids per culture In another embodiment, the encapsulated liver tissue is 3 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 In yet another embodiment, the subject comprises 2400, 2400, or 2500 liver organoids. Encapsulated liver tissue is cm 3 Hit, maximum 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 In yet another embodiment, the encapsulated liver tissue comprises a liver organoid of cm. 3 Win , 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 In yet another embodiment, the encapsulated liver tissue comprises a number of liver organoids between 0 and 100. cm 3 Each culture contains approximately 250-2500 liver organoids.

[0091] In some embodiments, the encapsulated liver tissue can be cultured or transplanted in vivo to produce hepatocytes. expressing genes and proteins associated with mesenchymal cells and any endothelial cells In a further embodiment, the encapsulated liver tissue can be (in vitro or in vivo) , producing albumin, synthesizing urea from ammonia, and activating CyP3A4. Indicate and / or use medications (e.g., tacrolimus and / or rifampicin) In some embodiments, the encapsulated liver tissue can metabolize liver organoids within the tissue. Per g of do, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Can produce 15, 16, 17, 18, 19, or 20 mg of albumin In another embodiment, the encapsulated liver tissue is subjected to one or more freeze-thaw cycles to remove hepatocytes, mesenchymal cells, and / or endothelial cells. expressing genes and proteins associated with albumin and any endothelial cells; producing amine, synthesizing urea from ammonia, and exhibiting CyP3A4 activity and / or drugs (such as tacrolimus and / or rifampicin) that are metabolized in the liver In some embodiments, the encapsulated liver tissue can metabolize the hydroxybenzoates (which are known to be metabolized by the hydroxybenzoates). After freezing, 1, 2, 3, 4, 5, 6, 7 per gram of liver organoids in tissue were obtained. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or , 2 0 mg of albumin can be produced.

[0092] The process that produces encapsulated liver tissue The process of creating encapsulated liver tissue begins with creating liver organoid(s). Then, the liver organoids are subjected to a first biocompatible crosslinking treatment. Encapsulating (at least partially) in a polymer (and optionally a second biocompatible crosslinker) polymer, and encapsulation in a further biocompatible cross-linked polymer).

[0093] Liver organoids consist of (i) a cellular core containing hepatocytes, mesenchymal cells, and optionally endothelial cells; (ii) a substantially spherical morphology; and (iii) a relative diameter of about 50 to about 500 μm. Hepatocytes, mesenchymal cells, and any endogenous cells were synthesized under the conditions necessary to obtain liver organoids. In some embodiments, the cells can be co-cultured with epithelial cells (all of which are described above) to produce These conditions include suspension culture of cells (e.g., ) to promote liver organoid formation. , ultra-low adhesion conditions).

[0094] Hepatocytes to be included in the encapsulated liver tissue are those that contain at least one of the phenotypes described herein. If subjected to a process, different origins (e.g., mammalian) and sources (e.g., primary cells) may be used. Hepatocytes can be derived from various sources (cell cultures, cell lines, differentiated stem cells). Differentially differentiated cells, such as posterior foregut cells, cells of the hepatocyte lineage, hepatocyte-like cells, and / or bile duct epithelial cells Hepatocytes derived from a single organoid can be of the same or different types. can be derived from different origins, from the same or different sources, and from the same or different types .

[0095] The mesenchymal cells to be included in the encapsulated liver tissue can be of different origins (e.g., mammalian) and sources (e.g., Mesenchymal cells can be obtained from various sources (primary cell cultures, cell lines, differentiated stem cells). They can be derived from different types of cells, such as adipocytes, muscle cells, or fibroblasts. Mesenchymal cells derived from a single organoid may be of the same or different origin, from the same or different sources. In one embodiment, the mesenchymal stem cells can be derived from different sources and can be of the same or different types. In yet another embodiment, mesenchymal stem / progenitor cells are used. In yet another embodiment, the cells are derived from mesenchymal stem cells / progenitor cells (such as pluripotent stem cells). Progenitor cells are derived from differentiated pluripotent stem cells (e.g., knockout serum replacement-supplemented group). Pluripotent stem cells were cultured on uncoated plastic in high-dose DMEM. The mesenchymal cells can be used fresh or used to form liver organoids. Therefore, the cells can be stored frozen until use.

[0096] The endothelial cells included in the encapsulated liver tissue, if present, may be of different origin (e.g., mammalian), and can be obtained from various sources (primary cell cultures, cell lines, differentiated stem cells). Endothelial cells can be derived from different types, such as endothelial progenitor cells and endothelial cells. In embodiments, endothelial progenitor cells are used. Endothelial cells derived from a single organoid can be obtained from the same may be derived from one or different origins, from the same or different sources, and from the same or different types In yet another embodiment, the endothelial progenitor cells are differentiated stem cells (pluripotent stem cells). In yet another embodiment, the endothelial progenitor cells are obtained from a differentiating pluripotent stem cell. (e.g., in combination with BMP4, bFGF, and / or VEGF) 99021 and / or Activin A) Endothelial cells can be used fresh or used to form liver organoids. It can be stored frozen until use.

[0097] In some embodiments, liver organoids are prepared from a single population of pluripotent stem cells. Sexual stem cells can be transduced using various methods, such as viral transduction (e.g., using the Sendai virus system). Induction using methods known in the art, or using synthetic mRNA approaches. Pluripotent stem cell populations can be derived from one or more induced pluripotent stem cells (iPSCs). Liver organoids can be obtained from the same pluripotent stem cell population. In embodiments where iPSCs are prepared, the populations include at least two (and in some embodiments, at least two) iPSC populations. At least three subpopulations are separated into hepatocytes and mesenchymal cells (and some In this embodiment, the cells are subjected to different culture conditions to generate endothelial cells.

[0098] Once each of the different cells is obtained, these cells are then used to generate liver organoids. Mix and culture in suspension. To control the size of liver organoids, 100-100 Under ultra-low adhesion conditions (e.g., suspension) using microcavities with a diameter of 0 µm In some embodiments, the cells can be cultured in 2 Approximately 500 μm There are a plurality of microcavities having a diameter and a depth. Once liver organoids are formed, they are grown in a bioreactor. It can be cultured in suspension (for growth purposes). In one embodiment, it contains 0.1-0.7% mesenchymal cells. Hepatocytes and mesenchymal cells are mixed prior to culture at a ratio of 1 part hepatocyte to 1 part endoderm cell. In this embodiment, when endothelial cells are present, the ratio is 0.2 to 1 endothelial cell to 1 endoderm cell. In yet another embodiment, the endothelial cells and endodermal cells are mixed prior to culturing in a ratio of: The ratio of hepatocytes, mesenchymal cells, and endothelial cells before culturing was 1:0.2:0.7. If cells are preferentially grown, they will differentiate preferentially, and the differentiation between different cells will be It is understood that the ratio may vary during culture. It is also understood that other ratios can be used to obtain liver organoids. During the process of creating the physical scaffold, or exogenous matrix material (tissue culture No other items are required (except the container).

[0099] Liver organoids can be used directly to generate encapsulated liver tissue. In some embodiments, the liver organoids are cryopreserved until they are introduced into the encapsulated liver tissue. It is possible.

[0100] Polymers that can be used in encapsulating liver tissue include liver organoid(s). As is known in the art, hydrogels are formed by dissolving water in water. Hydrogels are made of natural or synthetic polymers. In the context of the present disclosure, the term "hydrogel" refers to a polymer network. The encapsulation prevents the embedded liver organoids from leaking out of the polymer, There is a risk that cells from liver organoids will cause an immune response or tumor in the recipient's body upon transplantation. Eliminate or suppress the

[0101] In the context of the present disclosure, the polymer is non-toxic to the cells of the liver organoids. A substance is considered "biocompatible" if it is not toxic or does not exhibit toxicity when introduced into a subject (e.g., a human). In the context of this disclosure, a biocompatible polymer is a polymer that is compatible with the subject (e.g., It is not toxic to liver organoid cells when transplanted in vivo into a living organism (e.g., a human). Hepatotoxicity can be, for example, a decrease in the rate of apoptotic death of hepatocyte-like cells (e.g., , increased apoptosis indicates hepatotoxicity), transaminase levels (e.g., transaminase elevated adenosine adenosine levels indicate hepatotoxicity), hypertrophy of hepatocyte-like cells (e.g., hypertrophy of Increased levels of ATP are indicative of hepatotoxicity), microvacuolar degeneration in hepatocyte-like cells (e.g., increased levels of ATP are indicative of hepatotoxicity), Indicating hepatotoxicity), the rate of bile duct cell death (e.g., increased bile duct cell mortality indicates hepatotoxicity) ), gamma-glutamyl transpeptidase (GGT) levels (e.g., GGT levels The increase in the concentration of α-glucan is indicative of hepatotoxicity. Carbohydrates (hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratin Heparan sulfate, alginate, 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 poly(vinyl alcohol) (PVA)-based The biocompatible polymer may be a single polymer or a mixture of different polymers. mixtures of hydroxybenzoates (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 ingredients (such as chitosan, proteoglycans, or glycosaminoglycans (GAGs) (of which, alginate, collagen, thiolated heparin, and mixtures thereof are available. In some embodiments, the biocompatible polymer may be linear, branched, and optionally containing peptides (e.g., RGD), growth factors, It can also incorporate green or drugs.

[0102] In some embodiments, the polymer is a "low immunogenic polymer" and is non-immunogenic in the recipient. This low immunogenicity potentiates the immune response, but not the immune response itself. The primer blocks one or more antigenic determinants on the cell, and such antigenic determinants are not allogeneic. When introduced into a subject, it suppresses or prevents the immune response to that antigenic determinant. You can even do that.

[0103] The polymer present in the encapsulated liver tissue of the present disclosure is preferably crosslinkable, e.g. For example, the polymer can be crosslinked by thermal or chemical (e.g., VPMS, RGD, etc.). which one or more peptides are used), or the use of pH or light (e.g., UV light) It can be crosslinked by photopolymerization.

[0104] The polymers of the present disclosure may be biodegradable (e.g., easily hydrolyzed by the metabolism of a living organism). or be wholly or partially resistant to biodegradation (e.g., bioreplacement) Resistance to hydrolysis when subjected to chemical stress. An example of a degradable polymer is poly(ethylene glycol)-(maleimide) (PEG- Biocompatible and biodegradation-resistant polyamides include, but are not limited to, 8-armed Mal. An example of a mer is poly(ethylene-glycol)-vinyl sulfone (PEG-VS). However, it is not limited to this.

[0105] Once obtained, the liver organoids are at least partially (and in some embodiments, substantially) liver To coat the liver organoids, the liver organoids are coated with a first biocompatible crosslinkable polymer. The polymer can be used at various concentrations. The polymer concentration during contact with the liver organoids is between approximately 1% and 15% (weight / volume). In some embodiments, the concentration of the polymer when contacted with the liver organoids is at least Also about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% In yet another embodiment, the liver organoids are contacted with The polymer concentrations at these times were approximately 15%, 14%, 13%, 12%, 11%, 10%, 9%, and 8%. %, 7%, 6%, 5%, 4%, 3%, or 2% or less. Upon contact with the first polymer, the first polymer reacts (thermally, chemically, or by pH or The first biocompatible polymer is crosslinked using either a fluorine-containing polymer or a fluorine-containing polymer. Further bonds (and in some implementations) between different molecules and / or within the same molecule of the polymer In some embodiments, the first biomolecule is formed by forming an additional covalent bond. Cross-linking of compatible polymers creates additional bonds between the polymer molecules and the surface of liver organoids. In some embodiments, the covalent bond is formed by: The first polymer is at least partially biodegradable.

[0106] In some embodiments, the first biocompatible crosslinked polymer is (at least partially) coated or contacting the organoids or encapsulated liver organoids with a second biocompatible crosslinkable polymer; , at least partially (and in some embodiments, substantially) covering the encapsulated liver tissue. When the encapsulated liver organoids come into contact with a second polymer, the latter undergoes cross-linking (thermal (either electrically, chemically, or using pH or light). Crosslinking of synthetic polymers occurs between different polymer molecules and / or within the same polymer molecule. This is achieved by creating a further bond (and, in some embodiments, a further covalent bond) to In some embodiments, crosslinking of the second biocompatible polymer occurs by crosslinking the polymer molecules with the first biocompatible polymer. Between compatible cross-linked polymers and, in some embodiments, between polymer molecules and liver organelles. Further bonds (and in some embodiments further covalent bonds) between the surface of the nucleotide In some embodiments, the second polymer is at least partially resistant to biodegradation. It is resistant to

[0107] In some embodiments, this process involves the production of encapsulated liver organoids (first biocompatible scaffolds). bridge polymer / at least partially covered by a second biocompatible cross-linked polymer The encapsulated liver organoids are then coated with an additional biocompatible crosslinkable polymer to coat the organoids. When the liver organoids are contacted with the additional polymer, the latter undergoes crosslinking. (crosslinked either thermally, chemically, or using pH or light). The crosslinking of the biocompatible polymers is carried out between different molecules of the polymer and / or between the polymer molecules. This is achieved by creating additional bonds (in some embodiments, additional covalent bonds) within the same molecule. In some embodiments, cross-linking of the additional biocompatible polymer occurs by cross-linking the polymer molecules with the second and in some embodiments, between the polymer molecules and the first and / or a biocompatible cross-linked polymer between the surface of the liver organoid. A bond (and, in some embodiments, a further covalent bond) is formed.

[0108] This process involves the production of multiple monodispersed liver organoids within a first biocompatible crosslinked polymer. For example, a single iPSC can be used to generate a pre-hepatic cell line. progenitor cells, endothelial progenitor cells, and mesenchymal progenitor cells are obtained by mixing these cells, and and cultured in suspension to form liver organoids. In some embodiments, cells of the hepatic lineage are were differentiated into hepatocyte-like cells (before introducing liver organoids into the encapsulated liver tissue). The hepatocyte-like cells are essentially composed of a cell core formed by mesenchymal and endothelial progenitor cells. In a further embodiment, the liver organoids have a substantially spherical morphology and a size of about 150 Next, a cross-linking agent (UV light as shown in the examples) was used to perform the first matching. The liver organoids can be encapsulated in a crosslinkable matrix. The tissue is then incorporated into a regenerative medicine to produce transplantable liver tissue (e.g., tissue with a size of 5 mm to 10 cm). Alternatively, liver organoids can be cultured in a multi-well plate. Plates were designed to determine the metabolism or hepatotoxicity of the screened compounds. It can be used in drug development to determine

[0109] This process involves individually coating (at least partially) the nanoparticles with a first biocompatible cross-linked polymer. The organoids can then be designed to provide multiple liver organoids. A number of liver organoids were incorporated into a matrix made of a second biocompatible cross-linked polymer. In such an embodiment, the first biocompatible crosslinked polymer is first (at least partially) A plurality of liver organoids are formed, each individually covered with a different organelle. The ganoids are contacted with a second biocompatible crosslinkable polymer that crosslinks them.

[0110] This process involves mixing a first compatible cross-linked polymer and an optional second compatible cross-linked polymer. Designed to deliver multiple individual (e.g., monodisperse) liver organoids coated with In such an embodiment, the encapsulated liver tissue may be 2 Hit, at least Also 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 2 Hit, max about 500, 45 0, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90 , 80, 70, 60, or 50 liver organoids. In embodiments, the encapsulated liver tissue is 2 Per, approximately 50, 60, 70, 80, 90, 10 0, 125, 150, 175, 200, 250, 300, 350, 400, or 45 0 and approximately 500, 450, 400, 350, 300, 250, 200, 175, 150, The liver organoids may be between 125, 100, 90, 80, 70, or 60. In yet another embodiment, the encapsulated liver tissue is 2 Approximately 50 to 500 liver organs per In another embodiment, the encapsulated liver tissue comprises 3 At least about 250 per 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 this state, the encapsulated liver tissue is 3 Hit, maximum about 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, 300, In yet another embodiment, the encapsulated liver tissue comprises 250 liver organoids. m 3 Per, 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, Alternatively, the liver organoids may comprise between 300 and 300 cells / ml. Weave, cm 3 Each culture contains approximately 250-2500 liver organoids.

[0111] In some embodiments, the encapsulated liver tissue may be treated with the treatment methods and screening methods described herein. It can be used directly in the processing or frozen to extend its storage period. It can be saved.

[0112] Therapeutic Uses of Encapsulated Liver Tissue The encapsulated liver tissue described herein can be used as a medicine. The encapsulated liver tissue thus obtained exhibits some of the biological functions of the liver, making it suitable for use in subjects who require it. Can be used in vivo or ex vivo to restore or improve liver function Liver function is affected by, for example, the production of albumin and clotting factors (e.g., fibrinogen, protons, etc.). Rhombin, Factors V, VII, VIII, IX, X, XI, and XIII and protein C, protein S, and antithrombin synthesis. On the other hand, increased synthesis of albumin and / or clotting factors may improve liver function. Liver function is assessed by measuring the International Normalized Ratio, or INR. (e.g., a decrease in INR indicates recovery or improvement of liver function). It can also be assessed by measuring the detoxification of ammonia to urea (e.g., A decrease in serum urea levels and / or an increase in serum urea levels indicates recovery or improvement of liver function. .

[0113] In such embodiments, the encapsulated liver tissue is contacted with the biological fluids of the subject intended for treatment. In such embodiments, the encapsulated liver is intended to provide the necessary nutrients to the subject. Proteins and metabolites (albumin, clotting factors, and / or urea) in biological fluids Toxic substances (ammonia, unconjugated bilirubin, cholesterol) are released and metabolized. Encapsulated liver tissue can even absorb nutrients (e.g., ethanol, tyrosine) from biological fluids. It can be used to restore missing / decreased enzyme function in congenital anomalies of Xie .

[0114] To restore or improve liver function, subjects whose liver function has decreased to almost none or none The encapsulated liver tissue can be transplanted in vivo. The can be implanted into the peritoneal cavity, for example, so that it comes into contact with the ascites fluid. The liver tissue can be transplanted into the recipient's liver so that it is in contact with liver fluid. In another example, the encapsulated liver tissue is placed under the skin or in the muscle so as to be in contact with lymph or blood. can be implanted into

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

[0116] The encapsulated liver tissue can be used in a variety of subjects, and in these subjects, liver function can be restored. There are mammals, particularly humans, that are expected to benefit from the improvement of liver function. The tissue cells may be autologous, allogeneic, or xenogeneic to the subject for whom treatment is intended. However, encapsulated liver tissue is not amenable to the intended recipient's cells (especially can be designed to prevent physical contact with the intended recipient (immune cells) Autologous cells or immunosuppressants are used to prevent immunological recognition and response by the patient. This means that, for example, a subject comprising only one biocompatible cross-linked polymer does not need to be used. Using encapsulated liver tissue or a first biocompatible crosslinked polymer and a second biocompatible crosslinked polymer and / or by using encapsulated liver tissue containing both a polymer and a low immunogenic polymer. It can be used to implement.

[0117] In some embodiments, the encapsulated liver tissue is obtained using a surgical procedure, e.g., a laparoscopic procedure. Furthermore, liver tissue can be engineered and designed for introduction into a subject. , encapsulated in a biocompatible (and, in some embodiments, low immunogenic) polymer. Liver function is restored or the encapsulated liver tissue is no longer able to improve liver function. Once this has occurred, the encapsulated liver tissue can be removed from the subject.

[0118] Encapsulated liver tissue can be used to treat liver failure, which is a condition in which damage exceeds repair. This occurs when a large portion of the liver is subjected to excessive stress, and the liver is no longer able to function. Early symptoms of liver failure include nausea, loss of appetite, fatigue, and diarrhea. It can cause jaundice, bleeding, abdominal distension, confusion, and delirium (known as hepatic encephalopathy). Symptoms of liver failure include drowsiness, lethargy, and coma. Or it may be chronic with acute exacerbations. The most common causes of chronic liver failure are non-alcoholic fatty liver disease, hepatitis B, hepatitis C, long-term alcohol use, liver cirrhosis, hemochromatosis In chronic liver failure, hepatocyte transplantation is performed via the portal circulation. However, in cases where cirrhosis develops followed by chronic liver failure, Once the sinusoidal pores disappear (become capillary), the cells injected via the portal circulation can be transferred to the liver. They reach the parenchyma and prevent uptake into the liver lobule, thereby preventing the maturation and function of the transplanted cells. This can impede the function of the arteries, leading to complications such as sinusoidal and portal vein thrombosis. The encapsulated liver tissue provided does not require intraportal injection or immunosuppression, making it suitable for use in hundreds of thousands of liver transplants. Patients with cirrhosis, chronic (or acutely exacerbated chronic) liver failure, and those unsuitable for transplantation It also treats hepatic encephalopathy, prevents or suppresses serious complications (such as hepatic encephalopathy and coagulation disorders), and improves survival rates. It is believed that this can be improved.

[0119] The encapsulated liver tissue described herein can also be used to treat acute liver failure. The most common causes of chronic liver failure are reactions to prescription drugs and herbal remedies, or overdose, viral infections (including hepatitis A, B, and C), and Other causes include ingesting poisonous wild mushrooms, autoimmune hepatitis, or Wilson's disease. The onset of the disease is sudden, and the time to onset may be less than 48 hours. Furthermore, in acute liver failure, the targeting of fully mature and functional hepatocytes is difficult. Liver function is impaired to the point that transplantation is necessary. The tissue can be used to treat or alleviate symptoms of acute liver failure. Transplanting or processing the blood of subjects in need External (ex vivo) detoxification devices (extracorporeal liver support, bioartificial liver devices, or The number of liver organoids in the encapsulated liver tissue and the severity of the pathology were investigated. Depending on the severity, one or more encapsulated liver tissues can be used to treat the subject. The liver tissue(s) can be used simultaneously or sequentially. The use of encapsulated liver tissue to treat or alleviate liver disease provides an allogeneic response to the subject being treated. Cells of the system can be used.

[0120] Encapsulated liver tissue is a common cause of monogenic congenital disorders of liver metabolism (e.g., Crigler-Najjar syndrome). syndrome, familial hypercholesterolemia, urea cycle disorders, N-acetylglutamic acid Enzyme deficiency, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, citrullinemia, argininosuccinate lyase deficiency, arginase deficiency, etc. It can also be used to treat or reduce the symptoms of certain conditions, such as hypertyrosinemia type 1. In this embodiment, the encapsulated liver tissue provides the missing metabolic function and alleviates the symptoms. reduce the risk of heart disease, prevent or reduce complications, and / or reduce the need for lifelong medical treatment or dietary restrictions Reduce or eliminate the need to do so.

[0121] Encapsulated liver tissue is an implantable method for treating acute and chronic liver failure without the need for immunosuppression. Such tissues can be designed as self-contained products (e.g., encapsulated liver tissue sheets). In embodiments, the implantable tissue sheet is 2 Contains approximately several thousand liver organoids per sample In some embodiments, the encapsulated liver tissue sheet can be easily manipulated and fixed to the desired implantation site. The container can be placed in a container that makes it easy to do so (e.g., a custom-made permeable bag, etc.). In this embodiment, for ease of manipulation, the implantable tissue sheet has a thickness of at least 1 mm, and in some further embodiments, the width is at least 5 mm to 10 cm. The encapsulated liver tissue can be made into any shape or size desired. It can be shaped or cut during the procedure.

[0122] Screening method and screening kit for hepatic metabolism and hepatotoxicity The encapsulated liver tissue described herein is designed to retain at least some liver function. Investigating the metabolism of active substances (e.g., drug candidates) by organisms 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 whether or not a substance is hepatotoxic due to its nature. and the vast majority of (suspected) 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 purified liver tissue can be used to determine hepatotoxicity. Drugs, toxins, and other substances that have been reported to cause liver damage include: There are over 900 medicinal herbs, and drugs are the main cause of 20-40% of all cases of fulminant liver failure. 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., steroids) will streamline new drug discovery and development. It is useful in

[0123] The encapsulated liver tissue described herein actually exhibits 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 liver toxicity or liver toxicity. 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 liver organoid of encapsulated liver tissue Agents active against at least one (and, in some embodiments, two or three) cell types the agent to be tested under conditions sufficient to effect the agent, so as to obtain a test mixture; Alternatively, a combination of agents is brought into contact with the encapsulated liver tissue. The method comprises the steps of: (a) extracting at least one liver organoid from the encapsulated liver tissue; and (b) extracting at least one liver organoid from the encapsulated liver tissue. at least one (and in some embodiments, at least two or three) cell types of the or determining at least one agent-related hepatic metabolite of the agent in the test mixture. As used in connection with this disclosure, the expression "agent-related metabolite" refers to a metabolite of the agent being tested. refers to metabolites that can be formed by hydrolysis of

[0125] Alternatively, or in combination, at least one liver organoid of the encapsulated tissue At least one (and in some embodiments, at least two or three) cell types, or Alternatively, at least one liver parameter is determined in the test mixture. Liver parameters that can be assessed include albumin production, urea production, ATP production, and glutathione production. On production, cytochrome P450 (CYP) metabolic activity, liver-specific genes or proteins (e.g., CYP enzymes (CyP2C9, CyP3A4, CyP1A1, CyP1A2, C Expression of cytochrome P456 (cytochrome P456B6 and / or Cytochrome P456D6), response to liver toxins, cell death (e.g., for example, by measuring lactate dehydrogenase or transaminase in the test mixture), Cell apoptosis, cell necrosis, cell metabolic activity (e.g., live / dead assay, caspases) (e.g., Ze3 / 7 assay, MTT assay, or WST-1-based assay), mitochondrial At least one of the following may be involved: Once the liver parameter(s) are obtained, the corresponding control liver parameters are compared. In one embodiment, the liver parameters of a control are compared to those of the control. The parameters are determined by the activity of the screened agents (or the activity of the screened agents). or obtained in the absence of the agent (or combination of agents) screened The resulting solution (a combination of the active ingredients) can be obtained in the presence of a dissolving medium. The determination step can be performed on all or part of the cells of the encapsulated liver tissue. In some embodiments, the determining step is performed using hepatocyte-like cells of encapsulated liver tissue and / or bile duct-associated cells. It is performed on skin cells.

[0126] This method allows for determining whether the liver organoids of the encapsulated liver tissue metabolize an agent and / or or whether the agent is hepatotoxic to cells of liver organoids of encapsulated liver tissue; To do so, the agent-related liver function to be measured is compared to determine the Comparisons are made between metabolites and agent-related hepatic metabolites in controls. The active substance-related metabolites of the steroid are extracted in their intact (e.g., unhydrolyzed) form. It can be the agent itself, a control agent-related metabolite, or Once the presence of different agent-related metabolites has been determined, the metabolism of the agent in hepatocytes is then investigated. Determine the relationship between the liver parameters to be measured and those of the control. For example, liver parameters in controls may be compared with non-active substances. Liver parameters can be obtained in the presence of control and If it is determined that the differences are present, it is then determined whether the agent is hepatotoxic. .

[0127] In one embodiment, the method comprises: This is used to determine whether a compound (a combination of compounds) exhibits hepatotoxicity. In such embodiments, the screened agents (or the screened agents By contacting at least one of the encapsulated liver tissues with the liver organoids, To determine whether or not a compound induces toxicity in a single cell type (e.g., hepatocytes or bile duct epithelial cells), Toxicity can be measured, for example, by cell death (e.g., lactate dehydrogenase or transactivation of the test compound). metabolic viability of cells (e.g., by measuring hydroxylase activity, caspase activity, etc.); 3 / 7 assay, MTT assay, or WST-1-based assay), mitochondrial Mitochondrial function (e.g., impaired mitochondrial function indicates liver toxicity), cytochrome P450 system Modulation of the activity of one or more enzymes (e.g., CYP2E1, etc.) (e.g., cytochrome P4 Increased activity of the 50 enzyme(s) may indicate hepatotoxicity and / or regulation of bile acid production. The enzymes involved in the production of bile acids (e.g., increased bile acid production indicates hepatotoxicity) can be determined and measured. This method compares the toxicity results of the screened agents with those of a control agent (which is hepatotoxic). Toxicity results for compounds known not to induce liver toxicity or compounds known to induce liver toxicity This can include comparing with.

[0128] This method was applied to encapsulated liver tissue obtained using liver organoids with different metabolic activities. and contacting the screened agent (or agents) with the For example, different levels of specific metabolic functions can be achieved by differently controlling the metabolism of different cells. To generate liver organoids, cells from different origins and sources are used. (This allows for the variation observed among individuals in a typical population.) For example, the resulting encapsulated liver tissues differing in metabolic activity may be different from each other and from all other tissues. This allows the screening of active substances to be tested in comparison with other substances. In one embodiment, the liver Visceral organoids can be derived from different genders, races, and / or genotypes The effects of screening for these different genders, races, and / or genotypes Testing substances to determine metabolic differences or to determine whether sex, race, and / or genetic This allows for the determination of whether hepatotoxicity is present in all or only some of the types. The mesenchymal components and / or endogenous components of liver organoids were identified among multiple liver organoids. The epithelial components can be similar, but the hepatocyte-like cells and bile duct epithelial cells are of different sexes. As an example, each of the different encapsulated liver tissues may be derived from different genes, races, and / or genotypes. The tissues can be placed in different wells (as multiple replicates if desired), Contacting the same screened agent with each of the different encapsulated liver tissues can be done.

[0129] In some embodiments, the encapsulated liver tissue used in this screening method is a second living organism. does not contain a compatible cross-linked polymer or a further biocompatible cross-linked polymer, but instead The present invention relates to a method for producing a liver organoid from a first biocompatible crosslinked polymer, comprising: Become qualitative.

[0130] This screening method involves the use of individually encapsulated liver organoids or multiple Liver organoids encapsulated in a matrix containing liver organoids can be used. In the latter case, the encapsulated liver tissue is located at the bottom of the well, thereby allowing the screen The addition of the washed agent and washing of the encapsulated liver tissue prior to the determination step are very It becomes simple.

[0131] The present disclosure also provides a kit for determining hepatic metabolism or hepatotoxicity. Encapsulated liver tissue as described herein and instructions for carrying out the methods described herein. In some embodiments, the kit further comprises a tissue culture support, comprising: In a further embodiment, the encapsulated liver tissue may optionally comprise at least one well. The fabric is placed in the bottom of at least one well and, if desired, attached (covalently bound) to the surface of the well. The kit can be used to measure hepatic metabolism or hepatotoxicity. Measurement of (e.g., Live / Dead assay, caspase 3 / 7 assay, MTT assay, WST The present invention may also include reagents for performing the IL-1 assay (and / or LDH measurement). .

[0132] The present invention will be more readily understood by reference to the following examples, which are provided as illustrative examples only and are not intended to be limiting. are described to illustrate the present invention and are not intended to limit the scope of the present invention. . [Example]

[0133] Production and characterization of hepatocyte-like cells Hepatocyte-like cells (HLCs) were cultured using two different protocols: (referred to as Protocol B), a standard protocol described in PCT / CA2017 / 051404. The HLCs were then compared.

[0134] Differentiation Protocol (Protocol B) iPSC preparation (days -3 to 0). Three days before starting differentiation, trypLE iPSCs. Single cell passaging was performed. iPSCs were placed on laminin-coated plates and The cells were then cultured in Essential 8 Flex medium. Only Revita Cell™ (ThermoFisher Scientific) The medium was replaced daily.

[0135] Endoderm Detail (Days 1-2). The cells were washed with DMEM / F-12 medium. , insulin-free, containing 1% knockout serum replacement (KOSR), 100 ng / RPMI / supplemented with 1 ml Activin A and 3 μM CHIR99021 The cells were cultured in B27 at 37°C in an atmosphere of O2 / 5% CO2 for 2 days. The medium was changed every day.

[0136] Endoderm commitment (definitive endoderm, days 3-5). No insulin, 1% knockout blood RPMI / B27 containing supernatant substitute 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 6-10). Contains no insulin and 1% knockout serum replacer. , 20ng / ml BMP4, 5ng / ml bFGF, 4μM IWP2, and 1μ Cells were cultured in RPMI / B27 supplemented with M A83-01 at 37°C under ambient conditions. The cells were cultured for 5 days under O2 / 5% CO2 conditions, and the medium was changed daily.

[0138] Liver details (bipotent progenitor cells, days 11-15). Insulin, 2% knockout serum. Substitutes include 20ng / ml BMP4, 10ng / ml bFGF, 20ng / ml Cells were cultured in RPMI / B27 supplemented with HGF and 3 μM CHIR99021. The cells were cultured at 37°C in an atmosphere of O2 / 5% CO2 for 5 days. It was changed every day.

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

[0140] Hepatic maturation 2 (immature hepatocyte-like cells, days 21-25). Contains 1% knockout serum replacer. HBM / HCM medium supplemented with 20 ng / ml OSM and 10 μM dexamethasone. The cells were cultured in medium (without EGF or Lonza) at 37°C in an atmosphere of 5% O2 / C. The cells were cultured for 5 days in O2. The medium was changed every day. In addition, 1% knockout serum replacement and Primary Hepatocyte M maintenance Supplement(TM)(ThermoFisherSc Comparable results were obtained using William's E medium supplemented with entific (data not shown).

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

[0142] Table 1. Details of the 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 microscope (EVOS FL Cell Imaging Sys The differentiation process was carried out using a 3D mouse model (Thermo Fisher Scientific). The last living cells of the spores were observed and their morphology was studied.

[0144] Cell counting. Harvest cells from culture plates using TrypLE and automate cell counting. Counter Countess II FL Automated Cell Counter , and counted using Thermo Fisher Scientific.

[0145] Immunofluorescence. Cells were fixed in 4% paraformaldehyde and then in 0.2% TritoCl. The cells were permeabilized with n X-100 at room temperature for 5 minutes. Incubate with 3% blocking serum solution (corresponding to The fixed and permeabilized cells were then blocked with primary antibody solution (antibodies were in PBS- The cells were incubated with PBS (diluted to 2% with BSA) at room temperature for 1 hour. Incubate with labeled antibody solution (fluorescent) for 30 minutes at room temperature, protected from light During the last 15 minutes of incubation with the secondary antibody, a dye (Pureblu The nuclei were stained by adding nuclei staining (BioRad). The cells were fixed with anti-fade reagent (ProLong Gold). The day after this procedure, Fluorescence was analyzed. The following antibody was used: anti-human SOX17 in ABCAM diluted 1:100 ABCAM anti-human FOXA2 diluted 1:100; ABCAM anti-human CXCR4 diluted 1 DAKO anti-human AFP diluted 1:100; DAKO anti-human albumin (AL) B) Dilution 1:100; anti-human CK19 diluted 1:100 with ABCAM; and Anti-human CK7 diluted 1:200.

[0146] FACS analysis. 0.5–1 × 10 cells were added to each assay tube. 6 Cells were dispensed Incubate cells with 100 μL of fluorescent dye-conjugated primary antibody solution (membrane antigen) at room temperature, protected from light. The cells were then stained with 4% paraformaldehyde for 20 minutes at room temperature. The cells were fixed for 10 minutes. Then, the cells were permeabilized with 1% Triton X-100. Stain the cells with 1 μL of fluorescent dye-conjugated antibody solution (intracellular antigen) and incubate in the dark at room temperature. The cells were resuspended in 0.5 mL of PBS-BSA 1% and incubated for 20 minutes. The cells were 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 science), 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. Cultured cells were used as a template for the synthesis of single-stranded cDNA. Total RNA was extracted from cells or organoids using Rneasy Plus Mini Reverse transcription was performed to obtain cDNA. The PCR reaction mixture was prepared. The plates were sealed and centrifuged before loading into the instrument. Standard TaqMan qPCR reaction conditions were used. Relative quantification of gene expression was performed. Data were analyzed using the comparative C (ΔΔC) method to calculate the following TaqM an gene expression assay (obtained from Thermo Fisher scientific ) was 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 Al bumin 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 I, 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 Current assay and Hs00944626_m1 TAT Taqman gene expression assay Say.

[0148] Cyp 3A4 activity. Promega's "P450-Glo™ Assay" was used. Cyp3A4 activity was assessed using the manufacturer's instructions.

[0149] Urea synthesis. Gentaur's "Quantichrom urea assay kit" Urea synthesis was measured using a 't' according to the manufacturer's instructions.

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

[0151] Mitochondrial respiration analysis. Seahorse Bioscience XF96 analyzer (Seahorse Bioscience Inc.) was used to prepare a 96-well plate. Mitochondrial stocks were incubated at 37°C according to the manufacturer's instructions with minor modifications. Briefly, cells were cultured at 1 × 10 5 Cells seeded at 1000 cells / well , and 24 hours before the assay, different doses of acetaminophen (APAP-2, The cells were pretreated with 2, 4, 8, and 19 μM of amiodaron (AMIO-2, 4, 8, and 19 μM). On the day of testing, the growth medium was removed, washed twice, and XF assay medium (unbuffered DMSO) was added. EM, d5030 Sigma, 25 mM glucose, 2 mM glutamine, 1 mM pyruvate The plate was then incubated at 37°C for 1 hour in a CO atmosphere. Incubate in a 2-hour free incubator. Hydrate the cartridge sensor and add the appropriate Amount of mitochondrial modulator was loaded to achieve a final concentration in each well: Gomycin (2 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazo FCCP (2 μM), and rotenone / antimycin A (both 1 μM). The OCR values ​​were used to calculate basal respiration, A, and B as described in the manufacturer's protocol. The levels of TP production, proton leak, maximal respiration, and non-mitochondrial respiration were analyzed. Table 2. Abbreviations used.

[0152] [Table 2]

[0153] Endoderm induction treatment of hiPSCs for 5 days resulted in the expression of specific endoderm markers. SOX17, FOXA2, GATA4, CXCR4, and EOMES-expressing cells A homogeneous monolayer was obtained (Figure 1). The homogeneity of the population was confirmed by flow cytometry analysis. Over 80% of cells were triple positive for SOX17, FOXA2, and CXCR4 It was found that the cells did not express c-Kit (Fig. 2). The colors indicate that most cells express the definitive endoderm markers SOX17, FOXA2, and The results showed that the cells were positive for CXCR4 (Figure 3, lower panel). Similar results were obtained when human embryonic stem cells (hESCs, data not shown) were differentiated instead of PS cells. The results were obtained.

[0154] After endoderm induction, the cells were treated for 5 days to induce differentiation into the posterior foregut. In the second stage, signals such as FGF-2 and BMP4, which are normally expressed from the cardiac mesoderm, are observed. In addition, the Wnt / β-catenin and TGFβ signaling pathways (I, II, III, and IV, respectively) were upregulated. WP2 and A83-01), allowing expression of Hex and Proxl As shown in Figure 4, the cells expressed the foregut-specific markers FOXA2, SOX2, and F Enhanced expression of OXA1, HNF4A, AFP, and albumin.

[0155] Subsequently, FGF-2 and BMP4 signaling were maintained, HGF was added, and the liver By activating the Wnt pathway (using CHIR99021) which promotes liver growth For 5 days, liver specificity was induced (polygonal morphology of hepatoblasts). These cells were liver specific. The expression of the key markers AFP, albumin, CK19, CK7, and EpCAM was also shown. In addition, the iPSC-derived hepatic progenitor cell population did not contain undifferentiated cells. RT-qPCR also confirmed the expression of albumin, AFP, CK19, and CK. 7, PDX1, SOX9, PROX1, HNF4α, and HHEX, which are characteristic liver bud genes As shown in Figure 8, hepatic progenitor cells were expressed in the endothelial cells / hepatocyte markers (Figure 7). A significant increase in cell yield was observed compared to endoderm cells or undifferentiated iPSCs.

[0156] To further clarify the involvement in the liver, we inhibited TGFβ signaling (cholangiocytes) To avoid this, A83-01 was used), and the Wnt pathway was activated (CHIR 99021). FGF-2, BMP4, HGF, OSM, and dexamethasone At the final stage of differentiation (because hematopoiesis ceases in the liver after birth), OSM was removed and dexamethasone was maintained.

[0157] During differentiation, cell populations develop a large cytoplasmic to nucleus ratio, numerous vacuoles and vesicles, The cells gradually acquired the typical morphology of hepatocyte-like cells with prominent nucleoli. These cells were found to be binuclear (Fig. 9A). Immunofluorescence analysis showed that the expression of CK19 and IL-19 was significantly higher in the hepatoblast stage than in the hepatoblast stage (Fig. 9B). It showed increased expression of albumin and decreased expression of AFP and CK19. (Fig. 9B and data not shown). The majority of cells (98.5%) were albumin-positive (Figure 10). Analysis revealed the expression of specific liver genes, such as albumin, AFP, HNF4a, ASGR1, and SOX9. Gene expression was shown to be similar between HLC and FPHH (Fig. 11).

[0158] Figure 12 shows the HLCs obtained by Protocol B compared with primary human hepatocytes HepG2, undifferentiated iPSCs, and These results suggest that HLC-B and FPH cells are H showed similar CyP3A4 activity (Fig. 12A) and urea production (Fig. 12C). HLC-B cells express similar or lower levels of antigens compared to adult hepatocytes. They produce albumin (Figure 12B).

[0159] HLCs obtained by protocol B showed high expression of liver markers (Figure 13), significantly larger CyP 3a4 activity (Fig. 14A), albumin production (Fig. 14B), and cell yield (Fig. 14C) were measured. Compared with HLCs obtained with protocol A, a significant differentiation was achieved. It showed that.

[0160] Metabolic function of hepatocyte-like cells (obtained using Protocol B), mitochondrial respiratory capacity Force and ATP-related respiration were measured under basal conditions and after acetaminophen (APAP). ), and amiodarone (AMIO), after increasing doses of drugs metabolized specifically in the liver. The results shown in Example 15 show that when contacted with the drug, This indicates that the HLCs obtained in B regulate their respiration and are therefore metabolically active. are.

[0161] While the present invention has been described with reference to specific embodiments thereof, the claims are not intended to be construed as limiting the scope of the invention. The present invention is not limited to the preferred embodiments described above, but is consistent with the entire specification. It should be understood that the broadest interpretation should be given.

Claims

1. A process for producing posterior foregut cells from endoderm cells, said process comprising: contacting the cells with a first culture medium that does not contain insulin, and a first set of additives under conditions that allow differentiation of endoderm cells into said posterior foregut cells; wherein the first additive set does not include insulin, and • 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 beta (TGFβ) signaling pathway, including or The process qualitatively consists of them.

2. generating hepatic progenitor cells from the posterior foregut cells, and hepatocyte-like cells from the hepatic progenitor cells. The process of claim 1 further comprising:

3. 3. The process of claim 1 or 2, wherein the first culture medium comprises serum.

4. The activator of the BMP signaling pathway is a BMP receptor agonist. The process of any one of claims 1 to 3.

5. The process of claim 4 , wherein the BMP receptor agonist is BMP4.

6. wherein the activator of the FGF signaling pathway is an FGF receptor agonist.

6. The process of any one of claims 1 to 5.

7. 7. The process of claim 6, wherein the FGF receptor agonist is basic FGF.

8. The inhibitor of the Wnt signaling pathway inhibits the biological activity of porcupine. The process according to any one of claims 1 to 7, wherein

9. The method of claim 8, wherein the inhibitor of the Wnt signaling pathway is IWP2. Process.

10. The inhibitor of the TGFβ signaling pathway is ALK4, ALK5, or ALK6. Any of claims 1 to 9, which is capable of inhibiting at least one biological activity of K7.

10. The process according to any one of claims 1 to 9.

11. 11. The method of claim 10, wherein the inhibitor of the TGFβ signaling pathway is A83-01. The process described.

12. The endoderm cells express SOX17, GATA4, FOXA2, CXCR4, or EO The process according to any one of claims 1 to 11, wherein the process expresses at least one of MES. vinegar.

13. The endoderm cells of any one of claims 1 to 12, wherein the endoderm cells are substantially unable to express c-Kit.

10. The process of any one of claims 1 to 9.

14. The posterior foregut cells express SOX2, FOXA1, FOXA2, HNF4a, AFP, or The method according to any one of claims 1 to 13, wherein the strain expresses at least one of albumin. process.

15. Obtainable or obtainable by the process according to any one of claims 1 to 14. A population of posterior foregut cells obtained.

16. A process for producing hepatic progenitor cells from posterior foregut cells, said process comprising: contacting the foregut cells with a second culture medium, a second set of additives under conditions that allow differentiation of the cells into the second set of additives; The kit is: • 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 (HGF) signaling pathway; and - a pretreatment comprising, or consisting essentially of, an inhibitor of the Wnt signaling pathway; Process described.

17. 17. The process of claim 16, wherein the second culture medium comprises serum.

18. The activator of the insulin signaling pathway is an insulin receptor agonist.

18. The process of claim 16 or 17, wherein

19. 19. The process of claim 18, wherein the insulin receptor agonist is insulin. 。

20. The activator of the BMP signaling pathway is a BMP receptor agonist.

20. The process of any one of claims 16 to 19.

21. 21. The process of claim 20, wherein the BMP receptor agonist is BMP4.

22. wherein the activator of the FGF signaling pathway is an FGF receptor agonist.

22. The process of any one of claims 16 to 21.

23. 23. The process of claim 22, wherein the FGF receptor agonist is basic FGF.

24. 2. The method of claim 1, wherein the activator of HGF signaling is an HGF receptor agonist.

24. The process of any one of claims 16 to 23.

25. 25. The process of claim 24, wherein the HGF receptor agonist is HGF.

26. The activator of the Wnt signaling pathway inhibits the biological activity of GSK3. The process according to any one of claims 16 to 25,

27. 27. The method of claim 26, wherein the activator of the Wnt signaling pathway is CHIR99021. The process described in

28. The posterior foregut cells express SOX2, FOXA1, FOXA2, HNF4a, AFP, or The method according to any one of claims 16 to 27, wherein the strain expresses at least one of albumin. The process of.

29. The hepatocyte progenitor cells are derived from alpha-fetoprotein (AFP), albumin (ALB), and sucrose. Itokeratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, P At least one of ROX1, HHEX, HNF4a, or epithelial cell adhesion molecule (EpCAM) 29. The process of any one of claims 16 to 28, wherein at least one of the following is expressed:

30. obtainable by a process according to any one of claims 16 to 29, or Obtained population of posterior foregut cells.

31. 1. A process for producing mature hepatocyte-like cells from hepatic progenitor cells, comprising: (i) treating the hepatic progenitor cells with a third additive set under conditions to obtain cells of the hepatic cell lineage; and a third culture medium comprising a third set of additives, wherein the third set of additives comprises: • Activators of the insulin signaling pathway; • activators of the bone morphogenetic protein (BMP) signaling pathway; • Activators of the fibroblast growth factor (FGF) signaling pathway; • activator of the hepatocyte growth factor (HGF) signaling pathway; • Inhibitors of the Wnt signaling pathway; • Inhibitors of the transforming growth factor β (TGFβ) signaling pathway; • Cytokines; and - glucocorticoids, comprising or consisting essentially of them; (ii) subjecting the cells of the hepatocyte lineage to a fourth addition under conditions to obtain immature hepatocyte-like cells. and a fourth culture medium comprising a fourth set of additives, wherein the fourth set of additives comprises: • Cytokines; and glucocorticoids, comprising or consisting essentially of them; and (iii) subjecting the immature hepatocyte-like cells to a fifth culture under conditions for obtaining the mature hepatocyte-like cells. and a fifth culture medium containing a set of additives, wherein the fifth set of additives is 1. The composition of claim 1, wherein the composition is free of steroids and comprises or consists essentially of glucocorticoids. The process comprising:

32. 32. The method of claim 31 , wherein the fourth, fifth, and / or sixth culture medium comprises serum. process.

33. The activator of the insulin signaling pathway is an insulin receptor agonist.

33. The process of claim 31 or 32.

34. 34. The process of claim 33, wherein the insulin receptor agonist is insulin. 。

35. 2. The method of claim 1, wherein the activator of the BMP signaling pathway is a BMP receptor agonist.

35. The process of any one of claims 31 to 34.

36. 36. The process of claim 35, wherein the BMP receptor agonist is BMP4.

37. 2. The method of claim 1, wherein the activator of the FGF signaling pathway is an FGF receptor agonist.

37. The process of any one of claims 31 to 36.

38. 38. The process of claim 37, wherein the FGF receptor agonist is basic FGF.

39. The activator of the HGF signaling pathway is an HGF receptor agonist.

39. The process of any one of claims 31 to 38.

40. 40. The process of claim 39, wherein the HGF receptor agonist is HGF.

41. The activator of the Wnt signaling pathway inhibits the biological activity of GSK3. The process according to any one of claims 31 to 40, wherein

42. The activator of the Wnt signaling pathway activator is CHIR99021.

42. The process of claim 41.

43. The inhibitor of the TGFβ signaling pathway is ALK4, ALK5, or ALK6.

43. The method of claim 31, wherein the method is capable of inhibiting at least one biological activity of K7.

10. The process of any one of claims 1 to 9.

44. 44. The method of claim 43, wherein the inhibitor of the TGFβ signaling pathway is A83-01. process.

45. 45. Any of claims 31 to 44, wherein the cytokine is oncostatin M (OSM).

1. The process according to claim 1.

46. 45. The method according to any one of claims 31 to 44, wherein the glucocorticoid is dexamethasone. The process described.

47. The hepatic progenitor cells are derived from alpha-fetoprotein (AFP), albumin (ALB), cytochrome P454 (CYP454), and cytochrome P454 (CYP454). Keratin 7 (CK7), cytokeratin 19 (CK19), SOX9, PDX1, PRO Any of claims 31 to 46, wherein the vector expresses at least one of X1 and / or HNF4a.

10. The process according to any one of claims 1 to 9.

48. The immature hepatocyte-like cells and / or mature hepatocyte-like cells are derived from α-fetoprotein (AFP), albumin (ALB), ASGR1, HNF4a, or SOX9 48. The process of any one of claims 31 to 47, wherein at least one

49. The mature hepatocyte-like cells have detectable Cyp3A4 activity and The method according to any one of claims 31 to 48, wherein the method expresses albumin and / or urea. The process of.

50. Obtainable by a process according to any one of claims 31 to 49, or Obtained population of hepatocyte-like cells.

51. 1. A process for producing hepatic progenitor cells from endoderm cells, comprising: (a) performing the process of any one of claims 1 to 14 to obtain posterior foregut cells; Alternatively, providing a population of posterior foregut cells according to claim 15; and (b) subjecting the posterior foregut cells to the process of any one of claims 16 to 29 to produce the Obtaining hepatic progenitor cells, comprising or consisting essentially of:

52. 52. The method of claim 51, wherein the hepatic progenitor cells are obtainable or obtained by the process of claim 51. Group.

53. 1. A process for producing hepatocyte-like cells from hepatocyte progenitor cells, comprising: (a) performing the process of any one of claims 16 to 29 to obtain hepatic progenitor cells; Alternatively, providing a population of hepatic progenitor cells according to claim 30; and (b) subjecting said hepatic progenitor cells to a process as defined in any one of claims 31 to 49. obtaining hepatocyte-like cells, vinegar.

54. Hepatocyte-like cells obtainable or obtained by the process of claim 53. A group of.

55. 1. A process for producing hepatocyte-like cells from endoderm cells, comprising: (a) Obtaining posterior foregut cells by optionally carrying out the process of any one of claims 1 to 14. or optionally providing a population of posterior foregut cells according to claim 15; (b) subjecting the posterior foregut cells to the process of any one of claims 16 to 29 to produce prehepatic obtaining progenitor cells or providing a population of hepatic progenitor cells according to claim 30; and (c) subjecting the hepatic progenitor cells to the process of any one of claims 31 to 49 to produce the hepatic progenitor cells. The process comprising or consisting essentially of obtaining cell-like cells.

56. Hepatocyte-like cells obtainable or obtained by the process of claim 55. A group of.

57. 1. A process for producing encapsulated liver tissue, comprising: (a) providing a population of hepatocyte-like cells according to claim 54 or 56; (b) combining the hepatocyte-like cells, mesenchymal cells, and optional endothelial cells in a suspension; and culturing the cells to produce at least (i) hepatocyte-like cells and / or bile duct epithelial cells. a partially covered cell core comprising mesenchymal cells and optionally endothelial cells; (ii) (iii) at least one particle having a spherical morphology, and (iv) a relative diameter of about 50 to about 500 μm. Obtaining one liver organoid; and (c) cross-linking said at least one liver organoid with a first biocompatible cross-linked polymer. said process comprising at least partially covering said surface.

58. The endoderm cells and hepatocyte-like cells are combined in a ratio of 1:0.2-7 before culturing; 58. The process of claim 57.

59. The endoderm cells and endothelial cells are combined in a ratio of 1:0.2 to 1 before culturing.

59. The process of paragraph 57 or 58.

60. At least one of the hepatocyte-like cells, endoderm cells, and endothelial cells is subjected to stem cell differentiation.

60. The process of any one of claims 57 to 59, wherein the hydroxybenzoate is obtained from the polymerization of hydroxybenzoate.

61. 61. The process of claim 60, wherein the stem cells are pluripotent stem cells.

62. The process according to any one of claims 57 to 61, wherein the endothelial cells are endothelial progenitor cells. Seth.

63. At least one of the liver organoids is substantially crosslinked with the first biocompatible crosslinked polymer.

63. The process of any one of claims 57 to 62, comprising selectively covering the surface.

64. the first biocompatible cross-linked polymer comprises poly(ethylene) glycol (PEG); 64. The process according to any one of claims 57 to 63.

65. The first biocompatible crosslinked polymer is at least partially crosslinked with a second biocompatible crosslinked polymer.

65. The process of any one of claims 57 to 64, further comprising covering the portion.

66. said first biocompatible crosslinked polymer being substantially crosslinked with said second biocompatible crosslinked polymer; 66. The process of claim 65, including covering.

67. 58. The method of claim 57, wherein the first biocompatible crosslinked polymer is at least partially biodegradable.

67. The process of any one of claims 1 to 66.

68. The second biocompatible crosslinked polymer is at least partially resistant to biodegradation.

68. The process according to any one of claims 57 to 67.

69. the second biocompatible cross-linked polymer comprises poly(ethylene) glycol (PEG).

69. The process according to any one of claims 65 to 68.

70. obtainable by a process according to any one of claims 57 to 69; or Obtained encapsulated liver tissue.

71. A first set of additives as defined in any one of claims 1 to 14.

72. 72. A method for treating a vascular endothelial disorder comprising administering to a subject a first set of additives according to claim 71, ... and A first culture medium that does not contain an activator.

73. 73. The first culture medium of claim 72, further comprising endodermal cells.

74. 74. The first culture medium of claim 72 or 73, further comprising posterior foregut cells.

75. A second set of additives as defined in any one of claims 16 to 29.

76. 76. A second culture medium comprising a second set of additives as defined in claim 75.

77. 77. The second culture medium of claim 76, comprising posterior foregut cells.

78. 78. The second culture medium of claim 76 or 77, further comprising hepatic progenitor cells.

79. A third set of additives as defined in any one of claims 31 to 49.

80. 80. A third culture medium comprising a third set of additives as defined in claim 79.

81. A fourth set of additives as defined in any one of claims 31 to 49.

82. 82. A fourth culture medium comprising a fourth set of additives as defined in claim 81.

83. A fifth set of additives as defined in any one of claims 31 to 49.

84. A fifth set of additives as defined in claim 83, which is cytokine-free. Culture medium.

85. 1. A kit for generating posterior foregut cells, hepatic progenitor cells, or hepatocyte-like cells, comprising: An additive set as defined in any one of claims 71, 75, 77, 81 or 83. and / or at least one of At least one of the compounds defined in claim 72, 76, 80, 82 or 84 one culture medium; and - including instructions for producing posterior foregut cells, hepatic progenitor cells, or hepatocyte-like cells; The kit.

86. ●Endodermal cells, • Posterior foregut cells, and / or The kit of claim 85, further comprising: hepatic progenitor cells.

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