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WO2025146565A1 - Génération évolutive d'organoïdes cardiaques dérivés de cellules souches pluripotentes induites humaines - Google Patents

Génération évolutive d'organoïdes cardiaques dérivés de cellules souches pluripotentes induites humaines Download PDF

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WO2025146565A1
WO2025146565A1 PCT/IB2024/050075 IB2024050075W WO2025146565A1 WO 2025146565 A1 WO2025146565 A1 WO 2025146565A1 IB 2024050075 W IB2024050075 W IB 2024050075W WO 2025146565 A1 WO2025146565 A1 WO 2025146565A1
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ebs
differentiation
medium
cos
generation
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Fatma ALHASHIMI
Sarkawt HAMAD
Kurt Pfannkuche
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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
    • C12N2513/00—3D culture

Definitions

  • Embodiments of the present invention relates to the medicine and healthcare industry. Particularly, present disclosure relates to a method of scalable generation of human cardiac organoids from human induced pluripotent stem cells.
  • Cardiomyocytes generated from human pluripotent stem cells are important cell sources for disease models, regenerative treatments, and drug discovery.
  • hPSC-CMs human pluripotent stem cells
  • Organoids are multicellular structures that resemble organs and are thought to form spontaneously or systematically using stem cells' natural capacity for self-assembly. Organoids can imitate the salient features of the organs that serve as their models. Cardiovascular organoids (COs) are employed in the creation of new pharmaceutical medications via in-vitro drug validations. They allow researchers to study specific physiological pathways during phases of embryogenesis. Their application lowers costs and substitutes human test systems for animal cells.
  • COs Cardiovascular organoids
  • COs make it easier to conduct experimental transplantation experiments to repair the heart after a myocardial infarction, and they improve tissue engineering's 3-D bioprinting of human cardiac tissue. It remains a problem, nonetheless, to produce human cardiac organoids on a large scale in suspension culture. Such COs should ideally be made up entirely of cardiac fibroblasts, endothelial cells, and cardiomyocytes and not contain any cells from other organs, including the primitive intestine.
  • An object of the invention is to produce EBs in large numbers in a bioreactor.
  • An object of the invention is to produce EBs by centrifugation method in 96-well plates, and different size dishes.
  • Another object of the invention is to omit Matrigel and animal derived components to facilitate the development of GMP conform protocols in preparation of pre-clinical and clinical trials.
  • Yet another object of the present invention is to produce COs with high quality cardiac cells, less workload and lower cost.
  • Yet another object of the present invention is to generate COs containing pluripotent stem cells derived cardiomyocytes, endothelial cells, smooth muscle cells, and cardiac fibroblasts with mature cardiomyocytes action potential characteristics within 20 days.
  • EBs embryoid bodies
  • the method for generating EBs by centrifugation comprises the steps of: preparing a cell mixture; allowing centrifugation of the cell mixture; incubating the mixture to form embryoid bodies (EBs) transferring the EBs into a well; develop the EBs to mesodermal germ layer; exchange the medium completely and induce CO fate in medium containing growth factors inducing CO development by a specific growth factor composition; and characterize the COs.
  • hiPSCs are dissociated into single cells when the culture reaches confluency in the range of 70-80%.
  • predetermined quantity of hiPSCs is added in the range of 0.01 X 10 6 to 0.03 X 10 6 in 100-120 pl E8 medium.
  • the mixture is centrifuged at a temperature in the range of 15 °C to 30 °C for predetermined time in the range of 5 to 15 minutes.
  • the mixture is incubated at a predetermined temperature in the range of 30 °C- 37 °C for 24 to 72 hours.
  • embryoid bodies or singular spheroids are transferred into a single or multiwell plate containing a predetermined quantity of the medium.
  • a well of a U- shape ultra-low attachment 96 well plate is used in centrifugation.
  • wnt- signalling inhibitors such as IWP2 and XAV939
  • PKA protein kinase A
  • EBs embryoid bodies directly inside a bioreactor and differentiation to form human cardiac organoids.
  • the method for generating EBs using by inside a bioreactor comprises the steps of: culturing a predetermined quantity of hiPSC cells in a medium; washing the hiPSC cells with E8 medium; dissociating and filtering the cells; counting number of cells by an automatic cell counter; suspending the cells in the bioreactor; agitating the culture to obtain EBs; inducing mesodermal differentiation in a culture containing EBs; supplementing a medium without insulin and repeatedly incubating the culture for a pre-determined time period; exchanging the medium and inducing wnt- signalling; inducing development of COs; allowing the cardiac cells to be mature to obtain high quality COs; harvesting COs in a tube and allowing COs to settle at the bottom of the tube; and characterizing COs using an active reagent.
  • the COs are characterized using an activated papain reagent for dissociation.
  • the mixture is stimulated by human vascular endothelial growth factor namely VEGF and bFGF and protein kinase A (PKA) stimulator.
  • human vascular endothelial growth factor namely VEGF and bFGF
  • PKA protein kinase A
  • Dulbecco's Phosphate Buffered Saline (DPBS) is used to wash cells.
  • Fig. 1A illustrates a method of generation of embryoid bodies (EBs) by centrifugation and differentiation to form cardiac organoids (COs), in accordance with an embodiment of the present invention
  • Fig. IB illustrates a method of generation of embryoid bodies in a bioreactor and differentiation to form cardiac organoids, in accordance with an embodiment of the present invention
  • FIG. 2 illustrates experimental data, in accordance with an embodiment of the present invention.
  • FIG. 3 illustrates a diagram of single cell RNA sequencing of cardiac organoids at day 20, and selective markers, in accordance with an embodiment of the present invention.
  • figure 1A illustrates a method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form cardiac organoids (COs).
  • EBs embryoid bodies
  • COs cardiac organoids
  • hiPSCs human induced pluripotent stem cells
  • the method (100) starts at step 110, a cell mixture is prepared by adding a predetermined quantity of human induced pluripotent stem cells or hiPSCs, for example 0.01 X 10 6 to 0.03 X 10 6 hiPSCs in a medium poured in a 96 well plate. For smaller plate formats cell counts and medium volume are adapted.
  • hiPSCs are used for cell differentiation.
  • HiPSCs cells have the ability to develop into any form of a body cell. This property of hiPSCs makes them a fit for generation and regeneration of human cells.
  • Stem cells further have the ability to self-renew and have the ability to differentiate into different types of tissue or cells for example cardiac cells, hepatic cells, gastrointestinal cells, neural cells.
  • hiPSCs is dissociated into single cells when the culture reaches confluency in the range of 70-80%. Further, maintaining the hiPSC cultures utilized for CO generation at higher levels of pluripotency for example, over 90-99% pluripotency for repeatability.
  • E8 medium is a xenogen-free medium, which may be used for both clinical and academic purposes. This offers a simpler medium for the cultivation of pluripotent stem cells because it may contain the necessary elements for maintaining embryonic stem cells and induced pluripotent stem cells.
  • E8 medium may chemically be defined as a medium containing Dulbecco's modified Eagle's medium (DMEM)/F12, 64 mg/1 L-ascorbic acid-2- phosphate magnesium, 14 pg/1 sodium selenium, 100 pg/1 fibroblast growth factor (FGF)2, 20 mg/1 insulin, 543 mg/1 NaHCCE, and 10.7 mg/1 transferrin, 2 pg/1 transforming growth factor (TGF)pl or 100 pg/1 Nodal).
  • DMEM Dulbecco's modified Eagle's medium
  • FGF fibroblast growth factor
  • TGF transforming growth factor
  • the cell mixture in the plate may be centrifuged at up to 2400g for a predetermined time in the range of 3 to 15 minutes at a temperature in the range of 15°C to 30°C.
  • cells are collected into aggregates.
  • the cell aggregates may be incubated at a predetermined temperature in the range of 30°C- 37°C for about 24-48 hours to form one embryoid body (EB) per individual well of the multiwell plate. Incubation is carried out in the presence of carbon dioxide in the range of about 2-5%. In a preferred embodiment, the carbon dioxide is 5%. After about 24- 48 hours, one or more single spheroids or embryoid bodies or EBs are formed.
  • Embryoid bodies may be defined as, but not limited to, three-dimensional aggregates of pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).
  • PSC pluripotent stem cells
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • the EBs formed may be transferred using a cut tip of let’s say, a 1000 pl pipette into a single or multiwell plate containing predetermined quantity, let’s say for example 0.3 ml to 2 ml E8 medium.
  • EBs may be combined into a dish with roughly 3.5 cm diameter with 1-5 mL of medium.
  • the EBs are continuously maintained in individual wells of ultralow adhesive multiwell plates initially used in step 130 such as 96-wells or smaller formats. Plates may be cultured on a rocking table at a rate of 30-60 rotations per minute (rpm) at about 37 °C with 2-5% CO2 supply.
  • Well plates such as, but not limited to, 12 well plates, 24 well plates, 48 well plates may also be used.
  • embryoid bodies are generated from suspension instead by the centrifugation method.
  • pluripotent stem cells are diluted in an appropriate amount of medium.
  • 0.1 X 10 6 - 0.5 X 10 6 hiPSCs may be introduced to approximately 1 mL of E8 medium supplemented with ROCK inhibitor and transferred into a single or multiwell plate.
  • the amount of cell suspension is scaled up depending on the size of the single or multiwell plate.
  • the cells are cultivated in non-adhesive 6 or 10 cm plates and the cell suspension is agitated for 40-50 hours at 40 rpm at a temperature of about 37 °C, and 2-5% CO2.
  • E8 medium was supplemented with rho kinase inhibitor (ROCK inhibitor), and the plates or dishes were incubated inside a humidified incubator at 37°C and 5% CO2.
  • E8 medium may be supplemented with 2-10 pM ROCK inhibitor (an inhibitor to rho kinase, a kinase belonging to the AGC family of serine-threonine specific protein kinases). It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton).
  • step 150 involves developing the EBs to mesodermal germ layer.
  • the mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals.
  • the outer layer is the ectoderm, and the inner layer is the endoderm.
  • Cardiac tissues are derived from mesoderm. Density of EBs at day 0 may vary depending on cell line and medium agitation. In our hand 75-100 EBs per mL are a good starting point. Lower densities of EBs do not impair differentiation while too high numbers negatively affect the process.
  • the embryoid bodies may be suspended on day 0, into a mesodermal induction medium composed of RPMI 1640 with a lx B27 supplement without insulin and a 24- hour addition of CHIR99021 (small molecule to inhibit canonical Wnt signalling pathway) on day 0. Concentration of CHIR99021 may be optimized for individual stem cell lines. A typical concentration of CHIR99021 is 12pM.
  • the medium On day 1 (step 150), the medium may be changed into RPMI 1640 with a lx B27 supplement without insulin.
  • step 160 On day 2 (step 160), the medium was completely exchanged with RPMI 1640, lx B27 supplement (without insulin), 2-10 pM IWP2, 2-10 pM XAV939, 10 ng/mL bFGF, 10 ng/mL VEGF, and 0.2 mM 8 -Bromoadenosine 3', 5 '-cyclic monophosphate sodium salt monohydrate (8Bro), further inducing CO fate in medium containing growth factors.
  • the Wnt signalling pathway a critical signalling route involved in numerous biological processes, including cell proliferation, differentiation, and development, is specifically known for its function as an inhibitor of this pathway.
  • XAV939 is frequently used to control Wnt signalling in cell culture and molecular biology investigations to examine its impact on cell behaviour and gene expression. Later, cells may be kept on a rocking table at 30-60 rpm about 48 hours without having their medium changed.
  • VEGF growth factor is a powerful angiogenic factor identified as being crucial for the proliferation of vascular endothelial cells.
  • VEGF is crucial for cell proliferation and differentiation and bFGF is a crucial member of the family of heparin-binding proteins.
  • M-medium maturation medium
  • Ascorbate ascorbic acid
  • Esterified and non-esterified unsaturated fatty acids may be shielded against peroxidation by the water-soluble antioxidant ascorbic acid.
  • Antioxidant efficacy of ascorbate is well known. Glutathione, tocopherol, carotenoids, flavonoids, cysteine, methionine, polyamines, and dehydrins and annexins are among the other antioxidants that may be used in a cell culture media.
  • the medium is further supplemented with: Insulin, VEGF, bFGF, dexamethasone, elevated concentration of calcium ions, creatine, taurine, L-carnitine, oleic acid, palmic acid, T3 thyroid hormone, pencilline-streptomycine, B-mecarpoethanol, L-Glutamin, Heparin, 14 pg/1 sodium selenium, and 10.7 mg/1 transferrin. Cultivation in M-medium is performed till day 10, or 20 or longer depending on the demand and desired degree of maturation.
  • the cells may be matured and characterized to obtain high quality COs by continuous culture in M-medium.
  • Maturation of a cell may be defined as, but not limited to, a process of gaining profound phenotypic and functional changes as the cells convert from the processing to the costimulatory stages.
  • a mature cell refers to a cell that has differentiated, which means it has acquired a specific rather than a generalized function.
  • COs are used for any desired application usually from day 10 (early stage of cardiac electrophysiology) or day 20 (mature action potentials characteristics) onwards.
  • the cells are then isolated and allowed to settle in a container.
  • organoids may be dissociated by enzymatic digest.
  • Papain reagent may be used to dissociate the cells. Briefly, 2-10pL of 0.2 M L-cysteine may be added let’s say in 15pL of papain solution into 1 mL of DPBS and incubated at 37 °C for at least 15 minutes to activate the papain reagent.
  • DPBS also known as Dulbecco's Phosphate Buffered Saline may be a buffering agent, used to keep cell culture medium within the physiological range of 7.2-7.6 pH. Potassium chloride, monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate are all present in this well-balanced salt solution.
  • figure IB illustrates a method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form cardiac organoids (COs).
  • the CO differentiation process may be enhanced for scaling up.
  • a share stress free bioreactor of the clinostat type for example, ClinoStar (provided by Celvivo, Maryland, USA) may be used.
  • the process of CO differentiation and scaling up was carried out in a suspension bioreactor, Applikon, equipped with mass flow controllers and at least 250 mL multi-use glass vessel.
  • the growing and synthesis of various biological materials, such as cells, bacteria, or tissue cultures, in a suspended condition inside a liquid medium may take place in a suspension bioreactor, a type of bioreactor often used in biotechnology and bioprocessing.
  • the method starts at step 202, culturing a predetermined quantity of hiPSC cells in a medium.
  • the hiPSCs Prior to bioreactor inoculation, the hiPSCs may be cultured on Matrigel-coated 100 mm dishes (Coming Life Science, # 353003) and incubated in a humidified incubator at 35-37°C, with 2-10% CO2. Matrigel may be replaced by xenogen-free matrices such as GMP qualified vitronectin.
  • the hiPSCs were cultured in E8 medium for about one week with daily medium change until approximately 85% cell confluency is reached.
  • DPBS Dulbecco's Phosphate Buffered Saline
  • DPBS Dulbecco's Phosphate Buffered Saline
  • the usage of DPBS may be, but not limited to, as an irrigating, transporting or diluting fluid while it DPBS may also help in maintaining cell tonicity and viability for a limited period of time in a cell culture.
  • step 206 dissociating the cells and filtering the cell culture. For dissociation, cells were incubated with 1-2 mL ReLeSRTM (Stemcell Technologies, # 05872) per plate for 5-10 minutes at room temperature.
  • Dissociation may be done my methods such as, but not limited to, enzymatic dissociation, chemical dissociation and mechanical dissociation.
  • enzymatic dissociation cells may be treated with the enzymes such as, but not limited to, Accutase, TrypLE, or trypsin/EDTA, these enzymes followed by trituration, or a combination one of these enzymes followed by incubation with another enzyme, including DNase I.
  • Chemical approaches for dissociation of cells may include, but not limited to, in vitro and in vivo cell dissociation techniques and methods.
  • Mechanical dissociation refers to, but not limited to, the use of filters, chopping techniques, microfluidic devices, and various trituration strategies using a variety of pipettes.
  • the dissociated cells may be passed through a 30-40 pm filter (Greiner Bio-One, # 542040) and may be collected in a 50 mL Falcon tube with 5-15 mL E8 medium containing 5-15 pM ROCK inhibitor.
  • ROCK inhibitor may enhance survival of human pluripotent stem cells when they are dissociated to single cells by preventing dissociation-induced apoptosis, thus increasing their cloning efficiency.
  • Next step is to count (208) number of cells by an automatic cell counter.
  • Cells are counted by an automatic cell counter (NaNoEnTek, Korea).
  • an automatic cell counter NaNoEnTek, Korea.
  • the bioreactor glass vessel was placed under laminar airflow, for sterilizing (210) the bioreactor and equilibration E8 medium was discarded.
  • agitating (212) the culture to obtain EBs at a propeller speed of 120- 150 rpm for a standard impeller or about 50 rpm for a membrane impeller (Biothrust, Germany), gassed with approximately 95% air, and 2-10% CO2 followed by incubation at 37 °C for 24-48 hours in E8 medium supplemented with ROCK inhibitor.
  • Differentiation starts with the step of inducing (214) mesodermal differentiation in a culture containing EBs to COs fate.
  • the mesodermal differentiation was induced in a medium containing EBs.
  • the medium may be changed into to RPMI 1640 supplemented with lx B27 without insulin along with 80-100 pg/mL ascorbate and 10-15 pM CHIR9902L Ascorbic acid is a vital compound for cell growth and maintenance of healthy cells in vivo and in vitro. It is a water soluble antioxidant that may protect esterified and non-esterified unsaturated fatty acids from peroxidation.
  • RPMI 1640 containing lx B27 without insulin and 80-100 pg/mL ascorbate, and cells were incubated for another 24 hours.
  • 100 mL fresh RPMI 1640 supplemented with lx B27 without insulin, 80-100 pg/mL ascorbate, 10 pM IWP2, 10 pM XAV939, 10 ng/mL VEGF, 10 ng/mL bFGF, and 0.2 mM 8Bro were added into the vessel and culture was continued for another 48 hours.
  • the medium was replaced by 100 mL fresh RPMI 1640 supplemented with lx B27 without insulin and 80-100 pg/mL ascorbate, 10 ng/mL VEGF, and 10 ng/mL bFGF. From day 7 onward, every three days the medium was refreshed with 100 mL M- Medium.
  • the cells may be mature enough to obtain high quality COs. Maturation of a cell may be defined as, but not limited to, a process of gaining profound phenotypic and functional changes as the cells convert from the processing to the costimulatory stages.
  • a mature cell refers to a cell that has differentiated, which means it has acquired a specific rather than a generalized function.
  • Suspension bioreactors allow cells to grow freely in the liquid medium, typically swirled or agitated to give nutrients and maintain a uniform environment, in contrast to traditional adherent cell cultures, where cells grow attached to a surface (e.g., a petri dish or flask).
  • suspension bioreactors may be able to sustain a greater range of species and function on a much larger scale than immobilised cultures.
  • Suspension bioreactors may commonly be used in large-scale bioproduction processes, such as the production of biopharmaceuticals, vaccines, organoids etc.
  • step 224 harvesting COs in one or more 50 mL Falcon tube(s) and allowing COs to settle at the bottom of the tube by gravity. Cell harvesting may be done by, but not limited to, tools including pipettes, and centrifuges. Centrifuges are used for pelleting cells and separating them from culture media.
  • DPBS also known as Dulbecco's Phosphate Buffered Saline may be a buffering agent, used to keep cell culture medium within the physiological range of 7.2-7.6 pH. Potassium chloride, monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate are all present in this well-balanced salt solution.
  • FIG. 2 illustrates experimental data, in accordance with an embodiment of the present invention.
  • FIG. 1 Schematic illustration of differentiation; in section B. Bright field of cardiac organoids; in section C. Representative flow cytometric scatter plots analysis of dissociated cardiac organoids at day 10 of single cells stained for TNNT2-FITC cardiac maker: VE-Cadherin-PE endothelial marker; in section D. and E. Whole mount immunostaining of cardiac organoids where cardiac organoids were stained without prior dissociation with antibodies against a- actinin, CX43, VE-Cadherin, vWF; in section F. Electron microscopic images of cardiac organoid; in section G. Sharp electrode electrical activity of cardiac organoid.

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Abstract

L'invention concerne un procédé (100) de génération de corps embryoïdes (EB) et de différenciation pour former des organoïdes cardiaques humains, comprenant : la préparation (110) d'un mélange cellulaire, l'autorisation (120) de centrifugation du mélange cellulaire, l'incubation (130) du mélange pour former des corps embryoïdes (EB), le transfert (140) des corps embryoïdes (EB) dans un puits, le développement (150) des corps embryoïdes (EB) vers la couche germinale mésodermique, l'échange (160) complet du milieu et l'induction de l'évolution de CO dans un milieu contenant des facteurs de croissance, l'induction (170) du développement de CO par une composition de facteur de croissance spécifique et la caractérisation (180) des CO.
PCT/IB2024/050075 2024-01-04 2024-01-04 Génération évolutive d'organoïdes cardiaques dérivés de cellules souches pluripotentes induites humaines Pending WO2025146565A1 (fr)

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US20230212525A1 (en) * 2020-03-19 2023-07-06 Industry-Academic Cooperation Foundation, Yonsei University Cardiac organoid, manufacturing method therefor, and method for evaluating drug toxicity by using same
US20230295576A1 (en) * 2021-05-10 2023-09-21 Nexel Co., Ltd. Method for producing human pluripotent stem cell-derived cardiac organoids and human pluripotent stem cell-derived cardiac organoids produced thereby

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020132055A1 (fr) * 2018-12-19 2020-06-25 Regents Of The University Of Minnesota Procédés de production et d'utilisation d'organoïdes et de tissus
US20230212525A1 (en) * 2020-03-19 2023-07-06 Industry-Academic Cooperation Foundation, Yonsei University Cardiac organoid, manufacturing method therefor, and method for evaluating drug toxicity by using same
US20230295576A1 (en) * 2021-05-10 2023-09-21 Nexel Co., Ltd. Method for producing human pluripotent stem cell-derived cardiac organoids and human pluripotent stem cell-derived cardiac organoids produced thereby
KR20230067138A (ko) * 2021-11-09 2023-05-16 고려대학교 산학협력단 다능성 줄기세포 유래 성숙한 심혈관 오가노이드 제조 방법
KR20230090817A (ko) * 2021-12-15 2023-06-22 한국화학연구원 오로라 키나아제 억제제를 이용하는 심근세포 및 심장 오가노이드의 제조방법

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