BRPI0620049A2 - isolated liver stem cells - Google Patents

Abstract

ISOLATED STEM CELLS OF THE FIGURE The present invention relates to progenitor stem cells isolated from the liver, and cell population thereof, wherein said progenitor stem cells originate from an adult liver, especially from a human. The present invention also relates to the use of said isolated progenitor stem cells in medicines, hepatology, inborn errors of liver metabolism, transplants, infectious diseases, liver failure. The present invention also relates to methods of isolating these cells, their culture, characterization before and after differentiation, and their use in transplants, animal models of human disease, toxicology and pharmacology.

Description

FIELD ISOLATED STEM CELLS INVENTION

The present invention relates to isolated liver progenitor stem cells originating from an adult liver and their use of said isolated progenitor stem cells in medicines, hepatology, inborn errors of liver metabolism, transplantation, infectious diseases, liver failure. The present invention also relates to methods of isolating these cells, their culture, and characteristics before and after differentiation, and their use in transplantation, animal models of human disease, toxicology and pharmacology.

BACKGROUND OF THE INVENTION

The liver is a key organ that performs many vital functions, such as glucose homeostasis, xenobiotic detoxification or macromolecule synthesis. Thus, failure of one of the multiple functions of the liver can have a dramatic impact on health. The worldwide incidence of the set of acute or chronic liver diseases is between the 5th and 9th cause of death, according to the World Health Organization. To date, the only curative treatment for end-stage liver disease continues to be liver transplantation. liver. The results for patients undergoing liver replacement surgery are quite reasonable, with over 95% recovery. However, despite new surgical techniques, including liver donation and donor-related life, the growing organ shortage leads to high mortality on the waiting list. Therefore, an important objective in medical transplant research is to demonstrate the potential use of liver cells in liver regeneration and treatment of liver disease.

Liver cell transplantation (TCF) is an emerging process involving infusion of liver cell suspension into the receptor portal system. This is intended for the recovery of liver function of the recipient as a consequence of grafting and restocking of diseased parenchyma. TCF was first validated in animal models where syngeneic hepatocytes have been shown to survive indefinitely and to be able to correct various enzyme defects (for review, see Najimi and Sokal. 2005. Minerva Pediatr 57 (5): 243-57).

In humans, the first studies were designed to treat acute liver failure. These clinical studies have led to further increases in TCF indications, and so far at least 30 cases have been reported worldwide for having various defects (Strom et al. 1997. Transplant Proc 29 (4): 2103-6). . In the specific field of metabolic diseases, thirteen cases have been reported using hepatocytes to treat, among others, Crigler-Najjar type I syndrome, urea cycle defects or rare diseases such as infantile Refsum disease. These studies demonstrated hepatocyte grafting within the parenchyma and, consequently, an improvement in the patient's condition up to 18 months after transplantation.

However, because the supply of mature human hepatocytes for transplantation is still limited, in fact, about as limited as the availability of the entire liver, research also aims to obtain transplanted r® cells from other sources, such as stem cells. and progenitors, for example, embryonic or adult origin, which could be expandable, for example, in vitro, and able to differentiate into functional mature hepatocytes, esp. in vivo after transplantation. Thus, there is a great need to develop new means useful in the treatment of various diseases or conditions associated with liver-associated diseases, especially given the insufficiency of the treatments currently available for most of these diseases.

Historically, embryonic stem cells (ET) were thought to be involved only in organogenesis, due to their observed unlimited clonal division and pluripotent differentiation into whole tissue derived cells. On the other hand, in the regeneration processes of the adult organs it was typically attributed to the adult progenitor cells. However, this theory has been revised in view of the discovery of stem cells in adult organs that expresses known embryonic markers. Therefore, stem and progenitor cell characteristics are now based not only on the developmental process (embryonic versus adult), but also on the presence of specific cell markers in it. Indeed, expression of cellular markers, such as membrane proteins or transcription factors, may vary along differentiation of different pathways and the various reflected stimuli (eg, environmental stimuli) and cellular needs. It is often observed in the course of a differentiation process that a stem cell will gradually cease to display its pluripotent markers, eg Oct-Δ, and to express markers attributable to later stages, for example, markers of pluripotency. a specific lineage. As a non-limiting example, Oct-A may be progressively lost through maturation and, on the other hand, cells entering the endodermal lineage may begin to express alpha-fetoprotein.

With regard to liver regeneration through cell transplantation, various types of cell source can be considered. For example, it is expected that ES cells may be able to regenerate in any organ due to their pluripotency. In fact, this is widely explored in art. However, ES cells are prone to generate tumor growth when introduced into any tissue other than in utero. Therefore, its use in vivo remains limited by the risk of carcinogenic bypass. Even before in vitro success, ES cell differentiation may not be safe enough to consider human inoculation.

A safe alternative could be the use of adult progenitor cells which, unlike ES cells, tend to show limited capacity for clonal division and their differentiation gives rise to daughter cells with more limited fates. In the liver, adult progenitors such as oval cells (hepatocyte and cholangiocyte precursors) or small hepatocyte-like cells have been described. However, its medical use has become difficult due to its scarcity in normal adult organs.

Therefore, adult stem cells would show the ability to clone without risk or with little risk of cancerous shifts would represent a major improvement in source cell transplantation. Several types of adult stem cells are currently being evaluated in liver cell transplantation studies. For example, mesenchymal stem cells (MSCs) from umbilical or peripheral cord blood have been studied because of their ability to transdifferentiate into more mature cells of another strain. In addition, marrow hematopoietic stem cells have also been studied in terms of liver regeneration potencies.

Although in vitro characterization of adult stem cells still presents difficulties, it is currently accepted in the field that such characterization may advantageously involve detection (i) markers of their embryonic origin or lineage (esp., Mesodermal, endodermal, ectodermal or hematopoietic). , (ii) expression of markers that reflect the level of differentiation and therefore somewhat predictive of the different possible progenies, and (iii) fate in vitro or in vivo after differentiation. Therefore, the characterization and distinctiveness of adult stem cells obtained from a normal liver may advantageously involve assessing the presence or absence of the marker (s) reflecting the complex embryonic origin of this organ, (ii) differentiation marker (s) (eg, presence of albumin) and (iii) at least one marker indicative of target stem cells.

According to current knowledge, the liver comes mainly from the endoderm and hepatocytes that are part of the endodermal lineage. However, liver cell formation also involves the interaction between the endodermal epithelium and the cardiogenic mesoderm. In addition, in fetal development hematopoiesis also occurs in the liver. Given this interaction during development, it is necessary to have an open mind when contemplating present markers of adult liver stem cells, since endodermal, mesodermal and / or hematopoietic markers could be predicted.

In assessing the different levels and compromised type cells, various cell markers may be evaluated, as will be done later in this disclosure. For example, during the process of differentiation cells some markers may diminish or disappear, others may increase or may be acquired, and still others may be retained in all forms of a specialized and functional cell. By way of non-limiting example, during organogenesis, ie during fetal life, hepatoblasts are considered common progenitors of parenchymal forming cells (esp. Hepatocytes and biliary cells), and expressed inter alia cytokeratin-7 (CK-7 ) as well as CK-19, albumin and α-fetoprotein. In adult livers, a known common progenitor of hepatocytes and biliary cells is the oval cell, which expresses CK-19, albumin and α-fetoprotein. Following differentiation into bile cells, CK-19 and α-fetoprotein expression is maintained while CK-7 expression (considered a characteristic of more immature cells) tends to cease. On the other hand, hepatocytes maintain α-fetoprotein and albumin expression, but do not show CK expression above. Also from this example, it follows that the characterization of stem cells can be complex, but the evaluation of markers, which can be used to indicate the advantage of cell type or property.

To the inventors' knowledge, previous studies have described the isolation of adult normal liver progenitor cells, which demonstrated more than one target cell. An adult liver stem cell line capable of in vitro amplification and differentiation in vivo into hepatocytes and preferably only with target hepatocyte cells has not been described. In addition, previous studies use complicated techniques such as FACS, calcium-implemented medium, or specific density gradients to isolate your liver stem cells.

Thus, it is an object of the invention to provide a novel liver-derived parent or stem cell with improved properties, and particularly useful in, for example, liver cell transplantation. The invention also provides to provide a simple method for isolating said cells.

SUMMARY OF THE INVENTION

The invention provides adult liver progenitor or stem cells, stem cell lineages, or cell populations comprising the same populations obtained from normal liver tissue. Methods of isolating these cells, their culture, characterization before and after differentiation, and their use for transplantation, animal models of human disease, toxicology and pharmacology are also within the invention.

In one aspect, the present invention provides a novel stem cell or parent isolated from (a vertebrate, preferably a mammal, more preferably a human cell), originating from the adult liver, characterized in that it co-expresses ( ie positive for) at least one mesenchymal marker, esp. one, more than one, for example 2, 3 or 4, or all markers CD90, CD44, CD73, vimentin and α-actin smooth muscle (ASMA), with hepatocyte albumin marker (ALB) and possibly with one or more other hepatocyte or liver markers, preferably one, more than one or all of the CD29, alpha-fetoprotein (AFP), alpha-1-antitrypsin and / or MRP2 transporter. Said adult liver progenitors or stem cells may further express one, more than one or all of the following molecules indicative of similar hepatocyte functions or properties: G6P, CYPlBl, CYP3A4, HNF-4, TDO, TAT , GS, GGT, CK8, EAAT2. Said liver or stem cell progenitor may further be characterized by one, more than one, or all of the following: negative for at least the CD45 and CD34 hematopoietic markers and also, optionally, for one or more other hematopoietic markers such as , for example, CD105, HLA-DR, negative for epithelial marker cholangiolitis cytokeratin-19 (CK-19), and possibly for further epithelial markers; negative for at least the undifferentiated stem cell markers CD117 and Oct-4, and possibly also for one or more than one embryonic stem cell marker; low level AFP expression. Preferably, said liver stem or progenitor cell may have mesenchymal-like morphology, in particular involving one, more than one or all growth in flattened monolayers, broad cytoplasm and / or ovoid nuclei with one or two nucleoli.

In one embodiment, particularly the present invention provides for an isolated stem cell originating from the adult liver, which is CD90, CD29 positive and CD44 positive which is albumin positive, vimentin positive and alpha muscle positive smooth muscle actin. In one embodiment, stem cell isolation is also CK-19 negative, CD45 negative, CD34 negative, and CD117 negative. The present invention also provides a cell population comprising mesenchymal progenitor stem cells having at least three of the following characteristics: expression of detectable albumin antibodies; expression of detectable vimentin antibodies; expression of detectable smooth muscle alpha actin antibodies; absence of CK-19; absence of CD45, absence of CD45 marker, absence of CD34 markers, absence of CD117 marker, evidence of CD90 marker; CD2 marker tests 9, or CD44 marker tests. Preferably the cells have all of the above characteristics. In a preferred embodiment, the stem cells are human liver stem cells.

In one embodiment, isolation of adult liver stem cells originates from CD90, CD73, CD29 and CD44 positive and positive albumin, positive vimentin and positive alpha-actin smooth muscle.

The invention also provides a method for obtaining isolated parent or stem cell or a cell population comprising said parent or stem cell, the method comprising: (a) dissociating the adult liver or part thereof to form a primary cell population from said adult liver or part thereof, (b) plating the primary cell population onto a substrate that allows cells to adhere to it, and (c) culturing primary population cells, which adhere to said substrate, for at least 7 days, preferably for at least 10, preferably still for at least 13 or more preferably still for at least 15 days.

The invention also provides a method for obtaining isolated stem cells from the liver or its population according to the invention comprising the steps of culturing adult liver cells, isolating hepatocytes from them, plating hepatocytes and culturing said hepatocytes by at least 7 days, preferably for at least 10, preferably still for at least 13 or more preferably for at least 15 days.

In yet another aspect, the invention provides a progenitor isolated from the adult liver or stem cell, cell line thereof and / or a cell population comprising such may be obtained by or directly obtained using the methods of the invention.

In another aspect, the present inventors have established a certain cell population (cell line) of the human liver stem or progenitor cells according to the invention and deposited on February 20, 2006 said cell line isolated under the Treaty. of Budapest with the Belgian Consortium of Coordinated Microorganism Collections (BCCM / LMBP) under accession number LMBP 6452CB (given by the depositary International Authority; depositor reference id: ADHLSC). Thus, the present invention reports an isolated cell, cell line and cell population deposited with BCCM under accession number LMBP 6452CB (here, "LMBP 6452CB" cell line), underlining thereof includes clonal underline, and their progeny, including progeny differentiated from these, esp. hepatocytes or hepatocyte-like cells prepared therefrom, genetically modified or otherwise derived from them.

The present invention also provides a composition comprising the progenitor isolated from the liver or stem cell or the population thereof according to the invention. Preferably, liver cells are human liver cells, or mammalian liver cells.

Stem or progenitor cells according to the invention (specifically mentioning, although obviously not limited to the LMBP 6452CB strain) have several considerable advantages. For example, unlike embryonic cells, the present parent or stem cells are of adult origin and have a lower risk of growth control or malignant transformation when used in therapy.

Furthermore, the inventors have understood that stem or progenitor cells according to the invention substantially do not exhibit ability to differentiate into mesodermal cell types (e.g., osteocytes or chondrocytes, connective tissue cells), which decreases formation. ectopic examination of such tissues when cells are administered and implanted into the liver tissue.

The present inventors also understood that the stem or progenitor cells of the invention may have particular preference for the differentiation of hepatocytes or hepatocyte-like cells, which makes them particularly suitable for the reconstitution of hepatocyte functions in a liver.

The present stem or progenitor cells are clearly different from the liver-derived stem cells described above as oval cells, for example in their morphological characteristics and expression marker.

Adult liver stem cells or progenitors according to the invention are particularly useful in medicine, hepatology, inborn errors of liver metabolism, transplantation, infectious diseases, liver failure. Liver stem or progenitor cells according to the invention are particularly useful for liver cell transplantation (human), the elaboration of animal models of human liver cell transplantation, bio-artificial livers, in vitro liver cell line. liver cells and animal models of acquired human disease, liver metabolism screening tests (pharmacokinetics, cytotoxicity, genotoxicity) and targeted therapy of liver cell genetics. Liver stem or progenitor cells according to the invention may be further differentiated into hepatocytes.

The present invention also provides a pharmaceutical composition comprising liver stem or progenitor cells, cell line, or cell population, or descendants thereof, including differentiated progeny, esp. hepatocytes or optionally genetically engineered hepatocyte-like cells according to the invention and a pharmaceutically acceptable carrier. Preferably, the liver cells are human liver cells, or mammalian liver cells.

The present invention also provides a method of treating liver disease comprising administering an effective amount of liver stem or progenitor cells, a cell line, or cell population thereof, or descendants thereof, including differentiated progeny, esp. hepatocytes or optionally genetically engineered hepatocyte-like cells according to the invention. In one embodiment, liver diseases include, but are not limited to, phenylketonuria and other aminoacidopathies, haemophilia and other coagulation factor deficiencies, familial hypercholesterolemia, other lipid metabolism disorders, urea cycle disorders, glycogenesis, galactosemia, fructosemia, tyrosinemia, carbohydrate and protein metabolism deficiencies, organic aciduria, mitochondrial diseases, lysosomal and peroxisomal diseases, protein synthesis abnormality, liver cell carrier defects, glycosylation defect, hepatitis, cirrhosis, inborn errors of metabolism, acute liver failure , acute liver infection, acute chemical toxicity, chronic liver failure, cholangitis, biliary cirrhosis, Alagille syndrome, alpha-l-antitrypsin deficiency, autoimmune hepatitis, biliary atresia, liver cancer, liver cystic disease, liver fat, galactosemia, gallstones, yes Gilbert's syndrome, haemochromatosis, hepatitis A, hepatitis B, hepatitis C, and other liver viral infections, porphyria, primary sclerosing cholangitis, Reye's syndrome, sarcoidosis, tyrosinemia, glycogenosis type 1, or Wilson's disease.

The present invention also provides a method for treating gene expression errors comprising: (i) introducing into a liver stem or progenitor cell, including, including differentiated progeny, esp. hepatocytes or hepatocyte-like cells according to the invention a functional copy of a gene to provide a transformed population, and (ii) introducing into a liver patient who is ill in need of a functional copy of the gene at least one portion of the transformed population. Alternatively, the transformed population may be introduced into a non-human mammalian liver to produce a novel animal model of liver pathology.

The present invention also provides a composition for the treatment of errors of a gene expression comprising transformed liver stem or progenitor cells or cell line or cell population, or descendants thereof, including differentiated progeny, esp. hepatocytes or optionally genetically engineered hepatocyte-like cells according to the invention in which a functional copy of a gene has been introduced.

The present invention also provides a pharmaceutical composition for treating errors of a gene expression comprising transformed liver stem or progenitor cells or cell line, or cell population, or descendants thereof, including differentiated progeny, esp. hepatocytes or optionally genetically engineered hepatocyte-like cells according to the invention wherein a functional copy of a gene has been introduced and introduced and a pharmaceutically acceptable carrier.

The present invention also provides a method for enhancing the regeneration of diseased or injured liver which comprises administering to the liver an effective amount of liver stem or progenitor cells, cell line or population thereof, or descendants thereof, including differentiated progeny, esp. . hepatocytes or optionally genetically engineered hepatocyte-like cells according to the invention.

The present invention also provides a liver assisting device comprising a container for receiving liver stem or progenitor cells, their cell line or cell population, or descendants thereof, including their differentiated progeny, esp. hepatocytes or optionally genetically engineered hepatocyte-like cells according to the invention.

The present invention also provides a method for performing in vitro toxicity tests comprising: exposing to a test agent a liver stem or progenitor cell, cell line, or cell population, or descendants thereof, including differentiated progeny, esp. . hepatocytes or hepatocyte-like cells, optionally genetically modified according to the invention, and observing at least one effect, if any, of the test agent on the liver cell population. Preferably, the at least one effect includes an effect on cell viability, cell function, or both.

The present invention also provides a method of conducting in vitro drug metabolism studies comprising: (i) exposing the stem or progenitor cells of the liver, cell line or cell population or descendants thereof, including differentiated progeny, esp. hepatocytes or hepatocyte-like cells, optionally genetically modified according to the invention, to a test agent, and (ii) observing at least one change, where appropriate, involving the test agent after the predetermined test period. Preferably, at least one change includes a change in the structure, concentration, or both of the test agent.

The present invention also provides an effective method for performing a detection assay for agents for treating liver infections comprising: (i) infecting with an infectious agent of interest the liver stem or progenitor cells, cell line or cell population or descendants thereof , including differentiated offspring, esp. hepatocytes or hepatocyte-like cells, optionally genetically engineered according to the invention to provide an infected population, (ii) exposing the infected population to a certain amount of test agent, and (iii) observing the effects, if any. , of exposure on the infected population. In one embodiment, the infectious agent includes a microorganism. In one embodiment, the infectious agent will include one or more viruses, bacteria, fungi, or combinations thereof. In one embodiment, observed effects include effects on viral replication of a viral infectious agent. Preferably, the viral infectious agent will include a hepatitis virus.

The present invention also provides a method for producing a protein of interest which comprises (i) introducing into or stem cells or progenitors of the liver, cell line or population thereof, including the differentiated progeny, esp. hepatocytes or hepatocyte-like cells according to the invention a functional gene encoding a protein of interest, (hei) incubating said cell population under effective conditions for transcription, translation and, optionally, post-translational modification takes place, and (iii) collect the protein of interest. Preferably, the liver cells are human liver cells. In one embodiment, the protein of interest comprises an antigen vaccine.

The present invention also provides a method for conducting in vitro or in vivo studies of liver development and hepatocyte differentiation comprising: exposing a liver stem or progenitor cell, cell line, or cell population, or descendants thereof, including the differentiated progeny according to the invention, in vitro or in vivo, to conditions affecting differentiation conditions and to observe at least one effect on the cell population.

The present invention encompasses the cells, the preparation, the characterization, the culture, and the method of production thereof.

The present invention also encompasses the preservation of these cells by cryopreservation and cell culture.

The present invention also encompasses the technique of cell transplantation in animals and humans.

The present invention also encompasses the use of liver stem cells according to the invention for the preparation of a medicament for treating the aforementioned diseases. The present invention also encompasses the use of liver stem cells according to the invention in a kit or part of a kit.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the morphological appearance of an adult human stem cell or progenitor derived liver (ADHLSC) as prepared in example 1 after 1 month of culture using light microscopy (phase contrast) . A, in less confluence; B, in greater confluence. 100x magnification

Figure 2A: The presence of immunofluorescence staining of the adult human liver derived from stem or progenitor cells of the invention (ADHLSC) as prepared in example 1 after 1 month culture for alpha-smooth muscle (Al) actin , vimentin (A2) and albumin (A3, polyclonal, A4, monoclonal), B: RT-PCR gene expression profiles of said (ADHLSC, lane 1) cell line, compared to human hepatocytes (hHep, lane 2), cells human stellates (HSC, lane 3) and human hepatoblastoma (HepG2, lane 4).

Figure 3 shows the differentiation of the adult human liver derived from the stem cell or progenitor of the invention (ADHLSC) in hepatocyte-like lineage in vitro. The cells were differentiated as in example 1, photographs were taken on day 2 (J2), day 14 (J14) and day 30 (J30) of the process.

Figure 4 shows various hematoxylin and eosin staining images of uPA / SCID chimeric liver transplanted mice with undifferentiated ADHLSC cells, as detailed in example 1, demonstrating that these cells become differentiated hepatocytes.

Figure 5 showing various images of human albumin staining revealed the human origin of the human hepatocyte-like population 10 weeks post-transplantation as in example 1 with undifferentiated ADHLSC.

DETAILED DESCRIPTION

As used herein, the singular forms of "a", "um" and "o" include both singular and plural referring to the context unless clearly stated otherwise. By way of example, "one cell" refers to one or more cells.

The terms "comprising", "comprising" and "composed of" as used herein are used synonyms for "including", "includes" or "containing", "contain", and are inclusive of, or are open to, and not excluding further , non-recited elements, elements, or method steps.

All references cited in this report are incorporated by reference in their entirety.

In particular, the teachings of all references specifically cited herein are incorporated by reference.

Obtaining cells of the invention

In one aspect, the present invention provides a method for obtaining an isolated stem or progenitor cell or a cell population comprising said stem or progenitor cell, the method comprising: (a) dissociating the adult liver or part thereof to form a primary cell population from said adult liver or part thereof, (b) plating the primary cell population onto a substrate that allows cells to adhere to it, and (c) cultivate primary cell population cells, which adhere to said substrate. substrate for at least 7 days, preferably for at least 10, at least 13 or at least 15 days.

As used herein, the term "isolated cell" generally refers to a cell that is not associated with one or more cells or one or more cellular components with which the cell is associated in vivo. For example, an isolated cell may have been removed from its native environment, or it may result from propagation, for example, ex vivo propagation, of a cell that has been removed from its native environment.

The term "in vitro" as used herein denotes external or external to the human or animal body. The term "in vitro" as used herein should be understood to include "ex vivo". The term "ex vivo" usually refers to tissues or cells taken from a human or animal body or propagated outside the body, for example in a culture medium. The term "cell population" generally refers to a cell cluster. Unless otherwise indicated, the term refers to a cell grouping consisting of or comprising isolated cells as defined herein.

The cell population may be composed of cells with a common phenotype or may comprise at least a fraction of cells with a common phenotype. Cells are said to have a common phenotype when they are substantially similar or identical in one or more demonstrable characteristics, including but not limited to the morphological appearance, presence, absence or level of expression of certain cellular products or components, for example RNA. , proteins or other substances, the activity of certain biochemical pathways, proliferation and / or kinetic capacity, potential differentiation and / or response to signals of differentiation or behavior during in vitro cultivation (eg adherence, non-adherence, growth monolayer, proliferation kinetics, or the like). Such demonstrable characteristics may therefore define a cell population or a fraction thereof.

When a cell population is said to be "heterogeneous" herein, this usually denotes a cell population comprising two or more cells or cell fractions that do not have a common phenotype, for example, a cell population comprising cells of two or more different ones. cell types. By way of example and without limitation, a heterogeneous cell population may be isolated from the liver, and may include various types of liver cells, including, but not limited to hepatocytes (eg, small and large hepatocytes), cholangiocytes, Kupffer Cells. , liver stellate cells (Ito Cell) and liver endothelial cells.

When a cell population is referred to here as being "homogeneous", it is made up of cells that share a common phenotype. The cell population mentioned herein as being "substantially homogeneous" comprises a substantial majority of cells having a common phenotype. The "substantially homogeneous" cell population may comprise at least 70%, for example at least 80%, preferably at least 90%, for example at least 95%, or at least 99% of cells which have a common phenotype, such as the specifically referred phenotype (for example, the stem cell or progenitor cell phenotype). As used herein, the term "substantially homogeneous" as used herein may therefore also encompass a homogeneous population.

The term "cell population comprises stem or progenitor cells" refers to a cell population as defined herein comprises at least one stem or progenitor cell and usually a fraction of stem cell or progenitor cell as defined herein.

Usually, stem cells or progenitors of said fraction may have a common phenotype.

The term "progenitor cells" generally refers to a competent and relatively less specialized or unskilled proliferation cell, which or their descendants may give rise to at least one relatively more specialized cell type. By way of example and not limitation, a progenitor cell may give rise to offspring that may differentiate into one or more strains to produce more relatively more specialized cells, where such relatively more specialized growing and / or progeny cells may themselves be progenitor cells themselves. or even to produce terminally differentiated cells, that is, fully specialized cells, which may be post-mitosis. The term also encompasses the stem cells defined herein.

One parent cell is said to "give way" to one, relatively more specialized cells, when, by way of example and not limitation, differentiated parent cells become other cells without undergoing the first cell division, or to another cell. It is produced after one or more cycles of cell division and / or differentiation of progenitor or progenitor cells.

The term "stem cells" refers to one of the self-renewing progenitor cells, that is, they can proliferate without differentiation, whereby the progeny of one or at least part of the stem cells substantially retains the unskilled or relatively less specialized phenotype, potential differentiation, and competence of stem cell proliferation. The term embraces stem cells substantially capable of unlimited self-renewal, that is, where the ability of the offspring or part of it for further proliferation is not substantially reduced compared to the stem cell, as well as self-exhibiting stem cells. - limited renewal, ie where the ability of the offspring or part thereof to further proliferation is demonstrably reduced compared to the parent cell.

One of ordinary skill in the art knows that the above properties generally refer to the in vivo behavior of stem and progenitor cells and, and may under appropriate conditions be wholly or in part at least reproduced in vitro and / or ex vivo.

Based on the ability to give rise to different cell types, a stem or progenitor cell can generally be described as totipotent, pluripotent, multipotent or unipotent. A single "totipotent" cell is defined as being able to grow, ie develop, in an entire organism. The "pluripotent" cell is not able to grow in an entire organism, but is capable of giving rise to all types of cells from three germ layers, ie mesoderm, endoderm and ectoderm, and may be able to give rise to all cell types in an organism. The "multipotent" cell is capable of giving rise to at least one cell type from each of two or more different organs or tissues of an organism, wherein said cell types may be of the same or different germ layers, but it is not. able to give rise to all cell types in an organism. The "unipotent" cell is able to differentiate cells from only one cell line.

The terms "differentiation", "differentiating" or derivatives thereof are used herein to denote the process by which an unskilled or relatively less specialized cell becomes relatively more specialized. In the context of the ontogeny cell, the adjective "differentiated" is a relative term. Thus, a "differentiated cell" is a cell that progressively decreases a certain developmental path to which the cell is being compared. A differentiated cell may, for example, be a terminal of differentiated cells, that is, a fully specialized cell that performs specialized functions in different tissues and organs of an organism, and which may, but need not be, post-mitosis. In another example, a differentiated cell may also be a progenitor cell within a differentiated lineage, which may further proliferate and / or differentiate. Similarly, a cell is "relatively more specialized" if it has held back the progress of a certain developmental path to which the cell is being compared, where the latter is therefore considered "unskilled" or "relatively less specialized". " A relatively more specialized cell may differ from a relatively less specialized or unskilled cell in one or more demonstrable phenotypic characteristics, such as, for example, the presence, absence or level of expression of certain cellular products or components, for example RNA, proteins. or other substances, the activity of certain biochemical pathways, morphological appearance, proliferation and / or kinetic capacity, potential differentiation and / or response to signals of differentiation, etc., where such characteristics signify the progression of the even more relatively specialized cell. along this path of development.

Non-limiting examples of differentiation may include, for example, a change of pluripotent stem cells in a particular multipotent stem or progenitor type, a change in a multipotent stem cell or progenitors in a particular stem cell type or unipotent progenitors, or the shift from a unipotent stem cell or progenitor to more specialized cell types or to terminally specialized cells within a given cell line. Differentiation from a less specialized or unskilled cell to a more specialized cell can progress through the appearance of cells with intermediate degree of specialization.

Liver tissue dissociation

As mentioned, the method of the invention comprises a step of dissociating the adult liver or part thereof to form a primary cell population from said adult liver or part thereof.

The term "liver" refers to the liver organ. The term "liver part" generally refers to any part of the liver, without any limitation as to the amount of said liver part or region where it originates. Preferably, all cell types present in the liver may also be represented in said part of the liver. The amount of liver part may at least in part follow practical considerations, for example the need to obtain sufficient primary liver cells to reasonably perform the method of the invention. These considerations will be apparent to a person versed in view of the present teachings. Thus, by way of example and not limitation, a portion of the liver may represent (typically w / w) at least 0.1% of the liver, or at least 1%, or at least 10% or at least 20%. , or at least 30% or at least 40% or at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or more of the liver. In other non-limiting examples, a portion of the liver may be at least 1g, or at least 10g, 100g or at least 200g, or at least 300g, or at least 400g, or at least 500g, or at least 500g. 600g or at least 700g or at least 800g or at least 900g or at least 1000g or at least 1100g or at least 1200g or at least 1300g or at least 1400g or more. For example, a part of the liver may be a hepatic lobe, for example, right or left lobe, or segment IV resected during the liver splitting operation.

The term "adult liver" as used herein refers to the liver that has reached substantial maturity in the development of tissue organization and cell composition.

In particular, it is known that a person skilled in the liver may undergo changes over a period of time immediately after birth if he or she later attains a substantially mature organization. For example, in humans, the liver at birth contains a considerable hematopoietic cell population, which disappear substantially from the liver within about 1-2 weeks after birth. In addition, the liver of humans at birth contains a hepatic progenitor cell population, which is substantially replaced by mature hepatocyte and biliary cells within several months of birth.

Thus, in humans, "adult liver" refers to the liver of a subject who at any time after birth, preferably full time, and may be, for example, at least one month old, for example at least 2 months, at least 3 months, for example at least 4 months, at least 5 months, for example at least 6 months after birth, such as for example 1 year or more, 5 years or more, at least 10 years or more, 15 years or more, 20 years or more, or 25 years or more after birth. Thus, an "adult liver" or mature liver can be found in humans that would otherwise be described in the conventional terms "newborn", "infant", "juvenile", "adolescent" or "adult".

A skilled person will appreciate that the liver can attain substantial developmental maturity at different postnatal time intervals in different animal species, and may appropriately construct the term "adult liver" with reference to each species.

The liver or part thereof is obtained from a "subject" or "donor", referring to a vertebrate animal, preferably a mammal, more preferably a human.

The term "mammal" includes any animals classified as such, including but not limited to humans, domestic and farm animals, wild animals, pets, companion animals and experimental animals, such as rats, mice, rabbits, dogs, cats, cows, horses, pigs and primates, for example monkeys and gorillas.

In a particularly preferred embodiment, the adult liver or part thereof is from a human. As detailed elsewhere in this report, stem or progenitor cells or cell line or descendant thereof derived according to the invention of human livers can be advantageously used, for example, in patient research and therapy, esp. human patients suffering from liver disease.

In another embodiment, the adult liver or part thereof may be from a non-human animal, preferably a non-human mammal. Stem cells or progenitors or cell lines, or descendants, derived according to the invention from livers of non-human animal or non-human mammal may be advantageously used, for example, in the research and therapy of liver diseases in members thereof, related or other non-human animals or non-human mammalian species, or even in the therapy of human patients suffering from liver disease (e.g. xenotransplantation, artificial bio-liver comprising non-human or mammalian animal devices non-human). By way of example and not limitation, especially non-human mammalian cells suitable for use in human therapies may originate from swine.

A living or dead donor as determined by criteria of the adopted art, such as, for example, "heart-lung" criteria (usually involving a cessation of irreversible circulatory and respiratory functions) or to "brain death" criteria (usually involving a irreversible cessation of all brain functions, including the brain stem). Harvesting may involve procedures known in the art, such as biopsy, resection or excision.

A skilled person will appreciate that at least some aspects of liver harvesting or part of it from donors may be subject to ethical and legal standards respectively. By way of example and not limitation, liver tissue procurement from a human may need to be compatible with the donor's life organism. Thus, only a part of the liver can normally be removed from a human donor, for example using biopsy or resection, thus an adequate level of liver physiological functions is maintained in the donor. On the other hand, harvesting the liver or part of it from a nonhuman animal may, but need not necessarily be compatible with the survival of nonhuman animals. For example, nonhuman animals can be slaughtered after tissue harvesting. These analogous considerations will be apparent to a skilled person and reflect legal and ethical standards and are not substantially related to the essence of the invention.

In one embodiment, the liver or part thereof may be obtained from a donor, esp. human donor, who has prolonged circulation, for example, heartbeat, and prolonged respiratory functions, for example, pulmonary respiration or artificial respiration. Subject to ethics and legal rules, donors may or may not be brain dead (for example, the removal of all or part of the liver, which would not be compatible with the greater survival of a human donor, may be allowed in dead human being). Harvesting of the liver or part of it from such donors is advantageously, since the tissue does not undergo substantial anoxia (lack of oxygenation), which usually results in ischemia (cessation of circulation).

In another embodiment, and as surprisingly performed by the present inventors, the liver or part thereof may be obtained from a donor, esp. Hunano donor who, at the time of tissue procurement, has ceased circulation, for example without heartbeat, and / or has ceased respiratory functions, for example, without pulmonary respiration and without artificial respiration. While the liver or part thereof from such donors may have suffered at least some degree of anoxia, the present inventors have understood that the viability of stem cells or progenitors according to the present invention can also be obtained from such tissues. . The liver or part thereof may be harvested within about 24h after the donor circulation has ceased (e.g., heartbeat), for example within about 20h, for example within about 16h, more preferably within about 12h, for example, within about 8h, even more preferably within about 6h, for example, within about 5h, within about 4h or within about 3h, even more preferably within about 8h. 2h, and more preferably within about 1h, such as, within about 45, 30 or 15 minutes after the donor circulation has stopped (e.g., heartbeat).

Tissues harvested as above may be cooled to room temperature or below room temperature, but generally freezing of the tissue or parts thereof is avoided, especially as such freezing could result in nucleation or appearance of ice crystals. For example, the fabrics may be maintained at any temperature from about 1 ° C to room temperature, from about 20 ° C to room temperature, from about 3 ° C to room temperature, or from about 4 ° C to at room temperature, and may advantageously be kept at about 4 ° C. Fabrics can also be kept "on ice" as known in the art. Tissue may be cooled for all or part of the ischemic time, ie the period after donor circulation has ceased. That is, the tissues may undergo hot ischemia, cold ischemia, or a combination of hot and cold ischemia. Tissues may be harvested and stored for example up to 48h before processing, preferably for less than 24h, for example less than 16h, more preferably for less than 12h, for example less than 10h, less than 6h, less than 3h, less than 2h or less than Ih.

The harvested tissues may be advantageously, but need not be kept in, for example, fully or at least partially submerged in a suitable medium and / or may be, but need not be perfused in the appropriate medium, even prior to tissue processing. A skilled person is able to select an appropriate medium which can assist in the survival of tissue cells during the period prior to processing.

The method of the invention comprises disassociating the liver adult tissue as described above to form a primary cell population.

The term "disassociation" as used herein generally refers to the partial or total disruption of the cellular organization of a tissue or organ, i.e. partially or totally disrupting the association between cells and the cellular components of a tissue or organ. As can be understood by a skilled person, the purpose of disassociating a tissue or organ is to obtain a suspension of cells (a cell population) from said tissue or organ. The suspension may comprise single or solitary cells, as well as cells physically attached to form clusters or clumps of two or more cells. Preferably disassociation does not cause or cause the smallest possible reduction in cell viability.

A suitable method for disassociating the liver or part thereof to obtain a primary cell population (suspension) thereof may be any method known in the art, including, but not limited to, enzymatic digestion, mechanical separation, filtration, centrifugation and combinations thereof. In one embodiment, the method of disassociating the liver or part thereof may comprise enzymatic digestion of the liver tissue to release liver cells. In one embodiment, the method of disassociating the liver or part thereof may comprise mechanical disruption or separation of liver tissue to release liver cells. In one embodiment, the method of disassociating the liver or part thereof may include a combination of enzymatic digestion and mechanical disruption or separation of liver tissue to release liver cells.

Methods for disassociating the liver or part thereof as above are documented in the art. For example, isolation of liver cells from liver tissue has been well known since the mid-1960s (Howard et al. 1967. J Biol 35: 675-84). Rat hepatocytes were isolated using the combination of the enzymatic and mechanical digestion technique, subsequently modified by Berry and Friend (J Biol Cell 43: 506-20, 1969). This technique was developed by Seglen to become the widely used two-stage collagenase technique. (Methods Biol Cell 13: 29-83, 1976). Thus, in one embodiment, the method of disassociating the liver or part thereof to obtain a primary cell population (suspension) thereof is or comprising the two-step collagenase technique. One skilled in the art is aware that, since the publication of said above cited technique, various modifications have been described and / or conceivable, and are included in the invention.

By way of illustration and not limitation, here is a brief description of the two-stage collagenase technique. Throughout the liver, a cannula can be placed in the largest blood vessel in the liver, and placed and secured by sutures. As for parts or segments of the liver, the cannula can be placed in the opening of the patient's blood vessel on the cutting surface, and secured by sutures. In this case, the small openings of blood vessels usually need to be closed to prevent perfusion solutions from escaping from the cutting surface. Liver tissue is perfused with a pre-heated 3700C cation-free divalent buffer solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol tetracetic acid (EGTA).

Buffer solutions may comprise saline solutions such as, for example, N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), Williams E. Medium, Hank1S Balanced Saline, or Earl1S Balanced Saline, and may also include salts. as NaCl and KCI, among others. This leads to a disturbance of the desmosomal structures that hold the cells together. The tissue is then perfused with the buffer solution containing bivalent cations (s), such as Ca 2+ and Mg 2+, and the matrix of degrading enzymes that act on digesting the tissue. The primary liver cells, esp. Hepatocytes are usually released by gentle mechanical disruption, for example scraping with a comb, shaking, pressing through filters, for example stainless steel filters, cotton gauze or nylon fabric, to mechanically complete the cell dissociation process. . Such filters may have sieves with thicknesses which allow hepatocytes to pass therethrough, by way of example and not limitation, about 0.1 millimeters or more, about 0.25 millimeters or more, about 0.50 millimeters. or more, about 1 millimeter or more, or about 2 millimeters, 3 millimeters, 4 millimeters or 5 millimeters. A succession of screen filters with progressively shrinking pores may be used to gradually dissociate the detached tissue or cells. The dissociated cells are rinsed with a buffer containing a protease inhibitor, serum and / or plasma for collagenase deactivation and other enzymes used in the perfusion process, separated by low-speed centrifugation, for example, between 10 and 500 g χg (advantageously, all living cells can be substantially coated, while dead cells and cell debris are substantially eliminated), and the pellets obtained are washed with ice-cold buffer solution to purify the cell suspension.

The number and quality of isolated liver cells may vary depending, for example, on the quality of the tissues used, the perfusion compositions of the buffer solutions, as well as the type and concentration of the enzyme. Frequently, the enzymes used include, but are not limited to, collagenase, pronase, trypsin, dispase, hyaluronidase, thermolysin and pancreatin, and combinations thereof. Collagenase is most often used, made from bacteria (for example, from Clostridium histolyticum), and can often be a mixture of poorly purified enzymes, which may have inconsistent enzymatic action. Some of the enzymes have protease activity, which can cause undesirable reactions that affect the quality and quantity of healthy / viable cells. It is understood by those skilled in the art that enzymes of sufficient purity and quality must be used to achieve viable liver cell populations.

Other methods of harvesting primary liver cells may exclude enzymatic digestion techniques. Mechanical disturbance has been widely used, although the yield of liver cells produced by this form tends to be lower than by collagenase digestion and is less consistent. However, recent methods involving sucrose-EDTA perfusion in combination with controlled vibration in an ambient cooling have been developed with reasonable success (Kravchenko et al. 2002. Biol Int 26: 1003-1006). The liver is performed at the infusion site using a sucrose solution containing EDTA (pH 7.4). After perfusion, the liver is removed from the body, placed on a plate and finely divided into a small volume of ice medium. Liver fragment cells are released by controlled mechanical vibration disaggregation (MVD) using a motor to homogenize. The resulting mixture produced by this method can then be filtered through coarse mesh to give a first suspension of liver cells. The cells may be suspended in the medium, and recovered by centrifugation. Thus, in one embodiment, liver disassociation or part thereof may be mechanical rupture.

One of ordinary skill in the art is aware that the two-step collagenase technique can be especially adapted for the release of at least hepatocytes from liver tissue. Cell suspensions obtained using said technique may include a considerable portion of hepatocytes, and may also include other liver cell types. As mentioned, the present inventors have realized that such cell suspensions are particularly a suitable feedstock for obtaining stem cells or progenitors of the invention.

In one embodiment, the method of disassociating the liver or part thereof may form a cell suspension (which can be easily optimized by a skilled person) with at least 10%, for example at least 20%, at least 30%, for example at least 40%, at least 50%, for example at least 60%; at least 70%, for example at least 80%, or at least 90%, or up to about 100% of single cells, ie single cells.

As mentioned, the disassociation of liver tissue thus provides a primary cell population from said adult liver or part thereof.

As used herein, the term "primary cell" includes cells present in a cell suspension obtained from a subject's tissue or organ, for example by disassociating them (i.e., a cell population prior to plating) , cells present in a transplanted tissue, both previous and first-plated cell types, and cells from the cell suspension from these first-plated cells. The term "secondary cell" refers to cells in culture in all subsequent steps. Accordingly, when first plated primary cells are passed, for example, lifted from a surface substrate and plated, then they are referred to herein as secondary cells, as are all cells in subsequent passages.

The primary cell population as defined and obtained herein upon disassociation of the liver or part thereof may normally be heterogeneous, that is, it may comprise cells belonging to more than one cell type which are comprised in the liver. Examples of liver cell types include, but are not limited to hepatocytes, cholangiocytes (bile duct cells), Kupffer cells, hepatic stellate cells (Ito cells), oval liver cells, and endothelial cells. The above terms have meanings and established in the art and are broadly understood herein to encompass any cell type classified as such. Cell types that make up the liver still encompass both parenchymal and nonparenchymal liver cells.

By further but not limiting illustration, "hepatocytes" encompasses epithelial, parenchymal liver cells including, but not limited to, hepatocytes of different sizes (e.g., "small", "medium" and "large" hepatocytes), ploidy (for example diploid, tetraploid, octaploid) or other characteristics. For example, some authors propose that "large" hepatocytes, as defined herein, are parenchymal cells responsible for liver physiological functions, while "small" hepatocytes provide a reservoir of progenitor cells committed to the development of hepatocytes (see (e.g., Mitaka et al., Biochem Biophys Res Commun 214: 310-7, 1995). In addition, by illustration and not limitation, "cholangiocytes" encompass bile duct epithelial cells. Also by way of illustration and not limitation, "oval cell" encompasses cell of distinct morphology (e.g., core shape) and expression marker cell, as is known in the art, which is intended to be progenitor cells capable of, under certain conditions, give rise to hepatocytes and bile duct cells (Lowes et al. KN. 2003. J Gastroenterol Hepatol 18: 4-12; Yi et al 1999. J from Hepatology 31: 497-507).

Thus, in one embodiment, a heterogeneous primary liver cell population may comprise at least two cells, for example at least three or at least four or more liver cell types, for example constituting cells belonging to all of the liver cells. or nearly all liver cell types, including but not limited to the liver cell types listed above. One of skill will appreciate that population heterogeneity may include liver cell types that have been previously described as such, either in vivo or in vitro, as well as liver cell types, which have not previously been described, classified, isolated and / or featured in the art.

A skilled person will also appreciate that the heterogeneous cell population may, but need not understand, various liver cell types in the same or substantially the same relative proportions as present in the liver or part thereof having been dissociated. For example, one of ordinary skill in the art knows that particular ways in which liver tissue disassociation can lead to more effective isolation of one or more cell types compared to one or more other cell types, whereby cell suspension can be obtained purposely or inadvertently enrich the old or more cell types. Furthermore, some disassociation methods may differently affect the survival and / or viability of different liver cell types. In addition, one skilled in the art is well aware of methods for enriching a cell population obtained by disassociating the liver or part thereof to one or more desired liver cell types. Such methods include, but are not limited to differential centrifugation, density dynamism gradient centrifugation, filtration, cell purification, affinity purification, protease digestion, or the like.

In one embodiment, the heterogeneity of the primary liver cell population may include cells that belong to all or substantially all cell types that make up the liver. The present inventors have performed a method for obtaining a previously undisclosed type of liver stem or progenitor cell. However, the inventors do not wish to bind by any hypothesis as to the origin of said new stem or progenitor cells.

By way of example and not limitation, said stem cell or parent, or a predecessor thereof, may have been present in the liver, for example, in the parenchyma or non-parenchyma of the messo. For example, such a predecessor may have had similar, similar or different phenotypes from stem or progenitor isolation (eg, cultivating according to the invention may have altered the phenotype of the predecessor). Alternatively, or in addition, isolated stem or progenitor cells may have arisen due to alteration, for example, differentiation or differentiation, of one or more liver cell types, for example, a liver cell type that has or has not been previously known. In view, the method of the present invention may preferably start from a cell population representative of all or substantially all liver cell types.

The inventors have realized that stem cells or progenitors of the invention may advantageously be obtained from a population cell formed by disassociation of the liver or part thereof, wherein said cell population comprises hepatocytes. Thus, a suitable method for disassociating the liver or part thereof according to the invention represents a cell population comprising hepatocytes. Without being bound by any theory, the inventors thought that the stem or progenitor cells of the invention, or a predecessor, is co-released from liver tissue by disassociating the liver so that it releases at least liver hepatocytes.

In one embodiment, the method of disassociating the liver or part thereof may form a cell population comprising a proportion of hepatocytes that is at least about 10% at least about 20% at least 30% at least 40%, preferably, at least about 50%, for example at least 60%, more preferably at least about 70%, for example at least about 80%, more preferably at least about 90% or more such as at least about 95% at least about 96% at least about 97% at least about 98% or at least about 99%. The inventors have understood that the method of disassociating the liver or part thereof which forms a cell population comprising a substantial proportion of hepatocytes, for example at least about 50% or more as above, provides convenient starting cell populations for obtaining stem cells or progenitor cells of the invention.

In a preferred embodiment, a cell population comprising the above percentages of hepatocytes may be obtained by disassociating the liver or part thereof, without including steps to further enrich the hepatocyte cell population and / or other cell types.

In another embodiment, a cell population comprising the above percentages of hepatocytes may be obtained by disassociating the liver or part thereof and one or more new steps for enriching the hepatocyte cell population and / or other cell types, esp. for hepatocytes.

However, one of ordinary skill will appreciate that stem cells or progenitors of the invention or predecessors may be released from the liver by de-coupling under conditions suitable for releasing hepatocytes thereof, may, but do not always need to, co-purify with hepatocytes, or one or more. more hepatocyte subgroups (e.g., "large" or "small" hepatocytes), or other cell types, in methods for enriching a hepatocyte cell population, subgroups thereof, or other cell types. It is within the ability of a skilled person to select methods for enriching hepatocytes or other cell types, esp. by hepatocytes, which maintain the stem cells or progenitors of the invention, or predecessors, resulting in the cell population.

In addition, a person skilled in the art will understand that the stem or progenitor cells of the invention, or a predecessor, may have certain properties (e.g., physical properties or expression marker surface) that may allow their enrichment in the cell population obtained from the invention. of the liver using a suitable separation technique. It may be within the ability of a skilled person to determine which fraction of a cell population separated by one or more criteria comprises, or is enriched for, the stem cells or progenitors of the invention, or a predecessor. This can be done, for example, by culturing the cells of the various fraction tests according to the method of the invention and checking the yield of the stem or parent cell fraction or fractions of the invention.

An "enriched cell population" refers to a cell population in which one or more cell types are present in larger proportions than could be found in vivo or in the enriched cell population.

Primary cell plating from liver tissue

The method of the invention comprises culturing the primary cell population obtained by dissociating liver tissue as explained. For this purpose, the main population of liver cells is plated on a substrate that allows cell adhesion.

The term "electroplating" is used herein to mean seeding or inoculation, and generally refers to the introduction of an in vitro cell population into an environment capable of promoting the survival and / or growth of the introduced cells. Typically, said environment may be made in a system that is properly delimited from the surrounding environment, such that an undesirable exchange of matter between the environment and said surroundings can be avoided (thus avoiding, for example, contamination environment or escape from the culture medium or cells thereof, whereas it may permit the continuous or intermittent exchange of other components of useful material between said environment components and surroundings (for example, an occasional exchange of part or all of the culture medium, continuous gas exchange, or cell harvesting after culture, etc.) Normally, suitable cell culture environments can be generated in culture media well known in the art, such as vials for cell culture, plates and well plates of various shapes.

In the present invention, cells (e.g., primary liver cells) are plated on a substrate that allows cell adhesion, that is, a surface that is generally not repugnant to cell adhesion or attachment. This can be accomplished, for example, by plating the cells in a culture system (e.g., a culture medium), which exhibits one or more substrate surfaces compatible with cell adhesion. When said one or more substrate surfaces cantate the cell suspension (for example, the suspension in a medium) introduced into the cell culture system, adhesion between cells and substrate surfaces may be ensured. Thus, the term "plating on a substrate that allows cells to adhere to it" refers to introducing cells into a system that has at least one substrate surfaces that is generally compatible with cell adherence such that cells can contact the substrate surface. General principles of maintaining adherent cell cultures are well known in the art.

In general, a cell adhesion substrate can be substantially any hydrophilic substrate. As known in the art, culture media, for example, culture flasks, well plates, dishes, or the like, can usually be made from a wide variety of polymeric materials including, but not limited to polyacrylate, polymethylcrylate, polycarbonates, polystyrenes. , polysulfones, polyhydroxyacids, polyanhydrides, polyorthoesters, polyphosphates, polyphosphates, polyesters, nylon or mixtures thereof, etc. Generally, culture media made of such materials are made of surface treated after molding to provide hydrophilic substrate surfaces, thereby increasing the likelihood of effective cell binding.

The treatment surface may take the form of a coating surface, or may involve the use of surface-directed energy intended to generate chemical groups on the polymeric surface. These chemical groups will have a general affinity for water or otherwise sufficient polarity to allow stable adsorption to another polar group. These functional groups allow hydrophilicity and or an increase in oxygen surface and are recognized properties for increasing cell growth on modified substrate surfaces. Such chemical groups may include groups such as amines, amides, carbonyls, carboxylates, esters, hydroxyls, sulfhydryl and the like. Examples of directed energies include corona lightning, radiofrequency (RF) vacuum plasma treatment, DC plasma treatment brightness discharge (e.g. US 6,617,152). The standard of current adherent cell growth practices may involve the use of defined chemical media with the addition of animals from species whether bovine, human or other animal serum. The added serum, to provide nutrients and / or growth promoters, may also promote cell adhesion with plastic coated treated surfaces with a better adherent cell matrix layer.

An alternative substrate surface compatible with cell adhesion may be glass, optionally the surface treated to introduce functional groups, as, for example, listed above, to increase their hydrophilicity.

Other adherent substrate surfaces may be generated by surface coating, for example polymeric surface coatings or polymeric surfaces treated as above. In a non-limiting example it may involve polycations such as, for example, polyomithine or polylysine.

In another example, the preferred coating, and hence the substrate comprising one or more extracellular matrix components, for example ECM proteins, fibrin, laminin, collagen, preferably type 1 collagen, glycosaminoglycans, for example heparin or heparan sulfate , fibronectin, gelatin, vitronectin, elastin, tenascin, agrecana, agrina, bone sialoprotein, protein matrix cartilage, fibrinogen, fibrin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycine, SPARC / osteonectin, versicanad, thrombospon, or thrombospon adhesion cells molecules including cadherin, connexins, selectins, alone or in various combinations.

Preferred examples may include fibrin, laminin or collagen. Other preferred examples may involve compositions comprising ECM components, such as, for example, Matrigel® Basement Membrane Matrix (BD Biosciences), which is the solubilized basement membrane preparation extracted from mouse EHS sarcoma, a protein-rich ECM tumor, with laminin as an important component, followed by type 4 collagen, proteoglycan heparan sulfate, and entactin. A preferred embodiment includes coating consisting of or comprising collagen, esp. collagen type 1.

As appreciated by those skilled in the art, cells can be counted to facilitate subsequent plating of cells to a desired density. As in the present invention, when cells are plated they may first adhere to the substrate surface present in the culture system (for example in a culture medium), the density may be expressed in number of cells per mm2 or plates per cm2 of said substrate surface. In the present invention, the density of primary disassociation is obtained from or part of liver cells and can be between 1x10 ^ 6 cells / mm2, for example between 1x10 ^ 1 and 1x10 ^ 5 cells / mm2 between 1x10 ^ 2 and 1x10 ^ 5 cells / mm2, for example, between 1x10 ^ 3 and 1x10 ^ 5 cells / mm2, between 5x10 ^ 3 and 1x10 ^ 4 and 1x10 ^ 1 and 1x10 ^ 3 cells / mm2, between 1x10 ^ 2 and 1x10 ^ 4 , between 5x10 ^ 3 and 5x10 ^ 4 cells / mm2, between 1x10 ^ 1 and 1x10 ^ 3, between 1x10 ^ 2 and 1x10 ^ 4, for example, about 1x10 ^ 1, 5x10 ^ 1m, 1x10 ^ 2, 5x10 ^ 2.1x10 ^ 3, 5x10 ^ 3, 6x10 ^ 3, 5x10 ^ 1,7.7x10 ^ 3, 8x10 ^ 3, 1x10 ^ 4, 2x10 ^ 4, 3x10 ^ 4, 5x10 ^ 4, or 1x10 ^ 5 cells / mm2.

Normally, after primary liver cell plating, the cell suspension is left in contact with the adherent surface to allow cell population cells to adhere to said substrate. In contact with the adherent substrate primary liver cells, the cells may be advantageously suspended in an environment comprising at least one medium, in the methods of the invention typically a liquid medium, which supports cell survival and / or growth. Medium may be added to the system before, along with or after the introduction of the cells thereof.

The medium may be fresh, i.e. not previously used for cell culture, or may comprise at least a portion that has been previously conditioned by cell culture, for example, the culture of cells being plated or predecessors of these, or culture of distant cell-related or unreported cells to be plated.

To facilitate said adhesion, in embodiments, the main cell suspension may be contacted with the adhering surface for at least about 0h, for example at least about 1h, preferably for at least about 2h, for example at least 2h. at least about 4h, more preferably at least about 8h, for example for at least about 12h, even more preferably for at least about 16h, for example at least about 20h, and more preferably for at least about 20 hours or more, for example for at least about 30, 32, 36, 40, 44 or 48h.

In other preferred embodiments, the main cell suspension may be contacted with the adherent surface between about 2h and about 48h, for example between about 12h and about 48h, preferably between about 12h and about 36h, for example. between about 16h and about 32h, even more preferably between about 20h and about 28h, and most preferably for about 24h.

Although the above times are preferred, shorter or longer times may also provide for the attachment of cells compatible with the present invention, and one skilled in the art may optimize these times.

Following cells of the primary liver cell population that are allowed to attach to the adherent substrate as described above, non-adherent matter is removed from the culture system. Non-adherent materials may include, but are not limited to cells that have not been attached to the adherent substrate (such as cells that are not prone to adhesion, or cells that do not attach within the allowed time), so they are non-viable or dead cells, cell debris, etc. adherent materials cannot normally be removed by discarding the culture system, with adherent cells remaining attached to the substrate and optionally washing the adherent cells and culture system with appropriate media at one time or repeatedly. or isotonic buffer (e.g., PBS). Accordingly, the primary hepatic cell population cells, which have adhered to the substrate surface, are selected for further culture.

The environment to which cells may attach comprises at least one medium, in the methods of the invention typically a liquid medium, which supports cell survival and / or growth. Support may be added to the system before, along with, or after the introduction of the cells thereof. The medium may be fresh, i.e. not previously used for cell culture, or may comprise at least a portion that has been previously conditioned by cell culture, for example, the culture of cells being plated or predecessors of these, or culture of distant cell-related or unreported cells to be plated.

The medium may be a suitable culture medium as described elsewhere in this specification.

Preferably, the medium composition may have the same characteristics, may be the same or substantially the same as the medium composition used in the steps subsequent to culturing the attached cells. Otherwise, the medium may be different. Advantageously, the medium may comprise serum or plasma, which may further facilitate cell adhesion.

Primary cell culture from liver tissue

The primary population cells, which adhere to said substrate, preferably in said environment, are further cultured for at least 7 days, for example for at least 8 days or for at least 9 days, preferably for at least 10 days. for example at least 11 or at least 12 days, at least 13 days or at least 14 days, more preferably for at least 15 days, for example at least 16 days, or at least 17 days, or even for at least 18 days, for example at least 19 days or at least 20 days or more. The term "culture" is common in the art and broadly refers to the maintenance and / or growth of cells and / or descendants thereof.

In the embodiment, primary cells may be cultured for at least about 10 days to about 40 days, preferably for at least about 15 days to about 35 days, for example for at least about 10 days. 15 days and 20 days, such as for at least about 15, 16, 17, 18, 19 or 20 days. Preferably, the primary cells may then be cultured for a period no longer than 45 days, or no later than 50 days, or no later than 60 days. As appreciated by those skilled in the art, prolongation of cell culture in a culture system may necessitate a regular exchange of culture medium to a new medium. A skilled person is able to assess the need to change media by controlling cell culture parameters such as, for example, pH, cell density or cell appearance. Typically, the medium may be modified at regular intervals, for example, every 1 to 10 days, preferably between 16 and 32 hours (e.g., about 24 hours) after plating, and then preferably every 2 to 10 hours. 6 days, or more preferably every 2 to 4 days, for example about every 2, 3 or 4 days. The volume of all media may be changed or alternatively only part of the medium may be changed such that a portion of the conditioned medium for culturing previous cells is maintained. In one embodiment, the entire volume of the medium is substantially exchanged for a new medium. In another preferred embodiment, the medium is not exchanged during prolonged cell cultivation.

The primary cell suspension and more adherent cells are cultured in the presence of a liquid culture medium. Typically, the medium will have a basal medium formulation as known in the art. Many basal medium formulations (available, for example, from the American Type Culture Collection, ATCC, or from Invitrogen, Carlsbad, California, USA) may be used for primary cell culture herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), modified alpha Minimum Essential Medium (alpha-MEM), Basal Essential Medium (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb Medium, F-12 Nutrient Mixture (HAM), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle Medium (EMEM), RPMI-1640, Medium 199, Waymouth1S MB 752/1 or Williams Medium E, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and are within the ability in the art to modify or modulate media concentrations and / or media supplements when necessary for cell cultivation. In a preferred embodiment a basal medium formulation other than Williams Medium E, which is a rich formulation reported to support in vitro culture of adult liver cells. Other embodiments may employ more basal medium formulations, such as those chosen from above.

Such basal medium formulations containing ingredients necessary for mammalian cell development are known per se. By way of illustration and not limitation, such ingredients may include inorganic salts (particularly containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn salts), physiological buffers (eg HEPES , bicarbonate), nucleotides, nucleosides and / or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (eg glutathione) and carbon sources (eg glucose, pyruvate, eg sodium pyruvate, acetate, e.g. sodium acetate), etc. It will further be apparent that many media are available as low glucose formulations, with or without sodium pyruvate.

For use in culture, the basal medium may be provided with one or more components. For example, additional supplements can be used to provide cells with the necessary trace elements and substances needed for growth and expansion optimization. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components may be included in a salt solution, such as, but not limited to, Hank's Balanced Salt Solution (HBSS), Earle's Salt Solution. Other antioxidant supplements may be added, for example β-mercaptoethanol. While many basal media already contain amino acids, some amino acids may be supplemented later, for example, L-glutamine, which is known to be less stable when in solution. The medium may further be provided with antibiotics and / or antimycotic compounds, such as, usually, mixtures of penicillin and streptomycin, and / or other compounds, exemplified, but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin , mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin.

Hormones may also be advantageously used in cell culture and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin. , thyroxine, L-thyronine, epithelial growth factor (EGF) and hepatocyte growth factor (HGF). Liver cells may also benefit from cultivation with triiodithyronine, α-tocopherol acetate, and glucagon.

Lipids and lipid carriers may also be used to complement cell culture media. Such lipids and carriers may include, but are not limited to cyclodextrin, cholesterol, albumin-conjugated linoleic acid, oleic acid and albumin-conjugated linoleic acid, unconjugated linoleic acid, albumin-conjugated linoleic oleic acid, conjugated and unconjugated oleic acid albumin, among others. Albumin can also be used in formulations free fatty acids.

Supplementation of the cell culture medium with mammalian plasma or serum is also contemplated. Plasma or serum often contains cellular factors and components that are necessary for viability and expansion. Proper use of serum is also contemplated.

The term "plasma" is as conventionally defined. Plasma is usually obtained from a whole blood sample, which is supplied or contacted with an anticoagulant such as heparin, citrate (eg sodium citrate or citrate dextrose acid), oxalate or EDTA, during or soon after. blood collection to prevent clotting. Therefore, the term plasma refers to a composition that is not part of a human or animal body.

The term "serum" is defined as conventionally. Serum can generally be obtained from a whole blood sample, allowing the first coagulation to take place from the sample and thereby separating it to form the blood sample clot and cellular components from the liquid components (serum) with a proper technique, usually by centrifugation. An inert catalyst, for example glass beads or dust, may facilitate coagulation. Advantageously, the serum may be prepared using art-known serum separator tube (SST) which contain the catalysts to facilitate coagulation and further include gel of a density designed to be positioned between the liquid component and the clots and cellular components after centrifugation, simplifying separation. Alternatively, serum may be obtained from plasma by removal of anticoagulant and fibrin. The term "serum", therefore, refers to a composition that is not part of a human or animal body.

Isolated plasmas or sera can be used directly in the methods of the present invention. They may also be stored properly for later use of the method of the present invention.

Typically, plasma or serum may be stored for short periods of time, for example, up to about 1-2 weeks, at a temperature above their respective plasma or serum freezing points, but below room temperature. Typically such a temperature will be about 15 ° C or below, preferably about 10 ° C or below, more preferably about 5 ° C or below, for example about 5 ° C, 4 ° C, 3 ° C, 2 ° C or about 1 ° C, most preferably about 5 ° C or about 4 ° C. Alternatively, the plasma or serum may be stored under their respective freezing points, ie freezing storage. As is customary in the art, storage under freezing temperatures of plasma or serum may be about -70 ° C or below, for example, about -75 ° C or below about -8 ° C or below. These temperatures may advantageously prevent thawing of the stored plasma or serum, thereby preserving their quality. Freezing storage may be used regardless of the length of time for which plasma or serum should be stored, but may be particularly suitable when longer storage is required, for example for more than a few days or weeks for a longer period. at 1 or 2 weeks.

Prior to storage or use, isolated plasmas or serum may be heat inactivated. Heat inactivation is used in the art primarily to remove the complement. Heat inactivation usually involves incubating the plasma or serum at 56 ° C for 30 to 60min, for example 30min, with stable mixing, after which the plasma or serum is allowed to gradually cool to room temperature. A knowledgeable person should be aware that any modifications and requirements of said process may occur.

Optionally, the plasma or serum may also be sterilized prior to storage or use. Usual sterilization means may involve, for example, filtration through one or more filters with pores smaller than 1 μπι, preferably less than 0.5 μπι, for example less than 0.45 μπι, 0.40 τημ, 0.35 μπι, 0.30 μιτι or 0.25 μπι, more preferably 0.2 μπι or less, for example 0.15 μπι or less, 0.10 μπι or less.

Serums or plasmas suitable for use in the media of the present invention may include human serum or plasma, or serum or plasma from non-human animals, preferably non-human mammals, such as, for example, non-human primates (e.g., lemurs, chimpanzees). , monkeys), bovine fetuses or adults, horse, pig, sheep, goat, dog, rabbit, mouse or rat serum or plasma, etc. In another embodiment, the invention provides for the use of any combination of the above plasmas and / or serum.

Thus, in one embodiment, serum or plasma may be obtained from an organism of the same species as the species from which primary liver cells are obtained. In a non-limiting example, human serum or plasma may be used for human primary liver cell culture.

In another preferred embodiment, the medium comprises bovine serum or plasma, preferably bovine fetus (calf) serum or plasma, more preferably bovine fetus (calf) serum (FCS or FBS).

In another embodiment, the medium used for human primary liver cell culture comprises bovine serum or plasma, preferably bovine fetus (calf) serum or plasma, more preferably bovine fetus (calf) serum (FCS or FBS).

In the embodiments, the medium comprises from about 0.5% to about 40% (v / v) serum or plasma or replacement serum, preferably from about 5% to 20% (v / v), for example. from about 5% to 15% (v / v), more preferably from about 8% (v / v) to about 12% (v / v), for example about 10% (v / v) serum or plasma or replacement serum, esp. is the preferred serum or plasma as defined above.

In a new preferred embodiment, the medium used for human primary liver cells comprises bovine serum or plasma, preferably bovine fetus (calf) serum or plasma, more preferably bovine fetus (calf) serum (FCS or FBS), in the value. from about 0.5% to about 40% (v / v), preferably from about 5% to 20% (v / v), for example from about 5% to 15% (v / v) more preferably from about 8% (v / v) to about 12% (v / v), for example about 10%.

In still other embodiments, a medium may include plasma or serum from more than one species. For example, the medium may include a mixture of serum or plasma from one species corresponding to primary liver cell culture, and from another species. For example, a means for culturing human liver cells may include a mixture of human serum or plasma, preferably human serum, plasma or bovine serum, preferably bovine serum.

Additionally, the medium may preferably comprise at least one exogenous (i.e., in addition to plasma or serum) added growth factor. One of ordinary skill would appreciate that ordinary components of the basal medium (prior to the addition of serum or plasma), for example particularly saline, buffers, inorganic salts, amino acids, carbon sources, vitamins, antioxidants, pH indicators and antibiotics, are not. are considered growth factors or differentiating factors in the art. On the other hand, serum or plasma is a complex composition that possibly comprises one or more of these growth factors or growth factors.

The term "growth factor" as used herein refers to a biologically active substance that influences the proliferation, growth, differentiation, survival, and / or migration of various cell types, and may give effect to functional, morphological, and behavioral changes. development in an organism by itself or when modulated by other substances. A growth factor in can typically act by binding, as a ligand, to a receptor (e.g., surface or intracellular receptor) present in the cells responding to the growth factor. A growth factor herein may be particularly a protein entity comprising one or more polypeptide chains.

By way of example and not limitation, the term "growth factor" encompasses the elements of the fibroblast growth factor family (FGF), bone morphogenic protein family (BMP), platelet derived growth factor family (PDGF) , transformed beta growth factor (TGF-beta) family, nerve growth factor (NGF) family, epidermal growth factor (EGF) family, familial insulin-related growth factor (IGF) family, hepatocyte growth factor (HGF), hematopoietic growth factors (HeGFs), platelet-derived endothelial cell growth factor (PD-ECGF), angiopoietin, familial vascular endothelial growth factor (VEGF), glucocorticoids, and the like.

In a preferred embodiment, the medium comprises a growth factor which is an element of the familiar epidermal growth factor (EGF). In a further embodiment, said EGF family member in any choice of the group comprising anfiregulin, betacellulin, EGF, epiregulin, HB-EGF (heparin-linked growth factor as EGF), NRG1 (neuregulin-1) isoform GGF2, NRG1 SMDF isoform, NRG1-alpha, beta-NRG1, TGF-alpha, tomoregulin-1 and TMEFF2. In a particularly preferred embodiment, the medium comprises EGF.

In an additional embodiment, the medium comprises a growth factor which is an insulin element related to the growth factor family (IGF). In a further embodiment, said family member IGF is selected from the group consisting of insulin IGF1A (growth factor as isulin 1A), IGF1B, IGF2, INSL3 (as insulin 3), INSL5, INSL6 and relaxin. In a particularly preferred embodiment, the medium comprises insulin.

In an additional embodiment, the medium comprises a growth factor which is a glucocorticoid. In an additional embodiment, said glucocorticoid is any one chosen from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone, prednisone, triamcinolone, hydrocortisone, fluocinolone, cortisone, betamethasone. In a particularly preferred embodiment, the medium comprises dexamethasone.

In additional preferred embodiment, the medium may comprise a combination of two or more factors exogenously added to the above defined growth factors or preferred growth factors. By way of example and not limitation, the medium may comprise EGF and insulin, or EGF and dexamethasone, or insulin and dexamethasone, or each EGF, insulin and dexamethasone; A medium comprising these exogenously added growth factors may preferably comprise serum or plasma as defined in the above embodiment.

A skilled person generally knows the concentrations at which growth factors can induce an effect, esp. in in vitro cultured cells, and can use these concentrations for the growth factors mentioned above. By way of example and not limitation, EGF may normally be used at concentrations of about 0.1 ng / ml to 1 µg / ml and preferably between 1 ng / ml and 100 ng / ml, for example at about 25 ng. / ml; insulin may normally be used in concentrations between about 0.1 pg / ml and 1 mg / ml and preferably between about 1 pg / ml and 100 pg / ml, for example at about 10 μg / ml; dexamethasone may normally be used at concentrations of from about 0.1 nM to 1 μΜ, preferably from about InM to 100nM, for example to about 10nM.

In a preferred embodiment, esp. where the method is used for human liver cells, the growth factor used in the present method may be a human growth factor. As used herein, the term "human growth factor" refers to a growth factor substantially identical to what occurs naturally in a human growth factor. For example, when the growth factor is a protein entity, the constituent peptide (s) or polypeptide (s) thereof may have identical primary amino acid sequence for a naturally occurring human growth factor. The use of human growth factors in the present method is preferred, as such factors are expected growth to obtain a desirable effect on cell function.

As described, the present inventors have realized that primary liver cell culture for the duration of time as defined above, and preferably using medium compositions as described above, a stem cell or parent of the invention arises and proliferates. at the same time differentiated liver cell types become less prevalent in prolonged primary liver cell cultivation. Without being limited to any hypothesis, differentiated liver cell types, for example, may fail proliferation, die and / or be retro-differentiated during prolonged cultivation. As detailed in the experimental section, stem or progenitor cells can be distinguished from other cell types present in primary cell culture by their morphology among others, which according to the inventors' knowledge may be termed mesenchymal or mesenchymal-like morphology. and may usually include a flattened form, broad cytoplasm, and ovoid nuclei with one or two nucleoli.

The inventors have also realized that the emergence, proliferation and enrichment of the primary culture for said stem or progenitor cells can be promoted by altering the culture so that it further favors the elimination of one or more differentiated liver cell types, esp. of hepatocytes that may prevail in an isolated primary liver cell culture. Under such conditions, the stem or progenitor cells of the invention may advantageously proliferate and become a prevalent cell type in primary liver cell culture. Differentiated liver cell types may be lost, for example, because one or more differentiated cell types are physically reduced or eliminated during culture; alternatively, the differentiated phenotype may be diminished because one or more cell types are differentiated during culture.

Any medium that favors the elimination of one or more differentiated liver cell types, esp. Hepatocytes, while supporting the proliferation of the stem or progenitor cells of the invention (as can be readily assessed, for example, by visual inspection of cell culture for the prevalence of different cell types) can be used. By way of illustration, as exemplified by the inventors, a change may be used from a basal medium comprising high glucose concentration, for example, a concentration operation between 3000mg / l and 6000mg / l, preferably between 4000mg / l and 5000mg / l, and typically about 4500mg / l. Another change may be the absence of exogenous addition (ie in addition to those present in serum or plasma) to growth factors. By way of example, at least exogenously added insulin, dexamethasone and / or EGF may be absent from the medium. Yet an additional alteration may be used from the basal medium other than Williams Medium E (which may be particularly suitable for long-term culture primary liver cell types, esp. Hepatocytes). By way of example and not limitation, basal means such as MEM, DMEM, alpha-MEM or EMEM may be advantageously used. It is understood that the medium may be altered in one, more than one or all of the above ways. It is further understood that the medium comprises serum or plasma or replacement serum as described above, including the preferred embodiments detailed above. In one embodiment, a medium which favors the elimination of one or more differentiated liver cell types and promoting the stem cell or parent of the invention as described above may be added at the beginning of primary liver cell culture. In another embodiment, the medium may be altered during prolonged cultivation of primary liver cells. By way of example, the medium may be modified to begin at about 1 day, at about 2 days, 3 days, for example at about 4 or 5 days, or starting at about 6 days, such as , for example, about 7 or 8 days, or starting at about 9 days, such as about 10 or 11 days, starting at about 12 days, for example, about 13 or 14 days, plus preferably starting at about 15 days, for example at about 16, 17, 18, 19 or 20 days following primary liver cell plating. In an exemplary embodiment, the medium may be modified between 16 and 32 hours after plating, for example, about 24 hours. In mode, after the medium is then changed, it may be changed / renewed, for example, every 2 to 6 days, preferably every 2 to 4 days, for example, every 3 or 4 days.

Thus, in a preferred exemplary embodiment, primary liver cells may be cultured following plating in a medium favorable to primary liver cell types, including differentiated liver cell types such as hepatocytes, and at the time above the medium may be modified. for the elimination of a favorable medium from one or more differentiated liver cell types, incl. hepatocytes, and promoting the stem cell or parent of the invention. For example, primary liver cells may be grown in a medium with one, more than one or all of the following characteristics: comprising a rich basal medium, such as Williams Medium E, comprising low glucose, for example, between 500 mg / 1 to 2999 mg / l, and preferably between 1000 mg / l and 2000 mg / l, comprising at least one exogenously added growth factor and preferably one or more of one or all of insulin, dexamethasone and EGF. Thereafter, at the times above, the medium may be modified to include one, more or all of the following: comprising basal medium other than William Medium E, for example, basal medium such as MEM, DMEM, alpha-MEM or EMEM, comprising high glucose, not comprising exogenously added growth factors or not comprising at least dexamethasone, insulin and / or EGF. It is understood that the above average will comprise serum or plasma or replacement serum as described above, including the preferred embodiments detailed above.

As described, the above primary liver cell culture leads to the appearance of a stem cell or progenitor proliferation of the invention in the culture. Said culture may advantageously be continued until the appearance of stem cells or progenitors of the invention has sufficiently proliferated. For example, said culture may continue until the cell population reaches a certain degree of confluence, for example at least 40%, preferably at least 50%, more preferably at least 60% and most preferably. at least 70%, for example at least 80% or at least 90% or more confluence. The term "confluence" as used herein refers to a density of cultured cells where cells contact each other covering substantially all surfaces available for growth (i.e. fully confluent).

Cell passage

Following culture above the primary liver cells and emergence of stem or progenitor cell proliferation of the invention, the cell population can then be obtained can be passed at least once.

The inventors have surprisingly realized that the stem or progenitor cells of the invention have emerged in primary cell culture substantially conserving their proliferation capacity after passage, thus allowing to further enrich the cell population of such cells. For reasons of simplicity, the passage performed at this stage of the method is referred to herein as the "first pass" (or passage 1) within the scope of the method of the invention. The cells may be passed at least once and preferably two or more times. Each pass after 1 pass is referred to herein with an increasing number than 1, e.g. pass 2, 3, 4, 5, etc.

When passed, cultured cells are detached and dissociated from the culture substrate and each other. Cell detachment and dissociation may be performed as commonly known as in the art, for example by enzymatic treatment with proteolytic enzymes (e.g., chosen from trypsin, collagenase, for example, type I, II, III or IV, dispase, pronase, papain, etc.), treatment with bivalent ion chelators (eg, EDTA or EGTA) or mechanical treatment (eg by repeatedly pipetting through a small pipette hole or pipette tip), or any combination of these treatments. .

Preferably, detachment and dissociation of cultured cells would yield a substantial portion of the cells as single cells. For example, 40% or more of the cells may be recovered as single cells, for example at least 50%, preferably at least 60%, for example at least 70%, more preferably at least 80%, for example. For example, at least 90% or at least 95% of cells can be recovered as single cells. In addition, the remaining cells may be present in the clumps or clusters cells, most of which may contain a relatively small number of cells, for example on average, between more than 1 and 10 cells, for example less than 8 cells, of preferably less than 6 cells, more preferably less than 4 cells, for example less than 3 or less than 2 cells.

Typically, a suitable cell detachment and dispersion method should preserve cell viability. Preferably, a cell suspension obtained after detachment and dispersion may comprise at least 60% of viable cells, for example at least 70%, more preferably at least 80%, and most preferably at least 90% and up to 100% viable cells. Normally, one skilled in the art will know how to choose conditions that guarantee a desired degree of cell detachment and dispersion while preserving cell viability.

Then, dissociated and detached cells (usually as an isotonic suspension in buffer or isotonic medium) are re-plated onto a substrate that allows the cells to adhere to it, and then grown in a medium as described above supporting new cell proliferation. stem or progenitors of the invention. Normally, cells can be re-plated to density between 1x101 and 1x106 cells / cm2, for example between 1x102 and 1x106 cells / cm2 and preferably between 1x103 and 1x105 cells / cm2, for example at about 1x103 cells / cm2, at about 5x103 cells / cm2, about 1x104, about 5x104, about 1x105 cells / cm2, and preferably about 1x103 and 1x104 cells / mm2.

Alternatively, the cells may be re-plated by separation, for example from about 1/8 to 1/2, preferably from about 1/4 to 1/2, and more preferably about 1/2. or about 1/3. The split ratio denotes the fraction of past cells that are sown in an empty (usually new) culture tube of the same surface area as the vessel from which the cells were obtained.

The adherent substrate where cells are replated is as described elsewhere in this report. The substrate may preferably be of the same type as the substrate to which primary liver cells have been plated, including preferred embodiments of the substrate described above, or may be different. Preferably, this substrate is also collagen, esp. type I collagen as described above.

The passed cells are then cultured, advantageously until the cells become at least 50% confluent, for example at least 60%, preferably at least 70%, for example at least 80%, more preferably at least 90%, for example at least 95% or even fully confluent.

The present inventors have realized that while the cell population obtained at this stage of the method comprises a substantial fraction of the stem or parent cell of the invention, it may advantageously be passed at least one more time (i.e. at least a second pass), essentially as described above for the first pass. This number increases the proportion of stem cells or progenitors of the invention in the cell population, according to morphological evaluation and / or labeling analysis, and even a substantially homogeneous population of stem cells or progenitors of the invention can be obtained.

Thus, according to the invention, following the plating and culture of the primary liver cells leading to the emergence and proliferation of stem or progenitor cells in said culture, the cultured cells are passed at least once (i.e., first pass). and preferably at least twice (i.e. first and second pass) and optionally more times (i.e. first, second and each subsequent pass). For example, cells may be passed at least once, at least 2 times, at least 3 times, at least 4 times or at least 5 times in the sequence of the primary liver cell plaques. In another embodiment, cells may be passed between 2 and 10 times, for example, between 2 and 8 times, or between 2 and 5 times, following the primary liver cell plaques. Additional passages (eg, cell detachment and dispersion, replanting, substrate, etc.) and cultivation (eg, media, media changes, resulting confluence, etc.) may be carried out under conditions substantially identical or analogous to those of the first. passage, as described above, including preferred embodiments, and may include modifications that would be obvious to a skilled person.

The method of the invention may therefore provide for a cell population comprising a considerable fraction of stem or progenitor cells as defined in the present disclosure, as well as the fraction of stem or progenitor cells may be increased by one or more passages of the cell. prolonged culture of primary liver cells. Typically, the population will be comprised of at least about 10%, for example at least about 20% of said stem or progenitor cells, but the inventors have found that normally larger proportions of said stem or progenitor cells will be obtained, for example at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. Furthermore, the method may further yield a homogeneous or substantially homogeneous population of said stem or progenitor cells. The fraction of stem or progenitor cells may be evaluated by any appropriate standard method, for example by flow cytometry.

Keeping the cells obtained

When a population comprising liver-derived stem or progenitor cells is obtained by the method of the invention, and possibly further enriched by said stem or progenitor cells, the cell population may be maintained and / or propagated under conditions that permit growth. and the duplication of said stem or progenitor cells without differentiation. Such conditions may be, for example, those used to obtain stem or progenitor cells. A knowledgeable person who can assess the presence or absence of cell differentiation can easily create more conditions. This may advantageously increase the number of stem or progenitor cells available for reuse.

The present inventors have realized that the first or any later passage could be cryopreserved for later use, which is generally known in the mammalian cell art.

The present inventors have realized that the stem cells or progenitors of the invention that arose in primary cell culture substantially retained their ability to proliferate after freezing and thawing. Said cells may be stored as a frozen frozen thawed cell suspension, as is generally done in the art, and replated under the same conditions as described elsewhere in this specification.

Invention stem cells or progenitor cells The present inventors have found that the cell population obtained after culture and preferably also by passing the primary liver cells as described above comprises the liver derived stem or progenitor cells co-expressing ( that is, they are positive a) at least one mesenchymal marker, esp. one, more than one, for example 2, 3 or 4, or all markers CD90, CD73, CD44, vimentin and α-actin smooth muscle (ASMA), with the albumin hepatocyte marker (ALB) and possibly with a or more hepatocyte or liver markers, preferably one or both of alpha-1-antitrypsin and MRP2 transporter.

In a particular additional embodiment, the liver-derived stem or progenitor cells of the invention may co-express (i.e., are positive for) at least one mesenchymal marker, esp. one, more than one, for example 2, 3 or 4, or all of the markers CD90, CD73, CD44, vimentin and α-actin smooth muscle (ASMA), with the albumin hepatocyte marker (ALB) and possibly with one or more hepatocyte markers, preferably one or both of alpha-1-antitrypsin and MRP2 transporter and with at least one CD29 or alpha-fetoprotein (AFP) marker.

Any such adult liver stem or progenitor cell may further express one, more than one or all of the following molecules indicative of hepatocyte-like functions or properties: G6P, CYPlBl, CYP3A4, HNF-4, TDO, TAT, GS, GGT, CK8, EAAT2. Said adult liver stem cells or progenitors may further be comprised of one, more than one or all of the following: negative for at least the CD45 and CD34 hematopoietic markers and possibly also for one or more other hematopoietic markers such as such as CD105 &HLA-DR; negative for epithelial marker cholangiolitis cytokeratin-19 (CK-19) and eventually for more epithelial markers; negative for at least the undifferentiated stem cell markers CD117 and 0ct-4, and possibly also for one or more than one embryonic stem cell marker; low level alpha-fetoprotein (AFP) expression. Preferably, said liver stem or progenitor cell may have mesenchymal-like morphologies, especially involving one, more than one or all growth in flattened monolayers, broad cytoplasm and / or ovoid nuclei with one or two nucleoli.

Identification of particular cell surface molecules by their CD ("common determinant") is common in the art. Other names or designations for the molecules are also used in the present application as set forth in the art. Other specifications of certain molecules can also be found in the examples.

When a cell is said to be positive for a special marker, this means that a skilled person will perceive the presence or evidence of a distinct signal, for example detectable antibodies or eventual detection by PCR reverse transcription, so that the marker be transported to the proper extent compared to the proper controls. Where the method permits quantitative marker evaluation, positive cells may on average generate a signal that is significantly different from the control, for example, but without limiting at least 1.5 times the signal generated by such control cells. for example at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times higher, or more.

Expression of specific cell markers can be detected using any suitable immunological technique known in the art, such as flow cytometry, immunocytochemistry or adsorption affinity, Western Blot analysis, ELISA, etc., or by any suitable technique for measuring the mRNA amount of marker, e.g. Northern blot, semi-quantitative or quantitative RT-PCR, etc.

A skilled person will appreciate the cell population obtained by the present method which includes the above stem or progenitor cells can be harvested (e.g. by acceptable detachment technique) and optionally further enriched by cells exhibiting specific characteristics by generally known methods. in art (thus, these cells can be isolated from said population). By way of illustration and not limitation, cells exhibiting one or more of the surface characteristics of the stem or parent cell molecules of the invention, for example one or more of the markers listed above, may be recognized by specific antibodies (labeled). or other recognizing agents against such markers and classifying cells that do not exhibit such surface molecules, for example, using activated fluorescence cell separation or using binding affinity, for example, columns, beads or panning. Any other forms of cell enrichment are also included in the invention.

In another aspect, the present inventors have thus made a novel isolated stem or parent cell (a vertebrate, preferably a mammal, more preferably a human cell), originating from adult livers, being co-expressed (i.e. positive para) at least one mesenchymal marker, esp. one, more than one, for example 2, 3 or 4, or all markers CD90, CD29, CD44, vimentin and α-actin smooth muscle (ASMA), with the albumin hepatocyte marker (ALB) and possibly with one or more other hepatocyte markers. Said adult liver stem or progenitor cells may further comprise one, more than one or all of the following: negative for at least the CD45, CD34 and CD117 hematopoietic markers and also optionally for one or more other hematopoietic markers; negative for cytokeratin-19 (CK-19); morphology similar to mesenchymal and particularly involving some or all growth in flattened monolayers, broad cytoplasm and ovoid nuclei with one or two nucleoli.

Thus, in one embodiment (1), isolated stem cells or progenitors derived from the adult liver co-express CD90, CD73, CD44, vimentin and smooth muscle α-actin (ASMA) with ALB. In an additional embodiment (2), the isolated liver-derived stem or progenitor cell co-expresses CD90, CD44, vimentin and smooth muscle α-actin (ASMA) with ALB and alpha-1 antitrypsin. In an additional embodiment (3), the liver-derived isolated stem or parent cell co-expresses CD90, CD73, CD44, vimentin and smooth muscle α-actin (ASMA) with ALB and MRP2. In another embodiment (4), the liver-derived isolated stem or progenitor cell co-expresses CD90, CD44, vimentin and smooth muscle α-actin (ASMA) with ALB alpha-1 antitrypsin and MRP2. In additional embodiments (5), the liver-derived isolated stem or parent cell of any of the above embodiments of (1) to (4) still express CD29. In additional embodiments (6), the liver-derived isolated stem or parent cell of any of the above embodiments of (1) to (4) still express alpha-fetoprotein. In additional embodiments (7), the liver-derived isolated stem or parent cell of any of the above embodiments of (1) to (4) still express CD29 and alpha-fetoprotein.

In additional embodiments (8), the liver-derived isolated stem or progenitor cell of any of the above (1) to (7) embodiments are negative for CD45 and CD34. In additional embodiments (9), the liver-derived isolated stem or progenitor cell of any of the above embodiments of (1) to (7) are negative for CD117 and Oct-4. In additional embodiments (10), the liver-derived isolated stem or progenitor cell of any of the above (1) to (7) embodiments are negative for CD45, CD34, CD17 and Oct-4.

In additional embodiments (11), the liver-derived isolated stem or progenitor cell of any of the above embodiments of (1) to (10) are CK19 negative. In additional embodiments (12), the liver-derived isolated stem or progenitor cell of any of the above (1) to (10) embodiments are CK7 negative. In additional embodiments (13), the liver-derived isolated stem or progenitor cell of any of the above embodiments of (1) to (10) are negative for CK19 and CK7.

In additional embodiments (14), the liver-derived isolated stem or progenitor cell of either of the above (1) to (13) shows mesenchymal-like morphology, preferably involving some or all growth in flattened monolayers, broad cytoplasm and ovoid nuclei with one or two nucleoli.

In additional embodiments (15), the liver-derived isolated stem or parent cell of any one of the above (1) to (14) additionally expresses one, more than one, or all of the following molecules indicative of functions or properties. similar to hepatocytes: G6P, CYP1B1, CYP3A4, HNF-4, TDO, TAT, GS, GGT, CK8, EAAT2.

In additional embodiments (16), the liver-derived isolated stem or progenitor cell of any of the above (1) to (15) additionally expresses one or more of the following molecules: CD49e, CD13 , CD54, major histocompatibility complex (MHC) class I (HLA-ABC).

In additional embodiments (17), the liver-derived isolated stem or parent cell of any one of the above (1) to (16) is negative for one, more than one or all of the following molecules: CD105, HLA -DRi CD133, CD49b, CD49f & CD140.

In additional embodiments (18), the liver-derived isolated stem or progenitor cell of any of the above embodiments (6) to (17) express low level alpha-fetoprotein (AFP) expression. Preferably, said low level corresponds essentially to the expression level measured in normal hepatocytes. Preferably, it is lower than the level of measured expression human liver tumorigenic modified cell line, for example, HepG2.

The isolated liver-derived stem or progenitor cells of the invention may preferably exhibit characteristics of stem cells, esp. may exhibit at least limited to (and possibly substantially unlimited) self-renewal, ie the ability to propagate without differentiation. By way of example and not limitation, the stem or progenitor cells of the invention may be propagated by at least 4 passages, for example at least 6 passages, at least 10 passages, or at least 20 passages, at least 50 passages or more. .

In another aspect, the invention provides isolated liver-derived stem or progenitor cells, their cell line and / or a cell population comprising these obtained by or directly obtained using the method which includes: (a) dissociating, preferably in two steps of the collagenase method, the adult liver or part thereof from a subject, preferably a vertebrate, mammal and more preferably a human being, to form a primary cell population from said adult liver or part thereof; (b) plating the primary cell population on a Williams I Medium E substrate coated type I collagen comprising fetal calf serum, preferably 10% (v / v), EGF, preferably 25ng / ml, insulin, preferably 10 µg / ml, and dexamethasone, preferably 1 μΜ; (c) allowing cells to adhere from the primary cell population to said substrate for 24 hours and subsequently exchanging the medium for a new medium having the composition with in (b); (d) culturing the cells in said medium (c) for two weeks (preferably 15 days); (e) exchanging the medium for DMEM comprising high glucose and FCS, preferably 10%, and further culturing the cells, whereby said stem or progenitor cells emerge and proliferate; (f) optionally and preferably allowing the cells to become about 70% confluent and passing the cells at least once and preferably at least twice where the cells are plated on the substrate as in (b) and grown in a medium as in (e).

Step (a) includes disassociating the primary cell by passing through a sieve having 0.25 mm pores and preferably through a succession of sieves having gradually decreasing pores of 0.25 mm, for example. , as in example 1.

The medium of step (e) may preferably not comprise dexamethasone, insulin and EGF and in one embodiment. The medium may an additional embodiment do not comprise any exogenously added growth factor.

In one aspect, the invention also relates to the above methods.

In another aspect, the invention provides stem cells or progenitors isolated from the liver, cell line from this and / or from a population using this type of cell, obtained or directly obtained following the protocol set forth in example 1.

In another aspect, the present inventors have established a human liver stem cell or progenitor cell population (cell line) using the methods of the invention, in particular as documented in Example 1, and deposited on February 20, 2006 said lineage. isolated cell phone under the Budapest Treaty with the Belgian Consortium of Coordinated Microorganism Collections (BCCM) under accession number LMBP 6452CB (given by the depositary International Authority; reference id given by depositor: ADHLSC). Accordingly, the present invention reports an isolated cell, cell line and cell population, deposited with the BCCM under accession number LMBP 6452CB (here, "LMBP 6452CB" cell line), underlining of which includes clonal underlining, and their descent, including their differentiated offspring, esp. hepatocytes or hepatocyte-like cells prepared therefrom, genetically modified or otherwise derived from them.

One of ordinary skill will appreciate that stem cells or progenitor cell lines, comprising these, obtained in accordance with the methods of the invention of the adult human liver may have biological properties, esp. the proliferation and differentiation capacity, cell morphology and / or expression marker, identical or analogous to the cell line deposited above, although they may be genetically different (due to genetic variation among normal humans). Thus, the human stem cell or parent or cell population, esp. Liver derivatives having the same or analogous biological properties as the deposited cell population is also encompassed within the scope of the present invention.

In another aspect, the invention provides a cell population comprising the adult liver stem or progenitor cells having the above characteristics and optionally further modified, e.g., genetically modified. In one embodiment, said cell population may include about 5% or more, for example about 10% or more of said stem or progenitor cells, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more of the stem or progenitor cells, ie substantially homogeneous or a homogeneous population of said stem cell or parent.

In another aspect, the invention provides a cell line established by the propagation of the liver-derived isolated stem or parent cell of the invention, optionally further modified, for example, genetically modified. Such propagation may fend off one stem cell or progenitor (clonal cell line) or more than one cell.

In yet another aspect, the invention provides isolated liver-derived stem or progenitor cells, cell lines thereof and / or a cell population comprising them, may be obtained or directly obtained using the method of the invention described above, including preferred embodiments. .

In one embodiment, the present invention thus provides a method for isolating a viable progenitor stem cell population similar to the normal (human) liver mesenchymal (Figure 1). Stem cells are able to proliferate in a culture medium, and express markers of various liver cell types such as, for example, albumin (hepatocytes), vimentin (star cells), smooth muscle alpha-actin (ASMA) (figure 2A). They have no biliary phenotype as demonstrated by negative immunostaining and RT-PCR Assays for cytokeratin 19 (Figure 2B). When tested using flow cytometry, ADHLSC were negative for CD45, CD34 and CD117 indicating their non-contamination by hematopoietic lineage. In contrast, ADHLSC were positive for CD90, CD29 and CD44, markers for mesenchymal lineage. In one embodiment, cells have the ability to retain their proliferation capacity after trypsin / EDTA treatment. Incubation a defined medium allows these cells to specifically differentiate into hepatocytes (Figure 3). As specificity criteria additional criteria were not able to trans-differentiate into osteocytes or adipocytes, as would be observed using multipotent human bone marrow mesenchymal cells. Its proliferation capacity as well as its specificity leads to increased efficacy and safety for liver cell transplantation. In addition, due to their adult origin, these stem cells avoid the cancerous, ethical and immunological issues associated with embryonic cells.

Differentiation of stem or progenitor cells of the invention

In addition to detecting cell markers, in vitro and / or in vivo cell differentiation studies can be informative.

The present inventors further realized that liver-derived stem or progenitor cells obtained using the above methods may possess specific differentiation capabilities. In particular, cells may be differentiated into hepatocytes or hepatocyte-like cells. In a still further embodiment, the cells do not differ from mesodermal (mesenchymal) cell types, such as, for example, osteocytes, chondrocytes, myocytes, connective tissue cells, stromal cells or adipocytes.

The liver derived stem cell or parent progenitor of the invention, cell lines, or cell populations included therein (specifically mentioned, although not obviously limited to, LMBP 6452CB line), or descendants thereof, may be advantageously induced to differentiate from hepatocyte lineage, especially hepatocytes or hepatocyte-like cells. This differentiation may occur in vivo or in vitro or ex vivo. Thus, the invention further provides a method for generating hepatocyte or hepatocyte-like cells from the isolated stem or progenitor cells of the invention, and the resulting hepatocyte or hepatocyte-like cells.

In one embodiment, the isolated liver-derived stem or progenitor cell of the invention is defined by its ability to differentiate into hepatocytes or hepatocyte-like cells and lack of differentiation for mesodermal cell types such as, for example, osteocytes and adipocytes.

As understood by those skilled in the art, the ability to differentiate from a specific cell type or the absence of such ability can be observed in vitro, ex vivo or in vivo. By way of example, in vitro or ex vivo differentiation may be assessed by exposing the cell to a medium to induce specific differentiation as generally known in the art. On the other hand, differentiation can be assessed in vivo by following the fate of an introduction (e.g., transplanted, injected, or other administered cell). A skilled person is able to recognize differentiation for certain cell types by assessing phenotypic criteria, including but not limited to, cell morphology and / or expression marker, and / or specific metabolic activities or other physiological pathways.

Differentiation into hepatocyte lineage cells may advantageously be performed in the presence of cytokines and growth factors, which may be liver specific. By way of illustration, hepatocyte growth factor (HGF), or scattered factor, is a known cytokine that promotes hepatocyte phenotype differentiation. Similarly, a non-limiting list of cytokines further includes epidermal growth factor (EGF), basic FGF, insulin, nicotinamide, oncostatin M, dexamethasone, HDAC inhibitors (eg sodium butyrate), DIVISO, vitamin A, or matrix components, such as heparin sulfate, which have also been implicated in differentiation for hepatocytes. Protocols for inducing hepatocyte differentiation are generally known in the art and can still be optimized by a skilled person.

Identification and subsequent isolation of differentiated cells from their undifferentiated homologues can be accomplished by methods well known in the art. For example, cells that have been induced to differentiation can be identified by selectively cultured cells under conditions where differentiated cells outnumber undifferentiated cells. Similarly, differentiated cells can be identified by morphological changes and characteristics that are not present in their undifferentiated counterparts, such as cell size, shape, or complexity of intracellular organelle distribution. Methods of identifying cells differentiated by their expression of specific protein marker, such as surface cell markers, are also contemplated. Detection and isolation of these cells can be obtained, for example, by flow cytometry, ELISA, and / or magnetic beads. Reverse-PCR transcription (RT-PCR) can also be used to monitor changes in gene expression in response to differentiation. In addition, genome-wide analysis using microarray technology can be used to identify differentiated cells.

Genetic modification of stem cells or

parents of the invention

Said adult liver stem or progenitor cell may be further modified, e.g. genetically modified, as described above. The new invention relates to progeny, including differentiated progeny, from the stem cell or progenitor of the adult liver.

In one embodiment, to increase the replicative capacity of stem or progenitor cells obtained from the invention, cells may be generally known as "telomerated" in the art. A cell is described as "telomerated" if it has been genetically engineered with a nucleic acid encoding encoding telomerase reverse transcription (TERT) of any species such that TERT is transcribed and translated into the cell. The term also applies to progeny of originally altered cells that inherited the ability to express the TERT coding region at a high level. The TERT coding sequence is typically taken or adapted from a TERT mammalian gene, exemplified by human and rat TERT, as indicated below. Cells can be telomerasated by genetically altering them with an appropriate vector so that they express the catalytic component of telomerase (TERC) at a high level. Especially suitable is the catalytic component of human telomerase (hTERT), provided in WO 1998/14592. For some applications, other TERT sequences may be used. Other methods of cell immortalization are also contemplated, such as genetically modifying cells with DNA encoding the Epstein-Barr virus infected T-antigen SV40 (US 5,869,243, WO 1997/32972) by introducing oncogenes such as myc and / or by introducing viral replication genes such as Ela adenovirus, and by fusion of having desired phenotype with an immortalized cell line. Transfection with oncogenes or oncovirus products is generally less suitable when cells are used for therapeutic purposes.

In general, it should be preferable that the present stem or progenitor cells are not modified by telomerisation or other forms of immortalization when the use of said cells or progeny, including differentiated progeny, is contemplated in therapy, for example whenever such cells are introduced into the human or animal body, esp. human.

In the present invention, the isolated stem cell or progenitor derived from the liver or offspring thereof may be stable or transiently transfected or transformed into a nucleic acid of interest prior to other use, for example in therapy or research, in essentially as known in the art. art. Nucleic acid sequences of interest may include, but are not limited to, for example, those encoded gene products that enhance the growth, differentiation, and / or function of cell types useful in therapy, for example, derived cell types. stem cells or progenitors of the invention, and in particular hepatocytes or hepatocyte-like cells, or to emit a therapeutic gene to a site of administration or implantation of such cells.

By way of example and not limitation, the stem or progenitor cells obtained may be modified constitutively or inductively to express a polypeptide normally expressed by liver cells, esp. hepatocytes, but are defective or absent in a patient, this defect underlies a pathological state of the patient. Administration of the modified cells can then restore protein production and thus aid in patient treatment. For example, stem or progenitor cells or progenitors thereof may contain heterologous DNA encoding a metabolic protein such as ornithine transcarbamylase, arginosuccinate synthesis, argininosuccinate lyase, arginasa, carbamyl phosphate synthesis, N-acetyl glutamate synthesis, glutamine synthesis , glycogen glucose-6-phosphatase synthesis, succinate dehydrogenase, glucoquinase, pyruvate kinase, acetyl CoA carboxylase, fatty acid synthesis, alanine aminotransferase, glutamate dehydrogenase, ferritin, low density lipoprotein receptor (LDL), and P450 enzymes, and / or alcohol dehydrogenase. Alternatively, cells may contain DNA encoding a secreted plasma protein such as albumin, transferrin, complement, C3 component, α2-macroglobulin, fibrinogen, Factor XIII, Factor IX, al-antitrypsin, or the like.

The liver is a center of production of many secretory proteins. And it is anatomically connected to the circulatory system in such a way that it allows an efficient release of various proteins into the bloodstream. Therefore, genes encoding proteins that have systemic effects may be inserted into liver cells of the present invention, as opposed to the specific cell types that normally produce them, especially if such genes are difficult to integrate into these cells. For example, a variety of specific homologous genes or antibody genes may be inserted into the liver cells of the present invention for secretion of their gene products into the circulation.

Conventional gene transfer methods are used to introduce nucleic acids into cells. The precise method used to introduce a gene is not critical to the invention. For example, physical methods for introducing DNA into cells include microinjection and electroporation. Chemical methods such as calcium phosphate co-precipitation and incorporation of DNA into liposomes are also standard methods of introducing DNA into mammalian cells. Viral transfection is also contemplated. DNA is introduced using standard vectors such as murine and influenza retrovirus derivatives (see, for example, Gluzman et al., Viral Vectors, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Standard recombinant DNA methods are well known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989), and viral vectors for gene therapy have been developed and used clinically. successfully (see, for example, Rosenberg, et al., N. Engl. J. Med, 323: 370 1990).

Uses of stem or progenitor cells of the invention The isolated stem or progenitor cell originating from the liver, cell lines thereof or populations comprising them may be used for different purposes, it is understood that the following use and applications are broadly contemplated for any cell liver stem or progenitor as defined above, particularly for cells obtained or directly obtained using the method defined above, cells exhibiting the characteristics defined above, as well as for the LMBP 6452CB cell line, a particular embodiment, a lineage isolated adult liver derived stem cell, preferably an adult human liver derived stem cell line (ADHLSC); descendants of these; or genetically modified derivatives thereof, including but not limited to:

- liver cell transplantation to treat metabolic liver abnormalities, degenerative liver disease or fulminant liver failure using the isolated stem or progenitor cells or population thereof according to the invention,

- preparation of bio-artificial liver devices using isolated stem or progenitor cells or population thereof according to the invention,

- preparation of animal models of human liver disease by transplantation of isolated stem or progenitor cells or population thereof according to the invention into animals,

- preparation of in vitro animal models and toxicology, pharmacology using the isolated stem or progenitor cells or population thereof according to the invention,

testing new drugs on the isolated stem or progenitor cells or population thereof according to the invention, including antiviral drugs for the human hepatitis virus.

For example, analysis of uPA + / + -SCID livers and SCID mice transplanted intra-splenically with ADHLSC (LMBP 6452CB strain) demonstrated that these cells were able to graft and differentiate from mature hepatocytes (Figures 4 and 5). In addition, human albumin was detected in the serum of these transplanted mice 10 weeks post transplantation although alpha-fetoprotein levels were not detected.

In accordance with the present invention liver stem or progenitor cells (specifically mentioning, though obviously not limited to the LMBP 6452CB strain) can be used for inborn error metabolism transplantation, for transplantation in animal models of liver failure. or for the transplantation of animal models of human viral hepatitis. Therefore, cells may be used in the treatment of associated liver diseases, including but not limited to liver failure, hepatitis, inborn errors of metabolism.

In an exemplary embodiment, as shown in example 1, they demonstrate that the inventors of the human liver stem or progenitor cells of the invention, when administered by intrasplenic injection to an immunodeficient mammal, more preferably a rodent, esp. a mouse or rat to retain its ability to proliferate and graft within the host liver. Thus, in one embodiment, a cell population comprising liver stem cells or progenitors of the invention, preferably of human origin (specifically mentioned, although obviously not limited to the LMBP6452CB strain), is introduced, for example, injected and allowed to be grafted. in a genetically or chemically immunodeficient mammal to obtain an animal model. Preferably, such a population may comprise at least 2 x 106 cells of the invention. One of ordinary skill may appreciate other ways of administering the cell population of the invention to induce the grafting of said adult liver-derived stem or progenitor cells.

As specified in Example 1, non-grafted cells do not proliferate, thus evidencing their advantages in cell transplantation in humans or other animals, preferably mammals.

The present invention also encompasses the use of isolated stem cells or progenitors or the population thereof according to the invention (specifically mentioning, although of course not limited to the LMBP 6452CB strain), for the following purposes:

- transplantation of liver stem or progenitor cells to treat liver based on innate metabolic deficiencies: Non-exhaustive examples of such diseases include phenylketonuria and other aminoacidopathies, haemophilia and other coagulation factor deficiencies, familial hypercholesterolaemia, other lipid metabolism disorders, urea cycle disorders, glycogenesis, galactosemia, fructosemia, tyrosinemia, carbohydrate and protein metabolism deficiencies, organic aciduria, mitochondrial diseases, lysosomal diseases, peroxisomes, protein synthesis abnormality, liver cell transporter defects, glycosylation defect and the like ,

- transplantation of liver stem or progenitor cells according to the invention to treat acquired progressive degenerative liver diseases,

- use of liver stem or progenitor cells according to the invention to treat fulminant liver failure and acute or chronic liver failure,

- use of liver stem or progenitor cells according to the invention in an artificial bio-liver and liver aids,

- animal models of human liver disease by transplantation of liver stem or progenitor cells according to the invention into small and large animals,

the preparation of animal models of human hepatotropic virus infections (HBV, HAV, HCV, HEV, HDV, ...) to study natural history, transmission, resistance, treatment effects, use of antiviral drugs or any research using transplanted liver stem or progenitors according to the invention,

the in vitro preparation of animal models of toxicology, pharmacology and pharmacogenetics using liver stem or progenitor cells according to the invention, testing new drugs on liver stem or progenitor cells according to the invention,

- gene therapy by inserting viral sequences into the stem or progenitor cells of the liver according to the invention, which may be expanded in vitro,

- animal models for the study of human liver cell metabolism,

- tolerance of allogeneic cells, thanks to the use of liver stem or progenitor cells according to the invention, and / or - use of liver stem or progenitor cells according to the invention to prevent, prevent or treat the liver or liver cell transplant rejection.

The present stem or progenitor cells or progenitors thereof, including differentiated progeny, may, in one aspect of the invention, be intended for therapeutic applications, such as for tissue engineering and cell therapy.

One skilled in the art will appreciate that detailed use may involve the use of stem or progenitor cells, cell lines thereof, cell populations comprising these, as well as the use of such progeny, including differentiated progeny, esp. hepatocytes or genetically modified hepatocyte-like cells or derivatives thereof.

As noted above, the liver-derived stem or progenitor cells of the invention, cell line thereof, or cell populations comprising them (specifically mentioned, although of course not limited to the LMBP 6452CB line), or descendants thereof, optionally genetically modified, can be used in cell replacement therapies. Cells may be administered to a tissue of interest in a subject to complete or replace functioning cells that have lost function. Alternatively, methods of providing differentiated cells, particularly hepatocytes or hepatocyte-like cells, are also contemplated, where stem or progenitor cells are differentiated, in the presence of growth factors, isolated, and administered to a subject.

The disease state or type of liver mass loss and / or function deficiency, and which could benefit from stem cells or progenitors of the invention include those listed above, and further include, but are not limited to Alagille, alcoholic liver disease (alcohol-induced cirrhosis), al-antitrypsin deficiency (all phenotypes), dyslipidemias and other disorders of lipid metabolism, autoimmune hepatitis, Budd-Chiari syndrome, biliary atresia, familial progressive intrahepatic cholestasis types I, II and III, liver cancer, Caroli disease, Acrigler-Najjar syndrome, fructosemia, galactosemia, deficient carbohydrates, glycosylation defects, other carbohydrate metabolism disorders, Refsum Disease and other peroxisomal diseases, Disease of Niemann Pick, Wolman's disease and other lysosomal disorders, tyrosinemia, Triple H, amino acids and other metabolic disorders, Dubin-Jo syndrome hnson, liver fat (nonalcoholic steatohepatitis), Gilbert's syndrome, Glycogen storage disease I and III, hemochromatosis, hepatitis AG, porphyria, primary biliary cirrhosis, sclerosing cholangitis, tyrosinemia, coagulation factor deficiencies, hemophilia B, phenylketonuria, Wilson's disease, fulminate liver failure, post hepatectomy liver failure, mitochondrial respiratory chain disease. In addition, cells can also be used to treat liver dysfunction due to acquired viral infections.

Thus, their cell line is provided in one aspect to the adult liver stem or progenitor cells of the invention, or cell populations comprising them (specifically mentioned, although of course not limited to the LMBP 6452CB line), or descendants thereof, including differentiated offspring, esp. hepatocytes or hepatocyte-like cells, optionally genetically modified as detailed above, for use in their therapy and / or use in the manufacture of a medicament for treating liver disease. Such diseases may include diseases affecting liver tissue, diseases affecting viability and / or function of hepatocytes are specifically contemplated, and may represent, for example, inborn errors, the effects of a disease condition, the effect of trauma, toxins, viral infections, etc. The liver diseases listed in this report are specifically addressed. Administration of cells according to the invention may lead to reconstitution or regeneration of the subject's tissue. The cells are administered in a manner that allows grafting or migration to the tissue site to reconstitute or regenerate the functionally deficient area.

Another aspect of the invention is a method for the prevention and / or treatment of a liver disease comprising administering the adult liver stem or progenitor cells of the invention, cell line thereof, or cell populations comprising them (specifically, although it mentions not exclusively lineage LMBP 6452CB), or descendants thereof, including differentiated progeny, esp. hepatocytes or hepatocyte-like cells, optionally genetically engineered for a subject, esp. in need of treatment. Such administration is typically in therapeutically effective amount, i.e. generally in amount providing a desired site or systemic effect and performance. In another aspect, the invention relates to a pharmaceutical composition comprising the adult liver stem or progenitor cells of the invention, cell line, or population comprising them (specifically, but not exclusively, mentioning LMBP 6452CB), or descendants thereof including differentiated offspring, esp. hepatocytes or hepatocyte-like cells, optionally genetically modified as above.

By way of example and not limitation, stem cells or progenitors isolated from the adult liver of the invention, cell line thereof, or cell populations comprising them (specifically mentioning, although obviously not limited to the LMBP 6452CB line), or descendants of the They may be advantageously administered by injection (also including catheter administration) or implantation, for example, localized injection, systemic injection, intrasplenic injection (see also Gupta et al., Seminars, in Liver Disease 12: 321, 1992). , an injection into a portal vein, injection into the liver pulp, for example below the liver capsule, parenteral administration, or intrauterine injection into an embryo or fetus.

In a preferred embodiment, the liver-derived stem or progenitor cells of the invention, cell line thereof, or cell populations comprising them (specifically mentioning, although obviously not limited to the LMBP 6452CB line), or descendants thereof, optionally genetically modified, may be used for tissue engineering and cell therapy through liver cell transplantation (TCF). Liver cell transplantation (LSCT), and liver stem cell transplantation (LSCT) refer to the infusion of mature hepatocytes or liver progenitor cells, including the cells of the invention, in any manner that leads to hepatic access and cell grafting, preferably through the portal vein, but also by direct hepatic injection, or by intrasplenic injection.

For example, the cells may be provided as a cell suspension in any preservation medium, preferably containing human albumin, after the isolation process or after thawing followed by cryopreservation.

In one embodiment, the present invention may use the patient's own liver tissue to isolate the stem or progenitor cells of the invention. Such cells would be autologous to the patient and could easily be administered to the patient. Furthermore, if the patient has a genetic defect underlying a particular disease state, such defect can be prevented by genetic manipulation of the obtained cells.

In another embodiment, the stem or progenitor cells of the invention may be isolated from non-patient tissue. When administration of such cells from a patient is contemplated, it may be preferable that liver tissue subjected to the method of the present invention to obtain stem or progenitor cells is selected to maximize, or at least achievable within limits, compatibility. of tissue between the patient and the administered cells thus reduces the likelihood of rejection of the cells delivered by the patient's immune system (e.g., graft versus host rejection).

The ability of the immune system to differentiate itself from the non-self largely determined by major histocompatibility complex (MHC) products, whose genes are on chromosome 6 and, belong to the immunoglobulin superfamily gene. MHC class I products consist of HLA-A, HLA-B and HLA-C; they are widely distributed and present on the surface of virtually all cell and platelet nuclei. MHC class II products consist of HLA-D, HLA-DR7 HLA-DP, and HLA-DQ, as they have a more limited distribution, including B cells, macrophages, dendritic cells, Langerhans cells, and activated T cells ( but does not rest).

The HLA loci are generally multiallelic, for example using specific antibodies, at least 26 HLA-A alleles, 59 HLA-B alleles, 10 HLA-C alleles, 26 HLA-D alleles, 22 HLA-DR alleles, nine HLA-alleles. DQ and six HLA-DP alleles can be recognized. Because HLA loci is closely linked, HLA antigens may also be present as conserved haplotypes.

A subject in need of cell therapy of the present invention may be screened for the presence of anti-HLA antibodies and their HLA genotype and / or phenotype (e.g., in lymphocytes; for example, using serological methods or DNA genetic analysis). Stem or progenitor cells obtained in accordance with the present invention, or the source or donor liver tissue thereof, can usually be tested for their HLA phenotypes and / or genotypes and tissues or cells selected for administration, which still have HLA haplotypes. patient-identical, or have the most common HLA antigen alleles and none or fewer HLA antigens of which the patient contains pre-existing anti-HLA antibodies. The likelihood that the transplanted cells will be successfully accepted increases with the number of identical HLA antigens. A skilled person will understand a greater variation of all these considerations.

Other ways of obtaining the patient-like MHC profile are also contemplated, for example, by genetic manipulation of stem cells or progenitors obtained from the invention or descendants thereof.

If cells are obtained from heterologous (i.e. non-autologous) sources, concomitant immunosuppressive therapy may normally be administered, for example, using immunosuppressive agents such as cyclosporine or tacrolimus (FK506). Alternatively, cells may be encapsulated by a membrane that allows fluid exchange but prevents cell / cell contact. Microencapsulated cell transplants are known in the art, for example, Balladur et al., 1995, Surgery 117: 189-194; and Dixit et al., 1992, Cell Transplantation 1: 275-279. Preferably, the cells may be autologous, or show close HLA correspondence as explained.

In another preferred embodiment, the adult liver or part thereof is from a non-human animal, preferably a non-human mammal individual. Stem cells or progenitors or cell lines thereof or descendants thereof derived from livers of non-human animals or non-human mammalian animal individuals according to the invention may be advantageously used, for example, in the research and therapy of liver disease. in members thereof, related to other non-human animals or non-human mammalian species, or even in the therapy of a patient suffering from liver disease (e.g., xenotransplantation, bio-artificial liver devices comprising non-human mammary cells or non-human animals). By way of example and not limitation, especially non-human mammalian cells suitable for use in human therapies may originate from swine.

One issue regarding the therapeutic use of the stem or progenitor cells of the invention is the amount of cells required to achieve an optimal effect. Doses of administration may be variable, and may include an initial administration followed by other administrations, and may be determined by one of ordinary skill in the art. Typically, administered doses or doses will provide a therapeutically effective amount of cells, i.e. achieving a desired site or systemic effect and performance.

In current human studies of mononuclear autologous bone marrow cells, empirical doses between 1 and 4 X107 cells have been used with encouraging results. However, several scenarios may require optimizing the amount of cells administered. Thus, the amount of cells to be administered will vary according to the subject being treated. In a preferred embodiment, from 102 to 109, or from 103 to 109, or from 104 to 109, such as from 104 to 108, or from 105 to 107, for example, about 1x105, about 5x105, about 1 X 106, about 5 X 106, about 1 X 107, about 2 X 107, or about 3 X 107, about 4 X 107, about 5 X 107, about 6 X 107, about 7 X 107, about 8 X 107, about 9 X 107, about 1 X 10,8 may be administered to a human. However, the exact definition of an effective therapeutic dose may be based on individual factors for each patient, including their size, age, size of the injured tissue, and elapsed time of injury, and can be readily ascertained by those skilled in the art from this disclosure and knowledge of the technology.

Preferably, the purity of a cell population comprising stem or progenitor cells for the administration of the invention may be about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%. More preferably the purity may be from about 70 to about 75%, from about 75 to about 80%, about 80% to about 85%, and more preferably the purity may be from about 85 to about 80%. 90%, about 90 to about 95%, and about 95 to about 100%. Stem cell purity can be determined, for example, according to the cell surface marker profile within a cell population. Dosage can be easily adjusted by those skilled in the art (for example, lower purity may require increased dose).

Those skilled in the art can readily determine the amount of cells and optional additives, vehicles and / or carrier in compositions to be administered by the methods of the invention. Typically, any additives (in addition to active stem or progenitor cells and / or cytokine (s)) may be present in an amount from 0.001 to 50% (w / w or w / v) in saline buffer. phosphate, and active ingredient may typically be present in the order of micrograms to milligrams, such as about 0.0001 to about 5% (w / w or w / v), preferably about 0.0001 to about 1%, more. preferably about 0.0001 to about 0.05% or about 0.001% to about 20%, preferably about 0.01 to about 10%, and most preferably about 0.05 to about 5%. %.

When administering a therapeutic composition of the present invention, it may generally be formulated for injection in a single dose (e.g., solution, suspension, dispersion, emulsion). Suitable pharmaceutical formulations for injection include sterile aqueous solutions and dispersions. As used herein, solutions or dispersions include a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable. The carrier may be a pharmaceutically acceptable solvent or dispersing medium containing, for example, water, saline, phosphate buffer, polyol (e.g. glycerol, propylene glycol, polyethylene glycol liquid, and the like) and suitable mixtures thereof. In addition, various additives that increase the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers may be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.

In many cases, it will be convenient to include isotonic agents to ensure cell viability, for example sugars, sodium chloride, and the like. The desired isotonicity of the compositions of the present invention may be obtained using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other soluble organic or inorganic materials. Sodium chloride is particularly preferred for buffers containing sodium ions.

To potentially increase the survival of progenitor cells by introducing the differentiated stem cells or progeny of interest to the subject in need thereof, one may incorporate said cells into a biopolymer or synthetic polymers. Examples of acceptable biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. This can be constructed with or without cytokines, growth factors, differentiation factors or nucleic acid expression constructs, etc. Such biopolymers could be, for example, in suspension or a three-dimensional cell-soaked gel. These polymers may preferably be biodegradable.

Prolonged absorption of the injectable pharmaceutical form may be accomplished by the use of absorption delaying agents, for example aluminum monostearate and gelatin. However, according to the present invention any carrier, diluent, or additive used must be compatible with stem or progenitor cells.

Sterile ejectable solutions may be prepared by incorporating the cells used in the practice of the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients as desired.

Such compositions may further be mixed with an acceptable carrier, solvent, excipient, such as purified water, saline, glucose, dextrose, or the like. The compositions may contain auxiliary substances such as emulsifying agents, humectants, buffering pH agents, viscosity-increasing or gelling additives, preservatives, flavoring agents, colorants, and the like, depending on the route of administration and the desired preparation.

Standard texts, such as "Remington1S pharmaceutical caiense", 17th edition, 1985, are incorporated herein by reference, and may be consulted for suitable preparations without undesirable experimentation.

The viscosity of the compositions, if desired, may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is economically and readily available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of thickener will depend on the agent selected. The point is to use an amount, which will get the selected viscosity. Viscous compositions are usually prepared from solutions by the addition of such thickening agents.

Other uses of stem or progenitor cells of the invention

Other uses of the stem or progenitor cells of the invention, their cell line, or cell populations comprising them (specifically, but not exclusively, mentioning the LMBP 6452CB line), or descendants thereof, including differentiated progeny, esp. hepatocytes or optionally genetically modified hepatocyte-like cells are contemplated below.

Thus, differentiated stem or progenitor cells or their derivatives, esp. hepatocytes or hepatocyte-like cells may be used to detect cellular responses (e.g., toxicity) to bioactive (biological or pharmacological) agents, comprising contacting a cell culture, or differentiated derivatives thereof, with one or more biological agents or identifying one or more cellular responses to one or more biological or pharmacological agents, and comparing the cellular responses of the cell cultures to the control responses of the cell cultures. These responses can be determined by monitoring the activities of molecules such as, but not limited to, alkaline phosphatase, cytochrome P450, urea pathway enzymes, among others.

In addition, cytokines, chemokines, pharmaceutical and growth factor compositions, for example, may be screened using the stem or progenitor cells of the invention or their differentiated derivatives, esp. hepatocytes or hepatocyte-like cells, more clearly to elucidate their effects on the differentiation and function of these cells. The invention also provides for an organ prepared by tissue engineering, or specific portions or portions thereof, of tissue building device comprising a tissue of interest and optionally cytokines, growth factors, or growth factors, where the cells of the invention are used to generate tissues, esp. liver tissue, esp. tissue comprising hepatocytes. A tissue engineered organ can be used with a biocompatible polymeric support to support cell growth in a three-dimensional configuration that can be biodegradable. Tissue engineered organs from stem cells of the present invention may be implanted into subjects who need a new organ, portions, or specific parts thereof. The present invention also provides for the use of stem cells or differentiated cells as well as part of a bioreactor, e.g. g., a liver auxiliary device.

Organs, portions, or parts of stem cells obtained from the invention may be implanted into a host. The transplantation may be autologous such that the stem cell donor from which the organ or organ units are derived from the recipient of tissue engineering. The transplantation may be heterologous such that the stem cell donor from which the organ or organ units are not derived from the recipient of tissue engineering. Once transferred to a host, the tissue engineered organ can recap the function and architecture of the native host tissue. The organ produced by tissue engineering will benefit individuals in a wide variety of applications, including the treatment of cancer and other diseases disclosed herein, birth defects, or damage due to surgical resection.

As a tool for the drug development and testing process, liver cells and their offspring may be used to evaluate changes in patterns of gene expression caused by drugs that are considered for development. Changes in the pattern of gene expression of potential drugs could be compared to those caused by drugs that affect the liver. This would allow a pharmaceutical company to separate compounds from their liver effect before the development process, saving time and money. The complete liver cell line, from progenitor cells to mature cells, could also be used to test the toxicity of drugs in the liver and to study how the drug is metabolized. Pharmaceutical laboratories currently have difficulty obtaining a steady supply of liver cells for toxicity assays. The methods and cells of the present invention meet this need.

Additionally, adult liver stem or progenitor cells or progenitors thereof, including differentiated progeny, esp. Hepatocytes or hepatocyte-like cells are useful as biological components of detoxification devices, such as liver perfusion or liver aids.

A conventional liver assist device includes a hollow semipermeable membrane fiber and as a rigid plastic outer shell that is seeded with differentiated stem cells or hepatocytes or from derived stem cells. The fibers can be treated with collagen, lectin, laminin, or fibronectin for cell attachment or release of untreated cells. Body fluids are perfused through the detoxification device according to well known procedures and then returned to the patient. An example of a suitable LAD for the cells of the present invention is described in international patent application serial number.

The adult liver stem cells or progenitors of the invention or descendants thereof may be differentiated in vitro and further used in place of mature hepatocytes (ADMET administration, distribution, metabolism, elimination and toxicology) or cytotoxicity tests.

Hepatocytes or hepatocyte-like cells obtained by differentiating the adult liver stem or progenitor cells of the invention or descendants thereof may provide an in vitro model for studies of liver development, liver cell metabolism, liver cell biology, and for differentiated screening, toxic or proliferative molecules. Additionally contemplating genetic manipulation of such cells, they may be used to study genes involved in liver development, metabolism or biology of liver cells.

The invention will now be illustrated by way of examples, which does not limit the scope of the invention in any way.

Example 1

Liver cell isolation process Human liver cells were obtained from an entire liver or liver segments from a healthy cadaveric donor or without a heartbeat. Cells were isolated after withdrawal time (eg, between 6 and 12 hours after withdrawal) while livers were preserved in ice-cold medium at a University of Wisconsin until perfusion. The isolated hepatocytes were using a classic two-step perfusion technique (Seglen, 1976) (Stephenne, 2005). Sequentially perfused liver tissue in apparent blood vessels with an EGTA solution (Balanced Earl Solution without Ca ++ and Mg ++, 0.5 mM EGTA, 5 mM Hepes, 2 mg / l gentamicin, and 100,000 IU / I penicillin G) and a digestive enzyme solution for 9 to 12 minutes each at 37 ° C. The digestive solution (EBSS with Ca ++ and Mg ++, 5 mM Hepes, 2 mg / l gentamicin, and 100,000 IU / I penicillin G) included 0.9 mg / ml collagenase and 0.03 mg / ml soybean trypsin inhibitors. . The liver capsule was incised and the hepatocytes released by gentle shaking.

Digestion was stopped in cold medium (M199 medium, 5 mM Hepes, 2 mg / 1 gentamicin and 100,000 IU / I penicillin G), containing 0.03 mg / ml soybean trypsin inhibitors and 100 ml / 1 human plasma. . The cells were filtered and washed through 4 metal sieves with pore sizes of 4.5, 1, 0.5 and 0.25 mm respectively. The cells were washed 3 times by centrifugation at 1200 rpm for 3 minutes in a cold M199 wash medium.

Primary cell culture

Single cell suspensions were resuspended in Williams1 E medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Perbio, Hyclone), 25 ng / ml EGF (Peprotech), 10 pg / ml insulin, 1 μΜ dexamethasone and 1% penicillin / streptomycin (P / S) (Invitrogen). Cells were plated in collagen I rat tail (BD Biosciences) - coated vials or plates (eg 6-well plates) (Greiner Bio-one) and cultured at 37 ° C in a fully humidified atmosphere containing 5% CO2 - After 24 hours, the medium was changed to eliminate non-adherent cells and then renewed every three days. For two weeks, the culture was observed daily under a microscope and the culture medium was analyzed every three days. The culture medium was then switched to high glucose DMEM (Invitrogen) supplemented with 10% FCS (Perbio, Hyclone) and 1% P / S (Invitrogen) to accelerate the elimination of adult hepatocytes. A cell type with mesenchymal morphology then arose spontaneously, proliferating and filling the empty space in the plate as confirmed by phase contrast microscopy. These cells appeared between days 15 and 20 of culture and had a flattened shape, broad cytoplasm and ovoid nuclei with one or two nucleoli (Figure 1). When it reached 70% confluence, the cells were suspended with 0.25% trypsin and 1 mM EDTA and replated to the desired concentration. Analysis of cell suspension using flow cytometry showed that the population became homogeneous after passage 2. For each passage, cell suspension was also analyzed using RT-PCR and immunofluorescence.

Cell characterization

To avoid possible contamination with other cell types, immunocytochemistry was performed to study the cell phenotype. Due to their hepatic origin, specific expression markers, such as albumin, were analyzed in paraformaldehyde fixed cells. As indicated in Figure 2, albumin, which is exclusively expressed in hepatocytes, was detected using both monoclonal (Sigma A-11 clone) and polyclonal (Chemicon) antibodies. At the same time, the expression of mesenchymal cell markers was also evaluated by demonstrating that these cells are immuno-positive for smooth muscle vimentin and alpha-actin (Figure 2). So far the phenotypic characteristics have been studied over 7 passages with great stability.

Cell Differentiation

Cells were seeded at a density of 0.5 - 1x104 / cm2 in 6 rat tail type I collagen coated well plates in DMEM supplemented with FCS and P / S. The culture medium was exchanged 24 hours later for Iscove modifided Dulbecco's medium (IMDM) (Invitrogen). By induction, cells were incubated for 2 weeks with induction medium containing IMDM supplemented with 20 ng / ml HGF (Biosource), 10 ng / ml bFGF (Peprotech) and 0.61 g / l nicotinamide (Sigma). Subsequently, cells were incubated with maturation medium containing IMDM supplemented with 20 ng / ml oncostatin M (Sigma), 1 μΜ dexamethasone (Sigma), 50 mg / ml ITS (insulin, transferrin, selenium) (Invitrogen). For the induction and maturation stages, the medium was changed and analyzed every 3 days. Upon exposure to these cocktails, the cells began to lose their sharp edges, were progressively shrunk, and lost their initial morphology to adopt a polygonal shape (Figure 3).

Flow cytometry:

The cells were collected after centrifugation at 1200 rpm for 5 min and resuspended at a concentration of 500 to 1000 / μ1 in PBS. The cells were then incubated for 30 minutes at 4 ° C with antibodies. Corresponding control isotopes were used for the non-specific binding evaluation of monoclonal antibodies. The cells were then washed and resuspended in Isoton ® (Beckham Coulter) for a Beckham Coulter flow cytometer reading.

RT-PCR

Total RNA was extracted from cells cultured in 6-well plates using TriPure (Roche) isolation reagent and cDNA which were generated using the reverse transcription kit according to the manufacturer's instructions. PCR amplifications were performed using elongase polymerase in a final volume of 25 μl and appropriate primers. The samples were subsequently electrophoresed with 1% agarose gel and nucleic acids were visualized with ethidium bromide staining.

Immunofluorescence

For immunostaining, cells grown in collagen I rat tail - 12 mm round glass coverslips were adjusted with 4% (v / v) paraformaldehyde for 15 minutes at room temperature and then permeabilized with 1% Triton X100 (v / v) in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) for 15 min. Nonspecific immunostaining was prevented by incubating a TBS solution containing 3% non-fat milk dry matter at 37 ° C by incubation. The cells were then incubated successively in the same solution for 1h at room temperature with primary antibodies, washed 5 times with TBS and for 1h with secondary antibodies (1/500). The nuclei were stained for 30 min with nuclear DAPI dye (1/5000). After 3 washes, the preparations were mounted on Fluoprep media (BioMerieux, Brussels, Belgium) and analyzed using an Olympus 1X70 inverted microscope attached to a CCD camera (T.I.L.L photonic, Martinsried, Germany). Light excitation (552, 488 and 372 nm by Br-3, FITC and DAPI, respectively) was obtained from a Xenon coupled to a monochrome lamp (T.I.L.L photonic, Martinsried, Germany). Digital images were acquired using appropriate filters and combined using the TILLvision computer program.

Molecules detected in stem or progenitor cells of the invention

Using the above approaches, the following expression profiles from a series of cell markers have been created in an experiment for the cells created in this example (ADHLSC):

CD90 positive. CD90, or Thy-I, is a cell surface protein considered indicative of the mesenchymal lineage.

CD44 positive. CD44 is a molecular adhesion cell and is used to identify at least some types of mesenchymal stem cells (MSCs).

Positive vimentin. Vimentin is an intermediate type III filament commonly detected in mesenchymal cells and fibroblasts.

Positive albumin. Albumin is a plasma protein produced and secreted by the liver. Within hepatocytes, albumin is usually found as a cytoplasmic protein.

CD2 9 positive. CD29, also known as beta-1 integrin, is a transmembrane glycoprotein, also present in liver tissue, formed with alpha integrin a receptor complexes involved in interaction with the extracellular matrix.

CD73 positive. CD73 is a 5'-nucleotidase ecto considered a mesenchymal marker.

CD49b positive. CD49b is also called alpha-2 integrin or collagen receptor and is implicated in interaction with extracellular matrix.

HLA-ABC positive. HLA-ABC (human leukocyte antigens A, B and C) are the complexes of

major histocompatibility of class I antigens that form heterodimers.

Alpha-fetoprotein - low expression levels. Alpha-fetoprotein is a protein expressed during the development of primitive endodermis and throughout maturation, reflecting the endodermal lineage.

High levels of alpha-fetoprotein expression usually reveals tumorigenic shunt.

Alpha-1-antitrypsin positive. Alpha-1 antitrypsin is a plasma protein synthesized by the liver.

Glucose 6-phosphate (G6P) positive. G6P is a liver enzyme that hydrolyzes glucose 6-phosphate to glucose and inorganic phosphate, allowing liver glucose to enter the blood.

Cytochrome P450 IBl (CYP1B1) positive. CYP1B1 is an inducible cytochrome dioxin responsible for phase I metabolism of a wide range of structurally diverse substrates.

Cytochrome P450 3A4 (CYP3A4) positive. CYP3A4 is a crucial enzyme involved in xenobiotic metabolism.

Positive hepatocyte nuclear factor 4 (HNF-4). HNF4 is a nuclear receptor, is a transcription factor involved in regulating energy metabolism.

Positive tryptophan 2,3-dioxigenase (TDO). TDO is the first enzyme involved in tryptophan oxidation in the liver.

Positive tyrosine aminotransferase (TAT). TAT is a specific hepatic mitochondrial enzyme involved in amino acid metabolism and gluconeogenesis.

Positive glutamine (GS) synthesis. GS is a key enzyme for ammonium assimilation.

Glutamyl transpeptidase (GGT) positive range. GGT is an enzyme involved in glutathione metabolism.

Positive cytokeratin 8 (CK8). CK8 is a specific intermediate filament of epithelial cells.

Positive multidrug resistant protein 2 (MRP2). MRP2 is an anion transporter organ responsible for exporting intracellular organic anions of hepatocytes to the biliary tree.

Positive carrier glutamate 2 (EAAT2). The presence of the various above molecules that may participate in liver function and metabolism is indicative of the close binding of the ADHLSC cell line to liver phenotypes.

CD117 negative. CD117, also called c-kit, is a receptor on the surface of bone marrow cell type cells that identifies HSC MSC and thus characterizes undifferentiated stem cells.

CD34 negative. CD34 is a cell surface protein in bone marrow cells indicative of HSC and endothelial progenitors.

CD45 negative. CD45, also called antigen leukocytes, is a tyrosine phosphatase expressed from hematopoietic lineage cells, including hematopoietic stem cells.

CD105 negative. CD105, also called SH2 or endoglin, is an adhesion molecule. It is also considered a marker of mesenchymal stem cells.

CD133 negative. CD133 is a hematopoietic stem cell marker.

HLA-DR negative. These casse II antigens of the major histocompatibility complex are heterodimeric membranes expressed restrictively on the antigens present in the cells.

Oct-4 negative. Oct-4 is a transcription factor expressed only by pluripotent stem cells and essential for maintaining the undifferentiated state.

Cytokeratin 19 (CK19) negative. CK19 is widely used as a marker for bile cells, namely cholangiocytes.

Cytochrome P 2B6 (CYP2B6) negative. CYP2B6 is involved in endobiotic and xenobiotic metabolism.

CD54: Intercellular Adhesion Molecule - 1 (ICAM-I), a Glycoprotein Membrane

Without wishing to limit it in any way, the present inventors, based on their knowledge of marker cells, proceeded with a possible interpretation of the above data: This marker combination defines an original cell line, which expresses the mesenchymal lineage markers (CD90, CD73). , vimentin, CD44), as well as markers of hepatic differentiation pathway characteristics (CD29 and albumin, alpha-1-antitrypsin, HNF4, MRP2 transporter). The presence of albumin detected by both immunofluorescence and RT-PCR strongly respond against possible stellate cell contamination. ADHLSC does not appear to be undifferentiated mesenchymal and pluripotent stem cells (CD45 negative, CD34 negative, CD117 negative, Oct-4 negative), nor liver stellate cells (albumin positive). The ADHLSC strain appears to be bent on the liver strain (CD29 positive, expressing albumin and alpha-1 antitrypsin), but not typically expressed biliary (CK19 and 7 negative). Thus, the cells and cell lines of the invention may, without limitation, be called mesenchymal stem cells with characteristics of a parent hepatocyte.

Transplantation in uPA-SCID mice, histology and immunohistochemistry

One million ADHLSC (> 90% viability) was injected into the spleen from 6 to 14 days of uPA + / + - SCID mice. Prior to transplantation, mice were undetectable to albumin. Immunohistochemistry in mouse liver samples was performed on four μτη-thick liver pieces stained with hematoxylin and eosin (HE) for global histopathological evaluation. For immunostaining, liver slides were incubated overnight with primary antibodies at room temperature. Detection was performed after incubating the slides labeled with polymeric peroxidase and chromogenic substrate (Envision-DAB system, Dako, Carpinteria, CA).

Staining was performed using hematoxylin.

The transgenic mouse model used for the purpose of combining liver disease (uPA) with immunodeficiency (SCID). Following intra-splenic transplantation of the ADHLSC suspension, uPA + / + - SCID mice were left for recovery for 10 weeks. Liver analysis of the ADHLSC-transplanted uPA / SCID mice showed that these cells were able to graft (Figure 4) and differentiate into mature hepatocytes (Figure 5). In addition, human albumin was detected in the serum of these transplanted mice 10 weeks after transplantation while alpha-fetoprotein level is expressed, a marker of tumor development, which remained undetected.

Transplanted cells did not proliferate as demonstrated by microscopic observation, indicating the absence of tumorigenic colonies and normal level of expression of alpha-fetoprotein and Ki67 tumorigenic markers. The normal level of expression corresponds essentially to the level of expression measured in normal and lower hepatocytes than the level of expression measured in the tumorigenically modified human liver cell line, for example, HepG2.

Example 2

An example of treatment with liver-derived stem or progenitor cells of the invention, cell line thereof, or cell populations comprising them (specifically mentions, although of course not limited to the LMBP 6452CB line), or their optionally genetically engineered progeny, may be be as follows.

The cells are infused into a series of injections, preferably not greater than 25 to 50 χ 106 cells / kg, preferably at 4 hour or 8 hour intervals, or longer than 8 hours for up to one week, or more than one week. The total amount of cells of 250 χ 106 cells / kg, or 500 χ 106 cells / kg is infused over a few days, preferably one or two weeks. A series of infusions may be repeated as needed every month, every six months, or every year or more.

Access to the portal vein by direct puncture under radiographic and ultrasound guides, through a needle puncture, through a percutaneous catheter, through a port-a-cath R device, or through a Broviac R device surgically inserted into any drainage tube into the portal vein, preferably the inferior mesenteric vein, or a colonic vein. The catheter may be left in place for several hours, preferably several days, preferably a few weeks or preferably for several months to two years or preferably for repeated repeat infusions as needed.

Immunosuppression is initiated on the day of infusion, preferably the day before, preferably with Tacrolimus (FK506) and steroids. Through tacrolimus blood levels it is preferably 8 ng / ml initially, 6 ng / ml after 3 months, 4 ng / ml after 6 months, and then maintained at around 4 ng / ml.

Steroids are preferably given as prednisone or prednisolone, initially 5 mg / kg on day 1, 4 mg / kg on day 2, 3 mg / kg on day 3, 2 mg / kg on day 4, 1 mg / kg on day 5, and then progressively decreased to reach 0.25 mg / kg at 3 months, and discontinue at 6 months. Alternative immunosuppression may include, alone or in combination, Cyclosporine A, anti IL2 receptor antibodies, anti-thymocyte globulin, or any human lymphocytes monoclonal or polyclonal antibodies.

human antilymphocytes, azathioprine or mycophenolate mofetil any antimetabolite agent, cyclosporine, rapamune or any other calcineurin inhibitor.

Claims (34)

1. Stem cell or progenitor originating from the human adult liver CHARACTERIZED by the fact that: (a) it expresses at least one mesenchymal marker chosen from the markers CD90, CD73, CD44, vimentin and smooth muscle α-actin (ASMA), ( b) it expresses the hepatocyte albumin marker (ALB), and (c) is negative for cytokeratin (CK-19). An isolated human stem or progenitor cell according to claim 1, characterized in that: (a) it expresses the markers CD90, CD73, CD44, vimentin and ASMA, (b) it expresses ALB, and (c ) is negative for cytokeratin (CK-19). An isolated human stem or progenitor cell according to any one of claims 1 or 2, characterized in that it further expresses one or more hepatocyte or hepatic markers chosen from CD29, alpha-fetoprotein (AFP), alpha-1-antitrypsin, HNF-4 and MRP2 transporters. An isolated human stem or progenitor cell according to claim 3, characterized in that it expresses CD29, AFP, alpha-1-antitrypsin and MRP2 transporter. A human liver stem or progenitor cell according to any one of claims 1 to 4, characterized in that it is CD90, CD29 and CD44 positive, and is positive albumin and smooth muscle alpha-actin positive. and be negative for CD45, CD34, CD117, and CK-19. An isolated human stem or progenitor cell according to any one of claims 1 to 5, characterized in that said cell has a mesenchymal-like morphology involving any or all forms of flat, monolayer growth, broad cytoplasm and ovoid nuclei with one or two nucleoli. An isolated human stem or progenitor cell according to any one of claims 1 to 6, characterized in that said cell may differentiate into hepatocytes or hepatocyte-like cells and preferably not differentiate from mesodermal type. A cell line or cell population characterized in that it comprises a stem cell or progenitor isolated from the human liver as defined in any one of claims 1 to 8 and their descendants. 9. Isolated human liver stem cell or progenitor CHARACTERIZED for being as filed on February 20, 2006 under the Budapest Treaty with the Belgian Consortium of Coordinated Collections of Microorganisms (BCCM) under accession number LMBP 6452CB , underlining thereof including clonal underlines, and their progeny. A method for obtaining an isolated human stem or progenitor cell or a cell population comprising said stem cell or progenitor, the method being characterized by: (a) dissociating the adult liver or part thereof to form a primary cell population of the said adult liver or part thereof, (b) plating the primary cell population onto a substrate that allows cells to adhere to it, (c) culturing primary population cells, which have adhered to said substrate for at least -7 days, preferably for at least 10, at least 13 or at least 15 days, and (d) pass the cells of step (c) at least once and preferably at least twice. A stem cell or progenitor isolated from the human adult liver, its adult cell line and / or a cell population comprising them, characterized in that they are obtained by or directly obtained using the method of claim 10. A method for obtaining stem cell or progenitor isolated from the human adult liver, cell line thereof and / or a cell population comprising them as defined in claim 11, the method being characterized by comprising: (a) dissociating, preferably into two steps of the collagenase method, the adult liver or part thereof from a subject, to form a primary cell population of said adult liver or part thereof; (b) plating the primary cell population on a Williams I Medium E substrate coated type I collagen comprising fetal calf serum, preferably 10% (v / v), EGF, preferably 25ng / ml, insulin, preferably 10 µg / ml, and dexamethasone, preferably 1 μΜ; (c) allowing cells to adhere from the primary cell population to said substrate for 24 hours and thereafter exchanging the medium for a new medium having the composition with in (b); (d) culturing the cells in said medium (c) for two weeks (preferably 15 days); (e) exchanging the medium for DMEM comprising high glucose and FCS, preferably 10%, and further culturing the cells, whereby said stem or progenitor cells emerge and proliferate; (f) allow the cells to become about 70% confluent and pass the cells at least once and preferably at least twice where the cells are plated on the substrate as in (b) and grown in a medium such as in (e). Composition, characterized in that it comprises human liver stem cell or progenitor as defined in any one of claims 1 to 7, 9, 11, 12, or cell line or cell population as defined in any one of claims 8, 9. , 11, 12. A human liver stem or progenitor cell according to any one of claims 1 to 7, 9, 11, 12, or cell line or cell population as defined in any one of claims 8, 9, 11, 12, or descendants thereof, characterized in that they include differentiated progeny, preferably hepatocytes or hepatocyte-like cells, for use in therapy. Use of human liver stem cells or progenitors as defined in any one of claims 1 to 7, 9, 11, 12, or cell line or cell population as defined in any one of claims 8, 9, 11, 12 or descendants thereof, characterized by including differentiated progeny, preferably hepatocytes or hepatocyte-like cells, for the manufacture of a medicament for the treatment of liver disease. Use according to claim 15, characterized in that liver disease includes phenylketonuria and other aminoacidopathies, haemophilia and other coagulation factor deficiencies, familial hypercholesterolaemia, other disorders of lipid metabolism, urea cycle disorders, glycogenesis, galactosemia, fructosemia, tyrosinemia, carbohydrate and protein metabolism deficiencies, organic aciduria, mitochondrial diseases, lysosomal and peroxisomal diseases, protein synthesis abnormality, liver cell carrier defects, glycosylation defect, hepatitis, cirrhosis, inborn errors of metabolism , acute liver failure, acute liver infection, acute chemical toxicity, chronic liver failure, cholangitis, biliary cirrhosis, Alagille syndrome, alpha-l-antitrypsin deficiency, autoimmune hepatitis, biliary atresia, liver cancer, cystic liver disease, fat in the liver galactosemia c gallstones, Gilbert's syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, and other liver viral infections, porphyria, primary sclerosing cholangitis, Reye's syndrome, sarcoidosis, tyrosinemia, glycogenosis type 1, or Wilson's disease. Pharmaceutical composition, characterized in that it comprises the human liver stem cell or progenitor as defined in claims 1 to 7, 9, 11, 12 or cell line or cell population as defined in claims 8, 9, 11, 12 and a pharmaceutically acceptable carrier. An in vitro toxicity testing method. The method comprising: exposing to a test agent the liver stem or progenitor cell as defined in any one of claims 1 to 7, 9, -11, 12 or cell line. or cell population as defined in any one of claims 8, 9, 11, 12 and observing at least one effect, if any, of the test agent on the liver cell population. A method according to claim 18, characterized in that at least one effect includes an effect on cell viability, cell function, or both. An in vitro drug metabolism study method, characterized in that it comprises: (i) exposing the human liver stem or progenitor cell as defined in any of claims 1 to 7, -9, 11, 12 or cell line or cell population as defined in any one of claims 8, 9, 11, 12 to a test agent, and (ii) observe at least one change, if any, involving the test agent after a test period. predetermined. Method according to claim 20, characterized in that at least one change includes a change in the structure, concentration, or both of the test agent. Liver assisting device, characterized in that it comprises a container for harvesting human liver stem or progenitor cells as defined in any one of claims 1 to 7, 9, 11, 12 or cell line or cell population as defined in any one of claims 8, 9, 11, 12. Use of the human liver stem cell or progenitor as defined in any one of claims -1 to 7, 9, 11, 12 or cell line or cell population as defined in any one of claims 8, 9, -11, Characterized by the fact that a functional copy of a gene is introduced for the production of a medicament for treating gene expression errors, which medicament is introduced into the liver of a patient who is in need of a functional copy of the gene. Composition for treating errors of gene expression, characterized in that it comprises transformed human liver stem or progenitor cell as defined in any one of claims 1 to 7, 9, -11, 12, or transformed cell line or cell population, as defined in any one of claims 8, 9, 11, 12 wherein a functional copy of a gene has been introduced. Pharmaceutical composition for treating gene expression errors, characterized in that it comprises transformed human liver stem or progenitor as defined in any one of claims 1 to -7, 9, 11, 12, or transformed cell line or cell population. as defined in any one of claims 8, 9, 11, 12 wherein a functional copy of a gene has been introduced and a pharmaceutically acceptable carrier. Use of a human liver stem cell or progenitor as defined in any one of claims 1 to 7, 9, 11, 12 or cell line or cell population as defined in any one of claims 8, 9, -11, 12 CHARACTERIZED by the fact that it produces a medicine to improve the regeneration of a diseased or damaged liver. Method for performing a test with agents effective to treat liver infections, characterized in that it comprises (i) infecting with an infectious agent of interest the liver stem or progenitor cell as defined in any one of claims 7, 9, 11, 12 or cell line or cell population as defined in any one of claims 8, 9, 11, 12 to provide an infected population, (ii) exposing the infected population to a certain amount of test agent, and (iii) observe the effects, if any, of exposure on the infected population. A method according to claim 27, characterized in that the infectious agent includes a microorganism. A method according to claim 27, characterized in that the infectious agent includes one or more viruses, bacteria, fungi, or combinations thereof. A method according to claim 27, characterized in that the observed effects include the effects on viral replication of a viral infectious agent. A method according to claim 27, characterized in that the viral infectious agent includes a hepatitis virus. A method of producing a protein of interest, characterized in that it comprises introducing into the liver stem or progenitor cell as defined in any one of claims 1 to 7, 9, 11, 12 or the cell line or cell population as defined in any one of claims 8, 9, 11, 12 a functional gene encoding a protein of interest, incubating the liver cell population under conditions effective for transcription, translation and optionally leading to post-translational modification, and collecting the protein of interest. A method according to claim 32, characterized in that the liver cells are human liver cells. A method according to claim 33, characterized in that the protein of interest comprises an antigen vaccine.