Human mammary gland organoid derived from pluripotent stem cells and construction method of human mammary gland organoid

Abstract

According to the pluripotent stem cell-derived human mammary gland organoid and the construction method thereof, the human mammary gland organoid is obtained through the following multi-stage induction culture: first-stage culture: culturing for 1-3 days by using a first culture medium to obtain a surface ectoderm stage organoid expressing a surface ectoderm marker K8 / K18; second-stage culture: culturing for 1-3 days by using a second culture medium to obtain a mammary gland substrate-like organ which has bright epithelium and expresses mammary gland substrate markers EDAR and LEF1; third-stage culture: culturing for 3-5 days by using a third culture medium; fourth-stage culture: culturing for 3-5 days by using a fourth culture medium; a fifth culture medium is used for culture for 8-12 days to form an epithelial structure with basal cells and lumen cells, a mammary gland physiological microenvironment simulated by mesenchymal cells and vascular endothelial cells and a mature mammary gland organ with a lactation function. According to the invention, the mature mammary gland organ with a lactation function is successfully constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid construction, and specifically relates to a human breast organoid derived from pluripotent stem cells and a construction method thereof. Background Art

[0002] The mammary gland is one of the important organs of female mammals, and its main function is to secrete milk to feed offspring. Therefore, the mammary gland of mammals is crucial to the reproduction and survival of the entire species. There are certain differences between the mammary glands of different species, and the composition of milk is also different. Unlike other tissues and organs, mammary tissue has the characteristics of postnatal development, which also makes it a good model for studying the development of adult stem cells and tissues and organs. In recent years, organoid models have become an important tool for studying tissue development, disease model construction, and drug screening. Organoids are tissue-like models formed by using stem cells or specific tissue progenitor cells in three-dimensional culture to simulate the in vivo growth microenvironment, and then using specific growth factors, culture media and other bioactive substances in vitro to guide the self-organization and differentiation of cells. Organoid models can simulate the structure and function of tissues and organs to a large extent. In particular, with the development of organoid technology derived from pluripotent stem cells, many human organ models that are difficult to obtain tissue samples have been successfully constructed, such as brain, heart, kidney, etc., but the method for constructing human mammary organoid models is immature, especially the lack of embryonic mammary models. The differentiation pathways of pluripotent stem cells into organoids of different tissues mostly simulate the development process of their respective organs. Since the development processes of different types of organs are quite different, it is difficult to directly draw on the schemes of other types of tissue organoids to prepare human breast organoids.

[0003] Although the current mouse mammary organoid model, including mammary organoids derived from adult stem cells and pluripotent stem cells, has relatively mature construction methods, there are certain differences in the mammary development process and structure between humans and other species such as mice. For example, the lobule structure is the basic structural unit of the human mammary gland and the main location of breast cancer, but the mouse mammary gland does not contain this structure. In addition, the signal pathway regulation during the development of human mammary tissue is also significantly different from that in other species such as mice. Therefore, the mouse mammary organoid model cannot help us study the unique development process of the human mammary gland, especially the prenatal development process of the mammary gland and the occurrence of breast cancer. The construction of functional human mammary organoids has important significance and application value.

[0004] There are roughly three cell sources for the construction of human breast organoids reported so far, including immortalized cell lines, tissue somatic stem cells, and pluripotent stem cells. Currently, most of the breast organoids derived from immortalized cell lines are constructed based on the MCF10A cell line. These organoids mainly include the epithelial structure of the mammary gland and are quite different from real breast tissue. However, breast organoids derived from tissue somatic stem cells often face problems of sample acquisition and ethical restrictions. At present, although breast organoids induced from pluripotent stem cells can differentiate into basal cells and luminal cells, they have not yet formed the classic breast morphology, are not mature enough, and cannot fully simulate real breast tissue in structure and function. Moreover, their differentiation process cannot simulate the development process from the surface ectoderm, to the mammary placode, to the mammary lineage and mammary tissue, and staged organoids cannot be obtained. In addition, the development process, function and even disease formation of the mammary gland are inseparable from the epithelial-mesenchymal interaction. However, the currently constructed human mammary organoids fail to contain microenvironment cells, or rely on external introduction of adult or engineered mesenchymal cell lines, which is difficult to apply to the study of complex epithelial-mesenchymal interaction mechanisms in the mammary gland life cycle, limiting the application of these models in the study of mammary gland development and disease mechanisms. Therefore, new human mammary organoid models are still needed to solve these problems. Summary of the invention

[0005] The first object of the present invention is to provide a human breast organoid derived from pluripotent stem cells to address the problems in the prior art.

[0006] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions: A human mammary organoid derived from pluripotent stem cells, wherein the human mammary organoid is obtained by the following multi-stage induction culture: In the first stage, a first culture medium is used to induce pluripotent stem cells to differentiate into a surface ectoderm lineage, wherein the first culture medium includes a growth factor of the TGF-β superfamily and a TGF-β signaling regulator; In the second stage, a second culture medium is used to drive the surface ectoderm to differentiate into a mammary placode, wherein the second culture medium includes a Wnt signaling regulator; In the third stage, a third culture medium is used to simulate the embryonic mammary bud microenvironment to promote the production of mammary epithelial progenitor cell markers and mesenchymal cells, wherein the third culture medium includes growth factors of the TGF-β superfamily and paracrine signaling factors; In the fourth stage, a fourth culture medium is used to induce mammary epithelial lineage differentiation, and the fourth culture medium includes a Hedgehog signaling regulator; The fifth stage is cultured using the fifth culture medium to form an epithelial structure with basal cells and luminal cells, as well as a mammary physiological microenvironment simulated by mesenchymal cells and vascular endothelial cells, and mature mammary organoids with lactation function. The fifth culture medium includes a growth factor combination and metabolic regulatory factors that promote mammary epithelial morphogenesis.

[0007] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions: As a preferred technical solution of the present invention: before the first stage of culture, it also includes: cell aggregation culture: using cell aggregation medium to culture for 1-2 days to form cell aggregates, and the cell aggregation medium contains Essential 8 medium and 10 μM Y27632.

[0008] As a preferred technical solution of the present invention: the first culture medium includes MammoCult Human Medium Kit culture medium, 2% low growth factor matrix gel, BMP4 and SB431542, and the MammoCult Human Medium Kit culture medium is supplemented with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone; the concentration of BMP4 in the first culture medium is 2.5-10.0 ng / mL, and the concentration of SB431542 is 4-12 μM; In the first stage, the first culture medium is used for 1-3 days to obtain surface ectoderm stage organoids expressing surface ectoderm markers K8 / K18.

[0009] As a preferred technical solution of the present invention: the first stage and the second stage also include a transition culture stage: using P1 culture medium for 2-3 days, the P1 culture medium contains MammoCult Human Medium Kit culture medium and VEGFα, the MammoCult Human Medium Kit culture medium is supplemented with 4μg / mL heparin sodium and 0.48μg / mL hydrocortisone, and the concentration of VEGFα is 25-30 ng / mL.

[0010] As a preferred technical solution of the present invention: the second culture medium comprises MammoCult Human Medium Kit culture medium and CHIR99021, the MammoCult Human Medium Kit culture medium is supplemented with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone, and the concentration of CHIR99021 is 8-12 μM; In the second stage, the second culture medium is used for 1-3 days to obtain mammary placode-like organoids with bright epithelium and expression of mammary placode markers EDAR and LEF1.

[0011] As a preferred technical solution of the present invention: the second stage adopts the air-liquid interface culture method, and the mixed gel prepared with matrix gel and collagen I-C in a 1:1 ratio of the first stage organoids is resuspended, and then inoculated into a Transwell chamber pre-coated with the same mixed gel, and culture medium is added to the lower layer to form an air-liquid interface.

[0012] As a preferred technical solution of the present invention: the third culture medium contains MammoCult Human Medium Kit culture medium, BMP4 and PTHrP, the concentration of BMP4 is 37.5-75 ng / mL, the concentration of PTHrP is 100-200 ng / mL, and the third culture medium is used for culturing for 3-5 days in the third stage to obtain mammary placode-like organoids with bright epithelium and expressing mammary placode markers EDAR and LEF1.

[0013] As a preferred technical solution of the present invention: the fourth culture medium comprises EpiCult-B Human Medium Kit culture medium and SANT-1, the concentration of SANT-1 is 250 nM, and the fourth stage uses the fourth culture medium for 3-5 days.

[0014] As a preferred technical solution of the present invention: the fifth culture medium comprises EpiCult-B Human Medium Kit culture medium, FGF2, FGF10, HGF, EGF and Insulin.

[0015] The second object of the present invention is to provide a method for constructing human breast organoids derived from pluripotent stem cells in response to the problems in the prior art.

[0016] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions: A method for constructing human mammary organoids derived from pluripotent stem cells comprises the following steps: Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Inoculate the above single cells into an ultra-low adsorption well plate and culture them in a cell aggregation medium for 1-2 days to obtain cell aggregates; Step 3: Replace the cell aggregation medium with the first medium and culture for 1-3 days to obtain the first stage organoids; Step 4: Collect the first-stage organoids obtained in step 3 and continue to culture them using the air-liquid surface method. At the same time, replace the first culture medium with the second culture medium and culture for 1-3 days to obtain the second-stage organoids. Step 5: Replace the second culture medium with the third culture medium and culture for 3-5 days; Step 6: Replace the third culture medium with the fourth culture medium and culture for 3-5 days; Step 7: Replace the fourth culture medium with the fifth culture medium and culture for 8-12 days to obtain mammary organoids.

[0017] According to step three of the present invention, SB431542 is added to the first culture medium to promote the differentiation of pluripotent stem cells into ectoderm, and BMP4 is added to the first culture medium to promote the differentiation of pluripotent stem cells into surface ectoderm, avoiding the differentiation of pluripotent stem cells into neural ectoderm, so as to obtain the first germ layer origin of mammary gland development, namely, surface ectoderm.

[0018] According to step 4 of the present invention, the second culture medium is added with Wnt signal activator CHIR90021 to activate Wnt signal, activate the expression of key regulatory genes EDAR and LEF1 in the formation of mammary placode, and promote the differentiation of surface ectoderm into mammary placode cells.

[0019] According to step 4 of the present invention, an air-liquid surface method is used to promote the differentiation of surface ectoderm cells into mammary placode cells.

[0020] According to step five of the present invention, BMP4 and PTHrP are added to the third culture medium to activate BMP and PTH / PTHrP, the key regulatory pathways for the formation of mammary placode, thereby promoting the differentiation of surface ectoderm cells into mammary placode cells and avoiding the differentiation of surface ectoderm cells into epidermal cells.

[0021] According to step 4 and step 5 of the present invention, CHIR90021 and BMP4 act together to induce epithelial-mesenchymal transition and promote the production of mesenchymal cells.

[0022] According to step six of the present invention, the fourth culture medium uses EpiCult-B Human Medium Kit culture medium and adds SANT-1 to inhibit the activation of the hedgehog signaling pathway in the formation of mammary lineage, promote the formation of mammary lineage cells, and avoid the differentiation of placode cells into non-mammary lineage cells such as hair follicles and sweat glands.

[0023] According to step seven of the present invention, the fifth culture medium is added with FGF2, FGF10, HGF, EGF and Insulin to promote the generation of mammary gland branches and the maturation of mesenchymal cells.

[0024] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions: As a preferred technical solution of the present invention: in the step 2, the ultra-low adsorption well plate is selected from a 384-well plate, a 96-well plate, a 48-well plate, a 12-well plate or a 6-well plate; Or, the number of single cells seeded in the ultra-low attachment 6-well plate in step 2 is 2.0×10 5 -5.0×10 5The diameter of the obtained cell aggregates was 60-200 μm.

[0025] The third object of the present invention is to provide a mammary organoid to address the problems in the prior art.

[0026] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions: The mammary organoid is a second-stage organoid, which is a mammary placode organoid, has bright placode epithelium, expresses mammary placode markers EDAR and LEF1, and includes surrounding mesenchyme.

[0027] Compared with the prior art, the present invention provides a human mammary organoid derived from pluripotent stem cells and a construction method thereof. The human mammary organoid derived from pluripotent stem cells of the present invention completely simulates the human mammary embryonic process for the first time. Mammary development originates from the surface ectoderm, and goes through the formation stage of the mammary placode, a key structure of mammary development, mammary lineage orientation, branching structure formation, and then to the mature mammary formation stage. The human mammary organoid formed by the precise induction of five stages has the structural advantages of branching structure, basal / epithelial cell stratification and microenvironment cell components; it has the functional advantages of lactation function and the ability to simulate physiological and pathological processes; it solves the problem of shortage of human embryonic mammary research models, overcomes the species differences between mouse models and humans, and provides the application advantages of a dynamic development research platform.

[0028] The human mammary organoids derived from pluripotent stem cells and the construction method of the present invention obtain human mammary organoids at different stages of differentiation, provide new tools for mammary development research and disease mechanism analysis, provide an ideal model for drug screening and regenerative medicine, provide a new idea for the directed differentiation of human pluripotent stem cells into mammary organoids, and realize the in vitro reconstruction of the entire process of human mammary tissue development for the first time, filling the technical gap in this field and having great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A flowchart of the method for constructing human breast organoids derived from pluripotent stem cells of the present invention; Figure 2 The morphological diagrams at different differentiation time points of the method for constructing human mammary organoids derived from pluripotent stem cells of the present invention; Figure 3 The immunofluorescence identification results of organoids at different time stages in Example 1; Figure 4 The results of immunofluorescence identification of organoids at different time stages in Example 2; Figure 5 The results of immunofluorescence and real-time fluorescence quantitative PCR in Example 3, wherein Figure A is the immunofluorescence result and Figure B is the fluorescence quantitative PCR result; Figure 6 This is a bright field morphological image of the organoid in Example 4; Figure 7 The results of real-time fluorescence quantitative PCR identification of organoids in Example 5; Figure 8 To illustrate the bright field morphology and immunofluorescence identification results of mammary organoids in Example 6, Figure A is a bright field morphology image of mammary organoids, and Figure B is an immunofluorescence identification image; Fig. 9 The results of immunofluorescence identification of mammary microenvironment cells in Example 6; Fig.10 The results of immunofluorescence identification of the lactation function of mammary organoids in Example 6; Fig.11 The growth morphology and immunofluorescence identification results of human breast organoids obtained in Comparative Example 1, Figure A is a bright field morphology image of breast organoids, and Figure B is an immunofluorescence identification image; Fig.12 The growth morphology and immunofluorescence identification results of the human mammary organoids obtained in Comparative Example 2 are shown in Figure A, which is a bright field morphology image of the mammary organoids, and Figure B, which is an immunofluorescence identification image. DETAILED DESCRIPTION

[0030] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0031] A human mammary organoid derived from pluripotent stem cells, obtained through the following multi-stage induction culture: The first stage of culture: using the first culture medium to culture for 1-3 days to obtain surface ectoderm stage organoids expressing surface ectoderm markers K8 / K18, wherein the first culture medium contains MammoCult Human Medium Kit culture medium, 2% low growth factor matrix gel, 2.5-10.0 ng / mL BMP4 and 4-12 μM SB431542; Second stage culture: Use the second culture medium to culture for 1-3 days to obtain mammary placode-like organoids with bright epithelium and expression of mammary placode markers EDAR and LEF1. The second culture medium contains MammoCult Human Medium Kit culture medium and 8-12 μM CHIR99021; The third stage of culture: culture for 3-5 days using the third culture medium, wherein the third culture medium comprises MammoCultHuman Medium Kit culture medium, 37.5-75 ng / mL BMP4 and 100-200 ng / mL PTHrP; Stage 4 culture: culture for 3-5 days using a fourth culture medium, wherein the fourth culture medium comprises EpiCult-BHuman Medium Kit culture medium and 250 nM SANT-1; Stage 5 culture: Use the fifth culture medium to culture for 8-12 days to form an epithelial structure with basal cells and luminal cells, as well as a mammary physiological microenvironment simulated by mesenchymal cells and vascular endothelial cells, and mature mammary organoids with lactation function. The fifth culture medium contains EpiCult-B Human Medium Kit culture medium, FGF2, FGF10, HGF, EGF and Insulin.

[0032] Before the first stage of culture, the method further includes: cell aggregation culture: culturing with a cell aggregation medium for 1-2 days to form cell aggregates, wherein the cell aggregation medium comprises Essential 8 medium and 10 μM Y27632.

[0033] The first stage and the second stage also include: a transition culture stage: using P1 culture medium for 2-3 days, and the P1 culture medium contains MammoCult Human Medium Kit culture medium and 25-30 ng / mL VEGFα.

[0034] The MammoCult Human Medium Kit and EpiCult-B Human Medium Kit were both supplemented with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone.

[0035] The second stage of culture uses an air-liquid interface culture method: the first stage organoids are resuspended in a mixed gel prepared with matrix gel and collagen I-C in a 1:1 ratio, and then inoculated into a Transwell chamber pre-coated with the same mixed gel, and culture medium is added to the lower layer to form an air-liquid interface.

[0036] The present invention provides a mammary organoid.

[0037] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions: The mammary organoid is a second-stage organoid, which is a mammary placode organoid, has bright placode epithelium, expresses mammary placode markers EDAR and LEF1, and includes surrounding mesenchyme.

[0038] like Figure 1-Figure 2As shown, a method for constructing human mammary organoids derived from pluripotent stem cells of the present invention, the mammary organoids prepared by the method undergo multiple differentiation stages, and finally obtain a mammary organoid model with morphology and function similar to human mammary tissue, and also contain microenvironment cells related to mammary tissue. By this method, mammary organoids with a microenvironment can be obtained, which is expected to solve the problem of the current lack of stromal cells in human mammary organoids. By this method, organoids at various stages of different differentiation states can be obtained, providing a new idea for the directed differentiation of human pluripotent stem cells into mammary organoids.

[0039] Specific embodiments of the present invention include: Step 1: Digest the adherent cultured pluripotent stem cells into single cells to ensure uniform distribution of cells to facilitate the subsequent formation of cell aggregates; Specifically, when the confluence of adherent pluripotent stem cells reached 80%-90%, ACCUTASE enzyme was added for digestion at 37°C for 5 min, and the digested single cells were collected into a 15 mL centrifuge tube and centrifuged at 1000 r / min for 3 min. The supernatant was discarded, and the cells were resuspended in 2 mL of Essential 8 medium and counted.

[0040] Step 2: Inoculate the above single cells into an ultra-low adsorption well plate and culture them in a cell aggregation medium for 1-2 days to form uniform cell aggregates; Specifically, take 2.0×10 single cells from step 1. 5 -5.0×10 5 Transfer the cells to a new 15 mL centrifuge tube, add 2 mL of Essential 8 medium, and centrifuge at 1000 r / min for 3 min. Discard the supernatant, add 2 mL of cell aggregation medium to resuspend the cells, and spread the resuspended cell suspension into new wells of an ultra-low adsorption 6-well plate. Culture for 1-2 days to obtain cell aggregates. The cell aggregation medium is specifically Essential 8 medium, including 10 μM Y27632.

[0041] Step 3: Replace the cell aggregation medium with the first medium, culture for 1-3 days, induce the cells to differentiate into the surface ectoderm, and obtain the first stage organoids; Specifically, collect the cell aggregates obtained in step 2 into a new 15 mL centrifuge tube, centrifuge at 500 r / min for 1 min, discard the supernatant, add 2 ml of the first culture medium to resuspend the cell aggregates, and spread the resuspended cell aggregates into new wells of an ultra-low adsorption 6-well plate, culture for 1-3 days, and obtain the first stage organoids. The first culture medium is specifically MammoCult HumanMedium Kit culture medium, including 2% low growth factor matrix gel, 2.5-10.0 ng / mL BMP4 and 4-12 μM SB431542; Optional step: Replace the cell aggregation medium with P1 medium and culture for 2-3 days: Specifically, the first stage organoids obtained in step 3 were collected into a new 15 mL centrifuge tube, centrifuged at 300 r / min for 1 min, the supernatant was discarded, 2 ml of the second culture medium was added to resuspend the cell aggregates, and the collected first stage organoids were resuspended with a mixed gel of low growth factor matrix gel and collagen I, wherein the mixed gel ratio was 1:1, and then the resuspended organoids were spread into the Transwell cell culture chamber; wherein the Transwell cell culture chamber was pre-coated with the same mixed gel, and the gel had solidified. Then it was placed in a 37 ℃ incubator and allowed to stand for 30 min to allow the gel to solidify. After the gel solidified, the second culture medium was added to the lower layer of the Transwell cell culture chamber, ensuring that the liquid surface of the culture medium was in the lower gel area and did not reach the upper gel position to form a gas-liquid interface. The culture time was 2-3 days. The P1 culture medium was specifically the MammoCult HumanMedium Kit culture medium and 25-30 ng / mL VEGFα.

[0042] MammoCult Human Medium Kit is a culture medium launched by STEMCELL, with the catalog number 05622.

[0043] Step 4: Collect the first-stage organoids obtained in step 3 and continue to culture them using the air-liquid surface method. At the same time, replace the first culture medium with the second culture medium for 1-3 days to promote the branching structure and functional maturation of the organoids. Specifically, the first stage organoids obtained in step 3 were collected into a new 15 mL centrifuge tube, centrifuged at 300 r / min for 1 min, the supernatant was discarded, 2 ml of the second culture medium was added to resuspend the cell aggregates, and the collected first stage organoids were resuspended with a mixed gel of low growth factor matrix gel and collagen I, wherein the mixed gel ratio was 1:1, and then the resuspended organoids were spread into the Transwell cell culture chamber; wherein the Transwell cell culture chamber was pre-coated with the same mixed gel, and the gel had solidified. Then it was placed in a 37 °C incubator and allowed to stand for 30 min to allow the gel to solidify. After the gel solidified, the second culture medium was added to the lower layer of the Transwell cell culture chamber, ensuring that the liquid surface of the culture medium was in the lower gel area and did not reach the upper gel position to form a gas-liquid interface. The culture time was 1-3 days. The second culture medium was specifically the MammoCult HumanMedium Kit culture medium, including 8-12 μM CHIR99021.

[0044] Step 5: Replace the second culture medium with the third culture medium and culture for 3-5 days to promote the formation of mammary placode structure and functional maturation; Specifically, the second culture medium in the lower layer of the Transwell cell culture chamber is discarded, replaced with the third culture medium, and cultured for 3-5 days to obtain the second stage organoid. The third culture medium is specifically the MammoCult Human Medium Kit culture medium, including 37.5-75 ng / mL BMP4 and 100-200 ng / mL PTHrP.

[0045] Step 6: Replace the third culture medium with the fourth culture medium and culture for 3-5 days to promote the differentiation of mammary lineage cells; Specifically, the third culture medium at the bottom layer of the Transwell cell culture chamber is discarded and replaced with the fourth culture medium for 3-5 days. The fourth culture medium is specifically EpiCult-B Human Medium Kit culture medium including 250 nM SANT-1.

[0046] Step 7: Replace the fourth culture medium with the fifth culture medium and culture for 8-12 days to obtain mammary organoids.

[0047] Specifically, the fourth culture medium in the lower layer of the Transwell cell culture chamber was discarded and replaced with the fifth culture medium, and cultured for 3-5 days, and finally mammary organoids with epithelial and surrounding stromal cells were obtained, and the branching structure of the mammary organoids was clearly visible. The fifth culture medium is specifically EpiCult-B Human Medium Kit culture medium, including 25-30 ng / mL FGF2, 25-30 ng / mL FGF10, 25-30 ng / mL HGF, 25-30 ng / mL EGF and 10 μg / mL Insulin.

[0048] EpiCult-B Human Medium Kit is a culture medium launched by STEMCELL, with the catalog number 05602.

[0049] According to step three of the present invention, the pluripotent stem cell aggregates are finally successfully formed into surface ectoderm organoids through the combined culture treatment of MammoCult culture medium, 2% low growth factor matrix gel, BMP4 and SB431542 in the first culture medium.

[0050] According to the present invention, through the combined culture treatment of the second culture medium and the third culture medium, the surface ectoderm obtained in step three is differentiated into the mammary placode, and finally a mammary placode-like organoid having both epithelium and mesenchyme and expressing the mammary placode markers EDAR, p63 and LEF1 is obtained.

[0051] According to the present invention, through the combined culture treatment of MammoCult culture medium, CHIR99021, BMP4 and PTHrP, the surface ectoderm obtained in step three is differentiated into mammary placode, and finally a mammary placode-like organoid having both epithelium and mesenchyme and expressing mammary placode markers EDAR, p63 and LEF1 is obtained.

[0052] According to the present invention, through continuous combined culture treatment of the second culture medium, the third culture medium and the fourth culture medium, the surface ectoderm obtained in step three enters the mammary gland fate, and finally obtains mammary gland lineage cells, including luminal cells and basal cells.

[0053] According to the present invention, through continuous combined culture treatment of MammoCult medium, EpiCult-B medium, CHIR99021, BMP4, PTHrP and SANT-1, the surface ectoderm obtained in step three enters the mammary fate, and finally obtains mammary lineage cells, including luminal cells and basal cells.

[0054] According to the present invention, the second-stage mammary placode-like organoid obtained in step 5 is further differentiated to form mammary ductal cells and basal cells through the continuous culture treatment with the third culture medium and the fourth culture medium.

[0055] According to the present invention, the combined culture treatment of EpiCult-B culture medium and SANT-1 in the fourth culture medium ultimately allows mammary organoids to differentiate into mammary ductal cells and basal cells.

[0056] According to the present invention, through the continuous combined culture treatment of the second culture medium, the third culture medium, the fourth culture medium and the fifth culture medium, the surface ectoderm obtained in step three finally forms a mature mammary organoid, which has a cell arrangement and branching structure similar to the real mammary epithelium in vivo, as well as microenvironment cell components, including vascular endothelial cells and mesenchymal cells.

[0057] According to the present invention, through the consecutive culture treatments of the fourth culture medium and the fifth culture medium, the breast lineage cells continue to grow, proliferate, and arrange, and finally form a cell arrangement and branching structure similar to the real breast epithelium in vivo, as well as microenvironment cell components, including vascular endothelial cells and mesenchymal cells.

[0058] According to the present invention, mature mammary organoids with branched structures and vascular endothelial cell microenvironment cells are obtained by combined culture treatment of EpiCult-B medium, FGF2, FGF10, HGF, EGF and Insulin in the fifth culture medium.

[0059] According to the present invention, through the continuous combined culture treatment of the first, second, third, fourth and fifth culture media, the pluripotent stem cell aggregates are finally formed into mammary organoids, which have epithelial branching structures and microenvironment cell components similar to those of real mammary glands in vivo, including vascular endothelial cells and mesenchymal cells.

[0060] In the present invention, by using different culture media and growth factors in stages, the development process of the mammary gland is simulated, and by the staged differentiation of pluripotent stem cells, it is ensured that the cells gradually differentiate into mammary epithelial cells and mesenchymal cells to form a complex mammary structure; by gas-liquid interface culture, the branching structure and functional maturation of organoids are promoted; an optimized combination of culture media is provided, and the growth factors and small molecule compounds in the culture media are optimized according to the needs of different differentiation stages to ensure that cells can obtain appropriate signal stimulation in each step of differentiation. The mammary organoids obtained by the method of the present invention have mammary epithelial cells and microenvironment cells at the same time, and the organoids have ductal branching structures and lactation functions, similar to real mammary glands, and the entire differentiation and development process of organoids from stem cells to the formation of mammary organoids can be observed, and organoids at different differentiation stages can also be obtained. The mammary organoids obtained by the method can be used as a good in vitro model for studying the mechanism of mammary development and mammary disease, especially when it comes to embryonic mammary glands, the model has unique advantages.

[0061] The present invention provides a human mammary organoid derived from pluripotent stem cells and a method for constructing the same, including different stages of human mammary development. Currently, most of the insights into mammary embryonic development come from model organisms such as mice, whose mammary development process and mammary structure and function are different from those of humans. The organoid obtained by the present invention can track various stages of human mammary development, and provide an excellent model for detailed analysis of the key signals or regulatory mechanisms of human mammary prenatal development, while helping to analyze the pathogenesis and drug research of congenital mammary dysplasia. The present invention provides a method for constructing a human mammary organoid derived from pluripotent stem cells, and without the addition of additional stromal cells, stromal cells such as mesenchymal and vascular endothelial cells other than mammary epithelium can be directly obtained. These stromal cells are produced along with mammary epithelium, and no additional steps need to be introduced, which is simpler. At the same time, these stromal cells are similar to the specific microenvironment cells of mammary tissue in vivo. Example 1

[0062] like Figure 3 As shown, the method for constructing human breast organoids derived from pluripotent stem cells of the present invention comprises the following steps: Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Take 2.5×10 single cells from step 1 5 The cells were inoculated into ultra-low attachment 6-well plates and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0063] Step 3: Replace the cell aggregation medium with the first medium, culture for 3 days, and obtain the first stage organoids. In this embodiment, the first medium is the MammoCult Human Medium Kit medium, including 2% low growth factor matrix gel, 2.5 ng / mL BMP4 and 10 μM SB431542; Step 4: Collect the first stage organoids obtained in step 3 and continue to culture them by changing to the air-liquid surface method, while replacing the first culture medium with the second culture medium for 3 days. In this embodiment, the second culture medium is MammoCult Human Medium Kit culture medium, including 10 μM CHIR99021.

[0064] Step 5: Replace the second culture medium with the third culture medium, culture for 3 days, and obtain the second stage organoids. In this embodiment, the third culture medium is the MammoCult Human Medium Kit culture medium, which includes 75 ng / mL BMP4 and 200 ng / mL PTHrP.

[0065] Step 6: Replace the third culture medium with the fourth culture medium and culture for 3 days. In this embodiment, the fourth culture medium is EpiCult-B Human Medium Kit culture medium, including 250 nM SANT-1.

[0066] Step 7: Replace the fourth culture medium with the fifth culture medium, culture for 8 days, and obtain mammary organoids. In this embodiment, the fifth culture medium is EpiCult-B Human Medium Kit culture medium, including 25 ng / mL FGF2, 25 ng / mL FGF10, 25 ng / mL HGF, 25 ng / mL EGF and 10 μg / mL Insulin. Example 2

[0067] like Figure 4 As shown, the method for constructing human breast organoids derived from pluripotent stem cells of the present invention comprises the following steps: Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Take 2.5×10 single cells from step 1 5 The cells were inoculated into ultra-low attachment 6-well plates and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0068] Step 3: Replace the cell aggregation medium with the first medium, culture for 3 days, and obtain the first stage organoids. In this embodiment, the first medium is the MammoCult Human Medium Kit medium, including 2% low growth factor matrix gel, 2.5 ng / mL BMP4 and 10 μM SB431542; Step 4: Collect the first stage organoids obtained in step 3 and continue to culture them by changing to the air-liquid surface method, and replace the first culture medium with P1 culture medium for 3 days. In this embodiment, P1 culture medium is MammoCult Human Medium Kit culture medium, including 30 ng / mL VEGFα.

[0069] Step 5: Replace the P1 medium with the second medium and culture for 3 days. In this embodiment, the second medium is the MammoCult Human Medium Kit medium, including 10 μM CHIR99021.

[0070] Step 6: Replace the second culture medium with the third culture medium, culture for 3 days, and obtain the second stage organoids. In this embodiment, the third culture medium is the MammoCult Human Medium Kit culture medium, which includes 75 ng / mL BMP4 and 200 ng / mL PTHrP.

[0071] Step 7: Replace the third culture medium with the fourth culture medium and culture for 3 days. In this embodiment, the fourth culture medium is EpiCult-B Human Medium Kit culture medium, including 250 nM SANT-1.

[0072] Step 8: Replace the fourth culture medium with the fifth culture medium, and culture for 12 days to obtain mammary organoids. In this embodiment, the fifth culture medium is EpiCult-B Human Medium Kit culture medium, including 25 ng / mL FGF2, 25 ng / mL FGF10, 25 ng / mL HGF, 25 ng / mL EGF and 10 μg / mL Insulin. Example 3

[0073] Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Take 2.5×10 single cells from step 1 5 The cells were inoculated into ultra-low attachment 6-well plates and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0074] Step 3: Replace the cell aggregation medium with the first medium, culture for 3 days, and obtain the first stage organoids. In this embodiment, the first medium is the MammoCult Human Medium Kit medium, including 2% low growth factor matrix gel, 10μM SB431542 and 2.5-10.0 ng / mL BMP4, wherein the concentration of BMP4 is 2.5 ng / mL, 5.0ng / mL, 7.5 ng / mL, 10.0 ng / mL. The first stage organoids obtained are identified by immunofluorescence and real-time fluorescence quantitative PCR experiments, such as Figure 5 As shown, the results showed that surface ectoderm expressing K8 and K18 could be formed under different BMP4 concentrations. Example 4

[0075] Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Take 2.5×10 single cells from step 1 5 The cells were inoculated into ultra-low attachment 6-well plates and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0076] Step 3: Replace the cell aggregation medium with the first medium, culture for 3 days, and obtain the first stage organoid. In this embodiment, the first medium is MammoCult Human Medium Kit medium, including 2% low growth factor matrix gel, 2.5 ng / mL BMP4 and 4-12 μM SB431542, wherein the concentration of SB431542 is 4 μM, 8 μM, and 12 μM.

[0077] Step 4: Collect the first stage organoids obtained in step 3 and continue to culture them using the air-liquid surface method. At the same time, replace the first culture medium with the second culture medium for 3 days. In this embodiment, the second culture medium is MammoCult Human Medium Kit culture medium, including 10 μM CHIR99021. Figure 6 As shown, different SB431542 concentrations can form epithelial organoids after step 4. Example 5

[0078] Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Take 2.5×10 single cells from step 1 5 The cells were inoculated into ultra-low attachment 6-well plates and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0079] Step 3: Replace the cell aggregation medium with the first medium, culture for 3 days, and obtain the first stage organoids. In this embodiment, the first medium is the MammoCult Human Medium Kit medium, including 2% low growth factor matrix gel, 2.5 ng / mL BMP4 and 10 μM SB431542.

[0080] Step 4: Collect the first stage organoids obtained in step 3 and continue to culture them by gas-liquid surface method, and replace the first culture medium with the second culture medium for 3 days. In this embodiment, the second culture medium is MammoCult Human MediumKit culture medium, including 8-12 μM CHIR99021. The concentration of CHIR9921 is 8 μM, 10 μM, and 12 μM. The obtained organoids were identified by real-time fluorescence quantitative PCR experiment, and the results showed that Figure 7 As shown, different CHIR99021 concentrations can cause organoids to express mammary placode-related genes. Example 6

[0081] like Figure 8 As shown, the method for constructing human breast organoids derived from pluripotent stem cells of the present invention comprises the following steps: Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Take 2.5×10 single cells from step 1 5 The cells were inoculated into ultra-low attachment 6-well plates and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0082] Step 3: Replace the cell aggregation medium with the first medium, culture for 3 days, and obtain the first stage organoids. In this embodiment, the first medium is the MammoCult Human Medium Kit medium, including 2% low growth factor matrix gel, 2.5 ng / mL BMP4 and 10 μM SB431542; Step 4: Collect the first stage organoids obtained in step 3 and continue to culture them by changing to the air-liquid surface method, while replacing the first culture medium with the second culture medium for 3 days. In this embodiment, the second culture medium is MammoCult Human Medium Kit culture medium, including 10 μM CHIR99021.

[0083] Step 5: Replace the second culture medium with the third culture medium, culture for 3 days, and obtain the second stage organoids. In this embodiment, the third culture medium is the MammoCult Human Medium Kit culture medium, which includes 37.5 ng / mL BMP4 and 100 ng / mL PTHrP.

[0084] Step 6: Replace the third culture medium with the fourth culture medium and culture for 3 days. In this embodiment, the fourth culture medium is EpiCult-B Human Medium Kit culture medium, including 250 nM SANT-1.

[0085] Step 7: Replace the fourth culture medium with the fifth culture medium, culture for 8 days, and obtain mammary organoids. In this embodiment, the fifth culture medium is EpiCult-B Human Medium Kit culture medium, including 30 ng / mL FGF2, 30 ng / mL FGF10, 30 ng / mL HGF, 30 ng / mL EGF and 10 μg / mL Insulin.

[0086] The obtained mammary organoids were identified by immunofluorescence staining. Fig. 9 As shown, the human breast organoids derived from pluripotent stem cells of the present invention have two microenvironment cells: mesenchymal cells and vascular endothelial cells; Fig.10 As shown, the human mammary organoids derived from pluripotent stem cells of the present invention have basic lactation function and can secrete Milk protein and β-casein.

[0087] Comparative Example 1 This comparative example provides a method for preparing normal human breast organoids, which differs from the method in Example 1 in that an embedding method culture step is used in step 4 and subsequent steps. The specific method is as follows: Step 1 to step 3: Same as Example 1 Step 4: Wrap a first-stage organoid with 30 μL of low-growth factor matrix gel and collagen I mixed gel, where the mixed gel ratio is 1:1, and drip the gel wrapped with the first-stage organoid into a 24-well plate without tissue treatment to form a gel ball. Three gel balls are dripped into each well, and the gel balls do not touch each other. After dripping the third gel ball, turn the well plate upside down and let it stand for 5 minutes, and then put it in a 37°C incubator for 30 minutes to solidify the gel. After the gel solidifies, add the second culture medium along the wall of the well plate and culture for 3 days. The second culture medium in this embodiment is the same as that in Example 1.

[0088] Step 5 to step 7: same as in Example 1.

[0089] like Fig.11 As shown, the mammary organoids obtained in this comparative example had no obvious epithelial structure and no branching structure. Immunofluorescence identification was performed, and the results showed that the mammary organoids obtained in this comparative example had no basal cells and luminal cells, indicating that this method cannot successfully prepare mammary organoids.

[0090] Comparative Example 2 This comparative example provides a method for preparing normal human breast organoids, which is different from the method in Example 1 in that step 6 is different. The specific method is as follows: Step 1 to step 5: Same as Example 1 Step 6: Replace the fourth medium with MammoCult Human Medium Kit containing 250 nM SANT-1 and culture for 3 days.

[0091] Step 7: Same as Example 1.

[0092] like Fig.12 As shown, no vacuolar epithelial structure was observed in the mammary organoids obtained in this comparative example. Immunofluorescence identification showed that the mammary organoids obtained in this comparative example had almost no basal cells and luminal cells, indicating that this method cannot successfully prepare mammary organoids.

[0093] Experimental Example 1 In this experimental example, frozen sections and immunofluorescence staining were performed on the organoids at various stages prepared in the various embodiments and comparative examples, and the organoids were identified based on the frozen sections and immunofluorescence staining. The specific methods and results are described as follows.

[0094] (1) Use tweezers to peel off the polycarbonate membrane at the bottom of the chamber, and then use a blade or syringe needle to cut the mixed gel containing the organoid culture along the edge of the chamber and collect the sample into a 2 mL microcentrifuge tube.

[0095] (2) Fix the organoid cultures by immersing the samples in 4% paraformaldehyde fixation buffer overnight.

[0096] (3) Gently rinse the samples with PBS for 5 minutes each time, three times.

[0097] (4) Aspirate the supernatant, embed the sample with OCT embedding medium, and perform cryosectioning with a slice thickness of 7-10 µm.

[0098] (5) Place the sections at room temperature for 15 minutes to ensure that the samples have thawed. Permeabilize the sections with 100-200 µL of 0.5% PBST solution at room temperature for 10 minutes. Then, soak and wash the sections with 0.2% PBST solution for 10 minutes.

[0099] (6) Aspirate the supernatant and add 100-200 µL of blocking solution. Block at room temperature for 2 hours.

[0100] (7) After blocking, remove the supernatant, add 150 μL of prepared primary antibody, and incubate overnight in a 4°C refrigerator.

[0101] (8) After the primary antibody incubation is completed, wash the sample three times with 0.2% PBST solution for 10 minutes each time. After washing, aspirate the supernatant, add 100-150 μL of the prepared secondary antibody, and incubate at room temperature in the dark for 2 hours.

[0102] (9) After the secondary antibody incubation is completed, use 0.2% PBST solution to soak and wash 3 times, 10 minutes each time. After washing, remove the supernatant as much as possible, draw 20 μL of DAPI-containing mounting medium and drop it on the sample to ensure that the sample is completely covered, then carefully cover the slide on the mounting medium, and seal the slide at room temperature for more than 30 minutes before preparing for photography.

[0103] The immunofluorescence staining markers used in the above method are as follows: stage 1: K8 / K18 marks surface ectoderm cells; stage 2: EpCAM represents epithelial cells, EDAR represents mammary placode cells, and LEF1 represents mammary placode cells and mesenchymal cells around the placode; stage 3: K14 represents mammary basal cells, K8 represents mammary luminal cells, p63 represents mammary basal cells, CD31 represents vascular endothelial cells, VIM represents mesenchymal cells, β-casein represents β-casein, and Milk represents milk protein; Experimental Example 2 In this experimental example, RNA extraction and real-time fluorescence quantitative PCR experiments were performed on the organoids at various stages prepared in various embodiments and comparative examples, and the organoids were identified based on this. The specific methods and results are described as follows.

[0104] (1) Transfer the cultured organoids into a 15 ml centrifuge tube, add fresh PBS until the tube is full, and then centrifuge for 5 minutes at 1000 r / min. (2) Discard the supernatant, add 1 ml of Trizol, and pipette repeatedly to break up the sample; (3) Add 200 μl of chloroform, shake for 15 s, and leave at room temperature for 5 min; (4) Centrifuge at 12,000 g for 15 min at 4 °C; (5) Transfer the supernatant to a new 1.5 ml centrifuge tube without enzyme; add an equal volume of isopropanol, mix by inverting, and let stand at room temperature for 10 min; (6) Centrifuge at 4°C for 10 min at 12,000 g; (7) Discard the supernatant, slowly add 1 ml of 75% ethanol solution along the wall of the centrifuge tube, and wash by inverting the tube; (8) Centrifuge at 4°C for 5 min at 12,000 g; (9) Discard the supernatant and dry at room temperature for 5 min; (10) Add enzyme-free water to dissolve the precipitate, add reverse transcription reagent; incubate at 37°C for 15 min, then incubate at 85°C for 5 s to obtain sample cDNA; (11) The obtained cDNA was used as a template, the primers of the target gene were added, and the real-time quantitative fluorescence kit and real-time fluorescence quantitative PCR instrument were used for detection.

[0105] The mammary organoids prepared by the present invention have a branched structure, basal cells and epithelial cells that can simulate the complex morphology of the mammary gland in vivo, better simulate the physiological and pathological processes of the mammary gland; can simulate the physiological functions of the mammary gland, have a lactation function, and provide a more realistic model for mammary gland physiology and pathology in application. The human mammary organoids constructed by the present invention using pluripotent stem cells have morphological and structural microenvironment cells similar to those of the mammary gland in vivo, have higher tissue complexity, and can simulate the interaction between the mammary epithelium and the microenvironment during development.

[0106] The construction route of the mammary organoids of the present invention follows the principle of organogenesis, and its differentiation process is similar to the in vivo mammary development process, and also goes through the stages of surface ectoderm, mammary placode, mammary epithelial lineage, etc., which can fully simulate the entire development process of the mammary gland, and is expected to solve the current shortage of in vitro models of human embryonic mammary glands. The present invention can obtain organoids at different stages of differentiation, which can help study the mechanisms of mammary gland development and its lesions at different stages, and provide a dynamic model for mammary gland development and disease research. The present invention provides a new idea for the directed differentiation of human pluripotent stem cells into mammary organoids.

[0107] The resulting mammary organoids have basic lactation functions. Although a large number of studies have revealed the mechanisms of mammary gland development and its pathological changes, most of these studies are based on mouse models, and there are still certain differences between the mammary glands of mice and humans, which makes these conclusions not fully applicable to humans. Research on the mechanisms of human mammary gland development and its pathological changes is still lacking, especially for the embryonic development of the mammary gland. The mammary organoids derived from pluripotent stem cells constructed in this study can be used as a good in vitro model to assist in the study of human mammary gland development and pathological changes. Especially in the study of embryonic mammary glands, this model has its unique advantages. At the same time, this study also provides new guidance for the subsequent construction of mammary organoids.

[0108] The present invention provides a human mammary organoid derived from pluripotent stem cells and a construction method thereof, which simulates the development process of the mammary gland through staged culture and a specific culture medium combination to generate mammary organoids with complex structure and function. The constructed mammary organoids have higher tissue complexity, can better simulate the morphological structure and microenvironment of the mammary gland in vivo, and are suitable for studying mammary gland development, disease mechanisms and drug screening; the mammary gland development process is completely simulated, from the surface ectoderm to the mammary epithelial lineage, to solve the problem of shortage of in vitro models of the mammary gland in the embryonic period, and to provide an important tool for mammary gland development research; organoids at various stages of different differentiation time periods can be obtained, which helps to study the mechanisms of mammary gland development and its lesions at different stages, provides a dynamic model for mammary gland development and disease research, and helps to reveal the pathogenesis of breast diseases; the mammary organoids have a lactation function, can simulate the physiological functions of the mammary gland, and are suitable for studying the physiological functions of the mammary gland and their regulatory mechanisms. The present invention successfully constructed mammary organoids with complex structure and function through phased culture and optimized culture medium combination, combined with the gas-liquid surface method. It can simulate the development process, physiological function and pathological mechanism of the mammary gland, and provide an important tool for mammary gland development research, disease model construction and drug screening, and has great application prospects.

[0109] The above-mentioned specific implementation methods are used to explain the present invention and are only preferred embodiments of the present invention, rather than limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A human breast organoid derived from pluripotent stem cells, characterized in that: The human mammary organoids are obtained by the following multi-stage induction culture: In the first stage, a first culture medium is used to induce pluripotent stem cells to differentiate into a surface ectoderm lineage, wherein the first culture medium includes a growth factor of the TGF-β superfamily and a TGF-β signaling regulator; In the second stage, a second culture medium is used to drive the surface ectoderm to differentiate into a mammary placode, wherein the second culture medium includes a Wnt signaling regulator; In the third stage, a third culture medium is used to simulate the embryonic mammary bud microenvironment to promote the production of mammary epithelial progenitor cell markers and mesenchymal cells, wherein the third culture medium includes growth factors of the TGF-β superfamily and paracrine signaling factors; The fourth stage uses a fourth culture medium to induce mammary epithelial lineage differentiation, and the fourth culture medium includes a Hedgehog signaling regulator; In the fifth stage, a fifth culture medium is used to culture the epithelial structure having basal cells and luminal cells, as well as a mammary physiological microenvironment simulated by mesenchymal cells and vascular endothelial cells, and mature mammary organoids with lactation function. The fifth culture medium includes a growth factor combination and metabolic regulatory factors that promote mammary epithelial morphogenesis.

2. The human breast organoid derived from pluripotent stem cells according to claim 1, characterized in that: Before the first stage of culture, the method further includes: cell aggregation culture: culturing with a cell aggregation medium for 1-2 days to form cell aggregates, wherein the cell aggregation medium comprises Essential 8 medium and 10 μM Y27632.

3. The human breast organoid derived from pluripotent stem cells according to claim 1, characterized in that: The first culture medium includes MammoCult Human Medium Kit culture medium, 2% low growth factor matrix gel, BMP4 and SB431542, wherein the MammoCult Human Medium Kit culture medium is supplemented with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone; the concentration of BMP4 in the first culture medium is 2.5-10.0 ng / mL, and the concentration of SB431542 is 4-12 μM; In the first stage, the first culture medium is used for 1-3 days to obtain surface ectoderm stage organoids expressing surface ectoderm markers K8 / K18.

4. The human breast organoid derived from pluripotent stem cells according to claim 1, wherein: The first stage and the second stage also include a transition culture stage: culture for 2-3 days using P1 medium, wherein the P1 medium contains MammoCult Human Medium Kit medium and VEGFα, and the MammoCult Human Medium Kit medium is supplemented with 4 μg / mL sodium heparin and 0.48 μg / mL hydrocortisone, and the concentration of VEGFα is 25-30 ng / mL.

5. The human breast organoid derived from pluripotent stem cells according to claim 1, characterized in that: The second culture medium comprises MammoCult Human Medium Kit culture medium and CHIR99021, wherein the MammoCult Human Medium Kit culture medium is supplemented with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone, and the concentration of CHIR99021 is 8-12 μM; In the second stage, the second culture medium is used for 1-3 days to obtain mammary placode-like organoids with bright epithelium and expression of mammary placode markers EDAR and LEF1.

6. The human breast organoid derived from pluripotent stem cells according to claim 5, characterized in that: The second stage used the air-liquid interface culture method. The first stage organoids were resuspended in a mixed gel prepared with matrix gel and collagen I-C in a 1:1 ratio, and then inoculated into a Transwell chamber pre-coated with the same mixed gel. Culture medium was added to the lower layer to form an air-liquid interface.

7. The human breast organoid derived from pluripotent stem cells according to claim 1, characterized in that: The third culture medium comprises MammoCult Human Medium Kit culture medium, BMP4 and PTHrP, the concentration of BMP4 is 37.5-75 ng / mL, the concentration of PTHrP is 100-200 ng / mL, and the third culture medium is used for culturing for 3-5 days in the third stage to obtain mammary placode-like organoids with bright epithelium and expressing mammary placode markers EDAR and LEF1.

8. The human breast organoid derived from pluripotent stem cells according to claim 1, characterized in that: The fourth culture medium comprises EpiCult-B Human Medium Kit culture medium and SANT-1, the concentration of SANT-1 is 250 nM, and the fourth stage is cultured using the fourth culture medium for 3-5 days.

9. The human breast organoid derived from pluripotent stem cells according to claim 1, wherein: The fifth culture medium comprises EpiCult-B Human Medium Kit culture medium, FGF2, FGF10, HGF, EGF and Insulin.

10. The method for constructing human breast organoids derived from pluripotent stem cells according to any one of claims 1 to 9, characterized in that The following steps are involved: Step 1: Digest adherent cultured pluripotent stem cells into single cells; Step 2: Preparation of cell aggregates: Inoculate the single cells from step 1 into an ultra-low adsorption well plate, and culture them in a cell aggregation medium for 1-2 days to obtain cell aggregates; Step 3: Replace the cell aggregation medium with the first medium and culture for 1-3 days to obtain the first stage organoids; Step 4: Collect the first-stage organoids obtained in step 3 and continue to culture them using the air-liquid surface method. At the same time, replace the first culture medium with the second culture medium for 1-3 days. Step 5: Replace the second culture medium with the third culture medium and culture for 3-5 days to obtain the second stage organoids; Step 6: Replace the third culture medium with the fourth culture medium and culture for 3-5 days; Step 7: Replace the fourth culture medium with the fifth culture medium and culture for 8-12 days to obtain mammary organoids.

11. The method for constructing human breast organoids derived from pluripotent stem cells according to claim 10, characterized in that: The pluripotent stem cells in step 1 are human pluripotent stem cells, including human embryonic stem cells.

12. The method for constructing human breast organoids derived from pluripotent stem cells according to claim 10, characterized in that: In the step 2, the ultra-low adsorption well plate is selected from a 384-well plate, a 96-well plate, a 48-well plate, a 12-well plate or a 6-well plate; Or, the number of single cells seeded in the ultra-low attachment 6-well plate in step 2 is 2.0×10 5 -5.0×10 5 The diameter of the obtained cell aggregates was 60-200 μm.

13. A mammary organoid constructed by the method for constructing a human mammary organoid derived from pluripotent stem cells according to any one of claims 10 to 12, characterized in that: The mammary organoid is a second-stage organoid, which is a mammary placode organoid, has bright placode epithelium, expresses mammary placode markers EDAR and LEF1, and includes surrounding mesenchymal cells.

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