WO2020030822A1 - Hepatoblast organoids - Google Patents

Hepatoblast organoids Download PDF

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WO2020030822A1
WO2020030822A1 PCT/EP2019/071587 EP2019071587W WO2020030822A1 WO 2020030822 A1 WO2020030822 A1 WO 2020030822A1 EP 2019071587 W EP2019071587 W EP 2019071587W WO 2020030822 A1 WO2020030822 A1 WO 2020030822A1
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hepatoblasts
population
cells
medium
hepatoblast
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Ludovic Vallier
Alexander Ross
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Cambridge Enterprise Limited
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Definitions

  • a sixth aspect provides a method of treating a liver disease comprising administering an isolated population of the fourth aspect or a scaffold of the fifth aspect to an individual in need thereof.
  • a non-canonical Wnt signalling potentiator is a compound that stimulates, promotes or increases the activity of the non-canonical Wnt signalling pathway.
  • the non- canonical Wnt signalling pathway is a b-catenin- independent pathway involved in tissue polarity and morphogenetic processes in vertebrates (Komiya, Y. & Habas, R. Organogenesis 4, 68-75 (2008); Patel, V. et ai . Hum. Mol. Genet. 17, 1578-1590 (2008);
  • Hepatoblasts expanded as described herein may be cultured or maintained using standard mammalian cell culture techniques or subjected to further manipulation or processing.
  • the cell populations produced as described herein may be stored, for example by lyophilisation and/or
  • Hepatoblasts, hepatocytes or cholangiocytes for use in modelling and screening may be in the form of organoids (hepatoblast organoids, HOs), sub-organoid clusters or individual cells (hepatoblasts) produced, for example by disruption of organoids.
  • organoids hepatoblast organoids, HOs
  • sub-organoid clusters or individual cells (hepatoblasts) produced, for example by disruption of organoids.
  • the one or more media may be a 1x formulation or a more concentrated formulation, e.g. a 2x to 250x concentrated medium formulation.
  • a 1x formulation each ingredient in the medium is at the concentration intended for cell culture, for example a concentration set out above.
  • a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture.
  • Concentrated culture media are well known in the art. Culture media can be concentrated using known methods e.g. salt precipitation or selective filtration.
  • a concentrated medium may be diluted for use with water (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.
  • the single cell suspension was resuspended in the hepatoblast organoid media (HO-M) for liver samples.
  • HO-M hepatoblast organoid media

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Abstract

This invention relates to culture methods that allow the efficient long-term expansion of human hepatoblasts in the form of organoids. The methods comprise culturing primary immature human liver cells in a hepatoblast expansion medium comprising epidermal growth factor (EGF), a ΤΟΓβ inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor, to produce an expanded population of human hepatoblasts. The human hepatoblasts are bipotent and may be further differentiated into hepatocytes and cholangiocytes. Culture methods, cell populations and uses thereof are provided.

Description

Hepatoblast Organoids
Field
The present invention relates to the in vitro generation of organoids comprising hepatoblasts, for example for use in modelling liver disease or development, drug screening and regenerative medicine.
Background
The liver is unique as an organ in the broad spectrum of its functions during adult life (Gille et al., 2010). These functions include iron, vitamin and mineral homeostasis, and the detoxification of alcohol, drugs and other chemicals circulating in the bloodstream. The liver also synthesises bile for the digestion of fat and secretes blood clotting factors and serum proteins such as albumin (Alb) that represents the most abundant protein in the plasma. Finally, the liver plays an essential metabolic activity by storing glycogen and lipids (Gille et al., 2010). Most of these activities are managed by hepatocytes which constitute over 80% of the liver mass (Blouin, Bolender and Weibel, 1977). The cholangiocytes from the biliary tree also have an essential role in collecting waste, processing bile acids and possibly in hepatic regeneration after injury (Deng et al., 2018). Importantly, disorders affecting functions of these two cell types can be life-threatening and organ transplantation remains the only treatment for end stage liver diseases. However, the number of organ donors has remained constant for the past 10 years while the demand for liver transplantation has more than doubled in the meantime (Hopkinson and Allen, 2017). This situation is likely to worsen in the foreseeable future due to the pandemic of liver disease associated with obesity and non-alcoholic fatty liver disease (Younossi et al., 2016). Importantly, understanding liver organogenesis, especially the mechanisms by which hepatocytes and cholangiocytes are generated during development, could help to develop alternative regenerative approach such as cell-based therapies and also in vitro systems for disease modelling and drug screening.
Studies in the mouse and human have clearly established that hepatocytes and cholangiocytes originate from bi-potential progenitors named hepatoblasts (Yang et al., 2017)(Shiojiri et al., 2001 ). More precisely, hepatoblasts represent a proliferative population first detected in the liver bud at 5 weeks post conception in humans. These progenitors persist until 20 weeks of development playing a crucial role in liver
organogenesis. They are characterised by their capacity to express markers specific for both hepatocytes and cholangiocytes such as Epithelial Cell Adhesion Molecule (EPCAM), Albumin and Alpha-Fetoprotein (AFP). Importantly, the signalling pathways involved in hepatoblast self-renewal and differentiation remain to be fully uncovered as contradicting reports have suggested that Wnt signalling could block or promote differentiation of hepatoblasts toward hepatocytes and cholangiocytes in the mouse embryo (Decaens et al., 2008; Tan et al., 2008). On the other hand, TGF has been shown to direct differentiation of hepatoblasts toward the biliary lineage. However, such mechanisms have not been confirmed in human and other pathways may be involved (Clotman et al., 2005). Of note, transplantation of isolated human hepatoblasts have shown that these cells can colonise the rat adult liver in hepatic failure models thereby demonstrating their interest for regenerative medicine applications (Oertel et al., 2003). Thus, derivation of human hepatoblasts may provide a unique platform not only to study human liver development but also to produce cells with clinical interests. However, the development of robust protocols to grow human hepatoblasts in vitro have remained elusive, thereby limiting the study and utilisation of these progenitors. Organoid technology consists of growing stem cells, and more broadly a combination of progenitor and epithelial cells, in three-dimensional culture conditions. This technology was first developed using intestinal stem cells (Sato et al., 201 1 ) and then was rapidly applied to a diversity of adult organs including the liver, pancreas, prostate, mammary gland, biliary three and lungs (Huch and Koo, 2015). In addition, similar culture conditions have been combined with differentiation of human Pluripotent Stem Cells (hPSCs) to generate brain, kidney, gut and liver organoids which mimic the cellular complexity and architecture of native organs. The interest of organoids has been broadly demonstrated for basic studies, disease modelling (Schwank et al., 2013), drug screening (van de Wetering et al., 2015), and also regenerative medicine applications (Cruz-Acuha et al., 2017). Nonetheless, this technology has been more rarely applied to foetal tissues in the context of developmental studies. Indeed, primary organoids have only been derived so far from the foetal gut and lung tips. Interestingly, the resulting cells display capacity of differentiation in vitro (Kraiczy et al., 2017) and in vivo after co-culture with mesenchymal cells (Yui et al., 2018).
The existence of liver stem cells in the adult liver has been reported (Huch et al 2015, Hu et al 2018).
However, the resulting organoids maintain a strong biliary identity. More recently, human fetal hepatocyte organoids have been derived from livers of the second trimester. However, these cells are committed to a hepatocytic fate and no longer have the capacity to differentiate into cholangiocytes.
Summary
The present inventors have developed culture methods that allow the efficient long-term expansion of human hepatoblasts in the form of organoids. Populations of hepatoblasts expanded as described herein may be differentiated into hepatocytes and cholangiocytes and may be useful for example in regenerative medicine.
A first aspect of the invention provides a method for producing an expanded population of human hepatoblasts in vitro comprising:
(i) providing a population of primary immature human liver cells and;
(ii) culturing the population in a hepatoblast expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor, to produce an expanded population of human hepatoblasts.
Preferably, the immature human liver cells are cultured in three-dimensional culture in the expansion medium. The expanded populations of hepatoblasts may form organoids in the expansion medium.
In some embodiments, the method may further comprise disrupting the organoids to produce a population of isolated hepatoblasts. The isolated hepatoblasts may be further cultured in the expansion medium to expand or propagate the population.
The human hepatoblasts produced by the method of the first aspect are bipotent and are capable of differentiation into cholangiocytes and hepatocytes.
A second aspect of the invention provides a method for producing a population of human cholangiocytes in vitro comprising (i) producing an expanded population of human hepatoblasts by a method according to the first aspect; and,
(ii) culturing the expanded population in a differentiation medium comprising TGF to produce a population of human cholangiocytes.
A third aspect of the invention provides a method for producing a population of human hepatocytes in vitro comprising
(i) producing an expanded population of hepatoblasts by a method according to the first aspect; and,
(ii) culturing the expanded population in a hepatocyte culture medium to produce a population of human hepatocytes.
A fourth aspect of the invention provides an isolated population of human hepatoblasts, hepatocytes or cholangiocytes produced by a method according to any one of the first to third aspects.
A fifth aspect of the invention provides a biocompatible scaffold comprising an isolated population of the fourth aspect.
A sixth aspect provides a method of treating a liver disease comprising administering an isolated population of the fourth aspect or a scaffold of the fifth aspect to an individual in need thereof.
A seventh aspect of the invention provides a method of screening comprising;
contacting an isolated population of the fourth aspect or a scaffold of the fifth aspect with a test compound, and;
determining the effect of the test compound on the population or scaffold and/or the effect of the population or scaffold on the test compound.
Preferably, the population contacted with the test compound is in the form of organoids.
An eighth aspect of the invention provides a kit for the production of an expanded population of hepatoblasts comprising a hepatoblast expansion medium that comprises epidermal growth factor (EGF), a TGF inhibitor, non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor.
The kit may further comprise a hepatocyte culture medium and/or a differentiation medium comprising TGF .
Other aspects and embodiments of the invention are described in more detail below.
Brief Description of the Figures
Figure 1 shows the basic characterisation of Hepatoblast Organoid (HO). A) qPCR gene expression data comparing HO (n=25, p3-15, 9 donors) FBO (n=9, p3-15, 2 donors), PFL (n=3 donors) and PAH (n = 6, 3 donors, 6 plates) for the genes listed above. Expression is relative to housekeeping gene RPLP-0, mean+/- SEM. B) Representative brightfield images of HO (scale bar = 400uM, top, 200uM, bottom). C)
Immunofluorescent staining of HO for HNF4a (green, top left), AFP (red, top right), ALB (yellow, bottom left), and overlay image (bottom right). Nuclear staining (blue) was performed using Hoechst dye. D)
Concentration of secreted proteins in the culture medium of HO (n=16, 1 1 donors, p2-15) and FBO (n=4, p11-15, 2 donors) after 48 hours of freshly applied medium as detected by ELISA and normalised to cell number. E) CYP3A5/7 and CYP3A4 activity measured by chemiluminescent assay (relative luminescence units, RLU) with luciferin PFBE (top) and IPA (bottom), normalised to cell number for HO (n=3, p14), FBO, (n=2, p14), and PAH (n=6).
Figure 2 shows the characterisation of Primary Foetal Liver (PFL). Sections of an 8 post-conceptional week (pew) primary foetal liver, stained using haematoxylin and eosin (H&E, far left), immunohistochemistry for AFP (center left), KRT19 (center right), and hepatocyte marker Hep Par-1 (HEPPAR1 ) (far right). B) tSNE plot derived from whole primary foetal liver scRNAseq data (n=2, 6pcw, 1406 cells) demonstrating independent clustering into six groups, labelled according to cellular identity (stellate, hepatoblast, haematopoietic stem/progenitor cells (HSPC), megakaryocytes, lymphocytes, and Kupffer cells). C)
Heatmap comparing the top 10 differentially expressed genes differentiating each cluster as identified on the tSNE from B. D) Violin plots displaying markers distinct to each cluster of primary foetal liver cells. E) Feature plots displaying cellular gene expression for each gene listed, based upon the tSNE in E, with darker points representing higher gene expression in that cell, and lighter points demonstrating lower gene expression.
Figure 3 shows a comparison of HO to primary foetal liver. tSNE analysis based on scRNAseq data for HO (n=2 lines, p8, 1973 cells) and PFL hepatoblast (n=2 lines, 308 cells) demonstrating independent clustering based on original cellular identity (B) Feature plots displaying cellular gene expression for each gene listed, based upon the tSNE in A, with darker colored points representing higher gene expression in that cell, and lighter points demonstrating lower gene expression. C) Violin plots demonstrating the expression of key hepatic markers across each cellular population of PFL-hepatoblasts and HO-hepatoblasts. D) Principal component analysis of scRNAseq data from PFL-hepatoblasts (red), HO (green), CoP (turquoise) and Co (purple). E) Violin plots of scRNAseq data from PFL hepatoblasts, HO, CoP and Co, demonstrating the respective hepatoblast and cholangiocytic profiles. F) Heatmap comparing the top genes for each cluster identity for PFL hepatoblast, HO, CoP and Co. Yellow = higher expression, purple = lower expression.
Figure 4 shows In vitro differentiation of HO to cholangiocyte fate. 4A shows representative brightfield images of untreated HO (top), and TGF-beta (TGF-b) treated HO (bottom). B) Change in gene expression before and after treatment with TGF-b for HO (n=9, 7 lines, p4-p9) and FBO (n=4, 2 lines, p4&p9), measured by qPCR. C) Immunofluorescent staining for KRT19 expression (green, upper panel) and ALB expression (yellow, lower panel) in HO and HO treated with TGF-b (HO-TGF, center), with FBO as comparison (right).
D) scRNA-seq derived violin plots demonstrating distribution of gene expression in cells from different groups, comparing PFL hepatoblasts (PFL, n=308), HO (n=1976), HO-TGF (n=461 ), CoP (n=2238) and Co (n=913). E) Pseudotime analysis plot of sc-RNA sequencing data demonstrating a progression of cellular identity from a common time point (darker), and transition through two more differentiated branches. F) Identical plot from E, labelled according to cellular identity PFL hepatoblasts (orange), HO (yellow), HO-TGF (green), CoP (blue), and Co (purple), demonstrating two independent branches of CoP and Co, with HO- TGF aligning with Co. G) Plots of relative gene expression for each cell ordered according to pseudotime from left to right, demonstrating the trends in gene expression during pseudotime. Figure 5A shows representative brightfield images of HO (left) and HO treated with Hepatozyme (HZ, right). B) Immunofluorescent staining demonstrating ALB (yellow) and alpha-fetoprotein (AFP, red) in untreated HO (left) and HO treated with HZ (HO-HZ, right) C) Relative gene expression of selected hepatic markers measured by qPCR of HO (n=3 lines, p4) and HO after differentiation with WNT withdrawal and hepatozyme (n=3 lines, p4). Maturation markers SERPINA1 and G6PC were significantly upregulated, whilst AFP was downregulated, with no significant change in ALB. E) Violin plots demonstrating gene expression profiles across the scRNAseq data sets from PFL-hepatoblasts (n=308), HO (n=1986), and hepatozyme-treated HO (HO-HZ, n=4006). F) Pseudotime plots demonstrating trend of cells from earlier developmental time point (top, darker blue), to a later time point (light blue) (upper panel), with the same transition labelled according to cellular identity (lower panel, PFL (red), HO (green) and HO-HZ (blue). G) Plots of gene expression for each cell ordered according to pseudotime from left to right, demonstrating the trends in gene expression.
Figure 6 shows violin plots of normalised gene expression comparing PFL, HBO, BO and DBO.
Figure 7 shows a comparison of three human liver organoid systems. (A) Principal component analysis of differentiated biliary organoids (DBO, n =2), fetal hepatocyte organoids (FHO n = 6), hepatoblast organoids (HBO, n = 4), primary adult hepatocytes (PAH, n=2), and primary fetal hepatocytes (PFH, n=2). (B) Jitter plots comparing scaled expression values for DBO, FHO, HBO, PAH, and PAH for selected marker genes.
Figure 8 shows hepatoblast organoid differentiation into hepatocytes and cholangiocytes in vivo. (A) Human ALB in the serum of HBO recipient mice at the time of sacrifice (27 days after engraftment). (B)
Quantification (percentage) of KRT19-positive cells within KRT18-positive nodules following immunostaining for KRT19 (green) and KRT18 (red) identified nodules with no KRT19 (NEGATIVE), nodules that contained some KRT19-positive cells (MIX), and nodules in which cells self-assembled to resemble bile ducts and were nearly entirely positive for KRT19 (POSITIVE), (scale bars = 20uM).
Detailed Description
This invention relates to the expansion of primary hepatoblasts in vitro using a hepatoblast expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor. Optionally, the hepatoblasts may be further differentiated into hepatocytes or cholangiocytes. This may be useful for example in modelling liver development, drug screening and regenerative medicine, for example in the treatment of liver disease,
The hepatoblasts described herein may be expanded from immature liver cells. Immature liver cells may include neonatal and prenatal liver cells.
Immature liver cells may be primary cells isolated from a sample of immature liver tissue. Hepatoblasts are not present in mature liver tissue, for example in individuals of more than 2 years old. Hepatoblasts are present in immature liver tissue. In some embodiments, a sample of immature liver tissue may be obtained from an individual of 2 years or less, 1 year or less 6 months or less or 1 month or less (e.g. a neonate (Tolosa et al (2014) Cell Transplant. 23 (10) 1229-1242)). For example, neonatal liver cells may be employed. In other embodiments, immature liver cells may be obtained from a sample of foetal liver tissue pre-birth, for example a sample of tissue having a gestational age of 5 weeks or more, 6 weeks or more, 8 weeks or more or 12 weeks or more, for example to 5 to 20 weeks, or 6 to 12 weeks. Suitable samples of foetal liver tissue may, for example, be obtained from patients following elective terminations. Immature liver cells may express hepatic markers, such as ALB, AFP and EpCAM.
The immature liver cells may be isolated from a sample of immature liver tissue using any convenient technique. For example, a sample of immature liver tissue may be dissociated into a single cell suspension by enzymatic treatment, for example with collagenase and hyaluronidase, and the suspension sorted for EpCAM positive cells using anti-EpCAM coated microbeads.
Immature liver cells for use as described herein may be mammalian cells, for example human, mouse or rat cells. Preferably, the immature liver cells are human.
Populations of hepatoblasts may be expanded from the immature liver cells. Hepatoblasts are proliferative bipotent hepatic progenitor cells that are capable of differentiation into hepatocytes or cholangiocytes.
Hepatoblasts are involved in liver organogenesis in the developing mammalian foetus and express markers specific for both hepatocytes and cholangiocytes, such as Epithelial Cell Adhesion Molecule (EpCAM), Albumin and Alpha-Fetoprotein (AFP).
Preferably the hepatoblasts are human hepatoblasts.
The hepatoblasts are expanded in a hepatoblast expansion medium. This is a cell culture medium that supports the proliferation of hepatoblasts in the form of organoids (hepatoblast organoids or HOs).
The hepatoblast expansion medium is a nutrient medium which comprises epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor. In preferred embodiments, the hepatoblast expansion medium is a chemically defined medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor.
Epidermal Growth Factor (EGF; NCBI GenelD: 1950, nucleic acid sequence NM_001178130.1 Gl:
296011012; amino acid sequence NP_001 171601.1 Gl: 296011013) is a protein factor which stimulates cellular growth, proliferation and cellular differentiation by binding to an epidermal growth factor receptor (EGFR). EGF may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g. R&D Systems, Minneapolis, MN; Stemgent Inc, USA). Suitable concentrations of EGF for expanding cholangiocyte organoids as described herein may be readily determined using standard techniques. For example, the expansion medium may comprise 2 to 500ng/ml EGF, preferably about 20ng/ml.
A TGF inhibitor is a compound that reduces, blocks or inhibits TGF signalling through the TGF RI and TGF RII receptors. Suitable TGF inhibitors include A83-01 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4- quinoiinyi)-1 H-pyrazoie-1-carbothioamide), D4476 (4-[4-(2,3-Dihydro-1 ,4-benzodioxin-6-yl)-5-(2-pyridinyl)- 1 H-imidazoI-2-yl]benzamide), GW788388 (4-[4-[3-(2-Pyrid inyl)- 1 H-pyrazol-4-yl]-2-pyrid i nyl]-A/-(tetrahyd ro- 2H-pyran-4-yl)-benzamide), IN 1 130 (3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1 /-/-imidazol-2- yl]methyl]benzamide), LY364947 (4-[3-(2-Pyridinyl)-1 tf-pyrazol-4-yl]-quinoline), SB525334 (6-[2-(1 , 1- DimethyIethyl)-5-(6-methyl-2-pyridinyl)-1 H-imidazoI-4-yl]quinoxaline), SB431542 (4-(5-Benzol[1 ,3]dioxol-5-yl- 4-pyrldin-2-yl-1 H-imidazol-2-yl)-benzannide hydrate; Sigma, Tocris Bioscience, Bristol UK), SB-505124 (2-(5- benzo[1 ,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) and soluble protein factors, such as lefty (e.g. human lefty 2: NP_003231.2 Gl:27436881 ), cerberus (e.g. human Cerberus 1 : NP_005445.1 Gl:4885135) and follistatin (e.g. human foistatin: NP_006341.1 Gl:5453652). Suitable TGF inhibitors are available from commercial suppliers. In some embodiments, the TGF inhibitor may be A8301 , for example at 1 to 1000mM, preferably about 50mM.
A non-canonical Wnt signalling potentiator is a compound that stimulates, promotes or increases the activity of the non-canonical Wnt signalling pathway. The non- canonical Wnt signalling pathway is a b-catenin- independent pathway involved in tissue polarity and morphogenetic processes in vertebrates (Komiya, Y. & Habas, R. Organogenesis 4, 68-75 (2008); Patel, V. et ai . Hum. Mol. Genet. 17, 1578-1590 (2008);
Strazzabosco, M. & Somlo, S. Gastroenterology 140, (201 1 ).) Components of the non- canonical Wnt signalling pathway include Wnt4, Wnt5a, Wnt1 1 , LRP5/6, Dsh, Fz, Daaml , Rho, Rac, Prickle and
Strabismus. Suitable methods for determining the activity of the non- canonical Wnt/PCP signalling pathway are well known in the art and include ATF-2-based reporter assays (Ohkawara et al (201 1 ) Dev Dyn 240 (1 ) 188-194) and Rho-associated protein kinase (ROCK)-based assays. A non- canonical Wnt signalling potentiator may selectively potentiate non-canonical Wnt signalling or more preferably, may potentiate both the non-canonical Wnt signalling and the canonical Wnt signalling pathway (i.e. a Wnt signalling agonist).
Preferred non- canonical Wnt signalling potentiators include the Wnt signalling agonist R-spondin. R-spondin is a secreted activator protein with two cysteine-rich, furin-like domains and one thrombospondin type 1 domain that positively regulates Wnt signalling pathways. Preferably, R-spondin is human R-spondin.
R-spondin may include RSP01 (GenelD 284654 nucleic acid sequence reference NM_001038633.3, amino acid sequence reference NP_001033722.1 ), RSP02 (GenelD 340419 nucleic acid sequence reference NM_001282863.1 , amino acid sequence reference NP_001269792.1 ), RSP03 (GenelD 84870, nucleic acid sequence reference NM_032784.4, amino acid sequence reference NP_1 16173.2) or RSP04 (GenelD 343637, nucleic acid sequence reference NM_001029871.3, amino acid sequence reference
NP_001025042.2). R-spondin is readily available from commercial sources (e.g. R&D Systems,
Minneapolis, MN). Suitable concentrations of R-spondin for expanding heptoblasts as described herein may be readily determined using standard techniques. For example, the expansion medium may comprise 50ng/ml to 5pg/ml R-spondin, preferably about 500ng/ml.
A canonical Wnt potentiator is compound that stimulates, promotes or increases the activity of the canonical Wnt/ b-catenin-sig nailing pathway. The canonical Wnt signalling pathway is a b-catenin-dependent pathway involved in the regulation of gene expression (Klaus et al Nat. Rev. Cancer (2008) 8 387-398; Moon et al (2004) Nat. Rev. Genet. 5 691-701 ; Niehrs et al Nat Rev Mol. Cell Biol. (2012) 13 763-779). Suitable methods for determining the activity of the canonical Wnt signalling pathway are well known in the art and include the TOP-flash assay (Molenaar et al Cell. 1996 Aug 9; 86(3):391-9) and assays for b-catenin.
Suitable Wnt signalling activators include Wnt ligands, glycogen synthase kinase 3b (63K3b) inhibitors; b- catenin and activators of b-catenin. ObK3b inhibitors inhibit the activity of glycogen synthase kinase 3b (Gene ID 2932: EC2.7.11.26).
Preferred canonical Wnt potentiators include (hetero)arylpyrinnidines (Gilbert et al Bioorg Med Chem Lett. 2010 Jan 1 ;20(1 ):366-70), WAY-316606 (Bodine et al Bone. 2009 Jun;44(6): 1063-8), IQ1 (Miyabayashi et al PNAS USA 2007 104(13) 5668-5673), QS11 (Zhang et al (2007) PNAS USA 104(18) 7444-7448), and 2- amino-4-[3,4-(nnethylenedioxy)benzyl-annino]-6-(3-nnethoxyphenyl)pyhnnidine (Liu et al Angew Chem Int Ed Engl. 2005 Mar 18;44(13): 1987-90) 2-amino-4-[3,4-(methlyenedioxy)benzylamino]-6-(3- methoxyphenyl)pyrimidine (Wnt agonist), WAS P-1 , Wnt, for example Wnt3a, and GSK3 inhibitors, such as CHIR99021 (6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2- yl]amino]ethylamino]pyridine-3-carbonitrile), CHIR98014 (A/-6-[2-[[4-(2,4-Dichlorophenyl)-5-(1/-/-imidazol-1- yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine) alsterpaullone, kenpaullone, BIO(6-bromoindirubin- 3'-oxime (Sato et al Nat Med. 2004 Jan;10(1 ):55-63), SB216763 (3-(2,4-dichlorophenyl)-4-(1 -methyl-1 H- indol-3-yl)-1H-pyrrole-2,5-dione), and SB415286 ( 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H - pyrrole-2, 5-dione; Coghlan et al Chem Biol. 2000 Oct;7(10):793-803). Suitable canonical Wnt potentiators are available from commercial suppliers.
A ROCK inhibitor is a compound that reduces, blocks or inhibits Rho kinase (ROCK) (Liao et al (2007) J. Cardiovasc Pharmacol. 50 (1 ) 17-24). Suitable ROCK inhibitors include fasudil, Y39983 (4-[(1R)-1- Aminoethyl]-A/-1/-/-pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), azabenzimidazole-aminofurazans and Y-27632 (trans-4-[(1 R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide) and are available from commercial suppliers. For example, the hepatoblast expansion medium may comprise 1 to 100pM Y-27632, preferably about 10 mM.
The hepatoblast expansion medium may be devoid of growth factors other than the epidermal growth factor (EGF), TQRb inhibitor, non-canonical Wnt signalling potentiator, canonical Wnt potentiator and ROCK inhibitor.
Suitable hepatoblast expansion media may include HO-M (Table 1 ).
Preferably, the immature liver cells are cultured in the hepatocyte expansion medium in three-dimensional culture in the methods described above. For three-dimensional culture, the expansion medium further comprises a scaffold matrix which supports the growth and proliferation of cells in 3-dimensions and allows the hepatoblasts to assemble into organoids. For example, the expansion medium may comprise or consist of the scaffold matrix and the nutrient medium.
Suitable scaffold matrices are well-known in the art and include hydrogels, such as collagen,
collagen/laminin, compressed collagen (e.g. RAFT™, TAP Biosystems), alginate, agarose, complex protein hydrogels, such as Base Membrane Extracts, and synthetic polymer hydrogels (Gjorevski et al Nature (2016) 539 560-564), such as polyglycolic acid (PGA) hydrogels and crosslinked dextran and PVA hydrogels (e.g. Cellendes Gmbh, Reutlingen DE), inert matrices, such as porous polystyrene, and isolated natural ECM scaffolds (Engitix Ltd, London UK).
The scaffold matrix may be chemically defined, for example a collagen or densified collagen hydrogel, or non-chemical ly defined, for example a complex protein hydrogel. Preferably, the scaffold matrix in the expansion medium is a complex protein hydrogel. Suitable complex protein hydrogels may comprise extracellular matrix components, such as laminin, collagen IV, enactin and heparin sulphate proteoglycans. Complex protein hydrogels may also include hydrogels of extracellular matrix proteins from Engelbreth- Holm-Swarm (EHS) mouse sarcoma cells. Suitable complex protein hydrogels are available from commercial sources and include Matrigel™ (Corning Life Sciences) or Cultrex™ BME 2 RGF (Amsbio™ Inc). For example, the expansion medium may comprise 55% Matrigel™.
The hepatoblast expansion medium may comprise or consist of a scaffold matrix and a nutrient medium supplemented with epidermal growth factor (EGF), a TGF inhibitor, a non- canonical Wnt signalling potentiator, such as R-spondin, a canonical Wnt potentiator and a ROCK inhibitor.
A nutrient medium may comprise a basal medium. Suitable basal media include Iscove’s Modified
Dulbecco’s Medium (IMDM), Ham’s F12, Advanced Dulbecco’s modified eagle medium (DMEM) or
DMEM/F12 (Price et al Focus (2003), 25 3-6), Williams E (Williams, G.M. et al Exp. Cell Research, 89, 139- 142 (1974)), and RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508.
In some embodiments, DMEM/F12 medium may be preferred.
The basal medium may be supplemented with a media supplement, such as B27 (ThermoFisher Scientific) and/or one or more additional components, for example L-glutamine or substitutes, such as L-alanyl-L- glutamine (e.g. Glutamax™), nicotinamide, N-acetylcysteine, buffers, such as HEPES, and antibiotics such as penicillin and streptomycin. For example, the basal medium may be supplemented with 2% (v/v) B27, 20mM nicotinamide, 2mM N=acetylcysteine, 1 % L-alanyl-L-glutamine, 1 % HEPES, 1 % penicillin and 1 % streptomycin.
The nutrient medium may be a chemically defined basal nutrient medium. A chemically defined medium is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A chemically defined medium is devoid of undefined components or constituents which include undefined components, such as feeder cells, stromal cells, serum, serum albumin and complex extracellular matrices, such as Matrigel™. A chemically defined medium may be humanised. A humanised chemically defined medium is devoid of components or supplements derived or isolated from non-human animals, such as Foetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA), and mouse feeder cells. Conditioned medium includes undefined components from cultured cells and is not chemically defined. Suitable chemically defined nutrient media are well-known in the art and include DMEM/F12 supplemented with B27, nicotinamide, N-acetylcysteine, L-alanyl-L-glutamine, HEPES, penicillin and streptomycin.
The hepatoblasts described herein do not require the presence of HGF or FGF, such as FGF10 for long-term culture.
In some preferred embodiments, the primary immature liver cells are not subjected to any pre-culture before being expanded in the hepatoblast expansion medium.
The hepatoblasts may form organoids in the expansion medium. The hepatoblast organoids may be disrupted as required, such that the expanded population comprises individual cells.
The present methods allow the long-term proliferation of hepatoblasts. The hepatoblasts may be cultured in the expansion medium for multiple passages. For example, the hepatoblasts may be cultured for 25 or more, 30 or more, 40 or more or 50 or more passages. A passage may take 7-14 days, preferably about 10 days.
The hepatoblasts may be passaged by digesting the scaffold matrix, harvesting organoids by centrifugation and disrupting the organoids into individual hepatoblasts. The hepatoblasts may be re-suspended and cultured as described above in the expansion medium where they reform into organoids.
Suitable techniques for cell culture are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451 ; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN:
0471453293, Ho WY et al J Immunol Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) Basic Cell Culture Protocols’ by J. Pollard and J. M. Walker (1997),‘Mammalian Cell Culture: Essential Techniques’ by A. Doyle and J. B. Griffiths (1997),‘Human Embryonic Stem Cells’ by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside’ by A. Bongso (2005), Peterson & Loring (2012)Human Stem Cell Manual: A Laboratory Guide Academic Press and‘Human Embryonic Stem Cell Protocols’ by K. Turksen (2006). Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37°C, 21 % Oxygen, 5% Carbon Dioxide. Media is preferably changed every two days and cells allowed to settle by gravity
The population of hepatoblasts may be expanded 1010 fold or more, 1020 fold or more, 1030 fold or more, 104° fold or more or 105° fold or more as organoids in the expansion medium as described herein.
The population of hepatoblasts proliferates in the expansion medium and assembles into organoids.
Organoids are three-dimensional multicellular assemblies that comprise hepatoblasts linked by tight junctions. The organoids may display the morphology or physical characteristics of hepatoblasts and may for example form dense, branching or spherical structures. The morphology and physical characteristics of organoids may be determined by standard microscopic procedures. The hepatoblasts in the expanded population are bipotent progenitor cells and are capable of differentiation into either hepatocytes or cholangiocytes. The hepatoblasts may express genes important for hepatic function, including detoxification enzymes, such as cytochrome p450 3A7 (CYP3A7), and cytochrome p450 3A4 (CYP3A4), serum proteins, such as albumin, APOB, alpha-fetoprotein and TF and clotting factors, such as SERPINA1. The hepatoblasts may show very high expression of ALB, moderate expression of MUC20 and low or no expression of Factor II (prothrombin) and Factor X (Stuart-Prower factor) (see Table 2).
The hepatoblasts may express both hepatocytic and cholangiocytic markers. Hepatocytic markers may include ASGR1 , CYP3A4, albumin (ALB), alpha-1 -antitrypsin (SERPINA1 ), APOH, APOM, APOC1 ,
CYP1 A1 , RBP4, HP, AHSG and TTR. In some embodiments, the hepatoblasts may express similar levels of hepatocytic markers to primary adult hepatocytes. Cholangiocytic markers may include KRT19, EPCAM and SOX9. In some embodiments, the hepatoblasts may express lower levels of cholangiocytic markers than primary cholangiocytes.
The hepatoblasts in the expanded population may express one or more foetal markers. Foetal markers include alpha-fetoprotein (AFP), DLK1 , CYP2E1 and CYP3A7. The hepatoblasts in the expanded population may also express serum proteins, such as APOB and TF.
In some preferred embodiments, the hepatoblasts in the expanded population may express the markers ALB, AFP, SERPINA1 , APOE, APOC3, APOA1 , APOA2, TTR, AHSG, ARG1 , ASGR1 and ITIH1 and may not express the markers KRT7 and CTFR.
The expression of cell markers may be determined by any suitable technique, including
immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, fluorescence activated cell sorting (FACS), and enzymatic analysis. A cell may be considered to express a cell marker if the expression of the marker in the cell is detectable by one or more of the above techniques.
The hepatoblasts in the expanded population may display long term stability. For example, the hepatoblasts may be maintained in culture for at least 2 months without DNA copy number or other genetic abnormalities and with stable, preferably homogeneous, expression of hepatic markers, such as EpCAM, ASGR1 , AFP, A1AT, ALB, CYP3A4, CYP3A7, HNF4a, HNF1 b and APOB. The presence of genetic abnormalities may be determined for example by comparative genomic hybridisation (CGH).
The hepatoblasts in the expanded population may display high proliferative potential. For example, the hepatoblasts may display a doubling time of 2-4 days, for example about 3 days.
The expanded population of hepatoblasts, whether in the form of organoids or individual cells, may be free or substantially free from other cell types i.e. the population of hepatoblasts may be homogeneous or substantially homogeneous. For example, the population may contain, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more hepatoblasts, following culture in the medium. Preferably, the population of hepatoblasts is sufficiently free of other cell types that no purification is required. Alternatively, the hepatoblasts may be further purified, for example by magnetic bead sorting.
Hepatoblasts expanded as described herein may be cultured or maintained using standard mammalian cell culture techniques or subjected to further manipulation or processing. In some embodiments, the cell populations produced as described herein may be stored, for example by lyophilisation and/or
cryopreservation. The hepatoblasts may be stored as organoids, sub-organoid assemblies or individual cells. Suitable storage methods are well known in the art. For example, the hepatoblasts may be suspended in a cryopreservation medium (for example, Cellbanker™ (AMS Biotechnology Ltd, UK)) and frozen, for example at -70°C or below.
In some embodiments, the hepatoblasts in the expanded population may be further purified and/or isolated. For example, the hepatoblasts in the expansion medium may be separated from stellate cells. Conveniently, hepatoblasts may be distinguished from stellate cells by the expression of EpCAM and separated using routine techniques, such as flow cytometry.
In some embodiments, hepatoblasts expanded by the methods described above may be used directly, for example in regenerative medicine applications. In other embodiments, the hepatoblasts may be further differentiated in vitro.
The hepatoblasts may be differentiated into cholangiocytes by culturing in a differentiation medium that supports TGF signalling. For example, the differentiation medium may comprise TGF or a TGF ligand such as activin. In some embodiments, the hepatoblasts may be cultured in the absence of canonical Wnt signalling. For example, the differentiation medium may lack canonical Wnt potentiators, such as Wnt3a. Suitable differentiation media include CO-M that lacks Wnt3a and A8301 and is supplemented with TGF . The constituents of CO-M are shown in Table 1.
Culturing in the differentiation medium may increase the expression of cholangiocytic markers, such as KRT19, and reduce the expression of hepatocytic markers, such as ALB and AFP. For example, cholangiocytes produced as described herein may express cholangiocytic markers and not hepatocytic markers.
The hepatoblasts may be differentiated into hepatocytes by culturing in hepatocyte culture medium. The hepatoblasts may be cultured in the absence of canonical Wnt potentiators. The hepatoblasts may be cultured in the absence of canonical Wnt signalling. For example, the hepatocyte culture medium may lack canonical Wnt potentiators, such as Wnt3a. A hepatocyte culture medium is a medium that supports the culture of hepatocytes. In some embodiments, hepatocyte culture medium may be a chemically defined medium devoid of growth factors other than oncostatin-M. Suitable hepatocyte culture media are available from commercial suppliers and may include hepatocyte cell culture medium (#H1777 Sigma Aldrich), primary HEP medium (Cellartis) or Hepatozyme™ (Life Technologies), with supplementary oncostatin-M. Culturing in the hepatocyte culture medium may reduce the expression of cholangiocytic and foetal markers, such as AFP, and increase the expression of hepatocytic and mature markers, such as G6PC. For example, hepatocytes produced by differentiation of hepatoblasts as described herein may express hepatocytic markers and not cholangiocytic or foetal markers.
The hepatocytes and cholangiocytes produced by differentiation of hepatoblasts may be fully differentiated and may lack proliferative capacity in vitro. The hepatocytes and cholangiocytes produced by differentiation of hepatoblasts may display the functional activity of mature hepatocytes and cholangiocytes.
The population of hepatoblasts, cholangiocytes or hepatocytes produced as described herein may be admixed with other reagents, such as buffers, carriers, diluents, preservatives, and/or pharmaceutically acceptable excipients. Suitable reagents are described in more detail below. A method described herein may comprise admixing the population with a therapeutically acceptable excipient to produce a therapeutic composition. The admixed hepatoblasts may be in the form of organoids, sub-organoid assemblies or individual cells.
In some embodiments, the hepatoblasts, cholangiocytes or hepatocytes produced as described herein may be useful in therapy. For therapeutic applications, the hepatoblasts, cholangiocytes or hepatocytes are preferably clinical grade cells. Populations of hepatoblasts, cholangiocytes or hepatocytes for use in treatment are preferably produced from immature liver cells as described herein using chemically defined media. The hepatoblasts may be in the form of organoids, sub-organoid assemblies or individual cells, depending on the specific application.
The population of hepatoblasts, cholangiocytes or hepatocytes may be transplanted, infused or otherwise administered into the individual. Suitable techniques are well known in the art.
In some preferred embodiments, the population of hepatoblasts, cholangiocytes or hepatocytes produced as described herein may be admixed with a biocompatible scaffold.
A biocompatible scaffold may be seeded with hepatoblasts, cholangiocytes or hepatocytes expanded as described above. For example, individual hepatoblasts or sub-organoid assemblies of hepatoblasts may be injected on or into a scaffold or mixing into the scaffold during the manufacturing process. The scaffold containing the hepatoblasts may then be cultured in expansion medium, such that the hepatoblasts populate the scaffold. The hepatoblasts may proliferate within the scaffold and assemble into organoids.
Suitable biocompatible scaffolds may include hydrogels, such as fibrin, chitosan, glycosaminoglycans, silk, fibrin, fibronectin, elastin, collagen, glycoproteins such as fibronectin, or polysaccharides such as chitin, or cellulose collagen, collagen/laminin, densified collagen, alginate, agarose, complex protein hydrogels, such as Base Membrane Extracts, bio-organic gels, and synthetic polymer hydrogels, such as polylactic acid (PLA) polyglycolic acid (PGA), polycapryolactone (PCL) hydrogels, crosslinked dextran and PVA hydrogels (e.g. Cellendes Gmbh, Reutlingen DE), inert matrices, such as porous polystyrene, polyester, soluble glass fibres porous polystyrene, and isolated natural ECM scaffolds, for example decellularized gall bladder and bile duct scaffolds (Engitix Ltd, London UK). The scaffold may be biodegradable.
The size or shape of the scaffold is dependent on the intended application. Suitable scaffold shapes may for example include patches, sheets and tubes, including straight and branched tubes, with diameters up to for example 10-12 mm.
Hepatoblasts, cholangiocytes or hepatocytes cultured within a biocompatible scaffold organize into a functional hepatic tissue, for example functional hepatocytic or biliary tissue. For example, cholangiocytes cultured within a biocompatible scaffold may organize into a functional biliary epithelium that displays one or more properties of the biliary epithelium. Hepatoblasts or hepatocytes cultured within a biocompatible scaffold may organize into a functional hepatocytic tissue that displays one or more properties of hepatocytic tissue.
An aspect of the invention provides an isolated population of hepatoblasts, cholangiocytes or hepatocytes produced by a method described above. The hepatoblasts may be in the form of organoids, sub-organoid clusters or individual cells.
A population of hepatoblasts, cholangiocytes or hepatocytes generated as described herein may be substantially free from other cell types. For example, the population may contain 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more hepatoblasts, following culture in the expansion medium. The presence or proportion of hepatoblasts, cholangiocytes or hepatocytes in the population may be determined through the expression of biliary and/or hepatic markers as described above.
Preferably, the population of hepatoblasts, cholangiocytes or hepatocytes is sufficiently free of other cell types that no purification is required. If required, the population of cells or organoids may be purified by any convenient technique, including FACS.
In some embodiments, the hepatoblasts, cholangiocytes or hepatocytes may be engineered to express a heterologous protein, for example a marker protein, such as GFP, or an enzyme and/or to reduce or prevent expression of one or more endogenous protein, for example proteins associated with immunogenicity, such as HLA antigens.
The isolated population of hepatoblasts, cholangiocytes or hepatocytes may be within a biocompatible scaffold. Another aspect of the invention provides a biocompatible scaffold comprising an isolated population of hepatoblasts produced by a method described herein. Suitable scaffolds are described above.
Another aspect of the invention provides a collection of isolated populations of hepatoblasts as described herein, wherein each population in the collection comprise a different set of tissue markers. For example, each population in the collection may have a different antigenic profile or HLA type. Populations of hepatoblasts with different antigenic profiles may be generated from immature liver cells that display different antigenic profiles. A population in the collection may be matched to a recipient individual in which the hepatoblasts do not elicit a host immune response. This may be useful in allogenic cell therapy.
Aspects of the invention also extend to a pharmaceutical composition, medicament, drug or other composition comprising hepatoblasts, hepatocytes or cholangiocytes produced as described herein in solution or in a biocompatible scaffold, and a method of making a pharmaceutical composition comprising admixing such hepatoblasts with a pharmaceutically acceptable excipient, vehicle, carrier or biodegradable scaffold, and optionally one or more other ingredients.
A pharmaceutical composition containing hepatoblasts expanded in accordance with the invention or hepatocytes or cholangiocytes differentiated therefrom, may comprise one or more additional components. Pharmaceutical compositions may comprise, in addition to the hepatoblasts, hepatocytes or cholangiocytes, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabiliser, anti-oxidant, or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of the cholangiocytes. The precise nature of the carrier or other material will depend on the route of administration.
Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
The composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection. A composition may be prepared using artificial cerebrospinal fluid.
Another aspect of the invention provides a method of treatment of a liver disease comprising administering a population of hepatoblasts, hepatocytes or cholangiocytes produced as described herein to an individual in need thereof.
Another aspect of the invention provides a population of hepatoblasts, hepatocytes or cholangiocytes produced as described herein for use in a method of treatment of a liver disease in an individual in need thereof comprising administering the population to the individual.
Another aspect of the invention provides the use of a population of hepatoblasts, hepatocytes or cholangiocytes produced as described herein in the manufacture of a medicament for use in the treatment of a liver disease.
The hepatoblasts may be in the form of organoids, sub-organoid assemblies or clusters or individual cells. A liver disease is a condition in which liver tissue, such as hepatocytic tissue, in an individual is damaged, defective or otherwise dysfunctional, for example, disorders characterised by damage to or destruction of liver tissue, or aberrant liver tissue. Liver disease may include hepatitis (e.g. hepatitis A, B, C, D, E, G or K), cirrhosis, hepatocellular carcinoma, non-alcoholic fatty liver disease, drug induced liver injury, alcoholic liver disease, autoimmune liver disease or an inherited metabolic disorder such as Alpha 1 Antitrypsin deficiency, a Glycogen Storage Disease, for example Glycogen Storage Disease Type 1a, Familial
Hypercholesterolemia, Hereditary Tyrosinaemia, Crigler Najjar syndrome, ornithtine transcarbamylase deficiency, or factor IX deficiency or other haemophilia, haemochromatosis, Wilson's disease, Dubin- Johnson syndrome, familial amyloidosis, and Refsum’s disease.
In some preferred embodiments, the liver disease is haemophilia. A population of hepatoblasts, hepatocytes or cholangiocytes as described herein may express clotting factors, such as SERPINA1 , sufficient to rescue a haemophilia phenotype in an individual.
In some embodiments, the liver disease may be a biliary disorder. A biliary disorder is a condition in which the biliary tissue in an individual is damaged, defective or otherwise dysfunctional, for example, disorders characterised by damage to or destruction of bile ducts, aberrant bile ducts or the absence of bile ducts. Biliary disorders may include biliary tissue injury, ischaemic strictures, traumatic bile duct injury and cholangiopathies, for example inherited, developmental, autoimmune and environment-induced
cholangiopathies, such as Cystic Fibrosis associated cholangiopathy, drug induced cholangiopathy, Alagille Syndrome, polycystic liver disease, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), AIDS associated cholangiopathy, disappearing bile duct syndrome, biliary cancer, ductopenias such as adult idiopathic ductopenia, post-operative biliary complications, and biliary atresia.
In some embodiments, a population of hepatoblasts, hepatocytes or cholangiocytes may be administered to the individual in solution. The administration of a population of hepatoblasts or cholangiocytes in solution may be useful for example in the treatment of ductopenias, for example ischaemic ductopenia, congenital ductopenia, such as alagille syndrome, metabolic ductopenia, complex diseases, such as intrahepatic PSC and PBC, drug induced ductopenia, vanishing bile duct syndrome and conditions affecting the intrahepatic biliary tree
In other embodiments, a population of hepatoblasts, hepatocytes or cholangiocytes may be administered to the individual within a biocompatible scaffold. For example, a scaffold populated with hepatoblasts may be administered to the individual. For example, the administration of a population of hepatoblasts, in a scaffold may be useful for example in the treatment of biliary atresia, biliary strictures, traumatic or iatrogenic biliary injury and conditions affecting the extrahepatic biliary tree
Hepatoblasts, hepatocytes or cholangiocytes in solution or in scaffolds may be implanted into a patient by any technique known in the art (e.g. Lindvall, O. (1998) Mov. Disord. 13 Suppl. 1 :83-7; Freed, C.R., et al., (1997) Cell Transplant, 6, 201-202; Kordower, et al., (1995) New England Journal of Medicine, 332 1118- 1124; Freed, C.R.,(1992) New England Journal of Medicine, 327, 1549-1555, Le Blanc et al, Lancet 2004 May 1 ;363(9419):1439-41 ). In particular, cell suspensions may be injected or infused into the bile duct, gallbladder, portal vein, liver parenchyma, peritoneal cavity or spleen of a patient. A hepatic cell suspension may be administered intravenously, intraperitoneally or via an endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous cholangiography (PTC). A scaffold populated with hepatoblasts may be administered to the individual by surgical implantation.
Administration of a composition in accordance with the present invention is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors.
A composition comprising hepatoblasts, hepatocytes or cholangiocytes may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Populations of isolated hepatoblasts, hepatocytes or cholangiocytes produced as described above, may be useful in modelling the interaction of test compounds with hepatoblasts, for example in toxicity screening, modelling liver disorders or screening for compounds with potential therapeutic effects.
Another aspect of the invention provides the use of a population of hepatoblasts, hepatocytes or cholangiocytes produced as described herein for disease modelling and study of pathogenesis of liver diseases, such as biliary disorders.
Hepatoblasts, hepatocytes or cholangiocytes for use in modelling and screening may be in the form of organoids (hepatoblast organoids, HOs), sub-organoid clusters or individual cells (hepatoblasts) produced, for example by disruption of organoids.
A method of screening a compound may comprise;
contacting a population of hepatoblasts, hepatocytes or cholangiocytes produced by a method described herein with a test compound, and;
determining the effect of the test compound on said the hepatoblasts, hepatocytes or
cholangiocytes and/or the effect of said the hepatoblasts, hepatocytes or cholangiocytes on the test compound.
The proliferation, growth, apoptosis or viability of hepatoblasts, hepatocytes or cholangiocytes, protein production, metabolic activity of key enzymes, expression of stress response genes, or the ability of the hepatoblasts to perform one or more cell or organoid functions may be determined in the presence relative to the absence of the test compound.
A decrease in proliferation, growth, viability or ability to perform one or more cell or organoid functions is indicative that the compound has a toxic effect and an increase in growth, viability or ability to perform one or more cell or organoid functions is indicative that the compound has an ameliorative effect on the
hepatoblasts.
Gene expression may be determined in the presence relative to the absence of the test compound. For example, the expression of one or more hepatocytic or biliary marker genes may be determined. Suitable genes include genes encoding plasma proteins, such as albumin, APOB, APOA1 , FGG, C2, KNG1 , and FGA, enzymes, such as HA01 , RDH16, and ALDOB, bile proteins, such as AKR1 C4, SLC27A5, and BAAT, clotting factors, such as SERPINA1 and metabolic proteins. Combined decrease in expression is indicative that the compound has a toxic effect or can modify the functional state of the hepatoblasts. Gene expression may be determined at the nucleic acid level, for example by RT-PCR, or at the protein level, for example, by immunological techniques, such as ELISA, or by activity assays. Cytochrome p450 assays, for example, luminescent, fluorescent or chromogenic assays are well known in the art and available from commercial suppliers. The metabolism, degradation, or breakdown of the test compound by the hepatoblasts, hepatocytes or cholangiocytes may be determined. In some embodiments, changes in the amount or concentration of test compound and/or a metabolite of said test compound may be determined or measured over time, either continuously or at one or more time points. For example, decreases in the amount or concentration of test compound and/or increases in the amount or concentration of a metabolite of said test compound may be determined or measured. In some embodiments, the rate of change in the amount or concentration of test compound and/or metabolite may be determined. Suitable techniques for measuring the amount of test compound or metabolite include mass spectrometry.
This may be useful in determining the in vivo half-life, toxicity, efficacy or other in vivo properties of the test compound. Other aspects of the invention relate to kits and their use for production of expanded populations as described herein. A kit for the production of an expanded population of hepatoblasts may comprise a hepatoblast expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non- canonical Wnt signalling potentiator, Wnt and a ROCK inhibitor.
Suitable hepatoblast expansion media are described in more detail above.
A kit may further comprise a scaffold matrix, such as Matrigel™. The scaffold matrix may be provided as part of the expansion medium or may be provided separately.
The kit may further comprise a differentiation medium and/or a hepatocyte culture medium, as described above. The expansion medium may be formulated in deionized, distilled water. The expansion medium will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. The one or more media may be frozen (e.g. at -20°C or -80°C) for storage or transport. The one or more media may contain one or more antibiotics to prevent contamination.
The kit may further comprise a dissociation buffer to dissociate immature liver cells from sample tissue. Suitable buffers include Hanks Buffered Saline Solution (HBSS) supplemented with Liberase DH (Roche Applied Science) and Hyaluronidase (Sigma-Aldrich).
The kit may further comprise cryopreservation solution. Suitable cryopreservation media are described above.
The one or more media may be a 1x formulation or a more concentrated formulation, e.g. a 2x to 250x concentrated medium formulation. In a 1x formulation each ingredient in the medium is at the concentration intended for cell culture, for example a concentration set out above. In a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture. Concentrated culture media are well known in the art. Culture media can be concentrated using known methods e.g. salt precipitation or selective filtration. A concentrated medium may be diluted for use with water (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.
The one or more media in the kit may be contained in hermetically-sealed vessels. Hermetically-sealed vessels may be preferred for transport or storage of the culture media, to prevent contamination. The vessel may be any suitable vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag.
Another aspect of the invention provides the use of a hepatoblast expansion medium for the production of an expanded population of hepatoblasts.
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term“comprising” replaced by the term“consisting of and the aspects and embodiments described above with the term“comprising” replaced by the term’’consisting essentially of.
It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.
Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Experimental
Materials and Methods
Processing of human foetal liver tissue
Primary human foetal tissue was obtained from patients undergoing elective terminations (REC-96/085). The liver was dissected from the abdominal cavity and placed into a solution containing Hanks Buffered Saline Solution (HBSS) supplemented with 1.07 Wdnsch units/ml Liberase DH (Roche Applied Science) and 70U/ml Hyaluronidase (Sigma-Aldrich), and placed on a microplate shaker at 37°C, 750rpm, for 15 minutes. The sample was subsequently washed three times in HBSS using centrifugation at 400g for five minutes each time. The single cell suspension could then be sorted for EPCAM/CD326 positive cells using CD326 microbeads (Miltenyi), according to the manufacturer’s guidelines.
Establishment of hepatoblast organoids
The single cell suspension was resuspended in the hepatoblast organoid media (HO-M) for liver samples.
To the resuspended cells was added an equal volume of Growth Factor Reduced Phenol Free Matrigel (Corning), and the mixture pipetted into 48 well plates (20uL per well). The plates were placed at 37°C for fifteen minutes to allow the mixture to set, and subsequently 200uL of fresh HO-media applied to each well.
Maintenance of cell lines
The respective culture medium was changed every 48-72 hours, and organoids mechanically passaged every 7-10 days. Organoids were passaged by scraping the gel away from the plate, pipetting the resulting solution into 1.5ml tubes, and pipetting the solution up and down to break individual organoids into pieces. If a precise cell number was required, the organoids can alternatively be processed to a single cell solution as described below, and then re-plated at the required dilution in the 55% Matrigel-medium solution.
Dissociation of HO
Organoids established in culture were dissociated to single cell suspensions for splitting/ single cell sequencing/ cell counting by first removing the media from each well and replacing with Cell Recovery Solution (Corning). The organoids in the Matrigel could then be scraped off the plate and placed on ice for 30 minutes to remove the Matrigel. The organoids should then be washed with PBS, and placed in TrypLE (ThermoFisher Scientific) for 15 mins at 37°C. Cells should finally be washed three times in PBS or basal media.
Differentiation to choloangiocytes
CO-M lacking A8301 and supplemented with 2ug/ml TGF (Thermo Fisher Scientific) was applied to HO for 7d, applying fresh media every 48 hours. Differentiation to hepatocytes
Hepatozyme medium (Gibco) supplemented with oncostatin-M (Sigma Aldrich) was applied to HO for 7d, applying fresh media every 48 hours.
Imaging of organoids
The medium was removed and replaced with 4% Paraformaldehyde solution for 20 minutes at room temperature to fix the organoids in situ, followed by three washes in PBS (each for five minutes). Fixed organoids could then be stored in PBS for later Immunohistochemistry. Immunohistochemistry was performed by permeabilizing and blocking with 0.3% Triton X-100 (Sigma-Aldrich), and 10% donkey serum (Biorad) for three hours at room temperature, and then incubated overnight at 4°C, with the primary antibody in 1 % donkey serum. Samples were then washed with three one-hour PBS washes, followed by incubation with the secondary antibody for one hour at room temperature. The samples were finally washed three times in PBS, with 1 :10000 dilution Hoechst 33258 (ThermoFisher Scientific) added to the first wash. Images can then be obtained using an appropriate microscope. qPCR analysis
RNA was extracted from cells using GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s instruction. cDNA was synthesised from RNA using Superscript II (Invitrogen) according to the manufacturer’s instructions. qPCR was performed using SensiMix SYBR low-ROX (Bioline) and 150 nM forward and reverse primers (Sigma-Aldrich; primers listed in supplementary materials). All samples were run in technical duplicates and gene expression calculated relative to housekeeping gene Ribosomal Protein Lateral Stalk Subunit P0 (RPLP-0).
Single Cell RNA-sequencing
Primary Foetal Liver or organoids were dissociated to a single cell solution by the respective methods described above. The single cell solution from each was then processed using the Chromium single cell controller and reagents according to the manufacturer’s guidelines (10X genomics). Sequencing was performed on the HiSeq4000 sequencing system, and the subsequent raw data was aligned and used to generate matrices using the Cell Ranger platform (version 2.1.0). Basic analysis of the data was performed in R using the Seurat, ggplot2, and Monocle packages.
Assessment of genomic stability
Genomic stability of the organoids was assessed using the CytoScan 750k array, performed by the Medical Genetics Service at Cambridge University Hospitals.
Determination of cell line gender
Cell line gender was assessed by assessing RNA expression of the SRY gene (male), or Xist gene (female) based upon qPCR and/or single cell RNA sequencing analysis
Flow cytometry Organoids were dissociated to single cell solution as described above. The single cells were then fixed and permeabilised in FixPerm (ThermoFisher Scientific) for 20 minutes, then washed three times in PBS before resuspension in PermWash (ThermoFisher Scientific). Blocking was performed using 10% donkey serum (Biorad) in PBS for 30 minutes. Primary antibodies were diluted 1 : 100 in 1 % donkey serum in PBS and incubated with the cell suspension at room temperature for 1 hour. After three washes in PBS, cells were incubated with secondary antibodies diluted 1 :1000 in 1 % donkey serum in PBS. Data were collected using Cyan ADP flow cytometer, and analysed using FlowJo X.
Assessment of cell proliferation rates
Organoids were extracted and dissociated to single cells as described above. The cells were then counted using the countess II automated cell counter (Biorad), and the final number adjusted according to the number of wells used and the volume of resuspension.
Cytochrome P-450 assays
P450 Glo (Promega) assays were used according to manufacturer’s guidelines. Luciferin IPA (CYP3A4 specific), and Luciferin PFBE (CYP3A7 & CYP3A5 > CYP3A4), were added to media and incubated at 37°C for four hours, before using detection assay as per the manufacturer’s instruction. Luminescence RLU was then corrected for cell number.
Serum protein analysis
Albumin, alpha-fetoprotein, apolipoprotein-B, and alpha-1 -antitrypsin were detected in media by enzyme linked immunosorbent assay (performed by core biomedical assay laboratory, Cambridge University Hospitals). Concentrations were normalised to cell number.
Bodipy assay
BODIPY 493/503 (ThermoFisher Scientific) was diluted 1 :1000 in the organoid culture media, and applied to organoids for 30 minutes. After this time the organoids were washed with fresh media, and imaged in situ using a fluorescent microscope.
Freezing/ thawing of HO
Organoids were extracted from Matrigel using cell recovery solution as described above. The organoids were then washed in PBS and resuspended in Cell Culture Freezing Medium (ThermoFisher Scientific), placed in cryogenic vials (ThermoFisher Scientific), and cooled to -80oC using a“Mr. Frosty” Freezing Container (ThermoFisher Scientific). Organoids could then be kept long term in liquid nitrogen. To thaw, cryovials were kept at 4oC until the freezing medium had melted. Organoids were then washed three times in PBS to remove any residual freezing medium, and then replated as above.
Statistical analyses
Unless otherwise stated, statistical analyses of direct comparisons of two groups were performed using t-test with Welch correction. Those comparisons labelled * represent P<0.05, ** p <0.01 , *** P<0.001 , and **** p<0.0001. Results
Combination of WNT and reduced density ECM allows derivation of hepatobiast organoids from human hepatic foetal tissue
Organoids have been previously derived from the adult liver using two approaches. The first method consists of deriving adult stem cells from the intrahepatic biliary epithelium using WNT, Noggin and Rock-inhibitor for the first three days of in vitro culture (Huch et al., 2015). The resulting liver stem cell organoids (LSCO) mainly express biliary markers while displaying a limited capacity to differentiate into hepatocytes (Huch et al., 2015). The second method derived similar cells from the extra-hepatic biliary epithelium (Sampaziotis et al., 2017). These Extra-hepatic Cholangiocytes Organoids or ECOs closely resemble cholangiocytes and cannot differentiate into any other hepatic lineage.
We hypothesized that hepatobiast derivation would require the development of culture conditions different to those currently available to grow organoids from adult liver. Thus, we screened additional growth factors and basal media to notice that foetal liver tissue dissociated into single cells and grown in the presence of Wnt, R-Spondin, EGF, A83-01 , and Y-27632 (i.e. the same conditions as for FBO, but the addition of Wnt) gave raise to organoids with a markedly different morphology characterised by solid branching structures (Fig 1 C). These organoids were capable of regenerating after fragmentation or dissociation into single cells (Materials and Methods). Furthermore, a 55% dilution of Matrigel formed a permissive microenvironment for the organoids to expand. These“branching” organoids could be split every 10-14 days to a ratio of 1 :4-1 :6, thus allowing rapid expansion of individual cell lines. Crucially, these organoids express key
hepatoblast/hepatocyte markers such as the proteins albumin and alpha-1 -antitrypsin at similar levels to both the primary foetal liver and primary adult hepatocytes (Fig 2A). Genes associated with a foetal stage of development such as the serum protein alpha-fetoprotein or the p450 enzyme CYP3A7, were at comparable levels in organoid and primary cells, whilst being at low level in adult hepatocytes. Importantly, biliary markers such as KRT19, EPCAM and SOX9 were also detected albeit at low levels thereby suggesting that these cells could have the capacity to differentiate into both hepatocytes and cholangiocytes. Considered together, these data suggest that our culture conditions allow the derivation of hepatobiast organoid (HO). Importantly, this method was used to generate 12 HO lines derived from the liver of 5.5 to 10.2 pew embryos of different gender thereby demonstrating the robustness of our approach. The resulting HO lines have been maintained in culture for at least nine months, and all lines derived so far continue to proliferate and expand after each split while maintaining a homogenous expression of EPCAM, ASGR1 , AFP, and A1 AT as shown by flow cytometry. Dissociation and counting of individual cells in culture demonstrate a doubling time of approximately three days, and thus confirm the capacity of these cell line for large scale expansion. Analysis of genetic stability using comparative genomic hybridization in three lines, confirm the absence of DNA copy number abnormalities or any other clinically relevant abnormality. Of note, HO could be frozen and stored for several months while maintaining their characteristics (data not shown). Finally, ELISA analyses confirmed that HO secrete high levels of key cell type specific proteins such as albumin, alpha-fetoprotein, the enzyme alpha-1 -antitrypsin, and the lipoprotein apolipoprotein B (Fig 2C). Similarly, assessment of the function of key detoxification enzymes such as cytochrome p450 3A7 (CYP3A7; expressed in the immature liver), and cytochrome p450 3A4 (CYP3A4, expressed in the mature liver), demonstrated physiological function of both enzymes in the HO, whilst CYP3A7 is not highly active in the primary adult hepatocytes, reinforcing the difference in developmental state of the two cell types. Taken together these results show that HO can be derived from human foetal liver and that the resulting cells can be grown in vitro for prolonged period of time while maintaining hepatic functions in vitro.
Single cell analyses confirm the presence of hepatoblasts in the early human foetal liver
To fully validate the identity of HO, we decided to define the gene expression profile of their in vivo counterparts using scRNA-seq. For that, we isolated 2 foetal livers at 6 pew, performed single cell dissociation, and analysed the transcriptome of 1406 cells using 10X technology. tSNE analysis demonstrated independent clustering of non-parenchymal cell types including Kupffer Cells (CD68,
FCGR3A, CD33, ITGAM, SPI1 ) Stellate Cells (PDGFRa, ACTA2, VIM, NES), hematopoietic stem/progenitor cells (PTPRC, SPN, MYB, KLF1 , TFRC), lymphocytes (LSP1 , CD52, CD37, CD48) and megakaryocytes (NRGN, PPBP, ITGA2B, GP1 BA, GP6), as well as a homogeneous population of hepatoblasts (ALB, AFP, KRT8, KRT18, EPCAM, SERPINA1 , TTR) (Figure 3B).
Interestingly, these analyses could not identify cells with a biliary expression profile suggesting that these cells are extremely rare at this stage of development and that large scale scRNA-seq analyses might be required to establish a complete atlas of the human foetal liver at different stages of development.
Nonetheless, our data provides substantial novel information into the landscape of the liver at this developmental stage by demonstrating the early colonization of the liver by a diversity of cell types including immune cells, such as tissue resident macrophages and dendritic cells. Furthermore, IHC of an 8 pew liver demonstrates KRT19 positive cells near primitive ducts at this later stage (Fig 3A). Our data also confirm the production of MK cells by HSPC in the liver thereby confirming previous reports. Finally, the presence of stellate cells is also intriguing and suggests that these cells could colonise the liver earlier than initially thought and thus could have a function in early organogenesis. The population of hepatoblast cells showed great similarity between the biological replicates and showed a homogeneity in their gene expression signature, demonstrating the global ability of the liver parenchyma at this stage to expand the hepatoblast population. Importantly, this population of hepatoblasts did express a spectrum of known hepatic markers including albumin, alpha-fetoprotein, TTR and SERPINA1 and also new genes which were not identified previously such as RBP4, AHSG, APOH, APOM or FABP1. Interestingly, a majority of these genes are expressed at the protein level in adult hepatocytes based on the protein atlas with the exception of SPINK1 and FGB (Table S1 ). Based on this information, hepatoblast could be defined by the combined expression of ALB, SPINK1 , AFP, SERPINA1 , CYP3A7. Taken together, these data show that the human foetal liver contains a diversity of cell types including a homogeneous population of hepatoblast cells.
Hepatoblast organoids form a homogeneous population that closely resembles the in vivo counterpart In order to further characterise our novel foetal liver organoid culture we next applied scRNA-seq to our HO (n=2) and compared the generated profiles to those from primary tissue. Interestingly, we observed that HO culture contain two separate populations of cells. The first cluster consists of the majority of cells and includes cells homogeneously expressing hepatoblast markers contrary to the second small cluster which only express VIM, PDGFRa, and PDGFRb. Further analysis of the genes differentially expressed between these two populations confirmed that the first cluster contains a homogeneous population of hepatoblasts whilst the second cluster contains cells expressing markers specific to stellate cells. To confirm this observation, stellate cells grown in vitro were compared to their primary counterpart using single cell data generated on primary foetal liver. This analysis confirmed that HO culture contains a population of stellate cells (Fig S3B). To further understand the importance of these stellate cells, we isolated hepatoblast cells using EPCAM sorting and also derived new HO lines after EPCAM sorting of single cell from foetal tissue. In both cases, the isolation of EPCAM expressing cells resulted in the absence of stellate cells and the resulting HO maintain the same capacity to grow and to differentiate. Interestingly, stellate cells isolated by negative selection using EPCAM sorting were not able to grow independently more than a couple of passages thereby suggesting that these cells may require the presence of hepatoblasts for their
proliferation/survival while the reciprocity does not apply. Thus, these observations suggest that stellate cells development could rely on hepatoblast during liver organogenesis.
We next compared the single cell expression profiles of HO hepatoblast cells to PFL hepatoblast cells and the FBO. The Co and CoP populations maintain their distinct clustering, to each other and to the
hepatoblasts, with the most differentially expressed genes confirming the different identities of the hepatoblasts (SERPINA1 , APOB, ALB, ASGR1 , TF, ITIH1 ), CoP (CA4, CD24, CLDN6) and Co (KRT7, ADIRF, CEACAM6). Thus, HO and CoP clearly represent different types of progenitors despite sharing the expression of key genes such as AFP. Further detailed tSNE analyses refines these observations by showing that HO and primary hepatoblast can be distributed in separate clusters. Nonetheless, heat map visualisation reveals that HO and primary hepatoblasts broadly express the same markers but at different levels (fig 4F). Principal component analysis (PCA) demonstrated that HO and PH are highly similar (Fig 4D), expressing similar levels of genes such as apolipoprotein A1 (APOA1 ), asialoglycoprotein receptor 1 (ASGR1 ) and albumin (ALB). The genes driving the differences between the two populations included higher expression of genes from the Methallothionein sub-family of genes (MT1 H, MT1A, MT1G, MT1 E, MT1 F, MT2A, MT1X) and also higher expression of some markers of immaturity such as AFP. In comparison, the HO had higher levels of expression of metabolic enzymes such as CYP1 A1 and CYP2E1 , and metabolic proteins such as fatty acid synthase (FASN) and fatty acid desaturase (FADS1 ). GO term analyses show that these genes mainly correspond to hepatic function. Considered together these results show that HOs strongly resemble their in vivo counterparts and that expansion in vitro affects only a limited number of metabolic functions.
Hepatoblast Organoids can differentiate into cells expressing cholangiocyte markers after addition of TGFb in the absence of Wnt3a.
We then decided to explore the capacity of HO to differentiate into cholangiocytes. For that we studied the effect of growth factors that have been shown to drive differentiation of hepatoblasts into biliary cells in vivo especially TGFb. These analyses uncovered that withdrawal of TGFb inhibitor A8301 and Wnt3a, and the simultaneous addition of TGFb, resulted in a significant increase in the biliary marker KRT19. More precisely, HO grown for seven days in the presence of TGFb without WNT express biliary markers at a level similar to FBO while losing hepatoblast markers including ALB and AFP (Fig 4B). These results were further supported by the changes in protein expression shown by immunostaining and in secreted protein detected by ELISA (Fig 4C). Taken together, these data suggest that TGFbeta could drive differentiation of HO toward the biliary lineages. To further confirm this possibility, we compared the single cell transcriptomic profile of HO grown in the presence of TGFb with that of HO and FBO (Fig 5D). scRNAseq analysis demonstrated TGFb treated caused the cells to cluster separated from HO, and to adopt a profile similar to the FBO. Furthermore, HO, HO+TGFb and FBO cells ordered in pseudotime, with PFL and HO transitioning to CoP to Co and HO-TGF, confirming TGF beta treatment drives HO differentiation toward the biliary lineage following a precise temporal sequence of gene expression events (Fig 4E-G). Taken together, these results suggest that HO display the capacity to differentiate into cells expressing biliary markers in vitro. This process occurs in the absence of WNT signalling and in the presence of TGFb which promotes a biliary specification.
Absence of Wnt signalling drives differentiation of hepatoblast toward foetal hepatocytes.
In parallel to the experiments outlined above, we also explored culture conditions that could promote the differentiation of hepatoblast towards hepatocytes. To this end, we found that removing Wnt3a to promote differentiation and transferring into a hepatocyte specific medium (Hepatozyme with oncostatin-M), resulted in hepatoblast organoids undergoing a phenotypic shift (Fig 5A and B) and increased the expression of more mature hepatocytic markers such as G6PC, and in functional proteins such as alpha-1 -antitrypsin Figure 5C). We referred to these organoids as HO-HZ. Additionally, hepatoblast markers of the cholangiocyte lineage, such as cytokeratin-19 or gamma-glutamyl transferase, were further down regulated, demonstrating a robust change in cell fate from hepatoblast to a more foetal/mature hepatocyte (Fig 5C-G). Analysis of medium protein content also demonstrated a significant increase in proteins such as alpha-1 -antitrypsin, and down regulation in proteins associated with immaturity, such as alpha fetoprotein (Fig 5D). Of note, differentiated cell types lost proliferative capacity in vitro after treatment, even if treatment is withdrawn and original culture conditions are restored (data not shown). This further indicates a terminal differentiation of the cells in this system and mirrors the behaviour of primary cells cultured in vitro. Finally, we also observed that HO-HZ acquire functional activity absence in HO such as lipid uptake or CYP3A4 activity. To validate these results, HO-HZ were analysed at the single cell level and compared with HO and the PFL. PCA demonstrated that indeed absence of WNT induces differentiation of HO while heat map representation of the cells differentially expressed between each cluster suggest that the identity of HO-HZ differ from HO and PFL. Interestingly, genes specifically expressed in HO-HZ are in majority expressed at the protein level in adult hepatocytes (See table S1 ). GO analyses show that these genes are involved in metabolic pathways, serum and immune protein production, which all correspond to function fulfilled by the adult liver (Fig S6F). Markedly reduced expression of cell cycle genes such as CDK1 and Mi67 indicate that HO-HZ have stopped proliferating. Finally, pseudotime ordering revealed the precise gene expression signature corresponding to the transition between PFL/HO to foetal hepatocytes confirming dynamic decrease of key hepatoblast markers such as AFP and an induction of more mature markers such as G6PC and SERPINA1 (Fig 5F and G). Taken together, these results show that absence of WNT drives differentiation of HO towards a hepatocyte phenotype associated with the induction of specific marker, decrease in proliferation capacity and acquisition of specific function.
Hepatoblast organoids have a distinct identity from fetal hepatocyte and adult liver organoids.
Two previous reports have described culture systems to grow organoids from the liver. One approach consists of deriving organoids from the intrahepatic biliary epithelium of the adult liver (BO) (Huch M, et al. Cell. 2015;160(1-2):299-312) while the other method described organoids from the human fetal liver of the second trimester (fetal hepatocyte organoids or FHO; Hu H et al. Cell. 2018;175(6):1591-1606.e19).
Importantly, these organoids have different requirement for growth factors (See Table 2) while BO maintain a strong biliary identity and thus have a limited capacity to generate hepatocytes. To confirm this aspect, we derived two BO lines and then differentiated them toward hepatocyte-like cells them as described previously (Hu H et al supra, Huch et al supra). The resulting differentiated BO (DBO) secrete very low levels of ALB, AFP, APOB, and A1AT when compared to HBO (Fig. 6) thereby reinforcing recent reports (Hu H et al supra, Huch et al supra). Comparison at a single cell level demonstrated that BO and DBO overlap while forming distinct clusters from HBO or primary fetal liver hepatoblasts (PFL). The main difference between these populations was the high level of cholangiocytic markers such as KRT19 and KRT7 in BO/DBO in contrast to the high level of hepatoblast markers such as ALB and ARG1 in PFL/HBO. Next we compared the transcriptional profile of DBO and FHO, and HBO (Hu et al supra). PCA demonstrated separation into three categories; biliary (DBO), hepatocytic (primary adult hepatocytes, primary fetal hepatocytes and fetal hepatocyte organoids), and hepatoblast (HBO) according to genes associated with either biliary ( KRT19 , MUC20) or hepatocyte {ALB, AFP) identity. Interestingly, HBO occupied an intermediate position by coexpressing both categories of markers (Fig. 7A, 7B) while primary hepatocytes displayed adult markers involved in clotting (F2, F10, PROC, FGA), immunity (C2, C3), and metabolic processes (APOB, APOH, ALDOB, ABCA1 , PAH). Taken together these observations (summarized in Table 2) show that HBO have a unique transcriptional profile when compared to previously derived liver organoids and thus they provide a novel complementary model system for human fetal liver development
Hepatoblast Organoids can differentiate into biliary cells and hepatocytes in vivo
Capacity to differentiate into hepatocytes and cholangiocytes represents a hallmark of hepatoblast function. To confirm this key characteristic, we transplanted HBO into immunodeficient mice. We first generated HBO expressing a tdTomato reporter using lentiviral transduction. These fluorescent cells were transplanted into mice using an approach recently developed for primary hepatocytes (Stevens et al Sci Transl Med.
2017;9(399)). In sum,“human organoid tissues” were assembled by embedding HBO and human umbilical vein endothelial cells (HUVECs) in a fibrin-matrigel hydrogel (20:1 fibrimmatrigel mixture). The resulting organoid tissues were sutured into the inguinal fat pad of FRG mice which experience progressive liver failure unless administered nitisinone (NTBC) (Wilson et al Stem Cell Res. 2014;13(3 Pt A):404-412). The transplanted mice were cycled on and off NTBC over the course of the experiment to induce chronic liver disease, which has been shown to promote hepatic engraftment and growth (Stevens et al supra, Azuma et al Nat Biotechnol. 2007;25(8):903-910). After 27 days, implants were recovered and red fluorescent cells could be observed in all the grafts, indicating that HBO had engrafted efficiently. Hematoxylin & eosin staining of explanted tissue sections revealed the presence of numerous nodules resembling densely packed hepatocytes, as well as biliary epithelial-like cells assembled into structures reminiscent of bile ducts.
To assess the maturation state of grafted organoids, we then stained tissue sections for AFP and KRT18. Organoids in engineered tissues stained positively for both KRT18 and AFP at the time of implant, but AFP was either not identified or markedly decreased in KRT18-positive grafted organoids suggesting
differentiation of hepatoblasts into hepatocytes. Accordingly, numerous cells in hepatic nodule stained positively for ARG1 , A1 AT, and ALB while human albumin was identified in mouse blood serum suggesting functional activity and successful integration with the host vasculature (Fig. 8A). Finally, KRT18-positive nodules also contained cells expressing KRT19 (11 % had some cells that expressed KRT19 and 18% were nearly entirely positive for KRT19 with characteristic bile duct morphology; Fig. 8B). Together, our results demonstrate that HOs have the capacity to differentiate into cells characterized by hepatocyte and biliary markers after transplantation into mouse model for liver disease.
We studied the effect of growth factors that have been shown to drive differentiation of hepatoblasts into biliary cells in vivo, particularly TGF which is known to control formation of the ductal plate in vivo (Clotman et al Genes Dev. 2005;19(16):1849-1854; Seth et al Development. 2014;141 (3):538-54; Ober et al J Hepatol. 2018;68(5):1049-1062. These analyses demonstrated that the withdrawal of TGF inhibitor A83-01 and the simultaneous addition of TGF increased biliary markers such as KRT19 and decreased hepatoblast markers including ALB.
The above experiments demonstrate the derivation of the first human hepatoblast organoid model. This model offers an exciting opportunity to study human foetal liver development in vitro long term and in three dimensions, allowing assessment of the behaviour and characteristics of previously poorly understood cell types that have been difficult to investigate until now.
Hepatoblast organoids crucially exhibit high levels of function in vitro, continuing to express the key genes involved in hepatic function found in vivo and demonstrating their robustness as a representative in vitro model. Thus, this new model gives insight and opportunity to study human hepatoblast physiology, cell cycle characteristics, and potential insight to pathological mechanisms such as malignant transformation. A key concept of hepatoblast cells is the co-expression of hepatocytic and cholangiocytic markers. An interesting finding is that whilst the hepatocytic markers (ALB/A1 AT) are expressed at similar levels to in vivo, the cholangiocytic markers are more lowly expressed compared to cholangiocytes. In addition, human hepatoblast organoids transplanted into mice demonstrated the capacity to differentiate toward either hepatocytic or biliary fates while maintaining functional capacity.
The genetic and transcriptomic stability along with the high proliferative capacity of the organoids also allows for large scale expansion of a single biological sample, thus enabling in depth analysis of the system and allowing complex research questions to be investigated in the same system over long periods. Similarly, the homogeneity and high potential for cell expansion enables the potential of generating large scale biobanks of tissue that can be selectively utilised.
Single cell assessment of the primary foetal liver has allowed us to demonstrate the homogeneous character of the early hepatoblast, and also the early colonization of the liver by immune cells. This finding gives insight into how the early liver is populated, with a large population of bipotent dividing hepatoblast cells. Comparison with organoid demonstrated a similarly homogeneous population of self-organizing proliferative cells, underlining the broad nature of this cell type. The ability to differentiate these cells in vitro will allow future detailed analysis of how this process occurs, and greater interrogation of the pathways involved.
The ability to generate cells that could be expanded and transplanted into tissue matched recipients could represent an exciting opportunity to manage patients requiring liver transplant. The efficiency of organoid generation alongside a ready supply of tissue offers the potential of rapidly generating a large-scale biobank of hepatoblast organoids that could cover the majority of variations in tissue markers such as HLA. The ability of the organoids to produce serum albumin and differentiate to daughter cell types present an exciting prospect of developing a future bridge therapy for end stage liver disease, restoring some level of hepatic function through tissue matched donor organoids.
The controlled differentiation of hepatoblasts into either of the two specific daughter cell types offers a potentially unique insight into these mechanisms that can be further explored. Furthermore, the only current possibility to maintain hepatocytes in vitro is to await donor samples removed from deceased donors. The ability to generate large numbers of either cholangiocytes or hepatocytes on demand in vitro from multiple biological backgrounds, represents the first self-replenishing source of such cells. This again opens exciting possibilities to interrogate the development of these cells and opportunities for drug discovery/ screening. Overall, this new organoid system represents an exciting system to model development, cell fate decisions, and opportunities for regenerative medicine.
Reagent HO-M CO-M Stock concentration Final concentration
Basal medium:
DMEM/F12 500ml 500ml
Glutamax 5ml 5ml 100% 1 % (by vol) HEPES 5ml 5ml 100% 1 % (by vol)
Penicillin/streptomycin 5ml 5ml 100% 1 % (by vol)
B27 10ml 10ml 100% 2% (by vol)
Nicotinamide 10ml 10ml 1 M 20mM
N-acetylcysteine 2ml 2ml 0.5M 2mM
Complete medium:
Basal medium 19ml 19ml
DMEM 25ml
Wnt3a 25ml - 100% 50% (by vol.)
R-spondin 5ml 5ml 100% 10% (by vol.)
EGF 25uL 25ul 100ug/ml 50ng/ml
A8301 50uL 50uL 50mM 50uM
Y27632 50ul 50ul 10mM 10uM
Table 1
Figure imgf000032_0001
Key: - = low expression, + moderate expression, ++ high expression, +++ = very high expression
Table 2: Summary of human hepatic organoids
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Claims

Claims
1. A method for producing an expanded population of human hepatoblasts in vitro comprising:
(i) providing a population of isolated immature human liver cells and;
(ii) culturing the population in a hepatoblast expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor, to produce an expanded population of human hepatoblasts.
2. A method for producing a population of human cholangiocytes in vitro comprising
(i) producing an expanded population of human hepatoblasts by a method according to claim 1 ; and,
(ii) culturing the expanded population in a differentiation medium comprising TGF to produce a population of cholangiocytes.
3. A method for producing a population of human hepatocytes in vitro comprising
(i) producing an expanded population of hepatoblasts by a method according to claim 1 ; and,
(ii) culturing the expanded population in a hepatocyte culture medium to produce a population of human hepatocytes.
4. A method according to any one of the preceding claims wherein the expanded population forms organoids in the hepatoblast expansion medium.
5. A method according to any one of the preceding claims wherein immature human liver cells are foetal or neonatal liver cells.
6. A method according to any one of the preceding claims wherein the hepatoblast expansion medium is a chemically defined medium consisting of a basal medium, epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor,
7. A method according to any one of the preceding claims wherein the differentiation medium is a chemically defined medium consisting of a basal medium, epidermal growth factor (EGF), TGF , a non- canonical Wnt signalling potentiator, and a ROCK inhibitor.
8. A method according to any one of the preceding claims wherein the TGF inhibitor is A8301.
9. A method according to any one of the preceding claims wherein the non-canonical Wnt signalling potentiator is R-spondin.
10. A method according to according to any one of the preceding claims wherein the ROCK inhibitor is Y-27632.
1 1. A method according to any one of the preceding claims wherein the canonical Wnt potentiator is Wnt3a.
12. An isolated population of hepatoblasts, biliary cells, cholangiocyte progenitors or cholangiocytes produced by a method according to any one of claims 1 to 1 1.
13. A biocompatible scaffold comprising an isolated population according to claim 12.
14. A method of treating a liver disease, for example a hepatocytic or biliary disorder, comprising administering an isolated population according to claim 12 or a scaffold according to claim 13 to an individual in need thereof.
15. An isolated population according to claim 12 or a scaffold according to claim 13 for use in a method of treating a liver disease in an individual in need thereof.
16. A method according to claim 14 wherein the liver disease is haemophilia.
17. An isolated population or a scaffold for use according to claim 15 wherein the liver disease is haemophilia.
18. A method of screening comprising;
contacting an isolated population according to claim 12 or a scaffold according to claim 13 with a test compound, and;
determining the effect of the test compound on the population or scaffold and/or the effect of the population or scaffold on the test compound.
19. A method according to claim 18 wherein the population is contacted with the test compound is in the form of organoids.
20. A kit for the production of an expanded population of hepatoblasts comprising a hepatoblast expansion medium that comprises epidermal growth factor (EGF), a TGF inhibitor, non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor.
21. A kit according to claim 20 further comprising a hepatocyte culture medium and/or a differentiation medium comprising TGF .
22. Use of a hepatoblast expansion medium for the in vitro expansion of hepatoblasts, wherein the hepatoblast expansion medium comprises epidermal growth factor (EGF), a TGF inhibitor, non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor.
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