WO2020133436A1 - Procédé et milieu de culture permettant d'induire la différenciation d'une cellule souche embryonnaire en une cellule précurseur épithéliale glandulaire endométriale - Google Patents

Procédé et milieu de culture permettant d'induire la différenciation d'une cellule souche embryonnaire en une cellule précurseur épithéliale glandulaire endométriale Download PDF

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WO2020133436A1
WO2020133436A1 PCT/CN2018/125685 CN2018125685W WO2020133436A1 WO 2020133436 A1 WO2020133436 A1 WO 2020133436A1 CN 2018125685 W CN2018125685 W CN 2018125685W WO 2020133436 A1 WO2020133436 A1 WO 2020133436A1
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cells
endometrial
epithelial
embryonic stem
epithelial precursor
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PCT/CN2018/125685
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English (en)
Chinese (zh)
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江秀秀
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江秀秀
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Priority to CN201880010424.2A priority Critical patent/CN111642131B/zh
Priority to PCT/CN2018/125685 priority patent/WO2020133436A1/fr
Publication of WO2020133436A1 publication Critical patent/WO2020133436A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

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  • the present disclosure relates to the technical field of stem cell therapy, and in particular, to a method and medium for inducing embryonic stem cells to differentiate into endometrial glandular epithelial precursor cells.
  • the endometrium is a unique tissue that undergoes a periodic process of growth, differentiation, and exfoliation and is renewed during each menstrual cycle. Endometrial regeneration is essential for embryo implantation and maintenance of pregnancy. Uterine myomectomy causes severe endometrial damage, curettage, endometritis or endometrial tuberculosis resulting in thin endometrium, intrauterine adhesions, endometrial scar formation and fibrosis, which causes abnormal uterine bleeding, Abortion, pregnancy complications or infertility. Severe endometrial damage loses fertility and requires surrogacy or adoption.
  • the only way to treat uterine adhesions is to separate the adhesions under hysteroscopy while supplementing with IUD insertion, Foley balloon catheter, hyaluronic acid application or estrogen treatment, but severe adhesions relapse The rate is between 20% and 62.5%.
  • the pathological basis of intrauterine adhesions is the destruction of the basal lining of the endometrium, leading to the loss of endometrial epithelial stem cells, resulting in the proliferation of a large number of stromal cells, the formation of fibroblasts and even chondrocytes. Scar tissue replaced.
  • Stem cell therapy has broad prospects in the application of supplementary endometrial basal layer epithelial stem cells.
  • mesenchymal stem cells derived from bone marrow and umbilical cord mesenchymal stem cells, but these stem cells are derived from mesenchymal mesenchymal cells, and their differentiation potential is highly controversial.
  • mesenchymal stem cells derived from mesoderm can differentiate into endometrial glandular epithelial stem cells in vitro. Therefore, the mechanism of its clinical application in the treatment of intrauterine adhesions or thin endometrium is still unclear.
  • the tissue stem cells isolated from the endometrium proved to be the precursor cells of the endometrial epithelium.
  • the colony endometrial epithelial cells formed in vitro can provide a reliable source for the endometrial epithelial cells and can be passaged through the 3D culture system.
  • the yield is very low, and the isolation rate of epithelial stem cells colony from endometrial cells is about 1/10000. This severely limits the production of endometrial epithelial stem cells.
  • the purpose of the present disclosure is to provide a method of inducing human embryonic stem cells to differentiate into endometrial epithelial precursor cells.
  • the method can induce embryonic stem cells to differentiate into endometrial glandular epithelial precursor cells, and has the characteristics of simple operation, unlimited output and high purity of nearly 100%, and the endometrial glandular epithelial precursor cells obtained by the method are available It is used to treat endometrial loss, endometrial hyperplasia difficulties, intrauterine adhesions, early abortion and other related diseases.
  • the purpose of the present disclosure is to provide a culture medium for inducing human embryonic stem cells to differentiate into endometrial glandular epithelial precursor cells, which can be used to induce embryonic stem cells to form endometrial glandular epithelial precursor cells.
  • the purpose of the present disclosure is to provide an endometrial glandular epithelial precursor cell, which can only be differentiated into mature endometrial glandular epithelial cells, with the patient's own stromal cells as Niche cells, No immune rejection, safe and reliable, can be used to treat endometrial loss, endometrial hyperplasia difficulties, intrauterine adhesions, early abortion and other related diseases.
  • the purpose of the present disclosure is to provide the application of the aforementioned endometrial glandular epithelial precursor cells.
  • the endometrium mainly contains two types of cells.
  • One type is stromal cells, which differentiate into supporting tissues such as blood vessels, nerves, and fibers, derived from the mesoderm; the other Cell-like cells are epithelial cells that differentiate into mucous epithelium and luminal epithelium. They are functional cells of the endometrium, but are derived from endoderm or transformed from mesoderm.
  • endometrial stromal cells as Niche cells, which are not immunogenic, can be used in clinics, and are abundant in production, then induce the isolation of pure endometrial glandular epithelial precursors from embryonic stem cells Cells have broad clinical application prospects. But at present, there is a lack of methods to induce differentiation from embryonic stem cells to form endometrial glandular epithelial precursor cells.
  • the present disclosure provides a method for inducing human embryonic stem cells to differentiate into endometrial glandular epithelial precursor cells, which includes the following steps:
  • Epithelial cell differentiation step Put endothelial cells induced by human embryonic stem cells into epithelial cell differentiation medium to induce differentiation into epithelial cells;
  • Endometrial glandular epithelial precursor cell differentiation step the obtained epithelial cells are cultured in endometrial glandular epithelial precursor cell differentiation medium to induce differentiation to form endometrial glandular epithelial precursor cells;
  • the epithelial cell differentiation medium contains the following components: EGF (epidermal cell growth factor), FBS (fetal bovine serum) and GlutaMAX; wherein, the endometrial gland epithelial precursor cell differentiation medium contains the following components: EGF, FBS , GlutaMAX and WNT3A.
  • the concentration of EGF is 5-15 ng/ml.
  • the concentration of EGF may be any one of 10, 11, 12, 13, 14 or 50 ng/ml or a range between the two.
  • the content of FBS in the endometrial glandular epithelial precursor cell differentiation medium is 5%-15%.
  • the concentration of GlutaMAX is 1-3 mM.
  • the concentration of WNT3A is 150-250 ng/ml.
  • the concentration of WNT3A may be any one of 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ng/ml, or a range between the two.
  • the EGF concentration is 5-15 ng/ml
  • the FBS content is 5%-15%
  • the GlutaMAX concentration is 1-3 mM.
  • the epithelial cell differentiation medium is obtained by adding EGF, FBS, and GlutaMAX to the basic medium DMEM/F12.
  • the endoderm cells are endoderm cells with positive expression of CD324 and CD184.
  • the epithelial cells are pure epithelial cells with positive expression of SOX17.
  • the endometrial gland epithelial precursor cell differentiation medium contains the following components: 10 ng/ml EGF, 10% FBS, 2 mM GlutaMAX, and 200 ng/ml WNT3A.
  • the endometrial gland epithelial precursor cell differentiation medium is obtained from the basal medium DMEM/F12 supplemented with 10 ng/ml EGF, 10% FBS, 2 mM GlutaMAX, and 200 ng/ml WNT3A.
  • the time for inducing differentiation culture is 7-14 days.
  • the method before the step of inducing differentiation, the method further includes the step of inducing differentiation of endoderm;
  • the step of inducing differentiation of endoderm includes: using STEMdiff Definitive Endoderm Kit to induce differentiation of the human embryonic stem cells to form the endoderm cells.
  • the time for inducing differentiation culture is 4 days;
  • the time for inducing differentiation culture is 3 days.
  • the present disclosure provides a medium for inducing the differentiation of embryonic stem cells to form epithelial cells, layered with Fibronectin, which contains the following components: EGF, FBS and GlutaMAX, and the basic medium is DMEM/F12.
  • the present disclosure provides a medium for inducing embryonic stem cells to differentiate into endometrial gland epithelial precursor cells, layered with Matrigel, cultured in 3D medium, and stromal cells as Niche cells, medium Contains the following components: EGF, FBS, GlutaMAX and WNT3A.
  • the concentration of EGF in the culture medium is 5-15ng/ml
  • the content of FBS is 5%-15%
  • the concentration of GlutaMAX is 1-3mM
  • the concentration of WNT3A is 150-250ng/ml.
  • the culture medium is plated with Fibronectin and Matrigel, and endometrial stromal cells are used as niche cells.
  • the human embryonic stem cell is hESC9 (human embryonic stem cell H9).
  • the present disclosure provides endometrial glandular epithelial precursor cells cultured by the method for inducing differentiation of embryonic stem cells to form endometrial glandular epithelial precursor cells as described above.
  • the present disclosure provides a medicament for treating a disease, the medicament containing endometrial gland epithelial precursor cells as described above and a pharmaceutically acceptable excipient.
  • the present disclosure also provides a method of treating a disease, which is endometriosis, endometrial hyperplasia, and/or uterine adhesion disease, the method includes: The subject administers endometrial epithelial precursor cells as described above.
  • the subject is a human.
  • the method for inducing the differentiation of human embryonic stem cells to form endometrial gland epithelial precursor cells can induce the differentiation of human embryonic stem cells to form endometrial gland epithelial cells by scientifically and rationally matching the culture medium components and the ratio Somatic cells, and the resulting endometrial epithelial precursor cells have the characteristics of high yield and high purity up to 100% purity. This method solves the problem of purity and yield of clinical grade endometrial epithelial precursor cells.
  • the obtained endometrial glandular epithelial precursor cells can be used to treat endometriosis, endometrial hyperplasia, uterine adhesions and other related diseases, and can also be used to prepare endometriosis, uterus Endometrial endometrial hyperplasia, intrauterine adhesions and other related diseases.
  • FIG 1 shows H9 human embryonic stem cells (H9-hESCs/P44/SK), the embryonic stem cells at D0 days before differentiation induction.
  • Figure 2 shows the single cell confluent state before differentiation of embryonic stem cells.
  • Figure 3 shows differentiated endoderm cells.
  • Figure 4 shows CD184 and CD324 positive cells identified by FAS and FAC. Flow cytometric identification and sorting were performed on the 5th day of differentiation of H9 embryonic stem cells. CD184+ cells were 95.6% and CD324+ cells were 91.3%. CD184+/CD324+ endoderm cells were selected for epithelial cell differentiation.
  • Figure 5 is pure epithelial cells labeled with SOX17. Immunofluorescence was used to identify the differentiated epithelial cells. The epithelial cells were arranged in colony, blue for DAPI nuclear staining (A), red for SOX17 nuclear staining (B), and the two were purple after fusion (C).
  • A DAPI nuclear staining
  • B red for SOX17 nuclear staining
  • C purple after fusion
  • Figure 6 is the colony morphology formed by endometrial glandular epithelial precursor cells.
  • FIG. 7 is SSEA4 identifying the stemness of glandular epithelial precursor cells, and the differentiated glandular epithelial cells are pure epithelial cells.
  • Fig. 8 shows that the differentiated glandular epithelial precursor cells produce estrogen receptor (ER-alpha) under the induction of estrogen.
  • ER-alpha estrogen receptor
  • Fig. 9 is a model diagram of mouse uterine cavity adhesion.
  • Fig. 10 shows the morphology of endometrial cells regenerated in mouse endometrium 2 weeks after implantation of endometrial glandular epithelial precursor cells.
  • FIG. 11 shows that the P1 generation precursor cells were planted in the model mouse uterine cavity and the P2 generation precursor cells were planted in the model mouse uterine cavity, and the hyperplastic endometrial gland epithelial cells expressed the stem genes OCT and SSEA4.
  • Fig. 12 is a control in which the intimal thickening is clearly visible to the naked eye after implanting endometrial glandular epithelial precursor cells in mice.
  • Figure 13 is HE staining. Under the microscope, it can be seen that the endometrium surviving in the mouse uterine cavity can react to endogenous estrogen and progesterone, showing endometrial hyperplasia and secretion reactions.
  • the method for inducing the differentiation of embryonic stem cells to form endometrial epithelial precursor cells is as follows:
  • hESC Human embryonic stem cells H9 (hESC) were cultured in a 2 ml mTeSR1 medium (mTeSR TM 1, STEMCELL TECHNOLOGIES, Catalog No: 85850) in a 6-well culture plate in a 37°C, 5% CO 2 incubator.
  • mTeSR TM 1 STEMCELL TECHNOLOGIES, Catalog No: 85850
  • H9 embryonic stem cells become large and round, the germinal center is bright, and the peripheral cells are closely connected.
  • the cells are digested to form single cells, such as cell confluent as shown in Fig. 2, and then human embryonic stem cells H9 are cultured using endoderm cell induction differentiation medium, induced differentiation for 4 days to form endoderm cells. As shown in Figure 3, the cells are closely connected and form petals.
  • the endoderm cell induction differentiation medium used was prepared by STEMdiff TM Endoderm Basal Medium with STEMdiff TM Definitive Endoderm Kit ((#05110, STEMdiff TM Definitive Endoderm Kit)), the concentration of the added STEMdiff TM Definitive Endoderm Kit was 1 ⁇ g/ml .
  • Flow cytometry was used to detect CD324 and CD184 protein positive endoderm cells (CD324+/CD184+).
  • CD324 E-Cadherin
  • human 130-099-723Clone: 67A4, Miltenyl Biotec
  • PE Mouse Anti-Human CD184 (#561733Clone 12G5, Miltenyl Biotec).
  • Fig. 4 shows the results of flow cytometry to detect the content of two antibodies positive cells.
  • A is the proportion of CD184 and CD324 negative labeled cells
  • B is the proportion of CD184 antibody positive cells
  • C is the CD324 positive cell ratio.
  • Flow cytometric identification and sorting were performed on the 5th day of differentiation of H9 embryonic stem cells.
  • CD184+ cells were 95.6% and CD324+ cells were 91.3%.
  • CD184+/CD324+ endoderm cells were selected for epithelial cell differentiation.
  • the CD184+/CD324+ endoderm cells collected in step (3) were re-seeded in the wells covered with fibronctin (fibronctin), and epithelial cell differentiation medium was added to induce differentiation culture for 3 days.
  • the optimal number of cells per well in a 12-well culture plate is 200 ⁇ 10 3 cells/well.
  • 10 ng/ml fibronectin is used to make a gel, and the total volume of each well is 1 ml.
  • SOX17 antibody was used to identify the purity of epithelial cells. The results are shown in Figure 5.
  • red is SOX17 nuclear staining (A)
  • blue is DAPI and staining (B)
  • C purple after fusion
  • the epithelial cell differentiation medium is added with the following components on the basis of DMEM/F12: 10 ng/ml EGF, 2 mM GlutaMAX (Catalog No: 35050061, Thermo Fisher Scientific) and 10% FBS.
  • the differentiation-inducing endometrial glandular epithelial cell culture medium re-seed the SOX17+ epithelial cells collected in step (4) in an 8-well chamber slides 3D incubator layered with stromal cells, using 1:1 200 ⁇ 10 3 cells A mixture of epithelial cells and basement membrane matrix (Matrigel) with a total of 400 ⁇ l to make gel.
  • the endometrial glandular epithelial precursor cell differentiation medium is based on DMEM/F12 with the following components added: 10ng/ml EGF, 2mM GlutaMAX (Catalog No: 35050061, Thermo Fisher Scientific), 10% FBS and 200ng /ml WNT3A.
  • the cell morphology of the formed endometrial precursor glandular epithelial cells is shown in Figure 6. From day 3, the endometrial glandular epithelial precursor glandular epithelial cells can grow colony-like under the microscope and can be passaged after 2 weeks. Epithelial cells form colony, transparent and light and round.
  • Endothelial glandular epithelial precursor cells can be used for passage after 14 days of 3D culture. Cells were digested with TryplE 1ml, placed in an incubator for 3 minutes, and terminated with 4ml of DEME/F12. After cell counting and centrifugation, the cells were passaged with fresh medium suspension.
  • the primary endometrial gland epithelial precursor cells (P1 generation cells) obtained in the above step (4) and the endometrial epithelial precursor cells (P2 generation cells) passaged in the step (5) were identified.
  • the SSEA-4 antibody was used as a marker for endometrial gland epithelial precursor stem cells for identification. The results are shown in Figure 7. The cells were round and aggregated into colony with a purity of 100%. The expression of estrogen receptor was induced by estrogen (17 ⁇ -estradiol 2nM; Sigma-Aldrich), as shown in Figure 8.
  • the endometrium of the uterine cavity of one side of the mouse was scalded to remove the endometrium.
  • the modeling method is shown in Figure 9.
  • the side of the uterus of the mouse was scratched and the endometrium was removed after scald to form a scar.
  • Each uterine cavity was implanted with endometrial glandular precursor epithelial cells of Example 1 at 3, 6, 9, 12 o'clock in front of the uterus at 12 o'clock, and the implantation volume at each point was 300 ⁇ 103 , sutured wounds after implantation, conventional feeding in cages, 2 weeks after implantation, surgical resection of the endometrium at the implantation site was performed for control examination.
  • the method for inducing the differentiation of embryonic stem cells into endometrial epithelial precursor cells provided by the present disclosure is simple, high in purity, high in specificity, remarkable in effect, and free from the formation of malignant tumor cells. Therefore, the endometrial epithelial precursor cells provided by the embodiments of the present disclosure can be applied to the treatment of endometrial loss, adhesion and thin endometrial hyperplasia with different degrees, and has the prospect of application across the times.
  • the method disclosed in this disclosure for inducing the differentiation of human embryonic stem cells to form endometrial glandular epithelial precursor cells can induce the differentiation of human embryonic stem cells to form endometrial glandular epithelial precursor cells.
  • the operation is simple and one embryonic stem cell clone can be used Produce millions of high-yield and nearly 100% high-purity endometrial glandular epithelial precursor cells.
  • the resulting endometrial glandular epithelial precursor cells can be used to treat endometrial loss, endometrial hyperplasia, Related diseases such as intrauterine adhesions and early abortion of uterine origin.

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Abstract

L'invention concerne un procédé et un milieu de culture pour induire une cellule souche embryonnaire humaine à se différencier en une cellule précurseur épithéliale glandulaire endométriale. Le procédé comprend la culture d'une cellule épithéliale formée à partir d'une prolifération induite d'une cellule souche embryonnaire humaine dans un milieu de culture de différenciation pour une cellule précurseur épithéliale glandulaire endométriale pour induire la différenciation de la cellule épithéliale en cellule précurseur épithéliale glandulaire endométriale, le milieu de culture de différenciation pour une cellule précurseur épithéliale glandulaire endométriale comprenant les éléments suivants: EGF, FBS, GlutaMAX et WNT3A. Le procédé peut induire une cellule souche embryonnaire humaine à se différencier en une cellule précurseur épithéliale glandulaire endométriale. Le procédé est simple à mettre en oeuvre et peut produire, à partir d'un seul clone de cellules souches embryonnaires, des cellules précurseurs épithéliales glandulaires endométriales à un rendement élevé dans l'ordre de millions de cellules et avec une pureté élevée de presque 100 %. Les cellules précurseurs épithéliales glandulaires endométriales obtenues à partir du procédé peuvent être utilisées pour le traitement de maladies associées à l'absence d'endomètre, de difficulté d'hyperplasie endométriale, d'adhésions intra-utérines et d'avortement utérin précoce, entre autres.
PCT/CN2018/125685 2018-12-29 2018-12-29 Procédé et milieu de culture permettant d'induire la différenciation d'une cellule souche embryonnaire en une cellule précurseur épithéliale glandulaire endométriale WO2020133436A1 (fr)

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CN201880010424.2A CN111642131B (zh) 2018-12-29 2018-12-29 一种诱导胚胎干细胞分化形成子宫内膜腺上皮前体细胞的方法和培养基
PCT/CN2018/125685 WO2020133436A1 (fr) 2018-12-29 2018-12-29 Procédé et milieu de culture permettant d'induire la différenciation d'une cellule souche embryonnaire en une cellule précurseur épithéliale glandulaire endométriale

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834212A (zh) * 2017-03-24 2017-06-13 四川大学华西医院 一种用于肺组织3d培养的培养基
CN108396010A (zh) * 2017-02-06 2018-08-14 王琼 一种结直肠癌类器官的体外培养方法
CN109072179A (zh) * 2016-02-18 2018-12-21 学校法人庆应义塾 细胞培养基、培养方法以及类器官

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109072179A (zh) * 2016-02-18 2018-12-21 学校法人庆应义塾 细胞培养基、培养方法以及类器官
CN108396010A (zh) * 2017-02-06 2018-08-14 王琼 一种结直肠癌类器官的体外培养方法
CN106834212A (zh) * 2017-03-24 2017-06-13 四川大学华西医院 一种用于肺组织3d培养的培养基

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YU, W.Z. ET AL.: "Experimental and Therapeutic Medicine", CO-CULTURE WITH ENDOMETRIAL STROMAL CELLS ENHANCES THE DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO ENDOMETRIUM-LIKE CELLS, vol. 10, 31 December 2015 (2015-12-31), DOI: 20190914185059Y *
于文竹 (YU, WENZHU), 人胚胎干细胞向子宫内膜上皮样细胞诱导分化的相关研究 (THE RESEARCH OF HUMAN EMBRYONIC STEM CELLS DIFFERENTIATION TO ENDOMETRIAL EPITHELIAL-LIKE CELLS), 24 September 2015 (2015-09-24), DOI: 20190914184614Y *

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