Method for spontaneously assembling blastoid through pluripotent stem cell and use thereof

Abstract

Provided are a method for spontaneously assembling a blastoid through a pluripotent stem cell and a use thereof. The method comprises the following steps: (1) using GSK3 inhibitor A to induce and culture a primed-state human stem cell; (2) using GSK3 inhibitor B for transformation and culturing; and (3) transferring the transformed cell to a culture container, continuing to use the GSK3 inhibitor B to culture the transformed cell, and obtaining the blastoid. The method is simple and convenient, and can complete a spontaneous assembly culture system simply by using 5iLAF and a 5iLAF human naïve-state pluripotent stem cell derived from 5iLAF-C. The method has a good construction effect and a high yield and is suitable for various pluripotent stem cells. The obtained blastoids are highly similar to human real blastocysts in morphology, cell composition, and transcriptome characteristics and can exhibit exceptionally broad utility as a good model for studying human embryonic development in vitro.

Description

A method for spontaneously assembling blastocyst-like cells from pluripotent stem cells and its application Technical Field

[0001] The present invention belongs to the field of histology and embryology, and specifically relates to a method for spontaneously assembling blastocyst-like cells from pluripotent stem cells and its application. Background Art

[0002] Early embryonic development is a crucial research area in life sciences. Understanding the laws governing early human embryonic development and the regulatory mechanisms governing cell fate determination is crucial for the development of assisted reproductive technologies and the prevention of developmental disorders such as miscarriage and birth defects. However, the limited availability and accessibility of embryos for research, coupled with ethical restrictions on human embryo research, significantly limits basic research on early embryonic development. Recently, blastocyst-like reconstruction techniques have provided a new avenue for studying early embryos.

[0003] A blastoid is an embryo-like structure that is self-assembled using cellular materials, especially stem cells, and has similar structural, molecular, and developmental characteristics to a normal embryo. Blastoids can, to a certain extent, simulate the formation and developmental process of a real human embryo, providing a good in vitro model for the study of early human embryonic development. Although various methods for constructing human blastoids have been reported in the relevant art, the specific formation mechanism of human blastoids is still understudied, and the degree to which different methods simulate real human embryos is limited. Therefore, further exploration of new methods for constructing human blastoids and their synthesis mechanisms is of great significance for the application of human blastoid technology and basic research on early human embryonic development.

[0004] Embryonic development in mammals is a strictly programmed process of cell lineage specialization. After the fertilized egg is formed, the embryo begins to cleave, and then the first cell lineage separation occurs in the blastocyst period. The inner cell mass (ICM) maintains pluripotency, while the outer cells differentiate to form the trophectoderm (TE). TE will then develop into structures such as the placenta outside the embryo, and the ICM will further form the epiblast that eventually develops into the fetus and the hypoblast that develops into the yolk sac. Culturing ICM cells and epiblast cells in vitro can respectively obtain the original state. and primed embryonic stem cells (ESCs). Induced pluripotent stem cells (iPSCs) and ESCs have similar gene expression characteristics, epigenetic modification characteristics, and developmental potential to pre-implantation embryonic cells, making them a good model for studying embryonic cells in vitro.

[0005] In the related art, various methods for synthesizing mouse blastoids using pluripotent stem cells have been reported (Figure 1). The blastoids generated by the aggregation of endothelial stem cells (ESCs), trophoblast stem cells (TSCs), and extraembryonic endoderm stem cells (XEN) can develop into visceral endoderm similar to that of normal embryos. Blasoids formed using 2-cell embryo-like cells (2CLCs) or chemically induced extended pluripotent stem cells (EPSCs) can partially mimic post-implantation mouse embryonic development.

[0006] Classic People pluripotent stem cells (human The first 5iLA culture system for pluripotent stem cells (hnPSCs) was reported in 2014. Among them, the earliest 5iLA culture system included five chemical small molecule inhibitors (inhibitors of BRAF, MEK, SRC, GSK3β and ROCK) and leukemia inhibitory factors LIF and Activin A. The PXGL culture system obtained by optimizing the culture system includes MEK inhibitor PD0325901, WNT signaling pathway antagonist XAV939, protein kinase C inhibitor and LIF.

[0007] It can be seen that although systems for synthesizing human blastoids using hnPSCs have been disclosed in the relevant art, existing methods still have certain defects and shortcomings and cannot meet current usage needs. The common problem of all existing human blastoid construction systems is that they are too complex and require a multi-step process to gradually induce the production of blastoids. Their applicability and applicability are low and are only applicable to PXGL hnPSCs. Moreover, all methods have uncontrollable low efficiency issues when using 5iLAF hnPSCs.

[0008] Therefore, developing and establishing a new method based on 5iLAF hnPSCs to achieve direct spontaneous assembly of human blastoids in one step is of great significance to this field.

[0009] Summary of the Invention

[0010] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for spontaneously assembling blastocysts from stem cells and its application. The method of the present invention induces primed human stem cells into After the formation of stem cells, the GSK3 inhibitor was replaced to improve the 5iLAF system. The invention can enhance the potential of dynamic stem cells, thereby enabling them to more efficiently spontaneously assemble into human blastoids structures, thereby solving the problems of low blastoids structure output efficiency and poor applicability in the existing technology.

[0011] A first aspect of the present invention provides a method for self-assembly and constructing a blastocyst-like cell, comprising the following steps:

[0012] (1) Using a medium containing GSK3 inhibitor A to induce stem cells pluripotent stem cells;

[0013] (2) transferring the cells to a culture medium containing the GSK3 inhibitor B for transformation culture to obtain blastoids;

[0014] The GSK3 inhibitor A is different from the GSK3 inhibitor B.

[0015] In some embodiments of the present invention, step (2) comprises transferring the cells to a culture medium containing GSK3 inhibitor B for transformation culture, picking cell clones and then continuing to culture them in a culture medium containing GSK3 inhibitor B to obtain blastocyst-like structures; or

[0016] Cell clones were picked to establish cell lines and cultured in a medium containing GSK3 inhibitor B to obtain blastocyst-like structures.

[0017] In some embodiments of the present invention, the stem cells are selected from primed stem cells.

[0018] In some embodiments of the present invention, the primed stem cells are selected from human or non-human mammalian sources.

[0019] In some embodiments of the present invention, the primed stem cells are selected from primed embryonic stem cells (pESCs) and primed induced pluripotent stem cells (piPSCs).

[0020] In some embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cell lines.

[0021] In some embodiments of the present invention, the human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.

[0022] In some embodiments of the present invention, human pluripotent stem cells are cultured based on primed human pluripotent stem cells. After the pluripotent stem cells, human The human pluripotent stem cells are used as a model to construct blastoids. Human pluripotent stem cells have the ability to differentiate into trophectoderm cells, which makes The pluripotent stem cells have a unique advantage in self-assembly when constructing human blastoids. Under specific culture conditions, blastoids with structures, gene expression profiles, and lineage-specific characteristics highly similar to those of human blastocysts can be generated in vitro from 5iLAF-CtoI pluripotent stem cell lines (such as 5iLAF-CtoI niPSCs or 5iLAF-CtoI nESCs). A schematic diagram of the process is shown in Figure 2.

[0023] In the present invention, the term "primed state", also known as the excited state or the initial state, refers to the cells that will form a teratoma but cannot form a chimeric biological individual after being introduced into a preimplantation embryo.

[0024] In the present invention, the term " The "primed state", also known as the initial state or the original state, refers to another state of pluripotent embryonic stem cells relative to the primed state. ESCs in this state have stronger differentiation potential and germplasm transmission ability. They capture the stage of pre-implantation embryonic development in vivo and have stronger plasticity.

[0025] In some embodiments of the present invention, the GSK3 inhibitor A and GSK3 inhibitor B are selected from CHIR99021, IM-12, SB216763, AT7519, CHIR-98014, TWS119, Tideglusib, SB415286, 6-bromoindirubin-3-oxime, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, WAY-119064, PF-04802367, GSK-3βinhibitor 1. One or more of KY19382, BRD0705, Alsterpaullone, BIO-acetoxime, 1-Azakenpaullone, Indirubin, Indirubin-3'-oxime, Resibufogenin, 9-ing-41, 5-Bromoindole, CP21R7, or Bikinin.

[0026] In some embodiments of the present invention, the GSK3 inhibitor A is selected from CHIR99021.

[0027] In some embodiments of the present invention, the GSK3 inhibitor B is selected from IM-12.

[0028] In some embodiments of the present invention, the concentration of CHIR99021 in the culture medium is 0.5-1.5 μM; and / or the concentration of IM-12 in the culture medium is 0.5-1.5 μM.

[0029] In some embodiments of the present invention, the concentration of CHIR99021 in the culture medium is 1 μM.

[0030] In some embodiments of the present invention, the concentration of IM-12 in the culture medium is 1 μM.

[0031] In the present invention, the inventors found that based on the CHIR99021-induced Pluripotent stem cells can assemble into blastoids under specific culture conditions.

[0032] In some embodiments of the present invention, the culture medium includes DMEM / F-12 and Neurobasal as basic culture medium, supplemented with N2, B27 and other auxiliary components and induction factors.

[0033] In some embodiments of the present invention, the other auxiliary ingredients are selected from at least one of KnockOut SR, GlutaMAX, non-essential amino acids, trace elements, 2-mercaptoethanol, and bovine serum albumin.

[0034] In some embodiments of the present invention, the induction factor is selected from at least one of a MEK / ERK inhibitor, a BRAF inhibitor, a SRC inhibitor, a ROCK inhibitor, a leukemia inhibitory factor (LIF), activin A, and a fibroblast growth factor (FGF).

[0035] In some embodiments of the present invention, the trace elements include antibiotics.

[0036] In some embodiments of the present invention, the antibiotic is a double antibiotic. In some embodiments of the present invention, the double antibiotic is a penicillin-streptomycin double antibiotic.

[0037] In some embodiments of the present invention, the MEK / ERK inhibitor comprises PD0325901.

[0038] In some embodiments of the invention, the BRAF inhibitor comprises SB5900885.

[0039] In some embodiments of the present invention, the SRC inhibitor comprises WH-4-023.

[0040] In some embodiments of the invention, the ROCK inhibitor comprises Y-27632.

[0041] In some embodiments of the present invention, the fibroblast growth factor includes FGF2. In the art, FGF-2 (fibroblast growth factor 2), also known as bFGF, basic fibroblast growth factor or FGF-β, is a growth factor and signaling protein encoded by the FGF-2 gene.

[0042] In some embodiments of the present invention, the culture medium composed of the above formula is also called 5iLAF culture medium (based on the content or composition differences of the induction factors, different subspecies culture media will be produced, such as the 5iLAF culture medium and 5iLAF-C culture medium in this application). Of course, it should be understood that for the 5iLAF culture medium, its basic composition formula can be adjusted based on actual usage requirements, or reasonably replaced with ingredients with similar effects.

[0043] In the present invention, the inventors found that by rationally using 5iLAF, By processing pluripotent stem cells, blastocysts can be efficiently obtained in a one-step method in a simple culture medium.

[0044] In some embodiments of the present invention, the inventors have improved the 5iLAF human The potential of pluripotent stem cells enables them to efficiently and spontaneously assemble into blastoids structures in 5iLAF culture medium.

[0045] In some embodiments of the present invention, the 5iLAF culture medium is selected from a 5iLAF-C culture medium containing CHIR99021 and a conventional 5iLAF culture medium containing IM-12.

[0046] Among them, the specific composition of 5iLAF-C culture medium is:

[0047] The basal medium is DMEM / F-12 and Neurobasal medium, supplemented with N2 and B27 supplements. Supplementary components include KnockOut Serum Replacement (SR), GlutaMAX Supplement, MEM Non-Essential Amino Acids Solution, penicillin-streptomycin, 2-mercaptoethanol, and bovine serum albumin (BSA). Induction factors include PD0325901, CHIR99021, SB590885, WH-4-023, Y-27632, recombinant human leukemia inhibitory factor, activin A, and FGF2.

[0048] In some embodiments of the present invention, the volume ratio of the DMEM / F-12 medium to the Neurobasal medium is 1:1.

[0049] In some embodiments of the present invention, the added mass of the N2 supplement and the B27 supplement is 1%-2% of the total mass, respectively.

[0050] Of course, those skilled in the art can reasonably adjust the basic culture medium and the amount of N2 and B27 added according to actual usage requirements to meet the culture requirements under different environments.

[0051] In some embodiments of the present invention, the added mass of the N2 supplement is 1% of the total mass, and the added mass of the B27 supplement is 2% of the total mass.

[0052] In some embodiments of the present invention, the amounts of auxiliary components and induction factors added can be reasonably adjusted according to the type of stem cells actually induced and cultured.

[0053] In some specific embodiments of the present invention, the added amounts of auxiliary components are: based on the total mass of the culture medium, 0.5% of the total mass of KnockOut serum replacement (SR), 1 mM GlutaMAX additive, 1% of the total mass of MEM Non-Essential Amino Acids Solution, 1% of the total mass of penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 50 μg / mL of bovine serum albumin (BSA).

[0054] In some specific embodiments of the present invention, the amount of inducing factors added is: PD0325901 with a final concentration of 1 μM, CHIR99021 with a final concentration of 1 μM, SB590885 with a final concentration of 0.5 μM, WH-4-023 with a final concentration of 1 μM, Y-27632 with a final concentration of 10 μM, recombinant human leukemia inhibitory factor with a final concentration of 20 ng / mL (recombinant human LIF, purchased from Qkine), activin A with a final concentration of 20 ng / mL, and fibroblast growth factor (FGF2) with a final concentration of 8 ng / mL.

[0055] The specific composition of conventional 5iLAF medium containing IM-12 is:

[0056] The basal medium is DMEM / F-12 and Neurobasal medium, supplemented with N2 and B27 supplements. Supplementary components include KnockOut Serum Replacement (SR), GlutaMAX Supplement, MEM Non-Essential Amino Acids Solution, penicillin-streptomycin, 2-mercaptoethanol, and bovine serum albumin (BSA). Induction factors include PD0325901, IM-12, SB590885, WH-4-023, Y-27632, recombinant human leukemia inhibitory factor, activin A, and FGF2.

[0057] The difference between conventional 5iLAF medium containing IM-12 and 5iLAF-C medium is that IM-12 is used to replace CHIR99021 in 5iLAF-C medium.

[0058] In some embodiments of the present invention, in the conventional 5iLAF culture medium containing IM-12, the volume ratio of DMEM / F-12 culture medium to Neurobasal culture medium is 1:1.

[0059] In some embodiments of the present invention, the added amounts of N2 supplement and B27 supplement in the conventional 5iLAF culture medium containing IM-12 are 1%-2% of the total mass, respectively.

[0060] Of course, those skilled in the art can reasonably adjust the basic culture medium and the amount of N2 and B27 added according to actual usage requirements to meet the culture requirements under different environments.

[0061] In some embodiments of the present invention, the added mass of the N2 supplement is 1% of the total mass, and the added mass of the B27 supplement is 2% of the total mass.

[0062] In some embodiments of the present invention, the amounts of auxiliary components and induction factors added can be reasonably adjusted according to the type of stem cells actually induced and cultured.

[0063] In some specific embodiments of the present invention, the added amounts of auxiliary components are: based on the total mass of the culture medium, 0.5% of the total mass of KnockOut serum replacement (SR), 1 mM GlutaMAX additive, 1% of the total mass of MEM Non-Essential Amino Acids Solution, 1% of the total mass of penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 50 μg / mL of bovine serum albumin (BSA).

[0064] In some specific embodiments of the present invention, the amount of induction factors added is: PD0325901 with a final concentration of 1 μM, IM-12 with a final concentration of 1 μM, SB590885 with a final concentration of 0.5 μM, WH-4-023 with a final concentration of 1 μM, Y-27632 with a final concentration of 10 μM, recombinant human leukemia inhibitory factor with a final concentration of 20 ng / mL (recombinant human LIF, purchased from Qkine), activin A with a final concentration of 20 ng / mL, and fibroblast growth factor 2 (FGF2) with a final concentration of 8 ng / mL.

[0065] The preparation method of the culture medium is the same as the general preparation method of other conventional culture media in the art, and can be used after mixing the raw materials according to the proportion and then sterilizing them at high temperature.

[0066] In some embodiments of the present invention, the induction culture time in step (1) is 9-13 days, and the transformation culture time in step (2) is 4-11 days.

[0067] In some embodiments of the present invention, the conversion culture comprises transferring the cells to a culture medium containing GSK3 inhibitor B for conversion culture for 2-6 days, picking cell clones and then continuing to culture them in a culture medium containing GSK3 inhibitor B for 2-5 days.

[0068] In some embodiments of the present invention, step (1) includes the step of inoculating stem cells onto feeder cells and inducing culture using a culture medium containing GSK3 inhibitor A; and / or the step of dissociating stem cells is further included between steps (1) and (2).

[0069] In some embodiments of the present invention, the dissociation comprises dissociating the stem cells using an enzymatic or non-enzymatic digestion solution.

[0070] In some embodiments of the invention, the enzyme is selected from Accutase, Dispase, Collagenase or Trypsin.

[0071] In some embodiments of the present invention, the non-enzymatic digestion solution is selected from EDTA, NO-ZYME reagent, and Non-enzymatic digestion solution.

[0072] In some embodiments of the present invention, the cells used in step (2) are cells that have been induced and cultured in step (1) and passaged multiple times.

[0073] In some embodiments of the present invention, the cells used in step (2) are cells that have been passaged at least twice after induction culture in step (1).

[0074] In some embodiments of the present invention, the cells used in step (2) are cells that have been passaged 3-20 times after induction culture in step (1).

[0075] In some embodiments of the present invention, the method is specifically:

[0076] (1) Primed stem cells were induced and cultured for 9-13 days using 5iLAF-C medium containing CHIR99021 at a final concentration of 1 μM. pluripotent stem cells;

[0077] (2) The cells were inoculated into 5iLAF medium containing IM-12 at a final concentration of 1 μM and cultured for 4-11 days; or the cells were inoculated into 5iLAF medium containing IM-12 at a final concentration of 1 μM and cultured for 2-6 days, and then single clones were picked and cultured in a culture container containing 5iLAF medium containing IM-12 at a final concentration of 1 μM for another 2-5 days to obtain blastoids.

[0078] The second aspect of the present invention provides a blastocyst-like cell constructed by the method described in the first aspect of the present invention.

[0079] In some embodiments of the present invention, the blastocyst-like cells express markers of human true embryos, including SOX2, NANOG, GATA6, KRT18, and GATA3.

[0080] In the present invention, the inventors used immunofluorescence staining to identify embryo-specific markers in the obtained blastoids and found that the blastoids obtained in the present invention are similar to real human embryos. Inside the blastoid structure are epiblast-like cells expressing SOX2, and on the surface of the internal cells are some early hypoblast cells expressing GATA6, while outside the structure are trophectoderm-like cells expressing GATA6 and GATA3, which is extremely close to the cell composition of real human embryos.

[0081] The third aspect of the present invention provides use of the blastocyst-like embryo described in the second aspect of the present invention in preparing an embryo-like artificial mimic.

[0082] In some embodiments of the present invention, the embryo-like artificial mimic comprises:

[0083] (1) Artificial simulation of animal embryo blastocyst;

[0084] (2) Animal embryo in vitro cell model.

[0085] A fourth aspect of the present invention provides a product comprising a stem cell culture medium containing a GSK3 inhibitor A and a stem cell culture medium containing a GSK3 inhibitor B.

[0086] In some embodiments of the present invention, the product is used to prepare blastocysts.

[0087] In some embodiments of the present invention, the GSK3 inhibitor A and GSK3 inhibitor B are selected from CHIR99021, IM-12, SB216763, AT7519, CHIR-98014, TWS119, Tideglusib, SB415286, 6-bromoindirubin-3-oxime, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, WAY-119064, PF-04802367, GSK-3βinhibitor 1. One or more of KY19382, BRD0705, Alsterpaullone, BIO-acetoxime, 1-Azakenpaullone, Indirubin, Indirubin-3'-oxime, Resibufogenin, 9-ing-41, 5-Bromoindole, CP21R7, or Bikinin.

[0088] In some embodiments of the present invention, the GSK3 inhibitor A is selected from CHIR99021.

[0089] In some embodiments of the present invention, the GSK3 inhibitor B is selected from IM-12.

[0090] In some embodiments of the present invention, the concentration of CHIR99021 in the culture medium is 0.5-1.5 μM; and / or the concentration of IM-12 in the culture medium is 0.5-1.5 μM.

[0091] In some embodiments of the present invention, the concentration of CHIR99021 in the culture medium is 1 μM.

[0092] In some embodiments of the present invention, the concentration of IM-12 in the culture medium is 1 μM.

[0093] In some embodiments of the present invention, the culture medium includes DMEM / F-12 and Neurobasal as basic culture medium, supplemented with N2, B27 and other auxiliary components and induction factors.

[0094] In some embodiments of the present invention, the other auxiliary ingredients are selected from at least one of KnockOut SR, GlutaMAX, non-essential amino acids, trace elements, 2-mercaptoethanol, and bovine serum albumin.

[0095] In some embodiments of the present invention, the induction factor is selected from at least one of a MEK / ERK inhibitor, a BRAF inhibitor, a SRC inhibitor, a ROCK inhibitor, a leukemia inhibitory factor (LIF), activin A, and a fibroblast growth factor (FGF).

[0096] In some embodiments of the present invention, the trace elements include antibiotics.

[0097] In some embodiments of the present invention, the antibiotic is a double antibiotic. In some embodiments of the present invention, the double antibiotic is a penicillin-streptomycin double antibiotic.

[0098] In some embodiments of the present invention, the MEK / ERK inhibitor comprises PD0325901.

[0099] In some embodiments of the invention, the BRAF inhibitor comprises SB5900885.

[0100] In some embodiments of the present invention, the SRC inhibitor comprises WH-4-023.

[0101] In some embodiments of the invention, the ROCK inhibitor comprises Y-27632.

[0102] In some embodiments of the present invention, the 5iLAF culture medium is selected from a 5iLAF-C culture medium containing CHIR99021 and a conventional 5iLAF culture medium containing IM-12.

[0103] Among them, the specific composition of 5iLAF-C culture medium is:

[0104] The basal medium is DMEM / F-12 and Neurobasal medium, supplemented with N2 and B27 supplements. Supplementary components include KnockOut Serum Replacement (SR), GlutaMAX Supplement, MEM Non-Essential Amino Acids Solution, penicillin-streptomycin, 2-mercaptoethanol, and bovine serum albumin (BSA). Induction factors include PD0325901, CHIR99021, SB590885, WH-4-023, Y-27632, recombinant human leukemia inhibitory factor, activin A, and FGF2.

[0105] In some embodiments of the present invention, the volume ratio of the DMEM / F-12 medium to the Neurobasal medium is 1:1.

[0106] In some embodiments of the present invention, the added mass of the N2 supplement and the B27 supplement is 1%-2% of the total mass, respectively.

[0107] Of course, those skilled in the art can reasonably adjust the basic culture medium and the amount of N2 and B27 added according to actual usage requirements to meet the culture requirements under different environments.

[0108] In some embodiments of the present invention, the added mass of the N2 supplement is 1% of the total mass, and the added mass of the B27 supplement is 2% of the total mass.

[0109] In some embodiments of the present invention, the amounts of auxiliary components and induction factors added can be reasonably adjusted according to the type of stem cells actually induced and cultured.

[0110] In some specific embodiments of the present invention, the added amounts of auxiliary components are: based on the total mass of the culture medium, 0.5% of the total mass of KnockOut serum replacement (SR), 1 mM GlutaMAX additive, 1% of the total mass of MEM Non-Essential Amino Acids Solution, 1% of the total mass of penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 50 μg / mL of bovine serum albumin (BSA).

[0111] In some specific embodiments of the present invention, the amount of inducing factors added is: PD0325901 with a final concentration of 1 μM, CHIR99021 with a final concentration of 1 μM, SB590885 with a final concentration of 0.5 μM, WH-4-023 with a final concentration of 1 μM, Y-27632 with a final concentration of 10 μM, recombinant human leukemia inhibitory factor with a final concentration of 20 ng / mL (recombinant human LIF, purchased from Qkine), activin A with a final concentration of 20 ng / mL, and fibroblast growth factor (FGF2) with a final concentration of 8 ng / mL.

[0112] The specific composition of conventional 5iLAF medium containing IM-12 is:

[0113] The basal medium is DMEM / F-12 and Neurobasal medium, supplemented with N2 and B27 supplements. Supplementary components include KnockOut Serum Replacement (SR), GlutaMAX Supplement, MEM Non-Essential Amino Acids Solution, penicillin-streptomycin, 2-mercaptoethanol, and bovine serum albumin (BSA). Induction factors include PD0325901, IM-12, SB590885, WH-4-023, Y-27632, recombinant human leukemia inhibitory factor, activin A, and FGF2.

[0114] The difference between conventional 5iLAF medium containing IM-12 and 5iLAF-C medium is that IM-12 is used to replace CHIR99021 in 5iLAF-C medium.

[0115] In some embodiments of the present invention, in the conventional 5iLAF culture medium containing IM-12, the volume ratio of DMEM / F-12 culture medium to Neurobasal culture medium is 1:1.

[0116] In some embodiments of the present invention, the added amounts of N2 supplement and B27 supplement in the conventional 5iLAF culture medium containing IM-12 are 1%-2% of the total mass, respectively.

[0117] Of course, those skilled in the art can reasonably adjust the basic culture medium and the amount of N2 and B27 added according to actual usage requirements to meet the culture requirements under different environments.

[0118] In some embodiments of the present invention, the added mass of the N2 supplement is 1% of the total mass, and the added mass of the B27 supplement is 2% of the total mass.

[0119] In some embodiments of the present invention, the amounts of auxiliary components and induction factors added can be reasonably adjusted according to the type of stem cells actually induced and cultured.

[0120] In some specific embodiments of the present invention, the added amounts of auxiliary components are: based on the total mass of the culture medium, 0.5% of the total mass of KnockOut serum replacement (SR), 1 mM GlutaMAX additive, 1% of the total mass of MEM Non-Essential Amino Acids Solution, 1% of the total mass of penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 50 μg / mL of bovine serum albumin (BSA).

[0121] In some specific embodiments of the present invention, the amount of induction factors added is: PD0325901 with a final concentration of 1 μM, IM-12 with a final concentration of 1 μM, SB590885 with a final concentration of 0.5 μM, WH-4-023 with a final concentration of 1 μM, Y-27632 with a final concentration of 10 μM, recombinant human leukemia inhibitory factor with a final concentration of 20 ng / mL (recombinant human LIF, purchased from Qkine), activin A with a final concentration of 20 ng / mL, and fibroblast growth factor 2 (FGF2) with a final concentration of 8 ng / mL.

[0122] The preparation method of the culture medium is the same as the general preparation method of other conventional culture media in the art, and can be used after mixing the raw materials according to the proportion and then sterilizing them at high temperature.

[0123] In some embodiments of the present invention, the product includes a culture reagent or a culture kit.

[0124] The beneficial effects of the present invention are:

[0125] 1. The method of self-assembly to construct human blastocyst-like embryos in the present invention is simple and convenient. Human blastocyst-like embryos can be obtained by simple culture treatment, and the sources of cell materials and required reagents are simple. Only 5iLAF human embryos converted from 5iLAF-C are used. The self-assembled culture system can be completed by using pluripotent stem cells and 5iLAF.

[0126] 2. The human blastocyst-like self-assembly construction method of the present invention has good effect and high yield (can reach 55%), which has a significant efficiency improvement effect compared with conventional methods and is more widely applicable, and can be applied to various pluripotent stem cells such as 5iLAF-CtoI nESCs and niPSCs.

[0127] 3. The blastoids obtained by the method of the present invention are highly similar to real human blastocysts in morphology, cell composition and transcriptome characteristics, and can play a very wide role as a good model for in vitro research on human embryonic development. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] FIG1 is a schematic diagram of the process of preparing blastoid structures using five different technologies ((a)-(e)) in the related art.

[0129] FIG2 is a schematic diagram of the process of spontaneously assembling human blastoids according to the present invention.

[0130] FIG3 shows images of cells and blastoids at key time points in Example 1 of the present invention.

[0131] FIG4 is a partial blastoid display image in Example 1 of the present invention.

[0132] FIG5 is an image of immunofluorescence staining of blastoids to detect human blastocyst markers in Example 1 of the present invention.

[0133] FIG6 is a statistical diagram showing the efficiency of spontaneous assembly of cells into blastoids in different systems before and after transformation in Comparative Example 1 of the present invention.

[0134] FIG7A is a partial image of blastoids obtained using a suspension culture plate in Example 2 of the present invention.

[0135] FIG7B is a partial image of blastoids obtained using the AggreWell culture plate in Example 2 of the present invention.

[0136] FIG8A is a schematic diagram of a flow chart of a solution in Example 2 of the present invention.

[0137] FIG8B shows images of blastoids culture at key time points in different culture containers.

[0138] FIG9 is a statistical diagram of the diameters of blastoids in Example 2 of the present invention.

[0139] FIG10 is a statistical diagram of the spontaneous assembly efficiency of blastoids under different starting cell conditions in Example 2 of the present invention.

[0140] FIG11A shows the results of immunofluorescence staining of blastoids obtained from suspension culture in Example 2 of the present invention to detect human blastocyst markers.

[0141] FIG11B shows the results of immunofluorescence staining of blastoids obtained by 3D culture AggreWell in Example 2 of the present invention to detect human blastocyst markers.

[0142] FIG12 shows the results of other fluorescent staining such as NANOG, KRT18, and DAPI in suspension culture.

[0143] FIG13A is a graph showing the effects of natural assembly of blastoids by 5iLAF-CtoI nESCs and TJ-1#nESCs in 5iLAF or 5iLAF-C when cells assembled blastoids in different culture media before and after GSK3 factor replacement;

[0144] Figure 13B is a statistical graph showing the number of blastoids naturally assembled by 5iLAF-CtoI nESCs and TJ-1#nESCs in 5iLAF or 5iLAF-C when cells assembled blastoids in different culture media before and after GSK3 factor replacement. DETAILED DESCRIPTION

[0145] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0146] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0147] Material

[0148] In the following examples, the specific composition of the 5iLAF-C culture medium used is:

[0149] DMEM / F-12 medium and Neurobasal medium with a volume ratio of 1:1, N2 supplement with a total weight of 1%, B27 supplement with a total weight of 2%, KnockOut serum replacement (SR) with a total weight of 0.5%, GlutaMAX additive with a final concentration of 1 mM, MEM Non-Essential Amino Acids Solution with a total weight of 1%, penicillin-streptomycin with a total weight of 1%, 2-mercaptoethanol with a final concentration of 0.1 mM, and bovine serum albumin (BSA) with a final concentration of 50 μg / mL.

[0150] The following small molecules or cytokines were also added to the 5iLAF-C culture medium: PD0325901 at a final concentration of 1 μM, CHIR99021 at a final concentration of 1 μM, SB590885 at a final concentration of 0.5 μM, WH-4-023 at a final concentration of 1 μM, Y-27632 at a final concentration of 10 μM, recombinant human leukemia inhibitory factor (recombinant human LIF, purchased from Qkine) at a final concentration of 20 ng / mL, activin A at a final concentration of 20 ng / mL, and fibroblast growth factor (FGF2, purchased from Healthgen Biotechnology Corp) at a final concentration of 8 ng / mL.

[0151] In the following examples, the specific composition of the 5iLAF culture medium used is:

[0152] DMEM / F-12 medium and Neurobasal medium with a volume ratio of 1:1, N2 supplement with a total weight of 1%, B27 supplement with a total weight of 2%, KnockOut serum replacement (SR) with a total weight of 0.5%, GlutaMAX additive with a final concentration of 1 mM, MEM Non-Essential Amino Acids Solution with a total weight of 1%, penicillin-streptomycin with a total weight of 1%, 2-mercaptoethanol with a final concentration of 0.1 mM, and bovine serum albumin (BSA) with a final concentration of 50 μg / mL.

[0153] The following small molecules or cytokines were also added to the 5iLAF culture medium: PD0325901 at a final concentration of 1 μM, IM-12 at a final concentration of 1 μM, SB590885 at a final concentration of 0.5 μM, WH-4-023 at a final concentration of 1 μM, Y-27632 at a final concentration of 10 μM, recombinant human leukemia inhibitory factor at a final concentration of 20 ng / mL (recombinant human LIF, purchased from Qkine), activin A at a final concentration of 20 ng / mL, and fibroblast growth factor 2 (FGF2, purchased from Healthgen Biotechnology Corp) at a final concentration of 8 ng / mL.

[0154] Example 1

[0155] In this example, the inventors used primed STiPS O-XX1 iPSCs (purchased from Howard Hughes Medical Institute, USA) as an example of cell material to construct spontaneously assembled human blastoids.

[0156] The specific steps of Example 1 are as follows:

[0157] (1) 2×10 4 Primed STiPS O-XX1 iPSCs were seeded on feeder cells (inactivated mouse embryonic fibroblasts, feeders; in this example, the feeder cells were embryonic fibroblasts of ICR mice treated with Mitomycin C for 3 h) and induced to proliferate using 5iLAF-C medium containing the GSK3 inhibitor CHIR99021 (final concentration of 1 μM). During the induction of pluripotent stem cells, fresh 5iLAF-C medium was replaced every day, and the cells were cultured in a sterile cell culture incubator with 20% O2, 5% CO2 and 37°C.

[0158] (2) After 9-13 days of induction culture (12 days in this example, when the cell density is close to 90%), the cells were digested with Accutase cell digestion solution and passaged at a passage ratio of 1:2 (i.e., the ratio of the number of original cell plates / bottle to the number of passage cell plates / bottle) (denoted as P1). On the 6th day after passage, the cells were again passaged at 2×10 5 Cells / 35 mm culture dish were passaged (denoted as P2).

[0159] (3) From the third passage (P3), 5iLAF-C can be transformed into 5iLAF (CtoI) and blastoids can be spontaneously assembled. The specific operation is as follows: on the 3rd to 4th day after the P3 passage, the cells are cultured in 5iLAF-C medium. On the 3rd to 4th day, the medium is replaced with 5iLAF medium and cultured in 5iLAF medium for 3-5 days until a large number of dome-shaped, clear-edged, suspended clones (called 5iLAF-CtoI niPSCs) are observed (usually on the 4th day of CtoI). During this period, fresh 5iLAF medium is continuously replaced every day.

[0160] Cell images of 5iLAF-CtoI niPSCs are shown in Figure 3.

[0161] (4) Use a mouth pipette or a 10 μL pipette to pick up a single colony with clear and smooth edges suspended in the culture medium and place it in a 96-well U-bottom culture plate. Continue to culture in 5iLAF medium for 3-4 days to obtain human blastoids.

[0162] The obtained human blastoids structural image is shown in FIG4 .

[0163] The inventors used immunofluorescence staining to identify embryo-specific markers in the blastoids obtained in this example. The specific identification steps are as follows:

[0164] (1) Using a mouth pipette, transfer the blastoids constructed in the above example into a U-bottomed culture well containing Dulbecco's phosphate buffered saline (DPBS) and briefly rinse to remove any residual culture medium. The washed blastoids were then fixed with 4% paraformaldehyde (PFA) at room temperature for 30 minutes. Alternatively, the cells were fixed overnight at 4°C.

[0165] (2) Wash with DPBS three times to remove residual PFA. The washed blastoids were permeabilized in 0.75% Triton X-100 for 1 hour, and then blocked with blocking buffer containing 3% BSA and 0.1% Triton X-100 (in DPBS) at room temperature for 1 hour.

[0166] (3) The blastoids were transferred to blocking buffer containing diluted primary antibodies (anti-SOX2, 1:200 dilution, purchased from Invitrogen; anti-GATA6, 1:40 dilution, purchased from R&D systems; anti-GATA3, 1:250 dilution, purchased from Abcam) and incubated overnight at 4°C.

[0167] (4) After washing three times with DPBS containing 0.1% Triton X-100 to remove residual primary antibody, blastoids were transferred to blocking buffer containing diluted secondary antibodies (Donkey anti-Rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, labeled with Alexa Fluor 594; Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, labeled with Alexa Fluor 488; Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, labeled with Alexa Fluor 647; all secondary antibodies were purchased from Invitrogen, diluted 1:1000) and incubated in the dark at room temperature for 1 h.

[0168] (5) Wash the blastoids three times with DPBS to remove residual secondary antibody. Transfer the washed blastoids into a nuclear staining solution (DAPI) and incubate at room temperature for 20 min.

[0169] (6) The stained blastoids were imaged using a Zeiss LSM800 laser scanning confocal microscope.

[0170] The results are shown in Figure 5.

[0171] Based on the staining results, it can be found that the blastoids constructed by the GSK3 inhibitor replacement culture technology of the present invention are similar to real human embryos. The interior of the blastoids structure is composed of epiblast-like cells expressing SOX2, and there are some early hypoblast cells expressing GATA6 on the surface of the internal cells, while the outside of the structure is composed of trophectoderm-like cells expressing GATA6 and GATA3, which is very close to the cell composition of real human embryos.

[0172] Comparative Example 1

[0173] The culture method of Comparative Example 1 is the same as that of Example 1, except that in Comparative Example 1, the inventors no longer use 5iLAF culture medium to replace 5iLAF-C culture medium for subsequent culture in step (3).

[0174] The specific steps are:

[0175] (1) 2×10 4Primed STiPS O-XX1 iPSCs were seeded on feeder cells and induced to develop into During the induction of pluripotent stem cells, fresh 5iLAF-C medium was replaced every day, and the cells were cultured in a sterile cell culture incubator with 20% O2, 5% CO2 and 37°C.

[0176] (2) After 9-13 days of induction culture (12 days in this example, when the cell density is close to 90%), the cells were digested with Accutase cell digestion solution and passaged at a passage ratio of 1:2 (denoted as P1). On the 6th day after passage, the cells were again passaged at a ratio of 2×10 5 Cells / 35 mm culture dish were passaged (denoted as P2).

[0177] (3) After passage P3, the cells were cultured in 5iLAF-C medium (cell clones still cultured in 5iLAF-C medium were called 5iLAF-C niPSCs), and the total number of culture days was the same as in Example 1.

[0178] (4) Use a mouth pipette or a 10 μL pipette to pick up a single 5iLAF-C niPSCs clone with clear and smooth edges suspended in culture medium and place it in a 96-well U-bottom culture plate. Divide it into two groups: one group changes the culture medium to 5iLAF medium and culture for 3-4 days, while the other group continues to use 5iLAF-C medium for the same number of days (the cells obtained in this group are called 5iLAF-CtoI niPSCs).

[0179] The results are shown in Figure 6.

[0180] It was found that when the same 5iLAF-C niPSCs clones were cultured in 96-well U-bottom culture plates, a certain number of blastoids could still be obtained when the culture medium was changed to 5iLAF. However, for the 5iLAF-C niPSCs clones cultured in 5iLAF-C medium, no blastoids were effectively formed, whether before transformation (5iLAF-C niPSCs) or after transformation (5iLAF-CtoI niPSCs).

[0181] These results indicate that the replacement of GSK3 inhibitors during the construction of blastoids, especially the switch from CHIR99021 (5iLAF-C medium) to IM-12 (5iLAF medium), is crucial for the cloning of blastoids.

[0182] Example 2

[0183] In this example, the inventors used ESCs as an example of cell material to construct spontaneously assembled human blastoids.

[0184] Among them, the construction of ESCs cell line can refer to the inventors' previous paper: Bi Y, Tu Z, Zhang Y, et al. Identification of ALPPL2 as a Naive Pluripotent State-Specific Surface Protein Essential for Human Naive Pluripotency Regulation-ScienceDirect[J].2020.

[0185] The specific operation steps of Example 2 are the same as those of Example 1, except that ESCs cells (specifically primed TJ-1#ESCs) are used instead of the iPSCs cells in Example 1.

[0186] The stable cell line obtained after transformation was named 5iLAF-CtoI nESCs.

[0187] To demonstrate that the method of the present invention is also applicable to ESCs cell lines, the inventors used the constructed 5iLAF-CtoI nESCs as cell material to construct spontaneously assembled blastoids. The specific steps are as follows:

[0188] (1) Take 5 iLAF-CtoI nESCs, digest them with cell digestion solution, and then inoculate them into a gelatin-coated culture plate. Incubate at 37°C for 1 hour to remove feeder cells.

[0189] (2) Collect the cell culture supernatant to obtain non-adherent 5iLAF-CtoI nESCs. Centrifuge at 300g for 5 minutes, remove the supernatant, resuspend the cell pellet in 5iLAF medium, and count the cells to calculate the cell concentration.

[0190] (3) Take 4×10 4 5iLAF-CtoI nESCs after feeder removal were seeded in a 24-well low-adhesion suspension culture plate and cultured in 5iLAF medium for 6 days. 15-50 human blastoids were obtained in each well (as shown in Figures 7A and 7B).

[0191] The schematic diagram of the process in this embodiment is shown in FIG8A .

[0192] To further verify its feasibility, the inventors took an equal number of 5iLAF-CtoI nESCs after removing the feeder and inoculated them into a 24-well AggreWell culture plate. They used a 3D culture system and cultured them in 5iLAF medium for 6 days. Similarly, human blastoids structures with a yield of approximately 40-55% were obtained (as shown in Figure 8B).

[0193] Observation and measurement of the blastoids obtained in the above examples revealed that their diameters were mostly between 180 and 260 μm, similar to the size of a normal human embryo (as shown in Figure 9). Furthermore, by further adjusting the seeding volume of 5iLAF-CtoI nESCs in a 24-well AggreWell culture plate, it was found that 5iLAF-CtoI nESCs could efficiently and spontaneously assemble into blastoid structures regardless of the starting cell number (as shown in Figure 10).

[0194] The human blastoids structures obtained in the 24-well low-adhesion suspension culture plate were identified by embryo-specific markers using immunofluorescence staining. The specific identification steps were the same as those in Example 1.

[0195] The primary antibodies used for the markers NANOG and KRT18 were Nanog (1E6C4) Mouse mAb (1:1000 dilution) from Cell Signaling Technologies and Anti-Keratin 18 antibody produced in rabbit (1:200 dilution) from Sigma, respectively.

[0196] The results showed that the blastoids formed by 5iLAF-CtoI nESCs in 5iLAF medium also expressed the human real embryonic markers SOX2, NANOG, GATA6, KRT18 and GATA3 (as shown in Figures 11A, 11B and 12), indicating that it can also serve as an effective mimic of human blastocyst structure.

[0197] Comparative Example 2

[0198] The inventors referred to the operation of Comparative Example 1 and, on the basis of Example 2, did not use 5iLAF medium in step (3) but replaced it with 5iLAF-C medium for subsequent culture.

[0199] As shown in Figures 13A and 13B, it can be found that the results of Comparative Example 2 are similar to those of Comparative Example 1. TJ-1#nESCs that have not been transformed or cultured in 5iLAF-C medium cannot effectively assemble human blastoids structures, and the 5iLAF-CtoI nESCs obtained after transformation cannot be assembled into any blastoids structures in 5iLAF-C medium.

[0200] The above results indicate that the key to the successful construction of the blastoids structure in the present invention, which can serve as an effective mimetic of the human blastocyst structure, is the successive culture of 5iLAF-C and 5iLAF, the spontaneous assembly of blastoids by 5iLAF-CtoI nESCs after GSK3 factor replacement, and the use of 5iLAF culture medium.

[0201] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for self-assembly to construct blastocyst-like cells, comprising the following steps: (1) Using a medium containing GSK3 inhibitor A to induce stem cells pluripotent stem cells; (2) transferring the cells to a culture medium containing GSK3 inhibitor B for transformation culture to obtain blastocyst-like structures; The GSK3 inhibitor A is different from the GSK3 inhibitor B.

2. The method according to claim 1, characterized in that The step (2) comprises transferring the cells to a culture medium containing GSK3 inhibitor B for transformation culture, picking cell clones and then continuing to culture them in a culture medium containing GSK3 inhibitor B to obtain blastocyst-like structures; or Cell clones were picked to establish cell lines and cultured in a medium containing GSK3 inhibitor B to obtain blastocyst-like structures.

3. The method according to claim 1, characterized in that The stem cells are selected from primed stem cells; Preferably, the primed stem cells are selected from human or non-human mammalian sources; Preferably, the primed stem cells are selected from primed embryonic stem cells and primed induced pluripotent stem cells.

4. The method according to claim 1, wherein The GSK3 inhibitor A and the GSK3 inhibitor B are selected from one or more of CHIR99021, IM-12, SB216763, AT7519, CHIR-98014, TWS119, Tideglusib, SB415286, 6-bromoindirubin-3-oxime, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, WAY-119064, PF-04802367, GSK-3βinhibitor 1, KY19382, BRD0705, Alsterpaullone, BIO-acetoxime, 1-Azakenpaullone, Indirubin, Indirubin-3'-oxime, Resibufogenin, 9-ing-41, 5-Bromoindole, CP21R7, or Bikinin; Preferably, the GSK3 inhibitor A is selected from CHIR99021, and the GSK3 inhibitor B is selected from IM-12.

5. The method according to claim 3, characterized in that The concentration of the GSK3 inhibitor A in the culture medium is 0.5-1.5 μM; and / or the concentration of the GSK3 inhibitor B in the culture medium is 0.5-1.5 μM.

6. The method according to claim 1, wherein The culture medium includes DMEM / F-12 and Neurobasal as basic culture medium, supplemented with N2, B27 and other auxiliary components and induction factors; Preferably, the other auxiliary ingredients are selected from at least one of KnockOut SR, GlutaMAX, non-essential amino acids, trace elements, 2-mercaptoethanol and bovine serum albumin; Preferably, the induction factor is selected from at least one of a MEK / ERK inhibitor, a BRAF inhibitor, a SRC inhibitor, a ROCK inhibitor, a leukemia inhibitory factor, activin A and a fibroblast growth factor.

7. The method according to claim 1 or 2, characterized in that The induction culture time in step (1) is 9-13 days; the transformation culture time in step (2) is 4-11 days; Preferably, the conversion culture comprises transferring the cells to a culture medium containing GSK3 inhibitor B for conversion culture for 2-6 days, picking cell clones and then continuing to culture them in the culture medium containing GSK3 inhibitor B for 2-5 days.

8. The method according to any one of claims 1 to 7, characterized in that Step (1) includes the steps of inoculating stem cells onto feeder cells and inducing culture using a culture medium containing GSK3 inhibitor A; and / or A step of dissociating stem cells is also included between steps (1) and (2).

9. The method according to claim 8, characterized in that The dissociation includes dissociating the stem cells using an enzyme or non-enzymatic digestion solution; Preferably, the enzyme is selected from Accutase, Dispase, collagenase or trypsin; Preferably, the non-enzymatic digestion solution is selected from EDTA, NO-ZYME reagent, and Non-enzymatic digestion solution.

10. The blastocyst-like embryo constructed by the method according to any one of claims 1 to 9; Preferably, the blastocyst-like cells express markers of human true embryos, namely SOX2, NANOG, GATA6, KRT18 and GATA3.

11. Use of the blastocyst-like cell of claim 10 in preparing an artificial embryo-like mimic.

12. The use according to claim 11, characterized in that The embryo-like artificial mimic comprises: (1) Artificial simulation of animal embryo blastocyst; (2) Animal embryo in vitro cell model.

13. A product, characterized in that The product includes a culture medium containing GSK3 inhibitor A and a culture medium containing GSK3 inhibitor B; the product is used to prepare blastocyst-like cells; Preferably, at least one of the GSK3 inhibitor A, the GSK3 inhibitor B, the stem cells, and the culture medium is as defined in any one of claims 3 to 6; Preferably, the product includes a culture reagent and a culture kit.

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