Method for producing hepatocyte-like liver organoids

Abstract

The present invention relates to a method for the in vitro production of functional and proliferative hepatocytes and / or hepatocyte-like liver organoids (HELLOs). The present invention further relates to the hepatocyte-like liver organoids obtained via the present method and the use of said hepatocyte-like liver organoids in drug- and toxicity testing, and clinical trails.

Description

[0001] METHOD FOR PRODUCING HEPATOCYTE-LIKE LIVER ORGANOIDS

[0002] Description

[0003] The present invention relates to a method for the in vitro production of functional and proliferative hepatocytes and / or hepatocyte-like liver organoids (HELLOs). The present invention further relates to the hepatocyte-like liver organoids obtained via the present method and the use of said hepatocyte-like liver organoids in drug- and toxicity testing, and clinical trials.

[0004] The liver is a vital organ performing numerous functions. Most of the metabolic liver functions are performed by the hepatocytes, specialized epithelial cells that make up most of the liver. Dysfunction in hepatocytes is associated with many diseases, both inherited and noninherited. Since many metabolic functions are performed by hepatocytes, genetic metabolic diseases often manifest in the liver. Additionally, drug-induced liver injury (DILI) is one of the most important hepatocyte-related diseases. Often, DILI causes drugs to fail during and after clinical trials resulting in expensive and slow drug development.

[0005] Many in vitro models have been developed attempting to emulate hepatocyte functions. However, most hepatocyte models fall short in their capability to proliferate and retain hepatocyte functions for sufficient time. This lack of proliferation and long-term functional capacities lead to incomplete modelling of hepatocyte functions, limiting drug development, studies of disease mechanisms, and personalized medicine approaches. The liver is an essential organ involved in many important processes including blood detoxification and the production of serum proteins. In vitro, adult primary human hepatocytes (PHH) are the gold standard in toxicological studies and have the potential to treat patients with liver disease. However, PHHs have short-term functionality and limited expansion potential in vitro.

[0006] While hepatocytes are generally difficult to culture, cholangiocytes are highly proliferative in vitro. Cholangiocytes are specialized epithelial cells found lining the bile ducts within the liver and play a crucial role in the formation and secretion of bile that helps emulsify fats and aids in their digestion and absorption in the small intestine. Previously, cholangiocytes have been shown to trans-differentiate to hepatocytes under certain liver damage conditions in vivo. As such, cholangiocytes have been used as an alternative source of hepatocytes through in vitro transdifferentiation. One example of a cholangiocyte in vitro model is the intrahepatic cholangiocyte organoids (ICOs), which are highly proliferative and can be established from small pieces of liver tissue. This allows the generation of patient-specific cultures of ICOs supporting personalized medicine. Upon hepatic differentiation, the ICOs supposedly recapitulate important hepatocyte functions. ICOs represented a revolutionary innovation in terms of the ease of expansion and culturing while retaining some hepatic characteristics; the 3D organization allowing for specific transport studies; and their human origin, thereby representing the full (epi)genome of the person of origin allowing for personalized medicine.

[0007] However, a poor and incomplete hepatic differentiation of ICOs is a major drawback for numerous studies, including drug testing because of insufficient representation of several phase 1 and 2 drug metabolism, predicting DILI, modelling of important disease phenotypes (metabolic diseases, Hepatitis B, etc), and liver cell transplantations (poor engraftment). Furthermore, a recent comprehensive analysis of various hepatocyte-like cells has shown that ICOs still severely lack relevant hepatocyte phenotypes and the complex tissue microenvironment and much more closely resemble cholangiocytes (Ardisasmita et al., 2022), including their polarized structure, secretory function, and response to environmental cues. In terms of hepatocyte-derived organoids, so far, only hepatocyte organoids derived from fetal liver could be kept in long term cultures, thereby hampering personalized therapy testing.

[0008] Considering the above, there is a need in the art for an improved cell model or organoid for disease- and / or drug testing, personalized modelling, that more closely resembles in vivo liver cells or mature hepatocytes. There is a need for an improved liver model and method for providing an improved pre-clinical drug evaluation and DILI disease modelling, including liver cell transplantations.

[0009] It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

[0010] Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a method for the in vitro production of functional and proliferative hepatocytes and / or hepatocyte-like liver organoids (HELLOs), comprising the steps of; a) expanding cells, preferably liver cells, in cell culture in a expansion medium (EM), wherein EM is comprised of basal media comprising 50 to 500 ng / mL, preferably 100 to 300 ng / mL, more preferably 150 to 250 ng / mL of at least one R-spondin protein, and 1 to 50 ng / mL, preferably 5 to 40 ng / mL, more preferably 10 to 30 ng / mL most preferably 20 to 25 ng / mL EGF, and optionally at least one transforming growth factor P (TGF- ) inhibitor, preferably 0.1 to 50 pM, more preferably 1.5 to 25 pM, even more preferably 2 to 10 pM, most preferably 2.5 to 5 pM of at least one transforming growth factor (TGF-P) inhibitor, and 1 to 100 pM, preferably 2.5 to 50 pM, more preferably 5 to 25 pM, most preferably 7.5 to 10 pM forskolin, b) optionally, collecting the expanded cells of step a and transfer these cells to a scaffold matrix, c) culturing of the expanded cells in a differentiation medium (DM) comprised of basal media and 0.1 to 500 pM, preferably 0.5 to 250 pM, more preferably 2 to 100 pM, most preferably 5 to 50 pM of at least one transforming growth factor P (TGF- ) inhibitor , and 0.1 to 100 pM, preferably 1 to 50 pM, more preferably 5 to 10 M of a Notch inhibitor, preferably wherein the Notch inhibitor is DAPT, and 0.1 to 100 pM, preferably 1 to 50 pM, more preferably 5 to 30 pM glucocorticoid to generate functional and proliferative hepatocytes and / or HELLOs.

[0011] The hepatocyte-like liver organoids (HeLLOs) of present invention provide for a highly proliferative liver model resembling cholangiocytes during expansion, which can be differentiated to express important hepatocyte functions. Experiments show that the HeLLOs more closely resemble mature hepatocytes at the transcriptomic and functional levels than other hepatocyte models known, including the ICOs. More specifically, HeLLOs acquire mature drug metabolism, fatty acid metabolism, bile acid transport, and liver protein secretion function, especially when compared to ICOs. Furthermore, we show that ICO-started cultures can be adopted in the HeLLO medium to strongly improve functional differentiation. As used herein, ‘functional’ hepatocytes in cell culture refers to hepatocytes which retain key features and functions of hepatocytes in vivo, including key drug transporters, production and secretion of albumin; production and transport of bile. Protein expression and localization may be demonstrated, for example, through immunolocalization or immunohistochemistry. Protein function may be assessed using various commercially available kits, for example, albumin and CYP ELISA kits, which measure secretion of albumin and biotransformation by CYP enzymes, respectively.

[0012] Many scientific reports emphasize the need for growth factors to improve proliferation and differentiation of hepatocyte-like cells. The rationale being that hepatocyte-like cells require a basal level of liver pathway signaling to maintain hepatic fate. However, and surprisingly, the inventors found that the hepatocyte fate of hepatocyte-like liver organoids is highest under culture conditions without addition of growth factors. For example, removal of all growth factors increased expression of CYP3A4 10 to 100-fold. Proliferation and differentiation of cells are counteracting forces that continuously influence cell fate. In this regard, removing growth factors would inherently improve differentiation status of hepatocyte cells by decreasing proliferation signals. Therefore, the absence of growth factors can also be interpreted as both inhibiting growth factor-related signaling and inhibiting cell proliferation. Attenuation of cell proliferation signaling may be achieved by reducing the concentration of the growth factors in the media. For example, using EGF in a concentration less than 20 ng / ml including a concentration range of 0 to 15 ng / ml, such as 10, 5, 2.5, 1, 0.5, 0.25, 0.10, 0.05, 0.01 ng / ml.

[0013] The method of present invention comprises two separate cell cultivation steps, wherein first the cells are proliferated in expansion media, followed by differentiating the liver cells to generate functional and proliferative hepatocytes and / or HELLOs. The absence of growth factors in HeLLO differentiation medium played an important role in the improved hepatic differentiation of HeLLO. This was demonstrated by removing the growth factors in ICO differentiation medium which resulted in the increase of hepatocyte marker gene expression levels. Small molecules such as A8301, DAPT, dexamethasone, and BMP7 were also shown to be important since their removal inhibited acquisition of hepatocyte gene expression markers ALB, CYP3A4 and G6PC. Nevertheless, HeLLO expressed higher hepatocyte gene expression compared to ICOs without growth factors. This shows that expansion medium is also an important factor to attain better hepatocyte phenotypes. More importantly, whole transcriptome data analysis showed that HeLLOs in expansion medium had higher expression of hepatocyte-related genes although still retaining similarity to cholangiocytes.

[0014] The method of present invention includes forskolin in the EM cell culture media. Forskolin is a cell-permeable diterpene that directly activates adenylyl cyclase (ICso = 41 nM), the enzyme that produces cyclic adenosine monophosphate (cAMP), which as a result raises cAMP levels in the cell. Forskolin stimulates the activity of adenylyl cyclase which increases the intracellular level of cAMP. It has been reported that cAMP inducers promote proliferation of biliary cells in vivo and in vitro through ERK signalling. cAMP inducers may also be cell permeable cAMP analogs. Forskolin helps to grow the HeLLOs long-term. Organoids can still be grown without, but then the organoids will deteriorate much faster. Forskolin may preferably also be present in the DM cell culture media, as in EM media.

[0015] The method of present invention includes epidermal growth factor (EGF) in the EM cell culture media, and not in the DM media. EGF is a protein which induces cell proliferation through its binding with an epidermal growth factor receptor (EGFR). EGF has been shown to promote the proliferation of various cell types, either in vitro or in vivo. EGF is essential in the expansion medium due to its mitogenic properties. Without EGF, the liver cells will hardly proliferate and the HeLLO culture cannot be established.

[0016] The method of present invention includes R-sponding in the EM cell culture media. R- sponding is a Wnt agonist and stimulates or enhances the activity of the Wnt signalling pathway. The Wnt signalling pathway is a cascade of reactions which starts with the binding of a Wnt protein to a transmembrane receptor Frizzled protein. This binding leads to the translocation of P- catenin into the nucleus and subsequently the activation of target genes via TCF / LEF transcription factors. The activation of the Wnt signalling pathway is crucial to facilitate cell proliferation and survival. R-spondin amplifies the effect of Wnt protein and stimulates expansion. R-spondin 1 seems most effective. Preferably, R-spondin is human R-spondin. R-sponding may preferably also be present in the DM cell culture media, as in EM media.

[0017] The method of present invention includes TGF- inhibitor in the EM and DM cell culture media. A TGF- inhibitor inhibits the activity of TGF-P signaling pathway. This signaling pathway has been described to promote biliary and inhibit hepatocyte differentiation. We found that inhibiting the TGF-P signaling pathway increases the expression of some hepatic genes. This compound is added in higher concentrations in EM media (in comparison to DM media) due to the cells tending to differentiate towards more cholangiocyte-like phenotype during expansion, losing part of the hepatic differentiation capacity. Whereas in differentiation, the cells are being pushed towards hepatocyte phenotype already, hence lower concentration is sufficient to inhibit the biliary phenotype. Therefore, a fine balance is found between expansion and differentiation of the HeLLOs in EM and DM in view of the TGF-P inhibitor present. A preferred TGF-P inhibitor is A83-01.

[0018] The method of present invention includes glucocorticoid (for example dexamethasone) in the DM cell culture media. Glucocorticoid effect a wide range of processes, including survival, proliferation, and differentiation. The glucocorticoid has been shown to further assist in the differentiation of the HeLLOs towards a more functional hepatocyte and helps retain hepatic phenotype while proliferating.

[0019] The method of present invention includes DAPT in the DM cell culture media. The notch signaling pathway is important in the development of the biliary system. It has been reported that inhibition of the notch signaling promotes the conversion of cholangiocytes to hepatocytes in vivo. During HeLLO differentiation towards hepatocyte in DM, the presence of notch inhibitors increases the acquisition of hepatic phenotypes. Notch signaling inhibitors can be y-secretase inhibitors (e.g. DAPT, LY-411575, dibenzazepine, etc.).

[0020] Examples of a suitable basal medium that can be used in the method of present invention for both expansion medium (EM) and differentiation medium (DM) is preferably William’s E medium, but other basal media may be used e.g. DMEM, MEM, DMEM / F12, Advanced DMEM / F12, Ham’s F12, and RPML1640. Furthermore, the basal medica is preferably supplemented with Glutamine, for example with 2 mM glutamax,

[0021] The EM or / and DM media may further comprise 25 to 200 ng / ml, preferably 50 to 150 ng / ml, more preferably 75 to 100 ng / ml of a wnt inhibitor, preferably wherein the wnt inhibitor is Dickkopf-1 (DKK-1) and / or 1 to 20 mM, preferably 2.5 to 15 mM, more preferably 5 to 10 mM of Nicotinamide (NAM).

[0022] Furthermore, the EM and / or DM media may comprise antibiotics, such as penicillinstreptomycin, buffers such as HEPES and / or sodium bicarbonate, glucose, pyruvate, such as sodium pyruvate, 2-phospho-L-ascorbic acid trisodium salt, glucocorticoid, such as dexamethasone, insulin, transferrin, and selenous acid, such as lx ITS and / or a notch inhibitor, such as DAPT. According to a preferred embodiment, the present invention relates to the method, wherein the differentiation media (DM) is absent of one or more growth factors selected from the group consisting of EGF, HGF, FGF19, B27 and Gastrin, preferably none of said growth factors is present in the differentiation media. Growth factors in general assist in cell proliferation. However, it was found that proliferation inhibits the cell differentiation and therefore not adding further growth factors in the differentiation media benefits the cell differentiation of the liver cells into functional and proliferative hepatocytes.

[0023] According to another preferred embodiment, the present invention relates to the method wherein the cells are selected from the group consisting of cells originating from liver tissue cells, primary human hepatocytes, pre-cultured organoids, intrahepatic cholangiocyte organoids (ICOs), isolated liver cells, embryonic and induced pluripotent stem cells.

[0024] According to yet another preferred embodiment, the present invention relates to the method wherein the R-spondin is selected from the group consisting of R-spondin-1, R-spondin-3, R-spondin-2, or other protein family member of the R-spondin protein family, preferably R- spondin-1.

[0025] According to a preferred embodiment, the present invention relates to the method wherein the glucocorticoid is selected from the group consisting of hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone, preferably dexamethasone.

[0026] According to another preferred embodiment, the present invention relates to the method wherein the TGF-P inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947, or related ALK4-, ALK5-, ALK7- inhibitor, preferably A83- 01.

[0027] According to a preferred embodiment, the present invention relates to the method, wherein the differentiation media (DM) further comprises 1 to 100 ng / ml, preferably 5 to 50 ng / ml, more preferably 10 to 25 ng / ml of a BMP pathway activator, preferably wherein said BMP pathway activator is BMP-7. The method of present invention may include BMP-7 in the DM cell culture media to act as a multifunctional cytokine, regulating the growth and differentiation of many cell types including hepatocytes. BMP-7 has been shown to promote hepatocyte regeneration and function.

[0028] According to yet another preferred embodiment, the present invention relates to the method wherein the scaffold matrix is a laminin-rich extracellular matrix scaffold matrix, preferably Matrigel, Laminin-Entactin Complex, Ultimatrix, Cultrex, non-animal based scaffolds, synthetic hydrogels, such as polyethylene glycol, polyisocyanide, polyacrylamide, polyvinyl alcohol, polyglycolic acid, and polylactic acid, Polyethylene Glycol, Polyisocyanopeptide, or other basement membrane extract.

[0029] According to another preferred embodiment, the present invention relates to the method wherein the expansion of cells in EM is for about 1 to 60 days, preferably 3 to 30 days, more preferably for about 4 to 15 days, even more preferably for about 5 to 12 days, most preferably 6 to 10 days. However, cells may be cultured in EM for at most 1 year, preferably at most 6 months, preferably at most 2 months, even more preferably at most 4 weeks, without significantly affecting cell viability, cell expansion properties.

[0030] According to another preferred embodiment, the present invention relates to the method wherein the culturing of the expanded cells in DM is for at most 2 months, preferably at most 6 weeks, more preferably at most 4 weeks, even more preferably at most 2 weeks, most preferably at most 1 week.

[0031] According to yet another preferred embodiment, the present invention relates to the method wherein the method further comprises a step d) wherein the HELLOs are co-cultured with ICOs to improve functional differentiation of the ICOs and HELLOs. Alternative to co-cultering with HELLOs, the ICOs can also by themselves be transformed into HeLLOs. ICOs were grown for four passages, the medium was switched to HeLLO expansion medium in passage five, grown for several days, and subsequently differentiated using HeLLO differentiation medium. Therefore, ICOs still retain the potential to differentiate towards better hepatocyte -phenotypes, i.e. the HELLOs, when using HELLO medium and the method of present invention. Although this differentiation is partial, this shows that the medium conditions strongly affect the cell fate in vitro. Therefore, apart from starting from ICOs in the cell culture, alternatively HELLOs can also be cocultured with other liver cells, like ICOs, to improve functional differentiation towards better hepatocyte-phenotypes.

[0032] The present invention, according to a second aspect, relates to hepatocyte-like liver organoids (HELLOs), wherein said HELLOs express increased levels of one or more hepatocyte markers selected from the group consisting of ALB, CYP3A4, G6PC, SLC10A1, APOA1, CYP2E1, TF, as compared to the expression levels of said one or more hepatocyte markers in intrahepatic cholangiocyte organoids (ICOs). The hepatocyte markers are found in the public databases (www.ensembl.or ) under the following reference numbers; ALB (ENSG00000163631 ), CYP3A4 ( ENSG00000160868), G6PC (ENSG00000131482), SLC10A1 (ENSG00000100652), APOA1 (ENSG00000118137), CYP2E1 (ENSG00000130649), TF (ENSG00000091513).

[0033] According to a preferred embodiment, the present invention relates to the HELLOs, wherein the HELLOs express decreased levels of one or more cholangiocyte and / or progenitor markers selected from the group consisting of SSP1, ONECUT1, KRT19, EPCAM, KRT7, KRT8, DUOX2, TFF1, MUC5B, LYZ, as compared to the expression levels of said one or more cholangiocyte and progenitor marker in ICOs. A well-realized differentiation or transition process from one cell type to another is marked by both acquisition of the target cell identity and loss of original cell identity. During expansion, HeLLOs display immature cholangiocyte identity with high proliferative capacity. Therefore, upon differentiation into hepatocytes, HeLLOs not only need to acquire hepatocyte identity but also have to lose cholangiocyte and / or progenitor identity. Indeed, differentiated HeLLOs express decreased levels of one or more cholangiocyte and / or progenitor markers selected from the group consisting of KRT7, KRT8, DU0X2, TFF1, MUC5B, LYZ, LGR5, CD24, PR0M1, and S0X9. These are cholangiocyte markers, and since the present method uses starting cells from cholangiocyte-like cells which then differentiate towards hepatocyte-like cells, it was shown by RNA sequencing that these markers are reduced significantly. Furthermore, the extent of the reduction of these markers correlated with the extent of differentiation of the hepatocyte-like cell, see Table 1. The cholangiocyte and / or progenitor markers are found in the public databases (www.ensembl.or ) under the following reference numbers; KRT7 (ENSG00000135480), KRT8 (ENSG00000170421) DU0X2 (ENSG00000140279), TFF1 (ENSG00000160182), MUC5B (ENSG00000117983), LYZ (ENSG00000090382), LGR5 (ENSG00000139292), CD24 (ENSG00000272398), PR0M1 (ENSG00000007062), and S 0X9 (ENSG00000125398).

[0034] According to another preferred embodiment, the present invention relates to the HELLOs, wherein the expression levels of said one or more hepatocyte markers are comparable to their expression levels in mature human hepatocytes, such as (in vivo) mature human hepatocytes.

[0035] According to yet another preferred embodiment, the present invention relates to the HELLOs, wherein the HELLOs secrete at least 25 ug / day, preferably at least 100 ug / day, more preferably at least 200 ug / day, even more preferably at least 250 ug / day of albumin protein and / or secrete at least 1 ug / day, preferably at least 1,2 ug / day, more preferably at least 1.5 ug / day, even more preferably at least 1.7 ug / day of Al AT protein. The albumin and Al At protein are indicative of mature hepatocyte functioning. The HELLOs secrete relative high levels of both the Al AT and albumin proteins in comparison to other known liver cells including the intrahepatic cholangiocyte organoids (ICOs), indicating a more mature hepatocyte functioning, more closely resembling the mature human hepatocytes.

[0036] According to another preferred embodiment, the present invention relates to the HELLOs, wherein said HELLOs are obtained according to the method as disclosed herein of present invention.

[0037] According to a preferred embodiment, the present invention relates to the HELLOs, HELLOs according to any one of the claims 10 to 14, wherein the HELLOs comprise a mature drug metabolism, fatty acid metabolism, bile acid transport, and liver protein secretion function that closely resembles mature hepatocytes. Especially when compared to ICOs, the HELLOs outperform the ICOs in respect to their closer resemblance to mature liver cells. Although the abundance of intracellular bile acid in HeLLOs was similar to ICOs, experiments showed that the profile of the bile acid types in HeLLOs resembled the hepatocyte better with less cholic acid (CA) and taurocholic acid (T-CA), and more glycocholic acid (G-CA) and glycochenodeoxy cholic acid (G-CDCA). Moreover, HeLLOs were able to recapitulate bile acid transport function of the hepatocytes as observed by the accumulation of fluorophore-labeled bile acid in the lumen of the organoids. NTCP inhibition by zafirlukast exposure abolished the bile acid transport in HeLLOs, demonstrating an NTCP-dependent bile acid transport which also occurs in hepatocytes.

[0038] According to another preferred embodiment, the present invention relates to the HELLOs, wherein the HELLOs can be cultured for at least 3 weeks, preferably at least 4 weeks, more preferably at least 3 months, even more preferably at least 6 months, most preferably at least 1 year, without significantly affecting cell viability.

[0039] The present invention, according to a further aspect, relates to the use of HELLOs for drug testing, clinical trials, transplantation, toxicology studies, organ-on-a-chip, disease modelling, metabolic modeling. HeLLOs can predict drug-induced liver injury (DILI) due to their high level of drug metabolism activities. When exposed to acetaminophen, a known hepatotoxic compound, HeLLOs displayed higher sensitivity in cytotoxic response. The EC50 of acetaminophen for HeLLOs was 7.64 mM, a similar value to reported EC50 of cultured PHHs (10 mM), whereas the EC50 of ICOs was determined at about 20 mM. HELLOs combine all the advantages of ICOs, such as ease of generation, expansion and use for functional assays, 3D cultures and personalized modeling, with a more hepatic phenotype. Whole-transcriptome profiling shows that almost all drug metabolism genes are more highly expressed in HELLOs compared to ICOs. Functional analysis on drug metabolism also displays higher metabolism activities in HeLLOs. Thereby, HELLOs provide an improved model for preclinical drug screening, disease modeling, personalized therapy testing, and potentially also as a source for liver stem cell transplantations (as tried for ICOs, which showed insufficient engraftment putatively due to insufficient hepatic differentiation). Furthermore, these hepatic phenotypes are relatively maintained in HELLOs when cultured in 2D, giving more advantages when the presence of hydrogel may hamper certain functional analysis.

[0040] The present invention, according to a further aspect, relates to a differentiation medium for differentiating functional and proliferative hepatocytes and / or hepatocyte-like liver organoids (HELLOs), wherein said differentiation medium is comprised of basal media 0.1 to 500 pM, preferably 0.5 to 250 pM, more preferably 2 to 100 pM, most preferably 5 to 50 pM of at least one transforming growth factor P (TGF- ) inhibitor , and 0.1 to 100 pM, preferably 1 to 50 pM, more preferably 5 to 10 pM of a Notch inhibitor, and 0.1 to 100 pM, preferably 1 to 50 pM, more preferably 5 to 30 pM glucocorticoid, and 1 to 100 ng / ml, preferably 5 to 50 ng / ml, more preferably 10 to 25 ng / ml of a BMP pathway activator, and wherein the DM is absent of one or more growth factors selected from the group consisting of EGF, HGF, FGF19, B27 and Gastrin, preferably none of said growth factors is present in the differentiation media. Growth factors in general assist in cell proliferation. However, it was found that proliferation inhibits the cell differentiation and therefore not adding further growth factors in the differentiation media benefits the cell differentiation of the liver cells into functional hepatocytes.

[0041] According to a preferred embodiment, the present invention relates to a differentiation medium, wherein the TGF- inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947, or related ALK4-, ALK5-, ALK7- inhibitor, preferably A83-01. A TGF-P inhibitor inhibits the activity of TGF-P signaling pathway. This signaling pathway has been described to promote biliary and inhibit hepatocyte differentiation. We found that inhibiting the TGF-P signaling pathway increases the expression of some hepatic genes.

[0042] According to another preferred embodiment, the present invention relates to a differentiation medium, wherein the Notch inhibitor is selected from the group consisting of DAPT, LY-411575, dibenzazepine, or other related y-secretase inhibitors, preferably DAPT. The notch signaling pathway is important in the development of the biliary system. It has been reported that inhibition of the notch signaling promotes the conversion of cholangiocytes to hepatocytes in vivo. During HeLLO differentiation towards hepatocyte in DM, the presence of notch inhibitors increases the acquisition of hepatic phenotypes. Notch signaling inhibitors can be y-secretase inhibitors (e.g. DAPT, LY-411575, dibenzazepine, etc.).

[0043] According to yet another preferred embodiment, the present invention relates to a differentiation medium, wherein the glucocorticoid is selected from the group consisting of hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone, preferably dexamethasone. Glucocorticoid effect a wide range of processes, including survival, proliferation, and differentiation. The glucocorticoid has been shown to further assist in the differentiation of the HeLLOs towards a more functional hepatocyte and helps retain hepatic phenotype while proliferating.

[0044] According to another preferred embodiment, the present invention relates to a differentiation medium, wherein the differentiation medium further comprises 1 to 100 ng / ml, preferably 5 to 50 ng / ml, more preferably 10 to 25 ng / ml of a BMP pathway activator, preferably wherein said BMP pathway activator is BMP-7. The present invention may include BMP-7 in the differentiation medium to act as a multifunctional cytokine, regulating the growth and differentiation of many cell types including hepatocytes. BMP-7 has been shown to promote hepatocyte regeneration and function.

[0045] The present invention, according to a further aspect, relates to the use of the differentiation medium for differentiation of cells originating from liver tissue cells, primary human hepatocytes, pre-cultured organoids, intrahepatic cholangiocyte organoids (ICOs), isolated liver cells, embryonic and induced pluripotent stem cells.

[0046] The present invention will be further detailed in the following examples and figures wherein:

[0047] Figure 1: Shows that HeLLOs in expansion medium were highly proliferative and exhibited cystic organoid structures. HeLLOs in hepatic differentiation medium were dense and relatively small. HeLLOs exhibited a much denser and smaller morphology than ICOs after differentiation towards hepatocytes.

[0048] Figure 2: Shows that HeLLOs are highly expandable and can be cultured for a long period of time. HeLLOs retained their proliferative capacity as shown by their large cystic structure and that HeLLOs expanded better than ICOs, especially after 3 months of cell passaging.

[0049] Figure 3: Shows immunofluorescence staining on several hepatocyte protein markers on the

[0050] HELLOs and ICOs; CYP2E1, CYP3A4, and albumin (ALB). HeLLOs display robust hepatocyte protein marker expression which was significantly higher than in ICOs, indicating that HELLOs are more hepatic than ICOs.

[0051] Figure 4: Shows quantitative PCR results of the hepatocyte marker genes CYP3A4, G6PC,

[0052] SLC10A1, and albumin (ALB) in human liver tissue samples, HeLLOs, and ICOs. When comparing hepatocyte marker gene expression of HeLLOs to differentiated ICOs, an increased expression was observed in HELLO, and the expression of these markers in HeLLOs was comparable to the human liver cells.

[0053] Figure 5: Shows a heatmap of bulk RNA-seq data showing the expression of sets of hepatocyte, cholangiocyte, and progenitor marker genes in HeLLOs, ICOs, and several tissue controls (the liver, bile duct, and fibroblast). The heatmap shows that HeLLOs cluster closer towards the control liver tissues as compared to ICOs. These results indicate that HeLLOs have an overall better resemblance to hepatocytes than ICOs. Interestingly, HeLLO not only acquired more hepatic marker gene expression, but also expressed less cholangiocyte and progenitor marker genes. This suggests improved cell fate conversion towards hepatocytes in HeLLOs upon differentiation compared to ICOs. HeLLOs attain hepatocyte and lose both cholangiocyte and progenitor markers upon differentiation.

[0054] Figure 6: Show the protein levels of the albumin and Al AT protein secreted in the HeLLOs

[0055] (dark gray, left bars) and the ICOs (light gray, right bars). The albumin and Al AT secretion levels are indicative of mature hepatocyte functioning. The HELLOs secrete relative high levels of both the Al AT and albumin proteins in comparison to the ICOs, indicating a more mature hepatocyte functioning.

[0056] Figure 7 : Shows the metabolic capacity of the HELLOs (dark gray, left bars) and ICOs (light gray, right bars) for 6 drugs requiring different metabolic enzymes (CYP1A2, CYP2B6, CYP2D6, CYP2C9, CYP3A4 and UGTs). Enzymatic activities were determined by measuring the amount of metabolites (acetaminophen, bupropion- OH, dextrorphan, tolbutamide-OH, midazolam-OH, and 7-OH-coumarin glucuronide) in the media using LC-MS / MS. The results show that HELLOs exhibit mature hepatic functions. HeLLOs not only resembled hepatocytes better on the transcriptomic level but also on the functional level. The metabolic capacity for these six drugs requiring different metabolic enzymes show that HeLLOs greatly outperform ICOs.

[0057] Figure 8: Shows the fatty acid oxidation activity in HeLLOs (drak gray bars), fibroblasts

[0058] (white bars) and ICOs (light gray bars), in view of several important fatty acid oxidation enzymes; CPT2, VLCAD, MCAD, SCAD, Crotonase and SCHAD. HeLLOs are more capable in oxidizing several fatty acids requiring different components in the fatty acid metabolism pathway also when compared to fibroblasts, the current standard for clinical testing.

[0059] Figure 9: Shows the activities of bile acid synthesis enzymes in HeLLOs (drak gray bars), fibroblasts (white bars) and ICOs (light gray bars), in view of several important bile acid synthesis enzymes; DBP (hydration), DBP (dehydrogenation), SCPx and BAAT. Bile acid synthesis was also more active in HeLLOs compared to ICOs and fibroblasts.

[0060] Figure 10: Shows the hepatic gene expression levels of important hepatocyte marker genes (ALB, CYP3A4, and G6PC) in an organoid cell culture of HELLOs cultivated in differentiation media. The differentiation media varies in excluding one or more growth factors, EGF, HGF, and FGF19, . Removal of all growth factors from the differentiation media results in the strongest increase in gene expression levels of the hepatocyte marker genes indicating a significant increase in hepatic differentiation capacity of the cultivated cells.

[0061] Examples

[0062] Organoid establishment and HELLO cell culture

[0063] Organoids were established from liver biopsies as previously described (Huch et al., 2015). Liver biopsies were minced into small pieces and digested by incubation with 2.5 mg / ml Collagenase D (Sigma) in Hanks’ Balanced Salt Solution for 20 min at 37°C. The solution was then diluted using wash medium: cold Advanced DMEM / F12 medium (Gibco), supplemented with 2 mM GlutaMAX (Gibco), 10 mM HEPES (Gibco), 100 U / ml Pen-Strep (Gibco), and centrifuged at 300 x g, 4°C for 5 min. Supernatant was removed and cell pellet was resuspended in a 1:1 mixture of cold (~4°C) Cultrex BME and wash medium. The cell suspension was then plated by adding a volume of 30-50 pl droplet into each cell culture well plate. The droplet was incubated at 37°C for 15-30 min or until the matrix was solidified.

[0064] Hepatocyte-like liver organoids (HeLLOs) were grown by adding HeLLO expansion medium. HeLLO expansion medium is composed of William’ s E medium supplemented with 2 mM GlutaMAX, 10 mM HEPES, 100 U / ml Pen-Strep, 10 mM Nicotinamide (Sigma), 10% RSPO1 conditioned media, 20 ng / ml EGF (Peprotech), 50 pg / ml Primocin (Invivogen), 14 mM Glucose, lx ITS, 17 mM Sodium Bicarbonate, 6.3 mM Sodium Pyruvate, 0.2 mM 2-phospho-L- ascorbic acid trisodium salt, 0.1 pM Dexamethasone, 100 ng / ml DKK-1, 5 pM A83-01 (Tocris), 10 pM Forskolin (Tocris), and optionally at least one transforming growth factor (TGF- ) inhibitor. Hepatic differentiation was done by switching to HeLLO differentiation medium, which consists of William’s E medium supplemented with 2 mM GlutaMAX, 10 mM HEPES, 100 U / ml Pen-Strep, 10 mM Nicotinamide, 50 pg / ml Primocin, 14 mM Glucose, lx ITS, 17 mM Sodium Bicarbonate, 6.3 mM Sodium Pyruvate, 0.2 mM 2-phospho-L-ascorbic acid trisodium salt, 500 nM A83-01, 25 ng / ml BMP7 (Peprotech), 10 pM DAPT (Sigma), and 30 pM Dexamethasone (Sigma) for 8 days. To culture intrahepatic cholangiocyte organoids (ICOs), ICO seeding medium was added to the wells for the first 3-7 days after seeding of the biopsies or until organoids were formed (Huch et al., 2015). ICO seeding medium consists of Advanced DMEM / F12 medium (Gibco, 12634028) supplemented with 2 mM GlutaMAX, 10 mM HEPES, 100 U / ml Pen-Strep, 2% B27 without vitamin A (Gibco, 12587010), 10 mM Nicotinamide, 1.25 mM N-Acetylcysteine (Sigma, A9165), 10% RSPO1 conditioned media, 10 nM Gastrin (Tocris, 3006 / 1), 50 ng / ml EGF, 100 ng / ml FGF10 (Peprotech, 100-26), 25 ng / ml HGF (Peprotech, 100-39), 50 pg / ml Primocin, 5 pM A83-01, and 10 pM Forskolin, 30% Wnt conditioned media (homemade), 25 ng / ml Noggin (Peprotech, 120-10C), and hES cell cloning recovery solution (Stemgent, 010014500). After organoids were formed, the medium was changed into ICO expansion medium, which was based on ICO seeding medium but without Wnt conditioned media, Noggin, and hES cell cloning recovery solution. Differentiation towards hepatocyte was initiated by culturing the organoids using ICO expansion medium supplemented with 25 ng / ml BMP7 for 5-7 days followed by ICO differentiation medium, consisting of Advanced DMEM / F12 medium supplemented with 2 mM GlutaMAX, lOmM HEPES, 100 U / ml PenStrep, 2% B27 without vitamin A, 1.25mM N- Acetylcysteine, 10 nM Gastrin, 50 ng / ml EGF, 25 ng / ml HGF, 100 ng / ml FGF19 (Peprotech, 100- 32), 50 pg / ml, Primocin, 500 nM A83-01, 25 ng / ml BMP7, 10 pM DAPT, and 30 pM Dexamethasone for 8 days.

[0065] Cell culture was monitored in both expansion and differentiation media. The morphology of HeLLOs is distinct from ICOs upon differentiation. HeLLOs exhibited a much denser and smaller morphology than ICOs after differentiation towards hepatocytes, see Figure 1. HeLLOs are highly expandable and can be cultured for a long period of time, Figure 2. HeLLOs retained their proliferative capacity as shown by their large cystic structure even after 10 passages in culture, whereas with the ICOs a reduction in their proliferative capacity is observed.

[0066] Immunofluorescence staining

[0067] Organoids were isolated from BME and fixated using 4% paraformaldehyde for 20 min at room temperature, washed with PBS, embedded in 2% agarose, transferred into tissue cassettes, dehydrated, and embedded in into paraffin blocks. Sections were prepared using a microtome and hydrated. Antigen retrieval was performed by incubating the sections in either Tris-EDTA (pH 9.0) or citrate buffer (pH 6.0) for 30 min at 98°C followed by incubation for 30 min at room temperature. Sections were permeabilized using PBS supplemented with 0.1% Triton X-100 and blocked with 10% Normal Goat Serum (Thermo Fisher) for 30 min at room temperature. Sections were then incubated with primary antibodies at 4°C overnight, washed, incubated with secondary antibodies for 1 hour at room temperature, counterstained with DAPI, and mounted using FluorSave (Merck).

[0068] Immunofluorescence staining on several hepatocyte markers is performed on the HELLOs and ICOs; CYP2E1, CYP3A4, and albumin (ALB). Images were acquired using Leica Dmi8 Thunder Imager microscope (Leica) and processed using Leica Application Suite X, Adobe Photoshop 2023, or ImageJ. Results show that HeLLOs display robust hepatocyte protein marker expression, which was significantly higher than in ICOs, see Figure 3. The protein expression in HeLLOs was much higher than ICOs.

[0069] RNA isolation and RT-qPCR

[0070] Total RNA was isolated using PureLink™ RNA Mini Kit (Invitrogen) according to the manufacturer’s instructions and subsequently stored at -80°C until further processing. RNA concentration was determined using Qubit™ RNA Broad Range Assay Kit (Invitrogen) and Qubit™ 4 Fluorometer (Invitrogen). Synthesis of cDNA was performed using T100 Thermal Cycler (Bio-Rad) and iSripf™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s recommendation. Quantitative PCR was performed using 20 ng cDNA, 0.75 pM forward and reverse primers, and iQ™ SYBR® Green Supermix (Bio-Rad) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The PCR cycles consisted of 39 cycles of 95°C for 10 sec and 62°C for 30 sec. Gene expression was calculated using the AACt method normalized to the housekeeping gene heterochromatin protein 1 binding protein 3 (HP1BP3).

[0071] Expression levels of important hepatocyte marker genes in HeLLOs, ICOs and in human liver cells was measured. The hepatocyte markers CYP3A4, G6PC, SLC10A1, and albumin (ALB) were measured, see Figure 4. It was observed that in HeLLOs the hepatycyte markers were expressed similar to their expression in the human liver cells. The expression of these marker genes in ICOs were significantly lower.

[0072] To demonstrate that the removal of growth factors leads to improved hepatic phenotype in organoid culture, the effect of growth factors (EGF, HGF, and FGF19) in organoid differentiation media on hepatic differentiation capacity was screened. Expression levels of important hepatocyte marker genes in HeLLOs were measured upon removal of growth factors from the differentiation media, see Figure 10. The hepatocyte markers ALB, CYP3A4, and G6PC were measured upon removal of one or more of the growth factors EGF, HGF, and FGF19. Removal of these individual growth factor from the differentiation media resulted in a significant increase of the hepatocyte marker gene expression levels. Especially removal of EGF from the DM media provided a relatively large effect on the hepatocyte marker gene expression levels. Additionally, when all of the growth factors were removed, the hepatocyte marker gene expression levels increase sharply. This indicates that removal of growth stimuli is one of the most important factors for hepatic differentiation.

[0073] Bulk RNA sequencing and analysis

[0074] Messenger RNA was isolated from total RNA using Poly(A) Beads (NEXTflex). RNA integrity was assessed using Agilent RNA 6000 Nano Kit () and RNA concentration was determined using Qubit RNA High Sensitivity Assay Kit (). RNA samples with RIN > 8.0 were used for sequencing. Sequencing library was prepared using the Rapid Directional RNA-Seq Kit (NEXTflex) and sequenced on a NextSeq500 (Illumina) to produce 75 base reads (Utrecht Sequencing Facility).

[0075] Raw read processing was performed as previously described (Ardisasmita et al., 2022) using Galaxy web-based platform (https: / / usegalaxy.eu / ). Reads were assessed using FastQC tool (Galaxy Version 0.72) and trimmed using Cutadapt (Galaxy Version 1.66.6). Reads were mapped using RNA STAR tool (Galaxy Version 2.7.2b), Gencode human reference genome sequence release 33 (GRCh38.pl3), and Gencode comprehensive gene annotation v33 using default parameters. Count matrices were obtained by selecting the “-quantMode GeneCounts” option in the RNA STAR tool. Counts were normalized by applying the DESeq2 variance-stabilizing transformation (VST) from the “DESeq2” R package (Love et al., 2014). Differentially expressed genes were identified using the following parameters: “alpha=0.05” and “lfcThreshold=0”. Principal component analysis (PCA) was done using the “ggplot2” R package. Gene expression heatmaps were generated using the “pheatmap” R package and visualized as mean-centered expression per gene. Distance -based similarity score was calculated as previously reported (Ardisasmita et al., 2022). Enrichment analysis was performed in enrichR (Chen et al., 2013). CellNet analysis was done using Platform-Agnostic CellNet web application (Lo et al., 2023).

[0076] Whole transcriptome analysis was done wherein a heatmap of bulk RNA-seq data was made which shows the expression of sets of hepatocyte, cholangiocyte, and progenitor marker genes in HeLLOs, ICOs, and several tissue controls including the liver, bile duct, and fibroblast, see Figure 5. The HeLLOs cluster closer towards the control liver tissues based on the expression of the marker genes, as compared to ICOs. These results indicate that HeLLOs have an overall better resemblance to hepatocytes than ICOs. Interestingly, HeLLO not only acquired more hepatic marker gene expression, but also expressed less cholangiocyte and progenitor marker genes. This suggests improved cell fate conversion towards hepatocytes in HeLLOs upon differentiation compared to ICOs.

[0077] Marker expression data, both obtained via qPCR and RNA sequencing (Table 1 and 2, respectively) show that the HELLOs of present invention express increased levels of hepatocyte markers ALB, CYP3A4, G6PC, SLC10A1, APOA1, CYP2E1, TF, as compared to the expression levels of said markers in (ICOs). Hepatocyte marker levels were much higher than in ICO, and more close to the human hepatocyte cells. The hepatocyte markers included can be found in the public databases (www.ensembl.or ) under the following reference numbers; ALB (ENSG00000163631), CYP3A4 ( ENSG00000160868), G6PC (ENSG00000131482), SLC10A1

[0078] (ENSG00000100652), APOA1 (ENSG00000118137), CYP2E1 (ENSG00000130649), TF (ENSG00000091513).

[0079] Table 1. Comparison of expression of hepatocyte protein markers in HeLLO, ICO, and hepatocyte cells, obtained by RNA sequencing.

[0080] Table 2. Comparison of expression of hepatocyte protein markers in HeLLO, ICO, and hepatocyte cells, obtained by qPCR. Differentiated HeLLOs express decreased levels of cholangiocyte and progenitor markers of KRT7, KRT8, DUOX2, TFF1, MUC5B, LYZ, LGR5, CD24, PROMI, and SOX9. The cholangiocyte markers included can be found in the public databases (www.ensembl.org) under the following reference numbers; KRT7 (ENSG00000135480), KRT8 (ENSG00000170421 ) DU0X2 (ENSG00000140279), TFF1 (ENSG00000160182), MUC5B (ENSG00000117983), LYZ

[0081] (ENSG00000090382), LGR5 (ENSG00000139292), CD24 (ENSG00000272398), PR0M1 (ENSG00000007062), and S0X9 (ENSG00000125398). These are cholangiocyte markers, and since the present method uses starting, it was shown by RNA sequencing that these markers are reduced significantly, see Table 3. Results indicate that the HeLLO cells produced according to the method of present invention, starting from cholangiocyte-like cells, differentiate towards hepatocyte-like cells. Furthermore, the extent of the reduction of these markers correlated with the extent of differentiation of the hepatocyte-like cell.

[0082] Table 3. Comparison of expression of cholangiocyte protein markers in HeLLO, ICO, Hepatocyte and bile duct cells, obtained by RNA sequencing.

[0083] Albumin and alpha- 1-antitrypsin secretion assays

[0084] To measure albumin and Al AT secretion, culture medium was refreshed and subsequently collected after 24 hours. The amount of albumin and Al AT in the medium was then determined using Albumin Human ELISA Kit (Invitrogen, EHALB) and Human alpha- 1 -Antitrypsin ELISA Kit (AssayPro, EA5101-1) respectively.

[0085] Results show that HeLLOs secrete significantly more albumin and alpha- 1 -antitrypsin compared to ICOs, see Figure 6, a further sign of a more differentiating and mature hepatocyte. Secretion levels of both albumin and alpha- 1 -antitrypsin (Al AT) in HeLLOs (dark grey) and ICOs (light grey) are measured in the media after differentiation. The HELLOs secrete relatively high levels of both the Al AT and albumin proteins in comparison to the ICOs, indicating a more mature hepatocyte functioning.

[0086] CYP activity assay

[0087] Next, the ability of the HeLLOs to metabolize different drug compounds was examined by CYP activity assay. This to investigate if HELLOs exhibit mature hepatic functions and that. HeLLOs not only resembled hepatocytes better on the transcriptomic level but also on the functional level.

[0088] To measure CYP activity, a cocktail of CYP substrates was added to the cell culture medium: 5 pM midazolam (Sigma), 20 pM tolbutamide (Sigma), 20 pM bupropion (Sigma), 15 pM phenacetin (Sigma), 12 pM 7 -hydroxy coumarin (Sigma), and 15 pM dextromethorphan (Sigma). The cocktail was let to incubate for several timepoints (4, 8, 16, and 24 hours). After which, 400 pl conditioned medium was placed into a glass vial, mixed with 400 pl MeOH (0.1% (v / v) formic acid), and subsequently stored at -20°C until further processing.

[0089] The following compounds were used as standards for LC-MS / MS analysis: midazolam, 1- hydroxymidazolam (Sigma), tolbutamide, 4-hydroxytolbutamide (Sigma), bupropion, hydroxybupropion (Sigma), phenacetin, acetaminophen (Sigma), 7-hydroxy coumarin, 7- hydroxycoumarin glucuronide (Sigma), dextromethorphan, and dextrorphan (Sigma). All standards were prepared in the same matrix as the cell culture medium. The analysis was performed in a single run using a Shimadzu triple-quadrupole LCMS 8050 system with two Nexera XR LC-20AD pumps, a Nexera XR SIL-20AC autosampler, a CTO-20 AC column oven, an FCV-20AH2 valve unit (Shimadzu). Both substrates and metabolites were separated on a Synergi Polar-RP column (150 x 2.0 mm, 4 pm, 80 A) with a 4 x 2 mm C18 guard column (Phenomenex). The mobile phase consisted of 0.1% (v / v) formic acid in Millipore (A) and 0.1% (v / v) formic acid in MeOH (pH 2.7; B), and was set as 100% A (0-1 min), 100% to 5% A (1-8 min), 5% A (8-9 min), 5% to 100% A (9-9.5 min), and 100% A (9.5-12.5 min). The total run time was 12.5 min, and the flow rate was 0.2 ml / min. Peaks were integrated using LabSolutions software.

[0090] Results, Figure 7, show that drug metabolism-related enzymes are more active in HeLLOs than ICOs. The activities of important drug metabolizing enzymes are measured in HeLLOs (dark grey) and ICOs (light grey) after differentiation. Enzymatic activities were determined by measuring the amount of metabolites (acetaminophen, bupropion-OH, dextrorphan, tolbutamide - OH, midazolam-OH, and 7-OH-coumarin glucuronide) in the media using LC-MS / MS. The metabolic capacity for these six drugs requiring different metabolic enzymes show that HeLLOs greatly outperform ICOs.

[0091] Fatty acid oxidation and bile acid synthesis activity assays

[0092] The bile acid transport, and liver protein secretion function was examined of the HeLLOs to see of these resemble mature hepatocyte function. Also ICOs were included as well as fibroblasts as control, since fibroblast are the current standard for clinical testing. Enzymatic activities for both fatty acid oxidation and bile acid synthesis were all measured using the cell homogenates.

[0093] Briefly, cells were homogenized and sonicated in PBS. CPT2 activity was measured using palmitoyl-carnitine as substrate followed by detection of palmitoyl-CoA using ultra high- performance liquid chromatography (UHPLC) on a reversed-phase column (Wanders et al., 2010). VLCAD activity was measured using C16:0-CoA as substrate and ferrocenium hexafluorophosphate as electron acceptor, followed by detection of C16:l-CoA and 3-OH-C16- CoA using UHPLC (Wanders et al., 2010). MCAD activity was measured using 3- phenylpropionyl-CoA as substrate and ferrocenium hexafluorophosphate as electron acceptor, followed by detection of 3-phenylpropenonyLCoA using UHPLC (Wanders et al., 2010). SCAD activity was measured using butyryl-CoA as substrate and ferrocenium hexafluorophosphate as electron acceptor, followed by detection of crotonyl-CoA and 3-hydroxybutyryl-CoA using UHPLC (Schmidt et al., 2010). Crotonase (SCEH or ECHS1) activity was measured using crotonyl-CoA as substrate followed by detection of 3-hydroxybutyryl-CoA using UHPLC (Peters et al., 2014). SCHAD activity was measured spectrophotometrically using acetoacetyl-CoA as substrate (Wanders et al., 2010). DBP and SCPx were measured in a single assay using 24-ene- THC-CoA as substrate followed by detection of 24-hydroxy-THC-CoA, 24-keto-THC-CoA, and choloyl-CoA using UHPLC (Ferdinandusse et al., 2000). BAAT activity was measured using taurine () and choloyl-CoA as substrates followed by measurement of tauro-cholate by HPLC- tandem mass spectrometry (Ferdinandusse et al., 2005).

[0094] Results show that HeLLOs display higher fatty acid oxidation activity than ICOs, Figure 8. The activities of important fatty acid oxidation enzymes (CPT2, VLCAD, MCAD, SCAD, Crotonase and SCHAD) in HeLLOs (dark grey) ICOs (light grey) and fibroblasts (white). HeLLOs also display higher activities of bile acid synthesis enzymes (DBP (hydration), DBP (dehydrogenation), SCPx and BAAT) compared to ICOs but also compared to fibroblasts, see Figure 9.

Claims

CLAIMS1. A method for the in vitro production of functional and proliferative hepatocytes and / or hepatocyte-like liver organoids (HELLOs), comprising the steps of; a) expanding cells, preferably liver cells, in cell culture in an expansion medium (EM), wherein EM is comprised of basal media comprising 50 to 500 ng / mL of at least one R-spondin protein, 1 to 50 ng / mL EGF, 0.1 to 50 pM, and optionally at least one transforming growth factor (TGF- ) inhibitor, and 1 to 100 pM forskolin, b) optionally, collecting the expanded cells of step a and transfer these cells to a scaffold matrix, c) culturing of the expanded cells in a differentiation medium (DM) comprised of basal media and 0.1 to 500 pM of at least one transforming growth factor (TGF-P) inhibitor, and 0.1 to 100 pM of a Notch inhibitor, preferably wherein the Notch inhibitor is DAPT, and 0.1 to 100 pM glucocorticoid, and 1 to 100 ng / ml of a BMP pathway activator, preferably wherein said BMP pathway activator is BMP-7, and wherein the DM is absent of one or more growth factors selected from the group consisting of EGF, HGF, FGF19, B27 and Gastrin, preferably none of said growth factors is present in the differentiation media, to generate functional and proliferative hepatocytes and / or HELLOs.

2. Method according to claim 1, wherein the DM is absent of EGF.

3. Method according to claim 1 or claim 2, wherein the cells are selected from the group consisting of cells originating from liver tissue cells, primary human hepatocytes, pre-cultured organoids, intrahepatic cholangiocyte organoids (ICOs), isolated liver cells, embryonic and induced pluripotent stem cells.

4. Method according to any one of the claims 1 to 3, wherein the R-spondin protein is selected from the group consisting of R-spondin- 1, R-spondin-2, R-spondin-3, or other protein family member of the R-spondin protein family, preferably R-spondin- 1, and / or, wherein the glucocorticoid is selected from the group consisting of hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone, preferably dexamethasone, and / or, wherein the TGF-P inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947, or related ALK4-, ALK5-, ALK7- inhibitor, preferably A83-5. Method according to any one of the claims 1 to 4, wherein the differentiation media (DM) further comprises 1 to 100 ng / ml, preferably 5 to 50 ng / ml, more preferably 10 to 25 ng / ml of a BMP pathway activator, preferably wherein said BMP pathway activator is BMP-7.

6. Method according to any one of the claims 1 to 5, wherein the scaffold matrix is a laminin-rich extracellular matrix scaffold matrix, preferably Matrigel, Laminin-Entactin Complex, Ultimatrix, Cultrex, non-animal based scaffolds, synthetic hydrogels, such as polyethylene glycol, polyisocyanide, polyacrylamide, polyvinyl alcohol, polyglycolic acid, and polylactic acid, Polyethylene Glycol, Polyisocyanopeptide, or other basement membrane extract, most preferably Matrigel, even more preferably Laminin-Entactin Complex.

7. Method according to any one of claims 1 to 6, wherein the method further comprises a step d) wherein the HELLOs are co-cultured with ICOs to improve functional differentiation of the ICOs and HELLOs.

8. Hepatocyte-like liver organoids (HELLOs), wherein said HELLOs express increased levels of one or more hepatocyte markers selected from the group consisting of ALB, CYP3A4, G6PC, SLC10A1, APO Al, CYP2E1, TF, as compared to the expression levels of said one or more hepatocyte markers in intrahepatic cholangiocyte organoids (ICOs).

9. HELLOs according to claim 8, wherein the HELLOs express decreased levels of one or more cholangiocyte and / or progenitor markers selected from the group consisting of SSP1, ONECUT1, KRT19, EPCAM, KRT7, KRT8, DU0X2, TFF1, MUC5B, LYZ, as compared to the expression levels of said one or more cholangiocyte and progenitor marker in ICOs.

10. HELLOs according to claim 8 or 9, wherein the expression levels of said one or more hepatocyte markers are comparable to their expression levels in mature human hepatocytes.

11. HELLOs according to any one of the claims 8 to 10, wherein the HELLOs secrete at least 25 ug / day, preferably at least 100 ug / day of albumin protein, and / or secrete at least 1 ug / day, preferably at least 1,5 ug / day of Al AT protein.

12. HELLOs according to any one of the claims 8 to 11, wherein said HELLOs are obtained according to a method of any one of the claims 1 to 7.

13. HELLOs according to any one of the claims 8 to 12, wherein the HELLOs comprise a mature drug metabolism, fatty acid metabolism, bile acid transport, and liver protein secretion function that closely resembles mature hepatocytes.

14. HELLOs according to any one of the claims 8 to 13, wherein the HELLOs can be cultured for at least 3 weeks, preferably at least 4 weeks, more preferably at least 3 months, even more preferably at least 6 months, most preferably at least 1 year, without significantly affecting cell viability.

15. Use of HELLOs of any one of the claims 8 to 14 for drug testing, clinical trails, transplantation, toxicology studies, organ-on-a-chip, disease modelling, metabolic modelling16. A cell differentiation medium (DM) comprising basal media and 0.1 to 500 pM of at least one transforming growth factor P (TGF- ) inhibitor, and 0.1 to 100 pM of a Notch inhibitor, and 0.1 to 100 pM glucocorticoid, and 1 to 100 ng / ml of a BMP pathway activator, and wherein the DM is absent of one or more growth factors selected from the group consisting of EGF, HGF, FGF19, B27 and Gastrin, preferably none of said growth factors is present in the differentiation media.

17. Differentiation medium according to claim 16, wherein the DM is absent of EGF.

18. Differentiation medium according to claim 16 or claim 17, wherein the TGF- inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947, or related ALK4-, ALK5-, ALK7- inhibitor, preferably A83-01.

19. Differentiation medium according to any one of the claims 16 to 18, wherein the Notch inhibitor is selected from the group consisting of DAPT, LY-411575, dibenzazepine, or other related y-secretase inhibitors, preferably DAPT.

20. Differentiation medium according to any one of the claims 16 to 19, wherein the glucocorticoid is selected from the group consisting of hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone, preferably dexamethasone.

21. Differentiation medium according to any one of the claims 16 to 20, wherein the differentiation medium further comprises 1 to 100 ng / ml, preferably 5 to 50 ng / ml, more preferably 10 to 25 ng / ml of a BMP pathway activator, preferably wherein said BMP pathway activator is BMP-7.

22. Use of the differentiation medium according to any of the claims 16 to 21, for differentiation of cells originating from liver tissue cells, primary human hepatocytes, pre-cultured organoids, intrahepatic cholangiocyte organoids (ICOs), isolated liver cells, embryonic and induced pluripotent stem cells.

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