Method for producing embryo model, and embryo model

Abstract

The present invention addresses the problem of providing an embryo model that has an autonomously formed organ bud.　The present invention provides a method for producing an embryo model, the method including an induction step, an aggregation step, and an embryo formation step.

Description

Method for producing embryo model, and embryo model

[0001] The present invention relates to a method for producing an embryo model and an embryo model.

[0002] In research into diseases, etc., it is known to use models that mimic the organs of living organisms instead of living organisms. While such models are desirable from ethical and economical perspectives, it is difficult to establish models that reproduce the properties of organs, etc.

[0003] For example, Patent Document 1 discloses a method for producing a cardiomyocyte population, which includes culturing pluripotent stem cells under predetermined conditions. Patent Document 2 discloses a method for proliferating beating cardiomyocytes, which includes treating the beating cardiomyocytes with a WNT agonist or the like.

[0004] International Publication No. 2019 / 078278 Special Publication No. 2021-518113

[0005] However, conventional techniques have been limited to the formation of target organs (e.g., the heart) using differentiation inducers (e.g., cardiac differentiation inducers such as WNT agonists).On the other hand, embryo models in which organ primordia are autonomously formed through cell-cell interactions, as in living organisms, have not yet been fully established.

[0006] The present invention has been made in view of the above circumstances, and aims to provide an embryo model having autonomously formed organ primordia.

[0007] As a result of investigations, the present inventors have succeeded in producing an embryo model having autonomously formed organ primordia, leading to the completion of the present invention. More specifically, the present invention provides the following.

[0008] (1) A method for producing an embryo model, the method comprising: an induction step of culturing first pluripotent stem cells in the presence of a GSK-3 inhibitor to induce them into primitive streak-like cells; an aggregation step of co-culturing the primitive streak-like cells with second pluripotent stem cells to obtain co-aggregates; and an embryonization step of culturing the co-aggregates to obtain an embryo model having at least a cardiac primordium.

[0009] (2) The method according to (1), wherein the culture time in the induction step is 30 to 60 hours.

[0010] (3) The method according to (1) or (2), wherein the medium used in the induction step does not contain a cardiac differentiation inducer.

[0011] (4) The method according to any one of (1) to (3), wherein the medium used in the induction step does not contain any enzyme inhibitors other than a GSK inhibitor and a ROCK inhibitor.

[0012] (5) The production method according to any one of (1) to (4), wherein the medium used in the aggregation step and / or the embryonation step is a basal medium.

[0013] (6) The production method according to any one of (1) to (5), wherein in the aggregation step, the number of the primitive streak-like cells is greater than the number of the second pluripotent stem cells.

[0014] (7) The method according to any one of (1) to (6), wherein the second pluripotent stem cells are iPS cells or ES cells.

[0015] (8) An embryo model, comprising at least a cardiac primordium, a neural primordium, a pancreatic primordium, a vascular network, and a neural network, wherein the cardiac primordium is beating.

[0016] (9) The embryo model according to (8), wherein the cardiac primordium beats at a rate of 0.2 to 4 beats per second.

[0017] (10) The embryo model according to (8) or (9), wherein the embryo model is derived from a primate cell.

[0018] According to the present invention, an embryo model having autonomously formed organ primordia is provided.

[0019] 1 is a photograph of primitive streak-like cells induced from iPS cells (first pluripotent stem cells) in an example. 2 is a photograph of a co-aggregate of iPS cells (second pluripotent stem cells) and primitive streak-like cells prepared in an example. 3 is a photograph of an embryo model having a beating heart-like structure (cardiac primordium) prepared in an example. 4 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 5 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 6 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 7 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 8 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 9 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 10 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 11 is a photograph showing the results of tissue staining of an embryo model prepared in an example. 12 is a photograph showing the results of single-cell RNA sequencing analysis of an embryo model prepared in an example. 13 is a photograph showing the results of single-cell RNA sequencing analysis of a human embryo used in an example. 14 is a photograph showing the results of pulsation analysis of an embryo model prepared in an example. 15 is a photograph showing the results of pulsation analysis of an embryo model prepared in an example. 16 is a photograph showing the results of immunostaining of an embryo model prepared in an example.

[0020] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this.

[0021] (1) Definitions Each term used in the present invention includes, but is not limited to, the following meanings in one aspect.

[0022] (1-1) Embryo Model In the present invention, the term "embryonic model" refers to a roughly spherical cell mass obtained from pluripotent stem cells, which partially or entirely mimics the development of a fertilized egg (embryo).

[0023] The embryo model contains at least organ primordia (cell masses that are undifferentiated in terms of form and function as organs that constitute a living organism), and may further contain cells with differentiation potential (pluripotent stem cells, organ precursor cells, etc.), organs, etc.

[0024] In a preferred embodiment of the present invention, the embryo model may contain one or more of the following: Cell groups specific to the developmental stage (mesoderm, ectoderm, endoderm, etc.) Organ primordium (heart primordium, gut primordium, neural tube primordium, neural tube, lung primordium, liver primordium, pancreas primordium, etc.) Organs (blood vessels (vascular network), neural tube, etc.)

[0025] In a preferred embodiment of the present invention, the embryo model has the following structure: - The cardiac primordium is located near the outermost layer of the embryo model. - One or more (preferably all) of the lung primordium, liver primordium, and pancreatic primordium are formed adjacent to the cardiac primordium. - A vascular network is spread throughout the embryo model. In particular, a well-developed vascular network is formed around the cardiac primordium. - A neural network is spread throughout the embryo model. - It does not have a placenta or yolk sac.

[0026] In a preferred embodiment of the present invention, the embryo model does not fulfill the characteristics of a naturally occurring embryo, including the following: the cardiac anlagen is located near the outermost layer of the embryo model; the embryo model does not have a placenta and / or a yolk sac.

[0027] Whether or not an embryo model has been obtained can be determined by, but is not limited to, conventionally known tissue staining. For example, if organ primordia or the like are confirmed in the cultured cell mass by tissue staining, it can be determined that an embryo model has been obtained. The method of tissue staining is not particularly limited, but typically, the type and distribution of organ primordia contained in the embryo model can be analyzed using antibodies that can detect organ- or tissue-specific markers. For example, if the subject of analysis is "cardiac primordia," the marker used is a cardiac muscle gene (TNNT2, etc.).

[0028] In the present invention, the origin of the embryo model (i.e., the origin of all cells used to produce the embryo model) is preferably a primate, including humans and any monkeys belonging to Primates Linnaeus.

[0029] (1-2) Organ Primordium In the present invention, the term "organ primordium" refers to an organ at an early stage of development that will differentiate into a tissue or organ.

[0030] Organ primordia include the primordium of any tissue or organ, such as the heart primordium, intestinal primordium, neural tube primordium, lung primordium, liver primordium, and pancreatic primordium.

[0031] (1-3) Pluripotent Stem Cells In the present invention, the term "pluripotent stem cells" includes any cells that have the ability to self-proliferate and pluripotency.

[0032] The type of pluripotent stem cells is not particularly limited, but examples include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0033] The origin of the pluripotent stem cells is not particularly limited, but examples include humans and any non-human animals (primates, etc.).

[0034] (1-4) Primitive Streak-Like Cells In the present invention, the term "primitive streak-like cells" refers to cells found in a linear depression (primitive streak) formed in the posterior part of an embryo during early development.

[0035] Whether a cell is a primitive streak-like cell or not can be identified by checking the cell shape and the expression of a primitive streak gene (such as the T gene). A typical shape of a primitive streak-like cell is shown in Figure 1. Typically, a primitive streak-like cell is 10 to 50 μm in length and has a star-like shape.

[0036] (1-5) Autonomous formation of organ primordium In the present invention, the phrase "autonomously formed organ primordium" encompasses the formation of organ primordium during the culture of undifferentiated cells (such as pluripotent stem cells) without using a drug (differentiation inducer) that induces differentiation into a specific organ, tissue, or the like.

[0037] In the present invention, the term "differentiation inducer" encompasses any substance that has the effect of specifically differentiating undifferentiated cells (such as pluripotent stem cells) into predetermined cells. Examples of differentiation inducers include cardiac differentiation inducers (FGF, VEGF, Wnt inhibitors (XAV-939, IWR-1, etc.)). In one aspect of the present invention, organ primordia can be formed autonomously without the use of any of these cardiac differentiation inducers. Note that the "GSK-3 inhibitors" described below are excluded from the differentiation inducers referred to here.

[0038] Preferred embodiments of the present invention include those in which no differentiation inducer is used at all in the cell culture process, and those in which a differentiation inducer is used in an amount that does not exert a differentiation-inducing effect. The "amount that does not exert a differentiation-inducing effect" includes, for example, an amount that does not induce any differentiated cells when pluripotent stem cells are cultured in a basal medium in the presence of a differentiation inducer.

[0039] However, the present invention does not exclude embodiments in which a differentiation inducer is used.

[0040] (2) Method for Producing Embryo Model of the Present Invention A method for producing an embryo model according to one embodiment of the present invention (hereinafter also referred to as "the production method of the present invention") comprises the following steps: - an induction step of culturing first pluripotent stem cells in the presence of a GSK-3 inhibitor to induce them into primitive streak-like cells - an aggregation step of co-culturing the primitive streak-like cells with second pluripotent stem cells to obtain co-aggregates - an embryonization step of culturing the co-aggregates to obtain an embryo model having at least a cardiac primordium

[0041] Known methods for obtaining biological models include obtaining desired tissue primordia using differentiation inducers, etc. However, such methods only result in the formation of specific tissue primordia, and do not result in the coexistence of a variety of organs and organ primordia, as occurs in fertilized eggs.

[0042] Therefore, the present inventors conducted extensive research and discovered the extremely unexpected finding that when primitive streak-like cells induced in the presence of a GSK-3 inhibitor were co-cultured with pluripotent stem cells (second pluripotent stem cells), these cells formed aggregates and were able to autonomously form embryo models. Although no differentiation inducers (cardiac differentiation inducers, etc.) were used in the embryo model production process, organ primordia (heart primordium, gut primordium, neural tube primordium, etc.), organs (blood vessels, etc.), neural tubes, mesoderm, etc. were confirmed in the obtained embryo models.

[0043] Each step of the production method of the present invention will be described in detail below.

[0044] (2-1) Induction Step The induction step is a step of culturing pluripotent stem cells (first pluripotent stem cells) in the presence of a GSK-3 inhibitor to induce them into primitive streak-like cells.

[0045] Hereinafter, the term "first pluripotent stem cells" is used to distinguish them from the "second pluripotent stem cells" used in the aggregation step described below. The first pluripotent stem cells and the second pluripotent stem cells may be homogeneous or heterogeneous pluripotent stem cells.

[0046] As a result of investigations by the present inventors, it was unexpectedly found that when primitive streak-like cells are induced from pluripotent stem cells (first pluripotent stem cells) in the presence of a GSK-3 inhibitor, the obtained primitive streak-like cells form an embryo model having at least a cardiac primordium after undergoing coculture, etc., as described below. However, even when fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), and bone morphogenetic protein 4 (BMP4) were used in addition to a GSK-3 inhibitor, it was difficult to obtain an embryo model.

[0047] "GSK-3" (Glycogen synthase kinase 3) is a serine-threonine kinase, and two isoforms, α and β, are known. A "GSK-3 inhibitor" is a kinase inhibitor that targets any GSK-3.

[0048] The GSK-3 inhibitor of the present invention includes any substance known as a kinase inhibitor for GSK-3. For example, the GSK-3 inhibitor includes the following: LY2090314 (CAS number: 603288-22-8), BIO (CAS number: 667463-62-9), CHIR99021 (CAS number: 252917-06-9).

[0049] The conditions for the induction step are not particularly limited except that the pluripotent stem cells (first pluripotent stem cells) are cultured in the presence of a GSK-3 inhibitor, and any conditions known as culture conditions for pluripotent stem cells can be employed.

[0050] In a preferred embodiment of the present invention, the medium used in the induction step preferably does not contain a differentiation inducer, more preferably does not contain a cardiac differentiation inducer (WNT agonist, etc.). According to the present invention, an embryo model having cardiac primordium can be obtained even when a medium not containing a cardiac differentiation inducer is used.

[0051] In a preferred embodiment of the present invention, the medium used in the induction step preferably does not contain any enzyme inhibitors other than a GSK (Glycogen synthase kinase 3) inhibitor and a ROCK (Rho kinase) inhibitor. According to the present invention, cardiac primordium can be formed even in a medium with such a simple composition, and therefore, an embryo model in which not only cardiac primordium but also various organ primordium are autonomously formed can be obtained.

[0052] The type of GSK-3 inhibitor used in the induction step is not particularly limited, and one type may be used alone, or two or more types may be used in combination. In a preferred embodiment of the present invention, at least the GSK inhibitor LY2090314 is used in the induction step, and other GSK inhibitors may or may not be used in combination. However, even if only LY2090314 is used as the GSK inhibitor, a good embryo model can be obtained.

[0053] When a ROCK inhibitor is used in the induction step, the type thereof is not particularly limited, but examples thereof include Y27632. Y27632 is useful in that it can suppress cell damage when recovering the cells after culture.

[0054] The medium used in the induction step is not particularly limited, and any medium used in the culture of pluripotent stem cells can be used. In a preferred embodiment of the present invention, the medium used in the induction step is a basal medium (a medium containing the minimum nutrients necessary for cell survival). Examples of basal media include DMEM / F12, DMEM, RPMI 1640, and MEM. The basal medium may be formulated with serum (e.g., BSA), antibiotics (e.g., Penicillin-Streptomycin), and basal medium supplements (e.g., ITS-X, KSR, Geltrex). According to the present invention, cardiac primordium can be formed even in a medium with such a simple composition, making it possible to obtain an embryo model in which not only cardiac primordium but also various organ primordium are autonomously formed.

[0055] In a preferred embodiment of the present invention, the medium used in the induction step comprises a basal medium and a GSK-3 inhibitor, but does not contain a differentiation inducer or an enzyme inhibitor (other than a GSK inhibitor and a ROCK inhibitor). In a more preferred embodiment of the present invention, the medium used in the induction step comprises a basal medium and a GSK-3 inhibitor, but does not contain a differentiation inducer, an enzyme inhibitor (other than a GSK inhibitor and a ROCK inhibitor), an epidermal growth factor, a bone morphogenetic protein, or a BMP inhibitor. In a more preferred embodiment of the present invention, the medium used in the induction step consists only of a basal medium and a GSK-3 inhibitor.

[0056] The number of pluripotent stem cells (first pluripotent stem cells) used in the induction step is not particularly limited, but is preferably 10,000 to 200,000 cells per ml of medium.

[0057] The concentration of the GSK-3 inhibitor used in the induction step is not particularly limited as long as a sufficient induction effect is achieved, but is preferably 0.1 to 10 μmol / L.

[0058] The temperature conditions for the induction step can be any temperature at which cells can grow (for example, 32 to 39°C).

[0059] The atmosphere for the induction step is not particularly limited as long as it allows cells to grow, but is usually 2 to 7% CO ２ In this environment.

[0060] The culture time in the induction step is not particularly limited as long as it can sufficiently induce primitive streak-like cells from pluripotent stem cells (first pluripotent stem cells), and is preferably 30 to 60 hours, more preferably 38 to 44 hours. When the culture time in the induction step is within the above range, an embryo model can be stably produced.

[0061] The culture vessel used in the induction step is not particularly limited, and a well plate, well dish, etc. may be used.

[0062] The culture in the induction step is preferably static culture.

[0063] After the induction step is completed, the culture medium is appropriately centrifuged to obtain a cell population containing primitive streak-like cells. After the induction step, the obtained cell population may be directly subjected to the aggregation step or may be stored until use.

[0064] (2-2) Aggregation Step The aggregation step is a step of co-culturing the primitive streak-like cells obtained in the induction step with pluripotent stem cells (second pluripotent stem cells) to obtain co-aggregates.

[0065] In one embodiment of the present invention, the "primitive streak-like cells obtained in the induction step" may be the whole or a part of the cell population obtained after completion of the induction step. In one embodiment of the present invention, the "primitive streak-like cells obtained in the induction step" may be the primitive streak-like cells isolated from the cell population obtained after completion of the induction step using a flow cytometer or magnetic beads.

[0066] The conditions for the aggregation step are not particularly limited except that primitive streak-like cells and pluripotent stem cells (second pluripotent stem cells) are cultured, and any conditions known as culture conditions for pluripotent stem cells can be adopted.

[0067] In a preferred embodiment of the present invention, the second pluripotent stem cells are iPS cells or ES cells, from the viewpoint that a well-beating embryo model can be easily obtained.

[0068] In a preferred embodiment of the present invention, the aggregation step may be carried out in a concave depression. For example, a small hole-like well may be formed using agarose gel, a medium may be placed in the well, and the primitive streak-like cells and pluripotent stem cells (second pluripotent stem cells) may be cultured therein, thereby efficiently obtaining co-aggregates.

[0069] The culture vessel used in the aggregation step preferably has small pores (e.g., pores with a diameter of 200 to 1000 μm) to facilitate contact between the primitive streak-like cells and the pluripotent stem cells (second pluripotent stem cells). Primitive streak-like cells and the pluripotent stem cells (second pluripotent stem cells) are placed in such small pores, allowed to stand, and cultured for 1 to 3 days to obtain co-aggregates. The primitive streak-like cells and the pluripotent stem cells (second pluripotent stem cells) may be mixed in advance to form a cell suspension and then placed in the pores, or they may be placed separately.

[0070] In a preferred embodiment of the present invention, the medium used in the aggregation step is a basal medium (a medium containing the minimum nutrients necessary for cell survival). Examples of basal media include DMEM / F12, DMEM, RPMI 1640, and MEM. The basal medium may contain serum (BSA, etc.), antibiotics (Penicillin-Streptomycin, etc.), and basal medium supplements (ITS-X, KSR, Geltrex, etc.), as needed. According to the present invention, even using such a simple medium, organ primordia are formed autonomously, and an embryo model having a heart primordium can be obtained.

[0071] The temperature condition for the aggregation step can be any temperature at which cells can grow (for example, 32 to 39°C).

[0072] The atmosphere for the aggregation step is not particularly limited as long as it allows cells to grow, but is usually 2 to 7% CO ２ In this environment.

[0073] The incubation time in the aggregation step is not particularly limited as long as sufficient aggregation can be achieved, and is preferably 12 to 80 hours, more preferably 12 to 24 hours.

[0074] In the aggregation step, the numbers of primitive streak-like cells and pluripotent stem cells (second pluripotent stem cells) to be cultured are not particularly limited. In a preferred embodiment of the present invention, the number of primitive streak-like cells used in the aggregation step is 50 to 500, and the number of pluripotent stem cells (second pluripotent stem cells) is 50 to 500.

[0075] From the viewpoint of facilitating the production of co-aggregates, it is preferable to set the number of primitive streak-like cells to be greater than the number of pluripotent stem cells (second pluripotent stem cells). In a preferred embodiment of the present invention, the number of primitive streak-like cells in the aggregation step is 0.5 to 5 times the number of pluripotent stem cells (second pluripotent stem cells). The term "number of primitive streak-like cells in the aggregation step" preferably refers to the actual number of primitive streak-like cells. However, for convenience, the total number of cells contained in the portion of the cell population obtained in the induction step that is used in the aggregation step may be used as the "number of primitive streak-like cells in the aggregation step."

[0076] From the viewpoint of facilitating the production of coaggregates, the number of primitive streak-like cells per well in the aggregation step is preferably 50 to 500. In a preferred embodiment of the present invention, the number of primitive streak-like cells per well in the aggregation step is preferably 50 to 500.

[0077] Whether or not a coaggregate has been formed can be determined by microscopic observation. For example, if the primitive streak-like cells and pluripotent stem cells (second pluripotent stem cells) form a single cohesive cell mass, it can be determined that a coaggregate has been formed. A typical shape of a coaggregate of primitive streak-like cells and pluripotent stem cells (second pluripotent stem cells) is shown in Figure 2.

[0078] After the aggregation step is completed, the culture solution is appropriately centrifuged to obtain co-aggregates. The obtained co-aggregates may be subjected to the embryogenesis step as is or may be stored until use. Alternatively, after the aggregation step is completed, the wells may be inverted upside down, and the co-aggregates may be recovered from the culture vessel by gravity, transferred to a new culture vessel, and then the embryogenesis step may be carried out.

[0079] (2-3) Embryogenesis Step The embryoogenesis step is a step of culturing the co-aggregates obtained in the aggregation step to obtain an embryo model. The obtained embryo model has at least a cardiac primordium.

[0080] The conditions for the embryonation step are not particularly limited, and any conditions known as cell culture conditions can be employed.

[0081] In a preferred embodiment of the present invention, the medium used in the embryogenesis step is a basal medium (a medium containing the minimum nutrients necessary for cell survival). Examples of basal media include DMEM / F12, DMEM, RPMI 1640, and MEM. The basal medium may contain serum (e.g., BSA), antibiotics (e.g., Penicillin-Streptomycin), and basal medium supplements (e.g., ITS-X, KSR, Geltrex). According to the present invention, even using such a simple medium, organ primordia are formed autonomously, and an embryo model having a heart primordium can be obtained.

[0082] The temperature conditions for the embryonation step can be any temperature at which cells can grow (for example, 32 to 39°C).

[0083] The atmosphere for the embryogenesis step is not particularly limited as long as it allows cells to grow, but is usually 2 to 7% CO ２ In this environment.

[0084] The culture time in the embryonization step is not particularly limited and can be set appropriately depending on the degree of developmental progression of the embryo model to be obtained. The culture time in the embryonization step is preferably 2 days or more, more preferably 3 to 35 days, and even more preferably 3 to 5 days, from the viewpoint of forming a cardiac primordium that is sufficiently developed and exhibits clearly recognizable pulsation. The culture time in the embryonization step is preferably 2 days or more, more preferably 14 to 30 days, from the viewpoint of forming a clear organ primordium. The culture time in the embryonization step is preferably 2 days or more, more preferably 14 to 30 days, from the viewpoint of obtaining a well-developed vascular network.

[0085] During the culture period in the embryogenesis step, the medium may be changed as needed (for example, once every 1 to 2 days).

[0086] In the embryonization step, the number of coaggregates to be cultured is not particularly limited. In a preferred embodiment of the present invention, the number of coaggregates used in the embryonization step is 10 to 100 per well. In a more preferred embodiment of the present invention, the number of coaggregates used in the embryonization step is 10 to 100 per well. ２ ) is preferably 1 to 10.

[0087] The culture vessel used in the embryogenesis step is not particularly limited, and a well plate, well dish, etc. may be used.

[0088] In the embryonation step, shaking culture may be carried out as necessary.

[0089] After several days from the start of the culture in the embryogenesis step, pulsation of the embryo model can be observed. This pulsation indicates that an embryo model having at least a cardiac primordium has been obtained. In a preferred embodiment of the present invention, pulsation can be observed even after two months or more of culture has been continued.

[0090] (3) Embryo Model of the Present Invention The present invention also encompasses an embryo model obtained by the above-described production method.

[0091] The embryo model of the present invention has at least a cardiac primordium, and preferably has a cardiac primordium, a neural primordium, a pancreatic primordium, a vascular network, and a neural network.

[0092] The cardiac primordium in the embryo model preferably pulsates, and typically pulsates at a rate of preferably 0.2 to 4 beats per second, more preferably 0.5 to 2 beats per second.

[0093] The use of the embryo model is not particularly limited, and it can be used for any drug efficacy test or toxicity test. For example, since congenital heart disease can occur in the early stages of development, the embryo model of the present invention that resembles a state close to the early stage of development can be suitably used for research on congenital heart disease.

[0094] Since the embryo model of the present invention has organ primordia such as cardiac primordia, for example, the organ primordium can be removed from the embryo model and used as a material for organ primordia to be used in organ transplantation.

[0095] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0096] <Test 1: Preparation of Embryo Model> An embryo model was prepared from primitive streak-like cells and pluripotent stem cells (iPS cells in this example) by the following method, and its structure and pulsation were confirmed.

[0097] (1) Induction step: induction of primitive streak-like cells. "PS (primitive streak)-induction medium" (2 ml) was added to an empty 6-well dish (3810-006N, Iwaki) and heated at 37°C for 10 minutes or more. Next, iPS cells (HPS1005 strain, 1 x 10 ５ The cells were added and incubated at 37°C, 5% CO ２ Culture was initiated under this environment. These iPS cells correspond to the "first pluripotent stem cells." 40 to 48 hours after the start of culture, the cells were detached from the wells and collected using "TrypLE™" (Thermo Fisher Scientific).

[0098] The "LY2090314" contained in the "PS-induction medium" is a GSK-3 (glycogen synthase kinase 3) inhibitor. The ROCK inhibitor "Y27632" contained in the "PS-induction medium" was added to protect the cells from damage during detachment.

[0099]

[0100] The cells obtained were confirmed to be primitive streak-like cells because they had a star-like shape and expressed primitive streak genes (such as the T gene) (see Figure 1). The obtained primitive streak-like cells were isolated using a flow cytometer and used in the aggregation step described below.

[0101] (2) Aggregation Step: Aggregation of iPS Cells and Primitive Streak-Like Cells The primitive streak-like cells obtained in (1) above were aggregated with iPS cells (second pluripotent stem cells) by the following method. First, agarose gel was placed in a silicone resin mold (MicroTissues™ 3D Petri Dish™ micro-mold spheroid, MicroTissues Inc.) to form a well with a small hole (hereinafter referred to as an "agarose well"). 10 mM Y27632 (1 μl) was added to 1 ml of basal medium and heated at 37°C for 10 minutes or more (hereinafter referred to as "Basal medium-Y27632"). 650 μl of the heated medium was added to the lower layer of the agarose well. Next, iPS cells (HPS1005 strain, 5 × 10 ４ cells), and primitive streak-like cells (15 × 10 ４ The iPS cells were suspended in 160 μl of "Basal medium-Y27632" to obtain a cell suspension. These iPS cells correspond to the "second pluripotent stem cells." The entire volume of the obtained cell suspension was added to the upper layer of the agarose well and incubated at 37°C, 5% CO ２ The cells were cultured under this environment for 3 days to obtain coaggregates of iPS cells (second pluripotent stem cells) and primitive streak-like cells (see FIG. 2).

[0102]

[0103]

[0104] (3) Embryogenesis step: Shaking culture of co-aggregates "Basal medium" (2 ml) was added to a 35 mm dish and heated at 37°C for 10 minutes or more. The co-aggregates obtained in (2) above were transferred to a heated 35 mm dish (351008, Corning) and cultured while shaking approximately 60 times per minute using a shaker "NA-M301" (Nissin) to prepare embryo models. After the start of shaking culture, half of the medium was discarded once every three days, and fresh "Basal medium" (1 ml) was added.

[0105] Pulsation of the embryo model was observed from about the fifth day after the start of shaking culture. Figure 3 shows an embryo model (maximum length: approximately 2 mm) on the 14th day after the start of shaking culture. The area within the dashed line in Figure 3 pulsated up and down (approximately vertically in the image). The pulsation was at a constant pace, approximately 1 beat per second in this example. From the above characteristics, it was found that the embryo model obtained in this example had a heart-like structure (within the dashed line in Figure 3 ), i.e., a cardiac primordium, formed on its surface. It was also confirmed that myocardial genes (TNNT2, etc.) were expressed in this cardiac primordium.

[0106] When the embryo model was cultured under shaking, pulsation was observed for at least two months after the start of the culture.

[0107] When the embryo model was analyzed by histological staining, various organ primordia were confirmed in the embryo model obtained in this example, as shown below. As shown in Figure 4, the cardiac primordium was located near the outermost layer of the embryo model. Furthermore, in addition to a heart-like structure (cardiac primordium) containing myocardium, the embryo model also contained a neural tube-like structure (neural tube primordium), neural tube, mesoderm, etc. As shown in Figures 5 to 7, the lung primordium, liver primordium, and pancreatic primordium were formed adjacent to the cardiac primordium. As shown in Figure 8, a vascular network was present throughout the embryo model. In particular, a well-developed vascular network was formed around the cardiac primordium. As shown in Figure 9, a neural network was present throughout the embryo model.

[0108] In addition, no structures corresponding to the placenta or yolk sac were observed in the embryo model obtained in this example.

[0109] In this example, no differentiation inducers (e.g., cardiac differentiation inducers such as WNT agonists) were used in the embryo model production process. Nevertheless, it was a highly unexpected finding that various primordia (e.g., cardiac primordia) and organs (e.g., blood vessels) formed autonomously.

[0110] <Test 2: Consideration of Various Conditions> Based on the results of Test 1 above, various conditions for obtaining an embryo model were considered.

[0111] (1) Induction Conditions for Primitive Streak-Like Cells - 1 In the induction step of Test 1, iPS cells (first pluripotent stem cells) were cultured using a medium (PS-induction medium) containing the GSK-3 inhibitor "LY2090314." On the other hand, when epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), or the BMP inhibitor "LDN-193189" was added to the medium in addition to "LY2090314," induction of primitive streak-like cells was possible, but subsequent culture made it difficult to obtain embryo models with cardiac primordia and the like. These findings unexpectedly revealed that a medium consisting of a basal medium and a GSK-3 inhibitor is particularly preferable for the induction step.

[0112] Furthermore, when other GSK inhibitors (such as "BIO") were used instead of "LY2090314," results equivalent to those obtained when "LY2090314" was used were obtained. Therefore, it was found that the culture for the induction of primitive streak-like cells can be performed in the presence of a GSK-3 inhibitor.

[0113] (2) Conditions for Induction of Primitive Streak-Like Cells-2 In Test 1, iPS cells (first pluripotent stem cells) were cultured for 40 to 48 hours. On the other hand, when this culture time was changed to 24 or 72 hours, although it was possible to induce primitive streak-like cells, it was difficult to obtain embryo models having cardiac primordium and the like in subsequent culture. Therefore, it was inferred that a culture time of 30 to 60 hours is suitable for inducing primitive streak-like cells.

[0114] (3) Aggregation conditions for iPS cells and primitive streak-like cells In Test 1, 5 × 10 ４iPS cells (secondary pluripotent stem cells) and 15 x 10 ４ On the other hand, the number of primitive streak-like cells was increased to 5 × 10 ４ When the number of cells was less than 100, it was difficult to obtain an embryo model having cardiac primordium, etc. Therefore, it was inferred that it would be preferable to aggregate the primitive streak-like cells with iPS cells in a number greater than that of the iPS cells (for example, two times or more).

[0115] <Test 3: Single-cell RNA sequencing analysis of embryo model> For the embryo model obtained by the same method as in Test 1 above, single-cell RNA sequencing analysis was performed to comprehensively analyze the type and amount of RNA for each cell.

[0116] For the analysis, a single-cell RNA sequencing analysis tool from 10xGenomics was used.

[0117] The following two samples were selected for analysis: Human embryo model: a human embryo model obtained using the same method as in Test 1 above (with the exception that the culture time was changed as appropriate) Human embryo: a human embryo 15 to 40 days after fertilization The human embryo model was obtained by carrying out the "induction step" (approximately 2 days), "aggregation step" (approximately 3 days), and "embryogenesis step" (approximately 18 days) under the same conditions as in Test 1 above.

[0118] Based on the gene expression levels obtained as a result of the analysis, dimension reduction was performed using UMAP (Uniform Manifold Approximation and Projection).

[0119] The analysis results are shown in Figure 10 (human embryo model) and Figure 11 (human embryo). As shown in Figures 10 and 11, the human embryo model of the present invention and the human embryo each contained common cell types. On the other hand, the human embryo model of the present invention did not contain blood cells. This is thought to be because, as confirmed in Test 1 above, the model does not contain the yolk sac, which is a hematopoietic organ.

[0120] <Test 4: Pulse Analysis of Embryonic Model-1> Pulse analysis was performed by calcium imaging on an embryonic model obtained in the same manner as in Test 1 above.

[0121] In this example, the cells were cultured in the same manner as in Test 1 above (with the exception that the culture time was changed as appropriate), and on the ninth day of culture (the completion of the "induction step" (approximately 2 days), "aggregation step" (approximately 3 days), and "embryogenesis step" (approximately 4 days)), a calcium-binding dye (calcium fluorescent probe "Fluo-4") was added, and changes in calcium concentration were visualized by fluorescence observation.

[0122] The observation results of the heart-like structure (cardiac primordium) of the embryo model are shown in Figures 12 and 13. Figure 12 is a photograph showing the change in calcium concentration (fluorescence intensity) over 1 second. Figure 13 is a graph showing the change in calcium concentration (fluorescence intensity) over 60 seconds. The vertical axis "Ratio" in Figure 13 represents the fluorescence intensity of the calcium-binding dye, and the horizontal axis "Time" represents the elapsed time (unit: seconds). As shown in Figures 12 and 13, the cardiac primordium of the embryo model obtained in this example beat regularly at approximately 0.6 beats / second.

[0123] <Test 5: Pulse Analysis of Embryo Model-2> As in Test 4 above, it was confirmed that the embryo model obtained by the method of Test 1 pulsated well. In this example, furthermore, pulsation analysis was performed in the same manner as in Test 4 using an embryo model obtained using ES cells instead of iPS cells.

[0124] In this example, except for the type and combination of pluripotent stem cells, the culture was carried out in the same manner as in Test 1 above (however, the culture time was changed as appropriate), and on the 9th day of culture (the completion of the "induction step" (approximately 2 days), "aggregation step" (approximately 3 days), and "embryogenesis step" (approximately 4 days)), a calcium-binding dye (calcium fluorescent probe "Fluo-4") was added, and changes in calcium concentration were visualized by fluorescence observation.

[0125] The cells used in this example (first pluripotent stem cells or second pluripotent stem cells) were either of the following: iPS cells: HPS1005 strain (same as in Test 4) ES cells-1: SEES2 strain ES cells-2: SEES5 strain

[0126] In each test group, 50 embryo models were prepared, and the percentage of embryo models in which changes in calcium concentration were observed was calculated as the pulsation rate (unit: %). The results are shown in the table below. As shown in the table below, pulsating embryo models were obtained regardless of the type or combination of pluripotent stem cells.

[0127]

[0128] <Test 6: Developmental analysis using embryo model> The embryo model obtained in the same manner as in Test 1 above was observed for development into the heart.

[0129] In this example, the culture was carried out in the same manner as in Test 1 above (except that the culture time was changed as appropriate), and on the 36th day of culture (the completion of the "induction step" (approximately 2 days), "aggregation step" (approximately 3 days), and "embryogenesis step" (approximately 31 days)), antibodies were added to perform immunostaining, and the cells were visualized by fluorescent observation. The following two types of staining antibodies were used in this example. - Anti-MYH6 (myosin heavy chain 6) antibody: an antibody that detects MYH6, a marker for the atrium. - Anti-MYH7 (myosin heavy chain 7) antibody: an antibody that detects MYH7, a marker for the ventricle.

[0130] The results of immunostaining are shown in Figure 14. As shown in Figure 14, MYH6 and MYH7 were clearly detected, demonstrating that a heart having atria and ventricles was developed from the embryo model according to the present invention.

Claims

1. A method for producing an embryo model, comprising: an induction step of culturing first pluripotent stem cells in the presence of a GSK-3 inhibitor to induce them into primitive streak-like cells; an aggregation step of co-culturing the primitive streak-like cells with second pluripotent stem cells to obtain co-aggregates; and an embryonization step of culturing the co-aggregates to obtain an embryo model having at least a cardiac primordium.

2. The method of claim 1, wherein the incubation time in the induction step is 30 to 60 hours.

3. The method of claim 1, wherein the medium used in the induction step does not contain a cardiac differentiation inducer.

4. The method of claim 1, wherein the medium used in the induction step does not contain any enzyme inhibitors other than a GSK inhibitor and a ROCK inhibitor.

5. The method according to claim 1, wherein the medium used in the aggregation step and / or the embryonation step is a basal medium.

6. The manufacturing method described in claim 1, wherein in the aggregation step, the number of the primitive streak-like cells is greater than the number of the second pluripotent stem cells.

7. The manufacturing method described in claim 1, wherein the second pluripotent stem cells are iPS cells or ES cells.

8. An embryo model, comprising at least a cardiac primordium, a neural primordium, a pancreatic primordium, a vascular network, and a neural network, wherein the cardiac primordium is beating.

9. The embryo model according to claim 8, wherein the cardiac primordium beats at a rate of 0.2 to 4 beats per second.

10. The embryo model of claim 8, wherein the embryo model is derived from primate cells.

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