WO2008035169A2

WO2008035169A2 - Process for preparing hepatic cells from progenitor cells present in umbilical cord blood

Info

Publication number: WO2008035169A2
Application number: PCT/IB2007/002699
Authority: WO
Inventors: Annalisa Crema; Cristina Haas; Massimo Sanchez; Antonella Lisi; Antonio Ponzetto; Ercole Brunetti; Rodolfo Marchese; Guido Carloni
Original assignee: Consiglio Nazionale delle Richerche CNR; Universita degli Studi di Torino
Current assignee: Consiglio Nazionale delle Richerche CNR; Universita degli Studi di Torino
Priority date: 2006-09-18
Filing date: 2007-09-18
Publication date: 2008-03-27
Anticipated expiration: 2009-03-18
Also published as: ITMI20061772A1; WO2008035169A3

Abstract

The present invention concerns a process for obtaining cultures of differentiated hepatocytes starting from progenitor cells present in the umbilical cord blood of normal newborns. In particular the invention relates to a process for obtaining a long-term culturing system which allows the in vitro expansion and differentiation of purified hepatic precursors. Pluripotent progenitor cells are purified by immuno-selection, for example with magnetic beads and expanded in vitro starting from mononuclear cells present in umbilical cord blood. The hepatocyte culture obtained with the thus identified system is suitable for cell therapy which replaces diseased hepatocytes with healthy hepatocytes deriving from homologous stem cells. This hepatocyte culture can also be used for the development of bioartificial organs and, in a further embodiment, for the preparation of in vitro systems for the in vitro production of hepatotropic viruses and/or of their components (i.e. the HCV virus).

Description

PROCESS FOR PREPARING HEPATIC CELLS FROM PROGENITOR CELLS PRESENT IN UMBILICAL CORD BLOOD Field of the invention

The field of the invention is that of cell therapy with cells differentiated in vitro Prior art

Primary hepatocellular insufficiency is a serious health problem for our country. Fatalities due to hepatic cirrhosis in Italy are the main cause of death in the 20-39 age band and, between the ages 40 to 59 years, they are more than double those due to colon cancer in men. There is no current therapy able to arrest the progress of hepatic cirrhosis, except for liver transplant which is an extremely complex operation made more serious by the very high costs both to humans and the economy, and above all limited by the scarcity of available donors (each year in Italy, there are 15,000 deaths from cirrhosis and 10,000 from hepatocarcinoma). A possible alternative, identified as being promising for treating acquired or hereditary hepatic pathologies, is cell transplantation which is possible due to the great regenerative capacity of the hepatic cell. This technique, however, presents the disadvantage that the transplanted heterologous hepatic cells are subjected to a violent immune rejection reaction (Susick 2001). As another alternative, adult stem cells are found to be particularly promising, including so called "oval cells" which can be activated to proliferate and differentiate into cells with mature hepatic phenotype (Shafritz & Dabeva, 2002) though their activation, differentiation and proliferation mechanisms are still very incomplete. As yet, the specific cell markers of hepatic progenitors have not been defined with certainty, whereas the main markers of differentiated hepatocyte functionality are well known: production of albumin, urea, glycogen, cytokeratin-18, etc. Oval cells for example express some hepatocyte and bile ducts markers (cytokeratin-7 and -19) and also embryonic hematopoietic cell markers, such as: Thy1 , CD34, CD45, SCA1 , c-Kit and Flt-3. In any event, clinical use of hepatocyte precursors is limited both in terms of reproducibility and the histocompatibility problems in non-autologous transplants. Recent work has focused attention on the plasticity of poorly characterized pluripotent adult stem cells (PASC, see Brazelton et al., 2000), present in various tissues. Specifically, the capacity of PASC to "trans-differentiate" and to generate cells of non-hematopoietic type such as myocytes, cardiomyocytes (Orlic et al., 2001), neurons (Eglitis and Mezet, 1997) and hepatocytes (Petersen et al. 1999) appears to be interesting. With regard to man, hepatocytes have been obtained in vivo (Lagasse et al., 2000) or in vitro (Schwartz 2002) not only from bone marrow cells but also from cells isolated from peripheral blood (PBMCs) (Zhao 2003). This finding seems to offer unexpected therapeutic potential, though with some limitations; i) the low percentage of stem cells (ASC) in adult tissues; ii) the limited ex vivo expansion potential of ASCs; iii) the limited cell pool usable following selection of a sub- population of specific progenitors. An alternative source of stem cells usable in transplants are umbilical cord cells (UCB), from which both hematopoietic stem cells (HSCs) (Nakahata 1982) and mesenchymal stem cells (MSCs) (Huss 2000) can be extracted. Cord derived hematopoietic stem cells are as clonogenic as stem cells derived from bone marrow or from adult stem cells, responding to various growth factors and possessing the capacity to expand in vitro in long-term cultures (De Wynter 1998). It has been shown that umbilical cord derived stem cells are useful and effective substitutes for cells derived from bone marrow in allogeneic transplants during the course of therapy for malignant hemopathies. Umbilical cord cells also exhibit some embryonic characteristics (McGuckin 2005) which render them particularly interesting: they have a great potential for ex vivo expansion, are more immunotolerant and have longer telomeres than other adult stem cells. For these reasons cord derived cells are particularly indicated as a source of stem cells for cell transplant therapies in serious hepatic insufficiencies (Kakinuma 2003). The possibility of obtaining phenotypically pure progenitor populations which, once expanded, can be suitably cultivated and directed into specific differentiation lineages, is a further aspect of cord derived stem cells which can facilitate their experimental and therapeutic usage. The studies carried out on hematopoietic stem cells, purified on the basis of the expression of specific surface marker such as CD34 and CD45, have provided the opportunity to understand the in vitro culture conditions and the growth factors for differentiation necessary to achieve commitment to various differentiated cell lineages. It has been shown that medulla derived HSCs can give rise mainly to lymphoid, myeloid and myocytic cell lines, but also to cardiac and neuronal cell lines.

The mesenchymal cells present in cord blood are cells capable of prolonged proliferative activity having the substrate adhesion characteristics associated with a unique surface molecule expression profile (Lee 2004). This has enabled hepatocytes to be obtained in vitro from UCB- and bone marrow (BM)-derived human MSC cells, by varying and modulating the culturing conditions with suitable combinations of hormones and specific growth factors (Hong 2005). In this manner, even in the absence of a feeder layer, cells were obtained which exhibit the cuboidal morphology typical of hepatocytes, accompanied by the expression of differentiation markers and hepatic functionality which was analogous to that of fully differentiated hepatocytes. The isolation of MSCs from umbilical cord is not always possible and there exist some critical parameters which limit its applicability and suitability for therapeutic purposes: low yield, extremely short time margins (15 hours of useful time from collection), imperfect reproducibility (Bieback 2004). During embryonic development, the first sign of liver morphogenesis is a thickening of the ventral endoderm epithelium. Whether the umbilical cord also contains an endodermal cellular component is still the subject of controversy. Studying the differentiation potential of immature umbilical cord cells towards hepatic type cells, therefore, is of fundamental importance for cell replacement therapy for therapeutic purposes during the course of hepatic pathologies. As yet, little is known of the potential of immature umbilical cord cell populations, but interest is emerging regarding their use for the regeneration of non- hematopoietic tissues and for cell therapy. The works of Kakinum and Hong for example clearly show the hepatic differentiation potential of cord cells. Both have reported the presence in UCB of transplantable hepatic progenitors (Kakinuma 2003) which can be directed into the hepatic line (Hong 2005). This potential, however, refers to total MNCs or to cord blood MSCs; a specific hepatic progenitor population is neither identified nor isolated. The therapeutic use of a qualitatively and quantitatively heterogeneous cell population poses the problem of the reproducibility and efficiency of the response to the differentiation stimulus. On the contrary, the present invention concerns a process for obtaining hepatic cells in culture starting from a homogeneous population of CD133⁺/CD34⁺ precursors purified from human umbilical cord blood (UCB), which can be amplified while maintaining the same stem cell characteristics and the regenerative potential of the initial population. Summary of the Invention

The present invention concerns a process for obtaining cultures of differentiated hepatocytes starting from progenitor cells present in the umbilical cord blood of normal newborns. In greater detail, the invention relates to a process for obtaining a long-term culturing system which allows the in vitro expansion and differentiation of purified hepatic precursors. Pluripotent progenitor cells are purified by immuno- selection, for example with magnetic beads, and expanded in vitro starting from mononuclear cells present in umbilical cord blood. The present invention also relates to optimum growth conditions for achieving hepatocyte commitment. The hepatocyte culture obtained with the presently identified system is suitable for cell therapy replacing diseased hepatocytes with healthy hepatic cells deriving from homologous stem cells. This hepatocyte culture can also be used for the development of bioartificial organs and, in a further application, for the preparation of in vitro systems for the in vitro production of hepatotropic viruses and/or of their components (for example the HCV virus). Brief description of the drawings

Figure 1. Growth curve of purified CD133⁺/CD34⁺ cell population in 3 weeks of culturing. Figure 2. Phase-contrast photograph of selected CD133⁺ mononuclear cells (MNCs) at 9 days from the beginning of treatment with inducers of hepatocyte commitment. The cells become adherent to the substrate and elongated fibroblastoid forms are initially observed. Figure 3. Phase contrast micro-photograph of selected CD133⁺ MNCs at 13 days from the start of treatment with inducers of hepatocyte commitment. The cells assume stellate form. Figure 4. Phase contrast micro-photograph of selected CD133⁺ MNCs at 35 days from the start of treatment with inducers of hepatocyte commitment. In the bi- and polynuclear cells, cell volume increases substantially as in mature hepatocytes. Figure 5. As Figure 4 (different field). After 52 days there is a distinct increase in the presence of roundish cells that evolve over time into parenchymal cells similar to mature hepatocytes.

Figure 6. Kinetics of release and accumulation of albumin and urea in culture medium (immunoenzymatic assays) in response to treatment with the hepatic induction cocktail. Figure 7. Western-blot of the main proteins of hepatic synthesis in the cell supernatant. By means of specific antibodies, the presence of albumin and alpha- fetoprotein was detected together with their accumulation in the supernatant after induction, concurrent with the morphological changes upon differentiation. The days following induction are indicated in the figure above each lane (dθ, d27, d34). Detailed description of the invention The present invention is based on the finding that CD133⁺ cells derived from umbilical cord blood can be directed, by suitable conditions of growth and amplification, towards the hepatocytic lineage; they constitute a progenitor line which, with its high proliferative capacity, gives rise to cells expressing hepatic markers. It was hitherto known that neuronal and myocyte cells, and cardiomyocytes, could be derived from umbilical cord blood cells, but a homogeneous population of hepatic precursors with long-term stability has never been obtained. The in vitro process for obtaining hepatic cells from UCB-derived cells is divided into the following steps: a) purification of CD133⁺ precursors present among umbilical cord blood nucleated cells; b) in vitro expansion of said cells; c) induction of commitment and hepatocyte differentiation; d) obtaining a long-term culture from said cells suitable for therapeutic use. The selection of CD133⁺ precursors in step a) is preferably carried out after purification of nucleated cells from umbilical cord blood by means of a density gradient, such as Ficoll-Hypaque, in accordance with known methods. The selection of CD133⁺ ceils is preferably carried out by immunoaffinity and is based on the use of specific antibodies. In a preferred embodiment said antibodies are bound to a solid phase, for example magnetic beads, by i) positive immunomagnetic selection of CD133⁺ cells, ii) magnetic separation preferably on Miltenyi columns, iii) elution of the CD133⁺ cell population. By means of cytofluorometry, the purified CD33⁺ cells are found to be over 99% pure. Expansion of the purified CD133⁺ cell population preferably takes place in Iscove culture medium containing 20 ng/ml of Stem Cell Factor (SCF), with added 15- 30% fetal bovine serum (Bio Whittaker Australian FBS, Cambrex). Seeding takes place in accordance with known methods: preferably at an initial concentration of 0.25 x 10⁶ cells/ml. The expansion period is preferably between 5 and 15 days. After step b) CD133⁺ cells are obtained which are found to express, after immunophenotypization, the same cell surface antigens as hepatic stem cells, thus confirming their potential to differentiate into the hepatocyte line. In particular, said cells are found to be positive for CD133 as expected on the basis of the selection carried out, and also for CD33, CD34, CD43, CD44 but negative for CD90, CD10 and CD2, as indicated by immunophenotyping with specific antibodies. Induction to differentiate into the hepatocytic line c) is carried out in a culture medium, preferably EDM (Eagle's Dulbecco Modified) comprising two or more of the following growth and differentiation factors: FGF-α, FGF-β, HGF. The concentration of FGF (α and β) is preferably between 5 and 10 ng/ml, and that of HGF between 10 and 40 ng/ml. The medium also comprises preferably 5 to 10 ng/ml of the following components: EGF (Epidermal Growth Factor), LIF (Leukemia Inhibitory Factor), SCF (Stem Cell Factor), oncostatin-M (OSM) and the Clonetics Singe Quots mixture (CAMBREX) which contains insulin, transferrin, ascorbic acid according to the information given by the supplier, and preferably about 1 μM of hydrocortisone. Treatment with the induction cocktail is repeated each time the medium is changed and for at least 1 month but preferably for at least 7 weeks.

This treatment induces a characteristic morphological change in the selected population which is well documented in figures 2-5: firstly into elongated fibroblastoid forms and stellate forms (7-10 days), then into bi-and poly-nuclear cells (from 13 to 14 days) with a substantial increase in cell volume typical of the mature hepatocyte, and a distinct increase in the presence of roundish cells. In parallel with the morphological changes and with the substrate adhesion phenomenon, expression of specific hepatic markers is observed such as: alpha- fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18) and cytokeratin 19 (CK19). These markers are found to be positive only after treatment with the mixture of hepatic commitment inducers protracted over a few days or preferably for at least 120 hours or even more preferably for at least 1 week or for at least 5 weeks.

As summarized in table 3, the expression of specific hepatic markers continues for at least 7 weeks in the CD133⁺ cell population cultivated in Dulbecco medium, indicating that the obtained cells stably maintain their commitment to the hepatic line. Incidentally, a more thorough analysis of the experimental data in table 3 shows that it is actually the persistent expression of AFP after 5-7 weeks' induction which is indicative of said commitment, since AFP expression decreases then stops in cells which differentiate into the biliary line (cholangiocytes). The method of the present invention overcomes the reproducibility and efficiency problem of the response to the differentiation stimulus in that, under the selective conditions described, the cell population is qualitatively and quantitatively more homogeneous than that obtained with known methods in the prior art. This characteristic renders the population obtained according to the invention particularly suited to therapeutic use and renders the proposed method standardizable for preparing therapeutic quantities of cells. Additionally, the population obtained from UCB MNCs with the selection and differentiation method established in the present invention shows a higher ex vivo expansion potential than that obtainable with unselected mononuclear cells (MNCs), therefore allowing therapeutically useful quantities of cells to be obtained more easily, a lower rate of apoptosis, less donor risk and less incidence of graft- versus-host disease (GVHD). Finally, the CD133⁺/CD34⁺ subclass of adherent primitive stem cells has a higher GM-CSF production than that of non-adherent cultures. This factor is implicated in the mobilization of stem cells and plays a fundamental role in cellular regeneration processes.

In accordance with a further aspect, the invention relates to the use of the described process for preparing cell compositions with high regenerative potential for use in the hepatic regeneration required following cirrhosis, hepatectomy, post- trauma hepatic damage and hereditary or acquired hepatic pathologies, for example of viral etiology.

The method of the invention and the cells obtained therefrom are hence of therapeutic use in treating the damage caused by the aforesaid hepatic pathologies: cirrhosis, hepatectomy, post-trauma hepatic damage and hereditary or acquired hepatic pathologies, for example of viral etiology, by the administration of compositions of cells committed in vitro in accordance with the process of the invention, and administered in useful quantities to patients by infusion, injection or other known methods. The main embodiment of the hepatocyte culture obtained with the thus identified system is cell therapy which replaces diseased hepatocytes with healthy hepatic cells deriving from homologous stem cells. Further embodiments of the hepatocyte culture also include the development of bioartificial organs, and, in a further embodiment, the preparation of in vitro systems for the in vitro production of hepatotropic viruses and/or their components (for example HCV and HBV viruses etc).

The present invention will now be described by non-limiting illustrative examples, with reference being made to the figures and tables.

EXPERIMENTAL PART

Example 1. Enrichment of CD133⁺ cells from umbilical cord blood The blood (around 150 ml) obtained from a single umbilical cord was diluted 1 :4 with culture medium, then stratified on Ficoll-Hypaque in a 2:1 ratio and centrifuged for 30 minutes at 1800 rpm at ambient temperature. About 2/3 of the supernatant was removed by aspiration. The interface between the supernatant and the Ficoll-Hypaque, containing the mononuclear cells (MNCs), was collected by circular aspiration, washed twice with medium and once with PBS containing added 0.1% BSA and 2 mM EDTA. A batch of the mononuclear cells thus obtained (2.5 x 10⁶) was used for antigenic typing of surface markers.

The mononuclear cells (MNCs) obtained (about 8 x 10⁸ cells) were incubated with microbeads conjugated to anti-CD133 antibodies and subjected to positive immunomagnetic selection on small columns (Miltenyi, MACS Separation

Columns), by following the scheme below and in accordance with the company's instructions: a) positive immunomagnetic selection of CD133⁺ cells; b) magnetic separation on Miltenyi columns; c) elution of the CD133⁺ cell population. A batch of the thus purified CD133⁺ cells was used to check purification by means of cytofluorometry.

The purified CDT33⁺ cell population was placed in ISCOVE culture medium containing 20 ng/ml of Stem Cell Factor (SCF), at an initial concentration of 0.25 x

10⁶ cells/ml with added fetal bovine serum (Cambrex Bio Whittaker, Australian

FBS).

Table 1. Immunophenotvping of MNCs

In Table 1 , where typing of MNCs from umbilical cord blood is shown before and after purification, it is apparent how the enrichment system used has enabled a population containing 99% CD133⁺ cells, which co-express the CD34 marker in

100% of cases, to be isolated.

Example 2. In vitro expansion of CD133+/CD34+ cells

Figure 1 clearly shows the high capacity for in vitro expansion of said cells, with a 160-fold increase in cell numbers in 3 weeks of culturing. Table 2 shows that the expression profile of the cell surface markers of CD133⁺/CD34⁺ cells after 14 days of in vitro expansion is analogous to that of hepatic stem cells obtained starting from fetal livers (Barcena et al. 1995) confirming the hypothesized presence of hepatic precursors in the selected cell population.

Example 3. Antigenic typing of the selected cells

Antigenic typing of the selected and enriched cells was carried out by cytofluorometry.

Both the MNCs and the purified CD133⁺ cells were incubated for 30 minutes at 4°C with pretitrated saturating solutions of the following monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC): anti-CD133 (PE Miltenyi); anti-CD34 (APC Miltenyi); anti- CD7; anti-CD45 and anti-CD90 (FITC Immunotools); anti-OV6 (indirect with secondary FITC rat anti-mouse) (Table 1).

To verify the expression pattern prior to stimulation into the hepatocyte line, the CD133⁺ cells expanded in culture for 2 weeks were incubated with antibodies against the principal surface markers representative of the various differentiated cell lineages and/or of stem cells. For this purpose use was made of the following monoclonal antibodies conjugated with APC, PE, FITC: anti-CD133 (PE); anti- CD34 (APC); anti-CD43; anti-CD44; anti-CD10; anti-CD2; anti-CD33; anti-CD90; antic-Kit (all FITC) (Table 2). Table 2 shows the immunophenotyping results after in vitro expansion.

Table 2. lmmunophenotyping after in vitro expansion

Εarcena 1995; Lapidot, T. 2001 ; Nava 2005.

The data given in table 2 show that the expression pattern of the examined cell markers for the in vitro selected and expanded cell population corresponds to that described in the literature for hepatic stem cells derived from fetal livers, being rich irrprogenitors: Example 4. Induction of differentiation into the hepatocyte line After expansion in culture the pluripotent stem cells were induced towards hepatocyte differentiation by incubation in a medium comprising a mixture of specific growth factors. The expansion medium (ISCOVE) was replaced by Dulbecco's Modified Eagles Minimum Essential Medium (DME), containing 5-10% BIT 9500 serum substitute (Stem Cell Technology) or fetal serum with added growth factors and hepato-specific hormones in suitable concentrations. In particular the induction mixture contained the following as growth factors: EGF 10 ng/ml; FGFα 10 ng/ml; FGFβ10 ng/ml; LIF 10 ng/ml; OSM 10 ng/ml; HGF 15-50 ng/ml and SCF 10-30 ng/ml (Immunotools); Insulin hormones, transferrin and ascorbic acid contained in Clonetics HCM Single Quots (CAMBREX), in accordance with the producer's instructions, and hydrocortisone 1 μM (SIGMA). The cells were seeded onto 4 chamber slides (LAB-TEK) coated with 0.1% gelatin at an initial concentration of 0.4 x 10⁶ cells/ml and analysed for expression of hepatic markers.

The cell phenotype and the expression kinetics of the markers of hepatic functionality induced in culture by the differentiation cocktail were analysed up to the 52^nd day by a) observation with phase contrast microscope (Figures 2-5); b) direct and indirect immunofluorescence (Table 3); c) determining concentration of hepato-specific proteins and urea (Figure 6); d) immunoblotting for hepato-specific proteins (Figure 7). The data relating to the expression of hepatic markers are given in table 3. The changes in cell phenotype were photographed and recorded in figures 2-5.

Table 3. Expression of Hepatic Cell Markers.

p.i.: post-induction

Figures 2-5 clearly indicate that following administration of the hepato-specific induction cocktail, the cells change their morphology. They become adherent to the substrate and, initially, elongated fibroblastoid (Fig 2) and stellate (Fig 3) forms are observed then, later, bi- and polynuclear cells with a substantial increase in cell volume as is typical of the mature hepatocyte, with a distinct increase in the presence of roundish cells which over time evolve into parenchymal cells similar to mature hepatocytes (Figs. 4 and 5).

Table 3 shows that, as well as the morphological changes in the aforedescribed cells, a significant increase in positiveness for expression of hepatocyte-specific markers including Albumin (AIb), Alpha-fetoprotein (AFP), Cytokeratin-18 and 19 (CK18, CK19) were found. The persistent expression of AFP at later periods indicates a commitment to the hepatocytic lineage; indeed after the early stage (in which it is always expressed) production of this protein decreases then stops in cells that differentiate, instead, into the biliary line (cholangiocytes). Figure 6 documents the release and accumulation in culture medium of albumin and urea, their being the principle products of hepatic synthesis which according to the enzymatic assays carried out, start being detected in the medium at around 8 days after induction. The production of proteins and products of the hepatic line was also confirmed by the "western blot" method in the cell supernatant (Fig. 7) which clearly shows the presence of bands specific for albumin and alpha- fetoprotein as well as their increase over time.

In conclusion the data given herein show that the sub-population of CD133⁺/CD34⁺ cell precursors purified from umbilical cord blood has an increased capacity for expansion in long-term culture and a high potential for hepatic differentiation. Said population can be activated to proliferate and differentiate in vitro into cells with a mature hepatocyte phenotype (hepatocytes and cholangiocytes), which express the principle markers of hepatic functionality. The described process for the commitment of UCB derived precursor cells to differentiate in vitro into functionally active cells that are morphologically similar to mature hepatocytes can be applied for the purposes herein described. It is intended that variations and/or modifications can be made by experts of the art without leaving the relative scope of protection.

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Claims

1. A Process for the preparation of progenitor cells of the hepatocyte and biliary line from umbilical cord blood cells comprising the following steps: a) selection of a cell population of CD133* MNCs b) expansion c) in vitro induction of hepatocyte commitment.

2. Process according to claim 1 wherein the selection a) is preceded by the isolation of mononuclear cells (MNCs) from umbilical cord blood.

3. Process according to claim 2 wherein said isolation is carried out by separation on a density gradient.

4. Process according to claim 2 wherein said gradient is a Ficoll-Hypaque gradient.

5. Process according to claims 1-4 wherein the selection a) is carried out by immunoaffinity.

6. Process according to claim 1-5 wherein said CD133⁺ cells co-express the CD34 antigen.

7. Process according to claims 1-6 wherein the induction c) is carried out in a culture medium comprising two or more of the following growth factors and/or differentiation factors: FGF-α, FGF-β, HGF.

8. Process according to claim 7 wherein said medium also comprises the following components: EGF, LIF, SCF, OSM, insulin, transferrin, ascorbic acid and hydrocortisone.

9. Process according to claims 6-7 wherein the concentration of each of the factors FGF-α and FGF-β is between 5-10 ng/ml and that of HGF is between 10 and 40 ng/ml.

10. Process according to claim 8 wherein LIF and EGF are in a concentration of between 5-15 ng/ml, and OSM is in a concentration of 5-15 ng/ml.

11. Process according to claims 6-9 wherein the culture medium is EDM (Eagle's Dulbecco Modified).

12. Process according to claims 1-11 wherein the expansion b) is carried out in a culture medium comprising Stem Cell Factor (SCF) at a concentration between 10 and 40 ng/ml.

13. Process according to claims 1-12 comprising a final step of in vitro stem cell growth directed to the hepatic line as derived in step c) for at least one week.

14. Process according to claim 13 wherein said growth is for at least 5 weeks.

15. Cells induced towards hepatic differentiation, obtainable by the process of claims 1-14.

16. Cells according to claim 15 consisting of hepatocytes and/or cholangiocytes.

17. Use of the cells according to claims 15-16 for preparing compositions for hepatic regeneration.

18. The use according to claim 17 for hepatic regeneration of one of the following pathologies: cirrhosis, hepatectomy, post-trauma hepatic damage, hereditary or acquired hepatic pathologies.

19. Use of cells according to claims 15-16 for the preparation of viral components of hepatotropic viruses.

20. Use of cells according to claims 15-16 for the viral replication of hepatotropic viruses.

21. Use of cells according to claims 15-16 for the preparation of bioartificial organs.

22. Process for the preparation of CD133⁺ cell compositions for use in hepatic regeneration characterized by comprising the process claimed in claims 1-14.

23. Process for the preparation of CD133⁺ cell compositions for preparing viral components of hepatotropic viruses characterized by comprising the process according to claims 1-14.

24. Process for the preparation of CD133⁺ cell compositions for the viral replication of hepatotropic viruses characterized by comprising the process according to claims 1-14.

25. Process for the preparation of CD133⁺ cell compositions for the preparation of bioartificial organs characterized by comprising the process according to claims 1- 14.