Reprogrammed hepatocyte precursors

Abstract

The disclosure provides methods and media for generating, maintaining and / or expanding hepatocyte precursor cells, said methods and media comprising the use of one or more cAMP signalling pathway activators. Hepatocyte precursor cells generated by these methods or with the aid of the disclosed media, may be maintained in culture and used to provide functional hepatocytes for use in, for example, drug discovery, metabolism assays, hepatotoxicity tests, disease modelling as well as the development of hepatocyte transplantation therapies.

Description

[0001] Reprogrammed hepatocyte precursors

[0002] FIELD

[0003] The present disclosure provides methods for generating, maintaining and / or expanding hepatocyte precursor cells, cells derived from said methods and uses thereof. There is also provided a cell culture medium for generating hepatocyte precursor cells and a kit comprising the same.

[0004] BACKGROUND

[0005] Hepatocytes make up -80% of the liver mass and play a critical role in many physiological functions, including detoxification, drug metabolism, regulation of fats, glucose, and amino acid levels in the blood, synthesis of blood coagulation factors and complement proteins. Fully functional human hepatocytes are required for various research: in vitro toxicology tests, drug screening and disease modelling. Transplantation of hepatocytes, which can be cryopreserved, has also been proposed as an alternative therapy to liver transplantation (Dwyer et al., 2021), to address the limitations of whole organ liver transplantation, such as limited, unpredictable supply and high-risk surgery.

[0006] Mature hepatocytes rarely divide in the healthy liver but enter the cell cycle after liver injury to play a critical role in liver regeneration. However, when cultured in vitro, primary hepatocytes lose most of their synthetic and metabolic functions within -1 week in a standard 2-dimensional (2D) culture condition. While embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been expected to be an alternative source of hepatocytes, ESC / iPSC-derived hepatocyte-like cells (HLCs) are immature and have not reached a fully competent functional state to date (Bell et al., 2017; Gupta et al., 2021). Induced hepatocytes (iHeps) generated by reprogramming of fibroblasts with hepatic transcription factors (TFs) or immortalized hepatocyte cell lines also display limited hepatocyte functions (Gao et al., 2017). These have hampered ex vivo assays, as well as in vivo applications such as the development of hepatocyte transplantation therapy.

[0007] Previously researchers reprogrammed human hepatocytes into chemically reprogrammed liver progenitpors (CLiPs) or CLiP-like cells using inhibitors (Fu et al., 2019; Katsuda et al., 2019; Kim et al., 2019; Unzu et al., 2019; Zhang et al., 2018).

[0008] 55763880-1However, long-term maintenance of their re-differentiation capacity in vitro has been challenging. None of the cells showed global gene expression or metabolic activities similar to primary hepatocytes after >1 month of propagation. This has limited the large-scale production of functional human hepatocytes or the generation of genetically modified human hepatocytes.

[0009] It is amongst one of the objectives of the present disclosure to mitigate some of the abovementioned challenges associated with reprogrammed hepatocytes and hepatocyte progenitor cells and methods of producing such cells.

[0010] SUMMARY

[0011] The present disclosure provides a method of generating, maintaining and / or expanding hepatocyte precursor cells, said method comprising contacting hepatocytes with one or more cAMP signalling pathway activators.

[0012] In contrast to prior art methods, the methods of this disclosure provide hepatocyte precursors that maintain their differentiation capacity for an extended period of time (e.g. at least 1 month or more).

[0013] Prior art hepatocyte progenitor / precursor cells tend to very quickly lose their redifferentiation capacity, especially when subject to several passage in vitro. Indeed, none of the prior art methods yield cells which exhibit global gene expression and / or metabolic activities similar to primary hepatocytes after >1 month / 10,000-fold of propagation.

[0014] The hepatocyte precursor cells generated by the methods of this disclosure may be maintained in culture and used to provide functional hepatocytes for use in, for example, drug discovery, metabolism assays, hepatotoxicity tests, disease modelling as well as the development of hepatocyte transplantation therapies.

[0015] The one or more cAMP signalling pathway activators may be selected from the group consisting of:

[0016] (i) an adenylyl cyclase agonist;

[0017] (ii) cAMP or an analogue thereof;

[0018] (iii) Forskolin (or a functional analogue thereof); and

[0019] 55763880-1(iv) dibutyryl-cyclic AMP (db-cAMP).

[0020] In one teaching, the cAMP signalling pathway activators are forskolin and db-cAMP. Without wishing to be bound by theory, it has been shown that using forskolin and db-cAMP to maintain hepatocyte precursor cells, yields cells that maintain an ability to proliferate and re-differentiated into mature hepatocytes. Such maintenance of proliferation and re-differentiation capability exceeds any reported in the prior art.

[0021] In a further teaching, the disclosed method further comprises contacting hepatocytes with a gp130 signalling activator. The gp130 signalling activator may comprise IL-6 and / or Oncostatin M (OSM).

[0022] In a further teaching, any of the methods disclosed herein may further comprise the step of contacting hepatocytes with one or more additional factors selected from the group consisting of:

[0023] (i) a TGFp signalling inhibitor (which inhibitor can be a TGFp receptor or a TGFp ligand inhibitor);

[0024] (ii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi);

[0025] (iii) a WNT pathway activator (or a functional analogue);

[0026] (iv) a hepatocyte growth factor (HGF) or a functional analogue thereof;

[0027] (v) fetal calf serum (FCS) (or any suitable FCS replacement such as, for example, human platelet lysate (hPL) and / or B27);

[0028] (vi) LATS pathway inhibitor; and

[0029] (vii) a Src inhibitor.

[0030] In one teaching, the TGFp signalling inhibitor may be selected from the group consisting of:

[0031] (i) a TGF-p receptor ALK4 / ALK5 inhibitor;

[0032] (ii) Vactosertib (or a functional analogue); and

[0033] (iii) A-83-01 (or a functional analogue).

[0034] It should be noted that the term ‘ROCKi’ may embrace any compound which inhibits rho kinase (ROCK); the term may also more generally refer to anything which inhibits any of the pathways which involve ROCK or any of its effects or functions. Non-muscle

[0035] 55763880-1myosin II (NMII) is an actin-binding molecular motor with ATPase activity. A method of this disclosure may use a non-muscle myosin II (NMII) inhibitor, Blebbistatin or any functional analogue (e.g. Para-aminoblebbistatin). NMII is activated by ROCK or MLCK. Additionally, or alternatively, an inhibitor of MLCK (a MLCKi) may be used.

[0036] The WNT pathway activator may be selected from the group consisting of:

[0037] (i) a GSK3P inhibitor;

[0038] (ii) CHIR-99021 (a potent and highly selective inhibitor of glycogen synthase kinase 3 (GSK-3): or a functional analogue thereof); and

[0039] (iii) Wnt ligand.

[0040] The hepatocyte growth factor (HGF) analogue may comprise PG-001 (a c-Met agonist).

[0041] The term ‘Fetal calf serum (FCS)’ may embrace suitable alternatives which may include, human platelet lysate (hPL) and / or B27 supplement.

[0042] As used herein, a ‘LATS pathway inhibitor’ may embrace any agent which has an inhibitory effect on the LATS pathway, including any agent which directly inhibits LATS1 / 2 kinases or modulates an upstream or downstream component of LATS1 / 2 kinases. In one example, a YAP pathway activator may be used instead of, or in addition to, a LATS pathway inhibitor. A LATS pathway inhibitor may have the same or similar effect as a YAP activator. In one example, the LATS pathway inhibitor may comprise or consist of TDI-011536.

[0043] As used herein, a ‘Src inhibitor’ may embrace any agent which inhibits SRC family kinases, such as YES1 / SRC. In one example, the Src inhibitor may comprise or consist of eCF506.

[0044] In one teaching, the additional factors used in a method of this disclosure (additional to the cAMP signalling pathway activators) may comprise Vactosertib, Blebbistatin, CHIR-99021, PG-001, IL-6, FCS (or any suitable substitute), TDI-011536 and eCF506.

[0045] 55763880-1Without wishing to be bound by theory, hepatocyte precursor cells generated (or obtainable) by any of the methods described herein exhibit one or more (for example all) of the following characteristics / features:

[0046] (i) proliferation for at least 30 days

[0047] (ii) a maintenance of differentiation capacity as indicated by (upon differentiation):

[0048] (a) albumin secretion;

[0049] (b) urea synthesis; and

[0050] (c) CYP activities.

[0051] Moreover (and again without being bound to any specific parameters) cells maintained according to the methods disclosed herein, may be expanded over 1010-fold over 60 days. Furthermore, it is thought that when used in a method or cell culture medium of this disclosure, additional factors such as LATS pathway inhibitors may further promote proliferation of hepatocyte precursor cells, such as hepatocyte precursor cells derived from adult and / or juvenile hepatocytes. In addition, by supplementing Src inhibitors, the differentiation capacity of hepatocyte precursor cells, such as hepatocyte precursor cells derived from adult and / or juvenile hepatocytes, is maintained for an extended period (e.g. at least 30 days, at least 60 days).

[0052] It is further noted that hepatocyte precursor cells generated (or obtainable) using any of the methods described herein, may express the Asialoglycoprotein receptor 1. As such, within a population of hepatocyte precursor cells generated, maintained, obtainable or expanded using any of the methods described herein, a subset of those cells may be Asialoglycoprotein receptor 1 positive (ASGR1+) hepatocyte precursor cells. The ASGR1 + cells may represent hepatocyte precursors with differentiation capacity and those cells may exhibit superior re-differentiation capacity, indicated by robust CYP activities upon differentiation, and which can be maintained for a significant period of time - certainly over 30-60 days.

[0053] Accordingly, the methods of this disclosure may further comprise a step of enriching the hepatocyte precursor cells for Asialoglycoprotein receptor 1 positive (ASGR1+) hepatocyte precursor cells. The enriching step may comprise contacting a population of cells generated, maintained, expanded or obtainable using a method of this disclosure with a ASGR1 capture agent under conditions permitting binding between any

[0054] 55763880-1ASGR1+ve cells in the population and the ASGR1 capture agent. In one teaching, the ASGR1 capture agent may comprise and anti-ASGR1 antibody and / or poly-(N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-4]-D-gluconamide) (PVLA). ASGR1 capture and analysis methods may further comprise the use of FACS or magnetic beads.

[0055] This disclosure further provides a method of identifying hepatocyte precursor cells with enhanced proliferation and / or differentiation capacity, said method comprising probing hepatocyte precursor cells (generated or obtainable using a method of this disclosure) for ASGR1 expression, wherein a hepatocyte precursor cell that expresses ASGR1 exhibits superior differentiation capacity.

[0056] In view of the above, the disclosure provides hepatocyte precursor cells obtainable by the methods described herein. Cells made (or obtainable) using the methods of this disclosure may find a variety of applications. By way of example, the cells may be used as a source of cells from which to generate hepatocytes or mature hepatocytes. Cells made (or obtainable) using the methods of this disclosure may be used in assays (for example drug testing / discovery assays) or disease modelling systems.

[0057] A method of generating hepatocytes or mature hepatocytes, may comprise contacting hepatocyte precursor cells generated or obtainable by a method according to this disclosure with a cAMP signalling pathway activator so as to form spheroids or in suspension; and the further contacting the cells with small hepatocyte medium (SHM) (Chen et al., 2007) supplemented with one or more factors selected from the group consisting of:

[0058] (i) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi);

[0059] (ii) a TGFp signalling inhibitor (which inhibitor can be a TGFp receptor or a TGFp ligand inhibitor);

[0060] (iii) a WNT pathway activator;

[0061] (iv) a MEK inhibitor (MEKi: for example PD0325901);

[0062] (v) a hepatocyte growth factor (HGF) or a functional analogue thereof;

[0063] (vi) a gp-130 activator; and

[0064] (vii) fetal calf serum (FCS) or FCS replacement such as human platelet lysate (hPL) and / or B27.

[0065] 55763880-1In a further teaching, hepatocyte precursor cells may be cultured in a medium according to this disclosure; the cells may then be transferred to a medium based on SHM and supplemented with (i) a cAMP signalling pathway activator (for example Forskolin and / or db-cAMP), (ii) Vactosertib (or a functional analogue thereof), (iii) Blebbistatin (or a functional analogue thereof), (iv) CHIR-99021 (or a functional analogue thereof), (v) PG-001 (or a functional analogue thereof), (v) IL-6 (or a functional analogue thereof) and (vi) FCS or FCS replacement such as human platelet lysate (hPL) and / or B27. In such methods, the initial period of culture (in a medium of this disclosure) may be for a period of about 1 , 2, 3 or 4 days.

[0066] The disclosure also provides a cell culture medium for use in a method for generating hepatocyte precursor cells, said medium comprising a cAMP signalling pathway activator, which, in one teaching may be selected from the group consisting of:

[0067] (i) a cAMP signalling pathway activator;

[0068] (ii) an adenylyl cyclase agonist;

[0069] (iii) cAMP or an analogue thereof;

[0070] (iv) forskolin; and

[0071] (v) dibutyryl-cyclic AMP (db-cAMP).

[0072] The cell culture medium may further comprise one or more factors or supplements selected from the group consisting of:

[0073] (i) a TGFp signalling inhibitor;

[0074] (ii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi);

[0075] (iii) a WNT pathway activator;

[0076] (iv) a hepatocyte growth factor (HGF) or an analogue thereof;

[0077] (v) a gp-130 activator (IL-6, OSM or an analogue thereof);(vi) fetal calf serum (FCS) (or a suitable FCS replacement such as human platelet lysate (hPL) and / or B27);

[0078] (vii) a LATS pathway inhibitor; and

[0079] (viii) a Src inhibitor.

[0080] In one example, the cell culture medium of this disclosure may comprise one or more factors or supplements selected from the group consisting of: one or more cAMP

[0081] 55763880-1signalling pathway activator(s) described above, Vactosertib, Blebbistatin, CHIR-99021, PG-001, IL-6 and FCS.

[0082] In a further example, a cell culture medium of this disclosure may comprise one or more factors or supplements selected from the group consisting of: one or more cAMP signalling pathway activator(s) described above, Vactosertib, Blebbistatin, CHIR-99021, PG-001, IL-6, FCS and a LATS pathway inhibitor (e.g. TDI-011536).

[0083] In a yet further example, a cell culture medium of this disclosure may comprise one or more factors or supplements selected from the group consisting of: one or more cAMP signalling pathway activator(s) described above, Vactosertib, Blebbistatin, CHIR-99021, PG-001, IL-6, FCS, a LATS pathway inhibitor (e.g. TDI-011536) and a Src inhibitor (e.g. eCF506).

[0084] Any of the cell culture media described herein may further comprise small hepatocyte medium (SHM).

[0085] A cell culture medium of this disclosure may be used to generate hepatocyte precursor cells from ‘hepatocytes’, which term may embrace mature hepatocytes, human hepatocytes and / or cryopreserved human hepatocytes. The term hepatocytes may embrace human hepatocytes (or precursors of chemically expanded hepatocytes (pre-cHep)) which have been transplanted into murine liver for expansion. By way of example, a method of generating hepatocyte precursor cells may comprise contacting or maintaining hepatocytes (as described above) with, or in, any of the culture media described herein.

[0086] Without wishing to be bound by theory, the cell culture media described herein promotes the expansion / proliferation of hepatocyte precursor cells, such as pre-cHep derived from adult and / or juvenile hepatocytes. Further, and as detailed herein, the cell culture media of the present disclosure promotes the maintenance of the redifferentiation capacity of hepatocyte precursor cells, such as pre-cHep, over an extended period (e.g. at least 30 days, at least 60 days).

[0087] Any of the cell culture media of this disclosure may be used to facilitate the proliferation of pre-cHep lines from adult donors. A medium for this specific use may comprise one or more factors or supplements selected from the group consisting of:

[0088] 55763880-1(i) one or more cAMP signalling pathway activator(s) described above (e.g. Forskolin and / or dibutyryl-cyclic AMP (db-cAMP));

[0089] (ii) a TGFp signalling inhibitor (which in inhibitor can be a TGFp receptor or a TGFp ligand inhibitor) - for example Vactosertib;

[0090] (iii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi), for example Blebbistatin;

[0091] (iv) a WNT pathway activator, for example CHIR-99021;

[0092] (v) hepatocyte growth factor (HGF) analogue (for example PG-001);

[0093] (vi) a gp130 signalling activator, for example IL-6;

[0094] (vii) FCS (or any suitable alternative); and

[0095] (viii) a LATS pathway inhibitor (e.g. TDI-011536).

[0096] Optionally, a media for use in facilitating the proliferation of pre-cHep lines from adult donors may further comprise SHM.

[0097] By way of example, a media for use in facilitating the proliferation of pre-cHep lines from adult donors, may comprise:

[0098] (i) Forskolin and / or dibutyryl-cyclic AMP (db-cAMP));

[0099] (ii) Vactosertib;

[0100] (iii) Blebbistatin;

[0101] (iv) CHIR-99021;

[0102] (v) PG-001,

[0103] (vi) IL-6

[0104] (vii) FCS (or any suitable alternative);

[0105] (viii) TDI-011536; and

[0106] (ix) optionally, SHM.

[0107] In a further teaching, any of the cell culture media of this disclosure may be used to maintain pre-cHep re-differentiation capacity. A medium for this specific use may comprise one or more factors or supplements selected from the group consisting of:

[0108] (i) one or more cAMP signalling pathway activator(s) described above (e.g. Forskolin and / or dibutyryl-cyclic AMP (db-cAMP));

[0109] (ii) a TGFp signalling inhibitor (which in inhibitor can be a TGFp receptor or a TGFp ligand inhibitor) - for example Vactosertib;

[0110] (iii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi), for example Blebbistatin;

[0111] 55763880-1(iv) a WNT pathway activator, for example CHIR-99021;

[0112] (v) hepatocyte growth factor (HGF) analogue (for example) PG-001 ,

[0113] (vi) a gp130 signalling activator, for example IL-6

[0114] (vii) FCS (or any suitable alternative);

[0115] (viii) a LATS pathway inhibitor (e.g. TDI-011536); and

[0116] (ix) a Src inhibitor (e.g. eCF506).

[0117] Optionally, a media for use in maintain pre-cHep re-differentiation capacity may further comprise SHM.

[0118] By way of example, a media for use in facilitating the proliferation of pre-cHep lines from adult donors, may comprise:

[0119] (i) Forskolin and / or di butyryl-cyclic AMP (db-cAMP));

[0120] (ii) Vactosertib;

[0121] (iii) Blebbistatin;

[0122] (iv) CHIR-99021;

[0123] (v) PG-001,

[0124] (vi) IL-6

[0125] (vii) FCS (or any suitable alternative);

[0126] (viii) TDI-011536;

[0127] (ix) eCF506; and

[0128] (ix) optionally, SHM.

[0129] In one example, the culture media or any of the components and / or supplements described herein may be provided during the expansion phase and / or during culture of the hepatocyte precursor cells. In one example, the expansion phase and / or during culture of the hepatocyte precursor cells typically does not substantially involve differentiation and / or maturation of the hepatocyte precursor cells.

[0130] In one teaching, a method of generating hepatocyte precursor cells according to this disclosure may comprise seeding hepatocytes in any of the cell culture media described herein. A seeded medium may be contacted with a plate coated with, for example extracellular matrix proteins; such as, for example, fibronectin; and / or collagen. Hepatocytes may be seeded at a density of 106cells per 10 cm of plate. The cells may be maintained in the medium (and in contact with the plate) under hypoxic conditions and / or until confluent. Once confluent, the cells (which may be hepatocyte

[0131] 55763880-1precursor cells) may be harvested and used, stored or subject to a further round of maintenance, passage or culture in a medium of this disclosure.

[0132] In some examples, the method of generating, maintaining and / or expanding hepatocyte precursor cells detailed herein may comprise culturing hepatocytes and / or hepatocyte precursor cells in a 2D culture system. In one example, the hepatocytes and / or hepatocyte precursor cells are not cultured as organoids.

[0133] The disclosure further provides kits comprising a cell culture medium described herein. In one teaching, a kit may further comprise instructions for use.

[0134] DETAILED DESCRIPTION

[0135] The present disclosure will now be described in detail with reference to the following figures, which show:

[0136] Figure 1. The proliferation of precursor of chemically expanded hepatocytes (pre-cHep) over 90 days in culture.

[0137] Figure 2. ALBUMIN and Urea production by chemically expanded hepatocytes (cHep), differentiated from pre-cHep expanded for 60 days.

[0138] Figure 3. Lack of differentiation capacity towards cholangiocytes in pre-cHep.

[0139] Figure 4. Unsuccessful up-regulation of cholangiocyte genes in pre-cHep in cholangiocyte differentiation conditions.

[0140] Figure 5. Maintenance of ASGR1 expression by Forskolin, db-cAMP and IL-6.

[0141] Figure 6. CYP1A2, CYP2C9, CYP3A4 activities in cHep differentiated from 56 days-cultured pre-cHep.

[0142] Figure 7. Liver repopulation of pre-cHep lines, JO3 and JO6, with TK NOG hl L6 mice.

[0143] Figure 8. Global gene expression comparison with previously published hepatocyte-like cells (HLCs).

[0144] Figure 9. Expression of 269 liver-enriched genes in various HLCs.

[0145] Figure 10. Expression of hepatocyte marker protein and bile canaliculi formation in cHep.

[0146] Figure 11. Large-scale production of cHep with a spinner flask culture system.

[0147] Figure 12. Cryopreservation of cHep and direct differentiation of cryopreserved pre-cHep.

[0148] Figure 13. Clonal expansion of pre-cHep in L4 maintaining ALBUMIN and ASGR1 expression.

[0149] 55763880-1Figure 14. Modelling metabolic associated-steatotic liver disease (MASLD) with cHep.

[0150] Figure 15. Expansion of adult pre-cHep in L4 and L5 media. Adult primary human hepatocytes (PHH) from 3 independent donors (A01, A02, A03) were seeded in either L4 or L5 medium on day 0, and total viable cell numbers were enumerated at each passage time point. The cumulative fold expansion is depicted.

[0151] Figure 16. Expression of ALB, CYP1A2, and CYP3A4 in adult pre-cHep cell lines A01, A02, and A03. Following expansion in L4 medium for 20-30 days or in L5 medium for 30 days, with and without differentiation into cHep, expression of ALB, CYP1A2, and CYP3A4 was quantified by qRT-PCR. PHH samples from independent donors J08, J09, J 10, and A04 without culture served as references.

[0152] Figure 17. Global gene expression analysis of adult cHep. Following 30 days of expansion (~1 million-fold), adult pre-cHep line A04 was differentiated into cHep in vitro. Then global gene expression was compared with hepatocyte- 1 ike cells (HLCs) from 18 published studies (including Ardisasmita et al., 2022; Ma et al., 2022; Zhang et al., 2018; and Gupta, 2020). Principal component analysis (PCA) revealed that adult cHep clustered closely with cHep differentiated from pre-cHep lines established from juvenile PHHs (J03, J10, J13) and with 3D-cultured PHHs (3D PHH). Hep-HLC refers to HLCs derived directly from PHH, including PHH cultures maintained for 15 or 27 days in 2D with five small molecules without proliferation (Xiang et al., 2019), as well as HLCs redifferentiated after <5 passages of expansion. PSC-HLC indicates pluripotent stem cell-derived HLCs; FHep-HLC refers to fetal hepatocyte-derived HLCs (organoids); Fib-HLC denotes HLCs generated by reprogramming fibroblasts with transcription factors; Chol-HLC indicates cholangiocyte-derived HLCs. PHH, liver, HepG2, and fibroblast samples from each publication were included as references to mitigate batch effects.

[0153] Hierarchical clustering heatmap of 269 liver-enriched genes also demonstrated that the gene expression profile of adult cHep (A04) closely resembled that of cHep differentiated from pre-cHep lines established from juvenile PHHs (J03, J10, J13) and 3D-cultured PHHs (3D PHH). This indicates that adult cHep (A04) retains a gene expression signature characteristic of mature hepatocytes, comparable to both juvenile-derived cHep and 3D culture PHH models.

[0154] Figure 18. Identification of Src kinase activation in D60 pre-cHep compared to D30 pre-cHep. Activities of 144 serine / threonine kinases and 196 phosphotyrosine kinases were profiled in pre-cHep expanded for 60 days (D60) and 30 days (D30) using PamGene Kinase arrays. In both J03 and J06 lines, Src kinase activity was elevated in D60 samples, which corresponded with loss of liver repopulation capacity.

[0155] 55763880-1Figure 19. Maintenance of ASGR1 expression with Src inhibitor eCF506. (A) J13 pre-cHep expanded for 47 days in L4 medium were further cultured in L5 medium supplemented with varying concentrations of eCF506 for an additional 7 days. The proportion of ASGR1 -positive cells increased in an eCF506 concentration-dependent manner. L6 medium (L5 plus eCF506) provides an environment for prolonged functional expansion of pre-cHep lines.

[0156] Figure 20. Expression of ASGR1 in pre-cHep derived in L6 compared to in L4. ASGR1 expression of J09, J10, J13 pre-cHep lines was measured after 24-33 days (D24, D28, D30, D33) expansion in L4 and L6 medium starting from PHH.

[0157] Figure 21. Hepatic gene expression and CYP activity in J09, J 10, and J13 pre-cHep and cHep. (A) Expression of ARG1 and THRSP in pre-cHep expanded in L5 or L6, and cHep from pre-cHep expanded in L4, L5, L6 was measured by RT-qPCR. PHH samples without culture served as references. (B) CYP activities in cHep from J09, J10 pre-cHep from pre-cHep expanded in L5 and L6. All pre-cHep used here were expanded for 30 days before RNA extraction and differentiation. 3D PHH represents PHH cultured in 3D for 7-9 days in commercial hepatocyte maintenance medium before CYP assays to form spheroids.

[0158] Figure 22. Impacts of L6 medium on gene expression. pre-cHep from some donors reduce in vitro differentiation and liver repopulation capacity by day 30 of expansion in L4. This is reflected in principal component analysis (PCA) with global gene expression. Such lines (triangle, including J09 L4 D30, J10 L4 D30) cluster separately from more stable lines (circle) that maintained liver repopulation capacity after 30 days expansion. However, pre-cHep lines from the same donors expanded in L6 medium (square, J09 L6 D30, J10 L6 D30) cluster with pre-cHep lines with liver repopulation capacity. cHep from those pre-cHep (darker green circle and square) more closely aligned with 3D PHH (darker blue).

[0159] Example methods

[0160] Generation and maintenance of pre-cHep lines

[0161] pre-cHep culture was carried out with small hepatocyte medium (SHM) (Chen et al., 2007), comprised of DMEM / F12 (Sigma), 0.05% Bovine Serum Albumin (Gibco), 1:100 MEM Non-Essential Amino Acid Solution (Gibco), 10 ng / ml murine epidermal growth factor (EGF) (Peprotech), 1:100 insulin-transferrin-serine (ITS)-X (Life Technologies), 0.1 pM Dexamethasone (Sigma), 2.5 mM Nicotinamide (Sigma), 0.1 mM 2-phospho-L-ascorbic acid trisodium salt (Sigma), 2 mM L-glutamine (Invitrogen), 5 mM HEPES

[0162] 55763880-1sodium salt (Gibco), and penicillin / streptomycin solution (Invitrogen) with following supplements. For L1, 5 pM Blebbistatin (ApexBio), 1.1 pM Vactosertib (MedChemExpress), 3 pM CHIR-99021 (Stem Cell Technologies), 12.5 ng / ml PG-001 (Peptigrowth), 10% fetal calf serum (FCS) were supplemented to SHM. For L2, 10 pM forskolin (Tocris) and 50 pM Dibutyl cyclic AMP sodium salt (db-cAMP) (Sigma) were supplemented to L1. For L4, 10 ng / ml human IL-6 (R&D Systems) was supplemented to L2. For L5, 0.2 pM TDI-011536 was supplemented to L4. For L6, 1 pM eCF506 was supplemented to L5. For the derivation of pre-cHep, cryopreserved primary human hepatocytes were seeded in L2 or L4 at 106cells per 10 cm plates, coated with rat tail collagen type 1 (Corning). The cells were cultured in a hypoxic condition with 3% 02, 5% CO2 at 37 °C with medium change every 2-3 days until the cells became confluent (6-10 days). The resulting pre-cHep was harvested with TrypLE and passaged in the same culture conditions. Typically, 5x104pre-cHep seeded in L2 and L4 in a well of a 6-well plate become >2.5x105in 4 days with one medium change on day 2, before reaching a total of 30 days in culture after which the proliferation speed in L2 slows down gradually.

[0163] Enrichment of ASGR1+ pre-cHep

[0164] When the isolation of ASGR1+ pre-cHep is needed, poly-(N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-4]-D-gluconamide) (PVLA) (Kobayashi et al., 1992) (Funakoshi) is used. Briefly, 1 mg / ml PVLA was coated on a non-treated 6-well flat-bottom plate overnight at 37 °C. Subsequently, the wells were washed twice with PBS and then incubated with 1ml / well 0.5% BSA in PBS at 37 °C for 30 minutes to avoid non-specific binding of ASGR- cells. After washing the wells twice with PBS, dissociated pre-cHep were seeded at 106cells / well and incubated at 37 °C. One hour later, unbound cells were removed by washing the wells twice with SHM containing 10% FCS. To collect PVLA-bound pre-cHep, the remaining cells were incubated in 1 ml of 0.02% EDTA in PBS for 5 minutes, collected in a centrifuge tube with 4 ml 10% FCS SHM, and then centrifuged at 300 g for 3 minutes. After removing the supernatant, the cells were resuspended in a culture medium for subsequent use.

[0165] Differentiation of pre-cHep into cHep

[0166] pre-cHep were seeded in Elplasia Microcavity plates (Corning) at 100 cells / microwell in L2 or L4 and cultured for 3 days. The resulting cHep spheroids were collected for various assays or maintained in the microwells in cHep maintenance medium (cHMM) comprised of SHM supplemented with 5 pM Blebbistatin (ApexBio), 1.1 pM Vactosertib

[0167] 55763880-1(MedChemExpress), 3 pM CHIR-99021 (Stem Cell Technologies), 1 pM PD0325901 (Sigma), with or without 25 ng / ml human Oncostatin M (PeproTec). For the large-scale differentiation of pre-cHep, a 5 ml disposable ABLE Biott bioreactor (ReproCell) was precoated with Sigmacoat (Sigma) to prevent adhesion of the cells and washed thoroughly with 10% FCS SHM. 5 ml of pre-cHep suspension in L2 or L4 at 106cell / ml were cultured for 3 days at 120 rpm for differentiation. Aggregates were dissociated using TrypLE at 37 °C for 3-10 minutes and used for further experiments or cryopreservation with CELLBANKER 2 (AMSBIO) at 5x106cells per cryovial.

[0168] ALBUMIN ELISA

[0169] Three days after differentiation of pre-cHep, the culture medium was changed to cHMM and then cultured for 1 day before being used for Albumin sandwich ELISA assay (Proteintech). The assay was conducted following the manufacturer's instruction and the absorbance was measured using a Promega Glomax Explorer plate reader at 450 nm. The absorbance values were then compared to the standard curve generated to obtain the albumin concentration pg / day / million cells.

[0170] Urea detection

[0171] Three days after differentiation of pre-cHep, the culture medium was changed to cHMM and then cultured for 1 day before being used for measurement of urea using the QuantiChrom Urea Assay Kit (BioAssay Systems). 50 pl of each culture media supernatant was mixed with 200 pl of manufacturer working reagent in a 96 well plate and incubated for 20 minutes at room temperature. Then, optical density was measured using a Promega Glomax Explorer plate reader at 450nm. The optical densities were then compared to the standard curve generated to obtain the urea concentration pg / day / million cells.

[0172] CYP assay

[0173] CYP1A2, CYP2C9 and CYP3A4 activities were measured with P450-GIO™ CYP1A2, 2C9, and 3A4 assay kits (Promega) following the manufacturer’s instructions. Briefly, in a 96-well Elplasia plate, 100 pre-cHep per microwell were seeded for differentiation in the L2 medium. After 3 days, the spheroids were washed twice in 200 pl SHM. For the induction of CYP1A2, CYP2C9 or CYP3A4, the final concentration 100 pM Omeprazole (Sigma) or 1 mM Phenobarbital (Sigma) was supplemented in SHM. 48 hours later, the spheroids were washed twice in PBS with Mg2+ and Ca2+, and then incubated in 50

[0174] 55763880-1pl / well of CYPs substrate solution for 1 hour (CYP1A2, CYP3A4) or 4 hours (CYP2C9). After the incubation, 25 l of CYPs substrate solution was transferred to an opaque white 96-well plate, and 25 pl of luciferin detection reagent was added to each well. After incubation for 20 minutes at room temperature in the dark, the luminescence was measured using a Promega Glomax Explorer plate reader. The relative luminescence unit (RLU) / ml / million cells was calculated after averaging the raw luminescence values from the technical duplicate wells and subtracting the background luminescence from the control wells.

[0175] Differentiation capacity tests towards cholangiocytes

[0176] To test the differentiation capacity of pre-cHep to cholangiocytes, we have used two published protocols used for CLiP / CLiP-like cells (Katsuda et al., 2017; Kim et al., 2019), with human biliary epithelial cells (hBECs) derived from cholangiocytes as a positive control (Hallett et al., 2022). In the method by Kim, 2x105hBECs or pre-cHep were resuspended in 200ul cholangiocytes differentiation medium (CDM), comprised of DMEM / F12 containing 10% FCS and 12.5 ng / ml PG-001 (Peptigrowth). The cell suspension was mixed with equal volume of CDM supplemented with collagen type 1 (BD Biosciences). The mixture is then transferred into a 6-well plate and incubate for 30 min at 37 °C until the gel was set. Thereafter, 2 ml of CDM was overlayed, and which was changed every 2 days. At day 7, cells were subjected to Rhodamine 123 transport assay harvested for RNA extraction. In the method by Katsuda, one day before seeding pre-cHep or hBECs, 5x104irradiated mouse fetal fibroblast (iMEF) were plated onto a collagen-coated 12-well plate. On the following day (Day 0), 5x105pre-cHep or hBECs were resuspended in SHM with 10 pM Y-27632, 0.5 pM A-83-01, 3 pM CHIR99021 (YAC) and 5% FCS and seeded on iMEF. Next day (Day1), medium was changed to mTeSR™1 with YAC (Stemcell Technologies) (mTeSR1+YAC). Medium was changed every 2 days. At day 7, the medium was replaced with mTeSR1+YAC supplemented with 2% Matrigel. Thereafter, medium was changed every 2 days. At day 14, cells were subjected to Rhodamine 123 transport assay or harvested for RNA extraction. Rhodamine 123 transport assay was carried out by incubating the cells with 100 pM of Rhodamine 123 (Sigma-Aldrich) for 5 minutes at 37 °C, washed 3 times with media and then kept in fresh media for 1 hour. After 1 hour, the cells were washed twice with media before imaging.

[0177] 55763880-1Genetic manipulation of pre-cHep

[0178] PNPLA3 knockout pre-cHep lines were generated using CRISPR / Cas9. For each electroporation, 125 pmol of gRNA was combined with 122 pmol of Cas9 in a sterile RNase-free PCR tube and incubated at room temperature for 15 minutes before placed on ice. In the meantime, the cells were washed with PBS, and harvested with TrypLE at 37 °C for 5-10 minutes. 1x106cells were transferred to a sterile 1.5 ml tube and washed twice with Opti-MEM. Cells were gently resuspended in 100 pl of Opti-MEM containing the Cas9 / gRNA complex and transferred into an electroporation cuvette. Cell were electroporated using NEPA21 with the following settings: Poring pulse: voltage=200 V, pulse length=5 ms, pulse interval=50 ms, number of pulses=2, decay rate=10%, Polarity=+ and transfer pulse: voltage=20 V, pulse length=50 ms, pulse interval=50 ms, number of pulses=5, decay rate=40%, Polarity=+ / -. The cells were left in the cuvette for 20-40 minutes to recover before being seeded at clonal density in L4. After 10-14 days of culture, the colonies were picked and expanded in L4. Clones with the desired genotype and a high proportion of ASGR1+ cells (>50%) were chosen for further analyses.

[0179] In vitro steatosis model with cHep

[0180] pre-cHeps were plated at a density of 100-200 cells / microwell in an Elplasia plate to form spheroids in L2. After 3 days of differentiation, the medium was switched to lean or lipogenic cHMM. Lean cHMM contained 5.5 mM glucose and 172 pM insulin, with all other components in cHMM at the same concentrations, lipogenic cHMM contained 320 pM FFA (213 pM of oleic acid and 107 pM palmitic acid bound to BSA), 22 mM glucose and 1.7 pM insulin, with all other components in cHMM at the same concentrations. Media was changed on day 3, 5 and 7, and the spheroids were collected on day 10. The spheroids were used for BODIPY staining followed by imaging and flow cytometry or the quantification of triglyceride using an Adipogenesis Detection Assay Kit (Abeam).

[0181] Example 1

[0182] Cryopreserved human hepatocytes from 2 independent donors JO3 and JO6 were cultured in L4, L2, L1 medium containing key components in the table under the graph over 60 days, and fold expansion of the cells was recorded over 90 days (Figure 1). *Culture in L1 was introduced at day 35 by removing Forskolin and db-cAMP from L2, and stopped at day 76 when the cells hardly proliferated. This demonstrated the

[0183] 55763880-1importance of these two components, Forskolin and db-cAMP, for pre-cHep long-term proliferation. L4 with IL-6 in L2 further enhanced pre-cHep proliferation.

[0184] pre-cHep cultured in L1 , L2, L4 conditions for 60 days were differentiated into cHep in a 3D culture condition for 4 days. ALBUMIN production and Urea synthesis was measured using culture supernatant. As a reference, fresh HepaSH cells, human hepatocytes transplanted and expanded in TK-NOG mice (Uehara et al., 2023), were cultured in cHMM using Elplasia plates until they formed spheroids (9 days) before measuring the ALBUMIN and Urea production.

[0185] pre-cHep and human biliary epithelial cells (hBECs) derived from cholangiocytes were placed in the cholangiocyte differentiation condition described by Kim et al. or Katsuda et al (Huang et al., 2020; Katsuda et al., 2017; Kim et al., 2019). While hBECs formed the duct / spheroid structure with Rhodamine 123 transport activity, indicating differentiation into mature cholangiocytes, pre-cHep showed no structure changes nor Rhodamine 123 transport activity (Figure 3). These pre-cHep in the cholangiocytes-induction conditions maintained hepatocyte gene ALB expression and hardly up-regulated cholangiocytes genes, CFTR, AQP1 and GRHL2, unlike hBECs (Figure 4).

[0186] Example 4

[0187] A high proportion of ASGR1+ pre-cHep was observed in early passages in L2 (-until day 30) or in L4 condition (-60 days) with 2 independent cell lines, JO3 and JO6. Culture in L1 was introduced at day 35 by removing Forskolin and db-cAMP from L2, which resulted in the loss of ASGR1+ cells by day 56, demonstrating the importance of Forskolin and db-cAMP to maintain hepatocyte marker ASGR1 expression in pre-cHep (Figure 5). The proportion of ASGR1+ cells well correlated with CYP1A2, CYP2C9 and CYP3A4 activities after differentiation from those L4, L2 and L1 -cultured pre-cHep (Figure 6). When ASGR1+ pre-cHep were isolated at day 35 and expanded until day 56 in L4 and L2, inducibility of CYP activities in cHep was further improved (Figure 6), demonstrating the usefulness of ASGR1+ pre-cHep enrichment as a strategy to obtain pre-Hep with superior differentiation capacity.

[0188] 55763880-1pre-cHep lines, JO3 and JO6, expanded ~106-fold (cultured for 30 days) or ~1012-fold (cultured for 60 days), were transplanted into TK-NOG hlL-6 mice, and their liver repopulation was assessed at 5.0-9.3 weeks post transplantation (wpt) by immunostaining for a human mitochondria specific antibody (Figure 7). Both JO3 and JO6 pre-cHep demonstrated efficient liver repopulation. Primary human hepatocytes (PHH) were used as a control.

[0189] Example 6

[0190] Global gene expression of cHep from pre-cHep expanded for 56 days was compared with hepatocyte-like cells (HLC) from 16 published papers (Ardisasmita et al., 2022; Ma et al., 2022; Zhang et al., 2018) (Figure 8). The principal component analysis demonstrated that the closest samples to PHHs (within the dotted box) are cHep (red), Hep-HLC (HLC-derived from reprogrammed mature hepatocytes, like Kaji lab’s cHeps) with minimal (<1 month) expansion in vitro (orange), or PHH culture in 3D for 8 days or 5 small molecules in 2D (Xiang et al., 2019), with no proliferation (pink). This demonstrated that the expansion of pre-cHep with Forskolin and db-cAMP enabled the large-scale production of functional hepatocytes for the first time. PSC-HLC; pluripotent cell-derived HLC, FHep-HLC; foetal hepatocyte-derived HLC (organoid), Fib-HLC; HLC generated by reprogramming fibroblasts with transcription factors, Chol-HLC; cholangiocyte-derived HLCs. PHH, Liver, HepG2, and fibroblast samples from each publication were used as references to remove the batch effect. Hierarchical clustering heatmap with 269 liver-enriched genes also demonstrated that the samples most closely clustered with PHH and liver samples are PHH culture, early passage Hep-HLC and cHep from 56 days cultured pre-cHep (Figure 9).

[0191] Example 7

[0192] cHep generated from 60 days-cultured pre-cHep exhibited robust expression of hepatocyte marker ALBUMIN, HNF4a, CYP2E1, ASGR1 protein, as well as bile canaliculi formation visualized by Cholyl-lysyl-fluorescein (CLF) (Figure 10).

[0193] Example 8

[0194] pre-cHep were differentiated into cHep in static 3D culture with a microwell plate or spinner flask (SF) culture (Figure 11). RT-qPCR evidenced up-regulation of mature hepatocyte markers ALB, CYP1A2, CYP3A4 to similar levels to primary hepatocytes (PHH) in 3 days in both differentiation systems. Expression of the mature hepatocyte

[0195] 55763880-1genes was maintained after freeze & thaw, in cHep (Figure 12), indicating that the large-scale production and storage of cHep is possible for industrial use. In addition, cryopreserved pre-cHep also could induce those mature hepatocyte markers when defrosted and seeded into microwell plates directly (Figure 12). Thus, pre-cHep can be distributed and used for both expansion and direct differentiation into cHep.

[0196] Example 9

[0197] pre-cHep seeded at a clonal density in L4 formed ALBUMIN+ colonies (Figure 13A) and could expand as clonal lines maintaining ASGR1 expression (Figure 13B), making the establishment of genetically modified pre-cHep lines with differentiation capacity possible.

[0198] Example 10

[0199] When cultured in the presence of free fatty acids (FFAs), cHep accumulate triglycerides phenocopying metabolic-associated steatotic liver disease (MASLD) (Figure 14A). Thus, pre-cHep lines with knockout of PNPLA3 gene, a single nucleotide polymorphism in which is strongly associated with MASLD and metabolic dysfunction-associated steatohepatitis (MASH), was generated using nucleofection of CRISPR / Cas9 ribonucleoprotein (RNP) complex. cHep from the PNPLA3 knockout lines demonstrated a more severe steatosis phenotype in the presence of free fatty acids (FFAs) (Figure 14B). This presented the usefulness of pre-cHep / cHep for genetic modifications and disease modelling.

[0200] Example 11

[0201] A medium comprising the components of the L4 medium detailed herein supplemented with a LATS inhibitor may further enhance proliferation of adult precursors of chemically expanded hepatocytes (pre-cHep). A LATS inhibitor may encompass any LATS inhibitors and YAP pathway activators known in the art, such as TDI-011536 for example. L4 medium supplemented with a LATS inhibitor (referred to as L5 medium herein) may further enhance proliferation of adult pre-cHep (Figure 15). After 30 days of expansion, cHep differentiated from L5 adult pre-cHep showed higher expression of mature hepatocyte markers ALB, CYP1A2, and CYP3A4 (Figure 16), indicating that L5 promotes proliferation but also enhances the maintenance of the differentiation capacity of the cells. Global gene expression analysis confirmed that cHep from L5-expanded adult pre-

[0202] 55763880-1cHep closely resemble 3D cultured primary human hepatocytes (3D PHH) and cHep from L4-expanded juvenile cHep (Figure 17).

[0203] Studies were carried out to further improve pre-cHep culture media to enhance long-term maintenance of differentiation and liver repopulation capacity. Active kinase analysis via Pamgene kinase arrays on two juvenile pre-cHep lines (J03, J06) expanded for 30 and 60 days showed significant Src kinase activation at day 60, when the cells lost liver repopulation capacity (Figure 18). Supplementation of the Src inhibitor eCF506 in L5 medium enhanced maintenance of ASGR1 expression in a dose-dependent manner in a long-tern pre-cHep expansion over 50 days (Figure 19).

[0204] A higher proportion of pre-cHep expressed ASGR1 when derived and expanded in L6 from donors J09, J10, and J13 PHHs after 24-33 days expansion (Figure 20). Both L5 and L6 prolonged differentiation capacity, as indicated by robust ARG1 and THRSP expression in cHep differentiated from these lines, similar to those from stable L4 pre-cHep line J13 (Figure 21A). CYP1A2, CYP3A4, and CYP2C9 enzymatic activities were higher in cHep derived from L6-expanded pre-cHep versus L5, with CYP2C9 hardly detectable in J09, J10 cHep differentiated after 30 days expansion in L5 (Figure 21 B). Principal component analysis of global gene expression showed J09 and J10 L6 D30 pre-cHep (square) clustered with known repopulative lines J03, J06, and J13 (circle) (Figure 22). Similar clustering patterns were observed in differentiated cHep.

[0205] References

[0206] Ardisasmita, A. I., Schene, I.F., Joore, I.P., Kok, G., Hendriks, D., Artegiani, B., Mokry, M., Nieuwenhuis, E.E.S., and Fuchs, S.A. (2022). A comprehensive transcriptomic comparison of hepatocyte model systems improves selection of models for experimental use. Commun Biol 5, 1094. 10.1038 / s42003-022-04046-9.

[0207] Bell, C.C., Lauschke, V.M., Vorrink, S.U., Palmgren, H., Duffin, R., Andersson, T.B., and Ingelman-Sundberg, M. (2017). Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell-Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury. Drug Metabolism and Disposition 45, 419-429.

[0208] 10.1124 / DMD.116.074369.

[0209] 55763880-1Chen, Q., Kon, J., Ooe, H., Sasaki, K., and Mitaka, T. (2007). Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nat Protoc 2, 1197-1205.

[0210] 10.1038 / nprot.2007.118.

[0211] Dwyer, B.J., Macmillan, M.T., Brennan, P.N., and Forbes, S.J. (2021). Cell therapy for advanced liver diseases: Repair or rebuild. Journal of Hepatology 74, 185-199.

[0212] 10.1016 / J.JHEP.2020.09.014.

[0213] Fu, G.B., Huang, W.J., Zeng, M., Zhou, X., Wu, H.P., Liu, C.C., Wu, H., Weng, J., Zhang, H.D., Cai, Y.C., etal. (2019). Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens. Cell Res 29, 8-22. 10.1038 / S41422-018-0103-x.

[0214] Gao, Y., Zhang, X., Zhang, L., Cen, J., Ni, X., Liao, X., Yang, C., Li, Y., Chen, X., Zhang, Z., et al. (2017). Distinct Gene Expression and Epigenetic Signatures in Hepatocyte-like Cells Produced by Different Strategies from the Same Donor. Stem Cell Reports 9, 1813-1824. 10.1016 / J.STEMCR.2017.10.019.

[0215] Guo, R., Jiang, M., Wang, G., Li, B., Jia, X., Ai, Y., Chen, S., Tang, P., Liu, A., Yuan, Q., and Xie, X. (2022). IL6 supports long-term expansion of hepatocytes in vitro. Nat Commun 13, 7345. 10.1038 / s41467-022-35167-8.

[0216] Gupta, R., Schrooders, Y., Hauser, D., van Herwijnen, M., Albrecht, W., ter Braak, B., Brecklinghaus, T., Castell, J.V., Elenschneider, L., Escher, S., et al. (2021). Comparing in vitro human liver models to in vivo human liver using RNA-Seq. Archives of Toxicology 95, 573-589. 10.1007 / S00204-020-02937-6 / FIGURES / 10.

[0217] Hallett, J.M., Ferreira-Gonzalez, S., Man, T.Y., Kilpatrick, A.M., Esser, H., Thirlwell, K., Macmillan, M.T., Rodrigo-Torres, D., Dwyer, B.J., Gadd, V.L., etal. (2022). Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease. Cell Stem Cell 29, 355-371 e310.

[0218] 10.1016 / j.stem.2022.02.006.

[0219] Huang, Y., Sakai, Y., Hara, T., Katsuda, T., Ochiya, T., Gu, W.L., Miyamoto, D., Hamada, T., Kanetaka, K., Adachi, T., and Eguchi, S. (2020). Differentiation of chemically induced liver progenitor cells to cholangiocytes: Investigation of the optimal conditions. J Biosci Bioeng 130, 545-552. 10.1016 / j.jbiosc.2020.07.009.

[0220] Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M.M., Ellis, E., van Wenum, M., Fuchs, S.A., de Ligt, J., et al. (2015). Long-term culture of genomestable bipotent stem cells from adult human liver. Cell 160, 299-312.

[0221] 10.1016 / j. cell.2014.11.050.

[0222] 55763880-1Ise, H., Sugihara, N., Negishi, N., Nikaido, T., and Akaike, T. (2001). Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes. Biochem Biophys Res Commun 285, 172-182. 10.1006 / bbrc.2001.5139. Katsuda, T., Kawamata, M., Hagiwara, K., Takahashi, R.u., Yamamoto, Y., Camargo, F.D., and Ochiya, T. (2017). Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity. Cell Stem Cell 20, 41-55. 10.1016 / j. stem.2016.10.007.

[0223] Katsuda, T., Matsuzaki, J., Yamaguchi, T., Yamada, Y., Prieto-Vila, M., Hosaka, K., Takeuchi, A., Saito, Y., and Ochiya, T. (2019). Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes. eLife 8, 1-31.

[0224] 10.7554 / eLife.47313.

[0225] Kim, Y., Kang, K., Lee, S.B., Seo, D., Yoon, S., Kim, S.J., Jang, K., Jung, Y.K., Lee, K.G., Factor, V.M., et al. (2019). Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells. Journal of Hepatology 70, 97-107.

[0226] 10.1016 / j.jhep.2018.09.007.

[0227] Kobayashi, A., Kobayashi, K., and Akaike, T. (1992). Control of adhesion and detachment of parenchymal liver cells using lactose-carrying polystyrene as substratum. J Biomater Sci Polym Ed 3, 499-508. 10.1163 / 156856292x00466.

[0228] Ma, H., de Zwaan, E., Guo, Y.E., Cejas, P., Thiru, P., van de Bunt, M., Jeppesen, J.F., Syamala, S., Dall'Agnese, A., Abraham, B.J., et al. (2022). The nuclear receptor THRB facilitates differentiation of human PSCs into more mature hepatocytes. Cell Stem Cell 29, 795-809 e711. 10.1016 / j.stem.2022.03.015.

[0229] Ogawa, S., Surapisitchat, J., Virtanen, C., Ogawa, M., Niapour, M., Sugamori, K.S., Wang, S., Tamblyn, L., Guillemette, C., Hoffmann, E., et al. (2013). Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development 140, 3285-3296. 10.1242 / dev.090266.

[0230] Uehara, S., Higuchi, Y., Yoneda, N., Ito, R., Takahashi, T., Murayama, N., Yamazaki, H., Murai, K., Hikita, H., Takehara, T., and Suemizu, H. (2023). HepaSH cells: Experimental human hepatocytes with lesser inter-individual variation and more sustainable availability than primary human hepatocytes. Biochem Biophys Res Commun 663, 132-141.

[0231] 10.1016 / j. bbrc.2023.04.054.

[0232] Unzu, C., Planet, E., Brandenberg, N., Fusil, F., Cassano, M., Perez-Vargas, J., Friedli, M., Cosset, F.L., Lutolf, M.P., Wildhaber, B.E., and Trono, D. (2019). Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes. Hepatology 69, 2214-2231. 10.1002 / hep.30425.

[0233] 55763880-1Xiang, C., Du, Y., Meng, G., Yi, L.S., Sun, S., Song, N., Zhang, X., Xiao, Y., JieWan, J., Yi, Z., et al. (2019). Long-term functional maintenance of primary human hepatocytes in vitro. Science 364, 399-402. 10.1126 / science.aau7307.

[0234] Zhang, K., Zhang, L., Liu, W., Ma, X., Cen, J., Sun, Z., Wang, C., Feng, S., Zhang, Z., Yue, L., et al. (2018). In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell Stem Cell 23, 806-819. e804.

[0235] 10.1016 / j.stem.2018.10.018.

[0236] 55763880-1

Claims

CLAIMS:

1. A method of generating, maintaining and / or expanding hepatocyte precursor cells, said method comprising contacting hepatocytes with one or more cAMP signalling pathway activators.

2. The method of claim 1 , wherein the hepatocytes are maintained in the presence of:(i) extracellular matrix proteins; and / or(ii) fibronectin; and / or(iii) collagen.

3. The method of claim 1 or 2, wherein the hepatocytes precursor cells are maintained under hypoxic conditions.

4. The method of claim 3, wherein the cells are harvested, optionally at confluence, and again contacted with one or more cAMP signalling pathway activators for further propagation.

5. The method of any preceding claim, wherein the hepatocytes are mature hepatocytes, primary human hepatocytes, cryopreserved primary human hepatocytes or human hepatocytes expanded in the liver of animals.

6. The method of any preceding claim, wherein the one or more cAMP signalling pathway activators is selected from the group consisting of:(i) an adenylyl cyclase agonist;(ii) cAMP or an analogue thereof;(iii) forskolin; and(iv) dibutyryl-cyclic AMP (db-cAMP).

7. The method of any preceding claim, wherein the hepatocytes are contacted with forskolin and db-cAMP.

8. The method of any preceding claim, wherein the method further comprises contacting hepatocytes with a gp130 signalling activator.55763880-19. The method of claim 8, wherein the gp130 signalling activator comprises IL-6 or Oncostatin M (OSM).

10. The method of any preceding claim, wherein the method further comprises contacting the hepatocytes with one or more additional factors selected from the group consisting of:(i) a TGFp signalling inhibitor;(ii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NMI I) or an inhibitor of MLCK (MLCKi);(iii) a WNT pathway activator;(iv) a hepatocyte growth factor (HGF) or a functional analogue thereof; and(v) fetal calf serum (FCS) and / or human platelet lysate (hPL) and / or B27; (vi) a LATS pathway inhibitor and / or a YAP pathway activator; and (vii) a Src inhibitor.

11. The method of claim 10, wherein the TGFp inhibitor is selected from the group consisting of:(i) A TGF-p receptor ALK4 / ALK5 inhibitor;(ii) Vactosertib;(iii) A-83-01; and(vi) TGF-b ligand inhibitors.

12. The method of any of claim 10-11, wherein additional factor (ii) is selected from the group consisting of:(i) a non-muscle myosin II heavy chain inhibitor; and(ii) Blebbistatin or a functional analogue.

13. The method of any of claims 10-12, wherein the WNT pathway activator is selected from the group consisting of:(i) a GSK3p inhibitor;(ii) CHIR-99021; and(iii) a Wnt agonist or a functional analogue55763880-114. The method of any of claims 10-13, wherein the hepatocyte growth factor (HGF) analogue is PG-001.

15. The method of any one of claims 10-14, wherein the LATS pathway inhibitor and / or YAP pathway activator comprises TDI-011536.

16. The method of any one of claims 10-15, wherein the Src inhibitor comprises eCF506.

17. The method of any preceding claim, wherein the method further comprises enriching the hepatocyte precursor cells for Asialoglycoprotein receptor 1 positive (ASGR1+) hepatocyte precursor cells.

18. The method of any preceding claim, wherein the hepatocyte precursor cells:(i) maintain their proliferation and / or differentiation capacity; and / or(ii) exhibit proliferation capacity for at least 30 days; and / or(iii) maintain capacity to generate mature hepatocytes capable of:(iii-i) albumin secretion; and / or(iii-ii) urea synthesis; and / or(iii-iii) CYP activity.

19. Hepatocyte precursor cells obtainable by the method of any preceding claim.

20. Use of the hepatocyte precursor cells of claim 19 in a method of generating hepatocytes or mature hepatocytes in vitro and / or in vivo.

21. The use of claim 20, wherein the method of generating hepatocytes or mature hepatocytes comprises:contacting the hepatocyte precursor cells with a cAMP signalling pathway activators so as to form spheroids; andcontacting the spheroids with small hepatocyte medium (SHM) supplemented with one or more factors selected from the group consisting of:(i) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NMI I) or an inhibitor of MLCK (MLCKi);(ii) a TGFp inhibitor;55763880-1(iii) a WNT pathway activator;(iv) a MEK inhibitor;(v) a hepatocyte growth factor (HGF) or a functional analogue thereof; (vi) a gp130 signalling activator; and(vii) fetal calf serum (FCS) and / or human platelet lysate (hPL) and / or B27.

22. The use of claim 21, wherein the use further comprises using SHM additionally supplemented with one or more factors selected from the group consisting of:(i) a LATS pathway inhibitor and / or a YAP pathway activator; and(ii) a Src inhibitor.

23. A cell culture medium comprising a cAMP signally pathway activator.

24. Use of the cell culture medium of claim 23 in a method for generating and / or expanding hepatocyte precursor cells from hepatocytes, mature hepatocytes, human hepatocytes or cryopreserved human hepatocytes.

25. The cell culture medium of claim 23, or use of claim 24, wherein the cAMP signalling pathway activator is selected from the group consisting of:(i) a cAMP pathway modulator;(ii) an adenylyl cyclase agonist;(iii) cAMP or an analogue thereof;(iv) forskolin; and(v) dibutyryl-cyclic AMP (db-cAMP).

26. The cell culture medium of claim 23 or 25, wherein the medium further comprises one or more supplements or factors selected from the group consisting of:(i) a gp130-mediated signalling pathway modulator;(ii) IL-6;(iii) Oncostatin M (OSM);(iv) a TGFp signalling pathway inhibitor;(v) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi);(vi) a WNT signalling activator;(vii) a hepatocyte growth factor (HGF) or an analogue thereof;55763880-1(viii) fetal calf serum (FCS) or a FCS replacement;(ix) a LATS pathway inhibitor; and(x) a Src inhibitor / 27. A cell culture medium comprising:(i) one or more cAMP signalling pathway activator(s);(ii) a TGFp signalling inhibitor;(iii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi);(iv) a WNT pathway activator;(v) hepatocyte growth factor (HGF) analogue,(vi) a gp130 signalling activator(vii) FCS (or any suitable alternative); and(viii) a LATS pathway inhibitor (e.g. TDI-011536).

28. The cell culture medium of claim 27, wherein the medium further comprises SHM.

29. The cell culture medium of claims 27 or 28, comprising:(i) Forskolin and / or dibutyryl-cyclic AMP (db-cAMP));(ii) Vactosertib;(iii) Blebbistatin;(iv) CHIR-99021;(v) PG-001,(vi) IL-6(vii) FCS (or any suitable alternative); and(viii) TDI-011536; and(ix) optionally, SHM.

30. A cell culture medium comprising:(i) one or more cAMP signalling pathway activator(s);(ii) a TGFp signalling inhibitor;(iii) an inhibitor for ROCK (ROCKi), an inhibitor of non-muscle myosin II (NM 11) or an inhibitor of MLCK (MLCKi);(iv) a WNT pathway activator;(v) a hepatocyte growth factor (HGF) analogue;55763880-1(vi) a gp130 signalling activator;(vii) FCS (or any suitable alternative);(viii) a LATS pathway inhibitor; and(ix) a Src inhibitor.

31. The cell culture medium of claim 30, wherein the medium further comprises SHM.

32. The cell culture medium of any one of claims 30 or 31 , comprising:(i) Forskolin and / or dibutyryl-cyclic AMP (db-cAMP));(ii) Vactosertib;(iii) Blebbistatin;(iv) CHIR-99021;(v) PG-001,(vi) IL-6(vii) FCS (or any suitable alternative);(viii) TDI-011536;(ix) eCF506; and(ix) optionally, SHM.

33. A method of generating and / or expanding hepatocyte precursor cells, said method comprising contacting or maintaining hepatocytes with or in a medium according to any one of claims 23-31.

34. A method of extracting hepatocyte precursor cells with superior differentiation capacity from a cell population, said method comprising isolating Asialoglycoprotein receptor 1 (ASGR1) positive cells.

35. The method of claim 34, wherein the cell population is contacted with a ASGR1 capture agent under conditions permitting binding between any ASGR1+ve cells in the population and the ASGR1 capture agent, optionally wherein the ASGR1 capture agent comprises and anti-ASGR1 antibody and / or poly-(N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-4]-D-gluconamide) (PVLA).

36. The method of claim 35 wherein ASGR1+ve cells are isolated from the ASGR1 capture agent.55763880-137. A method of identifying hepatocyte precursor cells with enhanced proliferation and / or differentiation capacity, said method comprising probing hepatocyte precursor cells for ASGR1 expression, wherein a hepatocyte precursor cell that expresses ASGR1 exhibits superior differentiation capacity.

38. A kit comprising the medium of any of claims 23 and 25-32.55763880-1

Images