HK40026459A

HK40026459A - Use of a laminin for differentiating pluripotent cells into hepatocyte lineage cells

Info

Publication number: HK40026459A
Application number: HK42020016466.3A
Authority: HK
Inventors: T·H·阮; A·福尔埃
Original assignee: 国家健康科学研究所; Nantes Universite
Priority date: 2015-02-20
Filing date: 2020-09-18
Publication date: 2021-01-08

Description

Use of laminins for differentiating pluripotent cells into cells of the hepatocyte lineage

The present application is a divisional application of the chinese patent application having application number 201680010458.2, application date 2016, 2/19, entitled "use of laminin for differentiating pluripotent cells into cells of the hepatocyte lineage".

Technical Field

The present invention relates to the field of cell differentiation and cell therapy.

Background

Since their discovery, human induced pluripotent cells (hipscs) and human embryonic stem cells (hescs) have been intensively studied as a renewable source of any specialized cells of the body for replacing diseased cells [1,2 ]. They are expected to be useful in the treatment of a wide range of diseases such as severe heart disease [3], neurological diseases [4], liver disease [5] and retinal disease [6] as well as diabetes [7,8 ].

Liver disease affects millions of people worldwide. To date, liver transplantation is the only cure option when patients have severe metabolic liver disorders. Approximately 37,000 patients are on a waiting list for liver transplantation in europe and the united states. Over 5500 liver transplants are performed annually in europe, and inherited metabolic diseases account for 26% of the indications (european liver transplantation registration). However, patients on the liver waiting list who are less than 1/3 annually can receive liver transplantation, and the number of patients who die during the waiting list for liver transplantation (about 10%) has increased in recent years due to shortage of donor organs.

Transplantation of hepatocytes isolated from cadaveric livers into the liver of patients has emerged as a clinical alternative to liver transplantation over the past 15 years [9,10]In particular for such natural metabolic diseases: wherein liver function is maintained and a single enzyme deficiency exists. Definitive evidence of the metabolic function of transplanted human hepatocytes was demonstrated for the first time in patients with Crigler-Najjar type 1 (CN1) [11]. Following cell transplantation, the clinical status and quality of life of the patient were improved, and restoration of 5% UGT1a1 activity resulted in a 50% reduction in bilirubinaemia and phototherapy treatment. This potential study opened avenues for treating more than 40 patients affected by inherited metabolic liver disease who had primarily CN1 and urea cycle dysfunction, but also had glycogen storage disease type 1, refsum disease in infants, progressive familial intrahepatic bile stasis type 2, and hemophilia VII [9]]. Hepatocyte transplantation allows some children to avoid new neurological damage while awaiting liver transplantation. Relatively small amountThe input hepatocytes (1.5-2X 10)⁹Individual cells) are sufficient to ameliorate metabolic deficiencies in some affected patients. However, a persistent stabilizing effect of course requires repeated infusion of cells [12]. This approach has other important limitations for its general and routine use in clinical practice. Available liver transplants would prioritize liver transplantation, with only those with marginal quality being used for hepatocyte isolation. Thus, isolated hepatocytes are of variable quality and quantity. They differentiate rapidly and cannot be propagated in culture and are not well tolerated for cryopreservation. Collectively, these limitations underscore the need to explore other sources of functional human hepatocytes.

In recent years, several groups have reported the in vitro differentiation of hescs and hipscs into hepatocyte-like cells (HLCs), using different culture conditions that mimic the developmental stage of liver embryos [5,13-21 ]. HLC does not function as fully mature hepatocytes, but has a phenotype that more closely resembles embryonic human hepatocytes, expressing, for example, AFP [22 ]. They can be transplanted, to some extent propagated and maintained hepatic functionality after transplantation into the liver of immunocompromised animals that have been chemically damaged, irradiated or genetically manipulated to provide selective growth advantages to the transplanted hepatocytes, such as mice that overexpress urokinase-type plasminogen activator to induce constitutive recipient liver damage and regeneration stimulation [5,14,17,23-25,60 ].

Several studies explored the therapeutic potential of HLCs and successfully treated multiple murine models of chemically induced fatal liver failure [26-33 ]. Nevertheless, the liver has a surprising ability to regenerate itself from endogenous hepatocytes. Thus, transient support of liver metabolic function by HLCs is sufficient to salvage mice from acute liver failure. In contrast, for treatment of hereditary liver diseases, HLCs must be well differentiated in vivo and have long-term function to express missing mature hepatic metabolic functions in diseased animals. To date, successful metabolic correction has not been demonstrated for animals that model human genetic liver disease, where transplanted HLCs do not have selective survival and growth advantages in recipient liver.

Gunn rats (animals of CN1) have natural mutations in UGT1a1, resulting in complete loss of UGT1a1 activity and hyperbilirubinemia shortly after birth. The complete absence of bilirubin conjugates in bile would provide an easy, unambiguous and sensitive readout to assess recovery of UGT1a1 activity. Thus, gunn rats constitute a valuable and convenient model to follow hepatocyte maturation and the therapeutic potential of transplanted hepatocytes in vivo. Partial restoration of UGT1a1 activity following hepatocyte transplantation resulted in significant metabolic correction as in CN1 patients [37-39 ].

Before considering HLCs for clinical applications, a key issue was also to produce them using GMP (good manufacturing practice) compliant hepatic differentiation protocols. Current liver differentiation protocols typically contain feeder-conditioned media, serum, matrices of animal origin (such as matrigel (TM)), mouse feeder cells, viral vectors for improving liver differentiation, or small molecules not available in GMP [15,34-36 ]. They are both sources of unknown factors that make the resulting tissues unsuitable for future clinical use.

Therefore, there is a clinical need for human hepatocytes for allogeneic cell therapy. Pluripotent stem cells have been intensively explored as a renewable source of transplantable HLCs. However, it remains to be demonstrated that HLCs can treat inherited metabolic liver diseases without any selective growth advantage of the transplanted cells over the innate rodent hepatocytes. In addition, previously generated HLCs were produced using protocols that made them unsuitable for clinical use.

In recent years, laminins such as laminin-521 (LN-521) and laminin-111 (LN-111) have been described as relevant substrates for cell culture, more specifically for maintaining the functional properties of long-term in vitro cultures of cells of interest (such as pluripotent cells or HLCs), but there has never been disclosed nor suggested that laminins could be used to induce and/or improve hepatic differentiation. In addition, no studies suggest that the laminin matrix taken alone may support the initiation and subsequent differentiation of hepatocytes from pluripotent stem cell lines.

Thus, international patent application WO2012/080844 relates to a new use of laminin-521. In fact, laminin-521 can maintain stem cell pluripotency in vitro, achieve self-renewal, and enable single cell survival of human embryonic stem cells. When pluripotent human embryonic stem cells are cultured on plates coated with recombinant laminin-521 in the absence of differentiation inhibitors or feeder cells, the embryonic stem cells proliferate and maintain their pluripotency.

Furthermore, it has been demonstrated that human hepatocyte-like cells can be first produced from pluripotent cells on Matrigel-coated disks (induction of hepatic differentiation) and then cultured on laminin-111-coated disks for propagation of them for more than 3 months while maintaining the potential to differentiate into hepatocyte-like cells and cholangiocyte (cholangiocyte) -like cells [36 ].

Surprisingly, applicants have discovered a novel role for LN-111 and LN-521 in hepatocyte-like cell differentiation. This new role of LN-521 is different from the role of pluripotency maintenance described in WO 2012/080844.

Disclosure of Invention

In a first aspect, the present invention relates to the use of Laminin (LN) as a matrix for hepatic differentiation.

In a second aspect, the present invention relates to the use of LN for inducing and/or improving the differentiation of a population of pluripotent or multipotent cells or a Defined Endodermal (DE) cell population into a population of cells of the hepatocyte lineage.

In a third aspect, the present invention relates to a method of inducing human hepatic differentiation, said method comprising the steps of:

(i) providing a population of human DE cells, and

(ii) culturing the population in liver induction medium on a support coated with laminin to produce a population of human hepatocyte-like cells, and

(iii) optionally culturing the population of human hepatoblast-like cells in liver maturation medium on a support coated with laminin to produce a population of human embryonic hepatoblast-like cells.

In a fourth aspect, the present invention relates to a method of inducing hepatic differentiation, the method comprising the steps of:

(i) providing a population of human pluripotent cells,

(ii) culturing the population on a support coated with laminin in an endodermal induction medium to produce a population of human DE cells,

(iii) culturing the population of human DE cells in liver induction medium on a support coated with laminin to produce a population of human hepatocyte-like cells, and

(iv) optionally culturing the population of human hepatoblasts in liver maturation medium on a support coated with laminin to produce a population of human embryonic hepatocyte-like cells.

In a fifth aspect, the invention relates to a population of human hepatocyte-like cells obtained by the method of the invention.

In a sixth aspect, the invention relates to a population of human hepatocyte-like cells of the invention for use in a method of treatment of a human.

In a seventh aspect, the invention relates to a population of human embryonic hepatocyte-like cells obtained by the method of the invention.

In an eighth aspect, the invention relates to a kit for inducing human liver differentiation, the kit comprising a support coated with laminin, BMP4, and a member of the Fibroblast Growth Factor (FGF) family, such as FGF2 or FGF 10.

Drawings

FIG. 1: hepatic differentiation of human pluripotent stem cells in LN 111-coated disks.

(A) (B-D) liver differentiation was monitored over time by RT-qPCR analysis of mRNA expression for pluripotency markers (panel B, SOX2), endoderm markers (panel C, SOX17, FOXA2), and liver markers (panel D, HNF4a, AAT, AFP, ALB, CK19, CYP3a4, CYP3a7, UGT1a 1). (E) Secretion of the liver protein AFP in the cell supernatant was measured by ELISA. Data represent mean ± SEM. LN 111: results using hipscs differentiated on LN 111-coated disks; matrigel used in Matrigel^TMResults of differentiated hipscs on coated discs.

FIG. 2: transplantation of HLCS into the liver of Gunn rats serum bilirubin levels in Gunn rats.

Bilirubinemia was monitored over time in gunn rats, immunosuppressed with tacrolimus, who received IDHB 24 hours after 2/3 partial hepatectomy. Control rats received the same surgery but no transplantation.

FIG. 3: hepatic differentiation of human pluripotent stem cells in LN 521-coated disks.

Hepatic differentiation was monitored over time by RT-qPCR analysis of mRNA expression for endodermal (SOX17) and hepatic markers (HNF4a, AAT, AFP, ALB and CK 19).

FIG. 4 hepatic differentiation of iPSC cells in LN 111-coated disks.

Hepatic differentiation was monitored over time by RT-qPCR analysis for mRNA expression of endodermal markers (Nanog, OCT4, FOXA2, SOX 17).

FIG. 5 hepatic differentiation of iPSC cells in LN 111-coated disks.

Hepatic differentiation was monitored over time by RT-qPCR analysis for mRNA expression of hepatic markers (HNF4a, CYP7a1, AFP and CK 19).

FIG. 6 hepatic differentiation of iPSC cells in LN 111-coated disks.

Hepatic differentiation was monitored over time by RT-qPCR analysis for mRNA expression of hepatic markers (ALB, AAT).

FIG. 7 hepatic differentiation of iPSC cells in LN111 or LN521 coated disks.

Hepatic differentiation was monitored over time by RT-qPCR analysis for mRNA expression of hepatic markers (HNF4a, AAT, AFP and ALB).

FIG. 8 hepatic differentiation of hESCs in LN111 or LN521 coated disks with or without CHIR 99021.

Hepatic differentiation was monitored over time by RT-qPCR analysis for mRNA expression of hepatic markers (HNF4a, CK19, AFP and CYP3a 7).

FIG. 9 Effect of cell seeding on hepatic differentiation of hESCs in LN111 or LN521 coated disks with or without CHIR 99021.

Hepatic differentiation was monitored over time by RT-qPCR analysis for mRNA expression of hepatic markers (HNF4a, CK19, AFP and ALB).

FIG. 10 transplantation of fresh or frozen iPS hepatocytes into the liver of Gunn rats.

FIG. 11 detection of liver cells in spleen or liver of Gunn rat.

Immunohistochemical analysis of UGT1a1 in the liver and spleen of gunn rats immunosuppressed with tacrolimus, which received IDHB 24 hours after 2/3 partial hepatectomy.

Detailed Description

The present invention addresses these needs as it relates to the identification of chemically-defined media that can be used to differentiate a pluripotent cell population or a Defined Endodermal (DE) cell population into a cell population of the hepatocyte lineage. The inventors surprisingly demonstrated that laminin-111 alone is as effective as matrigel for initiating and supporting hepatic differentiation of hipscs in our culture conditions, but with recombinant matrices.

The inventors then demonstrated that laminin-521 (LN-521) can also serve as a matrix and allow the hipscs to better differentiate into definitive endoderm and HLCs.

The inventors have in fact produced hepatoblasts derived from human pluripotent stem cells (IDHB) in a xeno-free, feeder cell-free and chemically defined protocol using recombinant laminin-111 (LN-111) as the extracellular matrix for initiating and supporting the hepatic differentiation process. These IDHB with specific marker sets (combinations of expressed or unexpressed markers) were transplanted into the liver of gunn rats, an animal model of crigler-najal syndrome characterized by high levels of unconjugated bilirubin. After cell transplantation, they showed significant correction of hyperbilirubinemia, which remained stable in the 6 month study with no adverse events. They also showed that transplanted IDHB underwent further maturation in situ to repair defective metabolic liver function (bilirubin glucuronidation of UGT1a 1). Taken together, they demonstrate for the first time the efficacy of hipscs-based regeneration using GMP-compliant protocols for the treatment of inherited liver metabolic diseases without any selective growth advantage of the transplanted cells over the resident hepatocytes, as is the case in human hepatocyte transplantation.

Hepatic differentiation method and use of laminin

Thus, in a first aspect, the present invention relates to the use of Laminin (LN) as a matrix for hepatic differentiation.

The invention also relates to the use of LN for inducing and/or improving differentiation of a pluripotent cell population, a pluripotent cell population or a Definitive Endoderm (DE) cell population into a cell population of the hepatocyte lineage.

The term "laminin" (LN) as used herein denotes a protein of the heterotrimeric glycoprotein family that resides predominantly in the basal lamina. They act by binding interactions with adjacent cellular receptors on one side and by binding other laminin molecules or other matrix proteins such as collagen, nidogen or proteoglycans. Laminin molecules are also important signaling molecules that can strongly influence the behavior and function of cells. Laminins are important in maintaining cell/tissue phenotype, as well as in promoting cell growth and differentiation (in tissue repair and development). Laminins are large, multi-domain proteins with a common structural organization. Laminin molecules integrate multiple matrix and cell interaction functions into one molecule. Laminin molecules comprise an alpha-chain subunit, a beta-chain subunit, and a gamma-chain subunit, all joined together in a trimer by a coiled-coil domain. The 12 known laminin subunit chains can form at least 15 trimeric laminin types in native tissue. Within the trimeric laminin structure are identifiable domains that have binding activity to other laminin and basal lamina molecule and membrane bound receptors.

It should also be noted that the term "laminin" includes laminin intact, as individual chains or as fragments thereof. The term "intact" as used herein means that the protein is composed of all domains of the alpha-, beta-and gamma-chains, which are linked together to form a heterotrimeric structure. The proteins are not broken down into individual chains, fragments or functional domains. For example, laminin-111 and laminin-521 (described below) are intact proteins. The term "chain" as used herein denotes the entirety of the alpha, beta or gamma chain of a laminin protein. The term "fragment" as used herein denotes any protein fragment containing 1,2 or 3 functional domains, which has binding activity to another molecule or receptor. However, the chains should not be considered fragments, as each chain has more than three such domains. Similarly, intact laminin proteins should not be considered fragments. Examples of functional domains include domains I, II, III, IV, V, VI and G.

There are 5 different alpha, 3 beta and 3 gamma chains, which have been found in at least 18 different combinations in human tissue. These molecules are referred to as laminin-1 through laminin-15 based on their historical findings, but an alternative nomenclature describes isoforms based on their chain composition, such as laminin-111 (laminin-1) containing alpha-1, beta-1, and gamma-1 chains. 4 structurally defined families of laminins have been identified. The first set of 5 identified laminin molecules all have β 1 and γ 1 chains, and differ in their α -chain composition (α 1 to α 5 chains). A second set of 5 identified laminin molecules (including laminin-521) all have β 2 and γ 1 chains, and again, differ in their α -chain composition. The third set of identified laminin molecules has one identified member, laminin-332, with a chain composition of α 3 β 3 γ 2. The fourth set of identified laminin molecules has one identified member, laminin-213, with a newly identified γ 3 chain (α 2 β 1 γ 3).

At least 15 isoforms (or "isoforms") of Laminin (LN) have been identified based on their chain composition, including LN-111(α 1 β 1 γ 1), LN-121(α 1 β 2 γ 1), LN-211(α 2 β 1 γ 1), LN-213(α 2 β 1 γ 3), LN-221(α 2 β 2 γ 1), LN-311(α 3 β 3 γ 1), LN-321(α 3 β 2 γ 1), LN-332(α 3 β 3 γ 2), LN-411(α 4 β 1 γ 1), LN-421(α 4 β 2 γ 1), LN-423(α 4 β 2 γ 3), LN-511(α 5 β 1 γ 1), LN-521(α 5 β 2 γ 1), LN-522(α 5 β 2 γ 2), and LN-523(α 5 β 2 γ 3).

In one embodiment of the invention, the laminin is selected from the group consisting of laminin-111 (LN-111), laminin-211 (LN-211), laminin-332 (LN-332), laminin-411 (LN-411), laminin-421 (LN-421), laminin-511 (LN-511), and laminin-521 (LN-521).

In one embodiment of the invention, the laminin is human laminin.

In one embodiment of the invention, the laminin is human recombinant laminin.

In a preferred embodiment of the invention, the laminin is recombinant human laminin-111 (LN-111) and/or recombinant human laminin-521 (LN-521).

Laminin 521 (composed of α 5, β 2, γ 1 chains) is expressed during early embryonic development, is secreted by human pluripotent stem cells, and is known to stimulate their robust proliferation [63 ]. Laminin α 5 is present in the bile duct and hepatic vessels (hepatic artery, portal vein), and therefore laminin α 5 is not normally associated with normal hepatocytes, except in the sinusoids. In addition, laminin β 2 chain, and thus laminin-521, is not expressed in adult and normal animal livers [64 ].

Laminin alpha 1 chain, and thus laminin-111 (consisting of alpha 1, beta 1, gamma 1 chains), is not expressed in the adult animal liver [65 ].

Thus, laminin extracted from adult liver tissue does not contain laminin 521 or laminin-111.

The term "recombinant" polypeptide as used herein refers to a polypeptide produced by expression from an encoding nucleic acid molecule. Systems for cloning and expressing polypeptides in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably produced by expression from the encoding nucleic acid in a host cell. Any host cell may be used, depending on the individual needs of a particular system. Suitable host cells include bacteria, mammalian cells, plant cells, yeast and baculovirus systems.

Recombinant human laminins such as recombinant human LN-111 or LN-521 can be purchased from Biolamna, Sundbyberg, Sweden.

In one embodiment of the invention, the laminin is coated on a support such as a plate at 0.5-50 micrograms/milliliter (. mu.g/mL), preferably 1-10. mu.g/mL, more preferably at 5. mu.g/mL.

The term "matrix" as used herein means a component/substance (natural, synthetic or a combination thereof) that forms a polymeric network that provides a similar morphologically and physiologically relevant environment in vivo for cells cultured in vitro (e.g., on culture vessels such as flat plastic articles), enabling more realistic cell biology and better cell-cell interactions of cell cultures, thereby promoting cell attachment, growth, differentiation.

In one embodiment, the matrix comprises a laminin, such as recombinant human laminin-111 (LN-111) or recombinant human laminin-521 (LN-521), or a mixture thereof.

In another embodiment, the matrix comprises LN-521 and LN-111 in the following ratios: about 5%/95%; 10%/90%; 20%/80%; 25%/75%; 30%/70%; 40%/60%; 50%/50%, 60%/40%; 70%/30%; 75%/25%, 80%/20%; 90%/10%; 95%/5%.

In another embodiment, the matrix comprises a mixture of laminin and another component (such as matrix proteins, including collagen I and fibronectin).

The term "population" as used herein means a population of cells in which the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the target cells with respect to at least one target marker (e.g., a human hepatocyte-like cell population comprises at least about 60%, preferably at least about 70%, more preferably at least about 80% of cells having liver function and expressing a marker that is typically expressed by the human hepatocyte-like cells listed below, such as, for example, hepatocyte nuclear factor 4 α (HNF4 α)).

The term "marker" as used herein refers to a protein, glycoprotein or other molecule that is expressed on the surface of a cell or within a cell and that can be used to help identify the cell. The markers are typically detectable by conventional methods. Specific non-limiting examples of methods that can be used to detect cell surface markers are immunocytochemistry, Fluorescence Activated Cell Sorting (FACS) and enzymatic analysis, as well as RT-PCR and molecular biological methods for detecting mRNA for proteins.

The term "pluripotent" as used herein refers to cells that have the ability to produce progeny that can differentiate, under appropriate conditions, into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells may contribute to the tissue of a prenatal, postnatal or adult organism. A standard art-accepted test, such as the ability to form teratomas in 8-12 week old SCID mice, can be used to establish the pluripotency of the cell population. However, the identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells.

In one embodiment of the invention, the pluripotent stem cells are human pluripotent stem cells.

More specifically, human pluripotent stem cells may express at least some and optionally all of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-I-60, TRA-I-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-I, Oct4, Lin28, Rexl, and Nanog.

In one embodiment of the invention, the human pluripotent stem cells are human embryonic stem cells (hescs) or human induced pluripotent stem cells (hipscs).

The term "embryonic stem cell" as used herein means an embryonic cell that: it is capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state. Such cells may comprise cells obtained from: embryonic tissue formed after pregnancy (e.g., blastocyst), before embryo implantation (i.e., pre-implantation blastocyst), Expanded Blastocyst Cells (EBC) obtained from blastocysts at post-implantation/pre-gut formation stage (see WO2006/040763), Embryonic Germ (EG) cells obtained from the genital tissue of embryos at any time during pregnancy (preferably the first 10 weeks of pregnancy), and other methods using non-fertilized eggs, such as parthenogenesis methods or nuclear transfer.

Embryonic stem cells can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from pre-implantation embryos or In Vitro Fertilized (IVF) embryos in humans. Alternatively, single cell human embryos can be propagated to the blastocyst stage. To isolate human ES cells, the zona pellucida is removed from the blastocyst, and the Inner Cell Mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by aspiration. The ICM is then inoculated into a tissue culture flask containing an appropriate culture medium capable of effecting its stunning. After 9-15 days, the ICM-derived outgrowth is dissociated into pieces by mechanical dissociation or by enzymatic degradation, and the cells are then re-seeded into fresh tissue culture medium. Colonies exhibiting undifferentiated morphology were individually picked by micropipette, mechanically dissociated into clumps, and re-inoculated. The resulting ES cells were then routinely shunted every 4-7 days. For additional details on methods for making human ES cells, see Thomson et al, U.S. Pat. No. 5,843,780.

Commercially available stem cells may also be used. Human ES cells can be purchased from the NIH human embryonic stem cell registry (http:// escr. NIH. gov). Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, H9, WA09, Roslin cells (RC6, RC7, RC8, RC9 and RC10) and ESI-017, ESI-035, ESI-049, ESI-051, ESI-053 (BioTime).

The term "induced pluripotent stem cell" as used herein means a pluripotent stem cell derived artificially from a non-pluripotent cell. The non-pluripotent cells may be cells that have lower self-renewal and differentiation potential than pluripotent stem cells. The less potent cells may be, but are not limited to, adult stem cells, tissue-specific progenitor cells, primary or secondary cells. Ipscs have been reproducibly obtained by reprogramming different cell types as follows: forced expression of OCT4, SOX2, c-MYC, and KLF4 transcription factor mixture, or by an alternative combination of factors, replacing KLF4 and c-MYC, or adding NANOG and LIN28, or any method known to the skilled person to improve the reprogramming process (use of performing small molecules such as DNA methyltransferase (DNMT) inhibitors, miRNA, etc. …).

The term "reprogramming" as used herein refers to the process of changing the fate of a target cell to that of a different cell type caused by the expression of a small set of factors (or reprogramming factors) in the target cell.

Methods for the generation of induced pluripotent stem cells based on expression vectors encoding reprogramming factors have been described in the art; see, for example, WO2007/69666, EP2096169-A1 or WO 2010/042490.

Expression vectors for ectopic expression of reprogramming factors may be, for example, plasmid vectors, cosmid vectors, Bacterial Artificial Chromosome (BAC) vectors, transposon-based vectors (such as PiggyBac), or viral vectors.

Alternatively, the reprogramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc) or corresponding coding DNA or RNA are introduced into a target cell that does not incorporate exogenous genetic material into the host DNA, i.e., does not introduce nucleotide sequences into the genome of the cell. An expression vector (such as a plasmid vector) may be delivered into the cell in the form of naked DNA for ectopic expression of the reprogramming factors. Alternatively, chemically modified or unmodified RNA encoding the reprogramming factors may be introduced into the cells to reprogram them (see, e.g., Warren L, et al, 11/5/2010, Cell Stem Cell.; 7(5): 618-30). Other expression vectors have been described in e.g. WO 2009115295.

In one embodiment, the hipscs are derived from cells obtained from a healthy subject. In another embodiment, the hipscs are derived from cells obtained from a subject having a liver disease (such as a hereditary metabolic liver disease), and the hepatocyte-like cells exhibit a disease phenotype.

Alternatively, the target population of hepatocyte lineage cells can result from differentiation of pluripotent cells (such as mesenchymal stem cells) on a hepatic differentiation matrix comprised of laminins of the invention.

The term "pluripotent" as used herein denotes a cell capable of differentiating into at least two terminally differentiated cell types.

The term "mesenchymal stem cells" as used herein generally denotes mesenchymal cells found in differentiated (specialized) tissues and which are capable of producing identical copies of themselves (self-renewal) over the life of the organism and have a pluripotent differentiation potential (such as differentiation into osteoblasts, adipocytes, chondroblasts). Preferably, human mesenchymal stem cells that may be used in the context of the present invention thus include any suitable human pluripotent stem cells (i.e. cells that have the ability to self-renew and pluripotency) derived from any suitable tissue using any suitable isolation method. For example, human mesenchymal stem cells that may be used in the methods of the invention include, but are not limited to: adult Multispectral Inducible (MIAMI) cells (D 'Ipporito et al, J.Cell Sci.,2004,117:2971-2981), MAPCs (also known as MPCs) (Reyes et al, Blood,2001,98:2615-2625), umbilical cord Blood-derived stem cells (D' Ipportio et al, J.Cell Sci.,2004,117:2971-2981), MAPCs (also known as MPCs)G et al, J.Exp.Med.,2004,200(2): 123-; dellavall A et al, Nat. cell biol.,2007,9: 255-. In addition, cord blood banks (e.g., ethabelissement)du Sang, france) provides a safe and readily available source of such cells for transplantation.

Alternatively, the population of cells of the hepatocyte lineage can be derived from the differentiation of cells isolated from adult human liver (e.g., hepatocyte progenitors) on a hepatic differentiation matrix composed of laminin. Alternatively, the target population of hepatocyte lineage cells may be derived from somatic cells (such as fibroblasts), transformed on a hepatic differentiation matrix composed of laminin.

The term "hepatocyte lineage cell" or "hepatocyte-like cell" (HLC) as used herein denotes a cell obtained by differentiating a pluripotent cell, endodermal cell or other kind of cell, such as a pluripotent cell, in the manner described. The differentiated cells have at least one of a variety of distinguishing phenotypic characteristics of known hepatocyte progenitors, hepatoblasts, embryonic, neonatal, mature, adult and fully mature hepatocytes (as provided later in this disclosure). By using this term, no specific limitation in cell phenotype, cell marker, cell function or proliferative capacity is implied unless explicitly required. The hepatocyte lineage cells express hepatic markers including, but not limited to, hepatocyte nuclear factor 4 α (HNF4 α), Albumin (ALB), alpha-fetoprotein (AFP), cytochrome P450, and cytokeratin 19(CK 19).

In a particular embodiment, the population of human hepatocyte-like cells does not overexpress the gene. Preferably, these cell populations do not overexpress factors involved in hepatic differentiation. Examples of factors involved in hepatic differentiation include, but are not limited to, homeobox transgenes HEX, HNF-4, HGF, FGF4, OSM, activin, TGF- β, FOXA2, FGF2, HNF1 α, HNF1- β, HNF6, HNF3 β, SOX17, FOXa3, Foxa1, GATA4, ATF5, PROX1, CEBP α, or any other gene involved in hepatic development (i.e., endodermal induction, hepatic direction specification (specification) hepatic maturation, hepatic maintenance, hepatocyte proliferation factor).

In one embodiment, the population of human hepatocyte-like cells does not overexpress a HEX transgene.

The term "overexpression" as used herein means the expression of a gene product (RNA or protein) in an amount that is more than normal. Techniques for over-expressing a gene or protein are well known in the art and include, but are not limited to, microinjection of mRNA, injection, protein transfection, or transfection of expression vectors.

Methods for determining the expression level of a biomarker of the invention

The expression levels of markers, including liver markers (e.g., HNF4 α, ALB, AFP, CK19 genes) can be determined by a variety of techniques. Typically, the determined expression level is a relative expression level. For example, the determination comprises contacting the biological sample with a selective agent (such as a probe or ligand) and thereby detecting the presence or measuring the amount of the target nucleic acid or polypeptide originally in the biological sample. Contacting may be performed in any suitable device, such as a plate, microtiter plate, test tube, well, glass, column, and the like. In particular embodiments, the contacting is performed on a substrate (such as a nucleic acid array or a specific ligand array) coated with a reagent. The substrate may be a solid or semi-solid substrate, such as any suitable support, including glass, plastic, nylon, paper, metal, polymer, and the like. The substrate may have different forms and sizes, such as slides, films, beads, columns, gels, etc. The contacting can be performed under any conditions suitable for forming a detectable complex (such as a nucleic acid hybrid or an antibody-antigen complex) between the reagent and a nucleic acid or polypeptide of the biological sample.

In a particular embodiment, by determining the amount of mRNA, the expression level of the biomarker gene can be determined.

Methods for determining the amount of mRNA are well known in the art. For example, the nucleic acids contained in the target cells (such as the HLCs of the invention) are first extracted according to standard methods (e.g., using a lytic enzyme or a chemical solution), or extracted with a nucleic acid binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.

In another embodiment, the expression level of a biomarker gene may be determined by determining the amount of protein encoded by the gene.

Such methods comprise contacting a biological sample with a binding partner capable of selectively interacting with a protein present in the sample. The binding partner is typically an antibody, which may be polyclonal or monoclonal, preferably monoclonal.

For example, the levels of biomarker proteins, such as HNF4 α, ALB, AFP, and CK19, can be measured using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich-type assays. Such assays include, but are not limited to: western blotting; performing agglutination test; enzyme-labeled and mediated immunoassays, such as ELISA; biotin/avidin type assays; performing radioimmunoassay; performing immunoelectrophoresis; and (4) performing immunoprecipitation.

The cell culture conditions may include one or more additional components to provide a supportive environment during directed differentiation to a target differentiated cell type (e.g., a hepatocyte lineage cell). The method for obtaining a differentiated target cell population additionally comprises the step of inducing differentiation. Inducing differentiation may be achieved by altering the composition of growth factors in the culture medium at one or more stages of differentiation as described below.

Thus, in a second aspect, the present invention relates to a method of inducing human liver differentiation, said method comprising the steps of:

(i) providing a defined population of endoderm (DE) cells in humans, and

(ii) culturing the population in liver induction medium on a support coated with laminin to produce a population of human hepatocyte-like cells.

The invention also relates to a method for obtaining human hepatocyte-like cells, comprising the steps of:

(i) providing a population of human DE cells, and

The term "definitive endoderm cells" as used herein means cells that express characteristic biochemical markers (including, but not limited to Sox17 and FoxA2) and do not express nanog.

The term "hepatoblast-like cell" (HB) or "hepatic progenitor" as used herein is interchangeable and denotes a cell that: it expresses characteristic biochemical markers including, but not limited to, hepatocyte nuclear factor 4 α (HNF4 α), cytokeratin 19(CK19) and cytochrome P4503 a7(CYP3a7), and does not express or substantially express alpha-fetoprotein (AFP) and does not express Albumin (ALB), α -1 antitrypsin (AAT), cytochrome P4503 a7(CYP3a7) and Uridine Diphosphate (UDP) -glucuronyl transferase 1a1(UGT1a 1).

The term "substantially free" as used herein means a population of hepatocyte-like cells that express low levels of a protein of interest (such as AFP). Thus, such cells may only express low amounts or amounts of AFP protein that are not detectable by ELISA, such as a few picograms (pg), which is below the limit of detection of AFP by ELISA (LOD). However, it should also be noted that low amounts of AFP mRNA can be detected by RT-PCR (but such amounts are not sufficient for detecting the final expression of the AFP protein).

The expression "liver induction medium" or "medium inducing hepatic differentiation" as used herein means a medium capable of inducing definitive differentiation of endoderm into hepatocyte-like cells (and thus capable of inducing expression of hepatic markers such as HNF4 α, CK19 and CYP3a 7).

The term "culture medium" as used herein means any medium capable of supporting the defined growth of endoderm cells and differentiation into hepatic progenitor cells. Preferred media formulations that support defined endoderm cell growth and differentiation into hepatic progenitors include Chemically Defined Media (CDM).

The term "chemically defined medium" (CDM) as used herein means a nutrient solution for culturing cells which contains only the specified components, preferably components of known chemical structure. Chemically defined media are serum-free and cell-feeder free media. "serum-free" as used herein means a medium that does not contain added serum. As used herein, "feeder cells-free" refers to a medium that does not contain added feeder cells.

The medium used in the present invention may be an aqueous based medium comprising a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which are necessary for cell survival. For example, the medium according to the invention may be a synthetic tissue culture medium, such as RPMI (Roswell Park Medical Institute medium) or CMRL-1066(Connaught Medical research laboratory) for human use, as described further below (example section), supplemented as necessary with additives such as B27. The B-27 supplement (Invitrogen) contains, among other components, SOD, catalase and other antioxidants (GSH) and unique fatty acids such as linoleic acid, linolenic acid, lipoic acid.

The step of culturing the DE cells with the liver induction medium should be performed for a necessary time required for hepatocyte-like cell production. The duration of this incubation step can be readily determined by one skilled in the art. For example, during culture, one skilled in the art can monitor the cultured cells for the absence of expression of at least one marker that is solely determined by endoderm (DE) cell expression (e.g., Sox17 and FoxA2) and/or for expression of markers specifically expressed by hepatocyte-like cells (e.g., HNF4 α, CK19, CYP3a 7). The culturing with the liver induction medium may be stopped when the expression of one or more markers specific to DE cells and/or the expression of one or more markers specific to hepatocyte-like cells is not detected. Monitoring of these markers can be performed as follows: RT-PCR analysis using, for example, RNA extracted from cultured cells with specific primer pairs, immunofluorescence analysis using specific antibodies to markers, and FACS, or any method of detecting mRNA corresponding to the protein.

In general, step ii) may be carried out for 3 to 10 days, preferably 6 days.

The culture medium of the invention can be partially or completely renewed at regular intervals, if necessary. In general, the medium of the invention can be replaced with fresh medium of the invention every two days for 6 days.

The hepatocyte-like cells produced by the above methods may be isolated and/or purified by using any suitable method, for example FACS.

The term "FGF family growth factor" as used in connection with a method for obtaining human hepatocyte-like cells denotes any naturally occurring substance (e.g. protein) capable of stimulating cell growth, proliferation and cell differentiation by binding to a Fibroblast Growth Factor Receptor (FGFR). By binding to one FGFR, the substance increases, for example, tyrosine phosphorylation of the receptor.

In one embodiment of the invention, the liver induction medium is a chemically defined medium comprising Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF).

In one embodiment of the invention, the liver induction medium is a chemically defined medium comprising bone morphogenic protein 4(BMP4) and FGF.

In another embodiment, the liver induction medium is a chemically defined medium comprising BMP2 and FGF.

In another embodiment of the invention, the liver induction medium is a chemically defined medium comprising DMSO (dimethyl sulfoxide), KOSR (knockout serum replacement), HGF (hepatocyte growth factor), sodium butyrate. In a particular embodiment, the liver induction medium is a chemically defined medium comprising DMSO and KOSR. In another particular embodiment, the liver induction medium is a chemically defined medium comprising HGF and sodium butyrate.

Usually, DMSO is added to the medium of the invention at a concentration of about 0.5 to about 5%, preferably about 1%. DMSO may be purchased from Sigma. Generally, KOSR is added to the medium of the present invention at a concentration of about 5% to about 30%, preferably 20%. KOSR may be purchased from Life Technologies or Thermofeisher.

Typically, HGF is added to the media of the present invention at a concentration of about 1ng/mL to about 25ng/mL, preferably about 10 ng/mL. HGF is available from R & D Systems. Typically, sodium butyrate is added to the medium of the present invention at a concentration of about 1 to about 10mM, preferably about 2.5 mM. Sodium butyrate was purchased from Sigma.

In a preferred embodiment of the invention, the liver induction medium is a chemically defined medium containing bone morphogenic protein 4(BMP4) and family growth factor 10(FGF 10). The naturally occurring human FGF10 protein has the amino acid sequence as shown in Uniprot accession No. O15520. Generally, FGF10 is added to the medium of the present invention at a concentration in the range of 1-50ng/ml, preferably about 10 ng/ml. FGF10 is commercially available from Peprotech or Miltenyi Biotec.

The naturally occurring human BMP4 protein has the amino acid sequence as shown in Uniprot accession number P12644. In general, BMP4 is added to the medium according to the invention in a concentration in the range from 1 to 50ng/ml, preferably about 10 ng/ml. BMP4 is available from R & D systems.

The naturally occurring human BMP2 protein has the amino acid sequence as shown in Uniprot accession number P12643. In general, BMP2 is added to the medium according to the invention in a concentration in the range from 1 to 200ng/ml, preferably about 10 ng/ml. BMP2 is available from R & D systems or Thermofeisher.

In another embodiment of the invention, the liver induction medium is a chemically-defined medium comprising bone morphogenic protein 4(BMP4) and FGF2 (also known as basic fibroblast growth factor).

The naturally occurring human FGF2 protein has the amino acid sequence as shown in Uniprot accession No. P09038. Generally, FGF2 is added to the medium of the present invention at a concentration in the range of 1-50ng/ml, preferably about 10 ng/ml. FGF2 is commercially available from Peprotech or Miltenyi Biotec.

In certain embodiments of the invention, culturing the DE cell population may additionally comprise passaging the DE cell population prior to or during the differentiation process. The process of passaging the cell population may be repeated one or more times and may include dissociating the cells supported by the attachment matrix, diluting the dissociated cells in culture. Thus, step ii) may further comprise the step of passaging at least once and/or cell counting.

In certain embodiments of the invention, to increase colony formation, cells may be treated with a ROCK inhibitor 4 hours prior to dissociation and 24 hours after inoculation.

The term "Rho-associated protein kinase (ROCK) inhibitor" as used herein refers to a compound (natural or synthetic) that inhibits ROCK1 and/or ROCK2 activity, such as kinase activity.

In a particular embodiment of the invention, the ROCK inhibitor is Y27632(Watanabe et al, Nature Biotechnology 25,681-686 (2007)).

In general, the concentration of Y27632 in the medium may be 1-100ng/ml, preferably about 10 ng/ml.

In one embodiment of the invention, the laminin is selected from the group consisting of LN-111, LN-211, LN-332, LN-411, LN-421, LN-511, and LN-521.

In one embodiment of the invention, the laminin is human laminin.

In one embodiment of the invention, the laminin is human recombinant laminin.

In one embodiment of the invention, laminin is coated onto a support such as a plate at 0.5-50 micrograms/milliliter (. mu.g/mL), preferably 1-10. mu.g/mL, more preferably 5. mu.g/mL.

The support is typically a surface in a culture vessel.

In one embodiment of the invention, the support is selected from the group consisting of a plate, a slide, a flask, and the like. In a preferred embodiment of the invention, the support has at least a portion of a surface coated with a matrix of the invention, such as human recombinant LN-111 or human recombinant LN-521.

The present invention also relates to a method of inducing human hepatic differentiation, said method comprising the steps of:

(i) providing a population of human pluripotent cells, and

(ii) culturing the population on a support coated with laminin in an endoderm induction medium to produce a population of human DE cells.

The present invention also relates to a method for obtaining human DE cells, comprising the steps of:

(i) providing a population of human pluripotent cells, and

(i) providing a population of human pluripotent cells,

(ii) culturing said population in an endodermal induction medium on a support coated with laminin to produce a population of human DE cells, and

(iii) culturing the population of human DE cells in liver induction medium on a support coated with laminin to produce a population of human hepatocyte-like cells.

The present invention also relates to a method for obtaining human hepatocyte-like cells, the method comprising the steps of:

(i) providing a population of human pluripotent cells,

The expression "endoderm induction medium" or "medium which induces endoderm differentiation" as used herein means a medium which is capable of inducing differentiation of pluripotent stem cells into defined endoderm cells (and thus is capable of inducing expression of endoderm markers such as Sox17 and FoxA 2).

In a preferred embodiment of the invention, the endoderm induction medium is a chemically defined medium comprising at least activin a and optionally WNT 3A.

In another embodiment of the invention, the endoderm induction medium is a chemically defined medium further comprising CHIR 99021.

Activin A is well known in the art and is a dimeric polypeptide that exerts a range of cellular effects through stimulation of the activin/Nodal pathway (Vallier et al, Cell Science 118:4495-4509 (2005)). The naturally occurring human activin a protein has an amino acid sequence as shown in GeneBank accession No. NP _ 002183. Activin is readily available from commercial sources (e.g., Stemgent inc. ma USA or Miltenyi Biotec).

In general, the concentration of activin A in the medium can be from about 10 to about 1000ng/ml, preferably about 100 ng/ml.

The naturally occurring human WNT3A protein has the amino acid sequence as shown in Uniprot accession number P56704. Generally, the concentration of WNT3A in the medium can be 10-100ng/ml WNT3A, preferably about 50 ng/ml. WNT3A was purchased from Miltenyi Biotec.

CHIR99021 is an inhibitor of GSK3 that activates the WNT/β -catenin pathway. CHIR99021 is available from Tocris Bioscience, Stemgent.

Typically, the concentration of CHIR99021 in the medium is about 1. mu.M to about 10. mu.M, preferably about 3. mu.M.

The step of culturing pluripotent stem cells with endoderm induction medium should be performed for the time necessary to determine the production of endoderm (DE). The duration of this incubation step can be readily determined by one skilled in the art. For example, during culture, one skilled in the art can monitor cultured cells for expression of markers (e.g., Sox17 and FoxA2) specifically expressed by certain endoderm (DE). When expression of one or several markers specific to DE cells is detected, the culture using liver induction medium can be stopped. Monitoring of these markers can be performed as follows: using, for example, RT-PCR analysis of RNA extracted from cultured cells with specific primer pairs, immunofluorescence analysis using antibodies specific for the markers, ELISA and FACS, or any method of detecting RNA/protein/activity corresponding to a particular marker.

Typically, said step ii) may be carried out for 1 to 10 days, preferably 5 days.

The culture medium of the invention can be partially or completely refreshed at regular intervals, if necessary. In general, the medium of the invention can be replaced with fresh medium of the invention every two days for 5 days.

In one embodiment of the invention, the laminin is human laminin.

In one embodiment of the invention, the laminin is human recombinant laminin.

The support is typically a surface in a culture vessel.

In one embodiment of the invention, the support is selected from the group consisting of a plate, a slide, a flask, and the like. In a preferred embodiment of the invention, the support has at least a portion of a surface coated with a matrix of the invention (such as human recombinant LN-111 and/or human recombinant LN-521).

In other aspects, the invention relates to a method for obtaining human embryonic hepatocytes, the method comprising the steps of:

(i) providing a population of human pluripotent cells,

(iv) culturing the population of human hepatocyte-like cells in liver maturation medium to produce a population of embryonic hepatocyte-like cells.

The term "embryonic hepatocyte-like cell" or "differentiated hepatoblasts" as used herein are used interchangeably herein and refer to cells that are: it expresses characteristic biochemical markers including, but not limited to HNF4 α, CYP3a7, AFP and CK19, and can express some mature hepatic proteins including, but not limited to ALB, AAT, UGT1a1 and cytochrome P4503 a4(CYP3a 4).

The expression "liver maturation medium" or "medium that stimulates liver maturation" as used herein denotes a medium capable of inducing maturation of hepatoblast-like cells into embryonic hepatocyte-like cells.

In a preferred embodiment of the invention, the liver induction medium is a chemically defined medium comprising HGF and OSM.

The term "hepatic growth factor" (HGF) as used herein denotes a growth factor that modulates cell growth, cell motility and morphogenesis by activating the tyrosine kinase signaling cascade upon binding to the protooncogenic c-MET receptor. The naturally occurring human HGF protein has the amino acid sequence as shown in Uniprot accession No. P14210.

Typically, HGF is added to liver maturation media at a concentration in the range of 1-100ng/ml, preferably 5-50ng/ml, and even more preferably about 20 ng/ml. HGF is available from Peprotech.

The term "oncostatin M" (OSM) as used herein denotes a cytokine that inhibits the proliferation of many tumor cell lines. The naturally occurring human OSM protein has the amino acid sequence as shown in Uniprot accession number P13725.

Typically, oncostatin M is added to the liver maturation medium at a concentration in the range of 1-100ng/ml, preferably 5-50ng/ml, and even more preferably about 20 ng/ml. OSM is available from Miltenyi Biotec.

The step of culturing the hepatocyte-like cells with the liver maturation medium should be carried out for a necessary time required for the production of embryonic hepatocyte-like cells. The duration of this incubation step can be readily determined by one skilled in the art. For example, one skilled in the art can monitor cells in culture for the absence of expression of specific markers for pluripotent stem cells (e.g., Sox2 and Nanog), for the determination of the absence of expression of specific markers for endoderm (DE) (e.g., Sox17 and FoxA2), for hepatoblasts, and/or for the expression of markers specifically expressed by embryonic hepatocyte-like cells (e.g., AFP and ALB) during culture. The culturing with the liver maturation medium may be stopped when expression of one or several markers characteristic of the embryonic hepatocyte-like cells is detected. Monitoring of these markers can be performed as follows: using, for example, RT-PCR analysis of RNA extracted from cultured cells with specific primer pairs, immunofluorescence analysis using antibodies specific for the markers, ELISA and FACS, or any method of detecting RNA/protein/activity corresponding to a particular marker.

Typically, said step ii) may be carried out for 3 to 60 days, preferably 30 days.

The culture medium of the invention can be partially or completely refreshed at regular intervals, if necessary. In general, the medium of the invention can be replaced with fresh medium of the invention every two days for 30 days.

The embryonic hepatocyte-like cells produced by the above methods may be isolated and/or purified by using any suitable method, for example FACS.

In one embodiment of the invention, the laminin is human laminin.

In one embodiment of the invention, the laminin is human recombinant laminin.

The support is typically a surface in a culture vessel.

Human hepatocyte-like cell populations according to the invention and pharmaceutical compositions comprising them

In another aspect, the invention relates to a population of human hepatocyte-like cells obtained by a method as defined above.

The present invention also relates to a population of human hepatocyte-like cells obtained by a method as defined above, wherein the cells express hepatocyte nuclear factor 4 α (HNF4 α) and do not express or substantially express alpha-fetoprotein (AFP).

The present invention relates to a population of human hepatocyte-like cells obtained by a method as defined above, wherein the cells express HNF4 α, cytokeratin 19(CK19) and cytochrome P4503 a7(CYP3a7) and do not express or substantially express AFP, Albumin (ALB), α -1 antitrypsin (AAT), cytochrome P4503 a4(CYP3a4), Uridine Diphosphate (UDP) -glucuronyl transferase 1a1(UGT1a 1).

In another aspect, the invention relates to a population of human embryonic hepatocytes obtained by a method as defined above.

The invention also relates to a population of human embryonic hepatocyte-like cells obtained by a method as defined above, wherein the cells express HNF4 α, CK19, CYP3a7, AFP, ALB, AAT and UGT1a1, and cytochrome P4503 a4(CYP3a 4).

In one embodiment, the human hepatocyte-like cells exhibit a normal phenotype.

In another embodiment, the human hepatocyte-like cells exhibit genetic mutations, particularly genetic mutations affecting key proteins within human hepatocyte-like cells and associated with liver disease. It should be noted that mutations or defects in genes that cause disease phenotypes can be corrected in vitro or ex vivo. Various techniques are available for correcting gene mutations or defects in isolated mammalian cells.

Genetic modification of human hepatocyte-like cells of the invention

The term "genetically modified" indicates that a human hepatocyte-like cell comprises a nucleic acid molecule that does not naturally occur in an unmodified human hepatic progenitor cell, or that is present in the human hepatic progenitor cell in a non-natural state (e.g., amplified). The nucleic acid molecule may have been introduced into the cell or an ancestor thereof (such as an iPS containing a gene mutation associated with liver disease).

Many protocols can be used to genetically modify human hepatocyte-like cells, such as virus-mediated gene delivery, non-virus-mediated gene delivery, naked DNA, physical manipulation, and the like. For this purpose, the nucleic acid is often introduced into a vector (such as a recombinant virus, plasmid, phage, episome, artificial chromosome, etc.).

In a particular embodiment of the invention, the hepatocyte-like cells (HLCs) or pluripotent stem cells are genetically modified using viral vectors (or recombinant viruses) or non-viral methods. In this embodiment, the heterologous nucleic acid is, for example, introduced into a recombinant virus, which is then used to infect human hepatocyte-like cells (HLCs). Different types of recombinant viruses, especially lentiviruses, can be used.

In a preferred embodiment, the lentivirus encodes an immortalizing protein such as SV40T, hTERT, CDK4, and the like.

In a further preferred embodiment, the lentivirus encodes a wild-type protein (such proteins often exhibit disease-associated genetic mutations in patients suffering from liver disease), such as a wild-type α 1-antitrypsin (AAT) protein or a UDP glucuronosyltransferase family 1 polypeptide a1(UGT1a 1). Other gene mutations involved in liver disease have been described previously.

In one embodiment, the nucleic acid sequence encoding a protein of interest (e.g., an immortalizing protein or a wild-type protein for correcting a disease phenotype) is operably linked to a promoter.

Alternatively, the lentivirus encodes a reporter protein or a marker protein. The marker protein, which may be a fluorescent protein or a cell surface expressed protein, allows rapid identification and isolation of human hepatocyte-like cells (HLCs) of interest.

The marker protein may for example be selected from the group consisting of:

fluorescent proteins, in particular Green Fluorescent Protein (GFP) or derivatives thereof, such as enhanced green fluorescent protein, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), blue-green fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein (YFP, EYFP, Citrine, Venus, YPet); and

any cell surface expressed protein, which is not naturally expressed by HLCs.

The term "operably linked" as used herein means that the components are in a relationship that allows them to function in their intended manner. Thus, a nucleic acid sequence is "operably linked" when placed in functional association with another sequence nucleic acid sequence. For example, a promoter is "operably linked" to a coding sequence if the promoter causes transcription of the coding sequence. Generally, operably linked refers to the nucleic acid sequences being linked in proximity.

The term "promoter" as used herein means a DNA sequence that determines the transcription initiation site of RNA polymerase. The promoter may comprise an RNA polymerase III promoter, which may provide high levels of constitutive expression across a variety of cell types, and is sufficient to direct transcription of a distally located sequence linked to the 3' end of the promoter sequence in the cell. Suitable promoters include, for example, constitutive, regulated, tissue-specific, or ubiquitous promoters, which may be of cellular, viral, or synthetic origin.

In one embodiment, the promoter is a constitutive promoter such as human elongation factor-1 α (EF-1 α) or the like.

In another embodiment, the promoter is a liver-specific promoter, e.g., human 1-antitrypsin, albumin, and the like.

In another embodiment, the promoter is an endogenous sequence (constitutive or specific). In this case, the protein of interest is linked to the endogenous promoter by genome editing techniques based on the use of artificial nucleases (examples: CRISPR/cas, ZFNs, TALENs).

In another embodiment, genetic modification of human hepatocyte-like cells to modify the endogenous gene sequence is performed by genome editing techniques.

Importantly, Hepatocyte Lineage Cells (HLCs) are disease targets in a variety of disorders often referred to as "liver diseases" (also referred to as "conditions associated with liver damage"). These terms refer to any disease or clinical condition characterized by liver damage, impairment, dysfunction, defect or abnormality. Thus, the term includes, for example, lesions, degenerative diseases and genetic diseases.

Liver disease is becoming one of the most common causes of death in developing countries. The group of diseases targeting cells of the hepatocyte lineage, such as hepatocytes representing the major hepatocytes, includes inherited metabolic disorders (such as crigler-najal syndrome type I, glycogen storage disease, urea cycle defects, familial hypercholesterolemia, tyrosinemia and wilson's disease), chronic liver failure and acute liver failure, which may be caused by viral infections (especially HBV or HCV infections), toxins (alcohol) and drugs or autoimmune disorders (autoimmune chronic hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis).

In another aspect, the invention also provides a pharmaceutical composition comprising a population of human hepatocyte-like cells according to the invention.

The pharmaceutical composition may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents, solvents, and/or stabilizers. Such auxiliary substances may be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, and the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like. The pharmaceutical composition may contain further additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone, or other additives such as antioxidants or inert gases, stabilizers or recombinant proteins (e.g. human serum albumin) or vitamins suitable for in vivo administration.

The term "pharmaceutically acceptable" as used herein refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to a mammal, particularly a human, where appropriate. A pharmaceutically acceptable carrier or excipient represents any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.

Screening method of the present invention

In another aspect, the human hepatocyte-like cells of the invention obtained from healthy or diseased patients may also be advantageously used in screening applications in the pharmaceutical industry.

Such screening assays can be used for toxicological assay evaluation for finding hepatotoxicity of new drugs or compounds (such as drug candidate compounds) with clinical utility. The human hepatocyte-like cells according to the invention may for example be used to generate cell models of liver diseases as described above.

Accordingly, the present invention provides a method of screening for a compound useful for treating liver diseases, the method comprising the steps of:

(a) contacting a population of human hepatocyte-like cells or embryonic hepatocyte-like cells produced by the methods of the invention with a test compound, and

(b) determining the effect of the test compound on the human hepatocyte-like cell or embryonic hepatocyte-like cell.

Another aspect of the invention relates to a method for screening a compound having a hepatoprotective or hepatotoxic or liver proliferative effect, wherein the method comprises the steps of:

(b) comparing the survival of the cells of step a) with the survival of said population of cells as defined above cultured in the absence of said test compound.

The term "hepatotoxic" refers to a compound that causes a decrease in survival of hepatic progenitors. A compound is considered to have a hepatotoxic effect if the number of viable cells cultured in the presence of the compound is lower than the number of viable cells cultured in the absence of the compound.

The term "hepatoprotective" refers to a compound that results in an increase in survival of hepatic progenitors. A compound is considered to have a hepatoprotective effect if the number of viable cells cultured in the presence of the compound is higher than the number of viable cells cultured in the absence of the compound.

In general, hepatoprotection can be determined in the absence of a liver trophic factor. Alternatively, liver protection can be measured in the presence of known hepatotoxic drugs. Known hepatotoxic drugs include, but are not limited to, amiodarone, methotrexate, nitrofurantoin.

The term "hepatoproliferative" refers to compounds that cause increased proliferation of hepatic progenitor cells. The compound is considered to have a liver proliferating effect if the number of proliferating cells cultured in the presence of the compound is higher than the number of living cells cultured in the absence of the compound. In general, liver proliferation can be measured in the absence of growth factors.

Test Compounds of the invention

In one embodiment, the test compound may be selected from the group consisting of a peptide, a protein, a peptide mimetic, a small organic molecule, an aptamer, or a nucleic acid. For example, a test compound according to the invention may be selected from a library of previously synthesized compounds, or a library of compounds whose structure is determined in a database, or a library of compounds that have been synthesized de novo.

In particular embodiments, the test compound may be selected from small organic molecules. The term "small organic molecule" as used herein means a molecule of comparable size to those organic molecules typically used in pharmaceuticals. The term does not include biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000Da, and most preferably up to about 1000 Da.

In another embodiment, the test compound may be selected from a nucleic acid library including, but not limited to, shRNA, miRNA, mRNA.

Animal model

The availability of hepatocyte-like cells, which may be derived from human ES or iPS, further makes it possible to design in vitro and in vivo models of human liver disease and hepatotropic viruses, in particular hepatitis b or c. More specifically, by engraftment of human hepatic progenitors into the liver of a non-human mammal, in vivo models of human liver disease and hepatotropic viruses may be provided.

The invention therefore also relates to the use of human hepatocyte-like cells obtained by a method according to the invention for the production of a non-human mammalian host comprising functional human hepatocytes.

A suitable method of producing a chimeric non-human mammal comprising functional human hepatocytes may comprise the steps of: injecting the human hepatocyte-like cells according to the invention into the liver of said non-human mammal. To facilitate graft implantation of HLCs, the non-human mammal may receive anti-macrophage treatment to control non-compliant defenses. This can be achieved, for example, as follows: the administration of the bisphosphonic acid dichloromethylene ester is, for example, by intraperitoneal injection of the liposome-encapsulated bisphosphonic acid dichloromethylene ester. Other strategies that favor graft implantation of HLCs may rely on stimulating liver regeneration prior to cell injection. This can be done as follows: for example, by performing liver damage (e.g., partial hepatectomy, chemoembolization, liver irradiation, hepatotoxin, transgenesis), or by administering any compound that stimulates hepatocyte proliferation (hepatocyte growth factor, T3 hormone).

In another strategy, implantation of HLCs in grafts may also be facilitated by inhibiting the proliferative potential of endogenous hepatocytes in regenerating livers (young animals or injured livers). This can be achieved as follows: for example by administration of an altrekalide (intraperitoneal injection), mitomycin C (intraperitoneal injection) or naphthoxypropanol hydrochloride (drinking water). Finally, by administering a vasodilating compound, graft implantation of HLCs may be facilitated. This can be achieved, for example, by applying nitroglycerin.

The present invention also relates to a chimeric non-human mammal comprising functional human hepatocytes obtainable or obtainable by the method of the invention.

The non-human mammal of the present invention may be any non-primate mammal into which human hepatocytes may be introduced and maintained. This includes, but is not limited to, horses, sheep, cattle, cats, dogs, rats, hamsters, rabbits, gerbils, guinea pigs, and mice. Preferably, the host animal is a rodent, more preferably a mouse. It may also be a non-human primate (rhesus).

The non-human mammal may in particular be an immunocompromised mammal which is generally unable to generate a complete immune response against xenogeneic cells (human hepatocytes). An immunocompromised mammalian host suitable for implantation exists or may be established, for example, by administration of one or more compounds (e.g., cyclosporine, tacrolimus) or due to genetic defects that result, for example, in an inability to undergo germline DNA rearrangements at locations encoding immunoglobulins and T-cell antigen receptors.

The functionality of human hepatocytes can be monitored as follows: surrogate markers of hepatocyte activity of interest include physiological products of human hepatocytes distinguishable from their non-human mammalian (especially rodent) analogs by immunological or quantitative criteria, e.g., expression of human serum albumin, or serum bilirubin levels (also known as bilirubinaemia) in animals deficient in UGT1a1 activity, and the like. These markers can be used to determine the presence of cells without sacrificing the recipient.

Chimeric non-human mammals comprising functional human hepatocytes may in particular be used as in vivo models of human hepatitis b infection.

Methods of treatment and uses

One major field of application is cell therapy or regenerative medicine. Regenerative medicine can be used to potentially cure any disease caused by dysfunctional, damaged or failing tissues as follows: damaged tissue is regenerated in vivo by direct implantation in vivo of a pharmaceutical composition comprising the hepatocyte-like cells of the invention.

The invention also relates to a pharmaceutical composition for use in or for the treatment of liver disease, comprising, consisting of or consisting essentially of a population of hepatocyte-like cells as described above and at least one pharmaceutically acceptable excipient.

Another object of the invention is a medicament for treating or for treating liver disease, said medicament comprising, consisting of or consisting essentially of a population of hepatocyte-like cells.

As used herein, the term "consisting essentially of … …" with reference to a pharmaceutical composition or medicament means that at least one compound of the present invention is the only therapeutic agent or agent with biological activity within the pharmaceutical composition or medicament.

Pharmaceutically acceptable excipients mean any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. In one embodiment, the excipient comprises additive proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including mono-, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as polyhydric sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, including alone or in combination at 1-99.99% (weight or volume). Exemplary protein excipients include serum albumin such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.

Representative amino acid/antibody components that may also play a role in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended to be within the scope of the present invention, examples of which include, but are not limited to: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and polyhydric sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and inositol. For human administration, the article should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory authorities (e.g., FDA or EMA).

The invention also relates to human cell populations, which belong to the hepatocyte lineage but are not fully differentiated, for use in methods of treatment of the human body. The invention also relates to human cell populations for use in the treatment of liver diseases, which belong to the hepatocyte lineage but are not fully differentiated.

Examples of liver diseases include, but are not limited to, cirrhosis, fatty liver, nonalcoholic steatohepatitis (NASH), alcoholic hepatitis, fatty liver disease, fatty liver of pregnancy, virus-induced hepatitis a, b, c, d, and e, iron overload disorders, liver fibrosis, congenital liver diseases such as Crigler-Najjar type 1 (CN1), wilson's disease, urea cycle disorders, tyrosinemia, familial hypercholesterolemia, hemophilia, citrullinemia, progressive familial intrahepatic cholestasis, glycogen storage disease, acute liver failure fulminant hepatitis, subacute hepatitis, liver cancer, hypercholesterolemia.

Such diseases may be induced by environmental factors including, but not limited to, drugs, poisonous mushrooms, post-operative infections, viruses, bacteria, and alcohol. Another aspect of the invention thus relates to a population of hepatocyte-like cells (e.g. hepatoblast-like cells) of the invention for use in a method of treatment of the human body.

In one embodiment, the population of human hepatocyte-like cells is a population of cells that express hepatocyte nuclear factor 4 α (HNF4 α) and do not express, or substantially do not express, alpha-fetoprotein (AFP) as described above.

More specifically, one aspect of the invention relates to the hepatocyte-like cell populations of the invention for use in the treatment of liver disease. In one embodiment, the population of human hepatocyte-like cells is a population of cells that express hepatocyte nuclear factor 4 α (HNF4 α) and do not express, or substantially do not express, alpha-fetoprotein (AFP) as described above.

The present invention also relates to a method for treating liver disease, comprising the steps of: administering to a subject or patient in need thereof a pharmaceutically effective amount of a population of hepatocyte-like cells of the invention.

In the context of the present invention, the term "treatment" or "treating" as used herein denotes a method of: the purpose is to delay or prevent the onset of the condition, reverse, reduce, inhibit, slow or stop the progression, exacerbation or worsening of the symptoms of the condition, achieve an improvement in the symptoms of the condition, and/or cure the condition.

The term "pharmaceutically effective amount" as used herein means any amount of the population of human hepatocyte-like cells (or pharmaceutical compositions thereof) according to the invention sufficient to achieve the intended purpose.

Effective dosages and administration regimens can be readily determined by good medical practice based on the nature of the subject's symptoms, and depend on a number of factors, including, but not limited to, the route of administration, the degree of symptoms of the condition, the specific symptoms and degree of damage or degeneration of the target tissue or organ, and the characteristics of the subject (e.g., age, weight, sex, general health, etc.).

For treatment, the human hepatocyte-like cell populations and pharmaceutical compositions according to the invention may be administered by different routes. The dosage and number of administrations can be optimized in a manner known to the person skilled in the art.

In one embodiment, the human hepatocyte-like cell population, pharmaceutical composition, medicament of the invention is administered locally or systemically.

In one embodiment, the human hepatocyte-like cell population, pharmaceutical composition, medicament of the invention is administered topically and includes, but is not limited to, injection or infusion or implantation of the human hepatocyte-like cell population, pharmaceutical composition, medicament of the invention into, around or near the liver, in the parenchyma of the liver, under the Glisson's capsule of the liver, under the kidney tunica media, in the spleen, in the pancreas, in the peritoneal and omental sacs. Preferably, said local administration is injection or infusion or implantation via a blood vessel perfused into the liver (portal vein, artery, vein, mesenteric vein).

In another embodiment, the human hepatocyte-like cell population of the invention, the pharmaceutical composition or medicament is to be administered in the differentiated environment of the human hepatocyte-like cell population of the invention.

Such routes of administration may be achieved by surgical procedures, laparoscopic surgery, via catheter systems, or via intraperitoneal implantation.

In one embodiment, the human hepatocyte-like cell populations, pharmaceutical compositions, medicaments of the invention are administered systemically and include, but are not limited to, enteral or parenteral administration.

Examples of formulations suitable for injection or infusion or implantation include, but are not limited to: liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection. Examples of injections include, but are not limited to, intravenous, intra-aortic, intraperitoneal, subcutaneous, intramuscular, intradermal, and intraperitoneal injections or infusions. In another embodiment, the human hepatocyte-like cell population, the pharmaceutical composition or the medicament of the invention is sterile when injected. Methods for obtaining sterile pharmaceutical compositions include, but are not limited to, GMP synthesis (GMP stands for "good manufacturing practice").

In one embodiment, the human hepatocyte-like cell population, the pharmaceutical composition or the medicament of the invention is encapsulated. Examples of capsules include, but are not limited to, matrigel, hydrogel.

In one embodiment, the human hepatocyte-like cell population, the pharmaceutical composition or the medicament of the invention is administered in a sustained release form. In another embodiment, the human hepatocyte-like cell population, pharmaceutical compositions or medicaments of the invention comprise a delivery system for controlled release of the pharmaceutical agent.

In one embodiment, a therapeutically effective amount of a population of human hepatocyte-like cells, a pharmaceutical composition or a medicament of the invention is administered at least once or several times over the lifetime of a subject to obtain and/or maintain a therapeutic benefit in said subject.

In another embodiment, a therapeutically effective amount of a population of human hepatocyte-like cells or a pharmaceutical composition or medicament of the invention ranges from about 1 million to about 2 hundred million cells per kilogram body weight, preferably about 2500 million cells per kilogram body weight.

The term "about" as used herein before a number refers to the value of the number ± 10%.

In one embodiment, the subject is affected by a liver disease, preferably diagnosed with a liver disease.

In one embodiment, the human hepatocyte-like cells of the invention may be used for autologous regenerative therapy of patients suffering from liver diseases who require regenerative therapy due to specific disorders or treatments associated with such disorders, including, but not limited to, Crigler-Najjar type 1 (CN1) caused by a defect in one of the identified genes that can be replaced in vitro.

In one embodiment, the invention relates to the human hepatocyte-like cells of the invention for use as a cell therapy product for implantation into a human patient, as an allograft, or after genetic correction as an autograft (i.e. the cells have the same genotype as the cells of the subject/patient).

In one embodiment, the invention relates to the human hepatocyte-like cells of the invention for use as a helper liver. In one embodiment, the hepatocyte-like cells of the invention are localized in the spleen when administered. In one embodiment, the spleen containing the hepatocyte-like cells of the invention functions as a second liver.

In one embodiment, the human hepatocyte-like cells of the invention may be used in bioengineered livers as follows: by importing them into decellularized livers along with other liver-lineage cells, vascularized and functional human livers were prepared from human ipscs by transplantation of liver buds established in vitro (Takebe et al Nature 2013499), by 3D printing or every other method for preparing liver tissue. In another embodiment, the human hepatocyte-like cells of the invention may be used in bioartificial livers as follows: by introducing them into an extracorporeal setting or embedding them in a bio-matrix or hydrogel (such as alginate, silanized hydroxypropyl methylcellulose) prior to infusion into the body.

The figures and examples which follow further illustrate the invention. These examples and drawings, however, should not be construed as limiting the scope of the invention in any way.

Example 1: use of LN-111 as a substrate for differentiation of pluripotent cells into cells of the hepatocyte lineage and use of the hepatocyte-like cell-like cells so obtained in therapy.

Materials and methods

The study was conducted in accordance with french and european regulations.

Maintenance of hlsc: BBHX8 hipSC [46] was maintained in Matrigel (BD Biosciences, San Jose, Calif.) coated culture wells at 37 ℃ in a 5% CO2 incubator at mTeSR1(Stemcell Technologies, Vancouver, BC, Canada), with media changed daily. Cells were passaged every 5-7 days with Gentle CellDissociation Reagent (Stemcell Technologies, Vancouver, BC, Canada).

In vitro liver differentiation: according to a preliminary study [16,20,40 ]]The 3-step protocol of (1), with some modifications, was carried out for hepatic differentiation of human pluripotent stem cells. First, for determining endodermal differentiation, cells were harvested using TrypLE (Life Technologies, Carlsbad, CA, usa), counted using an ADAM automated cell counter (Labtech, Palaiseau, france), and then counted at 75x10³Individual cell/cm²The cell density of (D) was plated on plates (Costar; Corning Life Sciences, Acton, Mass.) precoated with 5. mu.g/ml laminin 111(Biolamina, Sundbyberg, Sweden) or 2mg/ml Matrigel (BD Biosciences, San Jose, Calif., USA). They were supplemented with B27 serum-free supplement (Life Technologies, Carlsbad, Calif., USA) (RPMI/B27), 100ng/ml activin A (Miltenyi Biotec, Paris, France), 50ng/ml WnT3A (R)&D systems, Minneapolis, MN, USA), 10 μ M rock inhibitor (Stemcell Technologies, Vancouver, BC, Canada) and optionally CHIR99021 in RPMI 1640 for 1 day. Rock inhibitor and Wnt3A were omitted from the medium on the next 2 and 3 days, respectively. On the next 4 days, cells were cultured in RPMI 1640/B27 containing 100ng/ml activin A and replaced daily. Second, for hepatic specialization (hepatic specialization), 10ng/ml Fibroblast Growth Factor (FGF)10(Miltenyi Biotec, Paris, France) and 10ng/ml Bone Morphogenetic Protein (BMP)4 (R) were then supplemented for 2 days&D systems, Minneapolis, MN, mg) were cultured in RPMI/B27 with daily medium changes. Then using 5 mu g/ml laminin 111 orTrypLE at 70X10 in 2mg/ml matrigel pre-coated plates³Individual cell/cm²The cell density of (a) shunts cells. They were cultured for 1 day in RPMI/B27 supplemented with 10ng/ml FGF10, 10ng/ml BMP-4 and 10. mu.M rock inhibitor, and the rock inhibitor was omitted from the medium the following day. Finally, for liver maturation, the cells were cultured in hepatocyte medium (Lonza,switzerland) (maturation medium) were cultured for 4 days. On the following days, HGF was omitted from the medium, and the medium was changed every 2 days.

Immunofluorescence assay: cultured cells were fixed with 4% paraformaldehyde at room temperature for 20min, permeabilized with 0.5% Triton X-100 in PBS for 15min, and blocked with 3% BSA in PBS for 15 min. Cells were incubated with primary antibody for 1 hour at room temperature. Primary antibodies against human AAT (1:100), CK19(1:50) and AFP (1:300) were purchased from DAKO (DakoCytomation, trappens, france); antibodies against human Oct4(1:100), HNF4 α (1:100) were purchased from TebuBio (Le Perray-en-Yvelines, France). After several washes with PBS 0.1% Triton, anti-mice, anti-goats (both from Life Technologies) or anti-rabbit (Abcam) IgG secondary antibodies (conjugated to ALEXA 488, 555 or 647) diluted in PBS 1% BSA 0.1% Triton 1/1000 were reacted for 30min at room temperature. Cells were then counterstained with DAPI diluted in PBS 1/10,000 for 1 minute at room temperature and read on an inverted fluorescence microscope (EvosFL from AMG). To evaluate the proportion of differentiated cells, random photographs from 10 independent differentiations were taken, and positive cells were counted for differentiation markers along with all cells.

Flow cytometry: for flow cytometry, differentiated cells were incubated with Accutase at 37 ℃ for 2 min. Dissociated cells were fixed with 0.25% paraformaldehyde at 4 ℃ for 30min, and then permeabilized with 0.2% tween-20 in PBS at 37 ℃ for 15 min. Cells were blocked with 3% BSA/PBS for 30min/4 ℃ in the presence or absence of primary antibody diluted in 0.5% BSA in PBS. After washing (with PBS), cells were incubated with 3% BSA/PBS followed by 20min at 4 ℃ with donkey anti-mouse Alexa Fluor 488-conjugated antibody. Flow cytometry analysis was performed using a BD LSR II flow cytometer (BD Biosciences).

Animal studies: animals were housed in animal facilities in the Nantes University Medical School (Nantes, France) and maintained under a 12-hour light cycle, fed ad libitum, and received human care according to the French Ministere de l' Agriculture guidelines. Homozygous (j/j) gunn rats of 120 + -30 g body weight (age: 8-9 weeks) were used for the study.

Liver function test: blood was drawn from the retro-orbital sinus. Serum total bilirubin and alanine and aspartate aminotransferases are measured by the conventional biochemical system at the Nantes University Hospital.

Histology and immunohistochemistry: immunohistochemical analysis involves the use of immunoperoxidase technology on fixed paraffin-embedded sections. Rabbit polyclonal anti-UGT 1A1(1: 50; Abgent Inc., San Diego, USA), anti-alpha-fetoprotein (1: 250; Dako, Glostrup, Denmark) and mouse monoclonal anti-serum albumin (1: 200; R&D SystemEurope; abingdon, uk) antibodies were used for human cell detection. Briefly, 4 μm thick liver sections were deparaffinized and pretreated in citrate buffer (pH 6, 10%, Dako) for 40min at 98 ℃. At H₂O₂After 5min of treatment in (Dako) and an additional 5min in TBS Tween (ScyTek Laboratories, West Logan, USA), sections were incubated with primary antibody, BSA 2% and TBS Tween for 30min at 37 ℃. Bound antibodies were detected using Envision second reagent (Dako) and DAB Liquid Substrate for immunoperoxidase (Dako).

RT-qPCR: RNeasy mini kit (QIAGEN) was used according to the manufacturer's instructions or according to the manufacturer's instructions(Life technology, Carlsbad, Calif.), Total mRNA was isolated from the cultured cells. Isolated mRNA was quantified using a Nanodrop, using a total of 10ng per reaction. Using Power EXPRESS One-StepGreenER^TMKit (Life Technologies, Carlsbad, Calif., USA), analysis of transcripts was performed with the ViiA7 sequence detection System (Life Technologies, Carlsbad, Calif., USA). The primer sequences are as follows:

name (R)	Sequence of	SEQ ID NO:
			AFP-F	GCT TGG TGG TGG ATG AAACA	1
AFP-R	TCC TCT GTT ATT TGT GGC TTT TG	2
			ALB-F	GCA CAG AAT CCT TGG TGA ACA G	3
ALB-R	ATG GAA GGT GAA TGT TTT CAG CA	4
			CK19-F	CTC CCG CGA CTA CAG CCA CT	5
CK19-R	TCA GCT CAT CCAGCA CCC TG	6
			CYP3A4-F	AGATGCCTTTAGGTCCAATGGG	7
CYP3A4-R	GCTGGAGATAGCAATGTTCGT	8
			CYP3A7-F	AAGGTCGCCTCAAAGAGACA	9
CYP3A7-R	TGCACTTTCTGCTGGACATC	10
			FOXA2-F	GCACTCGGCTTCCAGTATG	11
FOXA2-R	CACGTACGACGACATGTTCA	12
			GAPDH-F	AAT CCC ATC ACC ATC TTC CA	13
GAPDH-R	TGG ACT CCA CGA CGT ACT CA	14
			HNF4-F	TGG ACA AAG ACA AGA GGA ACC	15
HNF4-R	ATA GCT TGA CCT TCG AGT GC	16
			SOX2-F	CCTACTCGCAGCAGGGCACC	17
SOX2-R	CTCGGCGCCGGGGAGATACA	18
			SOX17-F	TTTCATGGTGTGGGCTAAGG	19
SOX17-R	CGGCCGGTACTTGTAGTTG	20

PCR method and use 2 as described previously^-ΔΔ^CtQuantitative methods analysis of relative gene expression data after normalization to GAPDH values. mRNA expression levels are defined as the fold change in mRNA levels in a given sample relative to levels in undifferentiated cells. mRNA expression levels were calculated as follows: mRNA expression level 2^-ΔΔ^CtWherein Δ Δ Ct ═ (Ct)_Target-Ct_GAPDH)_{Sample (I)}-(Ct_Target-Ct_GAPDH). Specific amplification was checked by amplicon melting curve.

Using AgPath-ID^TMOther analyses of transcripts were performed by One-Step RT-PCR Reagents (Life technologies, Carlsbad, Calif., USA) and appropriate primer pairs (Applied Biosystems) using the ViiA7 sequence detection System (Life technologies, Carlsbad, Calif., USA).

Gene	Suppliers of goods	Reference to
			OCT4(POU5F1)	Life Technologies	Hs00999632_g1
NANOG	Life Technologies	Hs04260366_g1
			FOXA2	Life Technologies	Hs00232764_m1
SOX17	Life Technologies	Hs00751752_s1
			HNF4A	Life Technologies	Hs00230853_m1
CK19(KRT19)	Life Technologies	Hs00761767_s1
			AFP	Life Technologies	Hs00173490_m1
CYP3A7	Life Technologies	Hs00426361_m1
			ALB	Life Technologies	Hs00910225_m1
AAT(SERPINA1)	Life Technologies	Hs01097800_m1
			GAPDH	Life Technologies	Hs99999905_m1

ELISA: the culture supernatants were evaluated for secretion of human alpha-fetoprotein (AFP) (Calbiotech) and albumin (Bethy Laboratories) by ELISA according to the manufacturer's instructions.

Transplanting the liver cells: cells (1X 10) Using TrypLE⁷Individual cells) were detached from the plate, washed, resuspended in 200. mu.l of physiological serum, and injected into the lower spleen pole of a gunn rat, which had undergone 2/3 partial hepatectomy before 24 hours, to establish an optimal environment for cell transplantation [48 ] using a 26-gauge butterfly needle]. Rats were immunosuppressed with tacrolimus at 0.2mg/kg daily for 3 days 1 day prior to cell transplantation and 0.1mg/kg thereafter.

Statistical scoreAnd (3) analysis: data are expressed as mean ± SEM. Using GraphPad for WindowsStatistical analysis was performed with Software (GraphPad Software, San Diego, CA). Statistical significance was assessed using the Mann-Whitney test for comparison between groups. Consider that<A p-value of 0.05 was statistically significant.

Results

We modified and combined the previously reported schemes [16,20,36,40 ]]To develop a GMP-compliant method in which HLCs are produced from hipscs using chemically defined conditions (xeno-free, feeder cell-free) and recombinant factors. Expression of laminin-111 (LN111) in embryonic liver [41]And laminin isoforms are mainly present in Matrigel^TMIn (1). We hypothesize that LN111 may constitute an efficient extracellular matrix for initiating and supporting hepatic differentiation of hipscs. Our 3-step differentiation protocol for the in vitro generation of HLCs is outlined in figure 1A. We used the human iPSC cell line BBHX8 as a representative cell line known to differentiate into HLCs [40,42]. When the hipscs were 70-90% confluent, cells were harvested and plated on disks coated with recombinant LN 111. To obtain a more reproducible hepatic differentiation process, cells were harvested enzymatically and single cell suspensions were counted and plated in new LN 111-coated disks, rather than using standard methods based on empirical cell density visualization to assess time and cell rate division to initiate HLC production. On day 0, hipscs were positive for the pluripotency marker (Nanog, Sox2) (fig. 1B) and negative for the definitive endoderm (Sox17, FoxA2) (fig. 1C) and hepatic markers (HNF4a, ALB, AFP, cytochrome P450) (fig. 1D). With AgPath-ID^TMOne-Step confirmed these experiments with more accurate RT-qPCR analysis (FIG. 4).

Cells were treated with activin a and basic fibroblast growth factor to produce definitive endoderm. The expression of endoderm and cell survival was determined after enzymatic cell harvest using short exposures to Wnt3a and rock inhibitors, respectively [16,36 ]. On day 5, over 80-90% of the cells strongly expressed the definitive endoderm markers SOX17 and FOXA2 and lost the expression of pluripotency genes (fig. 1B). The resulting definitive endoderm cells were then cultured in the presence of FGF10 and BMP 4(2 key factors involved in the initial signaling pathway for early embryonic liver development) [43] as described previously. After 3 days of cell treatment (day 8 of differentiation), cells were passaged enzymatically on new LN111 coated disks to propagate them and continue the hepatic specialization phase for 3 days.

On day 11, almost all cells were positive for markers expressed in hepatic progenitors during the early stages of hepatic development (HNF4a, CK19 and CYP3A7) (FIG. 1D.) they were negative for Alpha Fetoprotein (AFP), a marker for hepatoblasts, Albumin (ALB), AAT (α -1 antitrypsin) and cytochrome P4503A 4(CYP3A4), which are markers for mature hepatocytes, they did not express SOX17 and had reduced FOXA2 levels^TMOne-Step cells were tested and similar results were obtained (see fig. 5 and 6). In conclusion, the results show that they have a phenotype of hepatoblasts [44,45 ]]。

After day 20, hiPSC-derived hepatoblasts expressed HNF4a, CK19, AFP and CYP3a7, a CYP450 enzyme expressed in embryonic hepatocytes, and obtained expression of some markers of mature hepatocytes (ALB, AAT, UGT1a1, CYP3a4) and had lost FOXA2 expression as assessed by RT-qPCR analysis (fig. 1D). Based on these results, secretion of AFP in the cell supernatant was confirmed by ELISA assay. However, we did not detect albumin secretion, probably due to low expression levels of albumin mRNA (fig. 1E). Similar results were obtained when hipscs were differentiated on matrigel-coated disks.

Taken together, the results show that LN111 initiates and supports hepatic differentiation of hipscs as efficiently as matrigel in our culture conditions, but with recombinant matrix. Our study extended a recent study showing that LN 111-coated disks allowed to maintain hipscs at the hepatoblastoid cell stage, after hiPSC-endoderm and hepatic specification on matrigel-coated disks [36 ]. It is consistent with previous studies that showed poor maturation of HLCs in vitro. On days 20-30, our HLCs did not secrete albumin, probably because we were not cultured in the presence of dexamethasone or DMSO to increase HLC maturation. Highly mature HLCs similar to human primary hepatocytes can be produced as follows: they were cultured under co-culture conditions of 3D culture or micropatterning [34,46 ].

To explore the therapeutic effect, serum bilirubin levels were monitored over time following IDHB transplantation (day 11 of hepatic differentiation on LN 111-coated disks) in immunosuppressed gunn rats. As shown in fig. 2, there was a progressive decline in baseline bilirubin levels over the course of 60 days in the treated groups following cell transplantation. Thereafter, the decline in serum bilirubinaemia was maintained at a level of 28 ± 5% in the study and was statistically significant compared to pre-transplant values (P < 0.05). Serum bilirubin in the treatment group was 57 + -5 μ M at sacrifice compared to 84 + -11 μ M pre-transplant (p ═ 0.005). A similar decrease in bilirubinaemia was observed following transplantation of frozen IDHB (fig. 10). Serum bilirubin in the control group did not decrease and remained elevated relative to the treated rats, with values up to 150 μ M. These results were confirmed in fresh and frozen hepatocytes (fig. 10). Immunochemical analysis revealed the presence of cells expressing UDP-glucuronosyltransferase 1-A (UGT1A1), confirming the ability of transplanted IDHB to engraft into the liver parenchyma, albeit at very low levels (< 0.1%) (see arrows in FIG. 11). They are distributed throughout the liver parenchyma and act primarily as single cells. This is consistent with the low graft engraftment efficacy of normal hepatocytes in the liver of animals, where there is no selective growth advantage of the transplanted cells [37,47-50 ]. Most UGT1a 1-positive human cells were also detected in the spleen and accounted for approximately 1-2% of the spleen (fig. 11). No cells expressing human AFP were detected. These data indicate that the spleen promotes further maturation of IDHB to adult hepatocytes. The histology of the liver and other organs (not shown) was normal in all animals. Serum levels of aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT), defined markers of liver cell damage, in treated rats were 1.59. + -. 0.21. mu.kat/l and 1.07. + -. 0.16. mu.kat/l, respectively, at day 10 post-transplantation, and 2.07. + -. 0.85. mu.kat/l and 0.95. + -. 0.19. mu.kat/l, respectively, at sacrifice. These values did not differ from the preoperative values (AST: 2.44. + -. 1. mu. kat/l and ALT: 1.46. + -. 0.75. mu. kat/l) or the control rat values at 10 days after transplantation (AST: 1.59. + -. 0.12. mu. kat/l and ALT: 1.03. + -. 0.12. mu. kat/l) and at sacrifice (AST: 1.61. + -. 0.53. mu. kat/l and ALT: 1.13. + -. 0.33. mu. kat/l). Gamma-glutamyl transferase values were also normal (< 0.05. mu. kat/l) at these time points. These results indicate that the animals recovered well from the surgical procedure. To follow the in vivo functionality of the transplanted human cells, we measured human AFP and human albumin in animal serum over time. In one animal, we could detect significant levels of serum human AFP (above background of control rat serum) at day 11 post cell transplantation (16ng/ml) and gradually became undetectable thereafter. In two other animals, we could detect the presence of serum human albumin 5 months after transplantation, albeit at very low levels (6 and 23 pg/ml). Detectable levels of human albumin and human AFP were not detected in the serum of other engrafted gunn rats, probably due to the very low number of human cells in the liver.

In summary, we demonstrate for the first time the efficacy of hipscs-based regeneration for the treatment of inherited liver metabolic diseases without any selective growth advantage of the transplanted cells over the innate rodent hepatocytes, which is the case encountered in human hepatocyte transplantation [9 ]. After cell transplantation in adult gunn rats with jaundice, we demonstrated a lasting significant reduction in hyperbilirubinemia with freshly prepared and frozen therapeutic human cells. Therapeutic efficacy is equivalent to the effect obtained in gunn rats by ex vivo gene therapy using lentivirus-corrected primary hepatocytes [51 ]. It is noteworthy that it is as effective as transplantation of hepatocytes isolated from neonatal human liver and higher than that of hepatocytes isolated from adult human liver, which are gold standard cells [61] for human cell therapy for treating gunn rat. In this study, hipscs were differentiated into hepatoblast-like cells that did not express UDP-glucuronosyltransferase 1-a (UGT1a1) for cell transplantation. Our results indicate that in situ maturation has occurred to gain definitive evidence of UGT1a1 activity, which UGT1a1 activity reached a maximum at 8 weeks post-transplantation. We also detected transient expression of serum AFP and the appearance of serum albumin production in certain animals. Our results are consistent with previous studies showing that the liver will provide terminal differentiation of immature hepatocytes (i.e., hepatocytes isolated from embryonic livers) [52,53 ]. In recent years, it has been reported that hipscs directed to differentiated hepatoblasts (expressing AFP) are capable of further differentiation in murine liver, where they lose AFP expression [14,23,54,62 ]. Our studies now indicate that hipscs directed to hepatoblasts are able to engraft into the liver of another species and obtain sufficient levels of in situ maturation to ensure metabolic liver function in the context of normal liver regeneration. Furthermore, we demonstrated that the spleen also constitutes a suitable site for promoting further differentiation into adult hepatocytes, which was not reported in previous studies using HLCs.

Importantly, the in vivo functionality of liver graft implantation achieved by IDHB lasted long periods of time (6 months), with no signs of tumor formation (cancer or teratoma) and liver damage, as assessed by histological and blood parameter analysis. Like normal primary hepatocytes transplanted into adult liver [51], IDHB repopulates the liver as a single cell, suggesting that they do respond to anti-proliferative stimuli when liver regeneration has terminated and the liver has returned to mitotic quiescence. In addition, teratomas were not observed 2 months after direct injection of IDHB in the testis, confirming the absence of residual undifferentiated hipscs in HLC cell preparations.

Finally, our studies also provide an important step towards the clinical application of hipscs for the treatment of genetic liver diseases, since we have produced HLCs in a serum-free, xeno-free and chemically defined liver differentiation protocol that meets GMP production criteria. We chose to harvest HLCs at the hepatic progenitor stage (expression of HNF 4-alpha and some expression of AFP) because hepatic progenitors isolated from human embryonic liver have the ability to engraft and migrate within the liver of the transplanted recipient more efficiently than adult hepatocytes [55-57 ]. hiPSC-derived endoderm cells have also been shown to have higher graft engraftment capacity than AFP-expressing hepatic progenitor-like cells and mature hepatocytes (expressing albumin) [31 ]. In contrast, we did not transplant endoderm-like cells (day 5 of differentiation) to reduce the risk of residual undifferentiated hipscs or incompletely differentiated cells in the final cell preparation. Loh et al observed that hiPSC-derived hepatic progenitors did not engraft in the liver of murine newborns. This inconsistency with the above studies and our results may be related to different liver differentiation protocols leading to different graft engraftment capabilities of the produced HLCs (e.g., Loh et al use several small molecule inhibitors), or to differences in liver receptors (neonatal versus adult). In addition, in our study, little IDHB was detected in the liver, but this could be attributed to the fact that the spleen retains all IDHB upon intracellular cell injection; such splenic cell retention was not described in other studies [60-62 ].

Harvesting on day 11 of hepatic differentiation (end of hepatic specialization step) will also simplify and reduce culture time, and promote standardization, reproducibility, and reduce the cost of mass cell production.

In view of the foregoing, there is a clinical need for human hepatocytes for the treatment of hereditary liver diseases, including CN-1. Hepatic progenitors derived from hipscs or from hescs cultured on laminin-coated plates can be safe clinical alternatives to liver grafts and human hepatocytes isolated from cadaveric livers. While research has focused on the treatment of CN-1, it also provides an important advance in the treatment of other inherited liver diseases, such as urea cycle diseases. Indeed, these diseases can be treated by allogeneic hepatocyte transplantation [9] and are thus directly applicable to regenerative medicine protocols using normal human pluripotent stem cells. Since there is no limit to the access of HLCs, hepatocyte transplantation may be repeated to maintain or achieve satisfactory therapeutic benefit.

Example 2 use of LN-521 as a substrate for differentiation of pluripotent cells into cells of the hepatocyte lineage.

Interestingly, recombinant human laminin-521 (LN-521) can replace LN-111 as the extracellular matrix and allow the hipscs to better differentiate into definitive endoderm and HLCs derived from the hipscs as assessed by RT-qPCR analysis of HNF4 α, AFP, albumin, AAT expression gene expression. For example, we observed higher SOX17 (endoderm) expression at day 5 of differentiation, higher expression of HNF4 α, AFP and albumin from day 11 to day 30 as assessed by sybregreren based RT-qPCR (fig. 3). These results were confirmed using Taqman-based RT-qPCR for HLC derived from hiPSC (fig. 7) and HLC derived from hESC (ESI-017 cell line) (fig. 8).

The difference between LN-521 and LN-111 was more pronounced when CHIR99021 was added for 24h on day 0 of the hepatic differentiation process (FIG. 8).

Interestingly, hepatic differentiation of pluripotent stem cells (hESCs) was more efficient when we used a cocktail of LN-521 and LN-111 (25%/75%) compared to LN-521 or LN-111 alone (FIG. 9). Seeding at different cells (50X 10)³、75x10³And 100x10³Cell) observed the improvement. We again observed the efficacy of adding CHIR99021 in improving hepatic differentiation.

Reference to the literature

Throughout this application, various references describe the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Sequence listing

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(Note) = "ALB-R"

Biological = "artificial sequence"

<400>4

atggaaggtg aatgttttca gca 23

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Injection = "CK19-F"

Biological = "artificial sequence"

<400>5

ctcccgcgac tacagccact 20

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Injection = 'CK 19-R'

Biological = "artificial sequence"

<400>6

tcagctcatc cagcaccctg 20

<210>7

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..22

<223 >/molecular type = "unspecified DNA"

Injection = "CYP3A4-F"

Biological = "artificial sequence"

<400>7

agatgccttt aggtccaatg gg 22

<210>8

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..21

<223 >/molecular type = "unspecified DNA"

Injection = "CYP3A4-R"

Biological = "artificial sequence"

<400>8

gctggagata gcaatgttcg t 21

<210>9

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Injection = "CYP3A7-F"

Biological = "artificial sequence"

<400>9

aaggtcgcct caaagagaca 20

<210>10

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Injection = "CYP3A7-R"

Biological = "artificial sequence"

<400>10

tgcactttct gctggacatc20

<210>11

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..19

<223 >/molecular type = "unspecified DNA"

Note = "FOXA2-F"

Biological = "artificial sequence"

<400>11

gcactcggct tccagtatg 19

<210>12

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Note = "FOXA2-R"

Biological = "artificial sequence"

<400>12

cacgtacgac gacatgttca 20

<210>13

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Injection = 'GAPDH-F'

Biological = "artificial sequence"

<400>13

aatcccatca ccatcttcca 20

<210>14

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Injection = 'GAPDH-R'

Biological = "artificial sequence"

<400>14

tggactccac gacgtactca 20

<210>15

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..21

<223 >/molecular type = "unspecified DNA"

Note = "HNF4-F"

Biological = "artificial sequence"

<400>15

tggacaaaga caagaggaac c 21

<210>16

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Note = "HNF4-R"

Biological = "artificial sequence"

<400>16

atagcttgac cttcgagtgc 20

<210>17

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Note = "SOX2-F"

Biological = "artificial sequence"

<400>17

cctactcgca gcagggcacc 20

<210>18

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Note = "SOX2-R"

Biological = "artificial sequence"

<400>18

ctcggcgccg gggagataca 20

<210>19

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..20

<223 >/molecular type = "unspecified DNA"

Note = "SOX17-F"

Biological = "artificial sequence"

<400>19

tttcatggtg tgggctaagg 20

<210>20

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> sources

<222>1..19

<223 >/molecular type = "unspecified DNA"

Note = "SOX17-R"

Biological = "artificial sequence"

<400>20

cggccggtac ttgtagttg 19

Claims

1. Use of Laminin (LN) as a matrix for hepatic differentiation.

Use of LN to induce and/or improve a pluripotent cell population, a multipotent cell population or to determine the differentiation of an endodermal (DE) cell population into a cell population of the hepatocyte lineage.

3. A method of inducing human liver differentiation, the method comprising the steps of:

(i) providing a defined population of endoderm (DE) cells in humans, and

(ii) culturing a population of human DE cells in liver induction medium on a support coated with laminin to produce a population of human hepatoblast-like cells, and

(iii) optionally culturing the population of human hepatoblast-like cells in a liver maturation medium on a support coated with laminin to produce a population of hepatocyte-like cells, preferably human embryonic hepatocyte-like cells.

4. A method of inducing hepatic differentiation, the method comprising the steps of:

(i) providing a population of human pluripotent cells,

(iv) optionally culturing the population of human hepatoblast-like cells in a liver maturation medium on a support coated with laminin to produce a population of hepatocyte-like cells, preferably human embryonic hepatocyte-like cells.

5. A population of human hepatocyte-like cells obtained by the method of claim 3 or 4.

6. A population of human hepatocyte-like cells obtained by the method of claim 3 or 4, wherein the population expresses HNF4a and substantially expresses AFP.

7. The population of human hepatocyte-like cells of claim 5 or 6 for use in a method of treatment of a human.

8. The population of human hepatocyte-like cells of any one of claims 5-7, wherein the population is administered in the spleen.