Methods and compositions for obtaining liver cells

Abstract

Provided herein are methods and compositions for obtaining hepatocytes. Provided herein is a kit or cell culture medium composition for inducing hepatocytes from pluripotent stem cells, the composition comprising the following inducers: (1) Activin A, (2) a modulator of the BMP signaling pathway, (3) a modulator of the FGF signaling pathway, (4) a modulator of the Wnt signaling pathway, (5) a growth factor, (6) a TGFβ receptor / ALK5 inhibitor. Also provided herein are methods for preparing hepatocytes by the kit or cell culture medium composition, hepatocytes obtained by the methods, artificial liver devices containing the hepatocytes and / or use of the hepatocytes for preparing artificial liver devices, and methods for large scale culture of hepatocytes.

Description

Invention Field

[0001] This invention generally relates to methods and compositions for obtaining liver cells. More specifically, this invention relates to methods and compositions for obtaining liver cells from induced pluripotent stem cells. Background Technology

[0002] (I) Induced pluripotent stem cells

[0003] Pluripotent stem cells (IPS) can self-renew and differentiate into all somatic cell types. Somatic cells have been reprogrammed to be pluripotent through nuclear transfer into oocytes or through ectopic expression of limiting factors. For example, in August 2006, the laboratory of Professor Shinya Yamanaka at the Institute for Regenerative Medicine, Kyoto University, Japan, first announced the successful induction and reprogramming of mouse fibroblasts into pluripotent stem cells with properties similar to embryonic stem cells by introducing four genes (Oct4, Sox2, c-Myc, and KLF4). They found that transducing only Oct4, Sox2, c-Myc, and KLF4 could induce pluripotent stem cells (iPS cells). These iPS cells possess a normal karyotype, express molecular markers similar to those of embryonic stem cells, can be induced to differentiate into terminally differentiated cells of the endoderm, mesoderm, and ectoderm in vitro, and can form teratomas in nude mice containing differentiated cells from the endoderm, mesoderm, and ectoderm. Furthermore, similar to embryonic stem cells, iPS cells showed low methylation and high acetylation of H3 on Nanog and Oct4 promoters. The study also found that iPS cells could be obtained using only Oct4, Sox2, and KLF4, indicating that cMyc is not a necessary transcription factor for somatic cell reprogramming.

[0004] Subsequently, using the Yamanaka strategy, several new factors for generating induced pluripotent stem cells (iPSCs) were screened out. The Huck-Hui Ng research group in Singapore discovered that Nr5a2, Klf4, Sox2, and cMyc can be reprogrammed together (Jian-Chien Dominic Heng et al., 2009), and that Oct4, Esrrb, Klf4, and cMyc can also be reprogrammed together. Simultaneously, the study found that using only Oct4 and specific cell types, such as neural stem cells (Kim et al., 2009) and trophoblastic ectoderm cells (Tong Wu et al., 2011), can reprogram somatic cells into iPSCs. Furthermore, with the addition of small molecules, it is also possible to convert mouse fibroblasts into iPSCs (Yanqin Li et al., 2010) and human keratinocytes into iPSCs (Saiyong Zhu et al., 2010).

[0005] To date, different research groups have successfully experimented with reprogramming techniques on many different types of cells. Numerous methodological improvements have also been made in the induction process. In 2008, Hochedlinger's group successfully induced iPS cells using an adenovirus vector with a non-integrated genome (Matthias Stadtfeld et al., 2008); in the same year, Yamanaka also successfully induced iPS cells using a plasmid vector (Keisuke Okita et al., 2008); the piggyBac transposon system was later shown to allow for reprogramming by removing exogenously inserted genes (Woltjen K et al., 2009); later, Ding Sheng's group also successfully induced iPS cells using the protein of the four factors (Zhou H et al., 2009); recently, successful reprogramming has also been achieved using RNA (Luigi Warren et al., 2010) and microRNA (Frederick Anokye-Danso et al., 2011 and Alessandro Rosa et al., 2011).

[0006] The establishment of induced pluripotent stem cells (iPSCs) has effectively addressed the ethical issues surrounding embryonic stem cell research, holding significant importance for life sciences and human health. IPSC research can help elucidate developmental mechanisms and influencing factors in humans and animals; establish transgenic or gene-targeted animals to create human disease models; provide various types of human cells for pharmaceutical research through in vitro induced differentiation; and, under strict control, allow animals to derive certain organs from human cells for clinical transplantation through chimera formation. The most promising application is as target cells for cell and gene therapy, providing immunogenic materials for cell transplantation, and allowing genetically modified human cells to be transplanted back into the body to achieve disease cures, among other possibilities.

[0007] (II) Bioartificial liver system

[0008] The liver is one of the most vital metabolic organs in the human body. Abnormal liver function can significantly impact a patient's quality of life, and severe liver failure can even be life-threatening. Clinical statistics show that acute liver failure has a mortality rate as high as 80%, and the number of patients with abnormal liver function continues to rise. Currently, liver transplantation is the most effective treatment for liver failure. Unfortunately, due to the limited availability of liver donors, approximately 20%-25% of liver failure patients die before receiving a suitable donor.

[0009] Artificial liver systems are support systems developed for the clinical treatment of patients with acute liver failure. They can replace all or part of the liver's function, thus prolonging the patient's life until a suitable liver transplant donor is available. Furthermore, because the liver has the ability to proliferate and regenerate after acute injury, artificial liver systems can support the patient's survival, reduce the liver's metabolic burden, and promote liver self-repair to alleviate the condition. A bioartificial liver system is a cell-based perfusion device that directly or indirectly contacts the patient's serum with liver seed cells within the bioartificial liver device. Metabolic substances in the serum are then metabolized and detoxified by the liver seed cells, which simultaneously replenish important metabolic products synthesized by the liver, thereby improving the survival rate of patients with liver failure. Therefore, bioartificial liver devices combine the detoxification, synthesis, and regulatory functions of liver cells, making them more suitable for the clinical treatment of patients with liver failure. The core of a bioartificial liver system lies in the bioreactor, namely the selection of the seed cell line and its culture method.

[0010] The core of a bioartificial liver support system lies in the selection and preparation of seed cell lines. In the clinical treatment of liver failure, each patient requires no fewer than 10 [cells / cells / etc.]. 10Only suitable cells can effectively replace the functions of the liver. Meanwhile, the liver performs many important physiological functions, participating in protein synthesis (such as albumin and clotting factors), bile synthesis, metabolism of sugars, fats, and proteins, and detoxification of blood ammonia. Therefore, the cells must possess functions more complete than those of primary liver cells. Primary liver cells are the most ideal cell source, but the number of cryopreserved cells and freshly isolated cells is insufficient to meet the cell quantity requirements of bioartificial livers. Currently, the cell sources used in bioartificial liver systems mainly fall into the following categories:

[0011] Hepatocellular carcinoma cell lines: The HepG2 cell line is currently the most widely used hepatocellular carcinoma cell line in bioartificial livers. However, researchers compared bioartificial livers derived from HepG2 cells with those derived from isolated primary liver cells. The results showed that the metabolic efficiency of HepG2 cells differed by approximately two orders of magnitude from that of primary cells, and the ability of HepG2 cells to clear blood ammonia was significantly different from that of primary cells. Further comparison revealed differences in the metabolic patterns of HepG2 cells and primary cells. Primary liver cells exhibit gluconeogenesis by metabolizing lactate to produce glucose, while HepG2 cells consume glucose to produce lactate. The lactate produced can easily lead to acidosis in patients. In addition, the functions of HepG2 cells in albumin synthesis and drug metabolism differ significantly from those of primary liver cells, making it an unsuitable seed cell source for replacing primary liver cells.

[0012] Primary porcine hepatocytes: Due to their ease of acquisition and possession of the functions of primary livers, porcine hepatocytes are considered a relatively ideal source of seed cells for bioartificial livers. Experiments have shown that bioartificial liver systems derived from primary porcine livers have significant therapeutic effects on patients with acute liver failure. However, the clinical application of primary porcine hepatocytes in bioartificial liver treatment faces two main problems: First, the function of primary porcine hepatocytes is difficult to maintain in vitro. Currently, the treatment time for bioartificial liver systems is generally no less than 4 hours, and some treatment times exceed 24 hours. Primary porcine hepatocytes rapidly lose liver cell function within a few hours, including drug metabolism activity, protein synthesis capacity, and blood ammonia metabolism capacity, accompanied by a rapid decline in cell viability. Researchers attempted to transfer SV40-LT into primary porcine hepatocytes to establish immortalized porcine hepatocyte cell lines. Results showed that this method can effectively maintain the function and viability of primary porcine hepatocytes to some extent; however, the use of retroviruses poses significant risks from a clinical safety perspective, especially porcine retroviruses, which increase the risk of zoonotic disease transmission, limiting their subsequent application. Second, there are potential risks associated with the exchange of blood substances between species, particularly the unpredictable immune responses and disease transmission caused by porcine liver cells and the spread of pathogens between species. Therefore, some countries strictly prohibit the use of porcine liver cells in the treatment of human diseases.

[0013] Transdifferentiated cell origin: We and other research teams have demonstrated that human fibroblasts can be converted to human hepatocytes through transduction of liver-specific genes. Hepatocytes obtained through this method have a gene expression profile similar to primary hepatocytes, exhibiting the ability to secrete albumin, metabolize blood ammonia, and metabolize drugs. However, hepatocytes obtained through lineage reprogramming also present some challenges. First, current reprogramming techniques primarily use embryonic fibroblasts as starting cells; however, embryonic fibroblasts are unstable and cannot be stably expanded and passaged in vitro. Furthermore, transdifferentiated hepatocytes require in vitro expansion mediated by oncogenes such as Myc and SV40. Second, current reprogramming techniques mainly involve constructing expression vectors using retroviruses to overexpress key transcription factors intracellularly to alter cell fate, often accompanied by the introduction of tumor-related genes to enhance cell expansion potential, such as P53i, C-MYC, and SV40. Therefore, long-term passage can lead to genomic instability and may induce tumorigenesis, posing potential risks in clinical applications and making them unsuitable as seed cells for clinical bioartificial liver therapy. Furthermore, the transdifferentiated liver cells obtained so far still differ significantly in function from adult liver cells and are difficult to replace the function of primary liver cells. Summary of the Invention

[0014] Pluripotent stem cells (PSCs) can stably expand in vitro and can be directed to differentiate into various types of physiologically functional tissues, making them an ideal seed cell source for bioartificial liver systems. In this invention, the inventors have established a method and composition for the in vitro directed differentiation of human PSCs into liver progenitor cells with expansion capabilities. Based on this, the inventors have established a technique for the large-scale expansion of liver progenitor cells derived from human PSCs. Furthermore, the inventors have also established a novel method for the efficient and large-scale preparation of functionally mature liver cell clusters, ultimately enabling the acquisition of a sufficient quantity of liver seed cell banks derived from human PSC differentiation for application in bioartificial liver systems.

[0015] Therefore, in some embodiments, the present invention covers the technical solutions described below or variations thereof.

[0016] 1. A kit or cell culture medium composition for inducing liver cells from pluripotent stem cells, said composition comprising the following inducing agent:

[0017] (1)Activin A,

[0018] (2) BMP signal transduction pathway regulators

[0019] (3) Regulators of the FGF signal transduction pathway

[0020] (4) Regulators of the Wnt signaling pathway

[0021] (5) Growth factors

[0022] (6) TGFβ receptor / ALK5 inhibitor.

[0023] 2. The kit or cell culture medium composition described in Project 1 further comprises one or more of the following inducers:

[0024] (7) GSK-3β inhibitors

[0025] (8) cAMP agonists, and / or

[0026] (9) Nuclear receptor ligand,

[0027] Optionally, it may also contain one or more additional inducers, such as (10) LPA, (11) S1P, and / or (12) XMU-MP-1.

[0028] 3. The kit or cell culture medium composition described in item 1 or 2, wherein:

[0029] The (2) BMP signal transduction pathway regulators include one or more of BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, and anti-BMP receptor antibodies;

[0030] The (3) FGF signal transduction pathway regulators include one or more of FGF1, FGF2, FGF4 and FGF10;

[0031] The (4) Wnt signaling pathway regulators include one or more of Wnt3a, Wnt agonists, Dkk, and R-Spondin;

[0032] The growth factors (5) include one or more of KGF, IGF, EGF, and VEGF; and / or

[0033] The (6) TGFβ receptor / ALK5 inhibitors include one or more of SB431542, LY-364947, SB-505, and A-83-01.

[0034] 4. The kit or cell culture medium composition described in Project 2, wherein:

[0035] The (7) GSK-3β inhibitors include one or more of CHIR99021, TD114-2, BIO, Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A 014418, FRATide, Indirubin-3′-oxime, and L803;

[0036] The cAMP activator includes one or more of Forskolin, IBMX, Rolipram, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, dibutyryl-cAMP (DBcAMP), and Sp-8-Br-cAMPs; and / or

[0037] The nuclear receptor ligands of (9) include one or more of estradiol, all-trans retinoic acid, 13-cis retinoic acid, dexamethasone, clobetasol, androgens, thyroxine, troglitazone, pioglitazone, and prostaglandins.

[0038] 5. The kit or cell culture medium composition described in Project 1, wherein the inducer is present in an amount that induces the expression of marker proteins AFP, SOX9, and Ki67 in cells.

[0039] 6. The kit or cell culture medium composition described in Project 2, wherein the inducer is present in an amount that induces the expression of the marker proteins AFP and ALB in cells.

[0040] 7. A method for inducing liver cells from pluripotent stem cells, the method comprising contacting the pluripotent stem cells with a kit or cell culture medium composition of any one of items 1-6 to induce liver cells from the pluripotent stem cells.

[0041] 8. The method of Project 7, wherein the pluripotent stem cells are contacted with the kit or cell culture medium composition of Project 1 to induce hepatoblasts from the pluripotent stem cells.

[0042] 9. The method of Project 8, wherein the hepatoblasts are further contacted with the inducing agent of Project 2 to induce the generation of hepatocytes.

[0043] 10. A liver cell obtained by the method of any one of items 7-9, an artificial liver device containing the liver cell, and / or the use of the liver cell in the preparation of an artificial liver device, for example for the treatment of liver diseases, such as liver diseases requiring cell perfusion, such as liver failure, such as acute liver failure.

[0044] 11. A method for large-scale hepatocyte culture, the method comprising: 1) passage expansion of liver precursor cells in a 2D system, and 2) further culturing and maturing in a 3D system, wherein step 1) includes culturing and maturing at approximately 1 × 10⁻⁶ cells per cell line. 7 As the starting cell quantity, cells were stably and continuously passaged in culture flasks until the cell count was expanded to 1 × 10⁶ cells / year. 8 The samples were then transferred to a cell factory for further expansion to approximately 1 × 10⁻⁶. 9 At this scale, it is ready to be transferred to a rotary bottle for functional maturation.

[0045] 12. The method described in Item 11, wherein step 2) comprises placing approximately 1 × 10 9 Cells are digested from the cell factory, and single-cell suspensions are transferred to roller bottles for 3D stirred culture.

[0046] 13. The method described in Project 11 or 12, wherein the subculture ratio in step 1) is approximately 1:2 to 1:3, and the subculture frequency is approximately 2 to 3 days.

[0047] 14. The method described in any one of items 11-13, wherein in step 2), the amount of culture medium is about 1-1.5L, the rotation speed is about 60rpm, and the cells are cultured with the medium changed daily until the cells are functionally mature, for example, synthesizing liver cell functional proteins ALB and / or CYP3A4.

[0048] 15. The method of any one of items 11-14, wherein the liver progenitor cells are derived from pluripotent stem cells, such as induced pluripotent stem cells, such as liver progenitor cells induced by the kit or cell culture medium composition of any one of items 1-6 or the method of any one of items 7-9. Attached Figure Description

[0049] Figure 1 The morphology of hiPSC cells differentiated into hepatoblasts (HBs) is shown.

[0050] Figure 2 Immunofluorescence staining of hepatoblasts is shown.

[0051] Figure 3 This indicates the expansion of liver progenitor cell clones.

[0052] Figure 4 Flow cytometry analysis of liver precursor cells.

[0053] Figure 5 QPCR identification of liver precursor cells.

[0054] Figure 6 Immunofluorescence staining of liver precursor cells.

[0055] Figure 7 RNASeq analysis of liver precursor cells.

[0056] Figure 8 The expansion curve of liver precursor cells is shown.

[0057] Figure 9 This study demonstrates the passage stability of liver precursor cells.

[0058] Figure 10 This shows the cryopreservation and thawing of liver precursor cells.

[0059] Figure 11 This shows that liver precursor cells differentiate into mature liver cells.

[0060] Figure 12 QPCR detection of transcription factor-related genes in liver cells.

[0061] Figure 13 Detection of the liver precursor cell maturation process.

[0062] Figure 14 PAS staining and immunofluorescence staining of liver cells are shown.

[0063] Figure 15 QPCR detection of genes related to urea synthesis and coagulation factors in liver cells.

[0064] Figure 16 ELISA detection of liver cell albumin and urea.

[0065] Figure 17 RNASeq analysis of the cells showed that hiPSC1 and 2 were two different hiPSC cell lines, hMH1-3 were liver cell lines derived from three different hiPSC cell lines, and PHH1 and 2 were two different primary liver cell lines.

[0066] Figure 18 This diagram shows a large-scale liver cell culture model.

[0067] Figure 19 This image shows the morphology of large-scale cultured liver clones and immunofluorescence staining.

[0068] Figure 20 QPCR detection of liver clonal transcription factor-related genes.

[0069] Figure 21 QPCR detection of liver clones and related genes for liver characteristics.

[0070] Figure 22 QPCR detection of genes related to drug metabolism in the liver clone.

[0071] Figure 23 This study showed gene detection related to clonal urea synthesis and coagulation factors in the liver.

[0072] Figure 24 ELISA detection of liver clone albumin and urea synthesis.

[0073] Figure 25 This demonstrates the bioartificial liver treatment process.

[0074] Figure 26 This table shows the survival curves of miniature pigs with acute liver failure.

[0075] Figure 27 Photograph of a miniature pig with acute liver failure.

[0076] Figure 28 This indicates the detection of serum biochemical markers related to liver damage.

[0077] Figure 29 Hematoxylin and eosin (HE) staining of liver sections.

[0078] Figure 30 This diagram illustrates the differentiation of induced pluripotent stem cells (hiPSCs) into liver cells.

[0079] Figure 31 This diagram shows a large-scale culture and suspension culture of hepatocytes. Detailed Implementation

[0080] I. Definition

[0081] As used herein, “culture” means a population of cells grown in a culture medium and optionally passaged. A cell culture can be a primary culture (e.g., a culture that has not yet been passaged) or a passaged or subcultured culture (e.g., a population of cells that has been passaged or passaged once or multiple times).

[0082] As used in this article, “induced pluripotent stem cells” (iPSCs) are a type of pluripotent stem cells derived artificially from non-pluripotent cells.

[0083] The terms “isolated” or “purified” pluripotent stem cells refer to pluripotent stem cells that are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free of contaminated cell types such as non-pluripotent cells.

[0084] In this document, when the term “about” is used to describe a numerical value, it may refer to a suitable range of numerical fluctuations that are readily apparent to those skilled in the art. For example, in some embodiments, when the term “about” is used to describe a numerical value, it may refer to ±5%, 10%, 20%, etc., as understood by those skilled in the art as a suitable range of numerical fluctuations. In some embodiments, when the term “about” is used to describe a numerical value, it may refer to ±5%, as understood by those skilled in the art as a suitable range of numerical fluctuations. In some embodiments, even if the term “about” is not used, it may cover a suitable range of numerical fluctuations understood by those skilled in the art. As used herein, the term “pluripotent” (or pluripotent) refers to stem cells that have the potential to differentiate into any of the three germ layers: endoderm (e.g., internal gastric mucosa, gastrointestinal tract, lung), mesoderm (e.g., muscle, bone, blood, urogenital tract), or ectoderm (e.g., epidermal tissue and nervous system). The term “non-pluripotent” means that the cell does not have the potential to differentiate into all three germ layers. Pluripotent stem cells are less plastic and more highly differentiated, and can become one of many cell types within a given organ. For example, pluripotent hematopoietic stem cells can develop into erythrocyte progenitor cells, leukocytes, or platelet-producing cells. Adult stem cells are pluripotent stem cells. Adipose-derived stem cells are pluripotent.

[0085] As used in this article, “reprogramming” refers to converting one cell type into another. For example, non-pluripotent cells can be reprogrammed into pluripotent cells.

[0086] As used herein, “induction medium” refers to a cell culture medium containing one or more inducers.

[0087] II. Composition

[0088] A. Inducers or regulators

[0089] Inducing agents used in this article include those that, alone or in combination with other reagents, induce cell differentiation, such as differentiation from pluripotent stem cells into endoderm cells, hepatoblasts, liver progenitor cells, and mature liver parenchymal cells.

[0090] Therefore, agents that can be used to induce induced pluripotent stem cells to differentiate into endoderm cells, hepatoblasts, hepatic progenitor cells, mature hepatocytes, etc., have been identified. In some embodiments, the inducer or regulator includes one or more of the following: (1) Activin A, (2) BMP signaling pathway regulator, (3) FGF signaling pathway regulator, (4) Wnt signaling pathway regulator, (5) growth factor, (6) TGFβ receptor / ALK5 inhibitor, (7) GSK-3β inhibitor, (8) cAMP agonist, (9) nuclear receptor ligand, (10) LPA, (11) S1P, and / or (12) XMU-MP-1, and any combination thereof. The inducer or regulator may be provided alone or in combination as a composition of inducers or regulators. One or more other regulators may also be administered with the above-described agents.

[0091] (1) BMP signal transduction pathway regulators

[0092] BMP signaling pathway regulators may include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, and anti-BMP receptor antibodies. In some embodiments, the concentration of the BMP signaling pathway regulator is a concentration that can induce pluripotent stem cells to differentiate into endoderm cells. The concentration of BMP signaling pathway regulators such as BMP4 may be from about 1 ng / ml to about 200 ng / ml, preferably from about 1 ng / ml to about 100 ng / ml, more preferably from about 1 ng / ml to about 20 ng / ml. The BMP signaling pathway regulators may be added to, for example, culture media that induce iPSCs to differentiate into endoderm cells and culture media that induce endoderm cells to differentiate into hepatocytes.

[0093] (2) Regulators of FGF signal transduction pathway

[0094] FGF signaling pathway regulators may include FGF1, FGF2, FGF4, and FGF10. The concentration of an FGF signaling pathway regulator, such as FGF2 (bFGF), may be from about 1 ng / ml to about 200 ng / ml, preferably from about 5 ng / ml to about 100 ng / ml, more preferably from about 10 ng / ml to about 50 ng / ml. FGF signaling pathway regulators may be added to, for example, media for inducing the transformation of iPSCs into endoderm cells and media for the transformation of endoderm cells into hepatocytes.

[0095] (3) Wnt signaling pathway regulators

[0096] Wnt signaling pathway regulators may include one or more of the following: Wnt family proteins (e.g., Wnt1, Wnt3a, Wnt7a), Wnt receptors, Wnt receptor agonists, Dkk, and R-Spondin. The concentration of Wnt signaling pathway regulators such as Wnt3a may be from about 1 ng / ml to about 200 ng / ml, preferably from about 5 ng / ml to about 100 ng / ml, more preferably from about 10 ng / ml to about 50 ng / ml. Wnt signaling pathway regulators may be added to, for example, a culture medium for inducing the transformation of iPSCs into endoderm cells (e.g., added on day 1 of culture).

[0097] (4) Growth factors

[0098] The growth factors in the induction medium may include one or more of KGF, IGF, EGF, and VEGF. In some embodiments, growth factors such as KGF may be added to the medium for inducing the differentiation of endoderm cells into hepatocytes (e.g., added on days 4-8 of culture). In some embodiments, growth factors such as EGF may optionally be added to the medium for inducing the differentiation of hepatocytes into hepatic progenitor cells. The concentration of growth factors such as KGF and / or EGF may be, for example, from about 1 ng / ml to about 100 ng / ml, preferably from about 5 ng / ml to about 50 ng / ml, more preferably from about 10 ng / ml to about 40 ng / ml.

[0099] (5) TGFβ receptor / ALK5 inhibitors

[0100] The TGFβ receptor / ALK5 inhibitor in the induction medium may include one or more of SB431542, LY-364947, SB-505, and A-83-01. The concentration of the TGFβ receptor / ALK5 inhibitor, such as SB431542, may be, for example, from about 0.1 μM to about 100 μM, from about 1 μM to about 50 μM, or from about 1 μM to about 10 μM. The TGFβ receptor / ALK5 inhibitor may be added to the culture medium for inducing the transformation of endoderm cells into hepatocytes, the culture medium for inducing the transformation of hepatocytes into liver progenitor cells, and the culture medium for the transformation of progenitor cells into hepatocytes.

[0101] (6) GSK-3β inhibitors

[0102] The GSK-3β inhibitor in the induction medium may include one or more of CHIR99021, TD114-2, BIO, Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A 014418, FRATide, Indirubin-3′-oxime, and L803. The concentration of the GSK-3β inhibitor, such as CHIR99021, may be, for example, from about 0.1 μM to about 100 μM, from about 1 μM to about 50 μM, or from about 1 μM to about 10 μM. The GSK-3β inhibitor may be added to, for example, a medium used to induce the transformation of hepatoblasts into liver progenitor cells.

[0103] (7) cAMP activator

[0104] The cAMP activator in the induction medium may include one or more of Forskolin, IBMX, Rolipram, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, dibutyryl-cAMP (DBcAMP), and Sp-8-Br-cAMPs. The concentration of cAMP activator inhibitors such as Forskolin may be, for example, from about 0.1 μM to about 100 μM, from about 1 μM to about 50 μM, or from about 5 μM to about 20 μM. The cAMP activator may be added, for example, to the medium used to induce the transformation of hepatoblasts into hepatic progenitor cells, and to the medium used to induce the transformation of progenitor cells into hepatocytes.

[0105] (8) Nuclear receptor ligand

[0106] Nuclear receptor ligands in the induction medium may include one or more of estradiol, all-trans retinoic acid, 13-cis retinoic acid, dexamethasone, clobetasol, androgens, thyroxine, troglitazone, pioglitazone, and prostaglandins. Nuclear receptor ligands can regulate local gene expression or transcription at the delivery site. The concentration of nuclear receptor ligands such as dexamethasone may be, for example, from about 0.01 μM to about 100 μM, from about 0.1 μM to about 50 μM, or from about 1 μM to about 20 μM. Nuclear receptor ligands may be added, for example, to media for inducing the transformation of hepatoblasts into hepatic progenitor cells, and to media for the transformation of progenitor cells into hepatocytes.

[0107] (9) Other reagents

[0108] Other suitable reagents may optionally be added to the culture medium of the present invention to induce differentiation. For example, appropriate amounts of LPA, S1P, and / or Hippo signaling pathway modulators such as XMU-MP-1 may be added. In some embodiments, the concentration of LPA may be, for example, from about 0.01 μM to about 100 μM, from about 0.1 μM to about 50 μM, or from about 1 μM to about 20 μM. LPA may be added to, for example, a culture medium for inducing the transformation of hepatoblasts into liver progenitor cells. In some embodiments, the concentration of S1P may be, for example, from about 0.01 μM to about 100 μM, from about 0.1 μM to about 50 μM, or from about 0.5 μM to about 10 μM. LPA may be added to, for example, a culture medium for inducing the transformation of hepatoblasts into liver progenitor cells. In some embodiments, the concentration of the Hippo signaling pathway regulator, such as XMU-MP-1, can be, for example, from about 0.001 μM to about 10 μM, or from about 0.1 μM to about 1 μM. The Hippo signaling pathway regulator, such as XMU-MP-1, can be added to, for example, a culture medium used to induce the transformation of hepatoblasts into liver progenitor cells. The basal culture medium of the present invention is not particularly limited, and any suitable culture medium can be used, such as RPMI 1640 medium, DMEM / F12 medium, William'E medium, or a mixture thereof. In some embodiments, the culture medium of the present invention can be supplemented with other components suitable for cell culture, such as serum-free supplement B27, GlutaMax, etc.

[0109] B. Induced pluripotent stem cells

[0110] Induced pluripotent stem cells (iPSCs) derived from hepatocytes can be obtained by inducing partial or complete differentiation of cells from mammals, such as any mammal (e.g., cattle, pigs, dogs, cats, horses, primates), preferably humans. Sources include bone marrow, fibroblasts, fetal tissue (e.g., fetal liver tissue), peripheral blood, umbilical cord blood, pancreas, skin, or any organ or tissue. In a preferred embodiment, the iPSCs are obtained from self-induced fibroblasts, adipose-derived stem cells, neural stem cells, or cells derived from intestinal epithelium. In a more preferred embodiment, the iPSCs are obtained from self-induced neonatal (e.g., foreskin) or adult fibroblasts. However, iPSCs can be obtained from other cell types, including but not limited to: pluripotent stem cells, blood-derived cells, skin-derived cells, fibroblasts, adipocytes, epithelial cells, endothelial cells, mesenchymal cells, parenchymal cells, nerve cells, and connective tissue cells. iPSCs can be obtained from samples taken from mammalian subjects. Subjects can be any mammal (e.g., cattle, pigs, dogs, cats, horses, primates), including humans. Cell samples can be obtained from a variety of different sources, including bone marrow, liver, peripheral blood, cord blood, pancreas, skin, or any organ or tissue.

[0111] In a preferred embodiment, induced pluripotent stem cells are induced to obtain fibroblasts and adipose-derived stem cells. In a more preferred embodiment, induced pluripotent stem cells are induced to obtain fibroblasts, which can be nascent (e.g., foreskin fibroblasts) or adult fibroblasts.

[0112] Cells can be separated by breaking down a suitable organ or tissue serving as the cell source using techniques known to those skilled in the art. For example, tissues or organs can be mechanically broken down and / or treated with digestive enzymes and / or chelating agents that weaken the connections between adjacent cells, thereby dispersing the tissue to form a suspension of individual cells without perceptible cellular damage. Enzymatic dissociation can be accomplished by shredding the tissue and treating the shredded tissue with one or more enzymes such as trypsin, chymotrypsin, collagenase, elastase, and / or hyaluronidase, DNase, streptomycin, dispersant, etc. Mechanical destruction can also be achieved by a variety of methods, including but not limited to the use of grinders, mixers, sieves, homogenizers, pressure cells, or insonators.

[0113] III. Methods and Preparation

[0114] A. Inducing differentiation

[0115] Hepatocytes can be induced to differentiate into liver cells by providing induced pluripotent stem cells (iPSCs) (e.g., induced human pluripotent stem cells (hiPSCs)) with an inducing medium containing an inducing medium for a sufficient period of time. The iPSCs are then contacted with a medium containing an inducing agent that effectively induces and / or enhances the differentiation of iPSCs into liver cells for a sufficient period of time to induce the differentiation of the cells into liver cells.

[0116] B. Cell isolation

[0117] A substantially purified population of liver cells can be obtained by extraction from a culture source (e.g., by density gradient centrifugation and / or flow cytometry). Purity can be measured by any suitable method. For example, liver cells can be purified to 99%–100% purity by flow cytometry (e.g., FACS analysis). Liver cells can be isolated, for example, by using molecules that bind to markers on liver cells (e.g., antibodies, antibody derivatives, ligands, or Fc-peptide fusion molecules) and thus positively selecting the cells that bind to the molecules (i.e., positive selection). Other examples of positive selection methods include methods that preferentially promote the growth of desired cell types in a mixed population of desired and undesirable cell types. Alternatively, undesirable cells containing such markers can be removed from desired cells by using molecules that bind to markers that are absent on desired cell types but present on undesirable cell types (i.e., negative selection). Other negative selection methods include preferentially killing or inhibiting the growth of undesirable cell types in a mixed population of desired and undesirable cell types. Thus, rich populations of stem cells can be prepared by using negative selection, positive selection, or combinations thereof.

[0118] The separation process can include magnetic separation, the use of antibody-coated magnetic beads, affinity chromatography, cytotoxic agents linked to monoclonal antibodies, or such reagents used in combination with monoclonal antibodies, such as complement and cytotoxins, and “panning” of antibodies attached to a solid matrix (e.g., a plate), or other convenient techniques. Techniques providing accurate separation include fluorescently activated cell sorters, which can have varying degrees of complexity, such as multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels. Antibodies can be conjugated to labels such as magnetic beads that allow direct separation, biotin that can be removed using avidin or streptoavidin bound to a support, or fluorescent dyes that can be used with fluorescently activated cell sorters to allow for easy separation of specific cell types. Any technique that does not unduly harm the viability of induced pluripotent stem cells can be employed. In one embodiment, cells are incubated with an antibody against a label (e.g., TRA-1-81 antibody), and cells that are positively stained for the label are manually selected and passaged.

[0119] Combinations of enrichment methods can be used to improve the time or efficiency of purification or enrichment. For example, after an enrichment step that removes cells with markers that do not indicate the target cell type, cells can be further separated or enriched using a fluorescence-activated cell sorter (FACS) or other methods with high specificity. Multicolor analysis can be used in conjunction with FACS. Cells can be separated based on staining levels against a specific antigen or the absence of said staining levels. Antibodies specific to a specific antigen can be labeled using fluorescent dyes. Such fluorescent dyes include phycobiliproteins such as phycoerythrin and allophycocyanin, fluorescein, and Texas red. Any cell type-specific label can be used to select for or against a specific cell type.

[0120] C. Cell culture and preservation

[0121] Cells can be stored in culture for later recovery and use. According to known methods, such as those described in Doyle et al. (eds.), 1995, Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons, Chichester, cells can be cryopreserved for storage. For example, cells can be suspended in a “freezing medium,” such as a medium containing 15-20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO), with or without 5-10% glycerol, at a density of, for example, about 4-10 x 10⁻⁶. 6 Cells / ml. Cells are dispensed into glass or plastic vials, sealed, and transferred to the freezer compartment of a programmable or passive freezer. The optimal freezing rate can be determined empirically. For example, a freezing procedure providing a temperature change of -1°C / min via heat of fusion can be used. Once the vials containing cells reach -80°C, they are transferred to a liquid nitrogen storage area. Cryopreserved cells can be stored for periods of several years.

[0122] D. Large-scale cultivation methods

[0123] In some embodiments, the present invention also provides a method for culturing hepatocytes using the culture medium described herein, particularly a method for large-scale hepatocyte culture. In some embodiments, the large-scale culture method may include 1) passage expansion of a 2D system and 2) culturing and maturation of a 3D system. In some embodiments, step 1) includes culturing at approximately 1 × 10⁻⁶ cells / day. 7 As the starting cell quantity, stable and continuous cell passages are performed in culture flasks at a passage ratio of approximately 1:2 to 1:3, with a passage frequency of approximately 2 to 3 days, until the cell count is expanded to 1 × 10⁶ cells / year. 8 The samples were then transferred to a cell factory for further expansion to approximately 1 × 10⁻⁶.9 The scale is such that it is ready to be transferred to roller bottles for functional maturation. In some implementations, step 2) includes transferring approximately 1 × 10⁻⁶ units of water to a roller bottle. 9 Cells are digested in a cell factory, and single-cell suspensions are transferred to roller bottles for 3D stirred culture. In some embodiments, this document provides a method for large-scale hepatocyte culture, the method comprising 1) passage expansion of liver precursor cells in a 2D system, and 2) further 3D culture and maturation, wherein step 1) includes culture at approximately 1 × 10⁻⁶ cells / mL. 7 As the starting cell quantity, cells were stably and continuously passaged in culture flasks until the cell count was expanded to 1 × 10⁶ cells / year. 8 The samples were then transferred to a cell factory for further expansion to approximately 1 × 10⁻⁶. 9 The scale is such that it is ready to be transferred to roller bottles for functional maturation. In some implementations, step 2) includes transferring approximately 1 × 10⁻⁶ units of water to a roller bottle. 9 Cells are digested from a cell factory, and the single-cell suspension is transferred to roller bottles for 3D stirred culture. In some embodiments, the passage ratio in step 1) is approximately 1:2 to 1:3, and the passage frequency is approximately 2 to 3 days. In some embodiments, the culture medium volume in step 2) is approximately 1 to 1.5 L, the stirring speed is approximately 60 rpm, and the cells are cultured with the medium changed daily until the cells reach functional maturity, such as synthesizing the liver cell functional proteins ALB and / or CYP3A4. In some embodiments, the liver progenitor cells may be derived from pluripotent stem cells, such as induced pluripotent stem cells, for example, liver progenitor cells induced by the culture media and methods described herein.

[0124] IV. Methods and Applications

[0125] For therapeutic treatments, tissue engineering, and research, readily available sources of stem cells that can produce the desired cell type or morphology are important.

[0126] Once established, an artificial liver system (device) can be prepared using hepatocyte cultures. In some embodiments, it can be used to treat patients with, for example, acute liver failure, replacing all or part of the liver's function, thereby prolonging the patient's life until a suitable liver transplant donor is available. The artificial liver system can support the patient's survival, reduce the metabolic burden on the liver, and thus promote liver self-repair to alleviate the condition. In some embodiments, the artificial liver system comprises not less than 10 10These cells can effectively replace the functions of the liver. In some implementations, the artificial liver system is a cell-based perfusion device that directly or indirectly contacts the patient's serum with liver seed cells in the bioartificial liver device. The liver seed cells then metabolize and detoxify the metabolic substances in the serum, while simultaneously replenishing important metabolic products synthesized by the liver, thereby improving the survival rate of patients with liver failure. In some implementations, the bioartificial liver device can combine the detoxification function with the synthesis and regulatory functions of liver cells.

[0127] Hepatocytes can be formulated for administration, delivery, or contact with subjects, tissues, or cells to promote in vivo or in vitro / in vitro dedifferentiation. Additional factors can be combined, such as growth factors, other factors inducing differentiation or dedifferentiation, secretory products, immunomodulators, anti-inflammatory drugs, regression factors, bioactive compounds that promote innervation, vascularization, or enhance lymphatic networks, and pharmaceuticals.

[0128] In some implementations, cells are provided with or attached to a support structure. The support structure can be a mesh, solid support, scaffold, tube, porous structure, and / or hydrogel. The support structure can be wholly or partially biodegradable or bionon-biodegradable. The support can be formed from natural or synthetic polymers, metals such as titanium, bone or hydroxyapatite, or ceramics. Natural polymers include collagen, hyaluronic acid, polysaccharides, and glycosaminoglycans. Synthetic polymers include polyhydroxy acids such as polylactic acid, polyglycolic acid, and copolymers thereof, polyhydroxyalkanoates such as polyhydroxybutyrate, polyorthoesters, polyanhydrides, polyurethanes, polycarbonates, and polyesters. These can be in the form of implants, tubes, meshes, or hydrogels. The support structure can be a loosely woven or non-woven mesh in which cells are seeded in and onto the mesh. The structure can include a solid structural support. The support can be a tube, such as a regenerating long neural tube for nerve axons. The support can be a scaffold or valve. The support can be a joint prosthesis such as a knee or hip, or a portion thereof, having a porous interface that allows cells to grow inward and / or be seeded into the porous structure. Many other types of support structures are also feasible. For example, the support structure can be formed from sponges, foams, corals, or biocompatible inorganic structures with internal pores, or it can be a mesh of interwoven polymer fibers. These support structures can be prepared using known methods.

[0129] The supporting structure can be a permeable structure with a shaped, porous cavity or void that supports the hydrogel-cell mixture. For example, the supporting structure can be a porous polymer mesh, a natural or synthetic sponge, or a supporting structure formed of metal or materials such as bone or hydroxyapatite. The porosity of the supporting structure should allow nutrients to diffuse into the structure, thereby efficiently reaching the cell interior, and allow waste products generated by the cell to diffuse out of the structure.

[0130] Support structures can be shaped to fit spaces where new tissue is needed. For example, a support structure can be shaped to conform to an area of ​​burned skin or a portion of lost cartilage or bone. Depending on the material it is made of, the support structure can be shaped by cutting, molding, casting, or any other method that produces the desired shape. As described below, the support can be shaped before or after cell seeding or filling with a hydrogel-cell mixture.

[0131] An example of a suitable polymer is polyglactin, a 90:10 copolymer of glycolide and lactide, and used as a VICRYL protein. TM Braided absorbable sutures are manufactured (Ethicon Co., Somerville, NJ). Polymer fibers (such as VICRYL) can be used. TM Polymer fibers are woven or compressed into felt-like sheets, which can then be cut into any desired shape. Alternatively, polymer fibers can be compressed together in a mold to cast them into the desired shape of the support structure. In some cases, additional polymer can be added to the polymer fibers during molding to modify or impart additional structure to the fibrous network. For example, a polylactic acid (PLA) solution can be added to such a polyglycolic acid (PGA) fiber network sheet, and the assembly can be molded together to form a porous support structure. PLA binds to the crosslinks of the PGA fibers, thereby coating these individual fibers and fixing the shape of the molded fibers. PLA also fills the spaces between the fibers. Therefore, the porosity can vary depending on the amount of PLA introduced into the support. The pressure required to mold the fibrous network into the desired shape can be very moderate. All that is required is to hold the fibers in a position long enough to allow for the binding and coating of PLA.

[0132] Alternatively, or additionally, the support structure may include other types of polymer fibers or polymer structures prepared using techniques known in the art. For example, a fine polymer film can be obtained by evaporating a solvent from a polymer solution. These films can be cast into the desired shape by evaporating the polymer solution from a mold having a relief pattern of the desired shape. Polymer gels can also be molded into fine, permeable polymer structures using compression molding techniques known in the art. In another embodiment, cells are mixed with a hydrogel to form a cell-hydrogel mixture. The hydrogel can be applied by injection or catheter, or during the implantation of other support structures. Crosslinking can be performed before, during, or after application.

[0133] V. Reagent Kit

[0134] Kits are provided that contain the inducers disclosed herein. The inducers are as described above. These may be in the form of defined concentrations to facilitate addition to cell culture media to produce desired concentrations. The kit may include instructions for desired concentration ranges and application times. The kit may also contain a cell culture medium premixed with the inducer for culturing cells.

[0135] The invention will be further understood by referring to the following non-limiting examples.

[0136] Example

[0137] Experimental methods

[0138] 1. Induced differentiation of pluripotent stem cells (hiPSCs) into hepatocytes

[0139] 1) Inducing differentiation of human pluripotent stem cells into endoderm cells (DE)

[0140] (a) Day 1: RPMI 1640 medium supplemented with serum-free B27 (1:50), 100 ng / ml Activin A, 0.5 ng / ml BMP4, and 10 ng / ml bFGF, 20 ng / ml Wnt3a

[0141] (b) Days 2-3: RPMI 1640 medium supplemented with serum-free B27 (1:50), 100 ng / ml Activin A, 0.5 ng / ml BMP4 and 10 ng / ml bFGF.

[0142] 2) Inducing differentiation of endoderm cells into hepatoblasts (HBs).

[0143] (a) On days 4-5, serum-free supplement B27 (1:50), 20 ng / ml KGF, and 5 uM B431542 were added to RPMI 1640 medium and cultured for two days.

[0144] (b) On days 6–8, serum-free supplement B27 (1:50), 20 ng / ml KGF, 20 ng / ml BMP4, 10 ng / ml BMP2 and 10 ng / ml bFGF were added to RPMI 1640 medium.

[0145] 3) Inducing hepatoblasts to differentiate and proliferate into liver progenitor cells (participating in...) Figure 30 )

[0146] A 1:1 mixture of DMEM / F12 and William'E medium was added, along with serum-free supplement B27 (1:50), 10 μM Forskolin, 5 μM SB431542, 20 ng / ml EGF, 3 μM CHIR99021, 5 μM LPA, 0.5 μM S1P, and 1 μM DEX.

[0147] 4) Induce liver progenitor cells to differentiate into mature hepatocytes (MH) for 2-3 weeks.

[0148] Liver cell maturation medium: William'E medium supplemented with serum-free B27 (1:50), 50 uM Forskolin, 10 uM SB431542, 2 uM DEX, and GlutaMax (1:100).

[0149] 2. Large-scale culture and suspension culture of hepatocytes (see...) Figure 31 )

[0150] (1) The initial cell quantity for large-scale cell expansion was approximately 1 × 10⁻⁶ 7 Cells were cultured in T75 culture flasks. The cells were then stably passaged at a ratio of 1:2 to 1:3.

[0151] (2) After the cells were expanded to 12 T75 culture flasks, they were passaged into T225 culture flasks for further culture.

[0152] (3) After the cells were expanded to 12 T225 culture flasks, they were passaged into a 5-layer cell factory for further culture.

[0153] (4) Digest and count the cells from the two 5-layer cell factories, approximately 1 × 10⁻⁶. 9 Cells were transferred to magnetically stirred cell culture flasks for culture, with a corresponding culture medium volume of approximately 1-1.5 L and a stirring speed of 60 rpm.

[0154] (5) Change the culture medium daily. After culturing in the expansion medium for 3-5 days until cell clones are formed, switch to the mature medium and continue culturing for 2-3 weeks until the cells are functionally mature.

[0155] 3. Induction of Liver Failure in Miniature Pigs and Bioartificial Liver Therapy

[0156] One day prior to bioartificial liver treatment, pigs in all groups were intravenously injected with D-gal (0.4 g / kg)

[159] , and origin blood samples were collected. Piglets were continuously anesthetized by intravenous administration of propofol (10 mg / kg / h), and a catheter was inserted into the internal jugular vein of the pig, with the other end of the catheter connected to the bioartificial liver device. Whole blood samples were collected from piglets daily after treatment until 7 days after treatment for testing.

[0157] Experimental results

[0158] Differentiation of induced pluripotent stem cells into liver lineage

[0159] 1. Induced differentiation of pluripotent stem cells into hepatic progenitor cells

[0160] To simulate the developmental pathways and related signaling pathways of an embryo in vivo, and based on previous laboratory work, this study used human induced pluripotent stem cells (hiPSCs) as the starting cells to first obtain hepatoblasts. The differentiation of hiPSCs into hepatoblasts mainly consisted of three parts: First, induction of hiPSCs to the endoderm fate. High concentrations of Activin A effectively induced hiPSCs to transition to the endoderm fate. Experimental results showed that 100 ng / ml Activin A + 0.5 ng / ml BMP4 + 10 ng / ml bFGF + 20 ng / ml Wnt3a could induce hiPSCs to differentiate to the Primitive Streak stage on the first day and inhibit their transition to the ectoderm fate. Further induction of endoderm cell differentiation into the foregut was achieved by culturing cells in 100 ng / ml Activin A + 0.5 ng / ml BMP4 + 10 ng / ml bFGF for three days, effectively differentiating them into endoderm cells and inhibiting their transition to the mesoderm fate. Further induction of endoderm cell differentiation into the foregut was achieved by 20 ng / ml KGF + 5 μM SB431542. The induced foregut stage cells were then cultured for three days in conditions of 20 ng / ml KGF + 20 ng / ml BMP4 + 10 ng / ml BMP2 + 10 ng / ml bFGF to differentiate into hepatocytes. Figure 1 ).

[0161] Protein levels in induced hepatoblasts were detected by immunofluorescence staining. The staining results showed that differentiated hepatoblasts expressed AFP and SOX9, important marker proteins of liver progenitor cells, and also expressed Ki67, a cell proliferation-related protein, but did not express albumin ALB. Figure 2 .).

[0162] When hepatoblasts are cut into hepatic progenitor cell expansion conditions, they can continue to expand and differentiate into hepatic progenitor cells with self-renewal capacity. Immunofluorescence assays show that the induced hepatic progenitor clones exhibit an epithelial cell clonal morphology and express important marker genes for hepatic progenitor cells, AFP and ALB. Figure 3 ).

[0163] The cells exhibit an expanded epithelial cell morphology, with double-positive cells accounting for over 90%. Figure 4 ).

[0164] We first identified the properties of the obtained liver progenitor cells, including mRNA and protein level detection. qPCR results showed that hiPSC-derived liver progenitor cells expressed genes such as AFP and DLK1, and transcription factors such as HNF1B and FOXA2. Using hiPSC cells as a negative control and fetal liver cells as a positive control, the results indicated that the gene expression of hHPC cells was at the same level as that of fetal liver cells, but significantly different from that of hiPSC cells. Figure 5 (.). Among them, hiPSC is the iPS cell control, FHH is the fetal liver cell control, and hHPC corresponds to liver progenitor cells derived from three different hiPSC lines.

[0165] Based on this, immunofluorescence staining was used to detect the expression levels of cellular proteins. The immunofluorescence staining results were basically consistent with the qPCR results, showing that hiPSC-derived liver progenitor cells expressed important marker genes of liver progenitor cells, AFP, CK19, and HNF1B. Figure 6 It has liver precursor properties.

[0166] Preliminary analysis of cell characteristics showed that the cells expressed marker genes for liver precursors, exhibiting characteristics of liver precursor cells. To gain a more comprehensive understanding of gene expression in differentiated cells, we extracted RNA samples for analysis and performed RNA-Seq sequencing and data analysis. Among the RNA-Seq samples, hiPSC 1 and 2 were two different hiPSC cell lines, hHPC1-3 were three different liver precursor cell lines derived from hiPSC differentiation, and FHH 1 ​​and 2 were two different fetal liver cell lines. Sequencing results showed that the gene expression of hHPC cells was more similar to that of FHH cells, expressing specific genes related to the liver precursor stage; however, it differed significantly from hiPSC cells, not expressing stem cell genes. Figure 7 The above results indicate that hiPSC cells gradually acquire the fate of hepatocytes during differentiation and become hepatic progenitor cells similar to fetal hepatocytes.

[0167] Liver progenitor cells derived from hiPSC cells have the ability to be passaged and expanded in vitro over a long period. At a rate of 1×10⁻⁶... 6 Using these cells as the starting cells, the total number of cells and passage time were recorded at each passage, and cell expansion curves were plotted. Statistical results showed that the expansion rate of liver precursor cells remained relatively stable during passages P5-P9 and P20-P24. Figure 8 ).

[0168] Subsequent analysis focused on the mRNA expression of liver progenitor cell-related genes across different cell passages. HiPSC cells were used as a negative control, and FHH cells as a positive control. qPCR results showed no significant differences in key marker genes of liver progenitor cells across cell passages (P5, P15, and P25). Figure 9 This indicates that the liver precursor properties of early and late-generation cells were maintained during cell passage.

[0169] In addition, we performed cryopreservation and thawing on the cells. The results showed that there was no significant difference in cell morphology after thawing compared to before cryopreservation, and no significant changes were observed in the mRNA levels of liver progenitor cell-related marker genes. Figure 10 The above results indicate that liver progenitor cells derived from hiPSC cells can be cryopreserved and thawed, facilitating the subsequent large-scale preparation of seed cells and the establishment of a seed cell bank.

[0170] 2. Differentiation of liver progenitor cells into mature liver cells

[0171] Using hiPSC cells as the starting cells and differentiating them to obtain liver precursor cells, these cells can be further induced into functionally mature liver cells in vitro. The induced liver cells exhibit a strong morphological similarity to adult liver cells, with clearly defined nuclei, distinct cell boundaries, and a polygonal shape. Figure 11 ).

[0172] Cellular mRNA level detection results showed that the cells expressed key transcription factors of liver cells, and at the same level as primary liver cells. Figure 12 This indicates that the cells possess some of the properties of adult liver cells.

[0173] mRNA level detection during cell maturation showed that AFP, an important marker gene for liver progenitor cells, was downregulated to levels similar to those in adult liver cells after approximately 21 days of culture, indicating that the cells were destined to become mature liver cells. Figure 13 ELISA results showed that albumin secretion was not significantly affected during cell maturation, indicating that the liver properties of the cells remained stable to a certain extent. Figure 13 ).

[0174] Based on the established maturation conditions of hepatic progenitor cells, we conducted a comprehensive cellular functional identification and analysis of differentiated hepatocytes. PAS staining results showed that the cells can synthesize glycogen, and immunofluorescence assays showed that they can synthesize and secrete albumin and key drug metabolism-related CYP450 metabolic enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Figure 14 ).

[0175] Cellular mRNA level detection is mainly divided into three parts: drug metabolism-related genes ( Figure 15 ) and biosynthesis-related genes, including urea synthesis-related genes and coagulation factors ( Figure 15 hiPSC cells were selected as a negative control, and primary adult hepatocytes (PHH) were selected as a positive control. hMH represented three different mature hepatocyte cell lines derived from hiPSC differentiation. qPCR results showed that the gene expression of hepatocytes derived from hiPSC differentiation was at the same level as that of primary hepatocytes, showing significant differences from hiPSC cells, and possessing the functions of mature hepatocytes.

[0176] Based on mRNA level detection, we further detected and compared albumin secretion at the protein level using a Bethy reagent kit. The results showed that cellular albumin secretion capacity was at the same level as primary liver cells, significantly different from HepG2 liver cancer cells, while hiPSC cells secreted virtually no albumin. Figure 16 ELISA was then used to detect urea synthesis, and the results were consistent with those for albumin. These results further demonstrate that the cells possess the functions of mature liver cells.

[0177] RNASeq sequencing analyzed six modules: stem cell-related gene expression, liver transcription factor expression, glycogen synthesis-related gene expression, and drug metabolism-related gene expression. The results showed that the gene expression of mature liver cells derived from human pluripotent stem cell differentiation was more similar to that of primary liver cells, expressing genes related to liver cell functional modules; however, it differed significantly from hiPSCs, not expressing stem cell genes. Figure 17 This indicates that the differentiated cells possess the gene expression profile of mature liver cells.

[0178] 3. Large-scale culture of precursor cells and maturation of 3D systems

[0179] Bioartificial liver systems have the following requirements for seed cells: Firstly, there is the issue of cell quantity. Bioartificial liver systems require a large number of cells to meet therapeutic needs; the cell quantity required for liver failure treatment experiments in miniature pig models is approximately 1 × 10⁻⁶ cells / year. 9 -1×10 10 The number of cells required for the treatment of human liver failure is approximately 1 × 10⁻⁶. 10 -1×10 12 This requires us to culture a large number of cells in a limited volume, achieving high-density cell culture. The specific number of cells required for a bioartificial liver system depends mainly on the size of the liver in the treatment sample and the functional quality of the seed cells.

[0180] On the other hand, there is the issue of docking cells with bioreactor equipment. For ease of treatment, cells must first be easy to carry and transport, and relatively easy to transfer into the bioreactor; secondly, cell viability and functional levels must remain relatively stable during the high-speed perfusion culture process and treatment. Considering that adherent cells in 2D culture systems require digestion before being directly transferred into the reactor, which may affect cell viability, and that cell adhesion is difficult to maintain in perfusion systems, we prefer to dock 3D culture systems with bioartificial liver systems.

[0181] In summary, we have explored and established the following cell culture system ( Figure 18 )

[0182] The cell culture system mainly consists of two parts: the first step is the 2D passage expansion process of cells in a cell factory. This is done at a rate of 1×10⁻⁶ cells / year. 7 As the starting cell quantity, stable and continuous cell passages are performed in T75 or T225 culture flasks pre-coated with Matrigel, with a passage ratio of approximately 1:2 to 1:3 and a passage frequency of approximately 2 to 3 days. The cells can expand to 1 × 10⁶ cells in approximately 15 to 20 days. 8 The samples were then transferred to a cell factory for further expansion, reaching a scale of 1×10⁻⁶. 9The scale is significant, and it is ready to be transferred to a roller bottle for functional maturation.

[0183] The second step involves 3D stirred culture and maturation of the cells in roller bottles. 1×10 9 Cells were digested from the cell factory, and single-cell suspensions were transferred to roller bottles for 3D stirred culture. The culture volume was 1-1.5 L, with a stirring speed of 60 rpm / min. The culture medium was changed daily (a 1:1 mixture of DMEM / F12 and William's E medium, supplemented with serum-free B27 (1:50), 10 μM Forskolin, 5 μM SB431542, 20 ng / ml EGF, 3 μM CHIR99021, 5 μM M PA, 0.5 μM S1P, and 1 μM DEX). After 3 days, uniformly sized, well-defined clones were observed, and the cells were transferred to maturation medium. Continued culture in liver progenitor cell maturation medium for 21 days showed healthy cell clones and the ability to synthesize liver cell function-related proteins ALB and CYP3A4. Figure 19 The cells initially exhibited properties characteristic of mature liver cells. To identify the liver attributes of the suspension-cultured cells, we first assessed the expression of liver-related transcription factors at the mRNA level. The results showed that the cells expressed liver-related transcription factors at levels similar to those of primary liver cells, indicating properties characteristic of mature liver cells. Figure 20 ).

[0184] Due to the demand for bioartificial liver therapy, we further examined the expression levels of mRNA in genes related to cell and liver properties. The results showed that the expression levels of these genes were similar to those in primary liver cells, meeting the requirements for clinical application. Figure 21 ).

[0185] The detection of cell mRNA-level function-related genes is mainly divided into three parts: drug metabolism-related genes ( Figure 22 ) and biosynthesis-related genes, including urea synthesis-related genes and coagulation factors ( Figure 23 hiPSC cells were selected as a negative control, and primary adult hepatocytes (PHH) were selected as a positive control. 3D-hMH represented three different mature hepatocyte cell lines derived from hiPSC differentiation. qPCR results showed that the gene expression of hepatocytes derived from hiPSC differentiation was at the same level as that of primary hepatocytes, showing significant differences from hiPSC cells, and possessing the functions of mature hepatocytes.

[0186] Building upon mRNA level detection, we further used ELISA to detect and compare the albumin secretion and urea synthesis capabilities of mature hepatocytes derived from hiPSC cell differentiation in a 3D system. The results showed that the albumin secretion and urea synthesis capabilities of differentiated cells were at the same level as primary hepatocytes, but significantly different from the HepG2 hepatocellular carcinoma line. HiPSC cells, however, essentially did not secrete albumin or exhibit any urea metabolism-related functions. Figure 24 ).

[0187] 4. Bioartificial Liver Therapy in a Miniature Pig Model of Acute Liver Failure

[0188] Based on the establishment of large-scale cell culture volumes, we transferred hiPSC-derived cells into a bioartificial liver device for therapeutic experiments in a miniature pig model of acute liver failure. The acute liver failure model in miniature pigs was established by inducing acute liver failure with 0.4-0.45 g / kg D-galactosamine (D-gal), and the survival time of most miniature pigs was approximately 2 days.

[0189] Acute liver failure was induced in miniature pigs by injecting 0.4 g / kg D-gal on day 0. On day 1 post-induction, the treatment group miniature pigs received bioartificial liver therapy with approximately 1 × 10⁻⁶ cells. 9 Cells were collected and circulated in vitro for 4 hours; control group miniature pigs received no treatment. Blood samples were taken daily from piglets before modeling to measure various functional indicators of liver failure, continuing until day 7 after treatment or until piglet death. Piglets were considered viable if they survived more than 7 days. Figure 25 ).

[0190] We selected 12 miniature pigs to establish an acute liver injury model. Six pigs were randomly selected for treatment with a bioartificial liver system, while the remaining six served as controls and received no treatment. The results showed that the miniature pigs treated with the bioartificial liver system survived for more than 7 days, while the untreated miniature pigs all died within 4 days. Figure 26 ).

[0191] On the second day after D-gal injection, miniature pigs showed symptoms of liver failure, exhibiting lethargy. After treatment with a bioartificial liver system, the miniature pigs were able to stand the following morning and were in good spirits; the untreated control group showed no improvement in their condition and appeared to be on the verge of death. Figure 27 ).

[0192] Blood samples were continuously collected from six miniature pigs in each of the bioartificial liver treatment group and the acute liver failure control group for the detection of liver-related serum biochemical indicators. The following indicators were selected: aspartate aminotransferase (AST), alanine aminotransferase (ALT), ammonia, direct bilirubin (DBIL), clotting time (PT), and albumin. The results showed that both groups of miniature pigs exhibited a significant increase in liver failure-related indicators on the first day of induced failure. In the treatment group, these indicators gradually decreased after bioartificial liver treatment, reaching levels consistent with the baseline by day 7; in the control group, the indicators remained elevated or stabilized at a high level. Serum biochemical indicators in the treatment group were continuously monitored until day 7, while in the control group, monitoring continued until day 4, at which point all six miniature pigs died. Figure 28 The results indicate that the bioartificial liver system can effectively reduce liver failure indicators in miniature pigs with liver dysfunction, alleviate the degree of liver damage, and effectively maintain the survival of the pigs.

[0193] We subsequently collected liver tissue from miniature pigs, sectioned it, and stained it with hematoxylin and eosin (HE) to observe the damage to the pig livers. Samples were taken from pigs in the bioartificial liver treatment group on day 7, while samples were taken from pigs in the acute liver failure control group after death (days 2-4). HE staining results showed that the livers of pigs in the treatment group were in good condition, with no obvious vacuolar structures and good liver regeneration; the livers of pigs in the control group were in poor condition, with numerous vacuolar structures caused by hepatocyte necrosis, indicating severe liver tissue damage. Figure 29 This result indicates that bioartificial liver therapy can effectively alleviate liver failure symptoms and promote liver regeneration.

Claims

1. A method for inducing hepatoblasts from human pluripotent stem cells, the method comprising inducing the human pluripotent stem cells to generate hepatoblasts by: 1) Inducing human pluripotent stem cells to differentiate into endoderm cells, (a) Day 1: RPMI 1640 medium was supplemented with serum-free additive B27 at a ratio of 1:50, along with 100 ng / ml Activin A, 0.5 ng / ml BMP4, 10 ng / ml bFGF, and 20 ng / ml Wnt3a. (b) Days 2-3: RPMI 1640 medium was supplemented with serum-free additive B27 at a ratio of 1:50, along with 100 ng / ml Activin A, 0.5 ng / ml BMP4, and 10 ng / ml bFGF. 2) Inducing the differentiation of endoderm cells into hepatocytes, (a) On days 4-5, serum-free supplement B27 was added to RPMI 1640 culture medium at a ratio of 1:50, along with 20 ng / ml KGF and 5 μM SB431542. The medium was cultured for two days. (b) On days 6-8, serum-free supplement B27 was added to RPMI 1640 medium at a ratio of 1:50, consisting of 20 ng / ml KGF, 20 ng / ml BMP4, 10 ng / ml BMP2 and 10 ng / ml bFGF.

2. A method for inducing human liver cells from human pluripotent stem cells, the method comprising: Hepatoblasts are generated by the method of claim 1, and the hepatoblasts are induced to produce liver cells by the following: 3) Inducing hepatoblasts to differentiate and proliferate into liver precursor cells, A 1:1 mixture of DMEM / F12 and William 'E' medium was added, along with serum-free supplement B27 at a ratio of 1:50, 10 μM Forskolin, 5 μM SB431542, 20 ng / ml EGF, 3 μM CHIR99021, 5 μM LPA, 0.5 μM S1P, and 1 μM MEX. 4) Induce liver progenitor cells to differentiate into mature liver parenchymal cells for 2-3 weeks. Liver cell maturation medium: William 'E medium supplemented with serum-free B27 at a ratio of 1:50, 50 μM Forskolin, 10 μM SB431542, 2 μM DEX, and GlutaMax at a ratio of 1:

100.

3. The method of claim 2, wherein the method comprises contacting the pluripotent stem cells with the inducing agent to generate liver progenitor cells and hepatocytes, wherein the method is a large-scale hepatocyte culture method, the method comprising 1) passage expansion of the liver progenitor cells in a 2D system, and 2) further culturing and maturing in a 3D system, wherein step 1) comprises culturing and maturing the liver progenitor cells in a 1×10⁻⁶ molten iron solution. 7 As the starting cell quantity, cells were stably and continuously passaged in culture flasks until the cell count was expanded to 1 × 10⁶ cells / year. 8 The samples were then transferred to a cell factory for further expansion, reaching a scale of 1×10⁻⁶. 9 At this scale, it is ready to be transferred to a rotary bottle for functional maturation.

4. The method of claim 3, wherein step 2) comprises placing 1×10 9 Cells are digested from the cell factory, and single-cell suspensions are transferred to roller bottles for 3D stirred culture.

5. The method according to claim 3, wherein the subculture ratio in step 1) is 1:2-1:3 and the subculture frequency is 2-3 days.

6. The method of claim 3, wherein in step 2), the amount of culture medium is 1-1.5L, the rotation speed is 60rpm, and the cells are cultured with the medium changed daily until the cells are functionally mature.

7. The method of claim 3, wherein the cells are cultured with a daily medium change until the liver cell functional proteins ALB and / or CYP3A4 are synthesized.

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