A composition and method for culturing signal organizers and micro-organs using the same

Abstract

The present application discloses a kind of composition and the method for cultivating signal organizer and microorgan with it.The composition includes FGF2, BMP inhibitor, WNT signal activator and Notch pathway inhibitor.The method includes: using as described in the composition cultivate human pluripotent stem cells.The present application generates human signal organizer by the induction of the unique formula of composition, human signal organizer can spontaneously induce surrounding human pluripotent stem cells to generate each organ precursor, and then generate the combination of each microorgan.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a composition and a method for culturing signal organizers and microorganisms using the same. Background Technology

[0002] How different types of cells form, and why some stem cells in human embryos eventually become neurons instead of muscle cells, while others develop into cartilage tissue instead of heart tissue, have long been pressing questions for the scientific community. In the early 1920s, German embryologist Hans Spemann and his student Hilde Mangold began exploring how embryonic stem cells differentiate into any type of cell tissue in the body, including bone, brain, lungs, and liver. In their 1924 salamander embryo experiments, Spemann and Mangold discovered a unique group of cells. When transplanted into another salamander embryo, it induced nearby cells to form the rudiments of the brain and spinal cord—this new embryo grew into a pair of conjoined salamander twins. Later, scientists discovered similar cell groups in other amphibian and fish embryos. These cell groups play a crucial role in shaping the early structural development of tissues and are therefore called "signal organizers." They send out molecular signals that cause other cells to grow and develop in specific ways. When a signal organizer is transferred from one embryo to another, it stimulates its new host to gradually develop a central nervous system that includes the spinal cord and brain. The signal organizer itself develops into the skeleton and other tissues of the spine.

[0003] However, to observe a human signal organizer cell, scientists had to extend the embryo culture period to 15-16 days. But due to ethical and legal constraints, scientists cannot use human embryos that have developed for more than 14 days for experiments, as this is a critical point where the embryo can no longer divide and can be considered an individual. These ethical and related issues limit the direct use of human embryos for relevant experiments. Therefore, scientists have begun actively searching for various similar alternatives.

[0004] To verify its feasibility, Brivanlou and his team conducted a series of experiments using artificial human embryos. They cultured a small clump of cells, about one millimeter in diameter, from human embryonic stem cells in the lab. Although these artificial, simulated tissues differed greatly from natural embryos, they still contained many cells and tissues found in real human embryos and could serve as experimental substitutes. The researchers transplanted the artificial embryos into live chicken embryos for more human-like studies. When the human embryonic cells were transplanted into the avian host, the human cells began to induce basal development of the spinal cord and nervous system—clearly marking the formation of a truly human "signal organizer."

[0005] In chicken embryos, a second body axis can be induced by human signal organizers, leading to the development of the nervous system. It is possible that human spinal cord nerves and various organ tissues can also be induced under the influence of human signal organizers. Due to ethical issues, this process will be allowed to occur in vitro, using pluripotent stem cells as tool cells. These cells have the potential to develop into various organs, but how to obtain signal organizers and induce the generation of human organs is the most difficult technical challenge.

[0006] Organogenesis generally refers to the process in vertebrate ontogeny where organ primordia evolve into organs. Through organogenesis, various organs undergo morphogenesis and tissue differentiation, gradually acquiring specific morphologies and performing certain physiological functions. In 2014, Lancaster et al. systematically proposed the concept of organoids, which are 3D structures that, in vitro, can undergo cell differentiation and lineage orientation processes similar to those in vivo, and self-organize to form spatial structures and specific functions of human organs. Subsequently, more and more tissue-derived organoids have emerged, such as those resembling the intestine, retina, brain, and kidney.

[0007] However, existing, relatively mature human organoid systems only mimic specific functions of organs, such as the respiratory function of the lungs, the absorptive function of the intestines, and the filtration function of the kidneys. They have not yet achieved the goal of completely replacing treatment or transplantation, nor do they possess the morphology of fully formed organs. Furthermore, screening single organoids for anti-tumor drugs carries the risk of having strong adjuvant effects on other healthy organs, requiring lengthy trials before clinical application. In addition, the application of these single organoids is mainly concentrated in the later stages of development, with negligible effect on drug screening during early pregnancy (i.e., the implantation period).

[0008] Current research on early human embryos in domestic and international journals indicates that there is no research on human signal organizers inducing the formation of multiple human organs. A recent international article, "Self-organization of a human organizer by combined Wnt and Nodal signalling" (Martyn, I, et al., Nature, 2018, 558(7708): p.132-135), only involved transplantation onto chicken embryos, without inducing the formation of human organs and tissues. Furthermore, the resulting cells were only a flat, single-layered cell structure, lacking the morphology of a "signal organizer." Other technologies related to early embryos exist, but none relate to human signal organizers. Obtaining signal organizers and inducing the formation of human organs remains the most technically challenging aspect. Summary of the Invention

[0009] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies in obtaining signal organizers and inducing the formation of various organs, by providing a composition and a method for culturing signal organizers and micro-organs using the same. This invention provides a model for early pregnancy and pre- and post-implantation embryonic development studies, solves the problem of difficult embryo acquisition, provides a model platform for early pregnancy drug screening, and offers one source of organs for transplantation.

[0010] One of the technical solutions of the present invention is to provide a composition comprising FGF2, a BMP inhibitor, a WNT signaling activator, and a Notch pathway inhibitor.

[0011] In some preferred embodiments, the BMP inhibitor is CC, Noggin, or LDN193189, the WNT signaling activator is Wnt3a or CHIR99021, and the Notch pathway inhibitor is Compound E.

[0012] In some preferred embodiments, the composition includes FGF2, CHIR99021, Compound E, and LDN193189.

[0013] In some specific embodiments, the molar ratio of FGF2, CHIR99021, Compound E and LDN193189 is 0.00058:30:2:1.

[0014] The second technical solution of the present invention provides a method for preparing signal organizers, wherein the method includes: culturing human pluripotent stem cells using the composition described in the first technical solution, thereby harvesting signal organizers.

[0015] In some preferred embodiments, the method includes the following steps:

[0016] (i) Thoroughly mix the cell clump containing human pluripotent stem cells with Matrigel matrix gel; the cell clump containing human pluripotent stem cells is, for example, hESCs;

[0017] (ii) When the matrix gel solidifies, add the differentiation culture medium containing the composition to carry out differentiation.

[0018] In some preferred embodiments, in (i), the volume ratio of Matrigel to cell clusters containing human pluripotent stem cells is 1:(1-2), for example, 100 μL of Matrigel encapsulates approximately 100-200 cell clusters containing pluripotent stem cells; the concentration of the Matrigel is 50%-100%.

[0019] In (ii), the basic culture medium is mixed with the composition as described in technical solution 1 or 2 to prepare the differentiation culture medium; the basic culture medium is, for example, DMEM / F12 (1:1) and Neurobasal medium prepared at a ratio of 1:1, and 1×GlutaMAX, 1×N2 and 1×B27 are added; the differentiation is carried out in an incubator at 37°C.

[0020] In some preferred embodiments, the cell clumps undergo the following treatment steps before being mixed with Matrigel matrix gel:

[0021] 1) Add Dispase dispersing enzyme to digest cell clumps; for example, digest at 37°C;

[0022] 2) After digestion, discard the Dispase dispersing enzyme, wash with basal medium, and then add basal medium.

[0023] 3) Blow down the cell clumps and collect them in a container. Further blow down the cell clumps until they are 30-80 μm in diameter, for example, 50 μm. Take 2 mL of the middle segment of the cell clump, centrifuge to remove the supernatant and mix well.

[0024] For example, after agitating cells with 10 mL of culture medium and collecting them in a 50 mL centrifuge tube, allow them to settle naturally for 30 seconds. Then, transfer 2 mL of the cell clump to a 15 mL centrifuge tube and centrifuge. The "middle segment" cell clump refers to the cell clumps that, after settling naturally, are larger at the bottom and smaller at the top due to gravity; neither is the optimal size. The middle segment is the ideal size, with a diameter of approximately 50 μm.

[0025] In some preferred embodiments, 1) further includes washing the cell clump containing human pluripotent stem cells with basal culture medium and discarding the basal culture medium.

[0026] In step 2), the digestion time is 1-5 minutes, for example, 3 minutes;

[0027] In step 3), the centrifugation conditions are 1000-1500 rpm for 1-5 min.

[0028] The third technical solution of the present invention is to provide a signal organizer cultivated by the method described in the second technical solution.

[0029] The fourth technical solution of the present invention provides a method for preparing microorganisms, wherein the method includes transplanting the signal organizer described in the third technical solution into a mammal, such as a mouse, to generate it; the transplantation is performed, for example, subcutaneously or under the renal capsule of a mouse.

[0030] In this invention, the micro-organ is a structure that utilizes the pluripotency of human pluripotent stem cells and is assembled in an orderly manner by characteristic cells in vitro and in vivo under the induction of "signal organizers" and has corresponding functions.

[0031] The 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 formulations that generate organ precursor cells and / or microorganisms.

[0032] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0033] The reagents and raw materials used in this invention are all commercially available.

[0034] The positive and progressive effects of this invention are as follows:

[0035] This invention covers the generation of "signal organizers" (the primary technology of this invention is the induction of "signal organizers" through a unique formulation of small chemical molecules; the importance of "signal organizers" lies in their ability to spontaneously signal and induce surrounding human pluripotent stem cells to generate organ precursors, thereby generating combinations of various micro-organs); under their induction, more organ precursor cell combinations are obtained; there are relatively more complete organ formation morphologies; and the developmental process of various organs is simulated; multiple organs can be generated, allowing for more intuitive detection of the effects and influences of drugs on various organs; the developmental period is broadly covered, enabling direct drug screening in early pregnancy. Simultaneously, this invention realistically simulates the development of various human organs and obtains relatively complete organ morphologies, such as nerves, kidneys, intestines, and bones, overcoming the difficulty of obtaining early human embryos and providing an experimental source for drug screening in early pregnancy. Attached Figure Description

[0036] Figure 1 This is an immunofluorescence staining image of a cell cluster on the first day of differentiation.

[0037] Figure 2 This is an immunofluorescence staining image of cell clusters on the second day of differentiation.

[0038] Figure 3This is an immunofluorescence staining image of a cell cluster on the fourth day of differentiation.

[0039] Figure 4 This is an immunofluorescence staining image of a cell cluster on the seventh day of differentiation.

[0040] Figure 5 The image shows hematoxylin and eosin staining of a cell cluster transplanted subcutaneously into a mouse on day 14 of differentiation, 90 days later.

[0041] Figure 6 This is a comparative diagram of four combinations of compounds.

[0042] Figure 7 A statistical chart showing the results of the scheme that removes FGF2.

[0043] Figure 8 Immunofluorescence staining image of cells used to knock out β-catenin, an important downstream gene of WNT signaling. Detailed Implementation

[0044] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0045] Example 1

[0046] Raw materials: Matrigel, FGF2, CHIR99021, Compound E, LDN193189, human pluripotent stem cells, differentiation medium, DMEM / F12 (1:1) basal medium, Dispase dispersant, M-NSG mice, surgical instruments.

[0047] FGF2: Peprotech, 100-18B

[0048] CHIR99021: Selleck, CT99021

[0049] Compound E: MCE, HY-14176

[0050] LDN193189: STEMGENT, 040074

[0051] Dispase: Gibco, 17105041

[0052] Human pluripotent stem cells: WA09, WiCellAgreementNo.17-W0044

[0053] Part 1: Signal Organizers for Inducing Human Pluripotent Stem Cell Generation

[0054] step:

[0055] 1) Preheat the basal culture medium and Dispase dispersant in a 37°C water bath 10 minutes in advance. Precool 200 μL of yellow pipette tip at -20°C 10 minutes in advance. Take Matrigel out of the -80°C freezer and thaw it in a 4°C ice box 12 hours in advance.

[0056] 2) Remove the six-well plate containing human pluripotent stem cells from the 37℃ incubator, aspirate the original basal culture medium of human pluripotent stem cells, wash once with 2mL of DMEM / F12 (1:1) basal culture medium, add 1mL of dispersing enzyme, and place in a 37℃ incubator for digestion for 3min.

[0057] 3) Observe the digestion of human pluripotent stem cells under a microscope. When the edges around the human pluripotent stem cell clones curl up, it indicates that digestion is complete.

[0058] 4) After discarding the Dispase dispersant, wash with 2 mL of DMEM / F12 (1:1) basic culture medium to remove any remaining Dispase dispersant. Then add 1 mL of DMEM / F12 (1:1) basic culture medium to each well.

[0059] 5) Use a 10mL pipette to draw 10mL of DMEM / F12 (1:1) basic culture medium. For each well of human pluripotent stem cells, use the cross-cutting method to make 5 horizontal and 5 vertical cuts with the pipette, and blow off the cell clumps from three wells and collect them into a 50mL centrifuge tube.

[0060] 6) Set the pipette to medium speed and use an appropriate speed to blow the cells out at a certain angle from the bottom of the 50mL centrifuge tube to reduce the cell clumps. Repeat this process a certain number of times until the cells are in clumps about 50μm in diameter. Then, place the 50mL centrifuge tube vertically for 30 seconds to allow the large clumps of hESCs to precipitate. Transfer 2mL of the middle section of the cell clump to a 15mL centrifuge tube.

[0061] 7) Centrifuge at 1100 rpm for 1 minute to settle the hESCs clumps, discard the supernatant, and keep about 20 μL of liquid. Mix it with a 200 μL pipette.

[0062] 8) Take the melted Matrigel from the refrigerator and pipette 200 μL using a pre-cooled pipette tip; mix the cells thoroughly into the Matrigel in a 15 mL centrifuge tube, trying to avoid creating air bubbles. Add four drops to a 6 cm low-adsorption Corning gel culture dish and carefully place it in a 37°C incubator for 10 minutes to allow the gel to solidify.

[0063] 9) While waiting for the gel to solidify, prepare the differentiation medium. Pipette 50 mL of the 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, and mix well to prepare the differentiation medium. Remove the culture dish from the incubator, add 10 mL of the prepared differentiation medium, and then return it to the incubator to complete the differentiation process.

[0064] 10) For the first three days, change the medium daily with 10 mL of the prepared differentiation medium containing 4 small chemical molecules. Because the gel is quite fragile, be careful when using Pasteur tubes to aspirate the cells. Change the medium from day three to day seven. Obtain a 3D cell clump encapsulated in a single Matrigel.

[0065] The basal culture medium consisted of a 1:1 mixture of DMEM / F12 (1:1) and Neurobasal medium, with the addition of 1×GlutaMAX, 1×N2, and 1×B27. The differentiation culture medium was a basal culture medium supplemented with four small molecules (i.e., the composition of this invention).

[0066] Part Two: Transplanting organ precursor cells into mice to generate microorganisms

[0067] 1) After anesthetizing SPF-grade M-NSG mice, make a 1cm incision on the back of the mouse near the upper limb. Before making the incision, shave the hair at the surgical site with a razor and then spray with iodine for disinfection.

[0068] 2) Use flat-tipped forceps to transplant the entire 3D cell mass wrapped in Matrigel into the subcutaneous tissue.

[0069] 3) Perform suturing, and then apply iodine solution again for disinfection.

[0070] Experimental results

[0071] 1) Detection method: Cell immunofluorescence staining

[0072] Detection indicators: NANOG protein (representing cell pluripotency); FOXA2 protein (representing signal organizer); F-actin protein (representing the cytoskeleton outline).

[0073] Results Explanation: For example Figure 1As shown, the sample is a cell cluster from the first day of differentiation, indicating that under the induction of four small molecules, the generation of human pluripotent stem cells reduces cell pluripotency and begins to differentiate into signal organizers. Arrows pointing to NANOG protein-positive and FOXA2 protein-negative cells represent stem cells; arrows pointing to FOXA2 protein-positive and NANOG protein-positive cells represent "signal organizers"; and pentagrams pointing to NANOG and FOXA2 double-positive cells represent cells transitioning from stem cells to "signal organizers".

[0074] 2) Detection method: Cell immunofluorescence staining

[0075] Detection indicators: TBX6 protein (representing mesodermal precursor cells); SOX2 protein (representing neural precursor cells).

[0076] Results Explanation: For example Figure 2 As shown, the sample is a cell cluster from the second day of differentiation, indicating that under the induction of the "signal organizer" generated on the first day, mesodermal and neuroectodermal precursor cells began to be generated.

[0077] 3) Detection method: Cell immunofluorescence staining

[0078] Detection indicators: TBX6 protein (representing paraaxial mesodermal cells); SOX2 protein (representing neural progenitor cells).

[0079] The arrows point to a positive result for TBX6 protein and to a positive result for SOX2 protein. The pentagram represents ZO-1 protein.

[0080] ZO-1 represents tight junction proteins. The line in the middle of the structure indicates that these cells are polar. HOE means that Hoechst staining is applied to all cell nuclei.

[0081] Results Explanation: For example Figure 3 As shown, the sample is a cell cluster on the fourth day of differentiation, indicating that the mesodermal and ectodermal precursor cells generated on the second day further develop and differentiate, and the mesoderm migrates out of the structure to generate paraxial mesoderm, which surrounds the neural precursor cells.

[0082] 4) Detection method: Cell immunofluorescence staining

[0083] Detection indicators: PAX6 protein (representing spinal cord nerve progenitor cells); SIX1 protein (representing somatic cells).

[0084] The arrow indicates a positive result for SIX1 protein, and the arrow indicates a positive result for PAX6 protein.

[0085] Results Explanation: For example Figure 4As shown, the sample is a cell cluster on the seventh day of differentiation, indicating that the paraxial mesoderm generated on the fourth day further develops and differentiates into somites, and the neural progenitor cells in the middle further develop into spinal cord progenitor cells.

[0086] 5) Detection method: Hematoxylin and eosin staining method

[0087] Results Explanation: For example Figure 5 As shown, the sample consists of cell clusters (i.e., cell clusters of organ precursor cells) on day 14 of differentiation, which are transplanted subcutaneously (or under the renal capsule) into mice for 90 days, indicating the potential generation of glomeruli, bones, intestines, etc.

[0088] 6) Detection method: Microscopic observation

[0089] Results: To fully demonstrate the necessity of the four small molecule chemical combinations, the following six combination schemes were designed, as shown in Table 1. Scheme a is the control group of the normal four small molecule combinations; normal development was observed on days 1, 2, and 4. Figure 6 Schemes A, b, c, d, and e are designs that remove any one of the small molecules, and all result in developmental abnormalities, such as... Figure 6 As shown in B, C, D, and E. Scheme f added ActivnA to the four normal small molecules to activate the NODAL signaling pathway, resulting in developmental abnormalities, such as... Figure 6 As shown in F.

[0090] Table 1. Small molecule combination validation scheme:

[0091]

[0092] 7) Detection method: Biostatistics

[0093] Results indicate that: Figure 6 The results of D showed developmental abnormalities, but were similar to the control group. Therefore, the aspect ratio of the structures was statistically analyzed. The results showed that the structures of the d scheme without FGF2 were shorter, with a smaller aspect ratio, and the difference was statistically significant. Figure 7 As shown.

[0094] 8) Detection method: Immunofluorescence staining

[0095] Results Explanation: To further illustrate the importance of each signal, especially the WNT signal, in addition to Figure 6 In addition to removing the WNT activator CHIR99021 from the E sample, the important downstream gene β-catenin in the classic WNT signaling pathway was further knocked out. Even after knockout, developmental abnormalities were still observed under conditions involving the addition of four chemical combinations. The absence of TBXT protein, a marker of "signal organizers," further underscores the importance of WNT signaling. Figure 8 As shown.

[0096] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, The composition consists of FGF2, CHIR99021, Compound E and LDN193189.

2. The composition according to claim 1, characterized in that, The molar ratio of FGF2, CHIR99021, Compound E and LDN193189 is 0.00058:30:2:

1.

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

4. The method as described in claim 3, characterized in that, The method includes the following steps: (i) Thoroughly mix the cell clump containing human pluripotent stem cells with Matrigel matrix gel; the human pluripotent stem cells are, for example, hESCs; (ii) When the matrix gel solidifies, add the differentiation culture medium containing the composition to carry out differentiation.

5. The method as described in claim 4, characterized in that, In (i), the volume ratio of Matrigel to cell clumps is 1:(1-2); the concentration of Matrigel is 50%-100%. In (ii), the basal culture medium is mixed with the composition as described in claim 1 or 2 to prepare the differentiation culture medium; the differentiation is carried out in an incubator at 37°C.

6. The method as described in claim 5, characterized in that, The basal culture medium was prepared by mixing DMEM / F12 (1:1) and Neurobasal medium at a 1:1 ratio, with the addition of 1× GlutaMAX, 1× N2 and 1× B27.

7. The method according to any one of claims 4-6, characterized in that, The cell clumps undergo the following treatment steps before being mixed with Matrigel: 1) Add Dispase dispersant to digest cell clumps; 2) After digestion, discard the Dispase dispersing enzyme, wash with basal medium, and then add basal medium. 3) Blow down the cell clumps and collect them in a container. Further blow down the cell clumps until they are 30-80 μm in diameter. Take 2 mL of the middle segment of the cell clump, centrifuge to remove the supernatant and mix well.

8. The method as described in claim 7, characterized in that, In step 1), digestion is performed at 37°C; In step 3), the diameter is 50 μm.

9. The method as described in claim 7, characterized in that, 1) The process also includes washing the cell clumps containing human pluripotent stem cells with basal culture medium and discarding the basal culture medium; In step 2), the digestion time is 1-5 minutes; In step 3), the centrifugation conditions are 1000-1500 rpm for 1-5 min.

10. The method as described in claim 9, characterized in that, The digestion time is 3 minutes.

11. A method for preparing micro-organs, characterized in that, The method includes: cultivating a signal organizer by the method described in any one of claims 3 to 10, and transplanting the signal organizer into a mammal to generate it.

12. The method as described in claim 11, characterized in that, The mammal is a mouse; the transplant is performed subcutaneously or under the renal capsule of the mouse.

13. Use of the composition of claim 1 or 2 in the preparation of formulations that generate organ precursor cells and / or microorganisms.

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