CN111718890A

CN111718890A - Method for transdifferentiation of fibroblasts into glandular epithelial cells, culture system and application thereof

Info

Publication number: CN111718890A
Application number: CN201910208841.4A
Authority: CN
Inventors: 周琪; 李伟; 何正泉; 袁雪薇; 张映; 王柳
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2019-03-19
Filing date: 2019-03-19
Publication date: 2020-09-29
Anticipated expiration: 2039-03-19
Also published as: CN111718890B

Abstract

The invention relates to a method for transdifferentiating fibroblasts into glandular epithelial cells, a culture system and application thereof. The invention obtains chemically induced glandular epithelial cells (ciGE) with uterine glandular epithelial characteristics and response to ovarian hormones, which can be applied to the aspects of endometrial replacement and the like in clinical treatment. Obtaining ciGE by inducing fibroblasts using only chemical molecules has several advantages including cell permeability, ease of handling, lack of immunogenicity and ease of standardization, which make it an attractive strategy for clinical applications in the treatment of uterine disorders such as Absolute Uterine Factor Infertility (AUFI). Furthermore, the present inventors have found that upregulation of functionally related genes, including estrogen and progesterone response genes in ciGE, indicates that the obtained ciGE is an amplifiable functional uterine gland epithelial cell.

Description

Method for transdifferentiation of fibroblasts into glandular epithelial cells, culture system and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a method for transdifferentiating fibroblasts into glandular epithelial cells and a culture system and application thereof.

Background

Worldwide, approximately 15% suffer from infertility disorders, where infertility due to uterine abnormalities-Absolute Uterine Factor Infertility (AUFI) accounts for 3% -5% of the total female population. Currently, the primary method of treating AUFI is uterine transplantation (UTx) (see also

M., et al, transplantation 102, 569-577 (2018)). However, limited organ availability and ethical issues limit the widespread use of this technology. The artificial uterus can be used as an embryo incubator, and brings hopes to women with damaged or diseased uterus. The artificial uterus needs complete structure and sufficient cell sources, and the current cell sources are insufficient, so that the clinical application of the biological artificial uterus is hindered. Mammalian uterine tissue includes the endometrium, which is composed primarily of the luminal epithelium, glandular epithelium, and stromal cells, and the myometrium. Among them, glandular epithelium is essential for embryo implantation, and it responds to ovarian hormones such as estrogen (E2) and progesterone (P4), synthesizes and secretes growth factors such as leukemia inhibitory factor (Lif) to promote embryo implantation and development. A large number of studies have shownDefects in the glandular epithelium are indicative of female infertility.

Mammalian uterine Glandular Epithelium (GE) is essential for blastocyst implantation, and its deficiency can lead to infertility (see Gu, T.P., et al. Nature 477,606-610 (7366)). GE responds to ovarian hormones, estrogens and progesterone and induces the expression of embryonic implanted Lif (see Carson, D.D., et al. Dev Biol223, 217-684 (2000), and Demir, R., et al. Placenta 23, 672-684 (2002)). Reprogramming fibroblasts directly by specific Transcription Factors (TF) or chemical molecules has been previously reported (see Cieslar-Pobuda, A., et al, Biochim Biophys Acta Molcell Res 1864, 1359-. However, few reports have been made on obtaining uterine cells by fibroblast reprogramming.

Disclosure of Invention

The invention firstly utilizes a chemical method to directly transdifferentiate fibroblasts into glandular epithelial cells. The invention develops a culture system for expanding the epithelial cells of the uterine gland in vitro for a long time, and can provide a cell source for a mouse model for treating infertility caused by uterine factors and provide a cell source for in vitro reconstruction of the uterus.

The present invention has been completed based on the following findings of the inventors: the present invention has been accomplished by utilizing a culture system containing small chemical molecules to directly reprogram fibroblasts into glandular epithelial cells.

The invention therefore relates to a method for transdifferentiating fibroblasts into glandular epithelial cells, characterized in that a proline hydroxylase inhibitor is used in the induction process.

The invention relates to a non-therapeutic method for transdifferentiation of fibroblasts into glandular epithelial cells, characterized in that a proline hydroxylase inhibitor is used during the induction process.

The invention also relates to a culture system for in vitro amplification of uterine glandular epithelial cells, wherein the culture system comprises a proline hydroxylase inhibitor.

In the above embodiments, the proline hydroxylase inhibitor is a HIF prolyl 4-hydroxylase inhibitor.

In the above embodiments, the proline hydroxylase inhibitor is a HIF α prolyl 4-hydroxylase inhibitor.

In the above embodiment, the proline hydroxylase inhibitor is one or more of 1,4-DPCA, BAY87-2243, MK-8617, and JNJ-42041935. Wherein the structural formulas of the 1,4-DPCA, the BAY87-2243, the MK-8617 and the JNJ-42041935 are shown as follows.

Herein, the "proline hydroxylase inhibitor" is a substance capable of inhibiting the activity of Proline Hydroxylase (PH), and has the same mechanism of action to promote the stabilization of hypoxia inducible factor by inhibiting proline hydroxylase. The above proline hydroxylase inhibitors are known as common proline hydroxylase inhibitors.

In the above embodiment, an ALK inhibitor is also added during the induction process or in the culture system.

In the above embodiments, the ALK inhibitor is an ALK5 inhibitor.

In the above embodiments, the ALK inhibitor is one or more of a83-01, RepSox, SB 431542. Wherein, the structural formulas of A83-01, RepSox and SB431542 are shown as follows.

Herein, the "ALK 5 inhibitor" is a substance capable of inhibiting Aurora-like kinase (ALK-3 kinase5, ALK5, which is a transforming growth factor signal-beta (TGF- β) receptor), has the same action mechanism, and inhibits the transforming growth factor-beta signal pathway by inhibiting ALK 5. The ALK5 inhibitors described above are all known commonly used ALK5 inhibitors.

In the above embodiment, a GSK inhibitor is also added to the induction process or the culture system.

In the above embodiments, the GSK inhibitor is a GSK3 α/β inhibitor.

In the above embodiment, the GSK inhibitor is one or more of CHIR99021 and CHIR-98014. Wherein the structural formulas of CHIR99021 and CHIR-98014 are shown as follows.

Herein, a "GSK 3 α/β inhibitor" is a substance capable of inhibiting glycogen synthase (GSK 3 α/β), has the same mechanism of action, and activates Wnt signaling pathway by inhibiting GSK3 α/β. The GSK inhibitors are known as common GSK3 alpha/beta inhibitors.

In the above embodiment, any one or more of fibroblast growth factor 2(FGF2), bone morphogenetic protein 4(BMP4), and mouse leukemia inhibitory factor (mLif) is further added to the induction process or the culture system.

The invention also relates to the non-therapeutic use of any one of the above inhibitors or a combination of any two or three of them in stimulating fibroblast transdifferentiation.

The invention also relates to the application of any one of the inhibitors or the combination of any two or three of the inhibitors in preparing a medicament for stimulating the transdifferentiation of fibroblasts.

In one embodiment, said stimulated fibroblasts transdifferentiate into fibroblasts that transdifferentiate into glandular epithelial cells.

The invention also relates to the use of the above method and/or culture system to provide a source of cells for a mouse model for the treatment of infertility caused by uterine factors, or for the ex vivo reconstruction of the uterus.

In the present invention, the term "non-therapeutic" refers to the methods and/or uses of the present invention excluding the methods of diagnosis and treatment of diseases specified in article 25 of the Chinese patent Law.

In the present invention, the inventors obtained chemically induced glandular epithelial cells (ciGE) with characteristics of uterine glandular epithelium and responsive to ovarian hormones, which means that they can be applied in clinical treatment for endometrial replacement.

Obtaining ciGE by inducing fibroblasts using only chemical molecules has many advantages including cell permeability, ease of handling, lack of immunogenicity and ease of standardization, which make it an attractive strategy for clinical application in the treatment of uterine diseases such as AUFI.

The present inventors have discovered that upregulation of functionally-associated genes, including estrogen and progesterone response genes in ciGE, indicates that the obtained ciGE is an amplifiable functional uterine glandular epithelial cell.

The invention also relates to application of any one or the combination of any two or three of a proline hydroxylase inhibitor, an ALK inhibitor and a GSK inhibitor in preparation of a medicine for treating uterine factor infertility.

The invention also relates to the application of any one or the combination of any two or three of the proline hydroxylase inhibitor, the ALK inhibitor and the GSK inhibitor in the preparation of a medicament for promoting uterine regeneration.

The invention provides a brand new way for generating target cells by in-situ fibroblasts in damaged or aged uterus through chemical molecule induction. It also provides an in vitro model for embryo implantation and study of uterine structure or function loss. Meanwhile, the invention provides a new way for treating the uterine factor infertility and uterine regeneration.

Drawings

Figure 1a protocol for reprogramming Mouse Embryo Fibroblasts (MEFs) to ciges using small molecule induction medium (FBLDAC).

Representative morphology of MEF-derived clones induced by FBLDAC induction medium. b left, control; b, right, day 12; c left, growth on matrigel; c right, grown on 10% FBS.

Figure 1d chemically induced epithelial cell expansion over 20 passages exhibiting a "38 + XX" karyotype.

Figure 1e chemically induced immunofluorescent staining of the epithelial cell characteristic protein KRT19, EPCAM, CDH1, scale 50 μm. The marker protein KI67 of the proliferating cells was immunofluorescent-stained with a 100 μm ruler.

FIG. 2 (Fsp1) -Cre/ROSA26^mTmGFibroblasts are epithelial cells. (a) (Fsp1) -Cre/ROSA26^mTmGSchematic representation of fibroblasts. (b) Staining identifies epithelial cell fate of the starting fibroblasts. (c) GFP positive clones were generated after chemical induction. (d) Staining identifies epithelial cell fate after chemical induction.

FIG. 3. identification of chemically induced epithelial cells as glandular epithelial cell fates. (a) Heatmaps of RNA-Seq data and hierarchical clustering of genes. (b) Expression hotspot graph and Gene Ontology (GO) analysis of differentially expressed genes. (c-d) expression of genes specifically associated with uterine epithelial cells.

Figure 4. chemically induced uterine glandular epithelium has the ability to form glands. (a) Expression of adult stem/progenitor-related genes. (b) Self-assembled into a cavity structure, scale 75 μm.

Figure 5 chemically induced uterine glandular epithelium responds to stimulation by egg-derived hormones. (a-c) Progesterone response gene (5a), Estrogen response gene (5b) and uterine implantation related gene (5c) are all significantly upregulated. (d) MEF and chemistry induce uterine glandular epithelium in response to progesterone and estrogen stimulation.

Detailed Description

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not purposely formulated into the composition and/or is present only as a contaminant or in trace amounts. Thus, the total amount of a particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions wherein the amount of a particular component is not detectable by standard analytical methods.

As used in this specification, "a" or "an" may mean one or more. As used in the claims, the words "a" or "an" when used in conjunction with the word "comprising" may mean one or more than one.

The use of the term "or" in the claims is intended to mean "and/or" unless explicitly indicated to refer only to alternatives or that alternatives are mutually exclusive, although the disclosure supports definitions referring only to alternatives and "and/or". As used herein, "another" may mean at least a second or more.

Throughout this application, the term "about" is used to indicate that the value includes the inherent variation of error of the device, and the method is used to determine the value or variation that exists between subjects.

In this context, "differentiation" is the process by which less specialized cells become more specialized cell types. "dedifferentiation" is a cellular process in which partially or terminally differentiated cells revert to an earlier developmental stage, such as pluripotency or multipotency. "transdifferentiation" is the process of converting one differentiated cell type into another differentiated cell type. Typically, transdifferentiation occurs by programming without the cells going through an intermediate pluripotency stage-i.e., the cells are programmed directly from one differentiated cell type to another.

As used herein, the term "subject" or "subject in need thereof refers to a mammal, preferably a human, of any age, male or female, in need of cell or tissue transplantation. Typically, a subject is in need of a cell or tissue transplant (also referred to herein as a recipient) due to a disorder or pathological or undesirable condition, state or syndrome or physical, morphological or physiological abnormality that is amenable to treatment via cell or tissue transplant.

Some terms referred to herein are defined as follows:

FGF 2: fibroblast growth factor 2.

BMP 4: bone morphogenetic protein 4(bone morphogenetic protein4, bmp 4).

mLif: mouse leukemia inhibitory factor.

High-glucose DMEM: a high-sugar DMEM medium (DMEM), a commercially available medium containing various glucose and amino acids, was developed on the basis of MEM medium.

N2B 27: a cell culture solution which is prepared by mixing a DMEM/F12 basal medium and a Neurobasal medium at a ratio of 1:1 and comprises a N2 additive and a B27 additive and has definite components is reported to be beneficial to the neural differentiation of mouse embryonic stem cells.

DMEM/F12: a commercial basal medium prepared by mixing DMEM medium and F12 medium at a ratio of 1:1 is suitable for clone density culture.

Neurobasal: is favorable for the commercial basic culture medium of the nerve cell culture.

N2 additive: a commercial serum-free cell culture additive.

B27 additive: a commercial serum-free cell culture additive.

1,4-DPCA, BAY87-2243, MK-8617, JNJ-42041935 proline hydroxylase inhibitors, wherein proline hydroxylase is an important enzyme for hypoxia inducible factor hydroxylation.

A83-01: a selective TGF-beta inhibitor can obviously inhibit the activities of ALK4, ALK5 and ALK 7.

Repssox: a potent and selective TGF beta R-1/ALK5 inhibitor.

SB 431542: a potent and selective ALK5 inhibitor.

CHIR 99021: a potent GSK-3 alpha/beta inhibitor.

CHIR-98014: a potent GSK-3 alpha/beta inhibitor.

The invention relates to a method for transdifferentiating fibroblasts into glandular epithelial cells, characterized in that a proline hydroxylase inhibitor is used in the induction process.

In the above embodiment, the proline hydroxylase inhibitor is one or more of 1,4-DPCA, BAY87-2243, MK-8617, and JNJ-42041935.

In the above embodiment, an ALK inhibitor is also added during induction.

In the above embodiments, the ALK inhibitor is an ALK5/4/7 inhibitor.

In the above embodiments, the ALK inhibitor is one or more of a83-01, RepSox, SB 431542.

In the above embodiment, a GSK inhibitor is also added during induction.

In the above embodiments, the GSK inhibitor is a GSK3 α/β inhibitor.

In the above embodiment, the GSK inhibitor is one or more of CHIR99021 and CHIR-98014.

In the above embodiment, any one or more of fibroblast growth factor 2(FGF2), bone morphogenic protein 4(BMP4), mouse leukemia inhibitory factor (mLif) is also added during the induction process.

The invention also relates to the use of any one of the above inhibitors or a combination of any two or three of them for stimulating fibroblast transdifferentiation.

In the above embodiment, an ALK inhibitor is also added to the culture system.

In the above embodiments, the ALK inhibitor is an ALK5/4/7 inhibitor.

In the above embodiment, a GSK inhibitor is further added to the culture system.

In the above embodiments, the GSK inhibitor is a GSK3 α/β inhibitor.

In the above embodiment, any one or more of FGF2, BMP4, mLif is also added to the culture system.

The invention also relates to an induction culture solution for chemically reprogramming fibroblasts into glandular epithelial cells, which is characterized by comprising any one or more of 1,4-DPCA, A83-01, CHIR99021, FGF2, BMP4 and mLif.

The following detailed description illustrates and describes embodiments of the present invention with reference to specific examples, but the following should not be construed as limiting the invention in any way.

Examples

The embodiments of the present invention will be described and illustrated in detail with reference to the following specific examples, but the following should not be construed as limiting the present invention in any way, and the materials and the like used in the examples are commercially available products unless otherwise specified.

Example one

Separating mouse day 13.5 embryo to prepare fetal fibroblast (MEFs), inoculating MEF cell in culture medium of 0.1% gelatin for one day, culturing with inducing culture solution containing small molecular compound for 12 days, changing the culture solution every 3 days, cloning to appear in culture time of 8-12 days, changing the culture medium into expanding culture solution EFLAC, culturing, and continuously expanding the cell in inducing culture solution for 1:4-1:6 passages. As shown in fig. 1a.

After 12 days of FBLDAC medium induction, MEF cells will be reprogrammed to epithelial-like clones, as shown in figure 1b, which can be stably passaged for more than 20 passages in matrigel or 10% FBS-covered dishes, as shown in figure 1c, and which have a stable karyotype of "38 + XX", as shown in figure 1d. These induced cells expressed specific marker genes for epithelial cells, such as KRT19, EPCAM and CDH1, and the proliferation protein KI67, as shown in fig. 5.

The induction medium (FBLDAC) described above comprises DMEM/F12(Gibco, 12400-^TMSupplement (Gibco, 35050079, 200 ×), serum replacement (Gibco, 10828028, 10%), β -mercaptoethanol (Gibco, 21985, 55. mu.M), bovine serum albumin (sigma, A7906-100G, 0.002%), fibroblast growth factorSon (FGF2, R)&D, 233-FB-001MG/CF, 20ng/mL), bone morphogenetic protein 4(BMP4, R)&D, 233-FB-001MG/CF, 10ng/mL), mouse leukemia inhibitory factor (mLif, Millipore, ESG1007, 1000U/mL), streptomycin (Gibco, 15140-.

The amplification culture solution comprises: [ DMEM/F12 and neurobasal (3:1), serum replacement (Gibco, 10828028, 2%), epidermal growth factor (R & D, 2028-EG-200, 100ng/mL), fibroblast growth factor (R & D, 233-FB-001MG/CF, 10ng/mL), mouse leukemia inhibitory factor (Millipore, ESG1007, 1000U/mL), A83-01(Stemgent, 04-0014, 5. mu.M), CHIR99021(Stemgent, 04-0004, 3. mu.M), heparin (sigma, H4784, 1. mu.g/mL), beta-mercaptoethanol (Gibco, 21985, 55. mu.M), bovine serum albumin (sigma, A7906-100G, 0.002%), non-essential amino acids (Gibco, 11140-.

Example two

Next, to evaluate the tumorigenicity of induced epithelial cells, we individually measured 5x 10⁶Each of the cigE and mouse Embryonic Stem Cells (ESC) was transplanted subcutaneously into hind limbs of 5-week-old Balb/c male nude mice, and the tumorigenic effect was monitored three weeks later. Our results show that 8 out of 10 mice transplanted with ESCs developed teratomas after 3 weeks, and that mice transplanted with cigE had no tumor formation after two months, as shown in Table 1.

TABLE 1 ratio of tumor formation

EXAMPLE III

To more strictly demonstrate that epithelial cells do indeed consist of MEF cellsInduced by our induction, we carried fibroblast specific protein 1(Fsp1) -Cre/ROSA26^mTmGMEF was isolated from the transgenic mice to follow the fibroblasts. Fibroblasts permanently express membrane-targeted green fluorescent protein (mG) after Fsp1-Cre mediated excision of membrane-targeted tomato (mT) expression, as shown in FIG. 2 a. These tracking cells exclude contamination from epithelial cell fates and are induced according to the process shown in FIG. 1a. This result was more confirmed by the epithelial markers KRT19 and EPCAM staining, as shown in figure 2 b. The cells appeared green clones after FBLDAC induction as shown in figure 2 c. Induced epithelial cell fate was determined by staining for KRT19 and EPCAM staining, as shown in figure 2 d.

Example four

To further characterize these chemically induced epithelial cells, we performed whole transcriptome sequencing of ciGE, starting MEF and primary epithelial cells isolated from mouse uterus (priUterus), respectively. Clustering analysis showed that the gene expression pattern of ciGE is closely related to privters, but greatly different from MEF, as shown in fig. 3 a. By GO analysis, we found that the genes expressed in ciGE were associated with uterine development and estrogen response. Compared to MEF, ciGE is mainly enriched with GO terms related to epithelial cell fate and function, which was found to characterize ciGE with uterine glandular epithelium by comparison with reported data, as shown in fig. 3 b. The expression of the genes characteristic of the uterine gland epithelium (FOXA2 and SOX17) was verified by immunofluorescence staining and quantitative RT-PCR method, as shown in FIGS. 3c and 3 d.

And (3) immunofluorescence staining: cells were cultured on glass coverslips, then fixed with 4% PFA for 1 hour, followed by 3 washes with PBS. Cells were blocked with 0.1% Triton X-100 for 30 min and 2% BSA for 1 h at Room Temperature (RT). The cells were then incubated with the primary antibody overnight at 4 ℃ and then with the species-specific secondary antibody for 1 hour at room temperature. Cells were incubated with DAPI for 10 min at room temperature. Confocal microscopy (Leica TCS Sp8) images were taken. Antibody information used: anti-KRT19(Rabbit, Abcam, Ab52625, 1:500), anti-EPCAM (Rabbit, Abcam, Ab71916, 1:500), anti-CDH1(Rat, Sigma, U3254, 1:500), anti-KI67(Rabbit, Thermo Fisher scientific, PA5-19462,1:200), anti-FOXA2(Goat, Santa Cruz, Sc-9187, 1:200), anti-CD133(Rabbit, Abcam, Ab16518, 1:500), anti-SCA-1(Rat, Abcam, 51317, 1:500), anti-CDX2(Rabbit, Cell cloning Technology, Ab 7s, 1: 200).

Quantitative PCR: total cellular RNA was extracted using TRIzol reagent (Invitrogen, 15596-018). A high capacity cDNA reverse transcription kit (ABI, 4368814) was used to reverse transcription of cDNA. Relative gene expression was analyzed based on the 2- Δ Δ Ct method, GAPDH as an internal control. The information of the primers used is shown in Table 2.

TABLE 2 primers used in quantitative PCR in examples IV, V and VI

EXAMPLE five

We further tested the high expression of somatic stem/progenitor markers in ciGE as shown in figure 4 a. The ciGE was partially digested with 0.25% trypsin, and the cell aggregates were gently pipetted out and plated onto matrigel-pre-coated petri dishes for 7-14 days. ciGE is able to form glandular-like structures with cavities that express epithelial proteins, which means that glandular production of ciGE can occur, as shown in figure 4 b.

EXAMPLE six

Further analysis showed that progesterone (E2) responsive gene (fig. 5a), estrogen (p4) responsive gene (fig. 5b) and uterine implantation related gene (fig. 5c) were all significantly up-regulated in the chemically induced uterine glandular epithelium. Under physiological conditions, the epithelium of the uterine gland can secrete factors such as Lif under the stimulation of ovogenous hormones such as progesterone and estrogen during pregnancy, so as to promote implantation and embryonic development. To further investigate whether these cells have the ability to respond to progesterone and estrogen stimulation. We incubated cigE or MEF with 8nM estrogen (Sigma, E8875) and 200ng/mL progesterone (Sigma, p8811) or DMSO (Sigma, D2650) for 3 days in amplification medium without mLif, N2 and B27 and collected the mRNA to identify gene expression. The results show that: compared to MEF, ciGE was significantly upregulated after hormone treatment in E2 and p4 response genes, as shown in fig. 5 d. These results indicate that small molecule induction media can reprogram fibroblasts into expandable functional uterine gland epithelial cells.

The foregoing merely illustrates the principles of the invention and it is understood that the scope of the invention is not intended to be limited to the exemplary aspects described herein but is to include all equivalents that are currently known and that are developed in the future. In addition, it should be noted that several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be construed as the scope of the present invention.

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Claims

1. A non-therapeutic method of transdifferentiating fibroblasts into glandular epithelial cells, characterized in that a proline hydroxylase inhibitor is used in the induction process.

2. A culture system for expanding uterine glandular epithelial cells in vitro, wherein the culture system comprises a proline hydroxylase inhibitor.

3. The method according to claim 1 or culture system according to claim 2, wherein the proline hydroxylase inhibitor is a HIF prolyl 4-hydroxylase inhibitor;

preferably, the proline hydroxylase inhibitor is a HIF α prolyl 4-hydroxylase inhibitor;

more preferably, the proline hydroxylase inhibitor is selected from one or more of 1,4-DPCA, BAY87-2243, MK-8617 and JNJ-42041935.

4. The method according to any one of claims 1 to 3 or the culture system according to any one of claims 2 to 3, wherein an ALK inhibitor is further added during the induction process or to the culture system.

5. The method of any one of claims 1, 3 to 4 or the culture system of any one of claims 2 to 4, wherein the ALK inhibitor is an ALK5 inhibitor;

preferably, the ALK inhibitor is one or more of A83-01, RepSox and SB 431542.

6. The method of any one of claims 1, 3 to 5 or the culture system of any one of claims 2 to 5, wherein a GSK inhibitor is further added during the induction process or to the culture system.

7. The method of any one of claims 1, 3 to 6 or the culture system of any one of claims 2 to 6, wherein the GSK inhibitor is a GSK3 a/β inhibitor; preferably, the GSK inhibitor is one or more of CHIR99021 and CHIR-98014.

8. The method according to any one of claims 1, 3 to 7 or the culture system according to any one of claims 2 to 7, wherein any one or two or three of fibroblast growth factor 2(FGF2), bone morphogenic protein 4(BMP4) and mouse leukemia inhibitory factor (mLif) is further added during the induction process or the culture system.

9. Non-therapeutic use of any one of a proline hydroxylase inhibitor, an ALK inhibitor, a GSK inhibitor, or a combination of any two or three thereof, in stimulating fibroblast transdifferentiation.

10. The application of any one or the combination of any two or three of proline hydroxylase inhibitor, ALK inhibitor and GSK inhibitor in preparing a medicament for stimulating fibroblast transdifferentiation.

11. Use according to claim 9 or 10, wherein said stimulated fibroblasts transdifferentiate into fibroblasts that transdifferentiate into glandular epithelial cells.

12. The application of any one or the combination of any two or three of a proline hydroxylase inhibitor, an ALK inhibitor and a GSK inhibitor in preparing a medicament for treating uterine factor infertility and a medicament for promoting uterine regeneration.