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WO2013010965A1 - Génération de cellules mésodermiques à partir de cellules souches pluripotentes - Google Patents

Génération de cellules mésodermiques à partir de cellules souches pluripotentes Download PDF

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Publication number
WO2013010965A1
WO2013010965A1 PCT/EP2012/063853 EP2012063853W WO2013010965A1 WO 2013010965 A1 WO2013010965 A1 WO 2013010965A1 EP 2012063853 W EP2012063853 W EP 2012063853W WO 2013010965 A1 WO2013010965 A1 WO 2013010965A1
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cells
mps
cardiac
eomes
day
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PCT/EP2012/063853
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Pierre Vanderhaeghen
Jelle Van Den Ameele
Luca TIBERI
Cédric BLANPAIN
Antoine Bondue
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Universite Libre De Bruxelles
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • 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
    • C12N2501/60—Transcription factors
    • 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
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Embryonic stem (ES) cells including human embryonic stem (hES) cells spontaneously form spherical structures called embryoid bodies when they are cultured in suspension in serum-containing medium. Within these mixed populations of cells contracting areas with functional properties of cardiomyocytes can be found around day 8 of culture.
  • the cardiomyocyte yield using this approach can be rather low and/or inconsistent due to the heterogeneity among the aggregates. Furthermore, this method relies on culture media containing animal serum, which is a largely undefined component subject to batch-to- batch variations. Therefore, this approach does not readily lend itself to being a clinically useful and reproducible.
  • mesodermal cells particularly cardiac cells, more particularly cardiomyocytes, preferably techniques which are robust, simple, reproducible and/or which allow for consistent, and preferably comparatively high, yields of such cells.
  • Eomes Eomesodermin
  • cTNT cardiac-specific isoform of TroponinT
  • Eomes was initially identified as a key early gene in Xenopus mesoderm differentiation (Ryan et al., 1996. Cell 87: 989- 1000), in mammalian systems such as murine embryos (Arnold et al., 2008. Development 135: 501 -51 1 ; Teo et al., 201 1 . Genes Dev 25: 238-250) or embryonic stem cells (David et al., 201 1 . Cardiovasc Res; Lindsley et al., 2008. Cell Stem Cell 3:55-68; Teo et al., 201 1 .
  • the Eomes activity may be provided from day 2 to day 4 or from day 2 to day 3 following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent, and the duration of the methods may be between 9 and 1 1 days, even more preferably about 10 days following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.
  • mammalian pluripotent stem cell or "mPS” cell generally refers to a pluripotent stem cell of mammalian origin.
  • the terms “mammal” and “mammalian” refer to any animal classified as such, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes.
  • the mPS cells may be derived from a non-human mammal.
  • the present methods and protocols may preferably depart from pluripotent stem cell populations (e.g., mPS or hPS cell populations) which are "undifferentiated", i.e., wherein a substantial proportion (for example, at least about 60%, preferably at least about 70%, even more preferably at least about 80%, still more preferably at least about 90% and up to 100%) of cells in the stem cell population display characteristics (e.g., morphological features and/or markers) of undifferentiated mPS cells, clearly distinguishing them from cells undergoing differentiation.
  • pluripotent stem cell populations e.g., mPS or hPS cell populations
  • undifferentiated i.e., wherein a substantial proportion (for example, at least about 60%, preferably at least about 70%, even more preferably at least about 80%, still more preferably at least about 90% and up to 100%) of cells in the stem cell population display characteristics (e.g., morphological features and/or markers) of undifferentiated mPS cells, clearly distinguishing
  • Clumps or clusters of mPS cells present in such cell suspension may contain on average, e.g., between >1 and 1000 cells, between 1 and 500 cells, between 1 and 100 cells, between 1 and 50 cells or between 1 and 20 cells, e.g., about 5 cells, about 10 cells or about 15 cells.
  • Non-adherent matter may comprise, for example, cells that have not attached to the adherent substrate, non-viable or dead cells, cell debris, etc.
  • Non-adherent matter may be suitably removed by exchanging medium within the culture system, optionally including one or more washes of the attached cells with suitable medium or isotonic buffer.
  • suitable medium or isotonic buffer for example, cells from the mPS suspension which have adhered to the substrate surface are selected for further culturing.
  • the present methods comprise as a further step exposing the mPS cells that have attached to the adherent substrate to conditions (e.g., culturing the mPS cells that have attached to the adherent substrate in a medium) "permissive to differentiation of the mPS cells", which means that the medium may for example not contain components, in sufficient quantity, which would suppress mPS differentiation or would cause maintenance and/or proliferation of the mPS cells in undifferentiated or substantially undifferentiated state.
  • such components absent from the medium may include leukaemia inhibitory factor (LIF), basic fibroblast growth factor (b-FGF), and/or embryonic fibroblast feeders or conditioned medium of such feeders, depending on the particular mPS cell type.
  • LIF leukaemia inhibitory factor
  • b-FGF basic fibroblast growth factor
  • embryonic fibroblast feeders or conditioned medium of such feeders, depending on the particular mPS cell type.
  • Plasma is as conventionally defined. Plasma is usually obtained from an isolated sample of whole blood, provided or contacted with an anticoagulant, (e.g., heparin, citrate, oxalate or EDTA). Subsequently, cellular components of the blood sample are separated from the liquid component (plasma) by an appropriate technique, typically by centrifugation.
  • an anticoagulant e.g., heparin, citrate, oxalate or EDTA.
  • Exemplary human Eomes protein sequence may be as annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NP_005433.2 (sequence version 2, entered August 29, 2002).
  • Exemplary human Eomes mRNA (cDNA) sequence may be as annotated under NCBI Genbank accession number NM_005442.2 (sequence version 2, entered August 29, 2002).
  • Exemplary mouse Eomes protein sequence may be as annotated under NCBI Genbank accession number NP_034266.2 (isoform 1 ) (sequence version 2, entered December 23, 2005) or NP_001 158261 .1 (isoform 2) (sequence version 1 , entered September 18, 2009).
  • Exemplary mouse Eomes mRNA (cDNA) sequence may be as annotated under NCBI Genbank accession number NM_010136.3 (transcript variant 1 ) (sequence version 3, entered September 18, 2009) or NM_001 164789.1 (transcript variant 2) or (sequence version 1 , entered September 18, 2009).
  • said mesodermal cells may be positive for smooth muscle actin. In a further embodiment, said mesodermal cells may be positive for at least one vascular marker, indicating that said mesodermal cells may have vascular smooth muscle cell identity.
  • a skilled person will conclude the presence or evidence of a distinct signal (e.g., antibody- detectable or detection by reverse transcription polymerase chain reaction) for that marker when carrying out the appropriate measurement compared to suitable controls (e.g., cells known or expected not to express the marker, i.e., to be negative for said marker, i.e., "negative control").
  • a distinct signal e.g., antibody- detectable or detection by reverse transcription polymerase chain reaction
  • carrier or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like.
  • buffers such as, e.g., neutral buffered saline or phosphate buffered saline
  • solubilisers colloids
  • dispersion media vehicles
  • the cell preparation can be administered on a support, scaffold, matrix or material to provide improved tissue regeneration.
  • the material can be a granular ceramic, or a biopolymer such as gelatine, collagen, or fibrinogen.
  • Porous matrices can be synthesized according to standard techniques (e.g., Mikos et al., Biomaterials 14: 323, 1993; Mikos et al., Polymer 35:1068, 1994; Cook et al., J. Biomed. Mater. Res. 35:513, 1997).
  • Such support, scaffold, matrix or material may be biodegradable or non-biodegradable.
  • Results were normalized to the housekeeping gene TBP; primers used are summarized in Table 1 .

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Abstract

Cette invention concerne des procédés in vitro de production de cellules mésodermiques, plus particulièrement, des cellules cardiaques, plus particulièrement encore des cardiomyocytes, à partir de cellules souches pluripotentes de mammifères (mPS) à l'aide d'Eomes dans des conditions où la signalisation activine est sensiblement absente. Cette invention concerne en outre les cellules mésodermiques ou cardiaques ainsi obtenues; des populations cellulaires comprenant ces cellules; des compositions comprenant lesdites cellules mésodermiques ou cardiaques ou lesdites populations cellulaires; et leurs utilisations en aval, par exemple, dans des applications médicales telles que la cellulothérapie, le criblage de médicaments ou la recherche.
PCT/EP2012/063853 2011-07-15 2012-07-13 Génération de cellules mésodermiques à partir de cellules souches pluripotentes WO2013010965A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3444331A1 (fr) * 2017-08-14 2019-02-20 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Génération facilitée de cardiomyocytes par programmation future de cellules souches pluripotentes humaines
EP3740568A4 (fr) * 2018-01-18 2021-10-06 The Regents of The University of Michigan Compositions et procédés consistant à dériver des cellules de lignée mésodermique et des organoïdes de tissu mixtes à partir de cellules souches embryonnaires
CN114350599A (zh) * 2022-01-14 2022-04-15 内蒙古大学 一种增强牛胚胎干细胞的多能性和代谢活力的培养体系和培养方法

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EP3444331A1 (fr) * 2017-08-14 2019-02-20 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Génération facilitée de cardiomyocytes par programmation future de cellules souches pluripotentes humaines
WO2019034615A1 (fr) * 2017-08-14 2019-02-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Génération facilitée de cardiomyocytes par programmation vers l'avant de cellules souches pluripotentes humaines
EP3740568A4 (fr) * 2018-01-18 2021-10-06 The Regents of The University of Michigan Compositions et procédés consistant à dériver des cellules de lignée mésodermique et des organoïdes de tissu mixtes à partir de cellules souches embryonnaires
US12054742B2 (en) 2018-01-18 2024-08-06 Regents Of The University Of Michigan Compositions and methods to derive mesodermal lineage cells and mixed tissue organoids from embryonic stem cells
CN114350599A (zh) * 2022-01-14 2022-04-15 内蒙古大学 一种增强牛胚胎干细胞的多能性和代谢活力的培养体系和培养方法
CN114350599B (zh) * 2022-01-14 2024-01-19 内蒙古大学 一种增强牛胚胎干细胞的多能性和代谢活力的培养体系和培养方法

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