WO2019144605A1 - Procédé à haute performance pour la différenciation de hpsc en msc - Google Patents

Procédé à haute performance pour la différenciation de hpsc en msc Download PDF

Info

Publication number
WO2019144605A1
WO2019144605A1 PCT/CN2018/101076 CN2018101076W WO2019144605A1 WO 2019144605 A1 WO2019144605 A1 WO 2019144605A1 CN 2018101076 W CN2018101076 W CN 2018101076W WO 2019144605 A1 WO2019144605 A1 WO 2019144605A1
Authority
WO
WIPO (PCT)
Prior art keywords
differentiation
signaling pathway
mscs
activin
culture
Prior art date
Application number
PCT/CN2018/101076
Other languages
English (en)
Chinese (zh)
Inventor
王淋立
李强
陈月花
莫健
Original Assignee
皓昇莱生物制药有限公司
王淋立
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 皓昇莱生物制药有限公司, 王淋立 filed Critical 皓昇莱生物制药有限公司
Publication of WO2019144605A1 publication Critical patent/WO2019144605A1/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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/58—Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—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
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the invention relates to the field of cell culture, in particular to a method for efficiently differentiating hPSCs into MSCs.
  • the source of cells for clinical regenerative medicine transplantation mainly includes human pluripotent stem cells (hPSCs), namely human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
  • hPSCs human pluripotent stem cells
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cells
  • the general term and adult stem cells are two major categories, including adult stem cells including hematopoietic stem cells and mesenchymal stem cells.
  • Mesenchymal stem cells derived from mesoderm were originally isolated and identified by fibroblast-like cells from mouse bone marrow extracts by Friedenstein et al. in 1966. In 1991, Caplan initially referred to these cells as mesenchymal stem cells ( Mesenchymal stem cells, MSCs).
  • MSCs multipotent mesenchymal stromal cells' or 'mesenchymal stem cells' (MSCs); and define MSCs as 1) to grow adherently; 2) can differentiate into mesoderm lineages such as osteoblasts, cartilage and adipocytes; 3) high-level expression of CD105, CD90, CD73 and other MSCs positive markers; basically do not express CD45, CD34, CD14 or CD11b, CD79 ⁇ or CD19 and HLA-DR Such as MSCs negative markers.
  • ISCT International Society for Cellular Therapy
  • MSCs differentiate into the muscle, bone, cartilage, fat, tendon and ligament of the mesoderm lineage, and even transdifferentiate into the multi-lineage differentiation and immune regulation of neurons and islet cells, and avoid tumors.
  • the 2016 Clinical Trial Database shows that ⁇ 500 clinical trials are related to MSCs, many of which have evaluated MSC vs.
  • MSCs are widely available, clinical applications are generally obtained from bone marrow, umbilical cord and adipose tissue, but the isolation of MSCs from tissues will result in low yield, presence of mixed cells and purification of cells, and a more mature table. The longer time required for the type (about 1 month) is likely to cause cell aging, allogeneic immune rejection (the source of the umbilical cord) and the like. Therefore, in terms of clinical needs, the shortage of donor sources is still a problem that must be solved. In contrast, MSCs derived from autologous or immunomatch-matched and immortalized differentiation of hiPSCs are better at addressing these clinical cell therapies than MSCs derived from fat, cord blood, bone marrow, and ESCs. .
  • hPSC-MSCs MSCs
  • hiPSCs and hESCs the most classical method for the derivation of MSCs (hPSC-MSCs) from hPSCs (hiPSCs and hESCs) is to block the ALK5,4,7 kinase in the transforming growth factor TGF- ⁇ (Transforming Growth Factor) signaling pathway by the small molecule compound SB431542.
  • TGF- ⁇ Transforming Growth Factor
  • the object of the present invention is a method for efficiently differentiating hPSCs into MSCs.
  • a method for efficiently inducing differentiation of hPSCs into MSCs the steps of which are:
  • the new culture plate is coated with a gelatin-based extracellular matrix for cell screening.
  • the cells obtained by cell screening are cells having a spindle-type morphological feature, positive surface markers CD90, CD73, CD105, negative for CD34, CD45, CD14 or CD11b, CD79 ⁇ or CD19, and HLA-DR.
  • the gelatin-based extracellular matrix is gelatin for cell culture or type I, III, IV, V-type collagen and elastin, and laminin.
  • culture is carried out using a differentiation medium containing BMP-SMAD1/5/8 signaling pathway activator, TGF- ⁇ 1/Activin/Nodal-SMAD2/3 signaling pathway activator, Wnt activator, PI3K inhibitor.
  • the time is 12 to 72 hours.
  • step 1) at least one of BMP-SMAD1/5/8 signaling pathway activators BMP2, BMP4, BMP7.
  • the TGF- ⁇ 1/Activin/Nodal-SMAD2/3 signaling pathway activator is at least one of ActivinA, Activin B, TGF- ⁇ 1, and Nodal.
  • the Wnt activator is at least one of CHIR99021, BIO, WNT-3a, and R-spondin-2.
  • the PI3K inhibitor is at least one of TG100713, PIK90, and PI-103.
  • the TGF- ⁇ 1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor is at least one of SB431542, SB505124, A8301, and RepSox.
  • step 3 the culture is continued for 2 to 30 days.
  • step 2 culture with a differentiation medium containing BMP-SMAD1/5/8 signaling pathway activator, TGF- ⁇ 1/Activin/Nodal-SMAD2/3 signaling pathway activator, Wnt inhibitor .
  • step 2) the culture is continued for 2 to 10 days.
  • the Wnt inhibitor is at least one of Wnt-C59 and XAV-939.
  • the method of continuing the culture in step 4) is: culturing with a medium containing mesenchymal stem cells.
  • At least one medium contains basic fibroblast growth factor bFGF.
  • the obtained MSCs are obtained by inducing hPSCs to differentiate into MSCs through the middle stage or the mesoderm stage.
  • An MSCs cell prepared by the above differentiation method An MSCs cell prepared by the above differentiation method.
  • the beneficial effects of the present invention are as follows: 1) The present invention is the first method in the world to establish intact hPSCs in vitro to differentiate into MSCs through the middle stage or the lateral mesoderm stage. To provide a reliable technical resource for studying the differences between strains derived from different cells of human pluripotent stem cells and the differences between MSCs in vitro and in vivo.
  • the method for direct differentiation of hPSC-MSCs of the present invention has the advantages of simple steps, simple operation and high reproducibility, and phenotypic mature and high quality MSCs derived from mesodermal lineage can be obtained in only 12 days.
  • Figure 1 shows undifferentiated cells of hiPSCs.
  • Figure 2 shows undifferentiated cells of hESCs.
  • Figure 3 shows the differentiation of MSCs precursor cells obtained from the middle segment.
  • Figure 4 shows MSCs precursor cells obtained by differentiation through the mesoderm.
  • Figure 5 is a MSCs precursor cell obtained by adding bFGF to differentiate into a middle segment.
  • Figure 6 shows MSCs cells differentiated from hiPSCs into phenotypic and spindle-shaped forms.
  • Figure 7 shows MSCs cells differentiated from hESCs into phenotypic and spindle-shaped forms.
  • Figure 8 shows no cells found to adhere to growth.
  • hPSCs cells (Fig. 1 and Fig. 2) to matrigel (Matrigel, MG; one of extracellular matrices), coated with a 30-well adherent plate for 30 min, and differentiated with 0.5 ml of pluripotent stem cells.
  • the medium was cultured at 37 ° C, 5% CO 2 for 24 hours (Day 0), and changed to 0.5 ml of pluripotent stem cell differentiation medium containing no TGF- ⁇ 1.
  • the medium also contained 40 ng/ml BMP4 and 30 ng/ml Activin A, 6 ⁇ M CHIR99021, 100 nM PIK90.
  • BMP4 is bone morphogenetic protein 4
  • CHIR99021 is 6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidine-2 -ylamino]ethylamino]pyridine-3-carbonitrile (CAS: 252917-06-9)
  • Activin A is activin A
  • PIK90 is N-(2,3-dihydro-7,8-dimethoxy Imidazo[1,2-c]quinazolin-5-yl)-3-pyridinecarboxamide (CAS: 677338-12-4). Cultivate for 24 hours.
  • SB431542 is 4-[4-(1,3-benzothiazol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate (CAS: 301836-41 -9).
  • the expressions of pluripotency markers Oct3/4, Nanog, Sox2, etc. gradually decreased (Table 2), BRACHYURY decreased to no expression, and the mesodermal markers Nkx2.5, HAND1, FOXF1, etc. continued to be highly expressed. It can be concluded that the cells are in the stage of the mesoderm at this time (Table 1).
  • the cell confluence can reach ⁇ 90% by GT stage, and the cells are digested with 2ml of 0.05% Trypsin-EDTA for 3min, and then added with 4ml DMEM/F12 (containing 10% FBS). The 15ml centrifuge tube is recovered and centrifuged at 200g for 5min.
  • the cells can express MSC positive Markers such as CD105, CD90 and CD73 at high levels; they do not express MSC-negative Markers such as CD45, CD34, CD14 or CD11b, CD79 ⁇ , CD19 and HLA-DR.
  • MSC positive Markers such as CD105, CD90 and CD73 at high levels; they do not express MSC-negative Markers such as CD45, CD34, CD14 or CD11b, CD79 ⁇ , CD19 and HLA-DR.
  • the cells have a typical spindle-shaped morphology, and the cells can be identified as MSCs cells (Fig. 6 and Fig. 7).
  • BMP4 was replaced with BMP2 and BMP7;
  • Activin A was replaced with Activin B, TGF- ⁇ 1, Nodal, and CHIR99021 was used with BIO (CAS: 667463-62-9), WNT-3a (WNT signaling protein 3a), R-spondin- 2 (R-spondin signal protein 2) substitution;
  • SB431542 was replaced with SB505124 (CAS: 694433-59-5), A8301 (CAS: 909910-43-6), RepSox (CAS: 446859-33-2) PI-103;
  • a similar effect can also be produced by replacing PIK90 with TG100713 (CAS: 925705-73-3), PI-103 (CAS: 371935-74-9).
  • step 2) containing 20 ng/ml BMP4 (BMP-SMAD1/5/8 signaling pathway activator), 3 uM/ml SB505124 (TGF- ⁇ 1/Activin/Nodal-
  • the differentiation medium of SMAD2/3 signaling pathway activator and 1uM/ml Wnt-C59 (Wnt inhibitor) was further cultured for 24 hours, at which time the mesodermal markers Nkx2.5, HAND1, FOXF1 and the like were highly expressed (Table 4).
  • SB505124 is 2-[4-(1,3-benzobisazol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methylpyridine (CAS: 694433-59-5), Wnt-C59 is 4-(2-methyl-4-pyridyl)-N-[4-(3-pyridyl)phenyl]phenylacetamide (CAS: 1243243- 89-1).
  • step 4 typical spindle-shaped MSCs precursor cells are obtained, as shown in FIG. The remaining steps were unchanged. Finally, after FACS identification, the cells could express MSC-positive Markers such as CD105, CD90 and CD73 at high levels; MSC-negative Markers such as CD45, CD34, CD14 or CD11b, CD79 ⁇ or CD19 and HLA-DR were not expressed at all. 5 is shown.
  • MSC-positive Markers such as CD105, CD90 and CD73 at high levels
  • MSC-negative Markers such as CD45, CD34, CD14 or CD11b, CD79 ⁇ or CD19 and HLA-DR were not expressed at all. 5 is shown.
  • Step 1) Starting with the addition of basic fibroblast growth factor (bFGF) on the basis of the differentiation medium, at step 4) More and more typical spindle-shaped morphological MSCs precursor cells will be obtained, as shown in Figure 5. The remaining steps were unchanged. Finally, after FACS identification, the cells could express MSC-positive Markers such as CD105, CD90, CD73, etc.; MSC-negative Markers such as CD45, CD34, CD14, CD11b, CD79 ⁇ , CD19 and HLA-DR were not expressed at all. 6 is shown.
  • MSC-positive Markers such as CD105, CD90, CD73, etc.
  • MSC-negative Markers such as CD45, CD34, CD14, CD11b, CD79 ⁇ , CD19 and HLA-DR were not expressed at all. 6 is shown.
  • the undifferentiated hPSCs cells were transferred to a 24-well adherent culture plate coated with extracellular matrix (MG) for 30 min, and cultured with 0.5 ml of pluripotent stem cell differentiation medium at 37 ° C, 5% CO 2 for 24 hours ( Day 0), 0.5 ml of pluripotent stem cell differentiation medium containing no TGF- ⁇ 1, containing 30 ng/ml Activin A, 6 ⁇ M CHIR99021, and 100 nM PIK90 for 24 hours.
  • the expression levels of the protocolonary markers such as MIXL1, BRACHYURY, and TBX6 were detected, and it was found that high expression was obtained, and the cells were in the pre-segment phase (Table 7).

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)
  • Developmental Biology & Embryology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Reproductive Health (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Gynecology & Obstetrics (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé à haute performance pour induire la différenciation de hPSC en MSC, comprenant les étapes suivantes : 1) transfert de lignées cellulaires hPSC non différenciées sur une plaque de culture revêtue d'une matrice extracellulaire pour la culture ; puis culture de celles-ci à l'aide d'un milieu de culture de différenciation contenant un activateur de la voie de signalisation BMP-SMAD1/5/8, un activateur de la voie de signalisation TGF-β1/activine/Nodal-SMAD2/3, un activateur Wnt et un inhibiteur de PI3K ; 2) élimination de l'ancien milieu de culture et poursuite de la culture à l'aide d'un milieu de culture de différenciation contenant un inhibiteur de la voie de signalisation TGF-β1/activine/Nodal-SMAD2/3 ; 3) digestion et transfert des cellules sur une nouvelle plaque de culture, et poursuite de la culture à l'aide d'un milieu de culture de différenciation contenant un inhibiteur de la voie de signalisation TGF-β1/activine/Nodal-SMAD2/3 ; et 4) digestion des cellules et transfert de celles-ci sur une plaque de culture adhérente pour une culture continue, de façon à obtenir des MSC. La présente invention établit in vitro un procédé complet pour la différenciation dirigée de hPSC en MSC par le biais de l'étape de strie primitive centrale ou de l'étape de mésoderme latéral. Par rapport aux procédés classiques, les étapes techniques du présent procédé sont simples, faciles à utiliser et hautement reproductibles, et des MSC présentant un phénotype mature et une qualité élevée peuvent être obtenues en seulement 12 jours.
PCT/CN2018/101076 2018-01-26 2018-08-17 Procédé à haute performance pour la différenciation de hpsc en msc WO2019144605A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810075968.9A CN108570448B (zh) 2018-01-26 2018-01-26 一种高效的hPSCs向MSCs分化的方法
CN201810075968.9 2018-01-26

Publications (1)

Publication Number Publication Date
WO2019144605A1 true WO2019144605A1 (fr) 2019-08-01

Family

ID=63576718

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/101076 WO2019144605A1 (fr) 2018-01-26 2018-08-17 Procédé à haute performance pour la différenciation de hpsc en msc

Country Status (2)

Country Link
CN (1) CN108570448B (fr)
WO (1) WO2019144605A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022025598A1 (fr) * 2020-07-28 2022-02-03 삼성전자 주식회사 Composition pour induire la différenciation de cellules souches pluripotentes en cellules souches mésenchymateuses comprenant un activateur smad1/5/8 et utilisation associée

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592007B (zh) * 2019-09-19 2020-08-21 安徽中盛溯源生物科技有限公司 一种间充质干细胞及其制备方法和应用
CN111876380B (zh) * 2020-08-04 2022-06-28 浙江大学 使人胚胎干细胞分化成间充质干细胞的诱导培养基
WO2023011114A1 (fr) * 2021-08-06 2023-02-09 中国科学院动物研究所 Cellule ric et son procédé de préparation et son utilisation
CN113462642A (zh) * 2021-08-12 2021-10-01 呈诺再生医学科技(珠海横琴新区)有限公司 间充质干细胞的快速诱导分化方法、试剂盒及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1636054A (zh) * 2001-07-06 2005-07-06 杰龙公司 来自人胚胎干细胞的间充质细胞和成骨细胞
CN101712947A (zh) * 2009-11-12 2010-05-26 浙江大学 来源于胚胎干细胞的间充质干细胞的制备方法及其用途
CN101984048A (zh) * 2010-11-24 2011-03-09 中国人民解放军军事医学科学院放射与辐射医学研究所 一种培养间充质干细胞的培养基
CN102686724A (zh) * 2009-10-27 2012-09-19 财团法人首尔大学校产学协力团 由人类多能干细胞产生间充质干细胞的方法以及通过所述方法产生的间充质干细胞
CN104487568A (zh) * 2012-07-11 2015-04-01 爱姆斯坦生物技术公司 人胚胎干细胞衍生的间充质样干细胞、方法及其应用
CN105154393A (zh) * 2014-12-22 2015-12-16 浙江大学 一种胚胎干细胞分化间充质干细胞的方法
TW201819625A (zh) * 2016-08-23 2018-06-01 中央研究院 製備誘導型間質幹細胞及增進間質幹細胞之特質的方法及其應用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1636054A (zh) * 2001-07-06 2005-07-06 杰龙公司 来自人胚胎干细胞的间充质细胞和成骨细胞
CN102686724A (zh) * 2009-10-27 2012-09-19 财团法人首尔大学校产学协力团 由人类多能干细胞产生间充质干细胞的方法以及通过所述方法产生的间充质干细胞
CN101712947A (zh) * 2009-11-12 2010-05-26 浙江大学 来源于胚胎干细胞的间充质干细胞的制备方法及其用途
CN101984048A (zh) * 2010-11-24 2011-03-09 中国人民解放军军事医学科学院放射与辐射医学研究所 一种培养间充质干细胞的培养基
CN104487568A (zh) * 2012-07-11 2015-04-01 爱姆斯坦生物技术公司 人胚胎干细胞衍生的间充质样干细胞、方法及其应用
CN105154393A (zh) * 2014-12-22 2015-12-16 浙江大学 一种胚胎干细胞分化间充质干细胞的方法
TW201819625A (zh) * 2016-08-23 2018-06-01 中央研究院 製備誘導型間質幹細胞及增進間質幹細胞之特質的方法及其應用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HU JIABO ET AL.: "Preliminary investigation on the differantiation of human embryonic stem cells into mesenchymal stem cells", CHINSES JOURNAL OF CLINICAL LABORATORY SCIENCE, vol. 31, no. 4, 30 April 2012 (2012-04-30), pages 276 - 278 *
SHI LUNGANG ET AL.: "Induction and Identification of Mesenchymal stem lioke cells Huamn embryonic stem cells", JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY- MEDICAL SCIENCE, vol. 29, no. 3, 31 December 2009 (2009-12-31) - 311, pages 307 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022025598A1 (fr) * 2020-07-28 2022-02-03 삼성전자 주식회사 Composition pour induire la différenciation de cellules souches pluripotentes en cellules souches mésenchymateuses comprenant un activateur smad1/5/8 et utilisation associée

Also Published As

Publication number Publication date
CN108570448A (zh) 2018-09-25
CN108570448B (zh) 2019-04-02

Similar Documents

Publication Publication Date Title
WO2019144605A1 (fr) Procédé à haute performance pour la différenciation de hpsc en msc
Chen et al. Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells
Liu et al. Neural crest stem cells and their potential therapeutic applications
JP6678107B2 (ja) 膵前駆細胞の増殖方法
Techawattanawisal et al. Isolation of multipotent stem cells from adult rat periodontal ligament by neurosphere-forming culture system
Boyd et al. Human embryonic stem cell–derived mesoderm-like epithelium transitions to mesenchymal progenitor cells
Lee et al. Enhancement of osteogenic and chondrogenic differentiation of human embryonic stem cells by mesodermal lineage induction with BMP-4 and FGF2 treatment
JP7287948B2 (ja) 多分化能性幹細胞分化促進剤
Świerczek et al. From pluripotency to myogenesis: a multistep process in the dish
Mu et al. Enhanced differentiation of human amniotic fluid‐derived stem cells into insulin‐producing cells in vitro
WO2023106122A1 (fr) Procédé de production de cellules de crête neurale spécialisées pour la différenciation en lignée mésenchymateuse
Sauerzweig et al. A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells
WO2023127824A1 (fr) Procédé de culture de cellules de crête neurale et procédé de production
EP2882848B1 (fr) Procédé, combinaison et/ou composition pour induire la différenciation des cardiomyocytes
Perino et al. Cardiomyogenic stem and progenitor cell plasticity and the dissection of cardiopoiesis
WO2010108008A2 (fr) Différenciation de cellules bsc et leur utilisation en thérapie
JP7094567B2 (ja) 神経堤細胞および交感神経細胞の製造方法
Honda et al. N-cadherin is a useful marker for the progenitor of cardiomyocytes differentiated from mouse ES cells in serum-free condition
US9598675B2 (en) Method for preparing mesenchymal stem cell-like cells and cardiomyocyte-like cells
WO2019107485A1 (fr) Méthode de culture de cellules
Yamasaki et al. Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium.
KR101896803B1 (ko) 인간 만능줄기세포로부터 중간엽 줄기세포로의 분화 유도 생산율을 증가시키는 방법 및 이에 의해 생성된 중간엽 줄기세포
KR20110095218A (ko) ＰＩ３Ｋ／ＡＫＴ／ＧＳＫ３ 경로를 통해 성체줄기세포의 증식, 다분화능 및 재프로그래밍을 촉진하는 ＣＤ４９ｆ
Vasyliev et al. Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications
Wei et al. Lateral Mesoderm-derived mesenchymal stem cells with robust osteochondrogenic potential and hematopoiesis-supporting ability

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: 18902917

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18902917

Country of ref document: EP

Kind code of ref document: A1