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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/145—Thrombopoietin [TPO]
• • C—CHEMISTRY; METALLURGY
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/15—Transforming growth factor beta (TGF-β)
• • C—CHEMISTRY; METALLURGY
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)

Definitions

• the subject of the present invention is a method of multiplying stem cells.
• the subject of the invention is in particular a method making it possible both to rapidly multiply said stem cells and to maintain them in an undifferentiated state.
• the current cultures which allow stem cells to be maintained in an undifferentiated state, and in particular hematopoietic stem cells, and / or multiplying them, are long-term cultures in which the cells divide very slowly. More particularly, the cultures allowing for example a self-renewal (an identical multiplication) of stem cells, in particular of hematopoietic stem cells, are only cultures on medullary stroma, or cultures with slow multiplication, in the absence of medullary stroma .
• stem cells in particular hematopoietic stem cells
• medullary stroma which makes it possible to use only few cytokines but causes slow division of cells (and therefore slow multiplication of cells) in the presence of a mixture of progenitors with mature hematopoietic and stromal cells.
• stem cells in particular hematopoietic stem cells
• the Piacibello culture system In the case of long-term cultures, in the absence of a medullary stroma, the Piacibello culture system can be mentioned. In this culture system (Piacibello et al. 1997 Blood), the CD34 + cells containing hematopoietic stem cells are seeded at 20,000 cells per ml, and each week half of the culture is removed and replaced with fresh medium. After two weeks, the number of cells doubles at a rate of once a week, over several months. During the first two weeks of Piacibello culture, there is a strong loss of CD34 + cells which stabilize at less than 2%. Thus, current cultures are not very compatible with a production for clinical or biotechnological purposes of stem cells maintaining their undifferentiated and multipotent character.
• One of the main aims of the invention is to propose a method of culturing stem cells making it possible to obtain human stem cells in an undifferentiated state, quickly and in large quantities.
• One of the aims of the invention is to propose a rapid process for culturing stem cells, without medullary stroma, and making it possible to reduce the culture volumes.
• One of the other aims of the invention is to use the stem cells thus obtained to reconstitute tissues and organs to be transplanted.
• the subject of the invention is the use of a cell development inhibitor in a controlled manner to maintain the undifferentiated state of stem cells, in particular human stem cells, while allowing their cell division.
• an inhibitor of cell development can also be an inhibitor of cell differentiation of stem cells.
• TGF- ⁇ (“transforming growth factor”), previously known as an inhibitor of cellular development of stem cells, and in particular hematopoietic stem cells or primitive hematopoietic progenitors, appears to be, according to invention, an inhibitor of cell differentiation.
• cell development inhibitor includes both any substance that inhibits cell proliferation and / or cell growth, and / or cell differentiation.
• cell proliferation inhibitor also means cell division inhibitor, or cell cycle inhibitor.
• use of a cell development inhibitor in a controlled manner means that the cell development inhibitor is used in a modular manner in order to allow:
• the expression "use of a cell development inhibitor in a controlled manner” means, in other words, that the cell development inhibitor can be used in conditions such that stem cells are taken out of their resting state in order to divide, in controlled conditions preventing differentiation.
• stem cells means immature cells, or undifferentiated (or undifferentiated) cells, or primitive cells, or pluripotent cells or multipotent cells.
• the stem cells advantageously used according to the invention are human stem cells chosen from the group consisting of embryonic stem cells at the origin of somatic stem cells, and / or stem cells / somatic progenitors themselves themselves.
• blood and / or various solid tissues such as skin, liver, pancreas, heart, kidney, bone, or nervous tissue.
• embryonic stem cells at the origin of somatic stem cells is defined for example as a cell which can give rise to any one of the somatic stem cells.
• embryonic stem cells is defined for example as a cell which can give rise to a specific tissue.
• the stem cells according to the invention are in particular human hematopoietic somatic stem cells, also called primary hematopoietic progenitors.
• the cell development inhibitor advantageously used according to the invention is chosen from the group consisting of gene products controlling cell development with respect to cell differentiation and / or cell division , cyclin dependent kinase inhibitors, factors controlling apoptosis or aging, and cytokines (such as interferons, TGF- ⁇ ).
• cytokines such as interferons, TGF- ⁇ .
• retinoblastoma gene As inhibitors of dependent cyclin kinases, mention may especially be made of the retinoblastoma gene, the PI 5, PI 6, P21, P27 genes.
• the retinoblastoma gene is a tumor suppressor (anti-oncogenic) gene.
• the PI 5, PI 6, P21, P27 genes inhibit the cell cycle.
• telomerase As a factor controlling apoptosis, there may be mentioned in particular the genes bcl-2, bax, fas. As a factor controlling aging, mention may especially be made of telomerase.
• cytokines which may be inhibitors of cell development, mention may in particular be made of interferons and in particular TGF- ⁇ .
• the subject of the invention is the use of an inhibitor of cell development as defined above, in sequential association with an anti-inhibitor of cell proliferation, to trigger a number of cell divisions ranging from 1 to approximately 100, in particular from 1 to approximately 10, and in particular for triggering a single cell division, while maintaining the undifferentiated state of the stem cells, in particular human stem cells.
• the expression “sequential association” means that the anti-cell proliferation inhibitor is not used simultaneously with the cell development inhibitor.
• the subject of the invention is a method of multiplying stem cells in a culture medium, in particular human stem cells, characterized in that it comprises:
• the stem cells in particular human stem cells, in the resting state are brought out of their resting state by neutralizing the effect of an inhibitor of cell development, and in particular of an inhibitor of cell proliferation, produced by cells and / or present in the culture medium, so that there is triggering of a number of cell divisions ranging from 1 to approximately 100, in particular from 1 to approximately 10, and in particular of a single cell division,
• the subject of the invention is also a method of multiplying stem cells in a culture medium, in particular human stem cells, characterized in that it comprises: - a step according to which the stem cells, in particular the human stem cells, in the state of division are prevented from entering a state of differentiation, using a cell development inhibitor,
• the multiplication process according to the invention is characterized in that, during and at the end of said process, the stem cells thus multiplied are kept in an undifferentiated state.
• the expression “multiplied stem cells” has the same meaning as the expression “amplified stem cells”.
• the expression “multiplication process” has the same meaning as the expression “amplification process”.
• the resting state of the cells means that the cells do not differentiate or divide.
• the stem cells multiplied according to the method of multiplication of the invention are human stem cells chosen from the group consisting of embryonic stem cells at the origin of somatic stem cells, and the somatic cells they -same at the origin of blood and / or various solid tissues such as the skin, the liver, the pancreas, the heart, the kidney, the bone, or nervous tissue.
• the multiplication method according to the invention is characterized in that the stem cells, in particular human cells, are present at a cell concentration of approximately 1 to approximately 10 10 cells per ml, and in particular at a concentration ranging from approximately 10 3 to about 10 10 cells per ml, and more particularly about
• the cell development inhibitor is synthesized by stem cells, in particular stem cells human, and / or is added to the culture medium containing the stem cells, in particular the human stem cells.
• Said cell development inhibitor is synthesized by the stem cells and / or is added to the culture medium: (a) before the first cell division, and
• the cell development inhibitor When the cell development inhibitor is synthesized by stem cells, it may or may not be secreted.
• the quantity of cell development inhibitor synthesized by the stem cells, or added to the culture medium, must be sufficient so that said cells (a) are maintained in their resting state before the first cell division, and ( b) are placed in the state of rest when they were previously in a state of division.
• the amount of cell development inhibitor synthesized by the stem cells can vary from 0.01 pg to 1 mg / ml in the culture medium, and in particular from 0.1 pg to 10 ng / ml.
• the amount of cell development inhibitor added to the culture medium can vary from 0.1 pg to 1 mg / ml in the culture medium, and in particular from 1 pg to 10 ng / ml.
• the multiplication method according to the invention is characterized in that the cell development inhibitor is chosen from the group consisting of the gene products controlling cell development with respect to cell differentiation and / or cell division, inhibitors of dependent cyclin kinases, factors controlling apoptosis or aging, and cytokines (such as interferons, TGF ⁇ ).
• the cell development inhibitor is chosen from the group consisting of the gene products controlling cell development with respect to cell differentiation and / or cell division, inhibitors of dependent cyclin kinases, factors controlling apoptosis or aging, and cytokines (such as interferons, TGF ⁇ ).
• the cell development inhibitor is present at a low concentration in the culture medium containing the stem cells, and in particular at a concentration ranging from approximately 10 ⁇ 10 mg / ml to 1 mg / ml.
• the multiplication method according to the invention is characterized in that the neutralization of the effect of the inhibitor of cell development, and in particular of the inhibitor of cell proliferation, present in the culture medium, is carried out by: the addition to the culture medium of an anti-inhibitor of cell proliferation, and / or
• the effect of said inhibitor is neutralized by adding to the culture medium an anti-inhibitor of cell proliferation, which can penetrate into the cell, such as an antisense oligonucleotide.
• an anti-inhibitor of cell proliferation which can penetrate into the cell, such as an antisense oligonucleotide.
• the effect of said inhibitor is neutralized either by adding an anti-proliferation inhibitor to the culture medium. or by removing the culture medium from the cell development inhibitor.
• the removal of a cell development inhibitor from the culture medium can in particular be carried out using blocking antibodies or by washing in order to neutralize said inhibitor.
• the quantity of cell development inhibitor withdrawn from the culture medium containing the stem cells must be sufficient so that there is triggering of a number of cell divisions ranging from 1 to approximately 100, in particular from 1 to approximately 10, including a single cell division.
• the amount of cell development inhibitor removed from the culture medium varies from 0.01 pg to 1 ng / ml, and in particular from 0.1 pg to 10 ng / ml.
• the cell development inhibitor removed from the culture medium belongs in particular to the group of cytokines, antisense oligonucleotides blocking the expression of development genes.
• the amount of anti-cell proliferation inhibitor added to the culture medium containing the stem cells must be sufficient so that there is triggering of a number of cell divisions ranging from 1 to approximately 100, in particular from 1 to approximately 10, and in particular of a single cell division.
• the amount of anti-cell proliferation inhibitor added to the culture medium varies from 0.1 ⁇ g to 10 mg / ml for blocking antibodies or from 0.01 ⁇ M to 1 mM of antisense oligonucleotide in the culture medium , and in particular from 1 ⁇ g to 100 ⁇ g / ml for blocking antibodies or from 0.1 ⁇ M to 100 ⁇ M of antisense oligonucleotides.
• the anti-cell proliferation inhibitor is chosen from the group consisting of antisense oligonucleotides or blocking antibodies and in particular by the anti-
• the multiplication method according to the invention is characterized in that the anti-inhibitor of cell proliferation is present in a concentration ranging from approximately 10 "18 to approximately 10 " 3 g / ml, including 0.1 to
• the method of multiplication according to the invention of stem cells is characterized in that it comprises, in order to obtain a sufficient number of stem cells maintained in the undifferentiated state, a total number of cycles of divisions (or division states) of between 1 to 100 cycles, in particular between 5 to 20 cycles, and in particular 10 cycles.
• the total duration of all the rest states of the multiplication process according to the invention varies from 1 hour to 3 years, in particular from 20 hours to 200 hours, and the total duration of the whole division cycles of the multiplication process according to the invention varies from 10 hours to 3 years and in particular from 20 hours to 200 hours.
• the multiplication method according to the invention is characterized in that the duration of a single rest state ranges from approximately 1 hour to 3 years, and is in particular approximately 6 hours to 72 hours, and in that the duration of a single division cycle ranges from approximately 6 hours to 3 years, and is in particular from approximately 6 hours to 24 hours.
• the total duration of the multiplication process according to the invention comprising all of the rest and division states ranges from 1 day to 3 years, and is in particular from 1 day to 15 days.
• the set of rest and division cycles of a multiplication process lasts from 1 to 15 days, and the applications envisaged are cell therapy.
• the set of rest and division cycles of a multiplication process lasts from 1 day to 3 years, and the applications envisaged are experimental.
• the method of multiplying stem cells according to the invention makes it possible to obtain an amplification of the stem cells by a factor ranging from approximately 2 to approximately 10 12 , and in particular from approximately 2 to approximately 10 4 .
• the multiplication method according to the invention makes it possible to obtain a number of amplified stem cells and maintained in the undifferentiated state, from 2 to 10 12 times greater than the initial number of undifferentiated stem cells.
• the culture medium of the multiplication method according to the invention contains hematopoietic stem cells and comprises one or more cytokines (added to the culture medium) chosen from the group consisting of interleukins and CSFs , said cytokines being present at a concentration ranging from approximately 10 ⁇ 8 ⁇ g / ml to approximately 1 mg / ml, and in particular from approximately 10 ⁇ 5 ⁇ g / ml to 0.1 ⁇ g / ml, and possibly other factors growth.
• CSFs colony-stimulating factor
• GM-CSF factor stimulating granulocytic and monocytic colonies
• GCSF factor stimulating granulocytic colonies
• MCSF factor stimulating monocytic colonies
• SF Stepel factor
• TPO thrombopoietin
• FL Flt-3 ligand
• the method of multiplication according to the invention of stem cells, and in particular human somatic stem cells is more particularly characterized in that it comprises the following steps: a) the initiation of a first cycle of division of embryonic stem cells or undifferentiated somatic in a culture medium, and in particular of cells hematopoietic somatic strains, by seeding said undifferentiated stem cells in the resting state, at a high initial cell concentration, in particular at a concentration ranging from 10 to 10 cells per ml, in the presence of one or more cytokines, and by neutralizing the effect of the inhibitor of cell development, and in particular of the inhibitor of cell proliferation, present in the culture medium, so that the aforesaid cells come out of their state of rest by the triggering of a first cell division, b) the return to rest of the undifferentiated embryonic or somatic stem cells obtained in the previous step, using a cell development inhibitor, said inhibitor being synthesized by said stem cells, or
• the modalities of the different stages of the stem cell multiplication process according to the invention are those already described above.
• the quantity of anti-cell proliferation inhibitor added to the culture medium, or the quantity of cell development inhibitor withdrawn from the culture medium containing the stem cells, must be sufficient so that there is triggering of 'a first cell division.
• the cell development inhibitor is TGF- ⁇ ; TGF- ⁇ is synthesized by hematopoietic somatic stem cells themselves in an amount ranging from 0.01 pg to 1 ng / ml.
• the TGF- ⁇ is then neutralized by the addition to the culture medium of an anti-inhibitor of cell proliferation: the anti-TGF- ⁇ .
• the anti-TGF- ⁇ is added in an amount ranging from 0.1 ⁇ g to 100 ⁇ g / ml of antibody or 0.1 ⁇ M to 10 ⁇ M of oligonucleotides.
• the washing step ⁇ described above makes it possible in particular to neutralize the TGF- ⁇ inhibitor formed during or at the end of a previous division cycle in order to allow the following division.
• the dilution step (d) described above, maintaining an optimal cell concentration allows both: - the rapid return to the resting state of the stem cells, due to a rapid synthesis of said cells of the cell development inhibitor, in particular TGF- ⁇ ,
• TGF- ⁇ slows down the differentiation process of stem cells throughout the multiplication process, namely during and at the end of the different rest and division cycles.
• the stop step f) described above can be carried out by washing said stem cells thus multiplied, by freezing them, or by putting them in a differentiation medium.
• the differentiation medium in which the stem cells thus multiplied are placed and maintained in an undifferentiated state according to the method of the present invention is chosen as a function of the tissue or of the organ which it is sought to reconstitute.
• the multiplication process according to the invention makes it possible to obtain significant productions of stem cells, said stem cells being maintained in an undifferentiated state during the various rest and division cycles of said process. Large productions of immature cells are thus obtained in a reduced volume.
• the present invention relates to the use of undifferentiated and amplified human stem cells as obtained according to the method of the invention described above, for reconstituting human blood and / or solid tissues or human organs.
• undifferentiated and amplified human stem cells as obtained according to the method of the invention can in particular be used to amplify insufficient samples of umbilical cord blood, bone marrow or peripheral blood, or for the transplantation of hematopoietic stem cells.
• the undifferentiated and amplified human stem cells as obtained according to the method of the invention can also be used for studies on the human genome (expression and function of the genes for human development).
• Figures 1A and 1B represent the effect of TGF- ⁇ on the maintenance of the undifferentiated state of CD34 + cells.
• the PKH26 dye that attaches to the cell membrane allows you to determine the number of divisions that the cell has made: after doubling the cell, the intensity of the dye is divided by 2, after 2 doublings by 4 etc ... This gives different populations moving to the right.
• the concentration of TGF- ⁇ being higher (30 pg / ml of TGF- ⁇ ) (FIG. 1B), a higher percentage of cells is observed.
• stem cells used in the example below are more particularly hematopoietic human somatic stem cells originating from umbilical cord blood and characterized by the membrane marker CD34 +. In what follows, said cells will be called “CD34 + cells”.
• CD34 + cells are diluted in PBS (phosphate buffered saline) / BSA (bovine serum albumin) (0.2%) and incubated with anti-CD34 + antibodies conjugated to FITC (fluoroisothiocyanate) (clone 8G12; Becton Dickinson, San Jose, CA) for 30 minutes at 4 ° C. then washed twice with PBS / BSA.
• the control consists of cells incubated with non-specific IgG1 conjugated to the FITC.
• the CD34 + are labeled with the dye PKH26 (Sigma, France) according to the manufacturer's instructions. Cells with an average intensity of PKH26 are sorted in a window representing approximately 10% of the full width. This corresponds to approximately 20% of CD34 + cells. Four 400 cell aliquots are used in the HPP-Q test. The rest of the resting cells and the proliferating cells are sorted and cultured in a semi-solid medium. Calibrated beads (Coulter-Beckman) are used to standardize the analyzes between day 0 and day 3.
• HPP-Q test High Proliferative Potential-Quiescent Cell
• TGF- ⁇ the inhibitor of cell development
• stem cells in particular hematopoietic, at rest.
• HPP-Q test consists in determining the degree of maturity of stem cells, in particular hematopoietic cells, and is characterized in that it comprises the following steps:
• a first set of stem cells, in particular hematopoietic cells, is cultured in a medium suitable for their culture, said medium not containing means for blocking at least one inhibitor of cell development such as TGF- ⁇ , during about 14 to about 28 days, preferably 18 days,
• the culture of a second set of stem cells, in particular hematopoietic cells, of the same nature and of the same degree of maturity as those mentioned above, is carried out in an appropriate medium containing means for blocking at least less an inhibitor of cell development, these blocking means being present, in the culture medium at an effective concentration,
• HPP-CFC progenitor forming a colony with high potential proliferative
• HPP-MEG megakaryocytic progenitor with high proliferative potential
• HPP-GEMM granulocytic erythrocytic progenitor megakaryocyte with high proliferative potential
• the number and nature of the colonies are observed under a microscope: the number of mixed colonies made up of cells of the red line and one or more types of the white line is counted.
• the number of mixed colonies is higher when the cell population cultured is immature, that is to say made up of younger cells, some of which are at rest.
• the more immature the cell that gave birth to the colony, even at rest the greater its proliferation potential, so the colony from this cell once activated will be larger in size.
• CD34 + cells were seeded at a cell concentration of 10 6 cells per ml in 24-well culture plates at a rate of 1 ml per well at day -T0. Said cells were seeded in a serum-free medium containing cytokines and a TGF- ⁇ inhibitor (anti TGF- ⁇ ).
• Step Factor 10 ng / ml
• TPO Thrombopoietin 10 ng / ml
• IL6 Interleukin 6
• CD34 + cells are incubated at 37 ° C in an atmosphere with 5% CO2 and 97% humidity saturation. Every 2 days, the volume of the culture is measured, the number of CD34 + cells is counted, and the viability of said cells is determined.
• the medium is readjusted in volume, in cytokines or growth factors and in inhibitor of TGF- ⁇ .
• the protocol of the method according to the invention makes it possible to culture the cells in a microbioreactor which maintains the conditions of media constant with regard to catabolites and anabolites (cytokines and inhibitors excluded).
• the seeding of CD34 + cells at a high cell concentration makes it possible to create a bioreactor at high cell concentration, and thus to reduce the culture volumes.
• the microbioreactor consists of a culture chamber, of a size adapted to the number of cells and to their cell density (1 to 100 ml), separated from a dialysis chamber by a membrane, and optionally from a gas chamber by a another membrane, to keep the constituents of the medium constant.
• Micro detectors make it possible to control various parameters in the culture chamber: pH, CO, O 2 , glucose, lactate, etc.
• Inputs and outputs allow to renew or modify the culture media, or to add or remove cells.
• the entire microbioreactor can be automated by pumps and computerized programmers.

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