CN111642131B - Method and culture medium for inducing differentiation of embryonic stem cells to form endometrial gland epithelial precursor cells - Google Patents
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Abstract
一种诱导人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。该方法包括：将由人胚胎干细胞诱导分化形成的上皮细胞置于子宫内膜腺上皮前体细胞分化培养基中培养以诱导分化形成子宫内膜腺上皮前体细胞；子宫内膜腺上皮分化培养基含有：EGF、FBS、GlutaMAX以及WNT3A。该方法可以诱导人胚胎干细胞分化形成子宫内膜腺上皮前体细胞，操作简单、一个胚胎干细胞克隆即可产生百万级的高产量和近乎100％的高纯度的子宫内膜腺上皮前体细胞，且由该方法所得的子宫内膜腺上皮前体细胞可用于治疗子宫内膜缺失、子宫内膜内膜增生困难、宫腔黏连、子宫源性早期流产等相关疾病。
A method and medium for inducing human embryonic stem cells to differentiate into endometrial glandular epithelial precursor cells. The method comprises: culturing epithelial cells induced and differentiated from human embryonic stem cells in an endometrial gland epithelial precursor cell differentiation medium to induce differentiation to form endometrial gland epithelial precursor cells; endometrial gland epithelial differentiation medium Contains: EGF, FBS, GlutaMAX and WNT3A. This method 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 produce millions of high-yield and nearly 100% high-purity endometrial glandular epithelial precursor cells. , and the endometrial glandular epithelial precursor cells obtained by this method can be used for the treatment of related diseases such as endometrial loss, endometrial hyperplasia difficulty, intrauterine adhesion, and uterine-derived early abortion.
Description
Technical Field
The disclosure relates to the technical field of stem cell therapy, in particular to a method and a culture medium for inducing embryonic stem cells to differentiate to form endometrial gland epithelial precursor cells.
Background
The endometrium is a unique tissue that undergoes a periodic process of growth, differentiation and denudation and is renewed every menstrual cycle. Endometrial regeneration is critical for embryo implantation and for the maintenance of pregnancy. Hysteromyomectomy causes severe endometrial injury, uterine curettage, endometritis or endometrial tuberculosis resulting in endometriotic thinness, uterine cavity adhesion, endometrial scar formation and fibrosis, thereby causing abnormal uterine bleeding, abortion, pregnancy complications or infertility. Severe endometrial damage loses fertility and requires the infant to be fed. At present, the method for treating the intrauterine adhesion only comprises the steps of hysteroscopic adhesion separation and intrauterine device insertion, Foley balloon catheter, hyaluronic acid application or estrogen treatment, but the recurrence rate of intrauterine adhesion with serious adhesion is between 20 and 62.5 percent. The pathological basis for the occurrence of uterine cavity adhesion lies in the destruction of the basal layer of the intima, resulting in the loss of intimal epithelial stem cells, resulting in the proliferation of a large number of stromal cells, the formation of fibroblasts and even chondrocytes, and the loss of the mucosal surface of the intima, which is replaced by fibrous scar tissue.
The stem cell therapy has broad prospects in applications for supplementing endometrial basal lamina epithelial stem cells. At present, bone marrow and umbilical cord mesenchymal-derived mesenchymal stem cells exist in clinical experiments, but the stem cells are all derived from mesodermal mesenchymal cells, the differentiation potential of the stem cells is greatly controversial, and no experimental evidence indicates that the mesodermal mesenchymal stem cells can differentiate endometrial gland epithelial stem cells in vitro. Therefore, the mechanism of treating the uterine cavity adhesion or thin endometrium in clinical application is not clear. Tissue stem cells isolated from endometrium-proved to be precursor cells of endometrial epithelium, and the endothelial epithelial cells forming colony in vitro, through a 3D culture system, can provide a reliable source for endometrial epithelial cells and can be passaged, but the yield is very low, and the separation rate of the epithelial stem cells colony from the endometrial cells is about 1/10000. This makes the yield of endometrial epithelial stem cells severely limited.
In view of this, the present disclosure is set forth.
Disclosure of Invention
The present disclosure is directed to a method of inducing human embryonic stem cells to differentiate to form endometrial epithelial precursor cells. The method can induce the differentiation of the embryonic stem cells to form endometrial gland epithelial precursor cells, has the characteristics of simple operation, unlimited yield, high purity of nearly 100 percent and the like, and the endometrial gland epithelial precursor cells obtained by the method can be used for treating related diseases such as endometrial deletion, endometrial hyperplasia difficulty, uterine cavity adhesion, early abortion and the like.
The human embryonic stem cells described in this disclosure are stem cells isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.
The present disclosure aims to provide a culture medium for inducing differentiation of human embryonic stem cells into endometrial gland epithelial precursor cells, which can be used for inducing differentiation of embryonic stem cells into endometrial gland epithelial precursor cells.
The invention aims to provide an endometrial gland epithelial precursor cell, which can only be directionally differentiated into mature endometrial gland epithelial cells, takes stromal cells of a patient as Niche cells, has no immunological rejection, is safe and reliable, and can be used for treating related diseases such as endometrial deletion, endometrial hyperplasia difficulty, uterine cavity adhesion, early abortion and the like.
The purpose of the present disclosure is to provide the application of the endometrial gland epithelial precursor cells.
The present disclosure is implemented as follows:
although the uterus, which is a member of the reproductive system, is derived from the mesoderm, the endometrium contains two types of cells, one is stromal cells, and supporting tissues such as blood vessels, nerves and fibers are differentiated and derived from the mesoderm; another type of cell is an epithelial cell, which differentiates between the mucous epithelium and the luminal epithelium, is a functional cell of the endometrium, but is derived from or transformed by the endoderm. Because the precursor stem cells differentiated from the embryonic stem cells take the endometrial stroma cells of a patient as the Niche cells, have no immunogenicity, can be used clinically, and have rich yield, the pure endometrial gland epithelial precursor cells induced and separated from the embryonic stem cells have wide clinical application prospect. However, there is currently no method for inducing differentiation from embryonic stem cells to form endometrial glandular epithelial precursor cells.
Based on this, in one aspect, the present disclosure provides a method of inducing differentiation of human embryonic stem cells to form endometrial gland epithelial precursor cells, comprising the steps of:
epithelial cell differentiation step: placing endoderm cells formed by inducing differentiation of human embryonic stem cells into an epithelial cell differentiation medium to induce differentiation to form epithelial cells;
differentiation step of endometrial gland epithelial precursor cells: culturing the obtained epithelial cells in an endometrial gland epithelial precursor cell differentiation culture medium to induce differentiation to form endometrial gland epithelial precursor cells;
wherein, the epithelial cell differentiation medium contains the following components: EGF (epidermal growth factor), FBS (fetal bovine serum), and GlutaMAX; wherein, the differentiation culture medium of the endometrial gland epithelial precursor cells contains the following components: EGF, FBS, GlutaMAX, and WNT 3A.
Alternatively, in some embodiments of the present disclosure, the concentration of EGF in the differentiation medium of endometrial glandular epithelial precursor cells is 5-15 ng/ml.
For example, the concentration of EGF may be any one of 10, 11, 12, 13, 14 or 50ng/ml or a range between the two.
Alternatively, in some embodiments of the present disclosure, the FBS content in the differentiation medium of endometrial glandular epithelial precursor cells is 5% -15%.
Alternatively, in some embodiments of the disclosure, the concentration of GlutaMAX in the differentiation medium of endometrial glandular epithelial precursor cells is 1-3 mM.
Alternatively, in some embodiments of the disclosure, the concentration of WNT3A in the differentiation medium of endometrial glandular epithelial precursor cells is 150-250 ng/ml.
For example, the concentration of WNT3A may be in a range between any one or both of 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ng/ml.
Alternatively, in some embodiments of the disclosure, the epithelial cell differentiation medium has an EGF concentration of 5-15ng/ml, an FBS content of 5% -15%, and a GlutaMAX concentration of 1-3 mM.
Alternatively, in some embodiments of the disclosure, the epithelial cell differentiation medium is obtained from basal medium DMEM/F12 supplemented with EGF, FBS and GlutaMAX.
Alternatively, in some embodiments of the disclosure, the endoderm cells are endoderm cells that are positive for CD324 and CD184 expression during the epithelial cell differentiation step.
Alternatively, in some embodiments of the present disclosure, in the step of differentiation of endometrial glandular epithelial precursor cells, said epithelial cells are pure epithelial cells positive for SOX17 expression.
Alternatively, in some embodiments of the present disclosure, the endometrial glandular epithelial precursor cell differentiation medium contains the following components: 10ng/ml EGF, 10% FBS, 2mM GlutaMAX and 200ng/ml WNT 3A.
Alternatively, in some embodiments of the disclosure, the differentiation medium of endometrial glandular epithelial precursor cells is obtained by adding 10ng/ml of EGF, 10% FBS, 2mM GlutaMAX and 200ng/ml of WNT3A to DMEM/F12, a basal medium.
Alternatively, in some embodiments of the present disclosure, in the endometrial glandular epithelial precursor cell differentiation step, the time period for inducing differentiation culture is 7-14 days.
Optionally, in some embodiments of the present disclosure, prior to the inducing differentiation step, the method further comprises: endoderm inducing differentiation;
the endoderm inducing differentiation step comprises: using STEMdiffTMThe Definitive Endoderm Kit induces the human embryonic stem cells to differentiate to form the Endoderm cells.
Alternatively, in some embodiments of the disclosure, in the endoderm differentiation-inducing step, the differentiation-inducing culture is for 4 days;
preferably, in the step of inducing differentiation of epithelial cells, the time for inducing differentiation culture is 3 days.
The present disclosure provides a culture medium for inducing differentiation of embryonic stem cells into epithelial cells, which comprises Fibronectin layering, wherein the Fibronectin layering comprises the following components: EGF, FBS and GlutaMAX, and the basal medium is DMEM/F12.
In another aspect, the present disclosure provides a medium for inducing differentiation of embryonic stem cells to form endometrial gland epithelial precursor cells, wherein the medium is layered with Matrigel, cultured in a 3D medium, and stromal cells are used as cache cells, and the medium contains the following components: EGF, FBS, GlutaMAX, and WNT 3A.
Alternatively, in some embodiments of the present disclosure, the concentration of EGF in the culture medium is 5-15 ng/ml;
preferably, the content of FBS is 5% -15%;
preferably, the concentration of GlutaMAX is 1-3 mM;
preferably, the concentration of WNT3A is 150-250 ng/ml.
Alternatively, in some embodiments of the disclosure, the medium is plated with Fibronectin and Matrigel, and endometrial stromal cells are niche cells.
Alternatively, in some embodiments of the disclosure, the human embryonic stem cell is hESC9 (human embryonic stem cell H9).
In another aspect, the present disclosure provides endometrial gland epithelial precursor cells cultured by the method for inducing differentiation of embryonic stem cells to form endometrial gland epithelial precursor cells as described above.
In another aspect, the present disclosure provides a medicament for treating diseases, the medicament comprising the endometrial gland epithelial precursor cells as described above and pharmaceutically acceptable excipients.
In yet another aspect, the present disclosure also provides a method of treating a condition selected from the group consisting of endometrial loss, endometrial hyperplasia, and uterine cavity adhesion, the method comprising: endometrial epithelial precursor cells as described above are administered to a subject suffering from the above-mentioned diseases.
Optionally, in some embodiments of the disclosure, the subject is a human.
According to the method for inducing the differentiation of the human embryonic stem cells into the endometrial gland epithelial precursor cells, the components and the proportion of the culture medium are scientifically and reasonably matched, the human embryonic stem cells can be induced and differentiated into the endometrial gland epithelial precursor cells, the obtained endometrial gland epithelial precursor cells have the characteristics of high yield, high purity of 100% and the like, and the method solves the problems of purity and yield of clinical-grade endometrial gland epithelial precursor cells.
In addition, the obtained endometrial gland epithelial precursor cells can be used for treating related diseases such as endometrial deletion, endometrial hyperplasia difficulty and uterine cavity adhesion, and can also be used for preparing medicines for treating related diseases such as endometrial deletion, endometrial hyperplasia difficulty and uterine cavity adhesion.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows H9 human embryonic stem cells (H9-hESCs/P44/SK), which were induced to differentiate D0 days before differentiation.
FIG. 2 shows the status of single cell confluent before differentiation of embryonic stem cells.
FIG. 3 shows differentiated endoderm cells.
FIG. 4 shows CD184 and CD324 positive cells identified by FAS and FAC. Flow cytometric identification and sorting from day 5 of differentiation of H9 embryonic stem cells yielded 95.6% CD184+ cells and 91.3% CD324+ cells, and CD184+/CD324+ endoderm cells were selected for differentiation of epithelial cells.
FIG. 5 shows SOX 17-labeled pure epithelial cells. Immunofluorescence identifies the differentiated epithelial cells, which are in colony arrangement, with DAPI nuclear staining (A) in blue, SOX17 nuclear staining (B) in red, and purple (C) after fusion.
Fig. 6 shows the colony morphology formed by endometrial gland epithelial precursor cells.
FIG. 7 shows SSEA4 identifying the dryness of glandular epithelial precursor cells, and the differentiated glandular epithelial cells are pure epithelial cells.
FIG. 8 shows that differentiated glandular epithelial precursor cells produce estrogen receptor (ER-alpha) under estrogen induction.
FIG. 9 is a schematic representation of the molding of mouse intrauterine adhesions.
FIG. 10 shows the morphology of endometrial cells regenerated in endometrium of mouse observed under microscope 2 weeks after implantation of endometrial gland epithelial precursor cells.
FIG. 11 shows that precursor cells of generation P1 are planted in the uterine cavity of a model mouse and precursor cells of generation P2 are planted in the uterine cavity of the model mouse, and the hyperplastic epithelial cells of the intima gland have the expression of siccative gene OCT and SSEA 4.
FIG. 12 is a control in which the thickening of the intima is visible to the naked eye after the mice have been implanted with intimal gland epithelial precursor cells.
FIG. 13 shows HE staining showing that the viable endometrium of mice in the uterine cavity reacts to endogenous estrogen and progestin, indicating intimal hyperplasia and secretion.
Detailed Description
The method for inducing differentiation of embryonic stem cells to form endometrial epithelial precursor cells provided in this example is as follows:
(1) maintenance of embryonic stem cells:
2ml of mTeSR1 medium (mTeSR) in 6-well plates using human embryonic stem cells H9(hESC)TM1, STEMCELL TECHNOLOGIES, Catalog No: 85850) Medium, at 37 degrees, 5% CO2Cultured in an incubator.
The observation result of the morphology of the human embryonic stem cells before differentiation is shown in figure 1, H9 embryonic stem cells become large and round, the germinal centers are bright, and the marginal cells are tightly connected.
(2) Induction of endoderm:
after the step (1) is finished, digesting the cells to form single cells, for example, digesting the cells to form single cells as shown in figure 2, and then culturing human embryonic stem cells H9 by using an endoderm cell induced differentiation medium, and inducing and differentiating for 4 days to form endoderm cells. Cells are tightly connected, forming petals, see fig. 3.
The endoderm cell induction differentiation medium used was STEMdiffTMEndoderm basic Medium adding STEMdiffTM Definitive Endoderm Kit((#05110，STEMdiffTMDefinitive Endoderm Kit)), STEMdiff added theretoTMThe concentration of the Definitive Enoderm Kit was 1. mu.g/ml.
(3) And (3) FAC detection:
endoderm cells positive for CD324 and CD184 protein expression (CD324+/CD184+) were detected using flow cytometry.
Flow sorting markers used: CD324(E-Cadherin) -APC, human (130-; PE Mouse Anti-Human CD184, (#561733Clone 12G5, Miltenyl Biotec).
On day 5, the cells were digested to form single cells for Flow cytometry sortation, and CD184+/CD324+ endoderm cells were selected for subsequent differentiation of epithelial cells. FIG. 4 shows the results of flow cytometry for positive cell content for 2 antibodies. In the figure: the A graph shows the proportion of cells with negative markers for both CD184 and CD324, the B graph shows the proportion of cells with positive antibodies for CD184, and the C graph shows the proportion of cells with positive antibodies for CD 324. Flow cytometric identification and sorting from day 5 of differentiation of H9 embryonic stem cells yielded 95.6% CD184+ cells and 91.3% CD324+ cells, and CD184+/CD324+ endoderm cells were selected for differentiation of epithelial cells.
(4) Induced differentiation of epithelial cells
The CD184+/CD324+ endoderm cells collected in step (3) were reseeded in fibronectin (fibronectin) -covered wells, and epithelial cell differentiation medium was added to induce differentiation culture for 3 days. The optimum number of cells per well of 12-well culture plate is 200X 103cells/well, 12-well plate, gel with 10ng/ml fibronectin, total volume 1ml per well. The purity of the epithelial cells was identified with the SOX17 antibody. The results are shown in FIG. 5. In FIG. 5, red color is nuclear staining with SOX17 (A), blue color is nuclear staining with DAPI and staining with B, and the two are purple after fusion (C).
The experimental steps are all at 37 ℃ and 5% CO2Cultured in an incubator. Wherein the epithelial cell differentiation medium is prepared by adding the following components on the basis of DMEM/F12: 10ng/mlEGF, 2mM GlutaMAX (Catalog No: 35050061, Thermo Fisher Scientific) and 10% FBS.
(5) Induced differentiation culture of endometrial gland epithelial precursor cells:
in the medium for inducing differentiation of endometrial gland epithelial cells, the epithelial cells of SOX17+ collected in step (4) were re-seeded into 8-well chamber slides 3D culture box with stroma cell layer, and 200X 10 of 1: 1 was applied thereon3The mixture of cells epithelial cells and basement membrane matrix (Matrigel) was 400. mu.l for gel preparation. Wherein the endometrial gland epithelial precursor cell differentiation medium is added with the following components on the basis of DMEM/F12: 10ng/ml EGF, 2mM GlutaMAX (Catalog No: 35050061, Thermo Fisher Scientific), 10% FBS, and 200ng/ml WNT 3A.
The observation result of the cell morphology of the formed endometrial precursor glandular epithelial cells is shown in FIG. 6, the endometrial precursor glandular epithelial cells can grow like colony under a microscope at the 3 rd day, and can be passaged after 2 weeks. Epithelial cells formed colony, clear, light and round.
(6) Passage of endometrial glandular epithelial precursor cells
Endometrial gland epithelial precursor cells are available for passage after 14 days of 3D culture. The cells were digested with 1ml tryplE, placed in an incubator for 3 minutes, stopped with 4ml DEME/F12, centrifuged for cell counting, and suspended in fresh medium for passaging.
(7) Identification of endometrial pre-epithelial cells
And (3) identifying the primary endometrial gland epithelial precursor cells obtained in the step (4) (P1 generation cells) and the endometrial gland epithelial precursor cells passaged in the step (5) (P2 generation cells).
The SSEA-4 antibody was used as a marker for endometrial gland epithelial precursor stem cells for identification, and the results are shown in FIG. 7, where the cells are round and aggregated into colony with a purity of 100%. Estrogen receptor expression following induction with estrogen (17 β -estradiol 2 nM; Sigma-Aldrich)) is shown in figure 8.
Example 2
10 female mice were modelled by intrauterine adhesion, see FIG. 9, control: the uterine cavity is not treated after being mechanically adhered; model group: mechanical adhesion of the uterine cavity was followed by primary and post-passaging sub-scar implantation of endometrial precursor glandular epithelial cells from example 1, respectively.
(1) The molding method comprises the following steps:
the endometrium of uterus of one mouse is completely scalded to remove the endometrium, the molding method is shown in figure 9, one side of uterus of the mouse is scratched, and the endometrium is removed after the scald to form scar.
(2) Sub-scar implantation of endometrial precursor gland epithelial cells
Each uterine cavity is implanted with endometrial gland precursor epithelial cells of example 1 at 12 points, namely, right in front of the uterus, 3 points, 6 points, 9 points and 12 points, and the implantation amount of each point is 300 multiplied by 103And suturing the wound surface after implantation, conventionally feeding in cages, and performing surgical incision on the implantation point intima contrast examination 2 weeks after implantation.
The results are shown in FIG. 10, where the mature endometrial epithelial cells are enlarged and cylindrical, forming colony, with distinct intracellular lacunae. Cellular immunofluorescence staining showed that the implanted precursor stem cells of both P1 and P2 retained good dryness, as represented by OCT +/SSEA4+, as shown in fig. 11. The hyperplasia of the intimal cells is obvious, the uterine wound surface has no obvious adhesion, and the intimal thickening can be seen by naked eyes, as shown in figure 12. Immunohistochemical HE staining showed that the intima reacted with estrogen and progestin, hyperplasia of glands formed significantly, ductal structures formed, and the lumen of the glands of the intima increased significantly during the secretory phase, as shown in fig. 13.
In conclusion, it can be seen that the method for inducing differentiation of embryonic stem cells to form endometrial epithelial precursor cells provided by the present disclosure has the advantages of simple steps, high purity, high specificity, significant effect and no formation of malignant tumor cells. Therefore, the endometrial epithelial precursor cells provided by the embodiment of the disclosure can be applied to endometrial deletion, adhesion and thin-type endometrial hyperplasia treatment in different degrees, and have an epoch-spanning application prospect.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
Industrial applicability: the method for inducing the differentiation of the human embryonic stem cells to form the endometrial gland epithelial precursor cells can induce the differentiation of the human embryonic stem cells to form the endometrial gland epithelial precursor cells, is simple to operate, can generate the endometrial gland epithelial precursor cells with high yield of millions and high purity of nearly 100 percent by cloning one embryonic stem cell, and the obtained endometrial gland epithelial precursor cells can be used for treating related diseases such as endometrial deletion, endometrial hyperplasia difficulty, uterine cavity adhesion, early abortion of uterine origin and the like.
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