WO2012043814A1 - Procédé de différenciation de cellules souches pluripotentes humaines - Google Patents

Procédé de différenciation de cellules souches pluripotentes humaines Download PDF

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WO2012043814A1
WO2012043814A1 PCT/JP2011/072609 JP2011072609W WO2012043814A1 WO 2012043814 A1 WO2012043814 A1 WO 2012043814A1 JP 2011072609 W JP2011072609 W JP 2011072609W WO 2012043814 A1 WO2012043814 A1 WO 2012043814A1
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cells
human
derived
stem cells
cell
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辻　浩一郎
康博 海老原
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国立大学法人東京大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

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  • the present invention relates to a method for differentiating human-derived pluripotent stem cells. More specifically, the human-derived pluripotent stem cells are co-cultured with mesenchymal stem cells established from human-derived pluripotent stem cells. Relates to a method for inducing differentiation.
  • the present invention also relates to mesenchymal stem cells established from human-derived pluripotent stem cells used in this method, and cells differentiated from human-derived pluripotent stem cells obtained by this method.
  • pluripotent stem cells such as human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have pluripotency that can be differentiated into various functional cells
  • application to regenerative medicine is expected.
  • animal serum or animal-derived feeder cells are required. Therefore, when functional cells derived from human pluripotent stem cells are actually administered to humans, infection with unknown microorganisms, viruses, prions, etc. due to contamination with animal cells (heterologous cells) that are foreign antigens There is a danger and a way to avoid it is needed.
  • the present invention has been made in view of the above-mentioned problems of the prior art, and an object thereof is to provide a method for differentiating human-derived pluripotent stem cells without using heterogeneous cells.
  • mesenchymal stem cells established from human-derived pluripotent stem cells as feeder cells for co-culturing human-derived pluripotent stem cells. It has been found that human-derived pluripotent stem cells can be differentiated when (mesenchymal stromal cell, MSC) is used. This is a surprising finding in view of the fact that it has conventionally been difficult to differentiate human pluripotent stem cells in a co-culture system of human-derived pluripotent stem cells and human-derived feeder cells.
  • MSC mesenchymal stromal cell
  • the present inventors also perform the establishment of mesenchymal stem cells from human-derived pluripotent stem cells by using human-derived platelet lysate without using heterologous animal-derived serum or feeder cells. I found that I can do it. Furthermore, it has been found that even if the mesenchymal stem cell and the pluripotent stem cell are derived from the same person, the pluripotent stem cell can be induced to differentiate.
  • the present inventors have succeeded for the first time in the world in establishing a system for inducing differentiation of human-derived pluripotent stem cells by eliminating the use of heterologous cells, and have completed the present invention. .
  • the present invention provides the following inventions.
  • a method for differentiating human-derived pluripotent stem cells wherein the human-derived pluripotent stem cells are induced to differentiate under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. how to.
  • the pluripotent stem cell is at least one cell selected from the group consisting of ES cells and iPS cells.
  • the mesenchymal stem cell is a cell established from a human-derived pluripotent stem cell in the presence of a human-derived platelet lysate.
  • various cells induced to differentiate from human pluripotent stem cells can be produced without using heterogeneous cells. Therefore, according to the present invention, it is possible to provide various cells that have been induced to differentiate from human pluripotent stem cells in a form that is highly safe even when administered to humans.
  • FIG. 5 is a photomicrograph showing uniform spindle-shaped cells (mesenchymal stem cells) induced to differentiate from human-derived iPS cells (253G1 human iPS cells) in the presence of PL (platelet lysate).
  • FIG. 1 It is a histogram figure which shows the result of having analyzed the cell obtained by inducing differentiation from H1 human ES cell in PL presence by using FACS method by making marker protein expression into a parameter
  • FIG. 2 is an electrophoresis photograph showing the result of analyzing a cell (hESC-derived MSC) obtained by inducing differentiation from H1 human ES cells (hESC) in the presence of PL by RT-PCR. It is a microscope picture which shows formation of the undifferentiated colony of the H1 human ES cell co-cultured with the H1 human ES cell origin mesenchymal stem cell. It is a microscope picture which shows formation of the undifferentiated colony of 253G1 human iPS cell co-cultured with the H1 human ES cell origin mesenchymal stem cell.
  • FIG. 2 is a photomicrograph showing the result of May-Grunwald-Giemsa staining of a cytospin specimen prepared from an erythroid colony formed by culturing circular small cells in a blood colony.
  • FIG. 5 is a photomicrograph showing the result of May-Grunwald-Giemsa staining of a cytospin specimen prepared from a myeloid colony formed by culturing round small cells in blood colonies.
  • FIG. 2 is a photomicrograph showing the result of May-Grunwald-Giemsa staining of a cytospin specimen prepared from an erythroid colony formed by culturing circular small cells in a blood colony.
  • FIG. 5 is a photomicrograph showing the result of May-Grunwald-Giemsa staining of a cytospin specimen prepared from a myeloid colony formed by culturing round small cells in blood colonies.
  • FIG. 5 is a photomicrograph showing the result of May-Grunwald-Giemsa staining of a cytospin specimen prepared from a mixed colony formed by culturing a circular small cell in blood colony. After inducing differentiation of H1 human ES cells by co-culture with H1 human ES cell-derived mesenchymal stem cells, cytospin specimens were prepared from erythroid colonies formed using Methocult H4435, and Hb (upper) and ⁇ globin It is a microscope picture which shows the result of having analyzed the expression of (middle stage) by immuno-staining. The bottom row is a photomicrograph obtained by superimposing the upper and middle photos.
  • cytospin specimens were prepared from erythroid colonies formed using Methocult H4435, and expression of Glycophorin A (GPA) was observed. It is a microscope picture which shows the result analyzed by immuno-staining. In the figure, the arrows indicate erythroid cells expressing GPA. 253G1 human iPS cell-derived mesenchymal stem cells and 253G1 human iPS cells were co-cultured, and the culture solution was changed to a culture solution for inducing differentiation into blood cells, and was found in colonies derived from 253G1 human iPS cells.
  • FIG. 5 is a photomicrograph showing erythroid colonies formed using Method H4435.
  • 253G1 human iPS cell-derived mesenchymal stem cells and H1 human ES cells are co-cultured, and the culture solution is changed to a culture solution for inducing differentiation into blood cells.
  • 3 is a photomicrograph showing a mixed colony formed using Method H4435.
  • 253G1 human iPS cell-derived mesenchymal stem cells and H1 human ES cells are co-cultured, and the culture solution is changed to a culture solution for inducing differentiation into blood cells.
  • FIG. 6 is a photomicrograph showing a myeloid colony formed using Method H4435.
  • FIG. 1 It is a microscope picture which shows the co-culture state of the human iPS cell (SPH-0103) and mouse embryo fibroblast (MEF) which were established from the skin fibroblast derived from a healthy adult.
  • 2 is a photomicrograph showing mesenchymal stem cells derived from human iPS cells (SPH-0103) induced to differentiate by culturing in a medium containing autologous serum.
  • 2 is a micrograph showing undifferentiated human iPS cell (SPH-0103) colonies maintained on mesenchymal stem cells differentiated from human iPS cells (SPH-0103).
  • Oct-4 (Oct-3 / 4), Sox-2, Nanog and SSEA in undifferentiated colonies of human iPS cells (SPH-0103) co-cultured with mesenchymal stem cells derived from human iPS cells (SPH-0103) 4 is a fluorescence micrograph showing the expression of -4.
  • Human iPS cells (SPH-0103) -derived mesenchymal stem cells and human iPS cells (SPH-0103) are co-cultured, and the culture solution is changed to a culture solution for inducing differentiation into blood cells.
  • FIG. 2 is a photomicrograph showing the growth of small round cells (a panel) and cobblestone cells (b panel) found in colonies derived from cells (SPH-0103).
  • FIG. 2 is a photomicrograph showing a hematopoietic colony formed by subjecting small round cells collected from undifferentiated colonies of human iPS cells (SPH-0103) to blood colony culture using autologous serum.
  • a and d indicate erythroid colonies composed of erythroid cells.
  • b and e show myeloid colonies composed of myeloid cells such as neutrophils, macrophages and monocytes.
  • c and f represent mixed colonies composed of erythroid cells, myeloid cells and megakaryocytes.
  • Df shows the results of May-Grunwald-Giemsa staining of cytospin specimens prepared from each blood cell colony.
  • Photomicrograph showing the results of immunostaining analysis of ⁇ globin expression in erythroid cells contained in erythroid colonies derived from human iPS cells (SPH-0103) formed by blood colony culture using autologous serum It is.
  • the upper three panels show the results of analyzing the expression of human ⁇ globin, human ⁇ globin, and human ⁇ globin, respectively, and the middle three panels show the results of analyzing the expression of human hemoglobin.
  • the lower three panels show the results of superposing the upper panel and the middle panel, respectively.
  • the present invention is a method for differentiating human-derived pluripotent stem cells, wherein the human-derived pluripotent stem cells are differentiated under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. It is a way to guide.
  • the human-derived pluripotent stem cells to be differentiated in the present invention are cells having pluripotency capable of differentiating into various cells constituting human and self-replicating ability.
  • human-derived embryonic stem cells ES cells
  • human-derived artificial pluripotent stem cells iPS cells
  • human-derived embryonic tumor cells EC cells
  • human-derived embryonic germ cells EG cells.
  • at least one cell selected from the group consisting of ES cells and iPS cells is preferable from the viewpoint that the analysis of biological characteristics is progressing remarkably.
  • a human-derived iPS cell as the human-derived pluripotent stem cell according to the present invention.
  • iPS cells established from patient-derived somatic cells are used as human-derived pluripotent stem cells according to the present invention from the viewpoint that the blood type completely matches that of a patient transplanting cells differentiated from pluripotent stem cells. It is particularly preferable to use it.
  • the blood type in the present invention means not only the type of red blood cells (ABO, RH) but also the type of white blood cells (HLA).
  • mesenchymal stem cells for co-culture with human-derived pluripotent stem cells are themselves human-derived pluripotent stem cells (for example, the aforementioned iPS cells, ES cells, etc.) It is characterized by using mesenchymal stem cells established from The mesenchymal stem cells function as feeder cells when differentiation-inducing human-derived pluripotent stem cells.
  • Mesenchymal stem cells are cells having the ability to differentiate into various mesenchymal cells such as adipocytes, chondrocytes, osteoblasts, and myocytes, and self-replicating ability.
  • a mesenchymal stem cell used in the present invention when used for regenerative medicine and the like, from the viewpoint of preventing rejection of a patient transplanted with a cell differentiated from a pluripotent stem cell, in that respect, it is preferably a mesenchymal stem cell that is compatible with the human-derived pluripotent stem cell to be differentiated, and may be a mesenchymal stem cell derived from the same person as the human-derived pluripotent stem cell to be differentiated. More preferred.
  • the method for establishing mesenchymal stem cells according to the present invention from human-derived pluripotent stem cells is not particularly limited.
  • a mesenchymal stem cell was prepared using a culture solution containing serum derived from a heterologous animal.
  • mesenchymal stem cells can be established without using sera from different animals, human-derived sera, human-derived plasma, human-derived sera are used without using feeder cells.
  • a method for establishing mesenchymal stem cells from human-derived pluripotent stem cells using at least one blood component selected from the group consisting of platelet lysate (PL) is preferable.
  • the method is established using a platelet lysate, and the blood type (particularly the type of HLA) From the viewpoint of completely matching), it is particularly preferable to establish a method using a platelet lysate or serum derived from a patient transplanted with cells differentiated from pluripotent stem cells. Further, from the viewpoint of suppressing the proliferation of such mesenchymal stem cells, it is preferable to irradiate with radiation (for example, 15 to 18 Gy) in co-culture with human-derived pluripotent stem cells.
  • radiation for example, 15 to 18 Gy
  • the present invention also provides mesenchymal stem cells established from human-derived pluripotent stem cells used in the method of the present invention.
  • human-derived pluripotent stem cells are induced to differentiate under co-culture with mesenchymal stem cells thus established from human-derived pluripotent stem cells.
  • a factor that can be differentiated into desired blood cells may be added to the medium and cultured.
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • TPO thrombopoietin
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • GM-CSF granule Sphere / macrophage colony stimulating factor
  • EPO erythropoietin
  • basic fibroblast growth factor bFGF
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • LIF leukemia inhibitory factor
  • BMP-4 Bone morphogenetic protein 4
  • TNF- ⁇ Flt3 ligand, heparin, interleukin (IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-11, IL-15, IL-6, fusion protein-6 (complex of IL-6 and soluble IL-6 receptor), etc.)
  • Ri least one cytokine and the like are selected.
  • 10 to 500 ng / mL human stem cell factor (hSCF), 10 to 500 ng / mL human vascular endothelial growth factor (hVEGF), 10 to 1000 ng / mL human fusion protein-6 (human interleukin- (IL-)) 6 and human soluble IL-6 receptor complex, hFP-6), 5-100 ng / mL hIL-3, 5-100 ng / mL human thrombopoietin (hTPO), 5-100 ng / mL granulocyte colony-stimulating factor ( G-CSF), 1-20 U / mL human erythropoietin (hEPO), 1-100 ng / mL hbFGF, and 1-100 ng / mL human bone morphogenetic protein (hBMP) -4 cytokine cocktail method Is particularly preferred.
  • hematopoietic stem cells that are the basis of blood cells such as red blood cells, white blood cells, and platelets. Differentiation can be induced into various hematopoietic progenitor cells.
  • the obtained hematopoietic stem cells and hematopoietic progenitor cells were used, for example, a methylcellulose medium (for example, Methocult H4435 manufactured by StemCell Technologies Inc.) supplemented with serum, cytokines, and the like as shown in Examples described later.
  • Mature blood cells can be obtained by blood colony culture or suspension blood culture under serum-free conditions. Moreover, as shown in the below-mentioned Example, it can be set as a mature blood cell also by the blood colony culture method using the serum (autologous serum) etc. from the patient who transplants the cell which differentiated from the pluripotent stem cell.
  • blood colonies using autologous serum and the like are obtained from the viewpoint of obtaining mature blood cells not contaminated with not only allogeneic animal serum (FBS and the like) but also allogeneic (allogeneic) antigens. -Culture methods are preferred.
  • Examples include a method in which the separated OP9 cells are co-cultured, and then the VEGF-R2 (+) TRA1-60 ( ⁇ ) VE-cadherin (+) cells that appear are separated and cultured on the collagen IV coating. Further, in the case of inducing differentiation into cardiomyocytes, for example, mouse ES cells described in “Yamashita et al., FASEB J., 2005, Vol. 19, pages 1534 to 1536” contain LIF on a collagen IV coating. A method of culturing in an uncultured culture medium and then co-culturing Flk-1 (+) cells and OP-9 cells appearing thereafter.
  • mesenchymal stem cells instead of the feeder cells (PA6 cells and OP9 cells) derived from different animals used in these methods, human-derived pluripotency without using the different cells Stem cells can be differentiated into functional cells such as dopaminergic neurons, vascular endothelial cells, cardiomyocytes.
  • functional cells such as dopaminergic neurons, vascular endothelial cells, cardiomyocytes.
  • human-derived pluripotent stem cells are highly differentiated from the viewpoint of having high proliferative ability. It is preferable that “ ⁇ ” is formed.
  • the formation of undifferentiated colonies of human-derived pluripotent stem cells is, for example, in the case of using human-derived ES cells, in the HES1 culture medium described below, and in the case of using human-derived iPS cells, the below-described HES2 culture. It can be performed by culturing in a liquid for about 6 to 10 days.
  • the present invention also provides cells differentiated from human-derived pluripotent stem cells obtained by the above-described method of the present invention.
  • human ES cells H1 human ES cells (manufactured by WiCell Research Institute) or KhES-1 cells provided by Professor Norio Nakajo of Kyoto University were used.
  • human iPS cells human iPS cells (253G1 human iPS cells) provided by Professor Shinya Yamanaka of Kyoto University were used. These human ES cells and iPS cells were maintained by co-culture with mouse fetal fibroblasts (MEF), and passage was performed every 6 to 8 days.
  • MEF mouse fetal fibroblasts
  • HES1 culture medium As a culture solution for co-culture of H1 human ES cells, Dulbecco's modified Eagle's medium (DMEM) and Ham's nutrient mixture F-12 (manufactured by Sigma) were mixed in a 1: 1 ratio. 1 mM 2-mercaptoethanol (Wako), 200 mM L-glutamine (Invitrogen), 1M HEPES (Invitrogen), minimum essential medium (MEM) -non-essential amino acid solution (Invitrogen), 5 ng / mL human A recombinant basic fibroblast growth factor (bFGF) (manufactured by R & D) and 20% knockout serum substitute (KSR) (manufactured by Invitrogen) were used (hereinafter also referred to as HES1 culture medium).
  • bFGF basic fibroblast growth factor
  • KSR knockout serum substitute
  • DMEM and Ham's nutrient mixture F-12 were mixed at a ratio of 1: 1, and 0.1 mM 2 -Mercaptoethanol, 200 mM L-glutamine, 1 M HEPES, MEM-non-essential amino acid solution, 4 ng / mL human recombinant bFGF and 20% KSR were used (hereinafter also referred to as HES2 culture solution).
  • Platelet lysate is obtained by destroying platelets by freezing and thawing human platelet-rich plasma obtained by a platelet collection device at ⁇ 80 ° C. and then centrifuging at 900 g to collect the supernatant.
  • PL platelet lysate
  • human ES cells and iPS cells maintained on MEF are collected, or frozen human ES cells and iPS cells are thawed.
  • 5% PL and 10,000 U Novo-heparin are added to the HES1 culture medium or the HES2 culture medium (hereinafter referred to as “Mochida Pharmaceutical Co., Ltd.”). , Also referred to as PL culture solution), and cultured under conditions of 37 ° C. and 5% CO 2 .
  • the culture medium was exchanged twice a week, and subcultured every 2-3 weeks, and human ES cells and iPS cells were differentiated into uniform spindle-shaped cells at 6-8 weeks of culture. The obtained results are shown in FIGS.
  • ⁇ Analysis of characteristics of mesenchymal stem cells Cell surface markers of uniform spindle-shaped cells obtained as described above were detected using FACS (product name: FACSCalibur instrument, manufactured by BD Medical Systems), and using FlowJo software (manufactured by Tomy Digital Biology). Analyzed. In addition, gene expression of the obtained uniform spindle-shaped cells was examined by RT-PCR using the primers shown in Table 1. Further, using NH OsteoDiff Medium and NH AdipoDiff Medium (Miltenyi Biotec), differentiation of the obtained spindle-shaped cells into adipocytes and osteoblasts was performed, and oil red O (Oil red O) was used.
  • FACS product name: FACSCalibur instrument, manufactured by BD Medical Systems
  • FlowJo software manufactured by Tomy Digital Biology
  • human ES cells H1 human ES cells maintained on MEF were collected and seeded in a 10 cm culture dish coated with gelatin without using feeder cells.
  • a PL culture medium at 37 ° C. and 5% CO 2
  • uniform spindle-shaped cells ie, mesenchymal stem cells
  • mesenchymal stem cells were induced to differentiate after 6 to 8 weeks.
  • the uniform spindle-shaped cells are markers for blood cells, vascular endothelial cells, and undifferentiated ES cells.
  • CD45, CD34, CD14, CD31, and SSEA-4 were not expressed, and CD105 and CD166, which are markers for mesenchymal stem cells, were expressed.
  • differentiation was induced into osteoblasts and adipocytes, as clearly shown in the results shown in FIGS. 5 to 6, differentiation into ALP staining positive osteoblasts and oil red staining positive adipocytes, The cells established by the method were confirmed to be mesenchymal stem cells.
  • the mesenchymal stem cells differentiated from H1 human ES cells and 253G1 human iPS cells were subjected to chromosomal examination, and all of the analyzed 50 cells were normal karyotypes. It was confirmed.
  • human PL platelet lysate
  • differentiation from human ES cells to mesenchymal stem cells can be induced without using sera from different animals, and these are undifferentiated. It was confirmed that human ES cells and MEF were not mixed.
  • the differentiation induction method from human pluripotent stem cells to mesenchymal stem cells using human PL can be applied to both human-derived ES cells and human-derived iPS cells, and may cause chromosomal abnormalities. The sex is very low.
  • mesenchymal stem cells differentiated from human ES cells or iPS cells on 6-well plates were irradiated with 15-18 Gy and co-cultured with human ES cells or iPS cells. That is, as the culture medium at this time, if the cells to be seeded on the mesenchymal stem cells are H1 human ES cells, the HES1 culture medium is used. If the cells are 253G1 human iPS cells, the HES2 culture medium is used. Were co-cultured to form undifferentiated colonies of human ES cells and iPS cells. The obtained results are shown in FIGS.
  • the above method is used to induce differentiation of mesenchymal stem cells from human ES cells and iPS cells, and after irradiation with 15 to 18 Gy, self,
  • human ES cells or iPS cells that have been subcultured with MEF, or human ES cells or iPS cells that have just been thawed, Formation of undifferentiated colonies was observed.
  • all of the H1 human ES cells cultured on the mesenchymal stem cells derived from H1 human ES cells expressed an undifferentiated human ES cell marker. .
  • H1 human ES cells and 253G1 human iPS cells cultured on mesenchymal stem cells derived from H1 human ES cells are transplanted into NOD-scid mice, endoderm fistula, mesoderm, ectoderm Teratoma consisting of cells derived from the cells was formed, and it was confirmed that these human pluripotent stem cells were maintained undifferentiated.
  • Example 1 ⁇ Induction of differentiation from human ES cells and iPS cells into blood cells> As described above, on the 6th to 10th day of culture in which formation of undifferentiated colonies of human ES cells and iPS cells was observed, 2 mM glutamine and 4 ⁇ 10 ⁇ 4 M monothioglycerol (MTG, manufactured by Sigma) 100 ng / mL human stem cell factor (hSCF), 100 ng / mL human vascular endothelial growth factor (hVEGF) was added to StemPro-34 (Invitrogen) culture medium, which is a serum-free medium containing 50 mg / mL ascorbic acid (manufactured by Sigma).
  • MMG monothioglycerol
  • tissue immunostaining cells obtained as described above were fixed with 4% paraformaldehyde (PFA), then Oct-4, Sox-2, TRA-1-60, Nanog, glyccophorin A ( The expression of GPA), hemoglobin (Hb), and ⁇ globin was examined by immunostaining. In addition, nuclear staining was performed using Hoechst 33342 (Molecular Probes). The obtained results are shown in FIGS.
  • the cells contained in these erythroid colonies were erythroid cells expressing GPA and Hb, which are markers for erythroid cells. . Furthermore, when examining whether these erythroid cells are embryonic erythrocytes originating from immature primary hematopoiesis or adult erythrocytes originating from secondary hematopoiesis, more than 95% of these erythrocytes It was confirmed that it was an adult type erythrocyte expressing ⁇ -globin specific for secondary hematopoiesis (see FIG. 24). Although not shown in the figure, it was also confirmed that undifferentiated markers such as Oct-4, Sox-2, TRA-1-60 and Nanog were not expressed in these cells.
  • differentiated cells such as blood cells can be obtained by inducing differentiation of human-derived pluripotent stem cells under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. Became. Furthermore, adult erythrocytes specific for secondary hematopoiesis that were difficult to obtain by the conventional method of inducing differentiation from human pluripotent stem cells to blood cells via embryonic rods (see Non-Patent Documents 1 and 2). However, as described above, it has also been clarified that the method of the present invention can be obtained very efficiently at 95% or more.
  • the pluripotent stem cell that is induced to differentiate and the mesenchymal stem cell are derived from the same person (the above-mentioned co-culture of the H1 human ES cell-derived mesenchymal stem cell and the H1 human ES cell) And the example of co-culture of 253G1 human iPS cell-derived mesenchymal stem cells and 253G1 human iPS cells), and those derived from different people (the 253G1 human iPS cell-derived mesenchymal stem cells and It was also revealed that differentiated cells can be obtained from human-derived pluripotent stem cells in the same manner (see Example of co-culture with H1 human ES cells).
  • autologous mesenchymal stem cells are established from human iPS cells, and the human iPS cells are further converted into the mesenchymal system. Whether it can be differentiated into blood cells using autologous serum under co-culture with stem cells was verified as shown below.
  • Example 2 ⁇ Induction of differentiation from human iPS cells (SPH-0103) into blood cells> As described above, 100 ng / mL hSCF, 100 ng / mL hVEGF, 100 ng / mL hFP-6, 20 ng / day were observed on days 6 to 10 of culture in which formation of undifferentiated colonies of human iPS cells (SPH-0103) was observed.
  • FIG. 36 shows the results of the 10th to 14th days after changing the culture solution.
  • erythroid colonies composed of erythroid cells (FIGS. 37a and d), neutrophils, macrophages / monocytes, etc. by blood colony culture using the autologous serum.
  • Myeloid colonies (Fig. 37b, e) composed of multiple myeloid cells, and mixed colonies (Fig. 37c, f) composed of erythroid cells, myeloid cells and megakaryocytes were formed. It was.
  • Table 2 shows the results for blood colonies formed by co-culture with autologous mesenchymal stem cells, which were performed four times independently.
  • “number of iPS colonies” indicates the number of colonies of iPS cells subjected to blood colony culture using one autologous serum.
  • autologous serum is used to induce differentiation of mesenchymal stem cells, and by coculturing the differentiation-induced mesenchymal stem cells and autologous iPS cells, human iPS It was revealed that blood cells can be induced from cells without using heterologous animal serum (such as FBS) or allogeneic (allogeneic) serum.
  • blood cells such as red blood cells, white blood cells, megakaryocytes, etc. can be obtained from iPS cells (SPH-0103) established from a single donor without using animal cells or animal serum. It was also revealed that differentiation can be induced.
  • Functional cells such as blood cells obtained by inducing differentiation by the method of the present invention are free of foreign cell contamination, and further induced to differentiate under co-culture of mesenchymal stem cells established using human-derived serum lysate. In other words, no heterogeneous serum is mixed. For this reason, if various cells obtained by the method of the present invention are used, it is possible to realize extremely safe regenerative medicine without risk of contamination with animal cells, infection with unknown microorganisms, viruses, and prions. it can.

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Abstract

La présente invention concerne un procédé de différenciation de cellules souches pluripotentes humaines, le procédé comprenant l'induction de la différenciation de cellules souches pluripotentes humaines en co-culture avec des cellules souches mésenchymateuses établies à partir de cellules souches pluripotentes humaines.
PCT/JP2011/072609 2010-09-30 2011-09-30 Procédé de différenciation de cellules souches pluripotentes humaines WO2012043814A1 (fr)

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EP3045531A1 (fr) * 2013-09-12 2016-07-20 Kaneka Corporation Procédé permettant d'induire la différenciation de cellules souches pluripotentes induites et procédé permettant de sélectionner des cellules souches pluripotentes induites
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JPWO2020067439A1 (ja) * 2018-09-27 2021-10-07 国立大学法人大阪大学 心筋細胞のシート化方法
CN114642683A (zh) * 2022-02-25 2022-06-21 宁波一棵芽生物科技有限公司 一种具有抗光老化的牙髓间充质干细胞裂解液的制备方法

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Cited By (10)

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Publication number Priority date Publication date Assignee Title
EP3045531A1 (fr) * 2013-09-12 2016-07-20 Kaneka Corporation Procédé permettant d'induire la différenciation de cellules souches pluripotentes induites et procédé permettant de sélectionner des cellules souches pluripotentes induites
EP3045531A4 (fr) * 2013-09-12 2017-04-19 Kaneka Corporation Procédé permettant d'induire la différenciation de cellules souches pluripotentes induites et procédé permettant de sélectionner des cellules souches pluripotentes induites
JP2017507649A (ja) * 2014-01-21 2017-03-23 ザ メディカル カレッジ オブ ウィスコンシン インクＴｈｅ Ｍｅｄｉｃａｌ Ｃｏｌｌｅｇｅ Ｏｆ Ｗｉｓｃｏｎｓｉｎ， Ｉｎｃ． 多能性幹細胞の選択的な阻害のための方法
US11959096B2 (en) 2014-01-21 2024-04-16 The Medical College Of Wisconsin, Inc. Methods for selective inhibition of pluripotent stem cells
JP2015136349A (ja) * 2014-01-24 2015-07-30 国立大学法人 東京大学 プライム型多能性幹細胞からキメラ形成能を向上させた多能性幹細胞を製造する方法並びにそのための組成物および組合せ
US10246679B2 (en) 2015-03-02 2019-04-02 The University Of Tokyo Method for producing pluripotent stem cell having improved chimera forming ability from primed pluripotent stem cell, and composition and combination therefor
JPWO2020067439A1 (ja) * 2018-09-27 2021-10-07 国立大学法人大阪大学 心筋細胞のシート化方法
JP7256818B2 (ja) 2018-09-27 2023-04-12 国立大学法人大阪大学 心筋細胞のシート化方法
CN114642683A (zh) * 2022-02-25 2022-06-21 宁波一棵芽生物科技有限公司 一种具有抗光老化的牙髓间充质干细胞裂解液的制备方法
CN114642683B (zh) * 2022-02-25 2023-10-10 宁波一棵芽生物科技有限公司 一种具有抗光老化的牙髓间充质干细胞裂解液的制备方法

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