CN114085804B - Human embryonic stem cell in-vitro induced differentiation formed skin organoid and efficient culture method and application thereof - Google Patents
Human embryonic stem cell in-vitro induced differentiation formed skin organoid and efficient culture method and application thereof Download PDFInfo
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Abstract
The invention discloses a skin organoid formed by in vitro induced differentiation of human embryonic stem cells, and a high-efficiency culture method and application thereof, wherein the process comprises the following steps: pre-differentiation treatment; inducing the differentiation of the human embryonic stem cells by using a first differentiation medium to form epidermal ectoderm cells and cerebral neural crest ectoderm cells; sequentially adopting a second differentiation medium and an E6 medium to induce the brain neural crest ectodermal cells obtained in the first step to differentiate into dermis fibroblasts; and (3) sequentially inducing the development and maturation of the sensory nerve endings and hair follicles of the culture obtained in the step (II) by adopting a third differentiation medium and a fourth differentiation medium. The invention has the advantages that: the efficiency of three-dimensional differentiation from human embryonic stem cells into skin organoids is improved, the yield of the skin organoids is 55.30% +/-11.06%, and the culture cost is saved; the obtained skin organoid has layered epidermis, dermis, hair follicle, melanocyte and sensory nerve terminal, and can be used for research of diseases such as infectious and hereditary dermatoses and skin tumor, and for providing hair source for hair transplantation of alopecia patients.
Description
Technical Field
The invention relates to stem cell biology and regenerative medicine, in particular to a technology for obtaining skin organoids by in vitro induced differentiation.
Background
Human embryonic stem cells (Human embryonic stem cell, hESCs) are a class of cells isolated from early embryos or primordial gonads that have the properties of in vitro culture immortalization, self-renewal and multipotency. In an in vitro or in vivo environment, hESCs cells can be induced to differentiate into almost all cell types of the body.
Organoids (organoids) are a new technology for preparing organs similar to the body in vitro by 3D culture, and have been rated as annual technology in the journal of Nature in 2017. The structure and function of organoids are highly similar to those of body organs, and the editability of genetic information makes them important in human genetic disease model research. Organoids are structures that can be self-organized by 3D culture of one or more derived cells derived from embryonic stem cells, induced pluripotent stem cells and adult stem cells in vitro to form an organ-like structure. 3D cultured organoids have many advantages over human disease animal models and traditional cell cultures. The application range of organoids is quite wide: first, organoids can provide important tools for studies that are difficult to develop on humans, such as prenatal development and tissue maintenance models; second, organoids also provide an important vehicle for studying many biological processes, for example, migration of interneurons was first observed in organoids based on organoid studies; in addition, organoids can also be used to mimic diseases, including infectious diseases such as Zika virus infection, monogenic genetic diseases such as cystic fibrosis, and neoplastic diseases, among others. Since the starting stem cells can be compiled to carry the genetic mutation of interest, or derived from multiple genetic backgrounds, these can all be powerful models of the phenotypic outcome of genotype detection. Thus, disease models based on organoid preparation are an important supplement to animal models of human disease.
At present, the existing method for differentiating human embryonic stem cells into skin organoids is reported by Lee J et al in 2020 (Lee J, rabbani CC, gao H, steinhart MR, woodfoff BM, pflum ZE, kim A, heller S, liu Y, shipcanner TZ, koehler KR.hair-bearing human skin generated entirely from pluripotent stem cells.Nature.2020Jun;582 (7812): 399-404.doi: 10.1038/S41586-020-2352-3.) the skin organoids prepared by the culture method have 8-28% of yield and lower efficiency, and are difficult to popularize and apply on a large scale.
Disclosure of Invention
The invention aims to provide a skin organoid formed by in-vitro induced differentiation of human embryonic stem cells, and a high-efficiency culture method and application thereof, so as to solve the problem of low yield of cultured skin organoids in the prior art.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a high-efficiency culture method of human embryonic stem cells for inducing and differentiating skin organoids in vitro comprises the following steps:
step one: pre-differentiation treatment;
step two: inducing the differentiation of the human embryonic stem cells by using a first differentiation medium to form epidermal ectoderm cells and cerebral neural crest ectoderm cells;
the components of the first differentiation medium include E6 basal medium, FGF2, BMP4, SB-431542 and matrigel;
step three: sequentially adopting a second differentiation medium and an E6 medium to induce the brain neural crest ectodermal cells obtained in the second step to differentiate to form dermis fibroblasts;
the components of the second differentiation medium include E6 medium, FGF2 and LDN-193189;
step four: the third differentiation medium and the fourth differentiation medium are adopted in sequence to induce the development and maturation of the sensory nerve endings and hair follicles of the culture obtained in the third step,
the third differentiation medium comprises the following components: advanced DMEM/F12 culture medium, neurobasal culture medium, defined Keratinocyte-SFM culture medium, RA, N2, B27, beta mercaptoethanol, glutamax, matrigel,
the fourth differentiation medium comprises the following components: advanced DMEM/F12 medium, neurobasal medium, defined Keratinocyte-SFM medium, RA, N2, B27, beta mercaptoethanol, glutamax.
Further, the step one differentiation pretreatment comprises the following steps:
(1) Before differentiation, the pluripotency of the human embryonic stem cells needs to be checked, and the double positive rate reaches 97% on the premise that the expression modes of the pluripotency markers OCT4 and SSEA4 are correct, which indicates that the human embryonic stem cells have good differentiation potential;
(2) After 2 days before the initiation of differentiation, human embryonic stem cells were counted after digestion, diluted to 7000 cells/100 μl concentration with E8 medium, plated into a low-adhesion U-shaped 96-well plate, and the human embryonic stem cells were coagulated into cell pellets.
Further, the process of the second step includes: re-suspending the human embryonic stem cell agglutinated sphere obtained in the first differentiation medium step on the 0 th differentiation day, transferring the human embryonic stem cell agglutinated sphere to a new low-adhesion U-shaped 96-well plate, and continuously culturing for 5 days to obtain epidermic ectodermal cells and cerebral neural crest ectodermal cells;
the first differentiation medium is obtained by taking an E6 medium as a basic medium, adding FGF2 with the final concentration of 1-8ng/mL, BMP4 with the final concentration of 0.5-10ng/mL, SB-431542 with the final concentration of 1-15 mu M and matrigel with the final concentration of 1-5% by volume fraction.
Further, the process of the third step includes:
(1) On day 6 of differentiation, 25. Mu.L of the second differentiation medium was added to the culture of each culture well of the second step for cultivation,
the second differentiation medium is obtained by taking E6 medium as basic medium, and adding FGF2 with final concentration of 150-250ng/mL and LDN-193189 with final concentration of 0.5-4 μm;
(2) On day 8 of differentiation, 75 μl of fresh E6 medium was added to each of the above culture wells for culture;
(3) On day 10 of differentiation, 100. Mu.L of medium was aspirated from each of the above culture wells and 100. Mu.L of fresh E6 medium was added again for culture.
Further, the first differentiation medium is obtained by taking an E6 medium as a basic medium, adding FGF2 with a final concentration of 6ng/mL, BMP4 with a final concentration of 4ng/mL, MSB-431542 with a final concentration of 8 mu and matrigel with a final concentration of 1% by volume fraction;
the second differentiation medium was obtained by using E6 medium as a basal medium and adding FGF2 at a final concentration of 200ng/mL and LDN-193189 at a final concentration of 1.5. Mu.M thereto.
Further, the process of the fourth step includes: (1) On day 12 of differentiation, resuspending the organoid culture obtained in step three with a third differentiation medium and transferring it to a low-adhesion 24-well cell culture plate, which is put on a shaker rotating at 65rpm for suspension culture;
the volume ratio of the third differentiation medium is 1:1:1, mixing three culture mediums of Advanced DMEM/F12, neurobasal and Defined Keratinocyte-SFM as basic culture mediums, and adding RA with the final concentration of 2-10 mu M, N2 with the final concentration of 0.5% by volume, B27 with the final concentration of 1% by volume, beta mercaptoethanol with the final concentration of 0.1mM, glutaMAX with the final concentration of 1% by volume and matrigel with the final concentration of 1-5% by volume;
(2) Thereafter, changing the fresh fourth differentiation medium 1 time every 3 days, and culturing the culture medium until 140 days;
the fourth culture medium is prepared by the following steps of: 1:1, and mixing three media of Advanced DMEM/F12, neurobasal and Defined Keratinocyte-SFM as basic media, and adding RA with a final concentration of 2-10 mu M, N2 with a final concentration of 0.5% by volume, B27 with a final concentration of 1% by volume, beta mercaptoethanol with a final concentration of 0.1mM and glutamax with a final concentration of 1% by volume.
Further, the third culture medium is prepared by using a volume ratio of 1:1:1, mixing three media of Advanced DMEM/F12, neurobasal and Defined Keratinocyte-SFM as basic media, and adding 5 mu M RA (RA) with a final concentration of 0.5% N2 by volume fraction, 1% B27 by volume fraction, 0.1mM beta mercaptoethanol with a final concentration of 1% GlutaMAX by volume fraction and 1.5% matrigel by volume fraction;
the fourth culture medium is prepared by the following steps of: 1:1, neurobasal and Defined Keratinocyte-SFM were mixed as basal medium, and RA was added thereto at a final concentration of 5 μm, at a final concentration of 0.5% n2 by volume fraction, at a final concentration of 1% b27 by volume fraction, at a final concentration of 0.1mM beta mercaptoethanol, at a final concentration of 1% glutamax by volume fraction.
The invention also provides a skin organoid obtained by the culture method, which comprises epidermis with layering, dermis and melanocytes, sensory nerve endings, hair follicle structures and dermal papilla cells.
The invention also provides the application of the skin organoids obtained by the culture method in drug discovery and screening or the application of the skin organoids in preparation of regenerative medicine.
The invention also provides the application of the skin organoids obtained by the culture method in preparing medicines for treating infectious and hereditary skin diseases and skin tumors or in preparing medicines for providing hair sources for hair transplantation of patients suffering from alopecia.
The advantages of the invention include: the efficiency of three-dimensional differentiation from human embryonic stem cells into skin organoids is improved, the yield of the skin organoids is 55.30% +/-11.06%, and the culture cost is saved; the obtained skin organoid has stratified epidermis, dermis, hair follicle, melanocyte and sensory nerve terminal, etc., can be used for research of diseases such as infectious, hereditary dermatoses and skin tumor, etc., and can also provide hair source for hair transplantation of alopecia patients.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate and do not limit the invention, and together with the description serve to explain the principle of the invention:
FIG. 1 is a flow chart of the differentiation of human embryonic stem cells into skin organoids established in the present invention.
FIG. 2 is a white light plot of skin organoid cultures cultured in three dimensions at different time points.
FIG. 3 is a diagram showing immunofluorescent staining identification experiments of basal layer keratinocytes, intermediate layer keratinocytes and outermost layer keratinocytes in the skin organoid epidermis differentiated to day 50 into structures.
FIG. 4 is a diagram showing immunofluorescent staining of the epidermis layer of the organoid epidermis and dermis layer of the dermis structure differentiated to day 50.
FIG. 5 is a graph of experimental results of full-tissue immunofluorescence of sensory nerve endings and 3D scan identification of hair follicle structures of skin organoids differentiated to day 90.
FIG. 6 is a graph of experimental identification of dermal papilla cells differentiated into skin organoids on day 90, hair follicle structure, dermal papilla cell whole tissue immunofluorescence and 3D scanning.
FIG. 7 is a graph showing the evaluation of skin organoid hair coloring at various time points of differentiation.
FIG. 8 is a statistical plot of the skin organoid yields prepared by the method of the present invention and the method reported by Lee J et al.
Detailed Description
The present invention will now be described in detail with reference to the drawings and the specific embodiments thereof, which are illustrative embodiments and illustrations of the invention, but are not to be construed as limiting the invention.
The information of the main reagents used in the invention is shown in Table 1, and the information of the main antibodies is shown in Table 2. And the method can be realized by conventional technical means in the technical field unless otherwise specified.
The culture method for inducing and differentiating the high-efficiency human embryonic stem cells into the skin organoids in vitro is shown in figure 1, and comprises the following 3 stages:
stage 1: the differentiation of human embryonic stem cells into epidermal ectoderm and cerebral neural crest ectoderm cells (Day 0-6) was induced as follows:
(1) Before differentiation, H1 human embryo stem cells need to be checked for pluripotency, and the double positive rate reaches 97% on the premise that the expression modes of the pluripotency markers OCT4 and SSEA4 are correct, which indicates that the human embryo stem cells have good differentiation potential.
(2) After 2 days before the initiation of differentiation, human embryonic stem cells were counted after digestion, diluted to 7000 cells/100 μl concentration with E8 medium, plated into a low-adhesion U-shaped 96-well plate, and the human embryonic stem cells were coagulated into cell pellets.
(3) Resuspension of the human embryonic stem cell agglutinated spheres obtained in the step (2) by adopting a first differentiation medium on the 0 th day of differentiation, transferring the human embryonic stem cell agglutinated spheres to a new low-adhesion U-shaped 96-well plate, and continuously culturing for 5 days to obtain ectoderm-like cells (epidermal ectoderm and brain neural crest ectoderm cells);
the first differentiation medium is obtained by taking an E6 medium as a basic medium, adding FGF2 with a final concentration of 6ng/mL, BMP4 with a final concentration of 4ng/mL, SB-431542 with a final concentration of 8 mu M and matrigel with a final concentration of 1% by volume.
Stage 2: inducing differentiation of brain neural crest ectodermal cells to form dermal fibroblasts (Day 6-11) as follows:
(1) On day 6 of differentiation, 25. Mu.L of the second differentiation medium was added to the culture in each of the above-mentioned culture wells for cultivation,
the second differentiation medium was obtained by using E6 medium as the basal medium and adding FGF2 at a final concentration of 200ng/mL and LDN-193189 at a final concentration of 1.5. Mu.M thereto.
(2) On day 8 of differentiation, 75. Mu.L of fresh E6 medium was added to each of the above culture wells for culture.
(3) On day 10 of differentiation, 100. Mu.L of medium was aspirated from each of the above culture wells and 100. Mu.L of fresh E6 medium was added again for culture.
Stage 3: inducing the development and maturation of sensory nerve endings and hair follicles (Day 12-140) as follows:
(1) On day 12 of differentiation, the organoid culture obtained in stage 2 was resuspended in a third differentiation medium and transferred to a low-adhesion 24-well cell culture plate, which was placed on a shaking table rotating at 65rpm for suspension culture;
the volume ratio of the third differentiation medium is 1:1:1, and adding 5 mu M RA, 0.5% N2, 1% B27, 0.1mM beta mercaptoethanol, 1% Glutamax and 1.5% matrigel.
(2) Thereafter, changing the fresh fourth differentiation medium 1 time every 3 days, and culturing the culture medium until 140 days;
the fourth culture medium is prepared by the following steps of: 1:1, neurobasal and Defined Keratinocyte-SFM were mixed as basal medium, and RA was added thereto at a final concentration of 5 μm, at a final concentration of 0.5% n2 by volume fraction, at a final concentration of 1% b27 by volume fraction, at a final concentration of 0.1mM beta mercaptoethanol, at a final concentration of 1% glutamax by volume fraction.
Identification of skin organoid components
The specific scheme is as follows:
based on the above examples, the skin organoids differentiated to different time points were photographed, and the results are shown in fig. 2.
And collecting skin organoids at different differentiation time points for frozen section staining or full-tissue immunofluorescence staining identification.
The skin organoids differentiated on day 50 were collected, embedded with OCT, frozen at-80℃and prepared into sections of 7 μm thickness, immunofluorescent staining was performed with KRT5 and KRT15 antibodies, and after elution of the primary antibodies, fluorescent secondary antibodies, goat anti-Rabbit IgG (H+L) Secondary Antibody Alexa Fluor 568 and Rabbit anti-Mouse IgG (H+L) Secondary Antibody Alexa Fluor 488 were added. As shown in FIG. 3, KRT5+ and KRT15+ are observed as basal stratum corneum keratinocytes, KRT15+ positive as middle stratum corneum keratinocytes, and KRT5+ positive as outermost stratum corneum keratinocytes, indicating that the skin organoids prepared by this method have a stratified epidermal structure.
The skin organoids differentiated on day 50 were collected, embedded with OCT, frozen at-80℃and prepared into sections of 7 μm thickness, immunofluorescent staining was performed with KRT15 and PDGF alpha+ antibodies, and after elution of the primary antibodies, fluorescent secondary antibodies, goat anti-Rabbit IgG (H+L) Secondary Antibody Alexa Fluor 568 and Rabbit anti-Mouse IgG (H+L) Secondary Antibody Alexa Fluor 488 were added. As shown in FIG. 4, KRT15+ positive stratum corneum and PDGF+ positive dermis were observed, indicating that the skin organoids prepared by this method possess epidermis and dermis structures.
The skin organoids differentiated to day 90 were collected, whole tissues were fixed and permeabilized, were immunofluorescent stained with KRT17 and TUJ1 antibodies, and after elution of primary antibodies, were added with fluorescent secondary antibodies, goat anti-Rabbit IgG (H+L) Secondary Antibody Alexa Fluor 568 and Rabbit anti-Mouse IgG (H+L) Secondary Antibody Alexa Fluor 488. As shown in FIG. 5, KRT17+ positive hair follicle structure and TUJ1+ positive sensory nerve endings were observed, indicating that the skin organoids prepared by this method possess hair follicles and sensory nerve endings.
The skin organoids differentiated to day 90 were collected, whole tissues were fixed and permeabilized, were immunofluorescent stained with KRT17 and SOX2 antibodies, and after elution of primary antibodies, fluorescent secondary antibodies, goat anti-Rabbit IgG (H+L) Secondary Antibody Alexa Fluor 568 and Rabbit anti-Mouse IgG (H+L) Secondary Antibody Alexa Fluor 488 were added. As shown in FIG. 6, KRT17+ positive hair follicle structures and SOX2+ positive dermal papilla cells were observed, indicating that the skin organoids prepared by this method possess hair follicle and dermal papilla structures.
The skin organoids differentiated on days 65, 75, 85, 95, 105 and 120 were collected and observed under a white light microscope, as shown in fig. 7, and increased hair papilla staining and hair shaft darkening were seen, indicating that the skin organoids prepared by this method had melanocytes and were able to stain hair.
The comparison of Lee J in 2020 with the methods published by its colleagues (Lee J, rabbani CC, gao H, steinhart MR, woodfuff BM, pflum ZE, kim A, heller S, liu Y, shipchendler TZ, koehler KR.hair-bearing human skin generated entirely from pluripotent stem cells.Nature.2020Jun;582 (7812): 399-404.doi: 10.1038/S41586-020-2352-3.) was performed in parallel, and on differentiation to day 120, the skin organoids that successfully grew hair follicles were counted, and it was found that the present invention was effective in improving the yield of skin organoids, and the yield of skin organoids produced by the present method was 55.30% + -11.06%.
TABLE 1 list of reagents
Table 2 list of antibodies
The foregoing has described in detail the technical solutions provided by the embodiments of the present invention, and specific examples have been applied to illustrate the principles and implementations of the embodiments of the present invention, where the above description of the embodiments is only suitable for helping to understand the principles of the embodiments of the present invention; meanwhile, as for those skilled in the art, according to the embodiments of the present invention, there are variations in the specific embodiments and the application scope, and the present description should not be construed as limiting the present invention.
Claims (7)
1. A high-efficiency culture method of skin organoids formed by in-vitro induced differentiation of human embryonic stem cells is characterized by comprising the following steps:
the process comprises the following steps:
step one: pre-differentiation treatment, namely diluting the human embryonic stem cells with the double positive rates of the multipotent markers OCT4 and SSEA4 reaching 97% by using an E8 culture medium 2 days before the differentiation starts, plating, and agglomerating into cell spheres;
step two: on day0 of differentiation, inducing 5-day differentiation of human embryonic stem cells by using a first differentiation medium to form epidermal ectoderm and brain neural crest ectoderm cells;
the first differentiation medium comprises E6 basal medium, final concentration of 1-8ng/mLFG 2, final concentration of 0.5-10ng/mLBMP4, final concentration of 1-15 μM SB-431542 and final concentration of 1-5% matrigel by volume fraction;
step three: inducing the brain neural crest ectodermal cells obtained in the second step by adopting a second differentiation medium on the 6 th differentiation day, adding an E6 medium for continuous culture on the 8 th differentiation day, sucking out part of the medium on the 10 th differentiation day, adding a fresh E6 medium for continuous culture, and differentiating to form dermis fibroblasts;
the components of the second differentiation culture medium comprise E6 culture medium, FGF2 with the final concentration of 150-250ng/mL and LDN-193189 with the final concentration of 0.5-4 mu M;
step four: inducing the culture obtained in the third step by using a third differentiation medium on the 12 th day of differentiation, replacing the fresh fourth differentiation medium 1 time every 3 days, culturing by replacing the liquid until the 140 th day, inducing the development and maturation of the sensory nerve endings and hair follicles of the culture,
the third differentiation medium comprises the following components: the volume ratio is 1:1:1, advanced DMEM/F12, neurobasal and defined keratenocyte-SFM medium, final concentration of 2-10 μm RA, final concentration of 0.5% n2 by volume fraction, final concentration of 1% b27 by volume fraction, final concentration of 0.1mM beta mercaptoethanol, final concentration of 1% glutamax by volume fraction and final concentration of 1-5% matrigel by volume fraction;
the fourth differentiation medium comprises the following components: the volume ratio is 1:1:1, advanced DMEM/F12, neurobasal and defined basal-SFM medium, at a final concentration of 2-10 μm RA, at a final concentration of 0.5% n2 by volume fraction, at a final concentration of 1% b27 by volume fraction, at a final concentration of 0.1mM beta mercaptoethanol, at a final concentration of 1% glutamax by volume fraction.
2. The method for efficiently culturing the skin organoids formed by in vitro induced differentiation of human embryonic stem cells according to claim 1, wherein the method comprises the following steps:
the first differentiation pretreatment comprises the following steps:
(1) Before differentiation, the pluripotency of the human embryonic stem cells needs to be checked, and the double positive rate reaches 97% on the premise that the expression modes of the pluripotency markers OCT4 and SSEA4 are correct, which indicates that the human embryonic stem cells have good differentiation potential;
(2) After 2 days before the initiation of differentiation, human embryonic stem cells were counted after digestion, diluted to 7000 cells/100 μl concentration with E8 medium, plated into a low-adhesion U-shaped 96-well plate, and the human embryonic stem cells were coagulated into cell pellets.
3. The method for efficiently culturing the skin organoids formed by in vitro induced differentiation of human embryonic stem cells according to claim 1, wherein the method comprises the following steps:
the second step comprises the following steps: and (3) re-suspending the human embryonic stem cell agglutinated sphere obtained in the step one by adopting a first differentiation medium on the 0 th day of differentiation, transferring the human embryonic stem cell agglutinated sphere to a new low-adhesion U-shaped 96-well plate, and continuously culturing for 5 days to obtain the epidermal ectoderm and the cerebral neural crest ectoderm cells.
4. The method for efficiently culturing the skin organoids formed by in vitro induced differentiation of human embryonic stem cells according to claim 1, wherein the method comprises the following steps:
the process of the third step comprises the following steps:
(1) On day 6 of differentiation, 25. Mu.L of a second differentiation medium was added to the culture of each culture well of step two for cultivation;
(2) On day 8 of differentiation, 75 μl of fresh E6 medium was added to each of the above culture wells for culture;
(3) On day 10 of differentiation, 100. Mu.L of medium was aspirated from each of the above culture wells and 100. Mu.L of fresh E6 medium was added again for culture.
5. The method for efficiently culturing the skin organoids formed by in vitro induced differentiation of human embryonic stem cells according to claim 1, wherein the method comprises the following steps:
the first differentiation medium is obtained by taking an E6 medium as a basic medium, adding FGF2 with a final concentration of 6ng/mL, BMP4 with a final concentration of 4ng/mL, SB-431542 with a final concentration of 8 mu M and matrigel with a final concentration of 1% by volume fraction;
the second differentiation medium was obtained by using E6 medium as a basal medium and adding LDN-193189 to the basal medium at a final concentration of 200ng/mLFG 2 and a final concentration of 1.5. Mu.M.
6. The method for efficiently culturing the skin organoids formed by in vitro induced differentiation of human embryonic stem cells according to claim 1, wherein the method comprises the following steps:
the process of the fourth step comprises the following steps: (1) On day 12 of differentiation, resuspending the organoid culture obtained in step three with a third differentiation medium and transferring it to a low-adhesion 24-well cell culture plate, which is put on a shaker rotating at 65rpm for suspension culture;
(2) Thereafter, the fresh fourth differentiation medium was changed 1 time every 3 days, and the culture was changed to 140 days.
7. The method for efficiently culturing human embryonic stem cells in vitro induced differentiation into skin organoids according to claim 6, wherein the method comprises the steps of:
the volume ratio of the third culture medium is 1:1:1, mixing three media of Advanced DMEM/F12, neurobasal and defined Keratinoche-SFM as basic media, and adding RA with a final concentration of 5 mu M, N2 with a final concentration of 0.5% by volume, B27 with a final concentration of 1% by volume, beta mercaptoethanol with a final concentration of 0.1mM beta mercaptoethanol with a final concentration of 1% by volume, glutamax with a final concentration of 1.5% by volume;
the fourth culture medium is prepared by the following steps of: 1:1, neurobasal and defined Keratinoche-SFM as basal medium, and 5 μm RA was added thereto at a final concentration of 0.5% N2 by volume fraction, 1% B27 by volume fraction, 0.1mM beta mercaptoethanol at a final concentration of 1% Glutamax by volume fraction.
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