A pluripotent stem cell-derived human mammary organoid and methods of constructing the same

Abstract

The application discloses a human mammary gland organoid derived from pluripotent stem cells and a construction method thereof, and the 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 surface ectoderm markers K8 / K18; second stage culture: culturing for 1-3 days by using a second culture medium to obtain a mammary gland basement membrane-like organoid having a bright epithelium and expressing mammary gland basement membrane 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; and fifth stage culture: culturing for 8-12 days by using a fifth culture medium to form an epithelial structure having basal cells and luminal cells, a mammary gland physiological microenvironment simulated by mesenchymal cells and vascular endothelial cells and a mature mammary gland organoid having a lactation function. The application successfully constructs the mature mammary gland organoid having the lactation function.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organoid construction, and particularly relates to a human mammary gland organoid derived from pluripotent stem cells and a construction method thereof. BACKGROUND

[0002] The mammary gland is one of the important organs of female mammals, and its main function is to secrete milk to nurse offspring. Therefore, the mammary gland of mammals is crucial for the reproduction and survival of the entire species, and the mammary glands of different species have certain differences, and the milk components are also different. Unlike other tissues and organs, the mammary gland tissue has the characteristics of postnatal development, which also makes it a good model for studying adult stem cells and the development of tissues and organs. In recent years, organoid models have become an important tool for studying tissue development, disease model construction, and drug screening. An organoid is a tissue-like model formed by using stem cells or specific tissue progenitor cells to simulate the in vivo growth microenvironment in a three-dimensional culture, and then using specific growth factors, culture medium and other bioactive substances to guide the self-organization and differentiation of cells. The organoid model can simulate the structure and function of the tissue and organ to a large extent, especially with the development of pluripotent stem cell-derived organoid technology, many human organ models that are difficult to obtain tissue samples have been successfully constructed, such as brain, heart, kidney, etc., but the construction method of human mammary gland organoid is not mature, especially lacking an embryonic mammary gland model. Differentiation pathways of pluripotent stem cells into different tissue organoids are mostly simulated organ development processes, and since the development processes of different types of organs are quite different, it is difficult to directly use the schemes of other types of tissue organoids to prepare human mammary gland organoids.

[0003] Although the construction method of mouse mammary gland organoid models, including mammary gland organoids derived from adult stem cells and pluripotent stem cells, is relatively mature, the development process and structure of human and mouse mammary glands and other species have certain differences, for example, the lobular structure is the basic structural unit of human mammary glands and the main location of breast cancer occurrence, but the mouse mammary gland does not contain this structure, in addition, the signal pathway regulation in the development process of human mammary gland tissue is also significantly different from that in mice and other species, therefore, the mouse mammary gland organoid model cannot well help us study the unique development process of human mammary glands, especially the prenatal development process of mammary glands, and the occurrence of breast cancer, and the construction of functional human mammary gland organoids has important significance and application value.

[0004] Currently, there are three kinds of cell sources for the construction of human mammary gland organoids, including immortalized cell lines, tissue somatic stem cells and pluripotent stem cells. Most of the mammary gland organoids derived from immortalized cell lines are constructed based on MCF10A cell lines, and these organoids mainly include the epithelial structure of the mammary gland, and are quite different from the real mammary gland tissue. The mammary gland organoids derived from tissue somatic stem cells often face the problems of sample acquisition and ethical restrictions. At present, the mammary gland organoids induced from pluripotent stem cells can differentiate into basal cells and luminal cells, but they have not formed the classic mammary gland morphology, and the maturity is insufficient, and they cannot completely simulate the real mammary gland tissue in structure and function, and the differentiation process cannot simulate the development process from the surface ectoderm, to the mammary gland basement, to the mammary gland lineage and mammary gland tissue, and stage organoids cannot be obtained. In addition, the development process and function of the mammary gland cannot be separated from the epithelial-mesenchymal interaction, and the current constructed human mammary gland organoids cannot contain microenvironment cells, or rely on the external introduction of adult or engineered mesenchymal cell lines, which is difficult to apply to the research on the complex epithelial-mesenchymal interaction mechanism in the mammary gland life cycle, and limits the application of these models in the research on the development and disease mechanism of the mammary gland, so new human mammary gland organoid models are still needed to solve these problems. SUMMARY

[0005] The first object of the present application is to provide a human mammary gland organoid derived from pluripotent stem cells to solve the problems in the prior art.

[0006] To this end, the above object of the present application is achieved by the following technical solutions.

[0007] A human mammary gland organoid derived from pluripotent stem cells, wherein the human mammary gland organoid is obtained by the following multi-stage induction culture:

[0008] In the first stage, the pluripotent stem cells are directionally induced to differentiate into surface ectoderm lineage using a first culture medium, wherein the first culture medium comprises growth factors of the TGF-β superfamily and TGF-β signal regulatory factors;

[0009] In the second stage, the surface ectoderm is driven to differentiate into mammary gland basement using a second culture medium, wherein the second culture medium comprises Wnt signal regulatory factors;

[0010] In the third stage, the embryonic period mammary gland bud microenvironment is simulated using a third culture medium to promote the generation of mammary epithelial progenitor cell markers and mesenchymal cells, wherein the third culture medium comprises growth factors of the TGF-β superfamily and paracrine signal factors;

[0011] In the fourth stage, the mammary epithelial lineage is induced to differentiate using a fourth culture medium, wherein the fourth culture medium comprises Hedgehog signal regulatory factors;

[0012] The fifth stage uses a fifth culture medium to culture, form an epithelial structure with basal cells and luminal cells, and mesenchymal cells and vascular endothelial cells simulate the physiological microenvironment of the mammary gland, and have lactation function of mature mammary gland organoids, and the fifth culture medium comprises a growth factor combination and a metabolic regulatory factor that promote mammary epithelial morphogenesis.

[0013] While the above technical solutions are adopted, the application can also adopt or combine the following technical solutions:

[0014] As a preferred technical solution of the application: before the first stage culture, further comprising: cell aggregation culture: using cell aggregation culture medium to culture for 1-2 days to form cell aggregates, the cell aggregation culture medium comprises Essential 8 culture medium and 10 μM Y27632.

[0015] As a preferred technical solution of the application: the first culture medium comprises MammoCult Human Medium Kit culture medium, 2% low growth factor matrix glue, BMP4 and SB431542, the MammoCult Human Medium Kit culture medium is added 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.

[0016] The first stage uses a first culture medium to culture for 1-3 days, and obtains a surface ectoderm stage organoid expressing surface ectoderm markers K8 / K18.

[0017] As a preferred technical solution of the application: between the first stage and the second stage, further comprising a transition culture stage: using P1 culture medium to culture for 2-3 days, the P1 culture medium comprises MammoCult Human Medium Kit culture medium and VEGF alpha, the MammoCult Human Medium Kit culture medium is added with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone, and the concentration of VEGF alpha is 25-30 ng / mL.

[0018] As a preferred technical solution of the application: the second culture medium comprises MammoCult Human Medium Kit culture medium and CHIR99021, the MammoCult Human Medium Kit culture medium is added with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone, and the concentration of CHIR99021 is 8-12 μM.

[0019] The second stage uses a second culture medium to culture for 1-3 days to obtain a mammary basal plate-like organoid with a bright epithelium and expressing mammary basal plate markers EDAR and LEF1.

[0020] As a preferred technical solution of the present application: in the second stage, the air-liquid interface culture method is adopted, after the first stage organoid is resuspended in a mixed gel prepared by mixing Matrigel and collagen I-C at a ratio of 1:1, the organoid is inoculated into a Transwell chamber precoated with the same mixed gel, and a culture medium is added to the lower layer to form an air-liquid interface.

[0021] As a preferred technical solution of the present application: the third culture medium comprises a MammoCult Human Medium Kit culture medium, BMP4 and PTHrP, the concentration of BMP4 is 37.5-75 ng / mL, and the concentration of PTHrP is 100-200 ng / mL, the third stage uses the third culture medium to culture for 3-5 days to obtain a mammary basal plate-like organoid with a bright epithelium and expressing mammary basal plate markers EDAR and LEF1.

[0022] As a preferred technical solution of the present application: the fourth culture medium comprises an 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 to culture for 3-5 days.

[0023] As a preferred technical solution of the present application: the fifth culture medium comprises an EpiCult-B Human Medium Kit culture medium, FGF2, FGF10, HGF, EGF and Insulin.

[0024] The second object of the present application is to provide a method for constructing a human mammary gland organoid derived from pluripotent stem cells to solve the problems in the prior art.

[0025] To this end, the above object of the present application is achieved by the following technical solution:

[0026] A method for constructing a human mammary gland organoid derived from pluripotent stem cells, comprising the following steps:

[0027] Step one: digest the adherent cultured pluripotent stem cells into single cells;

[0028] Step two: inoculate the single cells into an ultra-low adsorption hole plate, and use a cell aggregation culture medium to culture for 1-2 days to obtain cell aggregates;

[0029] Step three: replace the cell aggregation culture medium with a first culture medium and culture for 1-3 days to obtain a first stage organoid;

[0030] Step four: continue to replace the first stage organoids obtained in step three with the air-liquid surface method, replace the first culture medium with the second culture medium, and continue to culture for 1-3 days to obtain second stage organoids;

[0031] Step five: replace the second culture medium with the third culture medium and culture for 3-5 days;

[0032] Step six: replace the third culture medium with the fourth culture medium and culture for 3-5 days;

[0033] Step seven: replace the fourth culture medium with the fifth culture medium and culture for 8-12 days to obtain mammary organoids.

[0034] According to the step three of the present application, the addition of SB431542 in the first culture medium promotes the differentiation of pluripotent stem cells into ectoderm, the addition of BMP4 in the first culture medium promotes the differentiation of pluripotent stem cells into surface ectoderm, and avoids the differentiation of pluripotent stem cells into neural ectoderm, so as to obtain the first step of embryonic layer origin of breast development, i.e. surface ectoderm.

[0035] According to the step four of the present application, the addition of Wnt signal activator CHIR90021 in the second culture medium activates Wnt signal, activates the expression of key regulatory genes such as EDAR and LEF1 of mammary basal plate formation, and promotes the differentiation of surface ectoderm into mammary basal plate cells.

[0036] According to the step four of the present application, the air-liquid surface method is used to promote the differentiation of surface ectoderm cells into mammary basal plate cells.

[0037] According to the step five of the present application, the addition of BMP4 and PTHrP in the third culture medium activates the key regulatory pathways of BMP and PTH / PTHrP of mammary basal plate formation, promotes the differentiation of surface ectoderm cells into mammary basal plate cells, and avoids the differentiation of surface ectoderm cells into epidermal cells.

[0038] According to the step four and step five of the present application, the combined action of CHIR90021 and BMP4 induces epithelial-mesenchymal transition and promotes the production of mesenchymal cells.

[0039] According to the step six of the present application, the fourth culture medium uses EpiCult-B Human Medium Kit culture medium and adds SANT-1, which inhibits the activation of the hedgehog signaling pathway in mammary lineage formation, promotes the formation of mammary lineage cells, and avoids the differentiation of basal plate cells into non-mammary lineage cells such as hair follicles and sweat glands.

[0040] According to the step seven of the present application, the fifth culture medium adds FGF2, FGF10, HGF, EGF and Insulin, which promotes the production of mammary branches and the maturation of mesenchymal cells.

[0041] In addition, the application can adopt or combine the following technical solutions:

[0042] As a preferred technical solution of the application: the ultra-low adsorption plate in step two is selected from 384-well plate, 96-well plate, 48-well plate, 12-well plate or 6-well plate;

[0043] Or, the number of single cells inoculated in the ultra-low adsorption 6-well plate in step two is 2.0*10 5 -5.0*10 5 The diameter of the obtained cell aggregate is 60-200 mu m.

[0044] A third object of the application is to provide a mammary gland organoid to solve the problems in the prior art.

[0045] To this end, the above objects of the application are achieved by the following technical solutions:

[0046] The mammary gland organoid is a second-stage organoid, which is a mammary gland basal plate organoid, has a bright basal plate epithelium, expresses mammary gland basal plate markers EDAR and LEF1, and includes surrounding mesenchyme.

[0047] Compared with the prior art, the pluripotent stem cell-derived human mammary gland organoid and the construction method thereof have the following advantages: the pluripotent stem cell-derived human mammary gland organoid first completely simulates the human mammary gland embryonic period, the mammary gland development originates from the surface ectoderm, and the mammary gland development goes through the formation stage of the mammary gland basal plate, the mammary gland lineage orientation, the formation of the branched structure, and the mature mammary gland formation stage, and the five-stage precise induction forms a human mammary gland organoid, which has the structural advantages of branched structure and basal / epithelial cell stratification and microenvironment cell components; has the functional advantages of lactation function and simulation of physiological and pathological processes; solves the problem of shortage of human embryonic mammary gland research models, overcomes the species difference between mice and humans, and provides application advantages of a dynamic development research platform.

[0048] The pluripotent stem cell-derived human mammary gland organoid and the construction method thereof obtain mammary gland organoids at different differentiation stages, provide a new tool for mammary gland development research and disease mechanism analysis, provide an ideal model for drug screening and regenerative medicine, provide a new idea for the directional differentiation of human pluripotent stem cells into mammary gland organoids, first realize the in-vitro reconstruction of the whole process of human mammary gland tissue development, fill the technical gap in this field, and have great application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 The flowchart of the construction method of the pluripotent stem cell-derived human mammary gland organoid;

[0050] Figure 2Morphology of different differentiation time points of the method for constructing human mammary gland organoids derived from pluripotent stem cells of the present application;

[0051] Figure 3 Results of immunofluorescence identification of organoids at different time stages in Example 1;

[0052] Figure 4 Results of immunofluorescence identification of organoids at different time stages in Example 2;

[0053] Figure 5 Results of immunofluorescence and real-time fluorescence quantitative PCR identification in Example 3, wherein Figure A is the result of immunofluorescence identification, and Figure B is the result of fluorescence quantitative PCR identification;

[0054] Figure 6 Bright field morphology of organoids in Example 4;

[0055] Figure 7 Results of real-time fluorescence quantitative PCR identification of organoids in Example 5;

[0056] Figure 8 Bright field morphology and immunofluorescence identification results of mammary gland organoids in Example 6, Figure A is the bright field morphology of mammary gland organoids, and Figure B is the immunofluorescence identification chart;

[0057] Figure 9 Results of immunofluorescence identification of mammary gland microenvironment cells in Example 6;

[0058] Figure 10 Results of immunofluorescence identification of lactation function of mammary gland organoids in Example 6;

[0059] Figure 11 Growth morphology and immunofluorescence identification results of human mammary gland organoids obtained in Comparative Example 1, Figure A is the bright field morphology of mammary gland organoids, and Figure B is the immunofluorescence identification chart;

[0060] Figure 12 Growth morphology and immunofluorescence identification results of human mammary gland organoids obtained in Comparative Example 2, Figure A is the bright field morphology of mammary gland organoids, and Figure B is the immunofluorescence identification chart. DETAILED DESCRIPTION

[0061] The present application will be further described in detail with reference to the accompanying drawings and specific examples.

[0062] A human mammary gland organoid derived from pluripotent stem cells is obtained by the following multi-stage induction culture:

[0063] First stage culture: culture for 1-3 days using a first medium comprising MammoCult Human Medium Kit medium, 2% low growth factor Matrigel, 2.5-10.0 ng / mL BMP4, and 4-12 μΜ SB431542 to obtain surface ectoderm stage organoids expressing surface ectoderm markers K8 / K18;

[0064] Second stage culture: culture for 1-3 days using a second medium comprising MammoCult Human Medium Kit medium and 8-12 μΜ CHIR99021 to obtain mammary basement layer-like organoids with a bright epithelium and expressing mammary basement layer markers EDAR and LEF1;

[0065] Third stage culture: culture for 3-5 days using a third medium comprising MammoCult Human Medium Kit medium, 37.5-75 ng / mL BMP4, and 100-200 ng / mL PTHrP;

[0066] Fourth stage culture: culture for 3-5 days using a fourth medium comprising EpiCult-B Human Medium Kit medium and 250 nM SANT-1;

[0067] Fifth stage culture: culture for 8-12 days using a fifth medium comprising EpiCult-B Human Medium Kit medium, FGF2, FGF10, HGF, EGF, and Insulin to form mature mammary organoids with epithelial structures of basal and luminal cells, and a physiological microenvironment of mammary gland mimicked by mesenchymal and vascular endothelial cells, and lactation function.

[0068] Further comprising, before the first stage culture: cell aggregation culture: culture for 1-2 days using a cell aggregation medium comprising Essential 8 medium and 10 μΜ Y27632 to form cell aggregates.

[0069] Further comprising, between the first stage and the second stage: a transition culture stage: culture for 2-3 days using a P1 medium comprising MammoCult Human Medium Kit medium and 25-30 ng / mL VEGFa.

[0070] Both the MammoCult Human Medium Kit culture medium and the EpiCult-B Human Medium Kit culture medium are added with 4 μg / mL of sodium heparin and 0.48 μg / mL of hydrocortisone.

[0071] The second-stage culture adopts a gas-liquid interface culture method: after the first-stage organoids are resuspended in a mixed gel prepared by mixing Matrigel and collagen I-C at a ratio of 1:1, the organoids are inoculated into a Transwell chamber precoated with the same mixed gel, and a culture medium is added to the lower layer to form a gas-liquid interface.

[0072] The present application provides a mammary gland organoid.

[0073] To this end, the above object of the present application is achieved by the following technical solutions.

[0074] The mammary gland organoid is a second-stage organoid, which is a mammary gland basal organoid, has a bright basal epithelium, expresses mammary gland basal markers EDAR and LEF1, and simultaneously includes surrounding mesenchyme.

[0075] As shown in Figures 1-2 The method for constructing a human mammary gland organoid derived from pluripotent stem cells provided by the present application, the mammary gland organoid prepared by the method undergoes multiple differentiation stages, and finally a mammary gland organoid model similar in morphology and function to human mammary gland tissue is obtained, which simultaneously contains microenvironment cells related to mammary gland tissue. The mammary gland organoid with a microenvironment can be obtained by the method, which is expected to solve the problem that the current human mammary gland organoid lacks mesenchymal cells. The organoids at different stages of differentiation can be obtained by the method, which provides a new idea for the directional differentiation of human pluripotent stem cells into mammary gland organoids.

[0076] The specific embodiments of the present application include:

[0077] Step one: digest the adherent cultured pluripotent stem cells into single cells to ensure uniform distribution of the cells for subsequent formation of cell aggregates;

[0078] Specifically, when the confluence of the adherent cultured pluripotent stem cells reaches 80%-90%, ACCUTASE enzyme is added for digestion at 37 ℃ for 5 min, the single cells after digestion are collected into a 15 mL centrifuge tube, centrifuged at 1000 r / min for 3 min, the supernatant is discarded, 2 mL of Essential 8 culture medium is used to resuspend the cells, and the cells are counted.

[0079] Step two: inoculate the single cells into an ultralow adsorption well plate, use cell aggregation culture medium to culture for 1-2 days to form uniform cell aggregates;

[0080] Specifically, 2.0×105 -5.0 x 10 5 The cell aggregates obtained in step two were collected into a new 15 mL centrifuge tube, and 2 mL of Essential 8 medium was added. The mixture was centrifuged at 1000 r / min for 3 min. The supernatant was discarded, and 2 mL of cell aggregation medium was added to resuspend the cells. The resuspended cell suspension was then plated into a new well of the ultra-low attachment 6-well plate, and cultured for 1-2 days to obtain cell aggregates. The cell aggregation medium was Essential 8 medium including 10 μM Y27632.

[0081] Step three: the cell aggregation medium was replaced with the first medium, and the cells were cultured for 1-3 days to induce differentiation of the cells into surface ectoderm to obtain first-stage organoids.

[0082] Specifically, the cell aggregates obtained in step two were collected into a new 15 mL centrifuge tube, centrifuged at 500 r / min for 1 min, and the supernatant was discarded. 2 mL of the first medium was added to resuspend the cell aggregates, and the resuspended cell aggregates were plated into a new well of the ultra-low attachment 6-well plate, and cultured for 1-3 days to obtain first-stage organoids. The first medium was MammoCult Human Medium Kit medium including 2% low growth factor Matrigel, 2.5-10.0 ng / mL BMP4, and 4-12 μM SB431542.

[0083] Optional step: the cell aggregation medium was replaced with P1 medium, and the cells were cultured for 2-3 days.

[0084] Specifically, the first-stage organoids obtained in step three were collected into a new 15 mL centrifuge tube, centrifuged at 300 r / min for 1 min, and the supernatant was discarded. 2 mL of the second medium was added to resuspend the collected first-stage organoids, and the resuspended first-stage organoids were plated into a Transwell cell culture chamber. The Transwell cell culture chamber was pre-coated with a mixed gel of low growth factor Matrigel and collagen I at a ratio of 1:1, and the gel had been solidified. Then, the mixture was placed in a 37 °C incubator for 30 min to allow the gel to solidify. After the gel solidified, the second medium was added to the lower layer of the Transwell cell culture chamber to ensure that the liquid level of the medium was in the lower gel area and did not reach the position of the upper gel, forming an air-liquid interface. The culture time was 2-3 days. The P1 medium was MammoCult Human Medium Kit medium and 25-30 ng / mL VEGFα.

[0085] MammoCult Human Medium Kit medium is a medium launched by STEMCELL Company, with product number 05622.

[0086] Step four: continue to culture the first-stage organoids obtained in step three by replacing the air-liquid interface method with a liquid-liquid interface method, and replace the first medium with a second medium, and culture for 1-3 days to promote the branching structure and functional maturation of the organoids;

[0087] Specifically, the first-stage organoids obtained in step three are collected into a new 15 mL centrifuge tube, centrifuged at 300 r / min for 1 min, the supernatant is discarded, 2 ml of the second medium is added to resuspend the cell aggregate, the collected first-stage organoids are resuspended with a mixed gel of low growth factor Matrigel and collagen I, wherein the ratio of the mixed gel is 1:1, and then the resuspended organoids are plated into a Transwell cell culture chamber; wherein the Transwell cell culture chamber is pre-coated with the same mixed gel, and the gel has been solidified. Then place it in a 37 ℃ incubator for 30 min to allow the gel to solidify. After the gel solidifies, add the second medium to the lower layer of the Transwell cell culture chamber to ensure that the liquid level of the medium is in the lower gel area and does not reach the position of the upper gel, forming an air-liquid interface. The culture time is 1-3 days. The second medium is specifically MammoCult Human Medium Kit medium, which includes 8-12 μM CHIR99021.

[0088] Step five: replace the second medium with a third medium, and culture for 3-5 days to promote the formation of mammary basal structure and functional maturation;

[0089] Specifically, the second medium in the lower layer of the Transwell cell culture chamber is discarded and replaced with a third medium, and cultured for 3-5 days to obtain a second-stage organoid. The third medium is specifically MammoCult Human Medium Kit medium, which includes 37.5-75 ng / mL BMP4, 100-200 ng / mL PTHrP.

[0090] Step six: replace the third medium with a fourth medium, and culture for 3-5 days to promote mammary lineage cell differentiation;

[0091] Specifically, the third medium in the lower layer of the Transwell cell culture chamber is discarded and replaced with a fourth medium, and cultured for 3-5 days. The fourth medium is specifically EpiCult-B Human Medium Kit medium, which includes 250 nM SANT-1.

[0092] Step seven: replace the fourth medium with a fifth medium, and culture for 8-12 days to obtain a mammary organoid.

[0093] Specifically, the fourth culture medium in the lower layer of the Transwell cell culture chamber is discarded, and the fifth culture medium is replaced, and cultured for 3-5 days, so as to finally obtain a mammary gland organoid with epithelial and surrounding interstitial cells, and the branch structure of the mammary gland organoid is clear. The fifth culture medium is specifically EpiCult-B Human Medium Kit culture medium, which includes 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.

[0094] The EpiCult-B Human Medium Kit culture medium is a culture medium launched by STEMCELL Company, with a product number of 05602.

[0095] According to the third step of the present application, the pluripotent stem cell aggregate is successfully formed into a surface ectoderm organoid through the combined culture treatment of the first culture medium, the MammoCult culture medium, 2% low growth factor Matrigel, BMP4 and SB431542.

[0096] According to the present application, the surface ectoderm obtained in step three is differentiated into a mammary gland substrate through the combined culture treatment of the second culture medium and the third culture medium, and finally a mammary gland substrate-like organoid is obtained, which has epithelial and mesenchymal cells, and simultaneously expresses mammary gland substrate markers EDAR, p63 and LEF1.

[0097] According to the present application, the surface ectoderm obtained in step three is differentiated into a mammary gland substrate through the combined culture treatment of the second culture medium and the third culture medium, and finally a mammary gland substrate-like organoid is obtained, which has epithelial and mesenchymal cells, and simultaneously expresses mammary gland substrate markers EDAR, p63 and LEF1.

[0098] According to the present application, the surface ectoderm obtained in step three is differentiated into a mammary gland substrate through the combined culture treatment of the second culture medium and the third culture medium, and finally a mammary gland substrate-like organoid is obtained, which has epithelial and mesenchymal cells, and simultaneously expresses mammary gland substrate markers EDAR, p63 and LEF1.

[0099] According to the present application, the surface ectoderm obtained in step three is differentiated into a mammary gland substrate through the combined culture treatment of the second culture medium and the third culture medium, and finally a mammary gland substrate-like organoid is obtained, which has epithelial and mesenchymal cells, and simultaneously expresses mammary gland substrate markers EDAR, p63 and LEF1.

[0100] According to the present application, the second stage mammary substrate-like organoids obtained in step five are allowed to continue differentiation to form mammary luminal cells and basal cells by sequential culture treatment with the third and fourth culture media.

[0101] According to the present application, the mammary organoids are finally allowed to differentiate to produce mammary luminal cells and basal cells by culture treatment with a combination of EpiCult-B medium and SANT-1 in the fourth culture medium.

[0102] According to the present application, the surface ectoderm obtained in step three is finally allowed to form mature mammary organoids with similar epithelial branching structure and microenvironmental cell components, including vascular endothelial cells and mesenchymal cells, by sequential combination culture treatment with the second, third, fourth and fifth culture media.

[0103] According to the present application, the mammary lineage cells are allowed to continue growth and proliferation and to arrange by sequential culture treatment with the fourth and fifth culture media, and finally form mature mammary organoids with similar epithelial branching structure and microenvironmental cell components, including vascular endothelial cells and mesenchymal cells, to the real mammary epithelium in vivo.

[0104] According to the present application, the mature mammary organoids with branching structure and vascular endothelial cell microenvironmental cells are obtained by combination culture treatment with EpiCult-B medium, FGF2, FGF10, HGF, EGF and Insulin in the fifth culture medium.

[0105] According to the present application, the pluripotent stem cell aggregates are finally allowed to form mammary organoids with similar epithelial branching structure and microenvironmental cell components, including vascular endothelial cells and mesenchymal cells, to the real mammary gland in vivo by sequential combination culture treatment with the first, second, third, fourth and fifth culture media.

[0106] In the present application, by using different culture media and growth factors in stages, the development process of mammary gland is simulated, and by the staged differentiation of pluripotent stem cells, the cells are ensured to be gradually differentiated into mammary epithelial cells and interstitial cells to form complex mammary structures; by air-liquid interface culture, the branched structure and functional maturation of the organoids are promoted; and an optimized combination of culture media is provided, and according to the needs of different differentiation stages, the growth factors and small molecule compounds in the culture medium are optimized to ensure that the cells can obtain appropriate signal stimulation in each step of differentiation. The mammary gland organoids obtained by the method of the present application have both mammary epithelial cells and microenvironment cells, and the organoids have duct branching structure and lactation function, which are similar to the real mammary gland, and the whole differentiation and development process of the organoids from stem cells to mammary gland organoids can be observed, and organoids at different differentiation stages can also be obtained. The mammary gland organoids obtained by the method can be used as a good in vitro model for studying the mechanism of mammary gland development and mammary gland diseases, especially when the embryonic mammary gland is involved, the model has unique advantages.

[0107] The pluripotent stem cell-derived human mammary gland organoid and the construction method thereof of the present application contain different periods of human mammary gland development. Most of the current insights into embryonic development of mammary gland are derived from model organisms such as mice, and there are differences between the development process and structure and function of mammary gland of mice and humans. The organoid obtained by the present application can track each period of human mammary gland development, and provide an excellent model for detailed analysis of key signals or regulation mechanisms of prenatal development of human mammary gland, and help to analyze the pathogenesis of congenital dysplasia of mammary gland and drug research. The construction method of the pluripotent stem cell-derived human mammary gland organoid of the present application can directly obtain stromal cells such as mesenchymal and vascular endothelial cells in addition to mammary epithelial cells without additional addition of stromal cells. These stromal cells are produced along with mammary epithelial cells, and do not need to be introduced by any additional steps, which is more convenient. Meanwhile, these stromal cells are specific microenvironment cells similar to in vivo mammary tissue. Example 1

[0108] As shown in Figure 3 , the construction method of the pluripotent stem cell-derived human mammary gland organoid of the present application comprises the following steps:

[0109] Step one: digest the adherent cultured pluripotent stem cells into single cells;

[0110] Step two: take 2.5x10 5 cells obtained in step one and inoculate into an ultra-low adsorption 6-well plate, and use cell aggregation culture medium to culture for 1 day to obtain cell aggregates.

[0111] Step three: replace the cell aggregation medium with a first medium, and culture for 3 days to obtain a first-stage organoid. In this embodiment, the first medium is MammoCult Human Medium Kit medium, including 2% low growth factor Matrigel, 2.5 ng / mL BMP4 and 10 μM SB431542.

[0112] Step four: collect the first-stage organoid obtained in step three, continue to replace it with an air-liquid surface method, and replace the first medium with a second medium, and culture for 3 days. In this embodiment, the second medium is MammoCult Human Medium Kit medium, including 10 μM CHIR99021.

[0113] Step five: replace the second medium with a third medium, and culture for 3 days to obtain a second-stage organoid. In this embodiment, the third medium is MammoCult Human Medium Kit medium, including 75 ng / mL BMP4 and 200 ng / mL PTHrP.

[0114] Step six: replace the third medium with a fourth medium, and culture for 3 days. In this embodiment, the fourth medium is EpiCult-B Human Medium Kit medium, including 250 nM SANT-1.

[0115] Step seven: replace the fourth medium with a fifth medium, and culture for 8 days to obtain a mammary gland organoid. In this embodiment, the fifth medium is EpiCult-B Human Medium Kit 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

[0116] As shown in the following, the method for constructing a human mammary gland organoid derived from pluripotent stem cells of the present application includes the following steps: Figure 4

[0117] Step one: digest the adherent cultured pluripotent stem cells into single cells;

[0118] Step two: take 2.5×10 5 cells obtained in step one, inoculate them into an ultra-low adsorption 6-well plate, and culture them with a cell aggregation medium for 1 day to obtain cell aggregates.

[0119] ​Step three: replace the cell aggregation medium with a first medium and culture for 3 days to obtain a first stage organoid. In this example, the first medium is MammoCult Human Medium Kit medium including 2% low growth factor Matrigel, 2.5 ng / mL BMP4 and 10 μΜ SB431542.

[0120] Step four: collect the first stage organoids from step three and continue culturing using air-liquid interphase method while replacing the first medium with a P1 medium and culturing for 3 days. In this example, the P1 medium is MammoCult Human Medium Kit medium including 30 ng / mL VEGFa.

[0121] Step five: replace the P1 medium with a second medium and culture for 3 days. In this example, the second medium is MammoCult Human Medium Kit medium including 10 μΜ CHIR99021.

[0122] Step six: replace the second medium with a third medium and culture for 3 days to obtain a second stage organoid. In this example, the third medium is MammoCult Human Medium Kit medium including 75 ng / mL BMP4 and 200 ng / mL PTHrP.

[0123] Step seven: replace the third medium with a fourth medium and culture for 3 days. In this example, the fourth medium is EpiCult-B Human Medium Kit medium including 250 nM SANT-1.

[0124] Step eight: replace the fourth medium with a fifth medium and culture for 12 days to obtain a mammary organoid. In this example, the fifth medium is EpiCult-B Human Medium Kit 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

[0125] Step one: digest adherent cultured pluripotent stem cells into single cells;

[0126] Step two: take 2.5 x 10 5 cells and seed into ultra-low attachment 6-well plates using cell aggregation medium and culture for 1 day to obtain cell aggregates.

[0127] Step 3: Replace the cell aggregation medium with the first culture medium and culture for 3 days to obtain the first-stage organoids. In this example, the first culture medium is MammoCult Human Medium Kit, which includes 2% low growth factor matrix gel, 10 μM SB431542, and 2.5-10.0 ng / mL BMP4, with BMP4 concentrations of 2.5 ng / mL, 5.0 ng / mL, 7.5 ng / mL, and 10.0 ng / mL. The obtained first-stage organoids were identified by immunofluorescence and real-time quantitative PCR experiments, such as... Figure 5 As shown, the results indicate that surface ectoderms expressing K8 and K18 can be formed under different BMP4 concentrations. Example 4

[0128] Step 1: Digest adherent pluripotent stem cells into single cells;

[0129] Step 2: Take 2.5 × 10⁻⁶ single cells from Step 1. 5 Cells were seeded into 6-well plates with ultra-low adsorption and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0130] Step 3: Replace the cell aggregation medium with the first culture medium and culture for 3 days to obtain the first-stage organoids. In this example, the first culture medium is MammoCult Human Medium Kit, which includes 2% low growth factor matrix gel, 2.5 ng / mL BMP4 and 4-12 μM SB431542, wherein the concentrations of SB431542 are 4 μM, 8 μM and 12 μM.

[0131] Step Four: Collect the first-stage organoids obtained in Step Three and continue culturing using the gas-liquid surface method, replacing the first culture medium with the second culture medium for 3 days. In this example, the second culture medium is MammoCult Human MediumKit, containing 10 μM CHIR99021. Figure 6 As shown, organoids with epithelium can be formed after step four at different SB431542 concentrations. Example 5

[0132] Step 1: Digest adherent pluripotent stem cells into single cells;

[0133] Step 2: Take 2.5 × 10⁻⁶ single cells from Step 1. 5 Cells were seeded into 6-well plates with ultra-low adsorption and cultured in cell aggregation medium for 1 day to obtain cell aggregates.

[0134] Step three: replace the cell aggregation medium with the first medium, and culture for 3 days to obtain the first-stage organoids. In this embodiment, the first medium is MammoCult Human Medium Kit medium, which includes 2% low growth factor Matrigel, 2.5 ng / mL BMP4, and 10 μM SB431542.

[0135] Step four: collect the first-stage organoids obtained in step three, continue to replace them with the air-liquid surface method for continuous culture, and replace the first medium with the second medium for culture for 3 days. In this embodiment, the second medium is MammoCult Human Medium Kit medium, which includes 8-12 μM CHIR99021. The concentration of CHIR9921 is 8 μM, 10 μM, and 12 μM. Real-time fluorescent quantitative PCR experiments are performed on the obtained organoids, and the results show that, as shown in Figure 7 , the organoids express breast basement membrane-related genes under different concentrations of CHIR99021. Embodiment 6

[0136] As shown in Figure 8 , the method for constructing human mammary gland organoids derived from pluripotent stem cells of the application includes the following steps:

[0137] Step one: digest the adherent cultured pluripotent stem cells into single cells;

[0138] Step two: take 2.5×10 5 cells obtained in step one and inoculate them into an ultra-low adsorption 6-well plate, and use a cell aggregation medium to culture for 1 day to obtain cell aggregates.

[0139] Step three: replace the cell aggregation medium with the first medium, and culture for 3 days to obtain the first-stage organoids. In this embodiment, the first medium is MammoCult Human Medium Kit medium, which includes 2% low growth factor Matrigel, 2.5 ng / mL BMP4, and 10 μM SB431542;

[0140] Step four: collect the first-stage organoids obtained in step three, continue to replace them with the air-liquid surface method for continuous culture, and replace the first medium with the second medium for culture for 3 days. In this embodiment, the second medium is MammoCult Human Medium Kit medium, which includes 10 μM CHIR99021.

[0141] Step five: replace the second medium with a third medium and culture for 3 days to obtain the second stage organoids. In this example, the third medium is MammoCult Human Medium Kit medium including 37.5 ng / mL BMP4 and 100 ng / mL PTHrP.

[0142] Step six: replace the third medium with a fourth medium and culture for 3 days. In this example, the fourth medium is EpiCult-B Human Medium Kit medium including 250 nM SANT-1.

[0143] Step seven: replace the fourth medium with a fifth medium and culture for 8 days to obtain the mammary organoids. In this example, the fifth medium is EpiCult-B Human Medium Kit medium including 30 ng / mL FGF2, 30 ng / mL FGF10, 30 ng / mL HGF, 30 ng / mL EGF and 10 μg / mL Insulin.

[0144] The obtained mammary organoids are subjected to immunofluorescence staining identification, as shown in FIG. 1, the human mammary organoids derived from pluripotent stem cells of the present application have two kinds of microenvironment cells of mesenchymal cells and vascular endothelial cells; as shown in FIG. 2, the human mammary organoids derived from pluripotent stem cells of the present application have basal lactation function and can secrete Milk protein and β-casein. Figure 9 Figure 10 The obtained mammary organoids are subjected to immunofluorescence staining identification, as shown in FIG. 1, the human mammary organoids derived from pluripotent stem cells of the present application have two kinds of microenvironment cells of mesenchymal cells and vascular endothelial cells; as shown in FIG. 2, the human mammary organoids derived from pluripotent stem cells of the present application have basal lactation function and can secrete Milk protein and β-casein.

[0145] Comparative Example 1

[0146] This comparative example provides a method for preparing human normal mammary organoids, which is different from the method of Example 1 in that the embedding method is used for culture in step four and subsequent steps, and the specific method is as follows:

[0147] Steps one to three: same as Example 1

[0148] Step four: wrap one first stage organoid with 30 μL low growth factor Matrigel and collagen I mixed gel, wherein the proportion of the mixed gel is 1:1, and drop the gel containing the first stage organoid into the 24-well plate without tissue treatment to form a gel ball. Drop 3 gel balls into each well, and the gel balls do not contact each other. After dropping the third gel ball, invert the plate and stand for 5 min, and then place it in a 37℃ incubator for 30 min to solidify the gel. After the gel is solidified, add the second medium along the wall of the well, and culture for 3 days. In this example, the second medium is the same as Example 1.

[0149] Steps five to seven: same as Example 1.

[0150] As​Figure 11 As shown, the mammary organoids obtained in this comparative example did not show obvious epithelial structures or branching structures. Immunofluorescence identification revealed that the mammary organoids obtained in this comparative example lacked basal cells and luminal cells, indicating that this method cannot successfully prepare mammary organoids.

[0151] Comparative Example 2

[0152] This comparative example provides a method for preparing normal human mammary gland organoids, which differs from the method in Example 1 in that step six is ​​different. The specific method is as follows:

[0153] Steps one through five: Same as in Example 1

[0154] Step 6: Replace the fourth medium with MammoCult Human Medium Kit medium containing 250 nM SANT-1 and incubate for 3 days.

[0155] Step 7: Same as Example 1.

[0156] like Figure 12 As shown, no vacuolar epithelial structures were observed in the mammary organoids obtained in this comparative example. Immunofluorescence identification revealed 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.

[0157] Experimental Example 1

[0158] In this experimental example, frozen sections and immunofluorescence staining were performed on organoids at various stages prepared using the various examples and comparative examples, and the organoids were identified based on this. The specific methods and results are described below.

[0159] (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 off the mixed gel containing organoid culture along the edge of the chamber and collect the sample into a 2 mL microcentrifuge tube.

[0160] (2) Immerse the sample in 4% paraformaldehyde fixation buffer overnight to fix organoid cultures.

[0161] (3) Rinse the sample gently with PBS for 5 minutes each time, and wash three times.

[0162] (4) Aspirate the supernatant, embed the sample with OCT embedding agent, and perform frozen sectioning with a section thickness of 7-10 µm.

[0163] (5) Place the slice at room temperature for 15 minutes to ensure the sample melts, then permeate with 100-200 µL of 0.5% PBST solution at room temperature for 10 minutes, and then soak and wash with 0.2% PBST solution for 10 minutes.

[0164] (6) After the supernatant is absorbed, 100-200 μL of blocking solution is added, and the blocking is performed at room temperature for 2 hours.

[0165] (7) After the blocking is completed, the supernatant is absorbed, 150 μL of prepared primary antibody is added, and incubation is performed in a refrigerator at 4°C overnight.

[0166] (8) After the incubation of the primary antibody is completed, the sample is soaked in 0.2% PBST solution for washing three times, each time for 10 minutes. After the washing is completed, the supernatant is absorbed, 100-150 μL of prepared secondary antibody is added, and incubation is performed at room temperature for 2 hours in the dark.

[0167] (9) After the incubation of the secondary antibody is completed, the sample is soaked in 0.2% PBST solution for washing three times, each time for 10 minutes. After the washing is completed, the supernatant is absorbed as much as possible, 20 μL of DAPI mounting agent is added on the sample, and the sample is ensured to be completely covered. Then, the glass slide is carefully covered on the mounting agent, and the sample is mounted at room temperature for more than 30 minutes, and then is ready for photographing.

[0168] The immunofluorescence staining markers used in the above method are as follows: in the first stage, K8 / K18 marks surface ectoderm cells; in the second stage, EpCAM represents epithelial cells, EDAR represents mammary basal cells, and LEF1 represents mammary basal cells and peribasal mesenchymal cells; in the third stage, K14 represents mammary basal cells, K8 represents mammary duct lumen 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.

[0169] Experimental Example 2

[0170] In this experimental example, RNA extraction and real-time fluorescent quantitative PCR experiments are performed on the organoids prepared in each of the examples and comparative examples, and the organoids are identified based on the results. The specific method and results are described as follows.

[0171] (1) The organoids obtained by culture are transferred into a 15 ml centrifuge tube, fresh PBS is added until the tube opening, and then centrifugation is performed at 1000 r / min for 5 min;

[0172] (2) The supernatant is discarded, 1 ml of Trizol is added, and the sample is broken by repeatedly blowing;

[0173] (3) 200 μl of chloroform is added, shaken for 15 s, and placed at room temperature for 5 min;

[0174] (4) Centrifugation is performed at 4°C for 15 min at 12000 g;

[0175] (5) Transfer the supernatant to a new enzyme-free 1.5 ml centrifuge tube; add an equal volume of isopropanol, mix well up and down, and stand at room temperature for 10 min;

[0176] (6) Centrifuge at 4 DEG C for 10 min at 12000 g;

[0177] (7) Discard the supernatant, and slowly add 1 ml of 75% ethanol solution along the wall of the centrifuge tube, and mix well up and down;

[0178] (8) Centrifuge at 4 DEG C for 5 min at 12000 g;

[0179] (9) Discard the supernatant, and dry at room temperature for 5 min;

[0180] (10) Add enzyme-free water to dissolve the precipitate, and add reverse transcription reagent; after 37 DEG C water bath for 15 min, 85 DEG C water bath for 5 s; obtain sample cDNA;

[0181] (11) Use the obtained cDNA as a template, add primers of the target gene, and use a real-time fluorescent quantitative PCR kit and a real-time fluorescent quantitative PCR instrument for detection.

[0182] The breast organoid prepared by the application has a branched structure, has basal cells and epithelial cells, can simulate the complex morphology of the in-vivo breast, better simulates the physiological and pathological processes of the breast, can simulate the physiological function of the breast, has a lactation function, and provides a more real model for the physiology and pathology of the breast. The human breast organoid constructed by the application by using pluripotent stem cells has a similar morphological structure microenvironment cell to the in-vivo breast, has higher tissue complexity, and can simulate the interaction between the breast epithelium and the microenvironment during the development process.

[0183] The construction route of the breast organoid of the application follows the principle of organogenesis, the differentiation process thereof is similar to the development process of the in-vivo breast, and the same process experiences surface ectoderm, breast basement, breast epithelial lineage, and the like, can completely simulate the entire development process of the breast, and is expected to solve the problem of shortage of in-vitro models of the breast during the embryonic period of human beings. The application can obtain organoids at different differentiation stages, can help to study the mechanism of breast development and lesions at different periods, and provides a dynamic model for the development and disease research of the breast. The application provides a new idea for the directional differentiation of human pluripotent stem cells into a breast organoid.

[0184] The final mammary gland organoids have basic lactation function. Although a large number of studies have revealed the mechanism of mammary gland development and its lesions, most of these studies rely on mouse models, and there are still some differences between mice and human mammary glands, so these conclusions cannot be completely applied to humans. The research on the mechanism of human mammary gland development and its lesions is still lacking, especially for the embryonic development of the mammary gland. The pluripotent stem cell-derived mammary gland organoids constructed in this study can serve as a good in vitro model to facilitate the research on human mammary gland development and its lesions. In particular, in the research involving the embryonic mammary gland, this model has its unique advantages. At the same time, this study also provides new guidance for the subsequent construction of mammary gland organoids.

[0185] The pluripotent stem cell-derived human mammary gland organoid and its construction method of the present application simulate the development process of the mammary gland through staged culture and specific medium combination, generate mammary gland organoids with complex structure and function, the constructed mammary gland organoids have higher tissue complexity, can better simulate the morphological structure and microenvironment of the in vivo mammary gland, are suitable for studying mammary gland development, disease mechanism and drug screening; completely simulate the mammary gland development process from the surface ectoderm to the mammary epithelial lineage, solve the problem of shortage of in vitro models of embryonic mammary glands, provide an important tool for mammary gland development research; can obtain organoids at different stages of differentiation, facilitate the research on the mechanism of mammary gland development and its lesions at different periods, provide a dynamic model for mammary gland development and disease research, and help reveal the pathogenesis of mammary gland diseases; the mammary gland organoids have lactation function, can simulate the physiological function of the mammary gland, and are suitable for studying the physiological function of the mammary gland and its regulation mechanism. The present application successfully constructs mammary gland organoids with complex structure and function through staged culture and optimization of medium combination combined with the gas-liquid surface method, which can simulate the development process, physiological function and pathological mechanism of the mammary gland, provide an important tool for mammary gland development research, disease model construction and drug screening, and have great application prospect.

[0186] The above specific embodiments are used to explain and illustrate the present application, and are only preferred embodiments of the present application, but not limit the present application, any modification, equivalent replacement, improvement, etc. made to the present application within the spirit of the present application and the protection scope of the claims, fall into the protection scope of the present application.

Claims

1. A human breast organoid derived from pluripotent stem cells, characterized in that, The human breast organoids were obtained through the following multi-stage induction culture: In the first stage, pluripotent stem cells were directed to differentiate into surface ectoderm lineages using a first culture medium. This first culture medium included growth factors from the TGF-β superfamily and TGF-β signaling regulators. The first culture medium consisted of MammoCultHuman Medium Kit medium, 2% low growth factor matrix gel, BMP4, and SB431542. The MammoCult HumanMedium Kit medium was supplemented with 4 μg / mL heparin sodium and 0.48 μg / mL hydrocortisone. The concentration of BMP4 in the first culture medium was 2.5-10.0 ng / mL, and the concentration of SB431542 was 4-12 μM. In the first stage, the organs were cultured in the first culture medium for 1-3 days to obtain surface ectoderm stage organoids expressing the surface ectoderm markers K8 / K18. In the second stage, a second culture medium is used to drive the differentiation of the surface ectoderm into the mammary basal plate. The second culture medium includes Wnt signaling regulators and contains MammoCult Human Medium Kit medium and CHIR99021. The MammoCult Human Medium Kit 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 breast basal plate-like organoids were cultured in a second culture medium for 1-3 days to obtain bright epithelium and express the breast basal plate markers EDAR and LEF1. The second stage uses the gas-liquid interface culture method. The organoids prepared in the first stage with a mixed gel of matrix gel and collagen I in a 1:1 ratio are resuspended and seeded into Transwell chambers pre-coated with the same mixed gel. Culture medium is added to the lower layer to form a gas-liquid interface. The third stage uses a third culture medium to simulate the microenvironment of mammary buds during the embryonic period, promoting the production of mammary epithelial progenitor cell markers and mesenchymal cells. The third culture medium includes growth factors and paracrine signaling factors from the TGF-β superfamily. The third culture medium contains MammoCult Human Medium Kit medium, BMP4, and PTHrP. The concentration of BMP4 is 37.5-75 ng / mL, and the concentration of PTHrP is 100-200 ng / mL. The third stage is cultured in the third culture medium for 3-5 days to obtain mammary bud-like organoids with bright epithelium and expressing mammary bud markers EDAR and LEF1. In the fourth stage, mammary epithelial lineage differentiation was induced using a fourth culture medium, which included Hedgehog signaling regulators. The fourth culture medium contained EpiCult-B Human Medium Kit medium and SANT-1 at a concentration of 250 nM. The fourth stage was carried out in the fourth culture medium for 3-5 days. In the fifth stage, a mature mammary organoid with lactation function was cultured using a fifth culture medium to form an epithelial structure with basal cells and luminal cells, as well as a mammary gland physiological microenvironment simulated by mesenchymal cells and vascular endothelial cells. The fifth culture medium included a combination of growth factors and metabolic regulators that promote mammary epithelial morphogenesis. The fifth culture medium contained EpiCult-B Human Medium Kit medium, FGF2, FGF10, HGF, EGF, and Insulin.

2. The human breast organoid derived from pluripotent stem cells as described in claim 1, characterized in that: Prior to the first stage of culture, the process also includes: cell aggregation culture: cell aggregates are formed by culturing in a cell aggregation medium for 1-2 days, wherein the cell aggregation medium contains Essential 8 medium and 10 μM Y27632.

3. The human breast organoid derived from pluripotent stem cells as described in claim 1, characterized in that: The first and second stages are further divided into a transition culture stage: cultured for 2-3 days in P1 medium containing MammoCult Human Medium Kit medium and VEGFα, wherein 4 μg / mL of heparin sodium and 0.48 μg / mL of hydrocortisone are added to the MammoCult Human Medium Kit medium, and the concentration of VEGFα is 25-30 ng / mL.

4. The method for constructing human breast organoids derived from pluripotent stem cells according to any one of claims 1-3, characterized in that... Includes the following steps: Step 1: Digest adherent pluripotent stem cells into single cells; Step 2: Preparation of cell aggregates: Seed the single cells from Step 1 into ultra-low adsorption plates and culture them in cell aggregation medium for 1-2 days to obtain cell aggregates; Step 3: Replace the cell aggregation medium with the first culture medium described in claim 1, and culture for 1-3 days to obtain the first-stage organoid; Step 4: Collect the first-stage organoids obtained in Step 3 and continue culturing them using the gas-liquid surface method, while replacing the first culture medium with the second culture medium described in claim 1, and culturing for 1-3 days; Step 5: Replace the second culture medium with the third culture medium as described in claim 1, and culture for 3-5 days to obtain the second-stage organoids; Step 6: Replace the third culture medium with the fourth culture medium as described in claim 1, and culture for 3-5 days; Step 7: Replace the fourth culture medium with the fifth culture medium as described in claim 1, culture for 8-12 days, and obtain mammary gland organoids.

5. The method for constructing human breast organoids derived from pluripotent stem cells as described in claim 4, characterized in that: The pluripotent stem cells in step one are human pluripotent stem cells, including human embryonic stem cells.

6. The method for constructing human breast organoids derived from pluripotent stem cells as described in claim 4, characterized in that: In step two, the ultra-low adsorption plate can be a 384-well plate, a 96-well plate, a 48-well plate, a 12-well plate, or a 6-well plate. Alternatively, in step two, the number of single cells seeded in the ultra-low adsorption 6-well plate is 2.0 × 10⁻⁶. 5 -5.0×10 5 The diameter of the obtained cell aggregates ranged from 60 to 200 μm.

Images