Composition and method for culturing signal organizer and micro-organ using same

Abstract

A composition and a method for culturing a signal organizer and a micro-organ using same. The composition comprises FGF2, a BMP inhibitor, a WNT signal activator and a Notch pathway inhibitor. The method comprises culturing a human pluripotent stem cell by using the composition. By means of induction by the composition, a human signal organizer is generated, and the human signal organizer is capable of spontaneously inducing the surrounding human pluripotent stem cells to generate various organ precursors, so that a combination of various micro-organs is generated.

Description

Composition and method for culturing signal organizer and micro-organ using the same

[0001] This application claims priority to Chinese patent application No. 2024101713264, filed on February 6, 2024. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to a composition and a method for culturing signal organizers and micro-organs using the composition. Background Art

[0003] How different cell types develop, and why some stem cells in human embryos eventually become neurons rather than muscle cells, while others develop into cartilage rather than heart tissue, have long been pressing questions in science. In the early 1920s, German embryologist Hans Spemann and his student Hilde Mangold began exploring how embryonic stem cells differentiate into every type of tissue in the body, including bone, brain, lungs, and liver. In 1924, while conducting experiments on salamander embryos, Spemann and Mangold discovered a unique group of cells. When transplanted into another salamander embryo, these cells coaxed nearby cells to form the rudiments of a brain and spinal cord—the new embryos grew into conjoined salamander twins. Similar cell groups have since been discovered in other amphibian and fish embryos. These cell groups play a crucial role in shaping the early structural development of tissues, earning them the name "signaling organizers." They send out molecular signals that direct other cells to grow and develop in specific patterns. When a signaling organizer is transplanted from one embryo to another, it stimulates its new host to gradually develop a central nervous system including a spinal cord and brain, and the signaling organizer itself develops into tissues such as the bones of the spine.

[0004] However, to observe a single human signaling organizer cell, scientists had to extend the embryo culture time to 15-16 days. However, ethical and legal constraints prevented scientists from conducting experiments on human embryos past 14 days of development, as this is the critical point at which the embryo can no longer divide and can be considered an individual. These ethical and other issues have limited the ability to conduct experiments directly with human embryos. Consequently, scientists have actively sought various alternatives.

[0005] To verify whether it is feasible, Brivanlou and his team conducted a series of experiments using artificial human embryos. They used human embryonic stem cells to culture a small mass of cell tissue about one millimeter in diameter in the laboratory. Although these artificial simulated tissues are far from natural embryos, they still contain many cells and tissues that exist in real human embryos and can be used as experimental substitutes. The researchers transplanted the artificial embryos into living chicken embryos for more human-like research. When human embryonic cells were transplanted into the avian host, the human cells began to induce the basic development of the spine and nervous system. Obviously, this behavior marked the formation of a true human "signal organizer."

[0006] Human signal organizers can be used to induce the second body axis of the chicken in chicken embryos, thereby promoting the development of the nervous system. Can the human spinal cord nerves and various organ tissues be induced under the action of human signal organizers? Due to ethical issues, this process will be allowed to occur in vitro, and pluripotent stem cells can be used as tool cells. They have the potential to develop into various organs, but how to obtain signal organizers and induce the generation of various human organs has become the most difficult technical point to overcome.

[0007] Organogenesis generally refers to the process by which organs evolve from primordia to organs during the development of vertebrate individuals. Through the organogenesis stage, various organs undergo morphogenesis and tissue differentiation, gradually acquiring specific morphologies and performing specific physiological functions. In 2014, Lancaster et al. systematically proposed the concept of organoids, which states that stem cells or organ progenitor cells can undergo cell differentiation and lineage commitment processes similar to those in vivo when cultured in vitro, and self-organize into 3D structures with the spatial structure and specific functions of human organs. Subsequently, an increasing number of organoids have been derived from tissues, such as the intestine, retina, brain, and kidney.

[0008] However, existing, more mature human organoid systems only simulate specific organ functions, such as the respiratory function of the lungs, the absorption function of the intestines, and the filtration function of the kidneys. They have not yet achieved the goal of completely replacing treatment or transplantation, and they also lack the morphology of complete organ generation. Moreover, screening for anti-cancer drugs from a single organoid carries the risk of having a strong auxiliary effect on other healthy organs, and clinical application requires a long period of testing. In addition, the application of these single organoids is mainly concentrated in the late developmental stage, and their role in drug screening in the early stages of pregnancy (i.e., the implantation period) is minimal.

[0009] According to technical research on human early embryos in domestic and foreign journals, there is no research on the induction of human multi-organ formation by human signaling organizers. A recent article published abroad: "Self-organization of a human organizer by combined Wnt and Nodal signaling" (Martyn, I, et al., Nature, 2018.558(7708): p.132-135) only transplanted into chicken embryos, without inducing human organ tissues, and the cells were flat and monolayer, without a "signaling organizer" morphology. There are also some other technologies related to early embryos, but none of them are related to human signaling organizers. How to obtain signaling organizers and induce the formation of various human organs has become the most difficult technical point to overcome.

[0010] Summary of the Invention

[0011] To overcome the existing limitations of obtaining signaling organizers and inducing the formation of various organs, the present invention provides a composition and a method for culturing signaling organizers and micro-organs using the composition. This invention provides a model for studying early pregnancy and embryonic development before and after implantation, addressing the difficulty of obtaining embryos. It also provides a model platform for early pregnancy drug screening and offers a potential source for organ transplantation.

[0012] One of the technical solutions of the present invention is to provide a composition, wherein the composition includes FGF2, a BMP inhibitor, a WNT signal activator and a Notch pathway inhibitor.

[0013] In some preferred embodiments, the BMP inhibitor is CC, Noggin or LDN193189, the WNT signal activator is Wnt3a or CHIR99021, and the Notch pathway inhibitor is Compound E.

[0014] In some more preferred embodiments, the composition comprises FGF2, CHIR99021, Compound E and LDN193189.

[0015] In some specific embodiments, the molar ratio of FGF2, CHIR99021, Compound E and LDN193189 is 0.00058:30:2:1.

[0016] A second technical solution of the present invention provides a method for preparing a signal organizer, wherein the method comprises: culturing human pluripotent stem cells using the composition described in one of the technical solutions, thereby harvesting the signal organizer.

[0017] In some preferred embodiments, the method comprises the following steps:

[0018] (i) thoroughly mixing Matrigel with a cell mass containing human pluripotent stem cells; the cell mass containing human pluripotent stem cells is, for example, hESCs;

[0019] (ii) When the matrix gel is solidified, a differentiation medium containing the composition is added to perform differentiation.

[0020] In some more preferred embodiments, in (i), the volume ratio of Matrigel to cell aggregates containing human pluripotent stem cells is 1:(1-2), for example, 100 μL of Matrigel can encapsulate approximately 100-200 cell aggregates containing pluripotent stem cells; and the concentration of the Matrigel is 50%-100%.

[0021] In (ii), the basal culture medium is mixed with the composition as described in one of the technical solutions to prepare a differentiation culture medium; the basal culture medium is, for example, DMEM / F12 (1:1) and Neurobasal medium prepared in a 1:1 ratio, and 1×GlutaMAX, 1×N2 and 1×B27 are added; the differentiation is carried out in a 37°C incubator.

[0022] In some preferred embodiments, the cell aggregate comprises the following processing steps before being mixed with Matrigel matrix gel:

[0023] 1) Add Dispase to digest the cell clumps; for example, digest at 37°C;

[0024] 2) After digestion is complete, discard the Dispase enzyme, wash with basal culture medium, and then add basal culture medium again;

[0025] 3) Blow off the cell clumps and collect them in a container. Further blow the cell clumps to a diameter of 30-80 μm, for example, 50 μm. Take 2 mL of the middle section of the cell clumps, centrifuge to remove the supernatant and mix.

[0026] For example, cells are pipetted with a total of 10 mL of culture medium, collected in a 50 mL centrifuge tube, and allowed to settle naturally for 30 seconds. Then, 2 mL of cell pellet is transferred to a 15 mL centrifuge tube and centrifuged. The intermediate cell pellet refers to the cell pellet that, after settling naturally, is larger at the bottom and smaller at the top due to gravity. Neither size is optimal. The intermediate cell pellet is approximately 50 μm in diameter.

[0027] In some more preferred embodiments, 1) further comprises washing the cell aggregate containing human pluripotent stem cells with a basal culture medium and discarding the basal culture medium.

[0028] In some further preferred embodiments, in 2), the digestion time is 1-5 min, for example, 3 min;

[0029] In some further preferred embodiments, in 3), the centrifugation condition is 1000-1500 rpm, 1-5 min.

[0030] The third technical solution of the present invention provides a signal organizer cultured by the method described in the second technical solution.

[0031] A fourth technical solution of the present invention provides a method for preparing organ precursor cells and / or micro-organs, wherein the method comprises transplanting the signal organizer described in the third technical solution into a mammal, such as a mouse, to generate the organ precursor cells and / or micro-organs; the transplantation is performed, for example, subcutaneously or under the renal capsule of the mouse.

[0032] In the present invention, the micro-organ utilizes the pluripotency of human pluripotent stem cells and is a structure with corresponding functions that is orderly assembled by characteristic cells in vitro or in vivo under the induction of a "signal organizer".

[0033] A fifth technical solution of the present invention provides the use of the composition as described in one of the technical solutions in the preparation of a preparation for generating signal organizers, organ precursor cells and / or micro-organs.

[0034] The sixth technical solution of the present invention provides an organ precursor cell and / or micro-organ, which is prepared using the method described in the fourth technical solution.

[0035] A seventh technical solution of the present invention provides a composition as described in one of the technical solutions, which is used to prepare signal organizers, organ precursor cells and / or micro-organs.

[0036] The eighth technical solution of the present invention provides a signal organizer as described in the third technical solution, which is used for preparing organ precursor cells and / or micro-organs.

[0037] A ninth technical solution of the present invention provides a method for treating a disease, which comprises transplanting the organ precursor cells and / or micro-organs as described in the sixth technical solution into a subject in need, thereby treating the disease.

[0038] The tenth technical solution of the present invention provides the use of the organ precursor cells and / or micro-organs as described in the sixth technical solution in drug screening.

[0039] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive progress effect of the present invention is:

[0042] The technology of the present invention covers the generation of "signal organizers" (the primary technology of the present invention is to generate "signal organizers" through the induction of a unique formula of chemical small molecules. The importance of "signal organizers" lies in that they can spontaneously signal to induce surrounding human pluripotent stem cells to generate various organ precursors, and then generate a combination of various micro-organs); under its induced tissue, more organ precursor cell combinations are obtained; there is a relatively more complete organ generation morphology; and the development process of each organ is simulated; a variety of organs can be generated, and the effects and impacts of drugs on each organ can be more intuitively detected; the development period is wide, and drugs can be directly screened in early pregnancy. At the same time, the present invention truly simulates the development of various human organs and obtains relatively complete organ morphology, such as nerves, kidneys, intestines, and bones, overcoming the problem of difficulty in obtaining early human embryos and providing an experimental source for early pregnancy drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 is an immunofluorescence staining image of cell clusters on the first day of differentiation.

[0044] FIG2 is an immunofluorescence staining image of cell clusters on the second day of differentiation.

[0045] FIG3 is an immunofluorescence staining image of cell clusters on the fourth day of differentiation.

[0046] FIG4 is an immunofluorescence staining image of cell clusters on the seventh day of differentiation.

[0047] FIG5 is a hematoxylin and eosin staining image of cell clusters on day 14 of differentiation transplanted into the subcutaneous tissues of mice for 90 days.

[0048] FIG6 is a comparative example of four compound combinations.

[0049] FIG7 is a statistical diagram of the results of the scheme in which FGF2 is removed.

[0050] Figure 8 is an immunofluorescence staining image of cells in which β-catenin, an important gene downstream of WNT signaling, was knocked out. DETAILED DESCRIPTION

[0051] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0052] Example 1

[0053] Materials: Matrigel, FGF2, CHIR99021, Compound E, LDN193189, human pluripotent stem cells, differentiation medium, DMEM / F12 (1:1) basal medium, Dispase, M-NSG mice, surgical instruments.

[0054] FGF2:Peprotech,100-18B

[0055] CHIR99021:Selleck,CT99021

[0056] Compound E: MCE, HY-14176

[0057] LDN193189:STEMGENT,040074

[0058] Dispase: Gibco, 17105041

[0059] Human pluripotent stem cells: WA09, WiCell Agreement No. 17-W0044

[0060] Part I: Inducing human pluripotent stem cells to generate signaling organizers

[0061] step:

[0062] 1) Preheat the differentiation induction basal medium and Dispase in a 37°C water bath 10 minutes in advance. Precool a 200 μL yellow pipette tip to -20°C 10 minutes in advance. Take Matrigel out of the -80°C freezer 12 hours in advance and thaw it in a 4°C ice box.

[0063] 2) Remove the six-well plate containing human pluripotent stem cells from the 37°C incubator, aspirate the original basal medium, and rinse with 2 mL of DMEM / F12 (1:1) basal medium. Add 1 mL of dispase and place in a 37°C incubator for digestion for 3 minutes.

[0064] 3) Observe the human pluripotent stem cell digestion under a microscope. Digestion is complete when curling edges form around the human pluripotent stem cell clones.

[0065] 4) After discarding the Dispase, wash the wells with 2 mL of DMEM / F12 (1:1) basal medium to remove residual Dispase. Then add 1 mL of DMEM / F12 (1:1) basal medium to each well.

[0066] 5) Use a 10 mL pipette to draw up 10 mL of DMEM / F12 (1:1) basal culture medium. Pipet the human pluripotent stem cells in each well five times horizontally and vertically using the cross-cross method. Blow off the cell clumps in three wells and collect them in a 50 mL centrifuge tube.

[0067] 6) Set the pipette to medium speed and pipette against the bottom of the 50 mL centrifuge tube at an appropriate speed and angle to dislodge the cells, reducing cell clumps. Repeat this several times until the cells are clumps approximately 50 μm in diameter. Then, place the 50 mL centrifuge tube upright for 30 seconds to allow the large hESC clumps to settle. Transfer 2 mL of the cell clump from the center of the tube to a 15 mL centrifuge tube.

[0068] 7) Centrifuge at 1100 rpm for 1 minute to sediment the hESCs clumps, discard the supernatant, and mix the remaining 20 μL of liquid using a 200 μL pipette.

[0069] 8) Remove the melted Matrigel from the refrigerator and aspirate 200 μL using a pre-chilled pipette tip. Thoroughly mix the cells in the Matrigel in a 15 mL centrifuge tube, minimizing the formation of bubbles. Place four drops of Matrigel into a 6 cm low-absorption Corning culture dish and carefully place in a 37°C incubator for 10 minutes to allow the gel to solidify.

[0070] 9) While the gel is solidifying, prepare the differentiation medium. Pipette 50 mL of basal medium into a 50 μL centrifuge tube. Add 50 μL of bFGF, 15 μL of CHIR99021, 10 μL of Compound E, and 5 μL of LDN193189 (molar ratio of 0.00058:30:2:1) to prepare the differentiation medium. Remove the culture dish from the incubator, add 10 mL of the prepared differentiation medium, and return it to the incubator to complete the differentiation.

[0071] 10) For the first three days, replace the prepared differentiation medium containing four small chemical molecules with 10 mL each day. Because the gel is fragile, be careful when pipetting through the Pasteur pipette to ensure it adheres to the wall. From the third to the seventh day, replace the basal medium. Obtain a complete Matrigel-encapsulated 3D cell mass.

[0072] The basal medium was a 1:1 mixture of DMEM / F12 and Neurobasal medium, supplemented with 1×GlutaMAX (5 mL of stock solution added to 500 mL of basal medium), 1×N2 (5 mL of stock solution added to 500 mL of basal medium), and 1×B27 (10 mL of stock solution added to 500 mL of basal medium). The differentiation medium was the basal medium supplemented with four small molecules (i.e., the composition of the present invention).

[0073] Part II: Transplanting organoid precursor cells into mice to generate micro-organs

[0074] 1) After anesthetizing SPF-grade M-NSG mice, make an incision of approximately 1 cm on the back of the mouse near the upper limb. Before incision, shave the surgical site with a razor and then spray with iodine for disinfection.

[0075] 2) Use flat-blade tweezers to transplant the entire Matrigel-encapsulated 3D cell mass into the subcutaneous tissue.

[0076] 3) Suture and then apply iodine again for disinfection.

[0077] Experimental results

[0078] 1) Detection method: Cell immunofluorescence staining

[0079] Detection indicators: NANOG protein (indicating cell pluripotency); FOXA2 protein (indicating signal organizer); F-actin protein (indicating cytoskeleton outline)

[0080] Results: As shown in Figure 1, the sample represents a cell cluster on the first day of differentiation. This indicates that, under the induction of the four small molecules, human pluripotent stem cells reduced their pluripotency and began to differentiate into signaling organizers. Arrows pointing to cells positive for NANOG protein and negative for FOXA2 protein represent stem cells; arrows pointing to cells positive for both FOXA2 and NANOG protein represent signaling organizers; and stars pointing to cells positive for both NANOG and FOXA2 represent cells undergoing the transition from stem cells to signaling organizers.

[0081] 2) Detection method: Cell immunofluorescence staining

[0082] Detection indicators: TBX6 protein (indicating mesoderm progenitor cells); SOX2 protein (indicating neural progenitor cells)

[0083] Result description: As shown in Figure 2, the sample is a cell cluster on the second day of differentiation, indicating that under the induction of the "signal organizer" generated on the first day, mesoderm and neuroectoderm precursor cells began to be generated.

[0084] 3) Detection method: Cell immunofluorescence staining

[0085] Detection indicators: TBX6 protein (indicating paraxial mesoderm cells); SOX2 protein (indicating neural progenitor cells)

[0086] The arrows point to TBX6 protein-positive areas, and the arrows point to SOX2 protein-positive areas. The five-pointed star indicates ZO-1 protein.

[0087] ZO-1 represents tight junction protein, the line in the middle of the structure indicates that these cells have polarity, and HOE means that Hoechst stains all cell nuclei.

[0088] Results: As shown in Figure 3, the sample represents a cell cluster on day four of differentiation, indicating that the mesoderm and ectoderm precursors generated on day two have further developed and differentiated, with the mesoderm migrating out of the structure to form the paraxial mesoderm, which surrounds the neural precursor cells. Figure 3 (a) is a top view, and Figure 3 (b) is a bottom view.

[0089] 4) Detection method: Cell immunofluorescence staining

[0090] Detection indicators: PAX6 protein (indicates spinal cord neural progenitor cells); SIX1 protein (indicates somite cells)

[0091] The arrow cluster indicates SIX1 protein positivity, and the arrowhead points to PAX6 protein positivity.

[0092] Results: As shown in Figure 4, the sample is a cell cluster on the seventh day of differentiation, indicating that the paraxial mesoderm generated on the fourth day further developed and differentiated into somites, and the intermediate neural progenitor cells further developed into spinal cord progenitor cells. Figure 3 (a) is a top view, and Figure 3 (b) is a bottom view.

[0093] 5) Detection method: Hematoxylin and eosin staining method

[0094] Result explanation: As shown in Figure 5, the sample is a cell cluster on the 14th day of differentiation (i.e., a cell cluster of organ precursor cells) transplanted into the subcutaneous tissue (or under the renal capsule) of mice for 90 days, indicating that cartilage (part a), bone (part b), intestine (parts c and d, where d is an enlarged view of c) and glomerulus (part e) can be generated.

[0095] 6) Detection method: Observation under a microscope

[0096] Results: To fully demonstrate the necessity of the four-molecule combination, the following six combinations were designed, as shown in Table 1. Plan a was a control group with the normal four-molecule combination. Development was normal on the first, second, and fourth days, as shown in Figure 6A. Plans b, c, d, and e were designed after removing any one of the small molecules. Developmental abnormalities were observed, as shown in Figure 6B, C, D, and E. Plan f, in addition to the normal four-molecule combination, added the Activn A small molecule to activate the NODAL signaling pathway. Developmental abnormalities were also observed, as shown in Figure 6F.

[0097] Table 1 Small molecule combination validation scheme:

[0098] 7) Detection method: biostatistics

[0099] Result explanation: In the result of D in Figure 6, it shows abnormal development but is similar to the control group, so the length-to-width ratio of the structure was calculated. The results showed that the structure of the D plan without FGF2 was shorter, with a smaller length-to-width ratio, and there was a significant difference, as shown in Figure 7.

[0100] 8) Detection method: Immunofluorescence staining

[0101] Explanation of results: To further illustrate the importance of each signal, especially the WNT signal, in addition to removing the WNT activator CHIR99021 in Figure 6E, the β-catenin gene, an important downstream gene of the classical WNT signaling pathway, was further knocked out. After the knockout, even when the four chemical compositions were added, the results still showed developmental abnormalities, and TBXT protein, one of the markers of the "signal organizer", was not expressed, further indicating the importance of the WNT signal, as shown in Figure 8.

[0102] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or modification made by those skilled in the art based on the present invention is within the protection scope of the present invention.

[0103] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A composition, characterized in that The composition comprises FGF2, a BMP inhibitor, a WNT signal activator and a Notch pathway inhibitor; preferably, the BMP inhibitor is CC, Noggin or LDN193189, the WNT signal activator is Wnt3a or CHIR99021, and the Notch pathway inhibitor is Compound E.

2. The composition according to claim 1, wherein The composition comprises FGF2, CHIR99021, Compound E and LDN193189; preferably, the molar ratio of FGF2, CHIR99021, Compound E and LDN193189 is 0.00058:30:2:

1.

3. A method for preparing a signal organizer, characterized in that: The method comprises: culturing human pluripotent stem cells using the composition according to claim 1 or 2, thereby harvesting signal organizers.

4. The method according to claim 3, wherein The method comprises the following steps: (i) thoroughly mixing Matrigel with a cell mass containing human pluripotent stem cells; the human pluripotent stem cells are, for example, hESCs; (ii) When the matrix gel is solidified, a differentiation medium containing the composition is added to perform differentiation.

5. The method according to claim 4, wherein (i), the volume ratio of Matrigel matrix glue and cell aggregate is 1: (1-2); the concentration of Matrigel matrix glue is 50%-100%; (ii), mixing the basal culture medium with the composition according to claim 1 or 2 to prepare a differentiation culture medium; The basal culture medium is, for example, DMEM / F12 (1:1) and Neurobasal medium prepared at a ratio of 1:1, and supplemented with 1×GlutaMAX, 1×N2, and 1×B27; the differentiation is performed in a 37° C. incubator.

6. The method according to claim 4 or 5, characterized in that The cell aggregates include the following processing steps before being mixed with Matrigel matrix gel: 1) Add Dispase to digest the cell clumps; for example, digest at 37°C; 2) After digestion is complete, discard the Dispase enzyme, wash with basal culture medium, and then add basal culture medium again; 3) Blow off the cell clumps and collect them in a container, and then further blow the cell clumps to a diameter of 30-80 μm For example, for a 50 μm sample, take 2 mL of the middle cell mass, centrifuge, remove the supernatant, and mix thoroughly.

7. The method according to claim 6, wherein 1) The step also includes washing the cell mass containing human pluripotent stem cells with a basal culture medium and discarding the basal culture medium; 2), the digestion time is 1-5 min, for example, 3 min; 3), the centrifugal conditions are 1000-1500 rpm, 1-5 min.

8. A signal organizer cultured according to the method of any one of claims 3 to 7.

9. A method for preparing organ precursor cells and / or micro-organs, characterized in that: The method comprises transplanting the signal organizer of claim 8 into a mammal, such as a mouse, to generate the organ precursor cells and / or micro-organs; the transplantation is performed, for example, subcutaneously or under the renal capsule of the mouse.

10. Use of the composition according to claim 1 or 2 in preparing a preparation for generating organ precursor cells and / or micro-organs.

11. An organ precursor cell and / or micro-organ prepared by the method according to claim 9.

12. A composition according to claim 1 or 2, which is used for preparing organ precursor cells and / or micro-organs.

13. A signal organizer according to claim 8, which is used for preparing organ precursor cells and / or micro-organs.

14. A method for treating a disease, characterized in that: Transplanting the organ precursor cells and / or micro-organs according to claim 11 into a subject in need thereof to treat the disease.

15. Use of the organ precursor cells and / or micro-organs according to claim 11 in drug screening.

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