An inducer for inducing mesenchymal stem cells to differentiate into estradiol-secreting cells

Abstract

The present application belongs to the field of biomedicine, and relates to an inducer for inducing mesenchymal stem cells to differentiate into estradiol-secreting cells. The inducer for inducing mesenchymal stem cells to differentiate into estradiol-secreting cells is based on a human mesenchymal stem cell serum-free culture medium, and is composed of the following components in a mass concentration ratio: bone morphogenetic protein-4 20-60 mg / L, bone morphogenetic protein-7 20-60 mg / L, tretinoin 2-8 mg / L, resveratrol 2-8 mg / L, icariin 2-8 mg / L, benzamide 2-8 ug / L, chloroplatinic acid hexahydrate 2-8 ug / L, ethanolamine 2-8 ug / L, erythropoietin 2-10 ug / L, and vascular endothelial growth factor 2-10 ug / L. The inducer for inducing mesenchymal stem cells to differentiate into estradiol-secreting cells has high induction efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an inducer for the differentiation of mesenchymal stem cells into estradiol-secreting cells. Background Technology

[0002] Estrogen plays a vital role in a woman's life that no other hormone can replace. It governs the development and maintenance of female secondary sexual characteristics, regulates the homeostasis of the female body, and controls the female life cycle. A woman's cyclical menstruation, fertility, and characteristic female physique all depend on the action of estrogen. There are three main physiologically present estrogens in the human body: estradiol (E2), estrone, and estriol. They are primarily produced by the ovaries, follicles, corpus luteum, and placenta during pregnancy. A deficiency in estrogen can lead to decreased function in multiple organs of the female body.

[0003] Premature ovarian failure (POF) refers to the cessation of menstruation before the age of 40 due to ovarian insufficiency. It is characterized by primary or secondary amenorrhea accompanied by elevated levels of gonadotropins and decreased levels of estrogen, along with varying degrees of hypoestrogenic symptoms such as hot flashes, night sweats, facial flushing, and decreased libido. Currently, hormone replacement therapy is generally used for women with POF to improve various complications caused by low estrogen levels. Exogenous steroid hormones are administered to POF patients to induce an artificial cycle, thereby promoting normal endometrial development and facilitating pregnancy. However, long-term hormone replacement therapy is difficult to achieve at a reasonable dosage, and may have side effects and potential risks, resulting in low ovarian function recovery efficiency. Therefore, there is an urgent need to find alternative solutions to this problem.

[0004] Germ cells (PCGs) are the collective term for cells in a multicellular organism capable of reproduction, encompassing everything from primordial germ cells to fully differentiated germ cells, including sperm and egg cells. In the later stages of embryonic development, germ cells cease to divide and proliferate; only a small portion of oogonia grow and differentiate into primary oocytes. These oocytes then undergo a series of processes to form primordial follicles, which then progress through the preantral follicle stage to become growing follicles, eventually maturing. After birth, the number of oocytes in a woman continuously decreases. When this number reaches a certain level, it leads to menopause. Menopause is accompanied by a decline in estrogen levels, which can increase the risk of cardiovascular, cerebrovascular, endocrine, bone, and nervous system diseases in women.

[0005] During the differentiation of stem cells into functional oocyte-like cells, the simultaneous development of newly formed follicles inevitably leads to the synthesis of sex hormones (estrogen, androgens, etc.), which in turn act on the oocytes, promoting their development. Because theca cells produce androgens, which are then transported to granulosa cells to produce estrogens, the hormones required for physiological activities are replenished through feedback and negative feedback mechanisms under the regulation of the patient's own hypothalamic-pituitary-target gland axis. This avoids the problems of overdose or underdose caused by hormone replacement therapy, a key difference from traditional exogenous hormone supplementation and its greatest advantage. Current stem cell induction methods mainly include in vitro induction, gene modification, protein transduction, and tissue microenvironment induction. In vitro induction uses different combinations of stimulating factors to induce stem cells to differentiate into target cells. However, the induction conditions vary across laboratories, the induction mechanisms are not yet fully understood, the induction efficiency is low, the induction process is complex and time-consuming, and the resulting cell numbers and functional levels are low.

[0006] Current research largely focuses on preparing embryoid bodies from mesenchymal stem cells and then co-culturing them with follicular fluid and leukemia suppressor factor (LIF). Professor Song Yangzhou of Sun Yat-sen University has used retinoic acid (RA) as an inducer to induce embryonic stem cells to differentiate into germ cells, and previous studies have also reported on RA inducing umbilical cord stem cells to differentiate into PCGs. However, only one study reported that RA can induce stem cells to differentiate into germ cells or promote the secretion of estrogen such as E2 from stem cells, and the differentiation rate was not significantly different from the control group.

[0007] Bone morphogenetic proteins (BMPs) are important regulators of estrogen and progesterone production. They can enhance the responsiveness of granulosa cells to follicular estrogen, promote E2 production, and inhibit progesterone production. Studies have found that bone morphogenetic protein-4 (BMP4) and bone morphogenetic protein-7 (BMP7) can inhibit progesterone secretion and increase E2 secretion through paracrine effects, and their mechanism of action may promote the transformation of primordial follicles into primary follicles. Kehkooi Kee and Katsuhiko et al. published similar papers in journals such as Nature and Science, respectively, using BMP4, BMP7, and BMP8b as inducers to induce embryonic stem cells to differentiate into PGCs.

[0008] In summary, this invention combines agents that induce the differentiation of embryonic stem cells into germ cells and mesenchymal stem cells into germ cells, aiming to find highly efficient agents that induce the differentiation of mesenchymal stem cells into E2 secretory cells. Through repeated experiments and in conjunction with literature, the inventors unexpectedly discovered that, based on BMP4, BMP7, and RA, the combination of benzamide, chloroplatinic acid hexahydrate, and ethanolamine can significantly improve the efficiency of inducing mesenchymal stem cells to differentiate into E2 secretory cells. Furthermore, the combination of antioxidants resveratrol and icariin, as well as growth factors erythropoietin (EPO) and vascular endothelial growth factor (VEGF), can significantly improve the viability and proliferation capacity of the induced E2 secretory cells. Summary of the Invention

[0009] The purpose of this invention is to address the aforementioned problems in the prior art by providing an inducer for the differentiation of mesenchymal stem cells into estradiol (E2)-secreting cells. Using serum-free human mesenchymal stem cell culture medium as a matrix, the inducer comprises the following components in the indicated mass concentrations: bone morphogenetic protein-4 (BMP4) 20-60 mg / L, bone morphogenetic protein-7 (BMP7) 20-60 mg / L, retinoic acid (RA) 2-8 mg / L, resveratrol 2-8 mg / L, icariin 2-8 mg / L, benzamide 2-8 μg / L, chloroplatinic acid hexahydrate 2-8 μg / L, ethanolamine 2-8 μg / L, erythropoietin (EPO) 2-10 μg / L, and vascular endothelial growth factor (VEGF) 2-10 μg / L. This inducer requires fewer steps, takes less time, and has high induction efficiency.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is: an inducer for inducing mesenchymal stem cells to differentiate into E2 secretory cells, which is composed of the following components: BMP4, BMP7, RA, resveratrol, icariin, benzamide, chloroplatinic acid hexahydrate, ethanolamine, EPO and VEGF.

[0011] The mass concentration ratio of each component of the inducer is as follows: BMP4 20-60 mg / L, BMP7 20-60 mg / L, RA 2-8 mg / L, resveratrol 2-8 mg / L, icariin 2-8 mg / L, benzamide 2-8 ug / L, chloroplatinic acid hexahydrate 2-8 ug / L, ethanolamine 2-8 ug / L, EPO 2-10 ug / L, VEGF 2-10 ug / L.

[0012] Preferably, the mass concentration ratio of each component of the inducer is as follows: BMP4 50 mg / L, BMP7 50 mg / L, RA 8 mg / L, resveratrol 6 mg / L, icariin 6 mg / L, benzamide 4 μg / L, chloroplatinic acid hexahydrate 6 μg / L, ethanolamine 4 μg / L, EPO 5 μg / L, and VEGF 5 μg / L.

[0013] The present invention provides an inducer for the differentiation of mesenchymal stem cells into E2 secretory cells, which has the following advantages: 1. No gene transfection is required, thus eliminating the risk of gene alteration and cancer, and ensuring high safety; 2. Fewer induction steps and shorter induction time; 3. Combining benzamide, chloroplatinic acid hexahydrate, and ethanolamine can significantly improve the efficiency of inducing mesenchymal stem cells to differentiate into E2 secretory cells; 4. Combining antioxidants resveratrol and icariin with growth factors EPO and VEGF can significantly improve the viability and proliferation capacity of the E2 secretory cells obtained after induction. Detailed Implementation

[0014] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. All equipment, instruments, and reagents used in the experiments are commercially available.

[0015] Example 1 This example describes an inducer for converting mesenchymal stem cells into E2 secretory cells, composed of the following components in the indicated mass concentrations: BMP4 40 mg / L, BMP7 50 mg / L, RA 8 mg / L, resveratrol 2 mg / L, icariin 6 mg / L, benzamide 4 μg / L, chloroplatinic acid hexahydrate 8 μg / L, ethanolamine 4 μg / L, EPO 10 μg / L, and VEGF 2 μg / L. The above components are added sequentially to serum-free human mesenchymal stem cell culture medium (or DMEM + 10% FBS or other commercially available mesenchymal stem cell culture medium) according to the indicated mass concentrations, mixed thoroughly, and then filtered for sterilization.

[0016] The inducing agents of this invention are all commercially available products: serum-free culture medium for human mesenchymal stem cells, brand LONZA, catalog number 00190632; BMP4, brand Gibco, catalog number PHC9533; BMP7, brand Gibco, catalog number PHC7204; RA, brand Sigma, catalog number R2625; resveratrol, brand Sigma, catalog number R5010-100MG; icariin, Shanghai Microcrystalline Biotechnology, catalog number 489-32-7; benzamide, brand Sigma, catalog number 135828; chloroplatinic acid hexahydrate, brand Sigma, catalog number 206083; ethanolamine, brand Sigma, catalog number 8008490100; EPO, brand PeproTech, catalog number CYT-201; VEGF, brand PeproTech, catalog number 96-100-20-2.

[0017] Example 2

[0018] This embodiment describes an inducer for converting mesenchymal stem cells into E2 secretory cells, comprising the following components in the indicated mass concentrations: BMP4 50 mg / L, BMP7 20 mg / L, RA 2 mg / L, resveratrol 2 mg / L, icariin 2 mg / L, benzamide 4 μg / L, chloroplatinic acid hexahydrate 6 μg / L, ethanolamine 4 μg / L, EPO 8 μg / L, and VEGF 8 μg / L. The above components are added sequentially to serum-free human mesenchymal stem cell culture medium (or DMEM + 10% FBS or other commercially available mesenchymal stem cell culture medium) according to the indicated mass concentrations, mixed thoroughly, and then filtered for sterilization.

[0019] Example 3

[0020] The inducing agent for mesenchymal stem cells to become E2 secretory cells in this embodiment consists of the following components in the following mass concentration ratios: BMP4 50 mg / L, BMP7 50 mg / L, RA 8 mg / L, resveratrol 6 mg / L, icariin 6 mg / L, benzamide 4 μg / L, chloroplatinic acid hexahydrate 6 μg / L, ethanolamine 4 μg / L, EPO 5 μg / L, and VEGF 5 μg / L. The above components are added sequentially to the serum-free culture medium for human mesenchymal stem cells (or DMEM + 10% FBS) according to the mass concentration ratio, mixed well, and then filtered for sterilization.

[0021] Example 4

[0022] Taking human adipose-derived mesenchymal stem cells as an example, this invention illustrates the effect of the inducing agent on the differentiation of mesenchymal stem cells into E2 secretory cells. Human adipose-derived mesenchymal stem cells, passaged for 3 generations, were used at a concentration of 1×10⁻⁶. 4 / cm 2 The cells were seeded in 96-well plates and induced to differentiate when they reached nearly 80% confluence and were growing vigorously. The induction conditions and groupings are shown in Table 1.

[0023] Table 1 Grouping of Induction Conditions

[0024]

[0025] Carefully observe the morphological changes of cells during the induction process. After 1-9 days of induction, collect culture media from each group, centrifuge at 2000 rpm for 10 min, and store the supernatant at -20℃. Detect the E2 content and perform statistical analysis. Follow the instructions for use of the estradiol ELISA kit (Shanghai Boco Biotechnology Co., Ltd.) to detect the E2 content. The results are shown in Table 2.

[0026] Table 2 Comparison of the induction effects of different inducers

[0027]

[0028] As can be seen from the induction results in Table 2, the inducing agent of this invention showed the highest efficiency in inducing human adipose-derived mesenchymal stem cells, resulting in cells that secreted the most E2. After adding the inducing agent of this invention, E2 secretion began to occur continuously after 2 days, and the amount of secretion gradually increased, reaching its maximum daily secretion around day 5 to 7 of induction, and continued to be secreted.

Claims

1. An inducer for the differentiation of mesenchymal stem cells into estradiol-secreting cells, characterized in that, The culture medium, based on serum-free human mesenchymal stem cells, consists of the following components in the indicated mass concentrations: bone morphogenetic protein-4 20-60 mg / L, bone morphogenetic protein-7 20-60 mg / L, retinoic acid 2-8 mg / L, resveratrol 2-8 mg / L, icariin 2-8 mg / L, benzamide 2-8 μg / L, chloroplatinic acid hexahydrate 2-8 μg / L, ethanolamine 2-8 μg / L, erythropoietin 2-10 μg / L, and vascular endothelial growth factor 2-10 μg / L.

2. The inducer for differentiating mesenchymal stem cells into estradiol-secreting cells according to claim 1, characterized in that, The culture medium, based on serum-free human mesenchymal stem cells, consisted of the following components in the indicated mass concentrations: bone morphogenetic protein-4 50 mg / L, bone morphogenetic protein-7 50 mg / L, retinoic acid 8 mg / L, resveratrol 6 mg / L, icariin 6 mg / L, benzamide 4 μg / L, chloroplatinic acid hexahydrate 6 μg / L, ethanolamine 4 μg / L, erythropoietin 5 μg / L, and vascular endothelial growth factor 5 μg / L.

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