WO2015125662A1 - Procédé de stimulation de la différenciation des cellules souches au moyen d'un milieu reformulé contenant des acides aminés, cellules souches traitées à l'aide dudit procédé, et milieu - Google Patents

Procédé de stimulation de la différenciation des cellules souches au moyen d'un milieu reformulé contenant des acides aminés, cellules souches traitées à l'aide dudit procédé, et milieu Download PDF

Info

Publication number
WO2015125662A1
WO2015125662A1 PCT/JP2015/053607 JP2015053607W WO2015125662A1 WO 2015125662 A1 WO2015125662 A1 WO 2015125662A1 JP 2015053607 W JP2015053607 W JP 2015053607W WO 2015125662 A1 WO2015125662 A1 WO 2015125662A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
cells
differentiation
cell
pluripotent stem
Prior art date
Application number
PCT/JP2015/053607
Other languages
English (en)
Japanese (ja)
Inventor
昭苑 粂
文夫 遠藤
伸明 白木
恭子 白木
和彦 粂
一徳 馬渡
Original Assignee
国立大学法人熊本大学
味の素株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人熊本大学, 味の素株式会社 filed Critical 国立大学法人熊本大学
Priority to JP2016504051A priority Critical patent/JP6351018B2/ja
Publication of WO2015125662A1 publication Critical patent/WO2015125662A1/fr

Links

Images

Classifications

    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids

Definitions

  • the present invention relates to a technique for inducing differentiation of pluripotent stem cells. More specifically, the present invention relates to a method and a medium for improving the differentiation induction efficiency of pluripotent stem cells such as ES cells and iPS cells.
  • ES cells embryonic stem cells
  • iPS cell artificial pluripotent stem cell
  • ES cells embryonic stem cells
  • iPS pluripotent stem cells
  • ES cells have a unique transcriptional circuit that maintains a pluripotent state. These cells are in a specific epigenetic state that is ready for cell fate decisions.
  • various histone methylation modes regulate ES cell pluripotency and plasticity.
  • ES cells are also characterized by a high growth rate and a shortened G1 phase. These unique molecular properties distinguish ES cells and iPS cells from somatic cells.
  • ES cells are in a special metabolic state.
  • ES cells and iPS cells are particularly dependent on glycolysis, while somatic cells utilize mitochondrial oxidative phosphorylation for energy production.
  • This unique metabolic requirement of stem cells appears to play a causative role rather than a result of gaining pluripotency.
  • Metabolism and intracellular signaling are linked, and these two processes have been shown to regulate each other and regulate cellular activities such as regulation of cell survival, proliferation, and stem cell function (Non-patent document 1: Wellen and Thompson, 2012) (Non-patent document 2: Takubo et al., 2013).
  • Non-patent Document 3 Ogaki et al., 2013
  • Non-patent document 4 Shiraki et al., Genes Cells, 2008
  • Non-patent document 5 Shiraki et al., PLoS One, 2011
  • Non-patent document 6 Shiraki et al., Stem Cell, 2008
  • Non-patented Literature 7 Osafune et al., 2008
  • Non-patent Literature 8 Kajiwara et al., 2012
  • This problem is a problem that must be overcome in regenerative medicine using ES / iPS cells.
  • a number of established ES / iPS cell lines are selected in advance from the ES / iPS cell lines that have good differentiation efficiency into the target tissue, and then differentiation induction is performed. It takes a lot of study period to prepare. If a method capable of overcoming this difference between cell lines can be developed, the time lag of cell line selection can be eliminated and efficient differentiation can be expected.
  • the essential amino acid methionine is a differentiation regulator of human iPS cells, and selecting undifferentiated cells by removing methionine from the differentiation-inducing medium during a certain period of the endoderm differentiation process. Cell death was induced, and efficient endoderm differentiation was succeeded (Patent Document 1: WO2012 / 056997). However, the above problem of cell line selection has not been solved.
  • An object of the present invention is to provide a method and a medium for improving the differentiation induction efficiency of stem cells such as ES cells and iPS cells.
  • an object of the present invention is to provide a method and a medium capable of inducing efficient differentiation of stem cells by eliminating or reducing the difference in differentiation efficiency between cell lines of stem cells.
  • the present inventors have cultured stem cells in a medium from which methionine has been removed (undifferentiated maintenance medium) before inducing differentiation by incubating in a differentiation induction medium. Thus, it was found that differentiation can be induced efficiently, and the present invention was completed.
  • the present invention includes the following. (1) A method for inducing differentiation of a pluripotent stem cell derived from a mammal, (A) a step of culturing the pluripotent stem cell in an undifferentiated maintenance medium of pluripotent stem cells not containing methionine; and (b) a differentiation-inducing medium containing the pluripotent stem cell cultured in the undifferentiated maintenance medium.
  • a differentiation induction method comprising: (2) The differentiation-inducing method according to (1), wherein the mammal-derived pluripotent stem cells are human or mouse-derived ES cells, or human or mouse-derived iPS cells.
  • the differentiation induction method according to (1) or (2) comprising culturing the cells in the undifferentiated maintenance medium for at least 5 hours.
  • the differentiation induction method according to (3) comprising culturing the cells in the undifferentiated maintenance medium for at least 10 hours.
  • the differentiation induction method according to any one of (1) to (4) comprising culturing the cells in the undifferentiated maintenance medium for a period not exceeding 24 hours.
  • the pluripotent stem cell derived from a mammal cultured in an undifferentiated maintenance medium for pluripotent stem cells not containing methionine is further induced to differentiate into any one of endoderm, mesoderm or ectoderm, 1) The differentiation induction method according to any one of (5).
  • any of (1) to (6), wherein the pluripotent stem cell is a stem cell resistant to differentiation into endoderm, and the differentiation induction medium is a differentiation induction medium into endoderm The method described in one.
  • the human iPS cells cultured in an undifferentiated maintenance medium of pluripotent stem cells not containing methionine are further cultured in a pancreatic differentiation induction medium to induce differentiation into pancreatic cells having insulin-producing ability.
  • the differentiation induction method according to any one of (5) (11) The human iPS cells cultured in an undifferentiated maintenance medium of pluripotent stem cells not containing methionine are further cultured in a liver differentiation induction medium to induce differentiation into liver cells having albumin secretion ability ( 2) The differentiation induction method according to any one of (5).
  • the mammal-derived pluripotent stem cell is a human or mouse-derived ES cell, or a human or mouse-derived iPS cell.
  • the cell according to (12) or (13), wherein the cell is a cell cultured in the undifferentiated maintenance medium for at least 5 hours.
  • a pluripotent stem cell differentiation induction medium kit comprising the undifferentiation maintenance medium according to (18) or (19) and a differentiation induction medium for pluripotent stem cells.
  • the present invention is a method capable of promoting differentiation into not only endoderm but also mesoderm / ectodermal by culturing pluripotent stem cells in an amino acid composition-changing medium, specifically, a methionine-removing medium in an undifferentiated state. It is.
  • FIG. 2 shows a schematic diagram of methionine metabolism in ES cells / iPS cells. It shows cell growth inhibition and cell number change when 201B7 cells are cultured for 2 days in a maintenance medium from which specific amino acids have been removed. Effects of culturing 201B7 cells for 2 days in maintenance medium from which Met or methyl donor (MD: methionine, folic acid, vitamin B12, betaine and choline) has been removed, and various methionine related metabolites (Met) on Met removal or MD removal , SAM, SAH, Hcy, Cys, and MTA). The upper row shows the cell morphology, and the lower row shows the cell number.
  • MD methionine, folic acid, vitamin B12, betaine and choline
  • the gene expression profiles of undifferentiated hiPS cells cultured for 5 hours in a complete medium or a Met-removed medium are compared, and genes whose difference is 1.5 times or more are shown. It is the figure which showed the change of the transcription
  • Methionine (Met) is an essential amino acid.
  • the metabolism of methionine is shown in FIG.
  • One of its important metabolites is S-adenosylmethionine (SAM), which is produced via metabolic reaction intermediates catalyzed by methionine adenosyltransferase (MAT).
  • SAM is an important methyl donor for gene regulation, and protein methylation, including DNA methylation and histone methylation, is catalyzed by methyltransferase using SAM as the methyl donor.
  • S-adenosylhomocysteine is produced as a product of transmethylation by methyltransferases including DNA methyltransferases (DNMTs) and then converted to homocysteine (Hcy) by SAH hydrolase (AHCY).
  • DNMTs DNA methyltransferases
  • Hcy homocysteine
  • AHCY SAH hydrolase
  • Homocysteine is remethylated and converted to methionine by 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR).
  • MTR 5-methyltetrahydrofolate-homocysteine methyltransferase
  • the reaction requires folic acid and vitamin B12 or betaine homocysteine methyltransferase (BHMT) as methyl group donors.
  • Hcy is converted to cystathionine by cystathionine ⁇ -synthase (CBS) and then further metabolized to cysteine by cis-tyonase (CTH).
  • CBS cystathionine ⁇ -synthase
  • CTH cis-tyonase
  • methionine salvage pathway in which S-methyl-5'-thioadenosine (MTA), a byproduct of polyamine biosynthesis, is enzymatically converted to methionine via several enzymatic steps.
  • the “pluripotent stem cell” used in the present invention refers to a cell having self-replicating ability, capable of being cultured in vitro, and capable of differentiating into cells constituting an individual. Specifically, embryonic stem cells (ES cells), fetal primordial germ cell-derived pluripotent stem cells (GS cells), somatic cell-derived induced pluripotent stem cells (iPS cells), and the like, In the present invention, iPS cells or ES cells are particularly preferably used, and human iPS cells and human ES cells are particularly preferable.
  • the ES cell used in the present invention is not particularly limited as long as it is an ES cell derived from a mammal.
  • mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cows, horses, goats, monkeys or humans, preferably mice or humans, more preferably Is a human.
  • ES cells are cultured by cultivating fertilized eggs at the blastocyst stage together with feeder cells, dissociating the proliferated cells derived from the inner cell mass, and further repeating the transplanting operation. It can be established as a stock.
  • ES cells are often obtained from fertilized eggs, but can be obtained from, for example, adipose tissue, placenta, testis cells other than fertilized eggs, and any ES cell is the subject of the present invention.
  • iPS cells artificial pluripotent stem cells
  • transcription factors differentiation
  • fibroblasts e.g, fibroblasts
  • a pluripotent factor is a cell that has acquired differentiation pluripotency equivalent to that of an ES cell by introducing a gene. Many factors have been reported as the “pluripotency factor” and are not particularly limited.
  • the Oct family eg, Oct3 / 4
  • the Sox family eg, Sox2, Sox1, Sox3, Sox15 and Sox17
  • Klf family eg, Klf4, Klf2, etc.
  • Myc family eg, c-Myc, N-Myc, L-Myc, etc.
  • Nanog LIN28 and the like.
  • the “undifferentiated cell” used in the present invention means a cell having pluripotency, and in the context, differentiation induction occurs even after differentiation induction treatment of a pluripotent stem cell such as an ES cell or iPS cell. It means cells that continue to have pluripotency.
  • mammalian ES cell culture methods can be performed by conventional methods.
  • mouse fetal fibroblasts are used as feeder cells, leukemia inhibitory factor, KSR (knockout serum substitute), fetal bovine serum (FBS), non-essential amino acids, L-glutamine, pyruvate, penicillin, streptomycin It can be maintained using a medium supplemented with ⁇ -mercaptoethanol, for example, DMEM medium.
  • iPS cells can also be cultured by a conventional method.
  • MEF cells as feeder cells and using a medium containing bFGF, KSR (knockout serum substitute), non-essential amino acids, L-glutamine, penicillin, streptomycin, ⁇ -mercaptoethanol, such as DMEM / F12 medium
  • KSR knockout serum substitute
  • non-essential amino acids L-glutamine
  • penicillin streptomycin
  • ⁇ -mercaptoethanol such as DMEM / F12 medium
  • Medium for culturing pluripotent stem cells includes “maintenance medium” or “undifferentiation maintenance medium” and “differentiation medium” or “differentiation induction medium” for pluripotent stem cells.
  • “maintenance medium” or “undifferentiated maintenance medium” have the same meaning as each other and can be used interchangeably.
  • “differentiation medium” or “ The “differentiation induction medium” has the same meaning as each other and can be used interchangeably.
  • the maintenance medium is a medium for maintaining and culturing pluripotent stem cells in an undifferentiated state, and when maintaining and culturing simply to preserve cells, and maintaining and culturing cells prior to differentiation induction
  • the maintenance medium used in the present invention means a medium for maintaining and culturing pluripotent stem cells prior to induction of differentiation of pluripotent stem cells.
  • Such a medium is not limited thereto, but can contain factors that contribute to maintenance of undifferentiation such as leukemia inhibitory factor (LIF) and bFGF used for maintenance culture of ES / iPS cells.
  • LIF leukemia inhibitory factor
  • bFGF used for maintenance culture of ES / iPS cells.
  • the undifferentiated maintenance culture of pluripotent stem cells such as ES cells or iPS cells in the present invention includes both a culture system containing feeder cells and a feeder-free culture system.
  • feeder cells include, but are not limited to, mmcMEF cells.
  • An example of the feeder-free culture system is Matrigel, but is not limited thereto.
  • a differentiation medium is a medium for inducing differentiation of pluripotent stem cells into either endoderm, mesoderm, or ectoderm, and a different medium is generally used depending on the desired differentiation direction. is there.
  • the medium used in the present invention can be prepared using a medium used for culturing animal cells as a basal medium.
  • a basal medium for example, BME medium, BGjB medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM medium, IMDM medium, Medium 199 medium, Eagles MEM medium, ⁇ MEM medium, DMEM medium, Ham medium, RPMI 1640 medium, Fischer 's medium, mixed media thereof, and the like can be mentioned, but there is no particular limitation as long as it can be used for animal cell culture.
  • the medium used in the present invention may be a serum-containing medium or a serum-free medium, but from the viewpoint of ensuring the safety of cell transplantation by eliminating heterogeneous components and / or sufficiently controlling Met removal, A serum-free medium is preferred.
  • the serum-free medium means a medium that does not contain unconditioned or unpurified serum, and a medium that contains purified blood-derived components or animal tissue-derived components (for example, growth factors) is serum-free. It shall correspond to the culture medium.
  • Examples of such serum-free medium include serum-free medium supplemented with an appropriate amount (for example, 1-20%) of commercially available KSR, serum-free medium supplemented with insulin and transferrin, medium supplemented with cell-derived factors, and the like. However, it is not limited to these.
  • the medium used in the present invention may or may not contain a serum replacement.
  • the serum substitute contains, for example, albumin (eg, lipid-rich albumin), transferrin, fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol or 3′-thiolglycerol, or an equivalent thereof as appropriate. Things are possible.
  • albumin eg, lipid-rich albumin
  • transferrin eg, transferrin
  • fatty acid e.g., transferrin, fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol or 3′-thiolglycerol, or an equivalent thereof as appropriate. Things are possible.
  • Such a serum substitute can be prepared, for example, by the method described in WO 93/30679, but a commercially available product can also be used. Examples of such commercially available serum substitutes include the above KSR.
  • the medium used in the present invention also includes any fatty acid or lipid, amino acid (for example, non-essential amino acid), vitamin, growth factor, cytokine, antioxidant, 2-mercaptoethanol, pyruvic acid, buffer, inorganic salts, and the like. It can contain ingredients. However, when methionine is removed from the medium according to the present invention, the medium does not contain methionine.
  • the term ⁇ differentiation '' or ⁇ differentiation induction '' of pluripotent stem cells as used in the present invention is used in the sense including differentiation induction of pluripotent stem cells into either endoderm, mesoderm or ectoderm, Furthermore, they are also used in the meaning including differentiation into organs or organ cells constituting any living body.
  • the differentiation induction of pluripotent stem cells, such as ES cells or iPS cells in the present invention includes both culture systems containing feeder cells and feeder-free culture systems. Examples of feeder cells include mmcM15 cells, but are not limited thereto. Examples of feeder-free culture systems include, but are not limited to, a pseudo basement membrane sBM (synthesized Basement Membrane substratum).
  • pluripotent stem cells such as ES cells and iPS cells
  • the pluripotent stem cells are treated with a maintenance medium (in this specification, containing methionine Cultivated (or maintained) by a medium having no medium ”,“ methionine (Met) -removed medium ”or“ ⁇ Met medium ”)
  • a maintenance medium in this specification, containing methionine Cultivated (or maintained) by a medium having no medium ”,“ methionine (Met) -removed medium ”or“ ⁇ Met medium
  • the induction efficiency can be improved.
  • culturing (or maintaining) pluripotent stem cells in ⁇ Met medium means placing pluripotent stem cells in ⁇ Met medium, and when cells proliferate or simply do not proliferate, they are simply placed in the medium for a certain period of time. It is meant to include any case.
  • the “methionine-removing medium” referred to in the present invention is a maintenance medium that does not contain methionine for culturing undifferentiated pluripotent stem cells in an undifferentiated state, which can increase the differentiation induction efficiency of undifferentiated pluripotent stem cells. Means. Therefore, even a medium for culturing pluripotent cells that does not contain methionine, a so-called differentiation medium that actively induces differentiation of pluripotent stem cells is included in the methionine removal medium referred to in this specification. Absent.
  • the methionine-removing medium referred to in the present specification is not particularly limited as long as it can maintain stem cells in culture and does not contain methionine.
  • a medium obtained by removing methionine from a known stem cell maintenance medium is used. I can give you.
  • it is a methionine-removing medium comprising a CSTI7 (Cell Science Laboratory) medium.
  • the differentiation medium referred to in the present specification is not particularly limited.
  • endoderm differentiation medium corresponds to, for example, differentiation into body cavities, blood vessels, heart, etc.
  • a mesoderm differentiation medium is applicable.
  • an ectoderm differentiation medium is applicable.
  • a medium that induces differentiation into specific organ cells for example, a liver differentiation medium or a pancreatic differentiation medium is also included.
  • the differentiation medium that can be combined with the methionine-removing medium of the present invention is not particularly limited, and in addition to a general differentiation medium, there is also a differentiation medium from which the specific amino acids reported by the present inventors described in Patent Document 1 are removed. Can be used.
  • the “methionine-free medium” or “methionine-removed medium” of the present invention includes the case where the medium contains a trace amount in addition to the case where it contains no methionine.
  • the medium contained in a trace amount means a medium that can increase differentiation induction efficiency by culturing undifferentiated pluripotent stem cells prior to differentiation induction because it contains methionine only at a low concentration.
  • the term “not containing methionine” in the present invention means that no methionine is contained in the medium at all, 10 ⁇ M or less, preferably 5 ⁇ M or less, more preferably 1 ⁇ M or less, and most preferably 0. It means containing 1 ⁇ M or less.
  • methionine is contained in a maintenance medium for pluripotent stem cells at a concentration of 100 ⁇ M or more.
  • the phrase “culturing pluripotent stem cells in a methionine-removing medium” in the present invention means culturing or maintaining pluripotent stem cells in an undifferentiated maintenance medium from which methionine has been removed for at least a certain time.
  • the lower limit of the time of culturing or maintaining in the methionine-removed medium is not particularly limited as long as the desired differentiation induction efficiency can be achieved in the subsequent differentiation induction, preferably at least 3 hours, particularly preferably at least 5 hours, more preferably at least 10 hours.
  • cultivating or maintaining with a methionine removal culture medium becomes like this. Preferably it is 24 hours or less.
  • differentiation induction efficiency can be improved by culturing pluripotent stem cells in a methionine-removing medium for the above period and then inducing differentiation of the pluripotent stem cells by replacing the medium with a differentiation induction medium.
  • resistant to differentiation in the present invention means that differentiation into a specific lineage occurs due to the differentiation direction of individual cells or cell populations of pluripotent stem cells (for example, ES cells or iPS cells). Means difficult.
  • resistance to endoderm differentiation means that the differentiation direction to ectoderm and mesoderm is stronger than the differentiation direction to endoderm, and the differentiation to endoderm is different from the differentiation to others. It means that it is hard to happen.
  • Non-Patent Document 4 (Shiraki et al., Genes Cells, 2008).
  • 201B7 cells which are undifferentiated cells cultured in a complete maintenance medium, were washed with Matrigel in complete CSTI-7 medium (Cytochemistry Laboratories (CSTI), Sendai, Japan) or Met-removed CSTI-7 medium. Cultured on coated plates.
  • iPS cells were seeded on a fibronectin-coated plate at 1.6 ⁇ 10 5 cells / cm 2 and then differentiated on the next day. Switched to.
  • As the differentiation medium RPMI 1640 medium supplemented with 100 ng / mL activin and 2% B27 was used. In the treatment by methionine removal, the cells were cultured for a certain period of time (for example, 5 hours or 10 hours) using a maintenance medium from which cells were removed before switching the medium to the differentiation medium.
  • Apoptosis analysis Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated d-UTP nick labeling (TUNEL) method using an in situ cell death detection kit (In situ Cell Detection Kit: Roche, Penzberg, Germany).
  • TUNEL terminal deoxynucleotidyl transferase-mediated d-UTP nick labeling
  • Hcy Concentration measurement of Hcy in culture medium Hcy was measured based on the description of Jiang et al. (Talanta 77, 1279-1284., 2009) based on tandem mass spectrometry, TQD (UPLC-MS / MS, Waters Corporation, Milford, Using high performance liquid chromatography with MA, USA). For the separation, an Acquity UPLC BEH C18 column was used. The cultured medium was collected and incubated with 10 mM DTT at 37 ° C. for 10 minutes to obtain a medium containing total Hcy secreted from the cells. The medium was deproteinized with 50% acetonitrile, filtered and then diluted with an equal volume of 50 mM Tris-HCl, pH 8.8. Each sample was injected, and the amount of each target product was calculated based on a standard curve obtained from a standard solution diluted in stages for each metabolite.
  • Example 1 Influence of amino acid removal from maintenance medium While maintaining culture of undifferentiated 201B7 cells, that is, an undifferentiation maintenance period before induction of differentiation in a differentiation medium
  • a single amino acid was removed from the maintenance medium for 2 days, and the effect on cell viability was examined.
  • 201B7 cells were seeded at a density of 5 ⁇ 10 4 cells / well in a 96-well plate (corning) coated with Matrigel (BD) for 24 hours.
  • undifferentiated maintenance culture medium Reproff Reproff (Reprocell, Japan) was used.
  • Example 2 Methionine metabolism in undifferentiated stem cells
  • Met removal methyl donor (MD: methionine, folic acid, vitamin B12, betaine) in a maintenance medium And choline) removal or Cys removal, and cultured for 48 hours.
  • MD methionine, folic acid, vitamin B12, betaine
  • a maintenance medium And choline a maintenance medium and choline
  • Met or MD removal inhibited the survival of pluripotent undifferentiated 201B7 cells, which was rescued by supplementation with Met or SAM (upper figure in FIG. 3).
  • the supplementation effect of Hcy or MTA was compared with supplementation of Met or SAM (lower figure in FIG. 3).
  • Met supplementation was most effective, followed by SAM.
  • Hcy supplementation was limited compared to Met and failed to rescue cell survival under MD removal conditions. Since Hcy cannot be converted to Met without MD, this result shows that the level of Met is important.
  • MTA supplementation rescued cell depletion under conditions where Met and MD were removed. This suggests that MTA was converted to Met through a salvage cycle.
  • Hcy or Cys supplementation saved cell depletion caused by Cys removal. This indicates that the effects of Cys and Met on undifferentiated cell survival occur through different mechanisms.
  • Example 3 Effect of methionine removal on extracellular excretion of Hcy ([Hcy] e medium) Human ES cells (khES3) were used in the experiment.
  • the transition of extracellular Hcy concentration when undifferentiated cells are cultured in a control complete medium is shown in the left diagram of FIG.
  • the right figure shows that Hcy when cultured for 24 hours in control complete medium (C) and methionine-removed medium ( ⁇ Met) using undifferentiated cells (white) and endoderm cells (gray) on differentiation day 5 as experimental materials. Shows total emissions.
  • the undifferentiated cells showed significantly higher Hcy extracellular excretion in the control state than the endoderm cells. In undifferentiated cells, Hcy extracellular excretion was remarkably suppressed by methionine removal, but such suppression was not observed in the endoderm.
  • Example 4 Irreversible inhibition of cell cycle arrest and survival by Met removal
  • Met concentration in the maintenance medium was examined.
  • the cells were cultured in complete maintenance medium and ⁇ Met medium for 48 hours, and changes in the number of cells were confirmed. The results are shown in FIG.
  • FIG. 5A When the Met concentration in the medium was lowered from 120 ⁇ M to 12 ⁇ M, cell proliferation decreased (FIG. 5A).
  • FIGS. 5B and C With time-tested, a significant decrease in cell number and cell proliferation was observed starting 5 hours after Met removal, consistent with a decrease in SAM levels (FIGS. 5B and C).
  • apoptosis increased significantly 24 hours after Met removal (FIG. 5D).
  • G0-G1 phase arrest as well as a decrease in cell population in S or G2-M phase, was observed following long-term Met removal of 24 hours, consistent with the timing of intracellular Met reduction (FIG. 5E).
  • the number of cells did not change at 24 hours, but decreased significantly at 48 hours, indicating that the cell cycle stopped, G0 / 1 It is thought to be caused by the occurrence of arrest.
  • FIG. 6 shows a culture schedule in a complete medium and a Met-removed medium under each of conditions a to h.
  • the removal of Met for 5 hours was reversible, and the cells began to grow when switched to complete medium (FIG. 6, c).
  • FIG. 6, right figure, g cell proliferation did not recover even when the cells were switched to complete medium.
  • SAM has shown that it functions as a sensor at an early stage. Met removal resulted in a decrease in SAM and a decrease in proliferation, and prolonged Met removal of 24 hours caused a decrease in intracellular Met concentration, leading to apoptosis.
  • Example 5 Met removal p53-p38 signaling pathway in undifferentiated hiPS cells The responsible signaling pathway that causes cell survival disorder was identified. The gene expression profiles of undifferentiated hiPS cells cultured for 5 hours in complete medium or Met-removed medium were compared. As a result, as shown in FIG. 7, cell cycle genes such as P21, GADD45A, GADD45B, MDM2, or p53-dependent genes involved in apoptosis, such as DHRS2 (gene encoding Hep27 protein), EGR1, FAS, A significant increase in the expression of TNFRSF10A, TNFRSF10D and RIPK2 was observed. When undifferentiated hiPS cells were used and Met was removed from the medium for 5 hours, it was confirmed that the transcription levels of EGR1, P21, and DHRS2 were up-regulated. The results are shown in FIG.
  • Nanog an undifferentiation marker
  • Oct3 / 4 is another undifferentiated marker
  • Example 7 Effect of short-term Met removal It was examined whether short-term Met removal for 5 to 10 hours increases the differentiation potential of hiPS cells. According to the above (A), (2) and (3), the cells were cultured in a ⁇ Met medium for 5 hours or 10 hours, and then transferred to a differentiation medium for endoderm or a differentiation medium for mesoderm or ectoderm for differentiation. The results are shown in FIG. The ratio of SOX17 + and Foxa2 expression levels (FIGS. 15A-C) increased with Met removal compared to control conditions, indicating that Met removal enhances differentiation into embryonic endoderm. Therefore, it was examined whether differentiation enhancement occurs in other germ layers.
  • Example 8 Effect of short-term Met removal on pancreatic differentiation It was examined whether short-term Met removal for 10 hours promotes pancreatic differentiation of hiPS cells.
  • HiPS cells (201B7) were pretreated with Met removal medium for 7 hours, and then pancreatic differentiation was performed. Pancreatic differentiation was performed based on a previously reported method (Shahjalal H et al., J Mol. Cell. Biol., 2014, PMID 24970864). After 22 days of culture, cells were fixed with 4% paraformaldehyde, stained with anti-insulin antibody, and nuclei were stained with DAPI. The results are shown in FIG. Compl shows the result of pancreas differentiation after pretreatment with complete medium and ⁇ Met for 10 hours with Met removal medium. The percentage of insulin positive cells increased to 6% in the complete medium and 43% in the ⁇ Met group.
  • Example 9 Effect of short-term Met removal on liver differentiation It was investigated whether short-term Met removal for 10 hours promotes liver differentiation of hiPS cells.
  • HiPS cells (Toe) were pretreated with Met-removed medium for 5 hours before liver differentiation.
  • Liver differentiation was performed based on a previously reported method (Yamazoe T et al., J Cell Sci., 2013, PMID 24101719).
  • Albumin secreted into the culture supernatant was quantified using ELISA.
  • FIG. Compl shows the result of liver differentiation after pretreatment with Complete medium and ⁇ Met for 10 hours with Met removal medium.
  • albumin secretion increased in the ⁇ Met group, suggesting that pretreatment with ⁇ Met promotes liver differentiation.
  • pluripotent stem cells such as ES cells and iPS cells can be efficiently induced to differentiate. Further, according to the present invention, the problem of differentiation directivity between cell lines in the induction of differentiation of pluripotent stem cells can be improved, so that the problem of cell line selection can be avoided and differentiation induction can be performed efficiently. Therefore, the present invention is useful in research and regenerative medicine using differentiation induction of pluripotent stem cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Reproductive Health (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Gynecology & Obstetrics (AREA)
  • Transplantation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention a pour objet un milieu et un procédé d'amélioration de la vitesse de différenciation induite des cellules souches telles que des cellules ES ou des cellules iPS. Au moyen de la présente invention, il est possible d'induire efficacement la différenciation par le biais de la mise en culture des cellules souches dans un milieu maintenant la non-différentiation dont la méthionine a été éliminée avant induction de la différenciation par mise en culture dans un milieu induisant la différenciation. La présente invention concerne un procédé d'induction de la différenciation de cellules souches pluripotentes dérivées de mammifères qui contient : une étape de culture des cellules souches pluripotentes dans un milieu de maintien de la non-différentiation des cellules souches pluripotentes et comprenant un milieu ne contenant pas de méthionine ; et une étape de mise en culture des cellules souches pluripotentes dans un milieu d'induction de différenciation pour induire la différenciation. La présente invention concerne également des cellules souches pluripotentes mises en culture au moyen du milieu maintenant la non-différentiation des cellules souches pluripotentes dérivées de mammifères et comprenant un milieu ne contenant pas de méthionine. La présente invention concerne en outre un milieu maintenant la non-différentiation des cellules souches pluripotentes et ne contenant pas de méthionine.
PCT/JP2015/053607 2014-02-21 2015-02-10 Procédé de stimulation de la différenciation des cellules souches au moyen d'un milieu reformulé contenant des acides aminés, cellules souches traitées à l'aide dudit procédé, et milieu WO2015125662A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2016504051A JP6351018B2 (ja) 2014-02-21 2015-02-10 アミノ酸組成変更培地を用いた幹細胞の分化誘導方法、及び該培地を含む培地キット

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2014032068 2014-02-21
JP2014-032068 2014-02-21
JP2014-184294 2014-09-10
JP2014184294 2014-09-10

Publications (1)

Publication Number Publication Date
WO2015125662A1 true WO2015125662A1 (fr) 2015-08-27

Family

ID=53878168

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/053607 WO2015125662A1 (fr) 2014-02-21 2015-02-10 Procédé de stimulation de la différenciation des cellules souches au moyen d'un milieu reformulé contenant des acides aminés, cellules souches traitées à l'aide dudit procédé, et milieu

Country Status (2)

Country Link
JP (1) JP6351018B2 (fr)
WO (1) WO2015125662A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019208713A1 (fr) 2018-04-26 2019-10-31 国立大学法人東京工業大学 Procédé pour favoriser la différenciation de cellules souches pluripotentes
WO2020054659A1 (fr) 2018-09-10 2020-03-19 国立大学法人東京工業大学 Procédé de production de cellules intestinales à partir de cellules souches pluripotentes
CN110997928A (zh) * 2017-08-08 2020-04-10 东京毅力科创株式会社 判定多能干细胞的未分化状态的方法、多能干细胞的传代培养方法及这些方法中使用的装置
WO2021006268A1 (fr) 2019-07-08 2021-01-14 国立大学法人東京工業大学 Procédé de production d'une cellule productrice d'insuline à l'aide de dérivés de dihydroindolizinone
US11530211B2 (en) 2018-01-18 2022-12-20 Daiichi Sankyo Company, Limited Dihydroindolizinone derivative

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009100702A (ja) * 2007-10-25 2009-05-14 Chiba Univ 胚性幹細胞から肝芽細胞を得る方法
WO2012056997A1 (fr) * 2010-10-28 2012-05-03 国立大学法人熊本大学 Procédé et milieu de culture pour améliorer l'efficacité d'induction de différenciation de cellules souches pluripotentes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009100702A (ja) * 2007-10-25 2009-05-14 Chiba Univ 胚性幹細胞から肝芽細胞を得る方法
WO2012056997A1 (fr) * 2010-10-28 2012-05-03 国立大学法人熊本大学 Procédé et milieu de culture pour améliorer l'efficacité d'induction de différenciation de cellules souches pluripotentes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Hito ES /iPS Saibo no Mibunkano Iji to Bunka ni Okeru Methionine Taisha no Yakuwari o Kaimei", 18 April 2014 (2014-04-18), Retrieved from the Internet <URL:http://www.kumamoto-u.ac.jp/daigakujouhou/kouhou/pressrelease/2014-file/release140418.pdf> [retrieved on 20150427] *
KYOKO NAKAMURA ET AL.: "Daekisen Yurai Zenku Saibo Oyobi ES Saibo no Zoshoku Bunka ni Okeru Glycine no Eikyo", JAPANESE JOURNAL FOR INHERITED METABOLIC DISEASES, vol. 25, no. 2, 2009, pages 79 *
SHIRAKI NOBUAKI ET AL.: "Methionine Metabolism Regulates Maintenance and Differentiation of Human Pluripotent Stem Cells", CELL METABOLISM, vol. 19, no. 5, May 2014 (2014-05-01), pages 780 - 794, XP055220770, ISSN: 1550-4131 *
SHYH-CHANG NG ET AL.: "Influence of Threonine Metabolism on S-adenosyl-methionine and Histone Methylation", SCIENCE, vol. 339, no. 6116, 2013, pages 222 - 226, XP055220769, ISSN: 0036-8075 *
ZHANG DONGHUI ET AL.: "Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells", CELL RESEARCH, vol. 19, no. 4, 2009, pages 429 - 438, XP055074920, ISSN: 1001-0602 *
ZHANG LIYUAN ET AL.: "Determination of eight amino acids in mice embryonic stem cells by pre-column derivatization HPLC with fluorescence detection", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 66, 2012, pages 356 - 358, XP028490228 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110997928A (zh) * 2017-08-08 2020-04-10 东京毅力科创株式会社 判定多能干细胞的未分化状态的方法、多能干细胞的传代培养方法及这些方法中使用的装置
CN110997928B (zh) * 2017-08-08 2023-11-14 东京毅力科创株式会社 判定多能干细胞的未分化状态的方法、多能干细胞的传代培养方法及这些方法中使用的装置
US11530211B2 (en) 2018-01-18 2022-12-20 Daiichi Sankyo Company, Limited Dihydroindolizinone derivative
WO2019208713A1 (fr) 2018-04-26 2019-10-31 国立大学法人東京工業大学 Procédé pour favoriser la différenciation de cellules souches pluripotentes
JPWO2019208713A1 (ja) * 2018-04-26 2021-05-13 国立大学法人東京工業大学 多能性幹細胞の分化促進方法
JP7470342B2 (ja) 2018-04-26 2024-04-18 国立大学法人東京工業大学 多能性幹細胞の分化促進方法
WO2020054659A1 (fr) 2018-09-10 2020-03-19 国立大学法人東京工業大学 Procédé de production de cellules intestinales à partir de cellules souches pluripotentes
WO2021006268A1 (fr) 2019-07-08 2021-01-14 国立大学法人東京工業大学 Procédé de production d'une cellule productrice d'insuline à l'aide de dérivés de dihydroindolizinone

Also Published As

Publication number Publication date
JPWO2015125662A1 (ja) 2017-03-30
JP6351018B2 (ja) 2018-07-04

Similar Documents

Publication Publication Date Title
US20210171912A1 (en) Method for scalable skeletal muscle lineage specification and cultivation
JP5938726B2 (ja) 多能性幹細胞の分化誘導効率を改善するための方法及び培地
Shiraki et al. Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells
CN104694570B (zh) 用于产生诱导的多能干细胞的组合化学遗传方法
JP6351018B2 (ja) アミノ酸組成変更培地を用いた幹細胞の分化誘導方法、及び該培地を含む培地キット
WO2018216743A1 (fr) Méthode pour induire la différenciation d&#39;une cellule mésodermique intermédiaire en une cellule progénitrice rénale, et méthode pour induire la différenciation d&#39;une cellule souche pluripotente en une cellule progénitrice rénale
US10196606B2 (en) Method of producing multipotent stem cells
JP6296399B2 (ja) 人工多能性幹細胞を腸管上皮細胞へ分化誘導する方法
JP7317323B2 (ja) 多能性幹細胞から腸管上皮細胞への分化誘導方法
JPWO2020022261A1 (ja) 新規腎前駆細胞マーカーおよびそれを利用した腎前駆細胞の濃縮方法
JP6677648B2 (ja) 細胞の増殖及び多能性の調節
KR102133621B1 (ko) 신경 전구 세포를 사용한 세포 치료에 있어서의 이식 보조제
JP2022019411A (ja) 人工多能性幹細胞由来の内胚葉細胞を製造する方法及びその利用
JP7058330B2 (ja) 腸管上皮細胞の製造方法および腸管上皮細胞
WO2022191130A1 (fr) Procédé de prolifération de cellules myocardiques, procédé de fabrication de cellules myocardiques, et agent de prolifération de cellules myocardiques
US20230126568A1 (en) Production method for intestinal epithelial cells and utilization thereof
KR101768581B1 (ko) 자발적 불멸화된 체세포의 단일 세포로부터 리프로그래밍 세포주의 제조방법
WO2020095879A1 (fr) Induction/différenciation de cellules souches pluripotentes en cellules épithéliales du tractus intestinal
Correia Mechanisms underlying metabolic shift in pluripotent stem cells: The potential role of sirtuins

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15751998

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
ENP Entry into the national phase

Ref document number: 2016504051

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15751998

Country of ref document: EP

Kind code of ref document: A1