WO2002034890A2 - Cellules souches pluripotentes - Google Patents

Cellules souches pluripotentes Download PDF

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WO2002034890A2
WO2002034890A2 PCT/GB2001/004747 GB0104747W WO0234890A2 WO 2002034890 A2 WO2002034890 A2 WO 2002034890A2 GB 0104747 W GB0104747 W GB 0104747W WO 0234890 A2 WO0234890 A2 WO 0234890A2
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stem cell
cell
somatic
cells
mammalian
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PCT/GB2001/004747
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WO2002034890A3 (fr
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Austin Gerrard Smith
Qi Long Ying
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University Of Edinburgh
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Publication of WO2002034890A3 publication Critical patent/WO2002034890A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system

Definitions

  • the invention relates to pluripotential stem cells that are capable of forming cells of all three of the somatic lineages of ectoderm, mesoderm and endoderm in the adult animal. Also provided are methods for generating these pluripotential stem cells. The invention further relates to derivatives and uses of such pluripotential stem cells.
  • the ultimate cell is the fertilised egg cell which is totipotent, in that as well as giving rise to all cells of the embryo and afterwards the organism, it also gives rise to the cells forming the placenta.
  • Embryonal stem cells are stem cells found in the embryo and which are able to give rise to all cells of the adult but have a dramatically reduced ability to form placenta - these cells are known as pluripotent stem cells. Embryonal stem cells give rise to three specialised cell lineages which in combination form all of the cells and tissues in the body except the gametes. These lineages are known as ectodermal, endodermal and mesodermal cells - while the gametes are derived from a fourth and separate cell lineage known as the germline. Ectoderm, endoderm and mesoderm cells supply all of the cell types comprising somatic tissue and are termed multipotent, as each lineage precursor or stem cell can produce a range, although not all, of the cell types found in the body.
  • Some of the cells formed from the embryonal stem cells are progenitor lineage stem cells and are capable both of forming a differentiated cell and of self-renewal, While other cells are terminally differentiated cells which no longer divide.
  • Cells which can self-renew or differentiate are known as stem cells, precursor cells or progenitor cells and play crucial roles in regeneration and homeostasis in virtually every tissue in the body.
  • Stem cells are found to have greater levels of potency the higher they are in the developmental cascade - i.e. the closer they are developmentally to the fertilised egg the more different cell types they are capable of forming .
  • Embryonic Stem (ES) cells were originally derived from the pluripotential inner cell mass (ICM) of the preimplantation mouse embryo, and retain the capacity for multilineage differentiation both in vitro and in vivo. With their potential for leading to all cell types, there is a desire to obtain and maintain embryonic stem cells from many species, in particular from humans.
  • ICM pluripotential inner cell mass
  • ES cells are typically isolated from the ICM by sorting, but even after careful segregation of cell types, these stem cell preparations consist of mixed cell types and while enriched for stem cells, include high proportions of more differentiated cells which are not categorised as ES cells. Further difficulties arise with regards culturing of these ES cells outside of the embryo, for example whilst there are various reports describing the isolation of ES cells from diverse species, ES cells from only two inbred strains of mice ( 1 29 and Black 6) can be efficiently cultured in vitro. Thus, a significant problem that exists in the art is that the range of ES cells that can be propagated efficiently in culture is limited to just a few types, mainly mouse ES cells, despite efforts to derive
  • ES cell has also been widely adopted and used to describe a variety of pluripotent stem cells including those which have been derived from cells of the mammalian germline, that is cells capable of giving rise to gametes.
  • ES cells are typically derived from the inner cell mass or primitive ectoderm, but other ES cells, which are more correctly described as EG (embryo-gonadal) cells, are isolated from primordial germ cells of the developing foetus.
  • An ES cell in its truest form is a cell which can be maintained indefinitely in culture whilst still retaining normal chromosome content and the ability to form any cell type found in the body, including the germline. While ES (and EG) cells have been isolated and definitively proven for some strains of mice, the term ES cell is also widely used to describe pluripotent cells derived from other species, including humans, which have not been shown or tested for germline competence.
  • ES cell chromosomally normal pluripotent stem cells capable of extended growth in vitro and differentiation into all of the three germ layers of ectoderm, mesoderm and endoderm.
  • EP-A-0695351 provides a method of isolating and/or enriching and/or selectively propagating pluripotential animal cells, and especially ES cells obtained from embryonic tissue. Whilst this represents a powerful method of selecting for stem cells of any given potential, it does not address the issue of the source of the starting material.
  • the present invention is based upon techniques developed by the inventors which have enabled cells having limited developmental potential to be taken back up the differentiation cascade, to yield cells having greater developmental potential. Specifically the methods of the invention enable production of a cell having the developmental capacity of an ES cell to be derived from a starting material that is not itself an embryo- or even germline-derived cell.
  • the present invention thus provides for the conversion of a somatic cell, having restricted developmental capacity, into a pluripotent stem cell that is able to form cells of the mesodermal, endodermal and ectodermal lineages.
  • a first aspect of the invention provides:- a mammalian stem cell, capable of acting as a progenitor for generating a plurality of mammalian cell types, wherein the stem cell is derived from a second stem cell of lower relative potency.
  • This cell has the capacity to form many different differentiated cells, but has been obtained from a cell hitherto believed to have the capacity to form fewer cell types - the parent of the cell of the invention is a cell that has been reversed up its developmental pathway, it has been de-differentiated.
  • a particular cell of the invention is a pluripotent stem cell, capable of stable regeneration in in vitro culture conditions, obtained by dedifferentiation of a somatic cell.
  • the invention further provides a method of generating a stem cell comprising : (a) obtaining a mammalian somatic stem cell; and (b) exposing the cell of (a) to conditions that promote conversion of the cell into a stem cell of greater relative potency.
  • the invention provides a somatic cell-derived pluripotential stem cell.
  • a somatic cell is a cell is not part of the germline, that is, does not have the ability to form gametes of either sex.
  • a fertilised oocyte develops into an embryo, which in turn develops into a foetus which is then born as a neonate and develops into an adult.
  • pluripotent stem cells of the developing embryo differentiate to form the foetus, cells are committed to either one of three somatic cell lineages (ectoderm, mesoderm and endoderm), or to the germline.
  • Undifferentiated pluripotent stem cells of the embryo have the capacity to form gametes and are therefore part of the germline.
  • the more specialised cells of the developing germline such as primordial germ cells, have the ability to form gametes and are also part of the germline.
  • Somatic cells are not part of the germline and are derived from one of three somatic cell lineages.
  • ES cells as hitherto described in the art are derived from germline cells either from the ICM (or primitive ectoderm) of embryos - usually by simply physically removing them from the ICM - or from the primordial germ cells of the developing foetal gonad .
  • ICM or primitive ectoderm
  • EG cells whilst functionally similar to cells of the present invention are clearly germline derived ES cells.
  • the cells of the present invention are termed 'soma-derived' ES cells or soma-derived pluripotential cells to clearly distinguish them from all prior art cells.
  • Pluripotential in relation to a stem cell refers to the ability of the stem cell to form cells of all three of the somatic cell lineages of mesodermal, endodermal and ectodermal.
  • a pluripotential stem cell is therefore capable of acting as a progenitor for all cell types found in the adult organism though capacity to form germ cells is often unproven or untested. This definition, it is emphasised, is not to be confused with multipotential, which in relation to a stem cell indicates it has the capacity to form daughter cells of a plurality but restricted number of somatic cell types..
  • a pluripotential cell is a multipotential cell, but a multipotential cell is not a pluripotential cell.
  • a stem cell has the capacity to self renew and also to form at least one type of differentiated progeny.
  • a soma-derived pluripotential stem cell is a chromosomally normal, pluripotential stem cell that has the ability to self renew and is also a progenitor for cells of all three of the mesodermal, endodermal and ectodermal lineages in the body of the adult, but which has not been obtained from an embryo or germline cell.
  • the soma-derived pluripotential stem cell also expresses the Oct4 gene, unlike other somatic stem cells, and specific cells of the invention have been found capable of stable culture in vitro for at least 20 cell divisions.
  • Somatic cells are cells of the adult that are not germ cells. Somatic stem cells are readily obtained from either foetal or adult tissue, in which sense foetal tissue refers to tissue that is post embryonic but pre-parturition.
  • Animal tissue encompasses all tissue in the fully formed organism and thus includes all tissues present in the organism post-parturition. Animal tissue therefore includes tissue taken from neonates and other animals through to fully grown as well as ageing adults.
  • the potency of a stem cell refers to its level of developmental commitment or how restricted the cell is to a particular call fate, and thus its ability to act as a progenitor for different derivative cell types.
  • the higher the potency of a cell the lower the restriction to a developmental pathway or fate.
  • a cell that is more restricted to a developmental fate is further down its developmental pathway and has a lower level of potency compared to a cell higher up the pathway which is less restricted.
  • a cell which is of relatively higher potency refers to a cell which is of relatively higher potency.
  • the developmental dogma of unidirectional differentiation teaches that cells higher up the developmental cascade become more and more restricted in developmental capacity or potency. Some tissue types are able to regenerate themselves (if they contain tissue specific stem cells) or simply progress towards a defined non-regenerative cell type. However, it is generally accepted that normal cells do not progress back up the differentiation pathway to a less differentiated state.
  • Dedifferentiation is a term used to describe a process whereby a cell moves back up the developmental pathway and thus becomes less restricted to a particular cell fate.
  • stem cells dedifferentiation expands their potency thereby increasing the number of cell types that they are capable of acting as progenitors for.
  • this process is characterised by reversion to a stem cell capable of forming differentiated cells of all three germ layers. Dedifferentiation of a somatic stem to a cell of higher potency typically results in a change of endogenous gene expression which in many instances is accompanied by a change in gross morphology.
  • a further aspect of the present invention provides methods to take cells having limited developmental potential back up the differentiation cascade, to yield cells having greater developmental potential.
  • the invention includes a method a method of generating a stem cell comprising:
  • the invention achieves this conversion by exposing the somatic stem cell to media and/or culture conditions that are believed to be suitable for the cell of greater potency but not in fact well tolerated by the somatic stem cell starting material.
  • the methods thus comprise "abusing" the starting material, using conditions believed in the art to be unfavourable to its growth.
  • the cells being cultured can be spread out on culture plates and even replated a number of times, each time spreading the progeny. It then becomes possible to isolate from the progeny, sometimes towards the outside of clusters of cells, cells that have been dedifferentiated by this process.
  • the invention provides for the conversion of a neural cell into a pluripotential stem cell. This is carried out by culturing the neural stem cell in the presence of medium that does not promote differentiation, and preferably medium that contains factors promoting growth only of pluripotential stem cells.
  • medium that does not promote differentiation and preferably medium that contains factors promoting growth only of pluripotential stem cells.
  • LIF Leukocyte Inhibitory Factor
  • somatic cells and particularly somatic stem cells are suitably used as starting material in the method of the invention.
  • haemopoietic cells and/or stem cells or even epithelial cells and/or stem cells are suitably used as starting material in the method of the invention.
  • a further aspect of the invention provides derivation of a pluripotent stem cell line, by:-
  • cells in the starting material posses a selectable marker that is differentially expressed in the desired target cells, the cells of higher relative potency, compared to its expression in the starting material.
  • selection for the selectable marker is used to purify the culture in respect of the target cells, and preferably substantially to remove all other cells.
  • Selectable markers suitable for application in this optional step may be present in the starting material or optionally introduced into the starting material by a range of techniques including but not limited to electroporation, lipofection, calcium phosphate precipitation, direct injection or ballistic missile.
  • a method used by the inventors and explained in more detail below resulted in a culture of cells containing cells of sufficiently high potency to act as progenitors for ectodermal, mesodermal and endodermal cell types. More generally, however, the invention is of application in obtaining cell lines of greater relative potency than the precursor cells.
  • the invention also provides for production of non-human animals using the pluripotential stem cells obtained as described.
  • a method of producing a non-human mammal comprising:
  • the resultant embryo can then be allowed to go to term, with birth of chimeric offspring having a range of cell types derived from that pluripotential cell including gametes.
  • a chimeric non-human animal, and a non-human mammal obtained by the fusion of gametes or the reproductive cloning of a cell produced by the method is a further feature of the invention.
  • the invention further relates to production of cells and organs and tissue, derived from cells and/or non-human embryos or animals obtained using the methods described.
  • the invention provides a method of generating a somatic mammalian cell or tissue, comprising:
  • the first and second cells are of different types. It is thus possible to start with a somatic cell of a selected patient mammal, dedifferentiate the cell to a second stem cell of higher relative potency and use that cell to derive a variety of different cells and/or tissues having the same genotype of the patient. This is of particular importance for human cell therapies, as rejection and disease transmission are thereby minimised and even potentially eliminated.
  • a hypothetical means of isolating pluripotent ES cells from a patient is to reprogramme a somatic nucleus from the patient using a Nuclear Transfer (NT) procedure wherein the nucleus is introduced into an enucleated human oocyte.
  • ES cells derived from any resulting embryos are used to isolate tissue of various types suitable for transplantation back into the original patient.
  • Other methods proposed include the introduction of oocyte or egg cytoplasm into somatic cells in an effort to reprogramme the somatic nucleus.
  • both the nuclear and cytoplasmic transfer/exchange procedures suffer from the disadvantage of the risk of disease transmission.
  • a preferred embodiment of the invention comprises:
  • the differentiation step can be suitably undertaken either in vitro or in vivo.
  • a further aspect of the invention provides a soma-derived pluripotential stem cell or soma-derived ES cell.
  • This cell is not obtained from an embryo or cells of the germline, but is obtained from a somatic cell and is nevertheless able to form all cells of the mesodermal, endodermal and ectodermal lineages.
  • This cell is a somatic cell-derived pluripotential stem cell.
  • These pluripotential stem cells of the invention are animal cells, preferably mammalian, and the term mammal is intended to encompass all such animals and include higher mammals, especially mice, rats, sheep, goats, cows, pigs, primates and humans.
  • a yet further aspect provides a somatic cell derived from a somatic cell-derived pluripotent stem cell.
  • This differentiated cell can be of mesodermal, endodermal or ectodermal type.
  • Another aspect of the invention is the conversion of the nucleus of a somatic cell into the nucleus of a cell that has greater developmental potential.
  • the invention provides a method of reprogramming a nucleus of a somatic cell, comprising:
  • a specific method described below in more detail comprises culturing said nuclear donor cell in the presence of conditions designed for maintenance of pluripotential stem cells; and isolating a cell or cells containing a nucleus or nuclei that have been reprogrammed. These conditions are preferably designed also to be relatively unsuitable for culture of somatic cells, even specifically unsuitable.
  • the methods described herein are thus suitable for reprogramming a nucleus of a somatic stem cell so as to be a nucleus of a soma-derived pluripotential stem cell or soma-derived ES cell.
  • the methods are characterised by steps comprising culturing said somatic stem cell in the presence of conditions designed for maintenance of an embryonic stem cell; and isolating a nucleus that has been reprogrammed.
  • a nucleus of a neural stem cell is reprogrammed so as to be a nucleus of a soma-derived pluripotential stem cell or soma-derived ES cell, by culturing the neural nuclear donor cell under conditions designed for maintenance of an ES cell and which conditions are not suitable for propagation of the neural stem cell.
  • a method of reprogramming a somatic cell so as to be a less differentiated somatic cell comprising culturing said somatic cell under conditions designed for maintenance of the less differentiated somatic cell.
  • a method of reprogramming a neural stem cell so as to be a non embryonic or soma-derived pluripotential stem cell or soma-derived ES cell comprises culturing said cell under conditions for maintenance of a pluripotential stem cell.
  • the invention provides the use of media for maintenance of an embryonic stem cell in conversion of a somatic stem cell into a soma-derived pluripotential stem cell or soma-derived ES cell, and use of embryonic stem cell culture conditions in conversion of a somatic stem cell into a soma-derived pluripotential stem cell or soma-derived ES cell.
  • the brains except forebrains were dissected from E14.5 embryos of Oct4-GFP mice (total 4 embryos) and were mechanically disassociated.
  • the cells were centrifuged for 5 min at 1 000 rpm and resuspended in N2B27 medium.
  • 5 x 1 05 cells were plated into PDL-laminin coated 5 cm Nunc. dish in 5 ml N2B27 medium with 20ng/ml bFGF, 1 00U/ml penicillin and 1 00 ⁇ g/ml streptomycin.
  • Two days after culture the medium was replaced with GMEM + 1 0%FCS medium supplemented with LIF and 20ng/ml bFGF.
  • the cells were left without changing medium for 5 days, then the medium as replaced with fresh
  • GMEM + 1 0%FCS medium supplemented with LIF GMEM + 1 0%FCS medium supplemented with LIF.
  • GMEM + 1 0%FCS medium supplemented with LIF GMEM + 1 0%FCS medium supplemented with LIF.
  • 7 of these aggregates were picked and trypsinized and plated in 0.1 % gelatin-coated 24-well plate in GMEM + 1 0%FCS medium supplemented with LIF.
  • One of these "cells aggregates” grew to be ES cell-like cells.
  • the cells in the 5cm dish were then trypsinized for 3 min at 37 °C and the dish was washed once with GMEM + 1 0%FCS medium. After trypsinization and washing, there were still some cells adhered to the bottom. These cells were then cultured in N2B27 medium with 20ng/ml bFGF without changing medium, and expanded to form numerous cell colonies.
  • Forebrains were dissected from E1 4.5 embryos of Oct4-GFP mice (total 4 embryos) and were mechanically disassociated. The cells were centrifuged for 5 min at 1 000 rpm and resuspended in N2B27 medium. 5 x 105 cells were plated into PDL-laminin coated 5 cm Nunc dish in 5 ml N2B27 medium with 20ng/ml bFGF, 1 00U/ml penicillin and 1 00 ⁇ g/ml streptomycin. Two days after culture, the cells were trypsinized and 1 X 1 06 cells were replated into non-coated 5cm petri dish in N2B27medium with bFGF. The cells grew in suspension and formed neurospheres (or aggregates) .
  • co-culture of ES cells and neural cells resulted in conversion of some neural cells into GFP-green colonies with ES cell morphology and a higher pluripotency consistent with pluripotent stem cells or so termed soma-derived pluripotent stem cells or soma-derived ES cells.
  • the brains except forebrains were dissected from E1 4.5 embryos of Oct4-GFP mice (total 4 embryos) and were mechanically disassociated.
  • the cells were centrifuged for 5 min at 1 000 rpm and resuspended in N2B27 medium.
  • 5 x 1 05 cells were plated into a PDL-laminin coated 5 cm Nunc. dish in 5 ml N2B27 medium with 20ng/ml bFGF, 1 00U/ml penicillin and 1 00 ⁇ g/ml streptomycin.
  • Two days after culture the medium was replaced with GMEM + 1 0%FCS medium supplemented with LIF and 20ng/ml bFGF.
  • the cells were left without changing medium for 5 days, then the medium was replaced with fresh
  • GMEM -I- 1 0%FCS medium supplemented with LIF GMEM -I- 1 0%FCS medium supplemented with LIF.
  • GMEM + 1 0%FCS medium supplemented with LIF 7 of these aggregates were picked and trypsinized and plated in 0.1 % gelatin-coated 24-well plates in GMEM + 1 0%FCS medium supplemented with LIF.
  • One of these "cells aggregates” grew to be pluripotent stem cells.
  • the cells in the 5cm dish were then trypsinized for 3 min at 37 °C and the dish was washed once with GMEM + 1 0%FCS medium. After trypsinization and washing, there were still some cells adhered to the bottom. These cells were then cultured in N2B27 medium with 20ng/ml bFGF without changing medium, and expanded to form numerous cell colonies.
  • soma-derived pluripotent stem cells displayed morphological characteristics and reporter gene expression profiles consistent with soma-derived pluripotent stem cells, or soma-derived pluripotent ES cells.
  • the cells were further tested for differentiation capacity in in vitro cell culture and found to be pluripotent.
  • the soma-derived pluripotent ES cells were shown to be capable of spontaneous differentiation into all three germ layers in the absence of additional cells including feeder or other co-culture cells frequently required for the induction of somatic cell differentiation in vitro.
  • a pluripotent mammalian stem cell capable of acting as a progenitor for generating a plurality of mammalian cell types, wherein the stem cell is derived from a second somatic stem cell of lower relative potency.

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Abstract

L'invention concerne des cellules souches de mammifères dérivées de cellules souches somatiques d'une puissance relativement inférieure. En particulier, les cellules souches pluripotentes sont obtenues à partir de cellules souches somatiques présentant une puissance relative inférieure. L'invention traite également de lignées cellulaires souches de mammifères qui peuvent se régénérer in vitro avec stabilité, lesquelles lignées sont dérivées de cellules souches somatiques de puissance relative inférieure. En outre les procédés selon l'invention sont prévus pour produire des cellules souches de mammifères, des lignées cellulaires et des cellules de mammifères non humains dérivées de cellules souches somatiques présentant une puissance relative inférieure. L'invention traite aussi de procédés permettant de reprogrammer un noyau d'une cellule somatique afin de devenir une cellule souche moins différentiée.
PCT/GB2001/004747 2000-10-26 2001-10-26 Cellules souches pluripotentes WO2002034890A2 (fr)

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WO2002102997A2 (fr) * 2000-11-30 2002-12-27 Stemron Inc. Cellules souches homozygotes isolees, cellules differenciees derivees de ces cellules souches, et materiaux et procedes permettant de les fabriquer et de les utiliser
EP1384775A1 (fr) * 2002-07-26 2004-01-28 Food Industry Research and Development Cellules somatiques pluripotentes
US7015037B1 (en) 1999-08-05 2006-03-21 Regents Of The University Of Minnesota Multiponent adult stem cells and methods for isolation
US7838289B2 (en) 2001-02-14 2010-11-23 Abt Holding Company Assay utilizing multipotent adult stem cells
US8252280B1 (en) 1999-08-05 2012-08-28 Regents Of The University Of Minnesota MAPC generation of muscle
US8409859B2 (en) 2005-10-14 2013-04-02 Regents Of The University Of Minnesota Differentiation of non-embryonic stem cells to cells having a pancreatic phenotype
US8609406B2 (en) 2010-08-24 2013-12-17 Regents Of The University Of Minnesota Non-static suspension culture of cell aggregates
US8609412B2 (en) 1999-08-05 2013-12-17 Regents Of The University Of Minnesota Mapc generation of lung tissue
US9005964B2 (en) 2006-11-24 2015-04-14 Regents Of The University Of Minnesota Endodermal progenitor cells
US9057051B2 (en) 2008-10-31 2015-06-16 Katholieke Universiteit Leuven Optimized methods for differentiation of cells into cells with hepatocyte progenitor phenotypes, cells produced by the methods, and methods of using the cells
US9090878B2 (en) 2010-06-17 2015-07-28 Katholieke Universiteit Leuven Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells
US9764044B2 (en) 2002-11-27 2017-09-19 Abt Holding Company Homologous recombination in multipotent adult progenitor cells
US9861660B2 (en) 2013-04-12 2018-01-09 Saverio LaFrancesca Organs for transplantation
US9937208B2 (en) 2010-05-12 2018-04-10 Abt Holding Company Modulation of splenocytes in cell therapy
US10006004B2 (en) 1999-08-05 2018-06-26 Abt Holding Company Multipotent adult stem cells and methods for isolation
US10253297B2 (en) 2008-01-18 2019-04-09 Regents Of The University Of Minnesota Stem cell aggregates and methods for making and using
US10638734B2 (en) 2004-01-05 2020-05-05 Abt Holding Company Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof

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US8252280B1 (en) 1999-08-05 2012-08-28 Regents Of The University Of Minnesota MAPC generation of muscle
US10226485B2 (en) 1999-08-05 2019-03-12 Abt Holding Company Multipotent adult stem cells and methods for isolation
US10006004B2 (en) 1999-08-05 2018-06-26 Abt Holding Company Multipotent adult stem cells and methods for isolation
US7015037B1 (en) 1999-08-05 2006-03-21 Regents Of The University Of Minnesota Multiponent adult stem cells and methods for isolation
US9974809B2 (en) 1999-08-05 2018-05-22 Abt Holding Company Multipotent adult stem cells and methods for isolation
US9974811B2 (en) 1999-08-05 2018-05-22 Abt Holding Company Multipotent adult stem cells and methods for isolation
US9974810B2 (en) 1999-08-05 2018-05-22 Abt Holding Company Multipotent adult stem cells and methods for isolation
US7659118B2 (en) 1999-08-05 2010-02-09 Abt Holding Company Multipotent adult stem cells
US8609412B2 (en) 1999-08-05 2013-12-17 Regents Of The University Of Minnesota Mapc generation of lung tissue
WO2002102997A3 (fr) * 2000-11-30 2003-12-24 Stemron Inc Cellules souches homozygotes isolees, cellules differenciees derivees de ces cellules souches, et materiaux et procedes permettant de les fabriquer et de les utiliser
WO2002102997A2 (fr) * 2000-11-30 2002-12-27 Stemron Inc. Cellules souches homozygotes isolees, cellules differenciees derivees de ces cellules souches, et materiaux et procedes permettant de les fabriquer et de les utiliser
US7838289B2 (en) 2001-02-14 2010-11-23 Abt Holding Company Assay utilizing multipotent adult stem cells
KR100879079B1 (ko) * 2002-07-26 2009-01-15 푸드 인더스트리 리써치 앤드 디벨롭먼트 인스티튜트 다능성 체세포
CN1470633B (zh) * 2002-07-26 2010-09-29 食品工业发展研究所 多能体细胞
JP2004057198A (ja) * 2002-07-26 2004-02-26 Food Industry Research & Development Inst 多能性体細胞
SG129236A1 (en) * 2002-07-26 2007-02-26 Food Industry Res & Dev Inst Somatic pluripotent cells
AU2002302089B2 (en) * 2002-07-26 2008-08-14 Food Industry Research And Development Institute Somatic Pluripotent Cells
US7422736B2 (en) 2002-07-26 2008-09-09 Food Industry Research And Development Institute Somatic pluripotent cells
EP1384775A1 (fr) * 2002-07-26 2004-01-28 Food Industry Research and Development Cellules somatiques pluripotentes
US10583202B2 (en) 2002-11-27 2020-03-10 Abt Holding Company Homologous recombination in multipotent adult progenitor cells
US9764044B2 (en) 2002-11-27 2017-09-19 Abt Holding Company Homologous recombination in multipotent adult progenitor cells
US10638734B2 (en) 2004-01-05 2020-05-05 Abt Holding Company Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof
US8409859B2 (en) 2005-10-14 2013-04-02 Regents Of The University Of Minnesota Differentiation of non-embryonic stem cells to cells having a pancreatic phenotype
US9005964B2 (en) 2006-11-24 2015-04-14 Regents Of The University Of Minnesota Endodermal progenitor cells
US10253297B2 (en) 2008-01-18 2019-04-09 Regents Of The University Of Minnesota Stem cell aggregates and methods for making and using
US10457914B2 (en) 2008-10-31 2019-10-29 Katholieke Universiteit Leuven Optimized methods for differentiation of cells into cells with hepatocyte and hepatocyte progenitor phenotypes, cells produced by the methods, and methods for using the cells
US9057051B2 (en) 2008-10-31 2015-06-16 Katholieke Universiteit Leuven Optimized methods for differentiation of cells into cells with hepatocyte progenitor phenotypes, cells produced by the methods, and methods of using the cells
US9937208B2 (en) 2010-05-12 2018-04-10 Abt Holding Company Modulation of splenocytes in cell therapy
US10758570B2 (en) 2010-05-12 2020-09-01 Abt Holding Company Modulation of splenocytes in cell therapy
US9777258B2 (en) 2010-06-17 2017-10-03 Katholieke Universiteit Leuven Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the method, and methods for using the cells
US9090878B2 (en) 2010-06-17 2015-07-28 Katholieke Universiteit Leuven Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells
US9447380B2 (en) 2010-08-24 2016-09-20 Regents Of The University Of Minnesota Non-static suspension culture of cell aggregates
US8609406B2 (en) 2010-08-24 2013-12-17 Regents Of The University Of Minnesota Non-static suspension culture of cell aggregates
US9861660B2 (en) 2013-04-12 2018-01-09 Saverio LaFrancesca Organs for transplantation
US11071752B2 (en) 2013-04-12 2021-07-27 Abt Holding Company Organs for transplantation

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