WO2016185281A1 - New hepatic cell lines and methods of making and using the same - Google Patents

Abstract

New celllines designated as HepaRP and Hepa RPHoriginating fromthehuman hepatoma line HEPARG® are disclosed. Methods of redirecting stem-like cells to hepatic reprogrammed cells, of getting stable production of single population of hepatocytes and of reaching a complete hepatocyte maturation status are also described.

Description

NEW HEPATIC CELL LINES AND METHODS OF MAKING AND USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of U.S. Serial Number 14/717,700, filed May 20, 2015, entitled NEW HEPATIC CELL LINES AND STEM-LIKE CELLS, METHODS OF MAKING AND USING THE SAME, incorporated by reference in its entirety herein.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to reprogrammed cells from stem-like cells of hepatic origin, and methods of making and using the same.

Description of Related Art

Up to now only human hepatic cell lines established from hepatocarcinoma cells are able to express part or all of the functional characteristics of mature hepatocytes. Some hepatoma cell lines have been extensively used such as HepG2, HuH7, etc. Such cells have drawbacks, including the lack of availability of a cell bank, and a progressive loss of many hepatic functions. HepaRG® cells (human hepatoma cell line deposit no. 1-2652, filed on 5 Apr. 2001 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15 in the name of Institut National de la Sante et de la Recherche Medicale (INSERM), U.S. Patent No. 7,456,018, incorporated by reference herein) are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many characteristics of primary human hepatocytes. Cryopreserved and differentiated HEPARG® cells (Biopredic, Inc.) are now widely used for many cell-based applications. Up to now, this line has been the best hepatic cell line in its ability to morphologically and functionally mimic primary human hepatocytes in vitro.

One crucial advantage of HEPARG® is the established cell bank, making possible long term high level of functional stability by regularly restarting new batches of cells from the original pool of cells. However, HEPARG® has 3 main limitations: 1) The cell bank has some limitations in number of frozen vials, 2) Although the cells have high reproducibility for 17 passages, the stability of the line is limited to 17-18 passages, which represents a strong limitation for long term experiments (requiring delivery to customers at passage 12 for preserving the bank); and 3) The mixed population of 2 cell types for which hepatocytes represent only half of the population. There remains a need for hepatic cell lines with functions mimicking primary human hepatocytes.

SUMMARY OF THE INVENTION

To overcome the limitation of the sustainability of the HEPARG® cell line represented by the limited number of remaining frozen vials, a strategy aiming to produce stem-like cells from HepaRG® cells has been developed. HepaSC cells (human hepatoma cell line deposit no. CNCM 1-4980, filed on May 19, 2015 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, in the name of Biopredic International, incorporated by reference herein) are undifferentiated proliferating hepatic stem-like cells derived from HepaRG® cells and sharing most properties of embryonic human stem cells without introducing additional genes into the genome. The HepaSC cells are described in detail in co-pending U.S. 14/717,700, filed May 20, 2015.

The HepaSC cells have been used for setting new HEPARG®-derived cell lines. The invention therefore aims to provide new HEPARG®-derived cell lines which could: 1 - preserve their unique capacity to reach a level of differentiation such that these cells could express practically all of the functions of the normal human hepatocyte whilst actively proliferating; 2- solve the cell bank limitation; 3- overcome the relatively short stability of HEPARG®; and 4- evidence high plasticity properties making them respond to new environmental conditions.

A strategy was developed for producing new HepaRP cell lines from the parental HEPARG® cell line taking advantage of the stem-like HepaSC cells reprogrammed from the HEPARG® cells and expressing properties of plasticity and indefinite cell renewal potentialities. These HepaSC cells were used to re-direct differentiation towards the hepatic lineage. Thus, described herein is the method of directing (re)differentiation of HepaSC cells into a population of hepatic reprogrammed cells. Differentiating cells of the new HepaRP lines are also described. The methods generally comprise culturing HepaSC cells under conditions preserving the stability of their sternness status, mainly indefinite cell renewal and plasticity. Then, the cells are submitted to a mechanical stress and in the presence of at least one differentiation factor for the target hepatic differentiation lineage for a time period sufficient for the HepaSC cells to commit to differentiation. The committed cells are then transferred to a culture medium comprising the differentiation factor and maintained in culture for growth. Thus, the techniques include methods for HepaSC culturing and expanding and for reconversion to hepatic differentiation lineage, such that a potentially unlimited number of reconverted new HepaRP cell lines could be obtained. The invention relates to obtaining new HepaRP lines originating from the parental HEPARG® cell line, in using a strategy of cell reprogramming to HepaSC so that HepaRP lines are the first hepatic lines derived from stem-like cells originating from a hepatoma cell line and able to produce mature human hepatocytes.

A normal human hepatocyte has the following morphological characteristics:

- A granular cytoplasm due to the fact that they are rich in organites such as mitochondria and rough endoplasmic reticulum vesicles, and a round and regular central nucleus with a very dense nucleolus.

- Cells are grouped and organized into typical trabecula, most often formed from cords of

2 to 4 cells, interlinked by joining structures of desmosome type, and communicating structures of "Gap"-type. This organization into trabeculae determines the double polarity which characterizes the hepatocyte: a basolateral domain corresponding to the sinusoidal pole on the side of the sinusoids and a biliary pole at the interface of the hepatocytes.

The biliary pole is associated with the detoxification function and more particularly with the elimination of biliary salts and conjugated metabolites. It is a dilated intercellular space, closed by characteristic complex junctions (tight junctions plus desmosomes) which delimit for each cell a specialized membrane zone on the one hand, at the functional level by the expression of specific proteins, and on the other hand, at the morphological level by the formation of numerous villi. Moreover, bile canaliculi are characterized by rhythmic dynamic movements. The latter make it possible to evacuate into the bile, the biliary salts originating from the hepatocytes.

The differentiated HepaRP cells may likewise have this polarized organization. Regarding Phase I detoxification function, the HepaRP cells may have one or more of the following characteristics:

Production of phenacetin O-deethylase >0.05 nmole/hr/M cells as an indication of

CYP1A2 function.¹

Production of midazolam 1 'hydroxylase >0.25 nmole/hr/M cells as an indication of

CYP3A4 function.¹

Production of buproprion hydroxylase >0.015 nmole/hr/M cells as an indication of CYP 2B6 function.¹

Production of dextromethorphan 0- >0.015 nmole/hr/M cells as an indication of demethylase CYP2D6 function.¹

V_max for [¾]MPP+ >15 pmole/min/M cells as an indication of uptake transporter OCT function.²

Vmax for [¾] taurocholate >1.5 pmole/min/M cells as an indication of uptake transporter NTCP function.²

Measured using LC-MS/MS following one hour treatment of cells with 200uM phenacetin, 50uM midazolam, lOOuM buproprion, or lOOuM dextromethorphan.

²Measured using a liquid scintillation counter following three-minute treatment of cells with 250uM of [³H]MPP+ or 25uM of [³H] taurocholate.

The invention also relates to the capacity of HepaRP cells to share similar features with HEPARG® or alternatively to express new biological properties making their functional behavior deeply changed, by modifying culture conditions.

Thus, a new hepatic cell line having the identifying characteristics of HepaRP is also described herein. Likewise, the invention is also concerned with a new culture medium for proliferating HepaRP cells comprising a basal nutrient medium, L-glutamine, insulin, and optionally at least one cortico-steroid and DMSO.

The invention is also concerned with cells derived from HepaRP but with characteristics deeply different from HepaRP cells, obtained by modified culture conditions. Thus, the HepaRP cell line, at any passage (preferentially after passages 7-9) are maintained and expanded in a new culture medium, this medium comprising a basal nutrient medium, L-glutamine, and insulin. The culture medium is, however, free of steroids (e.g., no cortico-steroid), and free of DMSO. Advantageously, this medium does not allow entry of HepaRP cells into differentiation even at confluence. The resulting cells are designated as "HepaRPH" (or RPH) cells. Thus, the invention also relates to these HepaRPH cells stably proliferating in a medium deprived of steroid for at least 25 passages, thus constituting a new HEPARG®-derived cell line. The invention also comprises a method of inducing a signal of commitment in the Hepa-RPH cells to differentiation toward mature hepatocytes. The invention is also concerned with the new functional behavior of these HepaRPH cells, the cells having distinct properties, mainly that of eliminating the primitive biliary cells during the differentiation process, thus constituting conditions for producing pure human hepatocyte cultures also described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure (Fig.) 1 shows phase contrast micrographs of proliferating Hepa-SCs at passage 4

(a) and 20 (b). Note the high homogeneity of the population and the richness in stem-like cells throughout numerous passages. White arrowheads indicate elongated cells during proliferation;

Fig. 2 shows phase contrast micrograph images of HepaRP cells during and after HepaSC reprogramming process, where the arrow indicates cells submitted to physical constraint by plating at high density for 20hrs and exposed to the signaling factor (hydrocortisone hemysuccinate), followed by re-seeding the cells at normal density and maintained in presence of corticoid;

Fig. 3 shows the relative quantification of the steady-state mRNA levels of three liver- specific markers and one progenitor marker in HepaSC, HepaRP3 and HEPARG® cells at the indicated passages, using the RT-PCR analysis;

Fig. 4 shows a graph of the growth of the new HepaRP3 cells at passages 12 and 20 as compared to HEPARG® cells at the indicated passages;

Fig. 5 shows images of HepaRP3 cells at a) passage 3, day 8; b) passage 11 , day 8, c) passage 14, day 6; and d) passage 19, day 8 + DMSO(1.5%), followed by (d) a phase contrast micrograph of the cells at passage 19; (e) expression of transferrin (green); and (f) expression of nuclear transcription factor HNF4 (light blue) and glutamine synthase (red), with the nuclei in dark blue;

Fig. 6 shows images of HepaRP3 cells at passage 29 and immunolocalization of (A) pGP transporter protein; (B) ZO-1 protein; (C) CYP3A4; and (D) HNF4 transcription factor, which all characterize the polarity and high differentiation status of the cells;

Fig. 7 shows (a) cell polarity with F-actin deposition at the biliary poles of mature Hepa- RP3 cells and (b) MRP2 transport activity with fluorescent MRP2-substrate CDFDA assay, with the cells at passage 19;

Fig. 8 shows graphs comparing three CYP450 enzyme activities for HepaRP3 and HepaRG® and their response to DMSO exposure at different passages;

Fig. 9 shows a graph of induction of 2 CYP enzymes by specific inducers in Hepa-RP cells: CYP3A4 induced by rifampicin and CYP1A2 by omeprazole. Note the weak induction of CYP1A2

Fig. 10 is a flowchart of the protocol used for production of the Hepa-RP cells and RPH cells from the HepaRG® cells, transitioned through stem-like HepaSC cells;

Fig. 11 shows micrograph images of hepatocyte differentiation process of RPH cells from HepaRP at passage 19; (a) RPH cells maintained in hydrocortisone-free medium; switch to medium + hydrocortisone for (b) 3 days, (c), 6 days, (d) 10 days + 0.4%DMSO; (e) 15 days +1.5%DMSO; (f) 20 days +1.5% DMSO;

Fig. 12 shows a graph of RPH cells from Hepa-RP at late passages (switching to corticoid-free medium at passage 18). Maintenance for 15 passages in corticoid- free medium; differentiation program at passage 15. Expression of the main liver specific CYPs enzymes in differentiated RPH for 12 or 21 days at passage 15. Comparison with their expression levels in the parental Hepa-RP3 at passage 16. The data demonstrates the possibility to greatly prolong the stability of Hepa-RP lines by switching to RPH conditions, reaching up to a total of 33-35 passages;

Fig. 13 shows effect of HDAC treatment (exposure to 5-Azacytidine) during the early steps of differentiation of RPH cells: (a) 4 days with 5-AZA; (b) One week post-treatment; (c) 3weeks post-treatment, in presence of 1.5% DMSO. Note heterogeneity during commitment to differentiation but a high purity in hepatocytes, a greater stability and a higher maturation level;

Fig. 14 shows the improved differentiation level of RPH cells when exposed to 5-AZA at the signal commitment to differentiation, and the role of 5-AZA epigenetic factor on the hepatocyte differentiation program as evidenced by the increased CYP expression level; and

Fig. 15 shows images using HepaRP3 cells and HPR cells to screen for cholestatic drugs such as chlorpromazine (CPZ) and fasudil. As expected, CPZ induced bile canaliculi constriction whereas Fasudil induced canalicular swelling, as observed with HepaRG®.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is concerned with new cell lines. A "cell line" is a population of cells of common origin cultured together after several passages in vitro, such that the cells share generally similar growth rates, morphology, nutritional requirements, and expression markers. The new cell lines are derived from the parental immortalized HEPARG® cell line through transition to stem cell-like cells, designated as HepaSC cells (or Hepa-SC cells). The term "derived," as used herein, refers to obtaining new cell types (i.e., progeny) that are distinct from the parental line, through a defined selection and manipulation of the parental cell line in cell culture, as described below. The invention is concerned with production of new hepatic cell lines, designated herein as HepaRP cells. The HepaRP cells are derived from the reprogrammed stem-like cells, Hepa-SC then, redirected to hepatic differentiation lineage, their phenotype being similar or distinct from the parental HEPARG® cells according to culture conditions. The invention is also concerned with pure hepatocyte monolayers of HepaRP cells, named HepaRPH cells, and culture conditions defined for obtaining the same. The HepaRPH cells are derived from the HepaRP cells by stably growing HepaRP cells as undifferentiated cells in a medium deprived of steroid and characterized by high plasticity. Hepatocyte differentiation is then induced by exposing the cells to the corticoid steroid differentiation factor. This invention relates to the uses of HepaRP and HepaRPH cells in biology, pharmacology, toxicology, and prophylactic applications.

The HepaSC cells are defined as stem-like cells derived from the HEPARG® cell line. The HepaSC cells are "stem-like," which means that they have characteristics of sternness. Sternness is an essential characteristic of a stem cell that distinguishes it from ordinary cells, and more specifically refers to undifferentiated (unspecialized) cells that have the potential to differentiate into specialized cells, and which are capable of renewing themselves through cell division. As such, cells exhibiting sternness are pluripotent or multipotent self-renewing cells. The term "undifferentiated" as used herein refers cells which have not developed a characteristic of a more specialized cell (e.g., a specific structure, purpose, function, etc.). In contrast, a "differentiated" cell has taken on characteristics (e.g., structure, function) of a more specialized cell type. Differentiated and undifferentiated cells are distinguished from each other by several well-established criteria, including morphological characteristics, expression characteristics and/or functional characteristics associated with the specialized function or purpose of a given cell.

One advantage of the invention is that setting Hepa-RP lines from reprogrammed HepaSC lines capable of expressing sternness properties, mainly self-renewal, represents a virtually infinite source of HEPARG®-like cells carrying the capacity to differentiate into mature human hepatocytes. The HepaSC cells are derived from HEPARG® cells through mechano- transduction techniques and have a characteristic morphology when seeded at low density, with round and flat shape, with a large regular nucleus, and a smooth wavy plasma membrane at the borderside. The proliferating cell population is composed of elongated (mesenchymal) and polygonal (epithelioid) cells. This property reflects a great plasticity of the cells. HepaSC cells are also characterized by a remarkable capacity to redirect a hepatic differentiation lineage after numerous passages (at least about 45 passages) in conditions allowing stem cell properties. For instance, under predefined culture conditions, they give rise to new permanent lines (HepaRPs) with recovered bipotent properties such as progression to both hepatocyte and biliary cell lineages, thus mimicking the parental HEPARG® cell line in that respect.

HepaSC cells have the capacity to react to mechanical forces such as shape constraint, when plated at very high density. Shape-constrained Hepa-SC cells are preferentially directed to hepatocyte differentiation in presence of insulin and at least one cortico-steroid such as hydrocortisone hemisuccinate, as discussed in more detail below. The Hepa-SC cells also have a stable karyotype which advantageously results from the epigenetic strategy used for HEPARG® reprogramming to Hepa-SC, thus, making Hepa-RP cells carrying unchanged karyotype.

Described herein are methods of expanding and producing the stem-like Hepa-SC cells specifically in preserving the stability of the "sternness" property. The methods also combine mechano-transduction signaling and hepatocyte differentiation inducers (such as cortico-steroids) for setting new Hepa-RP cell lines.

The methods generally comprise culturing the Hepa-SC cells in the absence of differentiation factors, but in the presence of an epigenetic factor such as Histone deacetylase (HDAC) inhibitors. As used here, a "differentiation factor" is an agent known to promote or induce differentiation signaling in the parental cell line. In one or more embodiments, Hepa-SC cells are cultured by exposing to a culture medium that is free of differentiation factors. The basal media for the culture media can include any suitable nutrient formulation, such as William's E, or Dulbecco's Modified Eagle's Medium (DMEM)/HamF 12, M199/DMEM, Roswell Park Memorial Institute (RPMI), and the like, which culture medium may be supplemented with insulin, L-glutamine, fetal calf serum (FCS), and HDAC inhibitor, combinations thereof, or equivalent media, and the like.

In one or more embodiments, differentiation factors include cortico-steroids, DMSO, retinoic acid, o-estrogens, thyroid hormones, and/or synthetic analogues thereof. The term "synthetic analogues" is used to refer to the functional analogues of non-natural origin. Exemplary cortico-steroids include hydrocortisone hemisuccinate and/or dexamethasone. As used here, the term "free of differentiation factors" means that such differentiation factors are not intentionally added or included as part of the culture medium, although it will be appreciated that some incidental impurities may exist (such as residual agents that may be present after washing the parental cells before seeding in the first culture medium free of differentiation factors). Thus, the amount of any residual differentiation factor that may be present in the culture media should be less than about 10^~6M and preferably less than about 10^~7M, for example in the case of cortico- steroid, and less than about 0.01% in the case of DMSO.

In one or more embodiments, the cells are subjected to a methylation inhibitor in an amount and for a time period sufficient to stabilize the sternness characteristics. In one or more embodiments, the cells are transferred to a container and cultured with culture medium in the presence of the selected modulator. Exemplary modulators include epigenetic factors belonging to the histone demethylase molecules, such as 5-azacytidine and the histone lysine methyltransferase EHMT2, a methylation inhibitor (BIX 01294). Other epigenetic factors belonging to the HDAC family could be also used, such as hydroxamic acids, benzamides, and the like. 5-aza-cytidine is a preferred modulator, when used at a dose which does not inhibit cell proliferation and does not induce visible cell toxicity. 5-azacytidine could be used in the stabilization medium at concentrations of about from about 1 to about 10 μΜ, preferably from about 5 to about 10 μΜ, and more preferably about 10 μΜ. 5-aza-2'-deoxycytidine can also be used as well. BIX 01294 can alternatively be used, but may induce higher cell toxicity.

The stabilized cells are then allowed to grow and proliferate. Conditions for growth and proliferation are favored by culturing the cells in the presence of exterior signaling that blocks the engagement of differentiation. In one or more embodiments, the cells are cultured in the presence of a differentiation inhibitor. In one or more embodiments, differentiation inhibitor is added to the stabilization culture medium. Suitable differentiation inhibitors include protein kinase inhibitors, such as RHO-kinase inhibitors (e.g., Y-27632, fasudil, effectin, etc.), GsK3 inhibitors (e.g., CHIR 99021), and the like. When Y27632 is used, the concentrations of about 5 and about 10μΜ can be used in the culture.

Advantageously, the selected culture conditions allow long term production of the stemlike cells without loss of their sternness fate. Many passages (at least 50 passages) have been successfully stably produced. Thus, in one embodiment, the method comprises continuously exposing the stem-like cells to a medium comprising at least one methylation/acetylation modulator and being free of a corticosteroid. In one embodiment, the invention also provides a maintenance medium suitable for maintaining the stability of the HepaSC cells. The HepaSC maintenance medium comprises a basic cell nutrient media, supplemented with L-glutamine, insulin (ΙΟμΜ), and 5-aza-cytidine, and is free of a differentiation factor (e.g., cortico-steroid). Exemplary basic cell nutrient media includes William's E, RPMI, DMEM/HamF 12 (3/1), or DMEM/MEM199 (3/1), and the like. This basic nutrient medium is added with L-Glutamine or preferentially, Glutamax, and contains insulin (from about 5 to about ΙΟμΜ, preferentially about ΙΟμΜ) and 5-aza-deoxycytidine (from about 2.5 to about ΙΟμΜ, preferentially about ΙΟμΜ). The nutrient medium can also contain about 10% FCS. Optionally bFGF (about 40ng/ml) and EGF (about 20ng/ml) plus essential fatty acids can be used when decreased FCS concentration (about 0.5 to 2% instead of 10%) is desired.

In one embodiment, methods of directing cell differentiation of HepaSC cells to hepatic differentiation are described. The methods generally comprise culturing the HepaSC cells in the presence of a differentiation factor and under mechanical stress to commit the cells towards the target cell population. More specifically, HepaSC cells in culture are first washed to remove the methylation/acetylation modulators and maintenance culture. The washed HepaSC cells are then subjected to mechanical stress, such as by plating at a high density (e.g., about 8X10⁵ per well of 24-well plate). The culture medium used for plating is preferably a proliferative medium for the target differentiated cell type. In the case of commitment towards differentiation into hepatic cells, the proliferative medium preferably comprises basic cell nutrient medium, supplemented with insulin, and at least one differentiation factor for hepatic cells. In one or more embodiments, the differentiation factor is a cortico-steroid at a non-toxic concentration which promotes differentiation. The phrase "non-toxic concentration which promotes differentiation" is used to refer to the cortico-steroid concentration promoting, during its addition to a culture of HepaSC, the differentiation of the cells towards a hepatic morphology and a functional state. This concentration is non-toxic, i.e. its addition does not lead to a cell mortality rate greater than approximately 10%. The cells are cultured under mechanical stress for a sufficient period of time (e.g., about 20 hours) to achieve commitment of the cells to differentiation. The cells are then transferred from the high density plating container and further cultured at low density (e.g., about 4X10⁵ per well of 24-well plate) in the same proliferative medium. The resulting cells are designated herein as HepaRP (or Hepa-RP) cells. Optionally, addition of differentiation factors such as activin A and BMP4 can improve the completion of the process.

According to the present invention, the HepaRP cells when cultured in conditions defined for HEPARG® (as described in U.S. Patent No. 7,456,018, incorporated by reference herein), have a functional behavior resembling HEPARG®, thus making sustainable production of HEPARG®-like cells for long term use. More specifically, the cells of the human HEPARG® line can express the functions characteristic of the hepatocyte, namely:

- the production of plasmatic proteins, in particular, albumin, transferrin, or proteins of the inflammatory response, such as a haptoglobin, ceruloplasmin, etc.

- the detoxification function, in particular:

- the expression of various forms of P450 cytochromes, such as CYP2E1, CYP3A and/or CYPIA, the expression of various forms of detoxification phase I and phase II enzymes (phases I and II (CYP2E1 , CYP3A, CYPIA and GSTa):

*activity expressed in nanomoles of metabo ites produced/h/mg of proteins +activity expressed in units/mg of proteins the Phase III detoxification function including uptake, conjugation, transport and efflux of biliary salts and efflux of conjugated metabolites to bile canaliculi, and

the elimination of urea,

energy regulation function:

the storage of sugar in the form of glycogen and the production of glucose by glycolysis, in particular, the expression of aldolase B and Pyruvate kinase L enzymes and those involved in neoglucogenesis, and hormones receptors controlling these functions, particularly the insulin receptor expression, and hepatic-specific metabolism of lipids. These new cell lines, designated herein as HepaRP, are characterized by a typical morphology described for proliferating hepatic progenitors at low density, with a mixed population of elongated and polygonal cells as found in the originating HEPARG® line. However, they are also characterized by the presence of numerous cells with round and fiat shape, with a large regular/round nucleus with a dense nucleolus in the center, and a floating plasma membrane at the periphery corresponding to the description of the parental HepaSCs. The HepaRP cells, like HEPARG® cells, have a cytoplasm very rich in mitochondria similar to human adult primary hepatocytes. In culture of a plurality of HepaRP cells, typical hepatocyte polarized morphology is observed in the HepaRP cells with cytoskeletal organization forming bile saccular and canalicular structures able to support intracellular trafficking towards this biliary pole, including bile salts transport using specific transporters such as NTCP, BSEP and MRP2. This polarized organization is closely associated with complete hepatocyte maturation, meaning a specific distribution of plasma membrane proteins such as receptors and transporters, at the plasma membrane which define baso-lateral domains and biliary poles, both supporting intracellular trafficking pathways characteristic of adult hepatocytes; mainly, the bile canaliculi formed at the biliary pole, characterized by tubular and saccular shapes, by the presence of specific transporters and by their dynamic movements made possible by the numerous F-actin fibers accumulated beneath the membrane and forming a ring for contraction or dilatation, all indicating functions characteristic of the hepatocyte, namely, active bile acids, conjugated bile salts and drugs clearance. HepaRP cells in culture form close contacts from one to the other through specific expression and localization of junctional proteins such as occludin and ZO-1 , constituting regular colonies of joined cells organized in a monolayer, which is advantageous for imaging and high throughput screening.

The HepaRP cells express characteristic features of mature adult liver cells, including those associated with sugar, lipid and drug metabolisms and detoxification function with a stability exceeding 2 weeks:

- sugar metabolism including enzymes involved in neoglycogenesis (PEPCK), glycogen accumulation and glycolysis (aldolase B and Pyruvate kinase L)

- hepatic lipid metabolism based on the expression of specific genes such as CYP4F3B and CYP 4Al l,

- drug metabolism and detoxification function supported by the expression of several forms of CYPs enzymes belonging to Phase I, for instance CYP1A, CYP3A, CYP4A, CP2E, also Phase II enzymes mainly GSTa, corresponding transcription factors (CAR, AhR and PXR), bile acids transport and bile salts conjugation, urea elimination (glutamine synthase), in the range of those characterizing HEPARG® cells above.

The HepaRP cells also have an active proliferation with a population doubling of 24h, but a delayed contact inhibition response compared to HEPARG® cells. The HepaRP cells are also characterized by bipotent properties so that cells can be further directed to hepatocyte or primitive biliary cell lineages and 2 distinct cell types: hepatocytes and primitive biliary cells. Likewise, the HepaRP cells have the capacity to undergo a complete differentiation program to mature hepatocytes as the originating HEPARG® line. The above-mentioned HepaRP characteristics are interesting given that they have been derived from early progenitors (e.g., HepaSC and HepaRP progenitors) devoid of polarity, of liver-specific functions, while active in proliferation. Further, the differentiated HepaRP cells have a weaker capacity to undergo reversion to progenitors, in contrast to HEPARG® cells.

In one or more embodiments, the Hepa-RP cells are maintained in medium comprising at least one cortico-steroid. The medium is further supplemented with DMSO in a quantity sufficient to induce differentiation. The term "quantity sufficient to induce the differentiation" is used to refer to the quantity of DMSO necessary to induce the differentiation of a culture of normal human hepatocytes. In one or more embodiments, the cells are cultured in the presence of a cortico-steroid, followed by exchanging the medium for one further supplemented with DMSO as described above. The cells are cultured for a sufficient time period such that the differentiation factor(s) directs maturation of the cells into the target population of hepatocytes. The differentiation factor is present at a non-toxic concentration which promotes the differentiation of hepatocytes (e.g. DMSO at from about 1% to about 2%, and preferentially about 1.5%). The resulting differentiated cells can then be maintained in this same medium.

The HepaRP cells also have the ability to express the different hepatocyte markers as in HEPARG® with minor variations in the expression levels such as APOAl . Thus, it will be appreciated that the HepaRP cells have preserved the same karyotype as HEPARG®.

However, transition through HepaSC status has introduced a few changes in the methylation/acetylation profile of some genes in the HepaRP cells. Thus, the new HepaRP cell line is distinct from HEPARG® cells in several characteristics. For example, the HepaRP cells do not exhibit multilayering, even after 2-3 weeks of differentiation, a characteristic which evidences new cell interaction properties. This feature will provide to HepaRP great advantages for all imaging analysis applications. The HepaRP cells also have a capacity to organize a gradient of differentiating hepatocytes around numerous circular empty zones randomly formed within the monolayer culture. This organization could advantageously mimic the gradient which is characteristic in the liver lobule in vivo and defining periportal and centra lobular zones. Importantly, this property has never been observed with HEPARG® cells. Cells with the highest level of differentiation surrounding the central empty zone seem to not divide and enter into the aging process, whereas primitive biliary cells remain alive as with HEPARG®.

The HepaRP cells have increased stability through passages which can be improved regarding their capacity to form hepatocyte colonies. This has been further improved through the addition of low concentration of DMSO into the medium early during the proliferation stage. At these low concentrations of DMSO, the cells preserve a high growth activity while occurrence of heterogeneous cell colonies is inhibited. In addition, enrichment in hepatocyte colonies is observed with the increase of passages number.

Accordingly, in one embodiment, the invention also covers the use of the new proliferating medium for HepaRP proliferation comprising a basal medium as defined above added with L-glutamine (preferably Glutamax), insulin, and at least one cortico-steroid, and containing low concentration (not exceeding 0.2%) of DMSO during the first stage of proliferation corresponding to the 3 first days post-seeding, followed by the same medium containing moderate concentrations (not exceeding 0.5%, preferentially 0.4%) of DMSO up to the use of the differentiation medium. This combination allows maintenance of HepaRP cells in conditions for obtaining behavior similar to that of HEPARG®.

Again, as noted above, the HepaRP cells are likewise distinct from HEPARG® cells, and have a high plasticity. The HepaRP cells are sensitive to environmental conditions in their ability to control differentiation programs. Unexpectedly, when maintained under culture conditions different from those established for HEPARG® cells, the HepaRP cell line achieve different fundamental features.

Advantageously, in contrast to HEPARG®, the HepaRP cells can be cultured and stably expanded in a medium that is free of differentiating factors e.g., free of any cortico-steroid and of DMSO. In the corticoid-free medium, the cells can be cultured for several passages (more than 20 passages, preferably more than 25-28 passages) without beginning to differentiate, even at confluence. This corticoid-free culture condition can be used for expanding HepaRP cells from few passages after reprogramming (e.g., from passages 7 or 9 up to passage 30), thus allowing production of RPH cells distinct from HepaRP by their specific behavior. This new corticoid-free culture condition and their functional properties can also be used for restoring stability of HepaRP cells after 20 passages thus, prolonging their uses up to 30-32 passages. This corticoid- free culture medium for HepaRP cell expansion is new. Moreover, HepaRP cells expanded in this corticoid-free medium evidence a highly stable and reproducible ability to rapidly respond to cortico-steroid signaling (preferentially hydrocortisone hemisuccinate) when added to the culture medium, in order to direct hepatocyte differentiation.

Methods herein also include a method of switching to corticoid-enriched medium. The methods include undifferentiated RPH cells preferentially chosen upon reaching confluence, to be switched to the differentiation medium. HepaRP cells are maintained in the proliferating medium for 2-3 days. This is followed by the addition of gradually increasing DMSO concentrations every 2 days (0.2%, 0.4%, 0.8% and 1.5%) preferentially within 1 week, then 1 additional week with 1.5% or 1.7% DMSO for completing the hepatocyte maturation program.

These new culture conditions advantageously allow improving the stability of HepaRP cells in very simple culture conditions, and keeping their capacity to respond to corticoid signaling and to undergo a complete differentiation program. Such stability was never reached before so that these culture conditions could represent a strategy for supporting or improving stability of these cell lines. The foregoing approach can be used to prolong the stability of HepaRP cultured in standard conditions beyond 18 passages and up to 32 passages. In the work carried out, the functional potentialities of the RPH hepatocytes appeared high and identical to those obtained from HepaRP cells cultured under permanent exposure to corticoids.

These new corticoid-free expanding conditions lead to a drastically increased proportion of hepatocytes in differentiated cultures obtained using the method as described above, reaching a near pure population in hepatocytes. This is a reproducible phenomenon through all passages. In addition, hepatocyte colonies through the culture are homogeneous regarding their differentiation level. Thus, HepaRP cell lines together with the new methods for expanding and differentiating RPH cells constitute new strategies for producing pure human hepatocyte cultures also described herein. When needed, completion of maturation is favored by short HDAC epigenetic treatment, preferentially with 5-azacytidine, for 4-5 days during differentiation commitment (figures 13 and 14).

In parallel and as consequence, the corticoid-free expanding conditions lead to a gradual reduction (in 2-3 passages) of the bipotent property when the RPH cells do receive the corticoid signal, which was a main characteristic of HEPARG®. It is surprising to observe a near complete disappearance of primitive biliary cells which slowly and lately re-appear only after 2-3 weeks of stable differentiation and when old hepatocytes are detaching. This event is also accompanied by a strongly delayed and reduced capacity of RPH hepatocytes to reverse to progenitor cells, in contrast to HEPARG® cells and Hepa-RP. This makes RPH a suitable cell model much more reproducible and easier to use for end-users. In one embodiment, these corticoid-free expanding conditions represent conditioning for producing Hepa-RP in an undifferentiated status, distinct from the progenitor cell status characteristic for HEPARG®, giving real advantages in successfully directing Hepa-RP and RPH cells to cholangiocyte differentiation program or other programs such as pancreatic or intestinal "routes," distinct from the hepatocyte one. Altogether, these new functional behaviors and new properties of Hepa-RP and RPH lines make them distinguishable and original from HEPARG®.

The new cell lines, HepaRP and RPH, have a variety of uses. For example, they can be used for the production of long term stably-recombined cell lines obtained by gene transfer into either Hepa-SC or progenitors from HepaRP or RPH.

The new cell lines, HepaRPs and RPHs, or differentiated cells derived therefrom can be used in the production of three dimensional spheroids using an easier and more scalable technique that relies on physiological influences, as compared to existing three-dimensional molding techniques (e.g., hanging drop method, etc.). For example, the techniques described herein use morphogens that make the cells "contract" the cytoskeleton and increase their cell-to cell contact affinity to form three dimensional spheroids.

The new Hepa-RP and RPH cell lines can be used for preparing bioreactors using a more scalable technique and taking advantage of higher stability of these cells in long term culture.

The cells can also be used in drug screening assays, assays related to infectious diseases

(e.g., HBV, HCV, Plasmodium), to studies on apoptosis and aging, and the like. The cells are also advantageous for procedures involving imaging analysis, since the cells form a regular monolayer. For instance, pure RPH hepatocytes are highly appropriate to screen genotoxic agents targeting specifically mature hepatocyte populations, in using micronuclei or Comet assays. Another application to screening potential cholestatic drugs will also get benefit of the pure hepatocyte population and of the regular cell monolayer supporting high imaging quality. The HepaRP cells can also be used as representative of the resident cancerous progenitor cells (also called "oval" cells) in the liver, for testing antitumoral drugs or toxic agents, in direct link with hepatocarcinogenesis. Likewise, the undifferentiated RPH cells cultured in the corticoid- free medium can be used to study anticancer drugs and evaluate therapeutic agents that can direct cancer oval cells to a differentiated phenotype and away from metastasis.

Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.

As used herein, the phrase "and/or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

EXAMPLES

The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention. EXAMPLE 1

Methods for stably producing the stem-like Hepa-SC cells in culture The new method for maintaining the Hepa-SC stem-like cells in conditions favoring their stability during long term culturing involves subjecting cells to a corticoid-free medium added with a factor inducing the stability of their stem cell fate by epigenetic mechanism. For instance, the corticoid-free medium contains methylation/acetylation modulators such as HDAC molecules. Epigenetic modifications refer to mitotically heritable changes in gene expression that are not coded in the DNA sequence itself (Levenson JM, Sweatt JD., 2005). The chosen epigenetic molecule should neither alter cell proliferation activity nor provoke toxicity effects and/or apoptotic induction. 5-azacytidine inhibits DNA methylation. It has been preferentially chosen a small molecule known to inhibit the histone H3 methylation. It is sometimes considered to replace oct 3/4 (Chang Y et al., 2009) - one of the four original genetic factors used for reprogramming of mammalian somatic cells into induced pluripotent stem (iPS) cells (Fig. 1).

Optionally, in addition to 5-azacytidine, it can be chosen for exposing the cells to exterior signaling, mainly related to protein kinase pathways. Two main factors have been tested: 1) Rho- kinase inhibitor which inhibits all differentiation processes, favors cell survival, growth and spreading. Different concentrations of ROCH (Y27632) have been tested, from 0.5 to 20μΜ. The concentration of 10μΜ has been preferentially chosen and added to the culture medium during Hepa-SC cell line establishment and maintained for the 5-6 first passages. Other forms of inhibitors could be used such as fasudil or effectin. 2) Another factor, CHIR 99021, known as a specific substrate of GsK3, is a protein kinase inhibitor that can be chosen for its capacity to inhibit the GsK3 pathway involved in differentiation through its relationship with the Wnt/β- catenin pathway (Ying Qi-L. et al., 2008).

Culture of Hepa-SC cells for at least 30 passages can be performed when conditions used are as followed:

- Absence of cortico -steroid and permanent exposure to an epigenetic factor capable of stabilizing the genome expression by controlling the methylation/acetylation levels of the genes, have been proposed. 5-aza-2'-deoxycytidine has been preferentially used at 10μΜ, a dose which does not inhibit cell proliferation and does not induce visible cell toxicity (Seeliger C. et al., 2013). - Moreover, cooperation between the epigenetic treatment and the RHO-kinase pathway inhibition (Y-27632) has been chosen for increasing the number of stem-like cells in the population, for improving their survival and for inhibiting their entry in any differentiation program (Watanabe K, Ueno M et al., 2007).

Cells are maintained in the William's E medium added with insulin, 5-azacytidine (ΙΟμΜ) and Y-27632 (ΙΟμΜ) and 10% Fetal calf serum) for the 10 first passages. Every 10 days, when cells are reaching subconfluence, the cultures are passaged by diluting the cells up to 5-6 fold. Fig. 1 shows the results of maintaining Hepa-SC cells in the absence (a) or presence (b) of 10μΜ Y-27632 at passage 4. Under these conditions, Hepa-SCs actively grow. Master and working banks have been settled. Four batches have been produced up to now (Hepa-SCl to Hepa-SC4). Stability of one of them, Hepa-SCl , has been verified up to passage 40 till now. EXAMPLE 2

Procedure for re-directing Hepa-SC to hepatic differentiation program -The general principle of the inventive technique:

Step 1 : Shape-constraint signal: Hepa-SCs are seeded at very high density (8X10⁵ per well of 24-well plate) in the medium used for their propagation, as described in Example 1 , except the epigenetic factor (5-aza) is discarded.

Step 2: Detachment of the cells by trypsin and reseeding at 4xl0⁵ per well either onto plastic or thick soft matrigel or other hydrogel, or stiff supports.

Step 3: Influence of corticoid, the main morphogen factor needed for directing the differentiation pathway to hepatocyte, added to the appropriate medium used for cell maintenance often enriched with various other growth factors.

-Example of differentiation reprogramming of Hepa-SC to hepatic lineage.

In this example, a specific reprogrammed Hepa-SC line, designated as Hepa-RP3, is described (Fig. 2). Fig. 10 illustrates the general process flow used for generating the reprogrammed hepatic lines (Hepa-RP) from the parental HepaRG® cells.

A. Redirecting the hepatic differentiation program in 1 -2 passages

Hepa-SC cells were submitted to a new method designed for redirecting the cells toward the hepatic differentiation route. Subconfluent Hepa-SC cells maintained in William's E medium added with 5μΜ 5-azacytidine were washed twice with PBS to eliminate serum and 5- azacytidine. The cells were then detached with 0.05% trypsin. The cells were collected in the proliferative medium used for HEPARG® cell line. Briefly, this medium includes the William's E medium as basic nutrient medium, added with L Glutamine or preferentially, Glutamax, and contains insulin (from 5 to 10μΜ, preferentially 10μΜ) plus 50μΜ hydrocortisone as cortico- steroid. The cells where seeded at high density (400,000/well in a 24-well plate) in order to induce mechanical stress. After 20h, the cells were detached and reseeded at a low density on plastic with the same proliferating medium.

The foregoing process allows the Hepa-SC cells to lose their stem-like status and to retrieve the hepatic lineage including both their bipotent property and their capacity to undergo a complete hepatocyte differentiation process.

The retrieval was performed in one step (one passage). The maximum of hepatocyte colonies is stabilized after 2 or 3 passages. Fig. 2 shows images of Hepa-RP3 cells at passage 1. Changes of the cell shape occur in several foci indicating commitment of some cell colonies toward hepatic cell differentiation program. Response to DMSO is also restored. Hepa-RP3 cells have been directed to undergo a complete process of hepatocyte differentiation from the Hepa-SC stem-like cells. This new cell line has recovered the main characteristics of the parental cells including a bipotent progenitor stage corresponding to a proliferative stage, and a differentiation stage leading to the organization of mature hepatocyte colonies. Although common properties are shared with the parental HEPARG® cells, Hepa-RP3 cells have developed their own characteristics. A master and a working banks have been performed by expanding the cells for 3 passages and cryopreservation in liquid nitrogen.

B. Cryopreservation for setting master and working banks

After detachment with 5 min trypsin treatment cells were put in suspension in proliferating medium and numbered. lxlO⁶ cells were distributed per vial in 1 mL of cold medium added with 10% DMSO. Vials were maintained on ice, rapidly placed in a freezer at - 80°C, then, put in liquid nitrogen for 6h (or more).

C. Flowchart for maintenance of Hepa-RP3

Hepa-RP3 cells were maintained using a basal medium composed of William's E medium added with antibiotics (peni/strepto), insulin, hydrocortisone at 50μΜ, and 10% FCS.

The cryopreserved Hepa-RP3 cells were rapidly thawed and dispersed in a 25 cm² flask containing 3 mL of basal medium. Six to 8h later cells were observed to have attached and medium was renewed with the proliferating medium described above. Medium was changed every 2-3 days thereafter.

1. Maintenance of the line in conditions for obtaining cell behavior similar to

HEPARG®.

The cells were maintained under conditions developed to achieve cell behavior similar to HEPARG® cells for up to 20 passages. At each passage, cells were seeded at a density of 26,500 cells per cm² in the proliferating medium containing a low concentration of DMSO (0.2%). The cells attached in 2-3 hours and grew actively for reaching confluence in 4-5 days. Two days after seeding and before reaching confluence, the proliferating medium was added with a higher concentration of DMSO, preferentially 0.5%. After 10-12 days, the cells were highly confluent and ready for replating or for undergoing differentiation.

Replating of the cells was performed every 10-12 days. Cells were washed once with PBS and then incubated with trypsin-EDTA (0.05%) at 37°C for 3-5 min. Detached cells were collected in the proliferating medium and the suspension was processed for cell numbering using trypan blue solution. Fig. 4 shows the Hepa-RP3 growth activity.

2. Hepatocyte differentiation protocol

10-12 day-old confluent cells were changed with the differentiation medium containing 1 or 1.5% DMSO. After 3-4 days, numerous colonies of cells had committed to differentiation and after one week, they were easily recognized by typical hepatocyte morphological features. The differentiation medium included the proliferative medium containing hydrocortisone and added with DMSO at 1 or 1.5% final concentration.

Because of the presence of DMSO in the medium, we have observed that a cell selection could occur for 4-5 days as evidenced by the numerous dead cells floating into the medium. After one week, the cell monolayer is stabilized and composed of 2 highly distinct cell types, granular differentiated hepatocytes surrounded by clear flat epithelial cells (primitive biliary cells). Maturation needs 4-5 more days to reach completion. Medium was renewed every 2-3 days. In such conditions the monolayer remains confluent and stable for 2 weeks or more.

D. Functional characteristics

1. Morphology of Hepa-RP3 cells at different stages of the culture

In good culture conditions, colonies of hepatocytes are expected to cover 50% of the surface of the monolayer and sometimes more. Hepatocytes are characterized by a dense cytoplasm when analyzed on phase contrast micrograph, with round nucleus and one dense nucleolus. Numerous typical formations of bile canaliculi were observed. As shown in Figs. 4A- 4B, Hepa-RP3 cells appear to grow faster than conventional HEPARG® cells. An active proliferation with a population doubling of 20-24h is found for Hepa-RP3. It is more rapid than with HEPARG® cells. A population doubling is seen in 24h as early as day 2 following cell seeding. In addition Hepa-RP3 cells have changed their response to contact inhibition by showing increasing number of cells per surface unit at confluence.

2. Differentiation status

The expression of 3 liver-specific functions has been analyzed at the mR A level using RT PCR quantification: CYP3A4 (detoxification function), aldolase B (specific glycolytic function) and APOAl (hepatic lipid metabolism). They are all highly expressed in Hepa-RP3, APOA1 being higher expressed than in Hear cells. A comparison has been performed with HEPARG® and Hepa-SC cells. As expected, these stem-like cells don't express liver markers. In contrast they express CK19, a cytokeratin weakly expressed in hepatocytes. The results are shown in Fig. 3.

3. Detoxification function

The Hepa-RP3 cells were analyzed for transporter activity and phase 1 enzymatic activities. Figs. 6 and 7 illustrate F-actin deposition at the biliary poles of mature Hepa-RP3 hepatocytes and MRP2 activity assay with the fluorescent MRP2-substrate (CDFA. Passage 19).

Fig. 8 shows comparison of three distinct CYP450 enzymes activities at indicated passages between the new Hepa-RP3 cell line and the parental HEPARG® measured using specific substrates. All the three were active in Hepa-RP3 cells and they are all induced by DMSO as in HEPARG® cells. In contrast, induction of CYP3A4 by corresponding inducers but not of CYP1 A2 was observed (Fig. 9).

E. Cell line stability

The Hepa-RP3 cell line was tested for its ability to maintain the capacity to differentiate into hepatocytes and to keep the ratio between hepatocytes colonies and flat primitive biliary cells close to 50% for up to passage 20. Results are shown in Fig. 5. The left hand panel is a phase contrast micrograph of Hepa-RP3 at passage 19. The middle panel shows the expression of transferrin. The right hand panel shows expression of both nuclear transcription factor HNF4 (light blue) and glutamine synthase (red). The nuclei are shown in dark blue. As can be see, there is organization of a gradient of differentiation around an empty lumen. Cells keep the capacity to form colonies of mature hepatocytes. Drastic increase in the richness in hepatocytes can be observed (Fig. 5 panel d).

Finally, karyotyping stability has been analyzed through passages: Both chromosomic classification and CGH array of the complete genome analysis have been performed on passage 20. The whole genome appears well preserved. EXAMPLE 3

Use of corticoid-free culture conditions for obtaining RPH cells from Hepa-RP cells:

Functional characteristics of the pure hepatocyte population when undergoing differentiation In this example, in contrast to all previous examples described above, Hepa-RP3 cells were grown in the proliferating medium deprived of corticoid. Expansion and differentiation characteristics of the Hepa-RP3 cells maintained in these new conditions were analyzed. They defined the RPH cells (see the flow chart Fig. 10).

Two strategies have been successfully explored:

1- Switching to corticoid-free medium was performed on Hepa-RP3 cells at early passages (passage 7 or 8). As shown in Fig. 11a, cells growing in the proliferating medium deprived of hydrocortisone actively proliferate, but cannot commit to a differentiation program even when reaching confluence. The images shown are for passage 23. Notably, there was no major change with number of passages up to around passage 25.

Commitment to differentiation was performed using the differentiation medium (containing 50μΜ hydrocortisone alone, then added with gradually increased doses of DMSO (from 0.2 to 1.5 % as described above) at each medium change (every 2-3 days) (Fig. 1 1). We note i) the very efficient differentiation process performed in most cells of the population; ii) the near complete disappearance of primitive biliary cells and the regular monolayer formed by hepatocytes (no piling up). In addition, in contrast to Hepa-RP3 cells cultured in proliferating medium with corticoid, weak capacity of RPH hepatocytes to reverse towards proliferating progenitors could be detected, thus, strongly improving the stability of the culture.

As shown in Fig. 1 1 , the morphological features are those of human primary hepatocytes and very similar to those obtained with HEPARG® cells. They correspond to a representative sequence of differentiation obtained at passage 19 with 12 days in presence of 1.5% DMSO. Note the numerous bile canalicular structures signing the high polarity of the cells. Cells expressed the main liver-specific functions as Hepa-RP3 cells including CYP enzymes characteristic of liver cells except induction of CYP1A2 which remains inhibited.

2- Switching to corticoid-free medium was performed on Hepa-RP3 cells at late passages (passage 17 or 18). Demonstration is provided that actively proliferating RPH can be obtained and maintained for up to 23-25 passages. They preserve the property to undergo a complete differentiation program and to form near pure populations of hepatocytes.

Four CYP enzymes were quantified for their activity after 15 passages in condition of corticoid-free medium: CYP3A4, 1A2, 2B6, 2D6 (Fig. 12). All were functional and support activity close to that of Hepa-RP3 at passage 16, suggesting high stability of the cell line. It is also of great interest to observe the capacity of CYPs to respond to specific inducers, property very important for applications in toxicology, except for CYP1A2 which remains poorly inducible in RPH as in Hepa-RP cells.

Notably, this second strategy offers the possibility to greatly prolong the stability of Hepa-RP cell lines by combining at passage 18, a switch to the corticoid-free medium for RPH production, thus, covering a period of hepatocyte production for up to 33-35 passages in total. This can be very useful for applications using permanent transfected cells.

EXAMPLE 4

Completion of RPH hepatocyte maturation by the epigenetic factor 5-AZA treatment during commitment to differentiation

As mentioned above Hepa-RP cells are characterized by a specific epigenetic signature regarding methylation/acetylation profiling of their genome which can be modulated. For instance, completion of hepatocyte maturation could be favored by using the 5-AZA HDAC inhibitor. A protocol was designed in order to get maximum effect, including 5-AZA concentration (lower than ΙΟμΜ, for example 5μΜ), conditions of exposure, addition just during commitment signal was more efficient than 2 or 3 days later, for 4- 5 days. Fig. 13 shows morphology of the cells and Fig. 14 shows quantification of 2 CYP enzymes evidencing improvement of their activity.

EXAMPLE 5

Applications for Hepa-RPs and RPH

A. Example of setting new culture models with HepaRP and RPH

-The HepaRP and RPH progenitor cells were used to produce hepatic cholangiocytes, taking advantage of the plasticity and bipotential properties of progenitors before they engage commitment to hepatocyte differentiation. The use of morphogens distinct from corticoids is explored as well as modulators of physical forces such as shape constraint relaxation.

-The HepaRP and RPH progenitor cells are used to rebuilt and produce hepatic functional units in 3D. Using appropriate environmental supports such as thick matrigel or network of fibers HepaRP and RPH progenitors, which are very plastic, can organize in 3D, forming regular spheroids. Alternatively, mature HepaRP and RPH hepatocytes can be used as well. These spheroids can be used as pure hepatocyte aggregates for various applications including drug toxicity testing; spheroids of hepatic cells mixed with non-parenchymal cells including cholangiocytes, sinusoidal cells, stellate and Kupffer cells can also be produced.

Benefit can be gained from both the high polarity of Hepa-RP and RPH cells, the presence of numerous bile canaliculi and the regular monolayer formed by the cells, in developing tests based on imaging analysis.

B. Example of cholestatic drug testing

Hepa-RPs and RPHs could be used for screening cholestatic drugs. As shown in Fig. 15, evidence of two opposite cholestatic effects (Chlorpromazine CPZ and Fasudil) was seen on the bile saccular canaliculi in RPH cells at passage 23 (10 passages with a corticoid-free medium). Alterations (constriction and dilatation) associated with delay in MRP2 transport of CDFDA compared to control cells were seen. These morphological and functional changes will constitute part of a cholestatic test aiming at screening drugs susceptible to induce adverse cholestatic disease.

C. Example of screening genotoxic drugs

Hepa-RP cells and mainly RPH cells could also be used for screening genotoxic compounds. Conditions allowing the production of pure populations of hepatocytes using corticoid-free proliferating medium will be very convenient. Until now no efficient and appropriate hepatic model was reported to be suitable for setting a genotoxicity test onto liver cells.

Claims

CLAIMS:

1. A hepatic cell derived from a stem-like cell deposited at CNCM, Institut Pasteur, Paris, France, under reference 1-4980 on May 19, 2015.

2. The cell of claim 1, wherein said hepatic cell is a differentiated cell.

3. The cell of claim 2, wherein said differentiated cell is a hepatocyte having morphology characteristic of a primary human hepatocyte.

4. A cell culture composition comprising a plurality of hepatic cells according to claim 1.

5. The cell culture of claim 4, wherein said hepatic cells are differentiated hepatocytes.

6. The cell culture of claim 5, wherein said hepatocytes are polarized having a biliary pole, said hepatocytes cooperatively forming in said cell culture a functional biliary canaliculi structure having a biliary space characterized by a lumen.

7. The cell culture of claim 6, wherein said bile canalicular structure functions for bile acids transport and drug clearance towards the biliary pole.

8. The cell culture of claim 5, wherein said hepatocytes express one or more hepatic lipid metabolism enzymes selected from the group consisting of CYP4F3B and CYP 4A1 1.

9. The cell culture of claim 5, wherein said hepatocytes express one or more hepatic drug metabolism enzymes selected from the group consisting of CYPIA, CYP3A, CYP4A, and CP2E.

10. The cell culture of claim 5, comprising a cell monolayer comprising said differentiated hepatocytes.

1 1. The cell culture of claim 5, further comprising primitive biliary cells, said biliary cells being derived from said stem-like cell deposited at CNCM, Institut Pasteur, Paris, France, under reference 1-4980, on May 19, 2015.

12. A method of directing differentiation of Hepa-SC cells deposited at CNCM, Institut Pasteur, Paris, France, under reference 1-4980, on May 19, 2015 into a hepatic cell according to claim 1, said method comprising:

culturing said Hepa-SC cells under conditions of mechanical stress and in the presence of at least one differentiation factor for said hepatic cell population for a time period sufficient for the Hepa-SC cells to commit to differentiation; and

transferring said committed cells to a second culture medium comprising said differentiation factor; and

maintaining said cells in culture without said mechanical stress to yield said hepatic cells, wherein said differentiation factor is a cortico-steriod.

13. The method of claim 12, wherein said hepatic cells are Hepa-RP cells.

14. The method of claim 13, further comprising culturing said Hepa-RP cells in culture medium comprising at least one of cortico-steriod and DMSO.

15. The method of claim 14, further comprising passaging said Hepa-RP cells in said culture medium for 17-19 passages.

16. The method of claim 14, further comprising passaging said Hepa-RP cells in said culture medium for 7-8 passages.

17. The method of claim 16, further comprising transferring said Hepa-RP cells after said 7-8 passages to a second culture medium that is essentially free of cortico-steroid and of DMSO.

18. The method of claim 17, further comprising maintaining said passaged Hepa-RP cells in said corticoid-free medium for up to 20-25 passages.

19. The method of claim 18, further comprising directing said passaged Hepa-RP cells to differentiate into mature hepatocytes by culturing said passaged Hepa-RP cells in a third culture medium comprising at least one cortico-steroid and DMSO in a quantity sufficient to induce differentiation.

20. The method of claim 17, further comprising maintaining said Hepa-RP cells in said corticoid-free culture medium, and subsequently directing said Hepa-RP cells to differentiate into mature hepatocytes by culturing said passaged Hepa-RP cells in a fourth culture medium comprising at least one cortico-steroid and DMSO in a quantity sufficient to induce differentiation at any passage.

21. A hepatic cell line having the identifying characteristics of Hepa-RP.

22. A culture medium for expanding Hepa-RP cells according to claim 21 , said medium comprising a basal nutrient medium, L-glutamine, and insulin.

23. The culture medium of claim 22, wherein said basal nutrient medium is selected from the group consisting of William's E, Roswell Park Memorial Institute, Dulbecco's Modified

Eagle's Medium/HamF12, and Dulbecco's Modified Eagle's Medium/MEM 199.

24. The culture medium of claim 22, further comprising a cortico-steroid and DMSO.

25. The culture medium of claim 22, being essentially free of cortico-steroid and of

DMSO.

26. A cell culture consisting essentially of a plurality of hepatocytes differentiated from the hepatic cell line of claim 21 , wherein said cell culture is essentially free of primitive biliary cells.