WO2016204298A1 - PROCÉDÉ DE PRODUCTION DE CELLULES iPS CANINES - Google Patents

PROCÉDÉ DE PRODUCTION DE CELLULES iPS CANINES Download PDF

Info

Publication number
WO2016204298A1
WO2016204298A1 PCT/JP2016/068194 JP2016068194W WO2016204298A1 WO 2016204298 A1 WO2016204298 A1 WO 2016204298A1 JP 2016068194 W JP2016068194 W JP 2016068194W WO 2016204298 A1 WO2016204298 A1 WO 2016204298A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
canine
cell
somatic cells
factor
Prior art date
Application number
PCT/JP2016/068194
Other languages
English (en)
Japanese (ja)
Inventor
俊夫 稲葉
俊哉 西村
恵里菜 田中
晋吾 鳩谷
喜久弥 杉浦
Original Assignee
公立大学法人大阪府立大学
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 公立大学法人大阪府立大学 filed Critical 公立大学法人大阪府立大学
Priority to JP2017524884A priority Critical patent/JPWO2016204298A1/ja
Publication of WO2016204298A1 publication Critical patent/WO2016204298A1/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for producing canine iPS cells.
  • Dogs are physiologically close to humans compared to rodents such as mice and rats, and are large and long-lived, so they can be administered relatively large amounts of drugs. Suitable for experiments. Moreover, it is more useful as a human disease model animal because it lives in the same environment as humans. Furthermore, since more than half of the inherited diseases seen in dogs are similar to human diseases caused by mutations in the same gene, and the canine genomic sequence has been elucidated, the demand for dogs as a disease model may further expand.
  • DMEM / F12 Dulbecco's Modified Eagle medium and Ham's ⁇ F-12 medium are mixed in equal amounts.
  • a culture medium to which serum such as FBS, a serum substitute such as KSR, and a differentiation inhibitor such as LIF were added was used.
  • MAPKK mitogen activated protein kinase kinase
  • PD0325901 ALK (activin receptor-like kinase) inhibitor A-83-01
  • GSK glycose kinase
  • a culture solution in which CHIR99021, a drug, LIF (leukemia inhibitory factor) and valproic acid, an inhibitor of HDAC (histone deacetylase), are mixed is used.
  • LIF leukemia inhibitory factor
  • HDAC histone deacetylase
  • a culture medium is used in which KSR, LIF, and bFGF (basic fibroblast growth factor), which are serum substitutes, are mixed in a basic medium.
  • Non-Patent Document 1 includes KSR, LIF and bFGF in the basic medium, GSK3 ⁇ (glycogen / synthase / kinase 3 ⁇ ) inhibitor, MEK (ERK-MAP kinase pathway blocker) inhibitor, TGF- ⁇ (beta type) A culture solution in which a mutant growth factor) antagonist is mixed is used.
  • Non-patent Documents 2 and 3 the basic medium DMEM / F12 medium is mixed with N2 supplement (trade name) used for neuronal cell culture, and neurobasal medium is B27 supplement.
  • N2 supplement trade name
  • neurobasal medium B27 supplement.
  • a medium in which LIF, MEK inhibitor, and GSK3 ⁇ inhibitor are added to a mixed medium of a medium mixed with (trade name) is used (Non-patent Documents 2 and 3). According to these documents, single cells can be obtained by enzymatic treatment of the obtained iPS colonies, and the single cells can be subcultured for about 50 generations.
  • Non-Patent Documents 4 and 5 it has been reported that it has a three-dimensional colony-forming ability similar to that of undifferentiated mouse iPS cells, but it can withstand passage methods using enzymes. The form cannot be maintained unless each inhibitor is added to the medium. In addition, it is considered that these iPS cells have not yet been established because long-term subculture is not possible.
  • somatic cells can be isolated from somatic cells in the presence of only cell growth promoting factors such as bFGF. There is no report that canine iPS cells have been produced and iPS cells that can be subcultured over a long period of time have been produced.
  • An object of the present invention is to produce a canine iPS cell line that can be stably passaged over a long period of time using a culture solution having an extremely simple composition.
  • the present invention relates to a method for producing a canine iPS cell line from canine somatic cells, comprising a step of introducing a nuclear reprogramming factor into a canine somatic cell, and a canine somatic cell into which a nuclear reprogramming factor has been introduced. It is a method comprising a step of culturing using a culture solution containing no differentiation pluripotency maintenance factor in the presence of a blast growth factor.
  • a canine iPS cell line that can be stably passaged over a long period of time with a culture solution having an extremely simple composition is produced. If this cell is induced to differentiate, canine mesenchymal stem cells (MSC), other canine stem cells, and somatic cells can be produced in vitro.
  • MSC canine mesenchymal stem cells
  • FIG. 1 is an image obtained by microscopic observation of a dog iPS colony cultured under various culture conditions on feeder cells.
  • FIG. 2 is an image obtained by microscopic observation of a canine iPS colony (when only bFGF is used) cultured for 50 passages on feeder cells.
  • (A) is an image at a magnification of 40 times
  • (B) is an image at a magnification of 400 times.
  • FIG. 3 is an image obtained by microscopic observation of a cell obtained by culturing a single cell obtained by enzymatic treatment of canine iPS cells obtained on feeder cells.
  • A) shows cells obtained by culture on feeder cells
  • (B) shows cells obtained by culture on Matrigel.
  • FIG. 4 is an image obtained by confocal laser scanning microscope observation of canine iPS cells (passage 30) cultured on feeder cells immunostained with an undifferentiated marker.
  • A shows OCT3 / 4
  • B shows NANOG
  • C shows SSEA4.
  • FIG. 5 is a graph showing the expression state of undifferentiated marker genes ( C-MYC , OCT3 / 4 , KLF4 , GBX2 , NANOG , SOX2 ) of canine iPS cells (passage 30) cultured on feeder cells.
  • FIG. 6 is a graph showing the expression state of an exogenous undifferentiated marker gene ( E2A-C-MYC ) in canine iPS cells cultured on feeder cells.
  • E2A-C-MYC exogenous undifferentiated marker gene
  • FIG. 7 is a diagram showing the differentiation potential of canine iPS cells
  • (A) is an image obtained by microscopic observation of embryoid bodies
  • (B) is Southern blotting showing gene expression in differentiated cells obtained from embryoid bodies. It is an image.
  • the iPS cell in (B) is a negative control.
  • FIG. 8 is a chromosomal image of canine iPS cells (passage 35).
  • FIG. 9 shows the influence of bFGF concentration on the maintenance of undifferentiated canine iPS cells.
  • FIG. 10 shows the influence of bFGF concentration on the proliferation rate of canine iPS cells.
  • FIG. 11 is an image obtained by microscopic observation of MSCs induced to differentiate from canine iPS cells.
  • FIG. 12 is an image obtained by microscopic observation of MSC subcultured after induction of differentiation from canine iPS cells.
  • A shows control adult dog MSC
  • B shows differentiation-induced MSC.
  • FIG. 13 is a graph showing the expression state of cell surface markers (CD44 and CD90) by flow cytometry of MSCs induced to differentiate from canine iPS cells.
  • FIG. 14 is a graph showing the expression state of cell surface markers (CD34 and CD45) by flow cytometry of MSCs induced to differentiate from canine iPS cells.
  • FIG. 15 is an image obtained by confocal laser microscope observation showing the expression state of cell surface markers (OCT3 / 4 and NANOG) of MSCs induced to differentiate from canine iPS cells.
  • FIG. 16 is an image obtained by confocal laser microscope observation showing the expression state of the cell surface marker of MSC induced to differentiate from canine iPS cells.
  • (A) shows NESTIN and (B) shows GFAP.
  • FIG. 17 is an image obtained by microscopic observation of osteoid cells further induced to differentiate from MSCs induced to differentiate.
  • A) shows unstained cells
  • (B) shows ALP-stained cells
  • (C) shows Von Kossa-stained cells.
  • FIG. 18 is an image obtained by microscopic observation of a neuron-like cell further induced to differentiate from MSC induced to differentiate.
  • (A) shows Neurosphere
  • (B) shows elongated neural stem cell-like cells.
  • the method for producing canine iPS cells comprises a step of introducing a nuclear reprogramming factor into a canine somatic cell, and differentiation of a canine somatic cell into which a nuclear reprogramming factor has been introduced in the presence of a basic fibroblast growth factor.
  • the canine somatic cell that can be used in the present invention is not particularly limited as long as it is a somatic cell collected from a dog.
  • it can be a fetal somatic cell or a mature somatic cell.
  • stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells and sperm stem cells, and already differentiated cells such as lymphocytes, epithelial cells, muscle cells and fibroblasts can be used.
  • Nuclear reprogramming factors are factors that induce nuclear reprogramming of somatic cells.
  • the nuclear reprogramming factor is not particularly limited as long as it has these functions, and is, for example, a nucleic acid (gene) represented by a transcription factor, a peptide, a protein, an organic compound, an inorganic compound, or a mixture thereof, preferably transcription. Is a factor.
  • the transcription factor is not particularly limited as long as it can reprogram canine somatic cells, and known transcription factors such as genes such as OCT3 / 4 , SOX2 , KLF4 , and C- MYC are shown.
  • the transcription factor is preferably a dog-derived factor, but may also be a transcription factor of animal species other than dogs such as humans and mice.
  • the method for introducing the nuclear reprogramming factor into the somatic cell is not particularly limited as long as it is a method capable of reprogramming the somatic cell, and a known method can be used.
  • a nucleic acid which codes a transcription factor the method of introduce
  • all the nucleic acids to be used may be incorporated into one vector and expressed in canine somatic cells at the same time. May be expressed simultaneously.
  • vectors examples include retrovirus (including lentivirus), adenovirus, adeno-associated virus, Sendai virus, herpes virus, vaccinia virus, pox virus, poliovirus, silbis virus, rhabdovirus, paramyxovirus, ortho Virus vectors such as myxoviruses; artificial chromosome vectors such as YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors; plasmid vectors; epis that can autonomously replicate in host cells Examples thereof include somal vectors.
  • retrovirus including lentivirus
  • adenovirus adeno-associated virus
  • Sendai virus herpes virus
  • vaccinia virus pox virus
  • poliovirus silbis virus
  • rhabdovirus paramyxovirus
  • ortho Virus vectors such as myxoviruses
  • somatic cells By artificially controlling the expression of the introduced transcription factor, somatic cells can be efficiently initialized, and by continuously expressing the transcription factor, iPS cells can be stably maintained in culture.
  • a drug-inducible vector that expresses a transcription factor by adding a drug for example, a doxycycline-inducible Tet-On lentiviral vector (TetO-FUW-OSKM: manufactured by Addgene) using doxycycline is shown.
  • the method for introducing the vector is not particularly limited, and a known method such as a lipofection method, a microinjection method, a DEAE dextran method, a gene gun method, an electroporation method, or a calcium phosphate method can be used.
  • Canine somatic cells into which a nuclear reprogramming factor has been introduced are cultured in a culture medium and initialized.
  • a culture solution containing no differentiation pluripotency maintenance factor such as LIF in the presence of a basic fibroblast growth factor is used.
  • the basal medium of the culture solution a medium used for culturing animal cells is used.
  • the basal medium is, for example, a BME medium, a BGJb medium, a GLASGOW MEM medium, an IMDM medium, an Eagle MEM medium, a DMEM medium, an F12 medium, or a Neuro Basal Medium.
  • the culture medium used in the present invention is a medium that does not contain a differentiation pluripotency maintenance factor, and more preferably a medium that does not contain a differentiation inhibitory factor.
  • the pluripotency maintenance factor refers to a factor that is artificially added to maintain the differentiation pluripotency of the obtained iPS cells, that is, to maintain the pluripotency of stem cells.
  • the differentiation pluripotency maintenance factor is a factor that activates a cell signal pathway necessary for stem cells to maintain differentiation pluripotency, and examples thereof include LIF and Activin that have been widely used so far.
  • the differentiation inhibitor refers to a substance that inhibits a cell signal necessary for a stem cell to differentiate, and the obtained iPS cell differentiates itself, that is, each cell exhibits various functions. Inhibits changing to a heterologous cell.
  • MEK inhibitors that have been widely used so far.
  • a differentiation pluripotency maintenance factor and a differentiation inhibitory factor need only be handled as a differentiation pluripotency maintenance factor when they can be used as a differentiation pluripotency maintenance factor or a differentiation inhibition factor in their action mechanism.
  • the culture medium which does not contain both it is not necessary to distinguish both from the property of this invention. This is because the object of the present invention is to produce iPS cells or maintain them as iPS cells without adding such factors.
  • the culture solution of the present invention is capable of maintaining pluripotency in terms of its action mechanism, such as MAPKK inhibitor, ALK inhibitor, GSK inhibitor CHIR99021, and HDAC inhibitor as described in Patent Document 1. It does not include a factor that exhibits the same action as the factor, and more preferably does not include a factor that exhibits the same action as the differentiation inhibiting factor.
  • basic fibroblast growth factor is a factor that falls into the category of pluripotency maintenance factor
  • bFGF basic fibroblast growth factor
  • the “pluripotency maintenance factor” does not include basic fibroblast growth factor (bFGF).
  • a serum-free culture solution means a medium containing no serum or an unpurified serum such as FBS or a serum substitute of unknown composition, such as KSR.
  • bFGF basic fibroblast growth factor
  • the amount of bFGF added can be appropriately determined by those skilled in the art.
  • the lower limit amount is, for example, 0.01 ng, 0.1 ng, 0.5 ng, and 1 ng with respect to 1 ml of the culture solution, for example.
  • the upper limit amount is, for example, 10,000 ng, 5,000 ng, 1,000 ng, 500 ng, or 200 ng. Regardless of the origin of bFGF, for example, it is human, bovine, and canine.
  • the culture solution may further contain factors related to the maintenance and growth of cells other than bFGF.
  • the factor can be, for example, fatty acids or lipids, amino acids (eg, non-essential amino acids), albumin, vitamins, growth factors, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts. Specifically, it is a so-called supplement and is a factor that can contribute to cell proliferation.
  • the culture solution contains one or more factors, and for example, a mixture such as N2 supplement (Invitorogen) and B27 supplement (Invitorogen) is preferably used.
  • the culture medium preferably used in the present invention is a culture liquid in which only bFGF, which is an essential component of the above-mentioned basal medium, is added. Furthermore, the DMEM / F12 medium contains at least one supplement of N2 supplement and B27 supplement, A culture solution in which only bFGF, which is an essential component, is added to an N2B27 medium containing two supplements (N2 and B27) is preferable, and a culture solution in which albumin is added to this is more preferable.
  • Nucleated canine somatic cells are cultured on feeder cells or without feeders.
  • the feeder cells various cells used in the production of conventional canine iPS cells, human iPS cells, and mouse iPS cells are used, and examples include canine fetal derived fibroblasts and mouse fetal derived fibroblasts.
  • Feederless means culturing on a culture instrument such as a well or a dish without using feeder cells.
  • a culture device coated with an artificial basement membrane matrix such as Matrigel is preferably used.
  • the canine iPS cell obtained by the present invention is a cell derived from a somatic cell of a dog, and is a cell in which expression of at least one of endogenous OCT3 / 4 , KLF4 , and SOX2 , preferably all genes and NANOG gene is observed. It is. More preferred are cells in which endogenous C-MYC or GBX2 gene expression is observed.
  • At least one of the four exogenous genes OCT3 / 4 eg, E2A-OCT3 / 4
  • SOX2 eg, E2A-SOX2
  • KLF4 eg, E2A-KLF4
  • C-MYC for example, E2A-OCT3 / 4
  • it is a cell in which expression of at least one of these four exogenous genes, preferably these four genes is observed.
  • Culture conditions for canine somatic cells into which nuclei have been introduced can be appropriately set by those skilled in the art.
  • the culture period is 5 days to 1 week, preferably 1 week to 2 weeks in the presence of about 1 to 10% CO 2 at a culture temperature of 30 to 40 ° C.
  • One example is culture at 37 ° C. for 2 weeks in the presence of about 5% CO 2 .
  • IPS cells obtained have self-renewal ability and differentiation pluripotency.
  • the self-replicating ability is the ability to proliferate in a state that retains pluripotency (undifferentiated state), and having self-replicating ability means that passage is possible.
  • canine iPS cells obtained by the production method of the present invention can be passaged longer than conventional canine iPS cells, that is, at least 10 generations, 30 generations, preferably 50 generations or more.
  • canine iPS cells obtained by the production method can divide the aggregate (cell mass) into single cells by enzyme treatment, and can be subcultured in a single cell state.
  • the enzyme treatment is, for example, treatment with trypsin, and can be treatment with actase.
  • the enzyme treatment is performed by bringing the enzyme solution into contact with the resulting cell mass of iPS cells.
  • Treatment conditions can be appropriately determined by those skilled in the art. For example, 0.01% to 1.0% trypsin solution, preferably a solution further containing EDTA is used.
  • the division by the enzyme treatment is a division method that changes to a physical method such as so-called pipetting or cell knife division.
  • the cell mass of canine iPS cells obtained by the method described in Patent Document 1 could not be divided into single cells by enzyme treatment, the production method of the present invention can be divided into single cells by enzyme treatment. A cell mass (cell population) is obtained, and efficiency in subculture is improved.
  • Pluripotency is the ability to differentiate into three germ layers (ectodermal, endoderm, mesoderm) stem cells, that is, any stem cell. Differentiating into pluripotency differentiates into any stem cell like canine ES cells It means to do. Therefore, various cells such as skeletal muscle cells, cardiomyocytes, blood cells, nerve cells, germ cells and the like can be produced by inducing differentiation of the obtained canine iPS cells. In addition, cells obtained by differentiating the prepared canine iPS cells, such as stem cells and somatic cells, can be further returned to iPS cells. This can be achieved, for example, by allowing the drug to act on cells into which the drug-inducible vector has been introduced, or by newly introducing a nuclear reprogramming factor.
  • the same culture broth as that used for the nuclear reprogramming can be used.
  • a differentiation pluripotency maintenance factor or a differentiation inhibitory factor may be used for the culture solution composition used for initialization. Regardless of the presence or absence of feeder cells, it can be on feeder cells or feeder-less conditions.
  • the differentiation induction method is not particularly limited, and a known differentiation induction method from iPS cells can be applied.
  • MSC mesenchymal stem cells
  • embryoid bodies were prepared by suspension culture in a medium containing fetal bovine serum (FBS) or non-essential-amino-acid (NEAA) in DMEM / F12 medium.
  • FBS fetal bovine serum
  • NEAA non-essential-amino-acid
  • MEM- ⁇ Glutamax trade name: manufactured by GIBCO
  • the prepared embryoid body is prepared in FMEM, Examples of the method include culturing in a bone differentiation medium containing ⁇ -glycerophosphate, but are not limited thereto.
  • canine iPS can be obtained from canine somatic cells without using a factor that is less than that of the conventional method, in particular, a pluripotency maintenance factor such as LIF, or a differentiation inhibitory factor.
  • a pluripotency maintenance factor such as LIF
  • a differentiation inhibitory factor such as LIF
  • the obtained canine iPS cells have a multi-generation passable self-renewal ability, a tridermal stem cell, and a pluripotency that differentiates into various somatic cells and germ cells.
  • [Preparation of canine iPS cell line] Gene induction was performed on canine fetal fibroblasts (CEF) using a drug-induced lentivirus capable of expressing a gene by addition of doxycycline to try to produce canine iPS cells.
  • the necessity of LIF, bFGF, GSK3 ⁇ inhibitor (CHIR99021) and MEK inhibitor (PD0325901) as additive factors was examined using N2B27 medium as a basal medium.
  • the necessity of feeder cells mouse embryonic fibroblast (MEF) was also examined. In the following, all reagent concentrations are final concentrations.
  • feeder cells mouse embryonic fibroblasts (MEF)
  • Fetal tissues obtained by extracting fetuses from 14.5 day-old ICR mice (Slc: ICR, manufactured by SLC, Japan) and following conventional methods 10% FBS (PAA Laboratories GmbH, Ontario, Canada), 2 mM L-glutamine (SIGMA), 1% penicillin / streptomycin mixed solution (SIGMA) was added to DMEM medium (SIGMA)
  • the culture was incubated at 37 ° C. and 5% CO 2 .
  • the primary cultured cells were subcultured after being proliferated, and subcultured cells within 3rd generation were used for the experiment.
  • mitomycin C treatment was performed by culturing at 37 ° C. under 5% CO 2 for 2.5 hours with the above culture solution to which 10 ⁇ g / ml mitomycin injection (manufactured by Kyowa Hakko Kogyo Co., Ltd.) was added.
  • the cells were washed with 5 ml of D-PBS ( ⁇ ) and collected with a 0.25% trypsin-EDTA solution (manufactured by SIGMA) to prepare feeder cells.
  • the cultured cells were recovered after growth using a 0.25% trypsin-EDTA solution.
  • the obtained 293FT cells were put into a 100 mm tissue culture dish, and the above-mentioned culture solution for 293FT cells was put in, and seeded so that the number of cells per dish was 2 ⁇ 10 6 , 37 ° C., 5% CO 2. Incubate overnight under.
  • TetO-FUW-OSKM lentiviral vector manufactured by Addgene
  • four pluripotency maintenance genes mouse OCT3 / 4, KLF4, SOX2, C-MYC
  • tetracycline response factor The protein expression vector was introduced into new 293FT cells as described above. After 24 hours, the respective culture solutions were replaced. After 24 hours, FUW-M2rtTA and TetO-FUW-OSKM virus solutions were collected using a 0.45 ⁇ m cellulose acetate filter (Whatman, Kent, ME).
  • CEF preparation and lentiviral infection The fetus (male) was extracted from a 30-week-old beagle dog (manufactured by SLC, Japan), and CEF was obtained from the fetal tissue obtained according to a conventional method under the same conditions as the MEF. Cultured. For the culture, a culture solution in which 10% FBS, 2 mM L-glutamine, 100 IU / ml penicillin-100 ⁇ g / ml streptomycin mixed solution was added to DMEM medium was used. CEF was seeded in a 35 mm tissue culture dish (IWAKI) so that the number of cells per dish was 9.5 ⁇ 10 4 and cultured overnight at 37 ° C. and 5% CO 2 . To the CEF grown on the entire dish, a mixture of the above two virus solutions with 8 ⁇ g / ml polybrene (Nacalai Tesque) added was added to infect the virus with CEF.
  • IWAKI tissue culture dish
  • Canine iPS cell production 1 Examination of canine iPS cell production conditions In order to examine canine iPS cell production conditions, substrate conditions and medium conditions (addition factors) were examined under the conditions shown in Table 1. Moreover, the conditions described in Patent Document 1 were adopted as a control. As a control, N2B27 medium supplemented only with doxycycline was used.
  • the culture solution was changed 24 hours after virus infection, and further 24 hours later, the cells were subcultured with 0.25% trypsin-EDTA solution, and prepared MEF cells (feeder cells) or Matrigel (Becton Dickinson Bioscience bioscience) On a dish coated with 2. Inoculate the infected CEF obtained in step 1 with a CEF culture solution so that the number of cells per dish for 35 mm tissue culture is 2 to 3 ⁇ 10 4 cells, at 37 ° C under 5% CO 2 In culture.
  • N2B27 medium [DMEM / F12 medium (manufactured by Lifetechnologies) supplemented with 1 ⁇ N2 (manufactured by Invitrogen) and Neuro basal medium with 1 ⁇ B27 (manufactured by Invitrogen) Medium mixed with (volume ratio)], 5 mg / ml albumin (bovine serum-derived albumin: BSA), 1 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 1% NEAA, 0.1 mM m 2-mercaptoethanol (SIGMA ), 4 ⁇ g / ml doxycycline (Clontech), and according to Table 1, 4 ng / ml basic ⁇ fibroblast ⁇ growth factor (bFGF: Peprotech Rocky), 1000 U / ml leukemia inhibitory factor (LIF: Wako Pure Chemical Industries) The medium was replaced with a medium supplemented with two inhibitors (3 ⁇ M GSK
  • bFGF and LIF were added every time the medium was changed.
  • commercially available dog iPS medium (DMEM / F12 medium, manufactured by SIGMA), 20% KSR (GIBCO), 2 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 0.1 mM NEA
  • the culture was carried out using a medium (see Patent Document 1) supplemented with 1.14 ⁇ M 2-mercaptoethanol, 4 ng / ml bFGF, and 1000 U / ml LIF.
  • Secondary antibodies are PE-labeled donkey anti-goat IgG antibody (NOVUS) for NANOG, Alexa546-labeled goat anti-mouse IgG antibody (Invitrogen) for OCT3 / 4, and Cy3 for SSEA4. Labeled goat anti-mouse IgM antibody (Chemicon) was diluted 1,000 times and used. After staining for 30 to 60 minutes, the cells were encapsulated using ProLong Gold antifade reagent DAPI (Invitrogen) and observed with a confocal laser microscope (Nikon Clsi type, Nikon). The results are shown in FIG.
  • OCT3 / 4 Accession: XM_538830.1, C-MYC: Accession: NM_001003246
  • KLF4 Accession: XM — 005626996.1
  • NANOG Accession: XM — 005642425).
  • cDNA was synthesized from the extracted total RNA by a reverse transcription reaction.
  • My Cycler (trade name: manufactured by Bio-Rad Laboratories) was used.
  • SsoFast TM EvaGreen (registered trademark) Supermix Bio Rad
  • 25 ⁇ mol / ⁇ l of extrinsic and endogenous undifferentiated markers 25 ⁇ mol / ⁇ l of extrinsic and endogenous undifferentiated markers
  • 0.2 ⁇ l each of the primer solution for the sense strand and the antisense strand The total volume was made up to 10 ⁇ l with sterile distilled water. Then, it was made to react using MiniOpticon (registered trademark) System (manufactured by Bio Rad).
  • iPS cell line 9: P15 was passaged and doxycycline was removed, and cDNA was prepared from mRNA obtained from the second, fourth and sixth day colonies.
  • ⁇ ACTIN was used as an internal standard marker.
  • CEF was used as a negative control.
  • the obtained canine iPS cells were cultured in suspension for 4 to 6 days.
  • As the culture solution a medium obtained by adding 20% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 0.1 mM NEAA, 1.14 ⁇ M 2-mercaptoethanol to DMEM / F12 medium was used.
  • the formed embryoid body was a 35 mm tissue culture dish coated with 0.1% gelatin and cultured for about 1 to 2 weeks using the same medium.
  • a cDNA was prepared from the recovered RNA by reverse transcription reaction and subjected to PCR, and then electrophoresed on a 2.0% agarose gel at 100 V for 20 minutes.
  • GATA4 , DESMIN and TUJ1 were used as oligonucleotide primers.
  • Primers for Desmin the base sequence of canine known (DESMIN: Accession: NM_001012394.1) were designed based on.
  • DESMIN the base sequence of canine known
  • a primer for GATA4 a primer reported by Vaags et al. (Stem Cells. 27: 329-40) was used.
  • TUJ1 a primer reported by Hayes et al. (Stem Cells. 26: 465-73) was used.
  • FIG. 7 (A) When the obtained canine iPS cells were cultured in suspension for 4 to 6 days, embryoid bodies were formed as shown in FIG. 7 (A). Further, by bonding culturing these embryos-like, differentiate into various cells, these differentiated cells, ectodermal marker TUJ1, a mesoderm marker Desmin, GATA4 gene is endodermal marker Expression was observed (Fig. (B)).
  • ALP activity For the obtained canine iPS cell line (cell line cultured on feeder cells), bFGF concentration was added at 4, 10, 20, 100 ng / ml, and the effect on the undifferentiated state was determined by alkaline phosphatase (ALP), an undifferentiated marker. ) The activity was used as an index. ALP activity was determined after enzyme staining using Stemgent (registered trademark) Alkaline Phosphatase Staining Kit II (manufactured by Stemgent). The result is shown in FIG. This result suggests that increasing the bFGF concentration increases the number of positive cells and improves the maintenance of the undifferentiated state.
  • FIG. 9 shows the case of canine iPS cells cultured on Matrigel. In culture on MEF, it can be said that culturing in a medium containing 100 ng / ml bFGF is preferable for maintaining an undifferentiated state (not shown). )
  • the obtained canine iPS cell line (cell line cultured on feeder cells) was added at bFGF concentration of 4, 10, 20, 100 ng / ml and cultured on Matrigel. Examined. The proliferation rate at 96 hours after single cell passage was measured with a Waken counter (manufactured by Wakken Pharmaceutical Co., Ltd.). The result is shown in FIG. Addition of 4 ng / ml or more did not affect the proliferation rate of canine iPS cells. In addition, even when cultured on MEF, addition of 4 ng / ml or more did not affect the proliferation rate of canine iPS cells (not shown).
  • canine iPS cells that can be passaged for a long time using a doxycycline-inducible lentiviral vector and a medium in which only bFGF is added to N2B27 medium, and that can be subcultured by single cells by enzyme splitting.
  • This iPS cell can be maintained on a feeder cell as well as without a feeder, and its morphology and proliferation ability were not changed by freezing and thawing (not shown). Since it has been reported that undifferentiated stem cells such as mouse iPS cells can be passaged by single cell passage, the obtained canine iPS cells are considered to have a high degree of undifferentiation in the same manner as mouse-type iPS cells.
  • Embryoid bodies were formed from canine iPS cells by suspension culture in the same manner as in Example 1, and after 4 days, a 60 mm cell culture dish (manufactured by IWAKI) was attached and cultured. At the time of adhesion, the medium was changed to MSC medium supplemented with 1% penicillin / streptomycin mixed solution, 1% MEM NEAA, 10% FBS, and 8ng / ml bFGF to MEM- ⁇ Glutamax (GIBCO), 37 ° C, 5% CO 2 and cultured under. The medium was changed once every two days, and bFGF was added every day.
  • BMSC bone marrow stromal cells
  • Fig. 11 shows an image of the MSC obtained by adhesion culture in the microscopic observation
  • Fig. 12 shows an image of the subcultured MSC in the microscopic observation. MSCs that were differentiated around the embryoid body were observed in the area surrounded by the dotted line shown in FIG. Moreover, as shown in FIG. 12, the change was not seen also in the passage of the canine iPS cell origin MSC obtained by repeated passage similarly to the passage culture of BMSC derived from an adult dog.
  • the suspended cells were washed with FCN solution (a solution of 2% FBS and 1 mg / ml sodium azide added to D-PBS (-)), and then streptavidin-PE- Cy5 (Becton Dickinson Bioscience) was added and allowed to stand for 30 minutes on ice for reaction. After staining, the expression of each cell surface marker was measured using a flow cytometer (FACSCalibur TM, manufactured by Becton Dickinson). As a control, measurement was also performed on canine bone marrow stromal cell-derived MSC (BMSC). The obtained canine iPS cells were positive for CD90 and CD44 (FIG. 13) and negative for CD45 and CD34 (FIG. 14), as in BMSC.
  • FCN solution a solution of 2% FBS and 1 mg / ml sodium azide added to D-PBS (-)
  • streptavidin-PE- Cy5 Becton Dickinson Bioscience
  • rabbit anti-OCT3 / 4 polyclonal IgG antibody (Santa Cruz), goat anti-NANOG polyclonal antibody (Abcam), rabbit anti-NESTIN polyclonal antibody (Millipore), and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (manufactured by Millipore) was reacted for 60 minutes at room temperature with anti-NANOG antibody diluted 100-fold and others diluted 1000-fold.
  • Secondary antibodies are 1000-fold diluted Alexa 546-labeled anti-rabbit IgG antibody (Invitrogen) for OCT3 / 4, NESTIN and GFAP, and PE-labeled donkey anti-goat IgG antibody (NOVUS) for NANOG. ) was diluted 100 times and allowed to react at room temperature for 30 minutes. After staining, it was encapsulated using ProLong (registered trademark) Gold antifade reagent with DAPI (Invitrogen) and observed with a confocal laser microscope (Nikon clsi type: Nikon). MSCs derived from canine iPS cells were positive for the neural stem cell marker NESTIN and negative for the glial cell marker GFAP (FIG. 15). Furthermore, it was negative for undifferentiated markers OCT3 / 4 and NANOG (FIG. 16).
  • MSCs obtained by differentiation induction to bone-like cells were seeded on a 12-well plate (manufactured by IWAKI) so that the number of cells was 4 ⁇ 10 3 cells / cm 2 . After 1 day, the medium was added to DMEM medium (1.0 g / L glucose) (Nacalai Tesque), 10% FBS, 1% penicillin / streptomycin mixed solution, 50 ⁇ M ascorbic acid (Wako Pure Chemical Industries), 10 mM ⁇ -glycerophosphate. (Nacalai Tesque) and 100nM Dexamethasone (SIGMA) were added.
  • DMEM medium 1.0 g / L glucose
  • FBS 1% penicillin / streptomycin mixed solution
  • penicillin / streptomycin mixed solution 50 ⁇ M ascorbic acid (Wako Pure Chemical Industries)
  • 10 mM ⁇ -glycerophosphate (Nacalai Tesque) and 100nM Dexamethasone (SIGMA
  • the medium was exchanged half a day once every two days, and cultured for 28 days at 37 ° C. and 5% CO 2 .
  • the differentiation-induced cells were subjected to alkaline phosphatase (ALP) staining that became positive in osteoblasts using Stemgent Alkaline Phosphatase Staining Kit II (manufactured by Stemgent).
  • ALP alkaline phosphatase
  • Stemgent Alkaline Phosphatase Staining Kit II manufactured by Stemgent
  • Von Kossa staining was performed to confirm calcification by osteoblasts.
  • the cells were fixed with 95% ethanol (SIGMA) for 15 minutes at room temperature.
  • the formed floating cell mass was transferred to a poly-L-lysine-coated 35 mm dish (IWAKI) coated with 10 ⁇ g / ml laminin (AGC Techno Glass), and 2 mM L-glutamine in Neurobasal Medium.
  • Adhesion culture was performed at 37 ° C. and 5% CO 2 in a medium supplemented with 1% penicillin / streptomycin mixed solution and B27 Supplement (50 ⁇ , manufactured by GIBCO). The medium was exchanged once and twice every two days, and bFGF and human recombinant epidermal growth factor (EGF, manufactured by R & D Systems) at a final concentration of 20 ng / ml were added every day.
  • EGF epidermal growth factor
  • Neuroshpere was obtained by culturing the obtained MSC in a neural stem cell differentiation medium (FIG. 18 (A)). By adhesion culture of this Neurosphere, cells showing a neural stem cell-like morphology appeared from around the Neurosphere (FIG. 18B).
  • canine iPS cells that can be passaged over a long period of time with a simple medium composition are produced.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le problème décrit par la présente invention est de produire, à l'aide d'une culture liquide ayant une composition extrêmement simple, une lignée de cellules iPS canines qui peut être mise en sous-culture pendant une longue période de temps. La solution de la présente invention consiste à obtenir des cellules iPS canines par mise en culture de cellules somatiques canines dans lesquelles ont été introduits quatre facteurs de transcription d'OCT3/4, SOX2, KLF4, et C-MYC qui sont des facteurs de reprogrammation nucléaire au moyen d'un vecteur pouvant être induit par médicament qui peut réguler l'expression des facteurs de transcription par l'utilisation de, par exemple, la doxycycline, dans un milieu liquide de culture contenant un facteur de croissance fibroblastique basique (bFGF), mais ne contenant pas un facteur de maintien de pluripotence (ne comprenant pas le facteur de croissance fibroblastique basique) tel que LIF, et de préférence ne contenant pas en outre un suppresseur de différenciation tel que le suppresseur de MEK et le suppresseur de GSK3β.
PCT/JP2016/068194 2015-06-19 2016-06-19 PROCÉDÉ DE PRODUCTION DE CELLULES iPS CANINES WO2016204298A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2017524884A JPWO2016204298A1 (ja) 2015-06-19 2016-06-19 イヌｉＰＳ細胞の作製方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2015123775 2015-06-19
JP2015-123775 2015-06-19

Publications (1)

Publication Number Publication Date
WO2016204298A1 true WO2016204298A1 (fr) 2016-12-22

Family

ID=57545953

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/068194 WO2016204298A1 (fr) 2015-06-19 2016-06-19 PROCÉDÉ DE PRODUCTION DE CELLULES iPS CANINES

Country Status (2)

Country Link
JP (1) JPWO2016204298A1 (fr)
WO (1) WO2016204298A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111534481A (zh) * 2020-05-18 2020-08-14 扬州大学 一种提高鸡胚成纤维细胞体外诱导重编程为iPS细胞效率的方法
WO2021209599A1 (fr) 2020-04-16 2021-10-21 Roslin Technologies Limited Induction de cspi
EP4170018A1 (fr) 2021-10-20 2023-04-26 Roslin Technologies Limited Induction ipsc et derivation de la descendance
WO2023067090A1 (fr) 2021-10-20 2023-04-27 Roslin Technologies Limited Induction de cspi et dérivation sur la descendance

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011050379A (ja) * 2009-08-07 2011-03-17 Kyoto Univ イヌｉＰＳ細胞及びその製造方法
JP2014161254A (ja) * 2013-02-22 2014-09-08 Osaka Prefecture Univ イヌのｉＰＳ細胞の作製方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011050379A (ja) * 2009-08-07 2011-03-17 Kyoto Univ イヌｉＰＳ細胞及びその製造方法
JP2014161254A (ja) * 2013-02-22 2014-09-08 Osaka Prefecture Univ イヌのｉＰＳ細胞の作製方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Jikken Igaku Bessatsu ES .iPS-saibo Jikken Standard", 5 March 2014 (2014-03-05), pages 202 - 216, 246-257 *
BRAMBRINK T ET AL.: "Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.", CELL STEM CELL, vol. 2, no. 2, 7 February 2008 (2008-02-07), pages 151 - 159, XP002560041 *
DEAN H. BETTS ET AL.: "Canine pluripotent stem cells: are they ready for clinical applications?", FRONTIERS IN VETERINARY SCIENCE, vol. 2, 7 October 2015 (2015-10-07), pages 1 - 4, XP055338834 *
DEANNE J. WHITWORTH ET AL.: "Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFbeta/Activin Signaling Pathway", STEM CELLS AND DEVELOPMENT, vol. 23, no. 24, 24 November 2014 (2014-11-24), pages 3021 - 3033, XP055338827 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021209599A1 (fr) 2020-04-16 2021-10-21 Roslin Technologies Limited Induction de cspi
CN111534481A (zh) * 2020-05-18 2020-08-14 扬州大学 一种提高鸡胚成纤维细胞体外诱导重编程为iPS细胞效率的方法
EP4170018A1 (fr) 2021-10-20 2023-04-26 Roslin Technologies Limited Induction ipsc et derivation de la descendance
WO2023067090A1 (fr) 2021-10-20 2023-04-27 Roslin Technologies Limited Induction de cspi et dérivation sur la descendance

Also Published As

Publication number Publication date
JPWO2016204298A1 (ja) 2018-04-05

Similar Documents

Publication Publication Date Title
JP7357086B2 (ja) 多能性幹細胞を培養するための新規な方法および培養培地
JP7053551B2 (ja) 多能性幹細胞を未分化状態で培養する培地、細胞培養および方法
Rajala et al. A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells
US20220177836A1 (en) Methods for differentiating cells
JP5896421B2 (ja) 多能性幹細胞から骨格筋または骨格筋前駆細胞への分化誘導法
US10100283B2 (en) Efficient chondrocyte induction method
US10557121B2 (en) Method for chondrogenic induction
WO2016204298A1 (fr) PROCÉDÉ DE PRODUCTION DE CELLULES iPS CANINES
KR20140050514A (ko) 세포 배양 기재, 및 그것을 사용한 세포 배양 방법 및 만능 줄기 세포의 분화 유도 방법
Saito et al. Human amnion–derived cells as a reliable source of stem cells
WO2020040166A1 (fr) Procédé de production d'une cellule précurseur de nerf du tractus intestinal
US8709805B2 (en) Canine iPS cells and method of producing same
Moraveji et al. Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis
JPWO2019107485A1 (ja) 細胞の培養方法
US20200248138A1 (en) Method for inducing differentiation of pluripotent stem cells into germline stem cell-like cells
KR101806401B1 (ko) 역분화 줄기세포로부터 중간엽 줄기세포로의 분화 유도용 조성물 및 그를 이용한 중간엽 줄기세포를 제조하는 방법
JP2014161254A (ja) イヌのｉＰＳ細胞の作製方法
JP2017216981A (ja) イヌ人工誘導胚体外内胚葉細胞様株の作製方法
LE PHUONG ANALYSIS ON TRANSCRIPTIONAL REGULATION IN THE EARLY PHASE OF SOMATIC CELL REPROGRAMMING
Yeola Generating multipotent stem cells from primary human adipocytes for tissue repair
JP2020174644A (ja) がん幹細胞又はそれを含む細胞集団、その製造方法並びにがん治療薬のスクリーニング方法
Cui et al. Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell‐like cells
Ortolano Activation of NF-κB Transcription Factor During In Vitro Differentiation of Mouse Embryonic Stem Cells
Megges Analysis of the applicability of iPS cell-based approaches to bypass senescence and to re-establish rejuvenation features in human MSCs from aged individuals
Rajala Development of human stem cell culture conditions for clinical cell therapy

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: 16811772

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017524884

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: 16811772

Country of ref document: EP

Kind code of ref document: A1