EP1824966A1

EP1824966A1 - Human stem cell lines derived from es cells and uses for production of vaccines and recombinant proteins

Info

Publication number: EP1824966A1
Application number: EP05816226A
Authority: EP
Inventors: Fabienne Guehenneux
Original assignee: Vivalis SA
Current assignee: Vivalis SA
Priority date: 2004-12-08
Filing date: 2005-12-08
Publication date: 2007-08-29
Also published as: US20080274125A1; FR2878860A1; WO2006061416A1

Abstract

The invention concerns the field of biology and virology. In particular, the invention concerns a method for obtaining human cell lines, in particular human stem cells derived from human embryonic stem cells, comprising separation from the serum, the feeder layer and at least one growth factor. Said lines are capable of proliferating indefinitely in a basic culture medium. The invention also concerns the use of the cells derived from such lines for virus replication, and more particularly for producing human or veterinary vaccines, as well as for producing recombinant proteins of therapeutic interest.

Description

Human stem cell lines derived from ES cells and uses for the production of vaccines and recombinant proteins

The present invention relates to the field of biology and virology. In particular, the invention relates to a method for obtaining human cell lines, in particular human stem cells derived from human embryonic cells, comprising a weaning of the serum, the feeder mat and at least one growth factor. These lines are able to proliferate indefinitely in a basic culture medium. The invention also relates to the use of cells derived from such lines for the replication of viruses, and more particularly for the production of human or veterinary vaccines, as well as for the production of recombinant proteins of therapeutic interest.

Historically, the vaccine industry has used, and still uses today, embryonated eggs to produce many vaccines such as the human flu vaccine. However, this system of production based on embryonated eggs has many limitations such as: (i) a variable biological quality of the eggs, because of the presence of accidental agents (viruses, toxins, ...) or problems infertility, (ii) lack of consistency in egg supply throughout the year, (iii) lack of flexibility in egg production in the event of a sudden increase in demand (ie in the case of an epidemic or sudden pandemic), (iv) a significant financial cost. A solution provided by the pharmaceutical industry to production problems in eggs has been to produce vaccines on cell culture. Indeed, viruses and viral vectors can be replicated and cultured in a large number of diploid primary cells, such as monkey kidney cells, calf kidney cells, hamster kidney cells and fibroblasts. chicken embryos. For example, the replication and propagation of certain viruses such as poxviruses are carried out in primary cell cultures of chicken embryo fibroblasts (CEFs for "chicken embryo fibroblasts"). However, these primary cells suffer from numerous disadvantages, such as contamination by accidental and / or pathogenic agents (bacteria, mycoplasma, yeasts, etc.), a variable quality. cells in culture, different sensitivities to variants of the same virus, low virus titers, high costs of virus production, the need to re-establish primary cells for each vaccine preparation, the need to use will be of animal origin to complement the culture medium, with the inherent risks of contamination by mycoplasmas, viruses or agents of bovine spongiform encephalitis (BSE) and finally the difficulties in obtaining and preparing such cells in culture.

This is why it has been proposed the use of immortalized continuous cell lines for the replication of viruses or viral vectors. Thus, continuous lines of animal origin, for example the MDCK cell line derived from the Madin-Darby dog kidney (Tobita et al., 1975, Med Microbiol, Immunol 162: 9-14), the VERO derived cell line African green monkey kidney (US 5,824,536), BHK21 cell line derived from baby hamster kidney were established. Human lines such as PER.C6 (WO 01/38362) have also been developed for vaccine production. However, the continuous cell lines currently available do not give complete satisfaction. Thus, some cell lines by their species specificity do not replicate, or poorly, some animal viruses. Also, certain cell lines do not make it possible to reach an economically profitable viral productivity. Moreover, the industrial exploitation of certain lines is sometimes difficult because it is necessary to have cells capable of growing in an asteric medium and in suspension. In addition, some of these continuous lines may not meet regulatory requirements because they have an unstable and abnormal karyotype, and / or are genetically transformed and / or are tumorigenic "in vivo". These regulatory considerations are particularly important when considering the use of a cell line from which vaccines will be isolated. Finally, it is also worth mentioning the existence of strong industrial protection on the most efficient continuous cell lines. For these reasons, pharmaceutical and veterinary companies in the vaccine field are looking for new continuous cell lines that do not have these disadvantages.

The problems encountered with the continuous cell lines in the field of the vaccine are also found in the field of the production of proteins recombinants in continuous cell lines. In addition to regulatory aspects related to the safety and stability of continuous lines, the major limitation encountered is the productivity of the cell. It is indeed necessary to have a cell capable of growing in suspension indefinitely in a medium without serum. Taken as a whole, the analysis of the prior art reveals an unfulfilled and long-awaited need to develop a cell capable of ensuring the replication of viruses and viral vectors, but also of production of recombinant proteins, in a system which do not have the disadvantages of existing production systems such as embryonated chicken eggs, diploid primary cells or continuous cell lines currently available.

This is the problem that the present invention proposes to solve by proposing a method for obtaining human stem cell lines suitable for use by the pharmaceutical industry. Indeed, various documents of the prior art describe the culturing and maintenance in culture of human embryonic stem cells primary. Thomson et al. (1995, Proc Natl Acad Sci USA 92: 7844, US 5,843,780) were the first to successfully cultivate primate stem cells. They are subsequently derived from human embryonic stem cell lines from blastocysts (1998, Science, 282: 114). Gearhart et al. derived human embryonic germ cell (hEG) cell lines from gonadal fetal tissues (WO 98/43679). However, to date, the human primary embryonic stem cells described are not adapted to their industrial use given the need to cultivate them in complete medium adhesion in the presence of growth factors, animal serum and feeder cells. The present invention intends to solve this problem by providing a method for obtaining human stem cell lines, said method comprising the following steps: a) culturing human stem cells in a complete culture medium containing all the factors enabling them to grow. growth, a layer of feeder cells, preferably inactivated by irradiation, and supplemented with serum; b) successive passages by modifying the culture medium so as to obtain a gradual or total weaning in feeder cells, and / or growth factors, and / or in serum. Preferably, the weaning sequence of the culture medium is: weaning in growth factors, then weaning in feeder cells, then weaning in serum; c) establishment of adherent and non-adherent human cell lines capable of proliferation in a basic culture medium in the absence of feeder cells, and optionally in the absence of exogenous growth factors and / or containing a low serum concentration, or even not containing of serum in the culture medium.

The method according to the invention further comprises the preliminary step of obtaining a population of human stem cells. For the purposes of the present invention, the term "stem cell" is intended to denote an undifferentiated cell derived from the embryo, the fetus or the adult. The stem cell is characterized by its capacity for self-renewal (that is to say, identical propagation to produce new stem cells), differentiation under certain culture conditions (in order to generate specialized cells) and proliferation in culture. According to a first embodiment, the human stem cell according to the invention is a totipotent stem cell derived from the first divisions of the fertilized egg until the fourth day of development. These totipotent stem cells potentially have the ability to regenerate a complete individual. According to a second embodiment of the invention, the human stem cell according to the invention is a pluripotent stem cell, also called ES embryonic stem cell. ES cells are present up to a hundred cells in the internal mass of the embryo at the blastocyst stage (5 ^ ⁶ to 7 ^th day after fertilization). ES cells are able to participate in the formation of all tissues of the body (more than 200 cell types). According to a third embodiment, the human stem cell according to the invention is a multipotent stem cell. These cells that are present in fetal or adult tissues have self-renewal capabilities and can give rise to several types of cells. These cells are involved in a specific tissue program, such as hematopoietic stem cells from bone marrow and cord blood. According to a fourth embodiment, the human stem cell according to the invention is a unipotent stem cell. These cells generate only one type of differentiated cells while keeping certain self-renewal and proliferation capacities (examples: hepatocytes of the liver, keratinocytes of the skin, etc.). According to one fifth embodiment, the human stem cell according to the invention is an intermediate progenitor cell. These cells have little or no capacity for renewal and divide only into differentiated cells. According to a preferred embodiment of the invention, the stem cell according to the invention is a human embryonic stem cell (ES) isolated or propagated from a human blastocyst.

Preferably, the human stem cell according to the invention has not been transformed chemically or with the aid of a biological agent (virus, nucleic acid,

...).

According to a preferred embodiment, the present invention relates to a process for obtaining continuous lines of human non-transformed, undifferentiated, and capable of proliferating indefinitely in cultured stem cells, characterized in that said method comprises the following steps: a) culture on a primary human stem cell feeder cell mat in a complete culture medium comprising at least:

animal serum;

an exogenous growth factor selected from: FGF (Fibroblast Growth Factor), stem cell factor (SCF) and IGF1 (insulin-like growth factor 1);

an exogenous growth factor selected from the ligands at the receptor which can form a heterodimer with the glycoprotein gp130. Among the ligands, Leukemia Inhibitory factor (LIF), interleukin 11 (ILI 1), interleukin 6 (IL6), interleukin-6 receptor (IL6R), ciliary neurotrophic factor (CNTF) )

Foncostatin, cardiotrophin; b) successive passages in an identical or different culture medium, so as to obtain a weaning of the feeder cell mat, a total or partial weaning of said exogenous growth factors and a total or partial weaning of the serum; c) optionally, selecting cell colonies having compact morphologies and composed of cells having a high nucleo-cytoplasmic ratio and a prominent nucleolus; d) establishing said continuous lines of human stem cells derived from embryonic cells capable of growing in the absence of feeder cells.

Preferably, said lines obtained in step d) are capable of growing in a culture medium completely depleted in growth factors. Even more preferably, said lines obtained in step d) are capable of growing in culture medium completely depleted in growth factors and completely depleted in serum.

The primary human stem cells are selected from totipotent stem cells, pluripotent stem cells, multipotent stem cells, unipotent stem cells, intermediate progenitor cells. Preferably, said primary human stem cell is a pluripotent stem cell, that is to say an embryonic stem cell (ES).

The method according to the invention may furthermore comprise a substep a) of step a) of dissociating the cell clusters formed in culture, characterized in that the dissociation is carried out enzymatically and / or mechanically. When carried out enzymatically, trypsin, pronase or collagenase is preferably used. The mechanical dissociation is carried out with a scraper or with the aid of a cutting object making it possible to split the compact clusters of cells into small cell groups facilitating the amplification of the cultures after transplanting or to individualize the cells composing the cell clusters.

The terms "growth factor" or "growth factor", used interchangeably in the present invention, mean a chemical or biological substance (usually a peptide or protein) necessary for survival and control. growth of human cells in culture. It is possible to distinguish schematically two families of growth factors: cytokines and trophic factors. The cytokines are essentially proteins whose action is via the receptor which is associated with the GPI 30 protein. Thus, the Leukemia Inhibitory Factor (LIF), Interleukin 11 (HH), Interleukin 6 (11-6), Interleukin 6 receptor (H-6R), Ciliary Neurotrophic Factor (CNTF), oncostatin and cardiotrophin have a similar mode of action with recruitment to the specific chain receptor and combination of the latter with GP 130 protein in the form monomeric or sometimes heterodimeric. Trophic factors are primarily SCF, IGF-1 and FGF.

By "complete culture medium" is meant a base medium supplemented with vitamins, nutrients, minerals, growth factors, serum, various compounds to ensure optimized growth of cells in culture. According to the present invention, a "base medium" means a medium whose formulation makes it possible to ensure the survival of cells in culture, and a minimum growth. Examples of basic media (or basic media) are, for example, BME (Basal Eagle Medium), MEM (Minimum Eagle's Medium), medium 199, DMEM (Dulbeco's Modified Eagle Medium), GMEM (Galsgow Modified Eagle's Medium), DMEM -HamF12, Ham-F12 and Ham-FlO, Iscove's Modified Dulbecco's Medium, MacCoy's 5A Medium, RPMI 1640. The base medium comprises inorganic salts (for example: CaCl ₂ , KCl, NaCl, NaHCO ₃ , NaH ₂ PO ₄ , MgSO ₄ , ...), amino acids, vitamins (thiamine, riboflavin, folic acid, calcium panthothenate, etc.) and other components such as glucose. It may be necessary to supplement the basal medium with at least one of the following compounds: animal serum, L-glutamine, sodium pyruvate, beta-mercaptoethanol, amino acids, vitamins, growth factors to generate a complete environment.

According to a preferred embodiment of the invention, said complete medium in step a) comprises serum, and at least FGF and LIF. According to another embodiment said complete medium in step a) comprises serum, FGF, LIF and at least one compound selected from IL-6, IL6R, IL1, CNTF and IGF1. According to yet another embodiment, said complete medium in step a) comprises serum, FGF, LIF, IL6, IL6R, IL1, CNTF and IGF1. Preferably, the FGF according to the invention is selected from among basic FGF, FGF3 and FGF4. The concentration of growth factors in the basal medium is between about 0.01 to 10 ng / ml, preferably 0.1 to 5 ng / ml, and most preferably about 1 ng / ml.

According to a preferred embodiment, the feeder cell mat of step a) consists of fibroblasts selected from primary human fibroblasts, human fibroblasts established in line, mammalian primary fibroblasts, mammalian fibroblasts established in line. Preferably, these are mammalian fibroblasts, more particularly mouse fibroblasts established in line, of preferably transformed STO cells or not. Preferably the feeder cell mat is inactivated. It can be chemically inactivated by, for example, mitomycin treatment or physically by exposure to physical rays such as X-rays or gamma rays.

The present invention is based on the discovery that the passage of a basic cell culture medium supplemented with growth factors containing animal serum and feeder cells to an aseric medium lacking growth factors can not be achieved by their simple removal of the basic culture medium. The method of deprivation according to the invention requires performing the deprivation growth factors, feeder cells and serum sequentially and progressively.

In order to successfully achieve growth factor deprivation, it is important that the complete starting medium in which the human stem cells harvested from an individual are cultured comprises at least two, at least 3, at least 4, at least 5, at least 6, at least 8 different growth factors so that the cell when it is deprived in one of its factors can adapt and compensate for this deprivation by developing an alternative metabolic path. Preferably, when the medium comprises at least two different growth factors, it is LIF and FGF.

The modification of the culture medium in step b) of the process of the invention, so as to obtain a progressive or total withdrawal of growth factors, serum and / or feeder cell layers can be performed simultaneously, so successive or separate in time. Said weaning cellular nurse feeder, exogenous growth factors, and serum are carried out successively or in a time-shifted manner, according to a sequence of weaning selected from:

- feeder cells / serum / growth factors;

- feeder cells / growth factors / serum;

- serum / growth factors / feeder cells; - serum / feeder cells / growth factors;

growth promoters / feeder cells / serum;

- growth factors / feeder cells / serum. The serum used is preferably animal serum, more preferably fetal calf serum. Alternatively, the serum may be a serum substitute as currently marketed by some companies (eg KO SR of GIBCO-BRL).

According to a preferred embodiment, the weaning sequence is 1) weaning in growth factors, 2) weaning in feeder cells and 3) weaning in serum.

During step a), the culture of the human stem cells is carried out for about 3 to 40 passages in complete medium, then the complete medium is then gradually and sequentially depleted in growth factors (step b). Preferably, for each growth factor, the depletion is carried out directly in a single step, from one passage to another. Alternatively, the depletion in growth factor is carried out gradually, by a gradual decrease in each passage of the concentration of the growth factor in the complete culture medium. According to a preferred embodiment, the depletion in growth factors is carried out simultaneously for at least two growth factors. Alternatively the depletion in growth factors is sequentially carried out growth factor after growth factor. Preferably, the LIF is removed first and directly from the complete culture medium, then after a few passages in culture in a complete medium free of LIF, the FGF is in turn removed directly from the culture medium. Preferably, the weaning in exogenous growth factors is total. Usually the medium is completely depleted of growth factors around passages 20 to 40.

Usually, deprivation in feeder cells is performed after deprivation in growth factors. The deprivation in feeder cells is progressive and carried out on several passages. The human stem cell is generally seeded in the flasks at a concentration lower than that carried out in step a) at about 4 × 10 ⁴ cells / cm ² up to 5 × 10 ⁴ cells / cm ² . The feeder cells are seeded in a flask at approximately 4 X 10 ⁴ cells / cm ² . Gradually, the concentration of feeder cells in the flasks is decreased. In practice, the same concentration of feeder cells is used for 2 to 5 passages, then the concentration of feeder cells is decreased for 2 to 5 passages, and so on. For example, the flange is seeded at about 4 X 10 ⁴ cells / cm ² , then about 3 X 10 ⁴ cells / cm ² , then about 2 X 10 ⁴ cells / cm ² , then about 1.5 X 10 ⁴ cells / cm ² , then about 10 ⁴ cells / cm ² , then about 0.5 X 10 ⁴ cells / cm ² . Finally the flask is seeded with 6 X 10 ⁴ human cells / cm ² to 10 ⁵ human cells / cm ² in the absence of feeder cells. Assuming that the human cells are not in good condition after decreasing the feeder cell concentration in the flask, then the human cells are cultured for a few additional passes with the same concentration of feeder cells in the flask before proceeding deprivation in feeder cells. In step b), a change in the nature of the plastic material of the cell culture dishes used is carried out simultaneously, successively or staggered in time with the weaning of the feeder cell mat. Said plastic material used is specifically treated so as to promote nonionic or hydrophobic interactions, so as to reduce the adhesion of the cells to said material. Step b) comprises at least 30, at least 50, at least 75, at least 100, at least 125, at least 130 successive passages in culture.

The method for obtaining human stem cell lines according to the invention also makes it possible to obtain lines capable of growing in an asteric medium. The serum weaning of the cells according to the invention is carried out by modifying or changing the culture medium of the cells in order to obtain a total weaning serum, either by progressive dilution, direct weaning, or progressive weaning. This method makes it possible to select clones that adapt to these new and increasingly stringent growing conditions, until stable cell lines are obtained which are capable of growing in a serum-depleted or serum-free environment.

Preferably, the basic culture medium of step c) further comprises a low serum concentration (i.e., a serum concentration in the culture medium less than or equal to 5%). The method according to the invention optionally comprises the additional step c) of change of the culture medium of step c). The medium used in step c) bis is thus chosen from:

basal medium (i) supplemented with the serum and diluted with a new asteric medium (ii). Then, the human cells are cultured by successive passages in a medium (i) in which the proportion of serum-free medium (ii) is gradually increased until the complete disappearance of the base medium (i) supplemented with serum (progressive dilution). ; - a new medium without serum (ii) supplemented with serum. Then, the human cells are cultivated by successive passages in a medium (ii) in which the proportion of serum is gradually decreased, until an asteric medium (progressive weaning) is obtained; - a new medium without serum (ii) not supplemented in serum. Then the human cells are directly cultured in the asteric medium (ii) (direct weaning).

Said weaning in serum is carried out by implementing a method chosen from progressive dilution, progressive weaning or direct weaning. In a preferred mode, serum weaning is achieved by progressive weaning.

According to the present invention, the term "aseric medium" or "serum-free medium" (SFM) means a ready-to-use cell culture medium, i.e., not requiring the addition of serum to enable cell survival and growth. The medium is not necessarily chemically defined and may contain hydrolysates of various origin, such as hydrolysates of plant origin, for example. Preferably, said SFM medium is qualified as "without an animal component", that is to say that it does not contain any components of animal or human origin (FAO status: "free of animal origin") . In the serum medium, native serum proteins are replaced by recombinant proteins. Alternatively, the SFM medium according to the invention does not contain protein (PF medium: "protein-free") and / or is chemically defined as CDM medium: "chemically-defined medium"). The SFM environment has several advantages: (i) the first being its ability to meet regulatory requirements (there is no risk of contamination by biological agents, such as animal prions or viruses); (ii) optimizing the purification process; (iii) the best reproducibility of the performances of the cell culture because the medium is better defined. Examples of commercially available SFM astral media are VP SFM (InVitrogen Catalog No. 11681-020, catalog 2003), Opti Pro (InVitrogen Catalog No. 12309-019, catalog 2003), Episerf (InVitrogen Catalog No. 10732-022, catalog 2003), Pro 293 S-CDM (Cambrex No. 12765Q, catalog 2003), LC 17 (Cambrex No. BESP302Q), Pro CHO 5-CDM (Cambrex No. 12-766Q, catalog 2003), HyQ SFM4CHO (Hyclone No. SH30515-02) , HyQ SFM4CHO-Utility (Hyclone P / N SH30516.02), HyQ PF293 (Hyclone P / N SH30356.02), HyQ PF Vero (Hyclone P / N SH30352.02), Ex cell 293 medium (JRH Biosciences 14570-1000M), Ex cell 325 PF CHO Protein free medium (JRH Biosciences 14335-1000M), Ex cell VPRO medium (JRH Biosciences 14560-1000M), Ex cell 302 free serum ( JRH Biosciences Ref 14312-1000M). The method for obtaining human stem cells described above may also comprise an additional step in which the cells obtained in step c) are subjected to selection and adaptation in a suitable culture medium so as to obtain useful cell clones. for the production of biological substances on a large scale. Preferably, the human stem cells, preferably the human stem cells derived from the human ES cells, established by implementing the method of the invention are cells which proliferate in an aseric medium, in the absence of feeder cells and do not require the addition of growth factors in the culture medium. The new human stem cell lines, preferably derived from embryonic stem cells, obtained by the process according to the invention can be maintained in culture in vitro for a long time, that is to say more than one hundred passages. . Advantageously, the embryonic stem cells obtained in step d) are capable of proliferating for at least 50 days, at least 100 days, at least 150 days, at least 300 days in culture and preferably at least 600 days in culture. culture. These 600 days are in no way a limit because the cells obtained will still be alive after that date. In fact, the stem cells according to the invention are considered to be capable of growing indefinitely in culture in a basic culture medium which does not comprise exogenous growth factors, serum and / or inactivated feeder cell layers. The terms "lineages" or "continuous lines", terms that are used interchangeably in this patent, mean that the cell population is capable of growing or proliferating indefinitely in vitro culture while essentially maintaining the same morphological and phenotypic characteristics. By "indefinite growth in culture" is meant to designate a property of cultured cell lines for long-term propagation. This characteristic is in contrast to those presented by most normal diploid cells isolated and cultured in vitro, such as the so-called "primary" cells that enter senescence after multiple passages. According to the present invention, the term "indefinite growth" includes a culture of at least 30 days, at least 60 days, preferably at least 6 months, more preferably at least one year.

The stem cells established according to the invention are preferably small, round, well individualized with a doubling period of between 20 and 40 hours, preferably between 24 and 30 hours. The cells obtained by the process according to the invention are at least in the passage p60, at least in the passage p70, at least in the passage p80, at least in the passage pi 00, at least in the passage pi 20, at least in the passage pi 40 or more. The cells thus established by the method according to the invention have the ability to proliferate for at least 50 days, at least 100 days, at least 150 days, at least 300 days in culture and preferably at least 600 days in culture in a basic medium such as DMEM, GMEM, HamF12, Optipro (GIBCO-BRL) or MacCoy supplemented with various additives commonly used by those skilled in the art. Among the additives, mention may be made of non-essential amino acids, vitamins, sodium pyruvate, beta-mercaptoethanol, etc.

The cells of the cell line obtained by the method according to the invention are derived from human stem cells, preferably embryonic (ES) cells, and possess at least one of the following characteristics: proliferate indefinitely in culture in a culture medium without feeder cell mat, optionally serum and optionally exogenous growth factors; and

- retain a normal diploid karyotype that is not altered by prolonged cell culture; and have a significant nucleo-cytoplasmic ratio; and

retain the ability to differentiate to form at least one differentiated cell type selected from a cell type of mesodermal, ectodermal and endodermal origin; and

- express at least telomerase and alkaline phosphatase. The cell line according to the invention is characterized in that the cells of said line also express the transcription factor Oct3 / 4 and exhibit a reactivity with at least one of the specific antibodies selected from the antibodies directed against SSEA4, TRA 1-60, TRA 1-81. The cell line according to the invention is further characterized in that the cells of said line do not exhibit reactivity with the antibody directed against SSEA1.

The cell line according to the invention preferably has a normal karyotype chosen from 46 XX and 46 XY).

The doubling time of the human stem cells obtained by the method according to the invention is characterized by a doubling time shorter than the doubling time of the human primary stem cells of step a) according to the method of the invention. The doubling time of the stem cells obtained by the process according to the invention is approximately between 20 and 40 hours, preferably between 24 and 30 hours.

Of course, the process described above makes it possible to obtain cellular clones derived from the cells obtained from these lines. These clones are cells that are genetically identical to the cells from which they divide.

The cells according to the invention are able to proliferate in adhesion on the support, but they can also be adapted for suspension culture. Preferably, the cells according to the invention have all the characteristics mentioned above.

The invention also aims to cover the cells according to the invention which have been genetically modified either stably or transiently by using techniques well known to those skilled in the art. The genome of said cell can thus be modified by: i. insertion of a pre-selected isolated DNA sequence; or ii. substitution of a fragment of the cellular genome by a pre-selected isolated DNA sequence; or iii. deletion of a pre-selected isolated DNA sequence; or iv. inactivation of a pre-selected isolated DNA sequence. The invention also aims to cover differentiated human cell lines obtained from the stem cells obtained by the process according to the invention. Said differentiated cell is preferably selected from neural cells, oligo-dendrocytes, glial cells, hematopoietic cells, exocrine cells, endocrine cells, epithelial cells, cells endothelial, cardiac muscle, skeletal muscle, bone marrow, fibroblasts, adipocytes, cartilage cells, bone cells.

According to a particular embodiment of the invention, the human stem cells obtained by the method of the invention are used as a medicament in cell therapy in vivo, in particular for the treatment of neurodegenerative diseases and hereditary or acquired genetic diseases.

The human stem cells established in lines by the method according to the invention are also useful for the production of biological substances, such as, for example, recombinant proteins and viral vaccines. More specifically, the human stem cells established in line according to the invention are useful for replicating viruses, viral vectors derived therefrom, and for producing the corresponding viral particles. More specifically, the human stem cells established in line according to the invention are useful for the production of live virus vaccines, live or attenuated, recombinant or not. The vaccines thus produced are intended for the prophylactic and / or therapeutic treatment of pathologies of viral etiology, acquired chronic diseases such as cancer and neurodegenerative diseases. Among the viruses, the viral vectors and the corresponding viral particles, mention should be made, in a non-exhaustive manner, of adenoviruses, hepadnaviruses, herpesviruses, orthomyxoviruses, papoviruses, paramyxoviruses, paramyxoviruses, picornaviruses, poxviruses , reoviruses, and retroviruses. Preferably, the virus is an orthomyxovirus, in particular human influenza virus. According to another preferred mode, the virus is a paramyxovirus, and more particularly the measles virus, and / or the mumps virus, and / or the rubella virus. According to another preferred embodiment, the virus is a human retrovirus, and more particularly the human immunodeficiency virus.

Alternatively human stem cells established in line according to the invention are useful for the production of recombinant proteins, especially proteins of therapeutic interest. In this regard, the cells obtained by the method according to the invention can be genetically modified, stably or transiently, using techniques available to those skilled in the art. According to a preferred embodiment, the protein of therapeutic interest is an antibody, preferably monoclonal, humanized or chimerised. Finally, the human stem cells established in line according to the invention are useful for carrying out sanitary diagnostic tests.

Claims

A method for obtaining continuous lines of undifferentiated, undifferentiated human stem cells capable of proliferating indefinitely in culture, characterized in that said method comprises the following steps: a) cultivation on a carpet of feeder cells of human stem cells in a complete culture medium comprising at least:

animal serum or a substitute for animal serum;

an exogenous growth factor selected from: the ligands at the receptor which can form a heterodimer with the glycoprotein gp130; b) successive passages in a different culture medium, so as to obtain a weaning of the feeder cell mat, total or partial weaning of said exogenous growth factors and total or partial weaning of the serum; c) optionally, selecting cell colonies having compact morphologies and composed of cells having a high nucleo-cytoplasmic ratio and a prominent nucleolus; d) establishing said continuous lines of human stem cells derived from embryonic cells capable of growing in the absence of feeder cells.

2. Method according to claim 1, characterized in that said lines obtained in step d) are capable of growing in culture medium depleted in growth factor.

3. Method according to claims 1 and 2, characterized in that said lines obtained in step d) are capable of growing in a cultured culture medium in serum.

4. Method according to claims 1 to 3, characterized in that said primary human stem cells are selected from totipotent stem cells, pluripotent stem cells, multipotent stem cells, unipotent stem cells, intermediate progenitor cells.

5. Method according to claim 4, characterized in that said primary human stem cell is a pluripotent stem cell, that is to say an embryonic stem cell (ES).

6. Method according to claims 1 to 5, characterized in that said ligand to the receptor which can form a heterodimer with the glycoprotein gp130 is selected from leukemia inhibitory factor (LIF), interleukin 11 (ILI 1), l interleukin 6 (IL6), interleukin 6 receptor (IL6R), ciliary neurotrophic factor (CNTF), funcostatin, cardiotrophin.

7. Method according to claims 1 to 5, characterized in that said complete medium in step a) comprises serum, and at least FGF and LIF.

The method according to claim 7, characterized in that said complete medium in step a) comprises serum, FGF, LIF, IL-6, IL6R, IL1, CNTF and IGF1.

9. The method of claims 1 to 8, further comprising a substep (a1) of step a) of dissociating the cell clusters formed in culture, characterized in that the dissociation is carried out enzymatically and / or mechanically.

10. Process according to claims 1 to 9, characterized in that the feeder cell mat of step a) consists of fibroblasts chosen from primary human fibroblasts, human fibroblasts established in line, mammalian primary fibroblasts, fibroblasts. of mammals established in lineage.

11. Method according to claim 10, characterized in that said mammalian fibroblasts are mouse fibroblasts established in line, preferably transformed STO cells or not.

12. Method according to claims 1 to 11, characterized in that step b) comprises at least 30, at least 50, at least 75, at least 100, at least 125, at least 130 successive passages in culture.

13. Method according to claims 1 to 12, characterized in that said weaning in feeder cell mat, in exogenous growth factors, and in serum are carried out successively or in a time-shifted manner, according to a sequence of weaning selected from: i . nurse cell mat / serum / exogenous growth factors; ii. nurse cell mat / exogenous growth factors / serum; iii. serum / exogenous growth factors / nourishing cell mat; iv. serum / nurse cell mat / exogenous growth factors; v. exogenous growth factors / serum / feeder cell mat; vi. exogenous growth factors / nurse cell / serum mat.

14. The method of claim 13, characterized in that the sequence of weaning is: weaning in exogenous growth factors, weaning in feeder cell mat, weaning serum.

15. Process according to claims 1 to 14, characterized in that the weaning of each of the exogenous growth factors is carried out by progressive reduction over several passages, preferably at least 3, of the concentration of each factor in the culture medium.

16. The method according to claims 1 to 15, characterized in that the weaning in exogenous growth factors is total.

17. Method according to claims 1 to 16, characterized in that said weaning serum is carried out by implementing a method selected from progressive dilution, progressive weaning or direct weaning.

18. An isolated human stem cell derived from a primary embryonic stem cell obtainable by the method according to claims 1 to 17, characterized in that said cell: i. proliferates indefinitely in culture in a culture medium lacking feeder cell layer, optionally serum and optionally exogenous growth factors; and ii. retains a normal diploid karyotype that is unaltered by prolonged cell culture; and iii. has a significant nucleo-cytoplasmic ratio; iv. retains the ability to differentiate to form at least one differentiated cell type selected from a cell type of mesodermal, ectodermal and endodermal origin; v. expresses at least telomerase and alkaline phosphatase.

19. Cell according to claim 18, characterized in that it also expresses the Oct3 / 4 transcription factor and exhibits reactivity with at least one of the Specific antibodies selected from antibodies to S SE A4, TRA 1-60, TRA 1-81.

20. Transgenic isolated human stem cell according to claims 18 and 19, characterized in that the genome of said cell has been modified by: i. insertion of a pre-selected isolated DNA sequence; or ii. substitution of a fragment of the cellular genome by a pre-selected isolated DNA sequence; or iii. deletion of a pre-selected isolated DNA sequence; or iv. inactivation of a pre-selected isolated DNA sequence.

21. Use of human stem cells according to claims 18 to 21

For virus replication, live or attenuated, recombinant or not, or viral vectors.

22. Use of human stem cells according to claims 18 to 22

21 for the production of human or veterinary vaccines.

23. Use of human stem cells according to claims 18 to 18

For the production of recombinant proteins or polypeptides, preferably of therapeutic interest.

24. Use of human stem cells according to claims 18 to 20 for carrying out sanitary diagnostic tests.