A culture medium for multidirectional induction differentiation of amniotic epithelial stem cells and preparation method thereof

Abstract

The present invention belongs to the field of stem cell induction differentiation, and specifically relates to a culture medium for multidirectional induction differentiation of amniotic epithelial stem cells and a preparation method thereof. The culture medium of the present invention relates to culture medium I and culture medium II, wherein culture medium I contains stem cell induction stimulator I, plant-derived isorhamnetin and a small amount of hepatocyte growth factor, and stem cell induction stimulator I is prepared from miRNA-141 and modified chitosan, and culture medium I is used to obtain hepatocyte-like cells; culture medium II contains stem cell induction stimulator II, plant-derived rosmarinic acid, bryostatin 1 and a small amount of glial cell-derived neurotrophic factor, and stem cell induction stimulator II is prepared from miRNA-206 and modified chitosan, and culture medium II is used to obtain neuron-like cells. The culture medium obtained by the present invention has a better induction differentiation effect, higher availability, lower toxicity, and less side effects.

Description

Technical Field

[0001] The invention belongs to the field of stem cell induction differentiation, and specifically relates to a culture medium for multi-directional induction differentiation of amniotic epithelial stem cells and a preparation method thereof. Background Art

[0002] During the development of the embryo, the fetal membrane is formed. The innermost layer of the fetal membrane is the amnion, which is composed of a thick basement membrane and avascular matrix. Under an electron microscope, the amnion can be divided into five layers from the inside to the outside: epithelial layer, basement membrane layer, dense layer, fibroblast layer and spongy layer. Human amniotic epithelial cells (hAECs) are applied in a single layer on the epithelial layer of the amnion surface, facing the amniotic fluid, and are easy to separate and culture. They develop from the epiblast on the eighth day of the embryo, before the three embryonic germ layers (external, middle and internal germ layers), so they still retain the molecular markers of the main characteristics of embryonic stem cells (such as OCT-4, SOX-2, NANOG, SSEA-3, SSEA-4, etc.). Since hAECs lack telomerase, they will not form teratomas in vivo or in vitro, so they are safe for transplantation in clinical practice; if a SOX-2 gene is transferred into it by electroporation technology, iPS clones will be formed after 7 days of culture, and teratomas will be formed after transfer culture. The differentiation potential of hAECs is even better than that of human amniotic mesenchymal cells (hAMSCs), which also exist in the amniotic tissue.

[0003] The stem cells of hAECs have the following characteristics: hAECs express almost all the main surface markers of embryonic stem cells, among which the expression of three genes, OCT-4, SOX-2 and NANOG, is necessary for maintaining the self-renewal and pluripotency of embryonic stem cells; hAECs have the characteristics of pluripotent stem cells. Under the induction conditions of in vitro culture, hAECs can differentiate into endoderm (pancreatic, liver) cells, ectoderm (neural) cells and mesoderm (cardiac) cells, etc.; hAECs also exhibit a mesenchymal stem cell (MSC)-like phenotype, that is, they meet the standards of three mesodermal lineage differentiation potentials (osteogenesis, adipogenesis and chondrogenesis), cell surface marker expression of CD90, CD105 and CD73, and lack of cell surface markers CD45, CD34, CD14, CD79 and HLA-DR.

[0004] In addition, hAECs stem cells have many advantages, such as no need for invasive collection procedures, minimal ethical constraints, easy to obtain sources, no need for expansion and can have a large number of cells, have immunosuppressive and regulatory effects, can curb immune rejection and inflammatory reactions, can secrete a variety of cytokines and growth nutrient factors, and help the host's own stem cells repair during cell therapy. At the same time, it does not involve DNA damage related to age or environment, and has genetic stability, etc. Therefore, hAECs stem cells are considered to be a promising source of stem cells in biological research and regenerative medicine. Therefore, the preparation of induced differentiation medium for amniotic epithelial stem cells can effectively promote the application of hAECs stem cells in clinical and medical research. Summary of the invention

[0005] Based on the above problems, the present invention provides a culture medium for multidirectional induction differentiation of amniotic epithelial stem cells, specifically inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells and neuron-like cells. Wherein culture medium I involves stem cell induction stimulator I, plant-derived isorhamnetin and a small amount of hepatocyte growth factor, and stem cell induction stimulator I is prepared from miRNA-141 and modified chitosan; culture medium II involves stem cell induction stimulator II, plant-derived rosmarinic acid, bryostatin 1 and a small amount of glial cell-derived neurotrophic factor, and stem cell induction stimulator II is prepared from miRNA-206 and modified chitosan. The culture medium for multidirectional induction differentiation of amniotic epithelial stem cells obtained by the present invention has better induction differentiation effect, higher usability, lower toxicity, and fewer side effects, and does not have the toxic effects and potential rejection problems caused by the large-scale use of recombinant growth factors.

[0006] The present invention provides a culture medium for multi-directional induction differentiation of amniotic epithelial stem cells, which relates to culture medium I and culture medium II;

[0007] Among them, culture medium I is a culture medium used to induce differentiation of amniotic epithelial stem cells into hepatocyte-like cells, specifically DMDM / F12 culture medium supplemented with 80-100 μL / mL of stem cell induction stimulator I, 10-15 ng / mL of plant-derived isorhamnetin, and 1-2 ng / mL of hepatocyte growth factor.

[0008] The preparation method of the stem cell induction stimulator I is as follows:

[0009] L1. miRNA-141-3p was obtained by chemical synthesis and stored in the form of lyophilized powder for future use. When used, sterilized RNase-free H2O was used to prepare a miRNA-141-3p solution with a mass volume concentration of 70-80 mg / L;

[0010] L2. Chitosan was dissolved in acetic acid solution to obtain a chitosan solution with a mass volume concentration of 4-5 mg / mL, myristic acid was dissolved in 75% ethanol to obtain a myristic acid solution with a mass volume concentration of 2-3 mg / mL, EDC.HCl was added to the myristic acid solution, stirred at a speed of 180-220 rpm for 30-40 min, and solution 1 was obtained. Solution 1 was dropped into the chitosan solution and kept at room temperature for 24-30 h to obtain solution 2. Solution 2 was dialyzed with deionized water for 28-32 h, and then vacuum freeze-dried to obtain substance 1;

[0011] L3. Dissolve the substance 1 obtained in step L2 in sterilized RNase-free H2O to obtain solution 3 with a mass volume concentration of 25-30 mg / L, mix the miRNA-141-3p solution obtained in step L1 with solution 3 at a volume ratio of 1:1-1.4, and treat with ultrasound at a power of 150-200 W for 2-4 minutes. After treatment, heat to 30-40°C and maintain for 15-20 minutes. After treatment, immediately bathe in ice water to obtain the stem cell induction stimulator, which is stored at 4°C for future use.

[0012] Preferably, the sequence of miRNA-141-3p in step L1 is 5'-UAACACUGUCUGGUAAAGAUGG-3'.

[0013] Preferably, in step L2, the chitosan is a product with a degree of deacetylation ≥ 95% and a MW in the range of 5000-6000Da, the mass percentage concentration of the acetic acid solution is preferably 1%, the amount of EDC.HCl added is 5-8% of the mass of the myristic acid solution, the volume ratio of solution 1:chitosan solution is 1:1-1.2, and the dialysis is preferably performed using a dialysis bag with a molecular weight cutoff of 3kDa.

[0014] The plant-derived isorhamnetin is extracted from natural plants. The purity of the isorhamnetin is ≥95%. The natural plants are preferably seabuckthorn or elaeagnus.

[0015] Among them, culture medium II is a culture medium used to induce differentiation of amniotic epithelial stem cells into neuron-like cells, specifically, DMDM / F12 culture medium supplemented with 70-90 μL / mL of stem cell induction stimulator II, 1-3 ng / mL bryostatin 1, 8-10 ng / mL plant-derived rosmarinic acid, and 0.8-1 ng / mL glial cell-derived neurotrophic factor.

[0016] The preparation method of the stem cell induction stimulator II is as follows:

[0017] S1. miRNA-206-3p is obtained by chemical synthesis and stored in the form of lyophilized powder for future use. When used, sterilized RNase-free H2O is used to prepare a miRNA-206-3p solution with a mass volume concentration of 70-80 mg / L;

[0018] S2. Obtain substance 1 by referring to step L2;

[0019] S3. The substance 1 obtained in step S2 is dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 25-30 mg / L. The miRNA-206-3p solution obtained in step S1 is mixed with solution 3 at a volume ratio of 1:1.2-1.6, and ultrasonically treated at a power of 150-200 W for 2-4 min. After the treatment, the solution is heated to 30-40°C and maintained for 15-20 min. After the treatment, the solution is immediately bathed in ice water to obtain the stem cell induction stimulator, which is stored at 4°C for future use.

[0020] Preferably, the sequence of miRNA-206-3p in step S1 is 5'-UGGAAUGUAAGGAAGUGUGUGG-3'.

[0021] The plant-derived rosmarinic acid is rosmarinic acid extracted from natural plants. The purity of rosmarinic acid is ≥92%. The natural plants are preferably mint, perilla or forget-me-not.

[0022] The present invention also provides a method for preparing a culture medium for multidirectional induction differentiation of amniotic epithelial stem cells, the specific steps of which are as follows:

[0023] Add stem cell induction stimulator I, plant-derived isorhamnetin, and hepatocyte growth factor to DMDM / F12 culture medium in sequence, and stir until evenly dispersed to obtain a culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells;

[0024] Alternatively, stem cell induction stimulator II, bryostatin 1, plant-derived rosmarinic acid, and glial cell-derived neurotrophic factor are added to DMDM / F12 medium in sequence, and stirred until uniformly dispersed to obtain a medium (medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells.

[0025] The beneficial effects of the present invention are as follows:

[0026] Long non-coding RNA (lncRNA) is a family of RNA with a length of more than 200 nucleotides, which is involved in a variety of biological processes, including cell proliferation, migration, invasion and apoptosis. lncRNA plays a role through a variety of mechanisms, including as a transcriptional regulator, a post-transcriptional regulator and an epigenetic modification. In addition, lncRNA also acts as a competitive endogenous RNA (ceRNA), and participates in tumorigenesis by acting as a sponge for miRNA. For example, studies have shown that the lncRNA HOTAIR / miR-1 / GOLPH3 regulatory axis affects the biological behavior of breast cancer cells. It is known that there is often a mutual regulatory relationship between lncRNA HOTAIR and miRNA. The present invention adopts overexpression of miRNA-141, thereby inhibiting the expression of endogenous lncRNA HOTAIR, promoting the transcription of the corresponding gene, and realizing the induction of differentiation of amniotic epithelial stem cells into hepatocyte-like cells. This process also involves a modified chitosan carrier, which has the advantages of low toxicity, easy availability of raw materials, and good surface reactivity. Considering that the target of miRNA-141 is not completely unique and that human amniotic epithelial stem cells are composed of heterogeneous cell groups, the present invention further adds plant-derived isorhamnetin to culture medium I. Some studies have used microarrays to show that isorhamnetin from natural plants may promote the differentiation of human amniotic epithelial stem cells, but there is a lack of experimental data to support this. On this basis, the present invention uses isorhamnetin from seabuckthorn or elaeagnus to conduct experiments. A single experiment shows that the efficiency of inducing differentiation by isorhamnetin is low (compared with the induction differentiation medium composed of recombinant growth factors, etc.), but the plant-derived isorhamnetin combined with stem cell induction stimulator I can effectively achieve the induction differentiation of amniotic epithelial stem cells into hepatocyte-like cells. The culture medium I finally obtained only involves a small amount of hepatocyte growth factor, and the overall culture medium has higher availability, lower toxicity, and fewer side effects, and does not have the toxic effects and potential rejection problems caused by the large-scale use of recombinant growth factors.

[0027] Similarly, the present invention still uses a modified chitosan carrier to overexpress miRNA-206, and induces the differentiation of amniotic epithelial stem cells into neuron-like cells through the miRNA-206 / lncRNA HOTAIR / YY1 regulatory axis. miRNA-206 also has similar problems as miRNA-141. The present invention selects plant-derived rosmarinic acid for addition, and adds bryostatin 1 and a small amount of glial cell-derived neurotrophic factor at the same time. The final culture medium II has a better use effect.

[0028] The culture medium I obtained by the present invention can effectively induce the differentiation of amniotic epithelial stem cells into hepatocyte-like cells. The obtained hepatocyte-like cells specifically express hepatocyte characteristic proteins, can promote cell cycle progression, and have the physiological functions of hepatocytes; the culture medium II obtained by the present invention can effectively induce the differentiation of amniotic epithelial stem cells into neuron-like cells. After testing, the obtained cells meet the characteristics of neuron-like cells in terms of cell morphology, gene expression, protein expression, immune response and functional detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0030] Figure 1 This is a phase contrast microscopy image of human amniotic epithelial stem cells;

[0031] Figure 2 Characteristic analysis of hematopoietic lineage markers CD34 and CD45, endothelial markers CD31 and CD144, and mesenchymal markers CD73 and CD90 of human amniotic epithelial stem cells;

[0032] Figure 3 This is the relative expression graph of NANGO, SOX-2, and OCT-4 genes;

[0033] Figure 4 This is the relative expression graph of ALB, CYP7A1, and AFP genes;

[0034] Figure 5 This is the relative expression graph of CYP3A4, p21, and p53 genes;

[0035] Figure 6 This is a diagram showing the Western blot detection results of hepatocyte-like cells obtained in Example 8;

[0036] Figure 7 This is a graph of the relative protein band intensities of the hepatocyte-like cells obtained in Example 8;

[0037] Figure 8 This is a graph showing the urea concentration of hepatocyte-like cells obtained in Example 8;

[0038] Fig. 9 This is a phase contrast microscope image of the neuron-like cells obtained in Example 9. DETAILED DESCRIPTION

[0039] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0040] Example 1: This example provides a method for obtaining human amniotic epithelial stem cells, and the specific steps are as follows:

[0041] Fetal placentas were sampled and transported to the laboratory with sterile Hanks' balanced salt solution (HBSS, without calcium and magnesium). The amniotic membrane was mechanically peeled from the chorionic layer beneath the placental body and washed thoroughly two to three times with HBSS to remove blood. Next, the amniotic membrane was treated with 0.25% trypsin-EDTA in a humidified incubator at 37°C with 5% CO2 for 60 minutes. The tissue was filtered through a 70 μm cell strainer (purchased from Shanghai Yubo Biotechnology Co., Ltd.) to obtain a single cell suspension, and the digestion was terminated by adding medium containing 10% FBS. The single cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in DMEM-high glucose supplemented with 10% FBS, 2 mM L-glutamine, 1% antibiotic-antimycotic, 1% sodium pyruvate, 1% non-essential amino acids, 55 μM β-mercaptoethanol, 100 U / mL penicillin and streptomycin, and 10 ng / mL epidermal growth factor (EGF; Peprotech) to obtain human amniotic epithelial stem cells. Human amniotic epithelial stem cells were cultured at 5 × 10 4 Cells / cm 2 The cells were placed in a cell culture dish (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) at a density of 1.5 μg / mL and placed in a 37°C, 5% CO2 incubator. Unattached cells and debris were removed after 3 days. In each experiment, cells grew to about 80% confluence, and cells between passages 2 and 5 were saved for subsequent induced differentiation. HBSS, 0.25% trypsin-EDTA, FBS, DMEM-high glucose, L-glutamine, antibiotics-antimycotics, sodium pyruvate, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, epidermal growth factor, etc. used in the experiment were purchased from Thermo Fisher Scientific (China) Co., Ltd. (Thermo Fisher).

[0042] Example 2: This example provides a method for preparing a culture medium for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells, the specific contents of which are as follows:

[0043] The culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells in this example is specifically DMDM / F12 culture medium supplemented with 80 μL / mL of stem cell induction stimulator I, 10 ng / mL of plant-derived isorhamnetin, and 1 ng / mL of hepatocyte growth factor (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number H980397);

[0044] The preparation method of stem cell induction stimulator I is as follows:

[0045] L1. miRNA-141-3p was obtained by chemical synthesis from Jiman Biotechnology (Shanghai) Co., Ltd. The sequence of miRNA-141-3p was 5'-UAACACUGUCUGGUAAAGAUGG-3' and was stored in the form of lyophilized powder for later use. When used, sterilized RNase-free H2O (Shanghai Yubo Biotechnology Co., Ltd.) was used to prepare a miRNA-141-3p solution with a mass volume concentration of 70 mg / L;

[0046] L2. Chitosan (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number C766421) was dissolved in 1% acetic acid solution to obtain a chitosan solution with a mass volume concentration of 4 mg / mL. Myristic acid (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number M3525) was dissolved in 75% ethanol to obtain a myristic acid solution with a mass volume concentration of 2 mg / mL. EDC.HCl (purchased from Sigma-Aldrich ( Shanghai) Trading Co., Ltd., product number E6383) was added to the myristic acid solution, the amount of EDC.HCl added was 5% of the mass of the myristic acid solution, and the mixture was stirred at a speed of 180 rpm for 30 min to obtain solution 1, and solution 1 was dropped into the chitosan solution, the volume ratio of solution 1:chitosan solution was 1:1, and the mixture was kept at room temperature for 24 h to obtain solution 2, and solution 2 was dialyzed with deionized water for 28 h, and a dialysis bag with a molecular weight cutoff of 3 kDa was used for dialysis, and then vacuum freeze-dried to obtain substance 1;

[0047] L3. The substance 1 obtained in step L2 was dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 25 mg / L. The miRNA-141-3p solution obtained in step L1 was mixed with solution 3 at a volume ratio of 1:1, and ultrasonically treated at a power of 150 W for 2 min. After the treatment, the solution was heated to 30°C and maintained for 15 min. After the treatment, the solution was immediately bathed in ice water to obtain a stem cell induction stimulator, which was stored at 4°C for future use.

[0048] Among them, the plant-derived isorhamnetin is extracted from sea buckthorn, and the purity of isorhamnetin is 95%;

[0049] The preparation method of the culture medium for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells in this example is as follows:

[0050] The stem cell induction stimulator I, plant-derived isorhamnetin, and hepatocyte growth factor are sequentially added to the DMDM / F12 culture medium, and stirred until uniformly dispersed to obtain a culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells.

[0051] Example 3: The culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells in this example is specifically DMDM / F12 culture medium supplemented with 90 μL / mL of stem cell induction stimulator I, 12 ng / mL of plant-derived isorhamnetin, and 1.5 ng / mL of hepatocyte growth factor (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number H980397);

[0052] The preparation method of stem cell induction stimulator I is as follows:

[0053] L1. miRNA-141-3p was obtained by chemical synthesis from Jiman Biotechnology (Shanghai) Co., Ltd. The sequence of miRNA-141-3p was 5'-UAACACUGUCUGGUAAAGAUGG-3' and was stored in the form of lyophilized powder for later use. When used, sterilized RNase-free H2O (Shanghai Yubo Biotechnology Co., Ltd.) was used to prepare a miRNA-141-3p solution with a mass volume concentration of 75 mg / L;

[0054] L2. Chitosan (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number C766421) was dissolved in 1% acetic acid solution to obtain a chitosan solution with a mass volume concentration of 4.5 mg / mL. Myristic acid (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number M3525) was dissolved in 75% ethanol to obtain a myristic acid solution with a mass volume concentration of 2.5 mg / mL. EDC.HCl (purchased from Sigma-Aldrich) (Shanghai) Trading Co., Ltd., product number E6383) was added to the myristic acid solution, the amount of EDC.HCl added was 6% of the mass of the myristic acid solution, and the mixture was stirred at a speed of 190 rpm for 34 min to obtain solution 1, and solution 1 was dropped into the chitosan solution, the volume ratio of solution 1:chitosan solution was 1:1.1, and the mixture was kept at room temperature for 28 h to obtain solution 2, and solution 2 was dialyzed with deionized water for 30 h, using a dialysis bag with a molecular weight cutoff of 3 kDa, and then vacuum freeze-dried to obtain substance 1;

[0055] L3. The substance 1 obtained in step L2 was dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 28 mg / L. The miRNA-141-3p solution obtained in step L1 was mixed with solution 3 at a volume ratio of 1:1.2, and ultrasonically treated at a power of 180 W for 3 min. After the treatment, the solution was heated to 35°C and maintained for 18 min. After the treatment, the solution was immediately bathed in ice water to obtain a stem cell induction stimulator, which was stored at 4°C for future use.

[0056] Among them, the plant-derived isorhamnetin is extracted from Elaeagnus chinensis, and the purity of isorhamnetin is 95%;

[0057] The preparation method of the culture medium for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells in this example is as follows:

[0058] The stem cell induction stimulator I, plant-derived isorhamnetin, and hepatocyte growth factor are sequentially added to the DMDM / F12 culture medium, and stirred until uniformly dispersed to obtain a culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells.

[0059] Example 4: The culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells in this example is specifically DMDM / F12 culture medium supplemented with 100 μL / mL of stem cell induction stimulator I, 15 ng / mL of plant-derived isorhamnetin, and 2 ng / mL of hepatocyte growth factor (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number H980397);

[0060] The preparation method of stem cell induction stimulator I is as follows:

[0061] L1. miRNA-141-3p was obtained by chemical synthesis from Jiman Biotechnology (Shanghai) Co., Ltd. The sequence of miRNA-141-3p was 5'-UAACACUGUCUGGUAAAGAUGG-3' and was stored in the form of lyophilized powder for later use. When used, sterilized RNase-free H2O (Shanghai Yubo Biotechnology Co., Ltd.) was used to prepare a miRNA-141-3p solution with a mass volume concentration of 80 mg / L;

[0062] L2. Chitosan (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number C766421) was dissolved in 1% acetic acid solution to obtain a chitosan solution with a mass volume concentration of 5 mg / mL. Myristic acid (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number M3525) was dissolved in 75% ethanol to obtain a myristic acid solution with a mass volume concentration of 3 mg / mL. EDC.HCl (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number M3525) was dissolved in 75% ethanol to obtain a myristic acid solution with a mass volume concentration of 3 mg / mL. The amount of EDC.HCl added was 8% of the mass of the myristic acid solution, and the mixture was stirred at 220 rpm for 40 min to obtain solution 1. Solution 1 was dropped into the chitosan solution at a volume ratio of solution 1 to chitosan solution of 1:1.2. The mixture was kept at room temperature for 30 h to obtain solution 2. Solution 2 was dialyzed with deionized water for 32 h using a dialysis bag with a molecular weight cutoff of 3 kDa. After the dialyzation, the substance 1 was freeze-dried in vacuum to obtain substance 1.

[0063] L3. The substance 1 obtained in step L2 is dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 30 mg / L. The miRNA-141-3p solution obtained in step L1 is mixed with solution 3 at a volume ratio of 1:1.4, and ultrasonically treated at a power of 200 W for 4 minutes. After the treatment, the solution is heated to 40°C and maintained for 20 minutes. After the treatment, the solution is immediately bathed in ice water to obtain a stem cell induction stimulator, which is stored at 4°C for future use.

[0064] The plant-derived isorhamnetin is consistent with that in Example 2;

[0065] The preparation method of the culture medium for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells in this example is as follows:

[0066] The stem cell induction stimulator I, plant-derived isorhamnetin, and hepatocyte growth factor are sequentially added to the DMDM / F12 culture medium, and stirred until uniformly dispersed to obtain a culture medium (culture medium I) for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells.

[0067] Example 5: This example provides a method for preparing a culture medium for inducing differentiation of amniotic epithelial stem cells into neuron-like cells, the specific contents of which are as follows:

[0068] The culture medium (culture medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells in this example is specifically DMDM / F12 culture medium supplemented with 70 μL / mL of stem cell induction stimulator II, 1 ng / mL of bryostatin 1 (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number B7431), 8 ng / mL of plant-derived rosmarinic acid, and 0.8 ng / mL of glial cell-derived neurotrophic factor (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number H980346);

[0069] The preparation method of stem cell induction stimulator II is as follows:

[0070] S1. miRNA-206-3p was obtained by chemical synthesis from Jiman Biotechnology (Shanghai) Co., Ltd. The sequence of miRNA-206-3p was 5'-UGGAAUGUAAGGAAGUGUGUGG-3' and was stored in the form of lyophilized powder for later use. When used, sterilized RNase-free H2O (Shanghai Yubo Biotechnology Co., Ltd.) was used to prepare a miRNA-206-3p solution with a mass volume concentration of 70 mg / L;

[0071] S2. Referring to step L2 in Example 2 to obtain substance 1;

[0072] S3. The substance 1 obtained in step S2 is dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 25 mg / L, the miRNA-206-3p solution obtained in step S1 is mixed with solution 3 at a volume ratio of 1:1.2, and ultrasonically treated at a power of 150 W for 2 min, and then heated to 30°C and maintained for 15 min. After the treatment, the solution is immediately placed in an ice water bath to obtain a stem cell induction stimulator, which is stored at 4°C for future use;

[0073] Among them, plant-derived rosmarinic acid is rosmarinic acid extracted from mint, and the purity of rosmarinic acid is 92%;

[0074] The preparation method of the culture medium for inducing differentiation of amniotic epithelial stem cells into neuron-like cells in this example is as follows:

[0075] The stem cell induction stimulator II, bryostatin 1, plant-derived rosmarinic acid, and glial cell-derived neurotrophic factor were sequentially added to the DMDM / F12 culture medium, and stirred until uniformly dispersed to obtain a culture medium (culture medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells.

[0076] Example 6: This example provides a method for preparing a culture medium for inducing differentiation of amniotic epithelial stem cells into neuron-like cells, the specific contents of which are as follows:

[0077] The culture medium (culture medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells in this example is specifically DMDM / F12 culture medium supplemented with 80 μL / mL of stem cell induction stimulator II, 2 ng / mL bryostatin 1 (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number B7431), 9 ng / mL plant-derived rosmarinic acid, and 0.9 ng / mL glial cell-derived neurotrophic factor (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number H980346);

[0078] The preparation method of stem cell induction stimulator II is as follows:

[0079] S1. miRNA-206-3p was obtained by chemical synthesis from Jiman Biotechnology (Shanghai) Co., Ltd. The sequence of miRNA-206-3p was 5'-UGGAAUGUAAGGAAGUGUGUGG-3' and was stored in the form of lyophilized powder for later use. When used, sterilized RNase-free H2O (Shanghai Yubo Biotechnology Co., Ltd.) was used to prepare a miRNA-206-3p solution with a mass volume concentration of 75 mg / L;

[0080] S2. Referring to step L2 in Example 3 to obtain substance 1;

[0081] S3. The substance 1 obtained in step S2 is dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 26 mg / L, the miRNA-206-3p solution obtained in step S1 is mixed with the solution 3 at a volume ratio of 1:1.4, and ultrasonically treated at a power of 180 W for 3 min, and then heated to 35°C and maintained for 18 min. After the treatment, the solution is immediately bathed in ice water to obtain a stem cell induction stimulator, which is stored at 4°C for future use;

[0082] Among them, plant-derived rosmarinic acid is rosmarinic acid extracted from Perilla frutescens, and the purity of rosmarinic acid is 94%;

[0083] The preparation method of the culture medium for inducing differentiation of amniotic epithelial stem cells into neuron-like cells in this example is as follows:

[0084] The stem cell induction stimulator II, bryostatin 1, plant-derived rosmarinic acid, and glial cell-derived neurotrophic factor were sequentially added to the DMDM / F12 culture medium, and stirred until uniformly dispersed to obtain a culture medium (culture medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells.

[0085] Example 7: This example provides a method for preparing a culture medium for inducing differentiation of amniotic epithelial stem cells into neuron-like cells, the specific contents of which are as follows:

[0086] The culture medium (culture medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells in this example is specifically DMDM / F12 culture medium supplemented with 90 μL / mL of stem cell induction stimulator II, 3 ng / mL bryostatin 1 (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥99%, product number B7431), 10 ng / mL plant-derived rosmarinic acid, and 1 ng / mL glial cell-derived neurotrophic factor (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number H980346);

[0087] The preparation method of stem cell induction stimulator II is as follows:

[0088] S1. miRNA-206-3p was obtained by chemical synthesis from Jiman Biotechnology (Shanghai) Co., Ltd. The sequence of miRNA-206-3p is 5'-UGGAAUGUAAGGAAGUGUGUGG-3' and is stored in the form of lyophilized powder for later use. When used, sterilized RNase-free H2O (Shanghai Yubo Biotechnology Co., Ltd.) was used to prepare a miRNA-206-3p solution with a mass volume concentration of 80 mg / L;

[0089] S2. Referring to step L2 in Example 4 to obtain substance 1;

[0090] S3. The substance 1 obtained in step S2 is dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 30 mg / L, the miRNA-206-3p solution obtained in step S1 is mixed with the solution 3 at a volume ratio of 1:1.6, and ultrasonically treated at a power of 200 W for 4 minutes, and then heated to 40°C and maintained for 20 minutes. After the end, it is immediately iced and bathed to obtain the stem cell induction stimulator, which is stored at 4°C for future use;

[0091] Among them, plant-derived rosmarinic acid is rosmarinic acid extracted from forget-me-not, and the purity of rosmarinic acid is 95%;

[0092] The preparation method of the culture medium for inducing differentiation of amniotic epithelial stem cells into neuron-like cells in this example is as follows:

[0093] The stem cell induction stimulator II, bryostatin 1, plant-derived rosmarinic acid, and glial cell-derived neurotrophic factor were sequentially added to the DMDM / F12 culture medium, and stirred until uniformly dispersed to obtain a culture medium (culture medium II) for inducing differentiation of amniotic epithelial stem cells into neuron-like cells.

[0094] Example 8: This example provides a method for culturing hepatocyte-like cells, the specific contents are as follows:

[0095] The third generation human amniotic epithelial stem cells were cultured in IMDM medium containing 10% fetal bovine serum for 7 days, and then induced to differentiate using medium I obtained in Example 2. The amount of cells added was 1×10 4 / mL, culture medium I was replaced every 3 days, the culture conditions were 37°C, 5% CO2, and the culture medium I obtained in Example 2 was cultured for 21 days to finally obtain hepatocyte-like cells.

[0096] Example 9: This example provides a method for culturing neuron-like cells, the specific contents are as follows:

[0097] The 4th generation human amniotic epithelial stem cells were cultured in IMDM medium containing 10% fetal bovine serum for 7 days, and then induced to differentiate using the medium II obtained in Example 5. The amount of cells added was 1×10 4 / mL, culture medium II was replaced every 3 days, the culture conditions were 37°C, 5% CO2, and the culture medium II obtained in Example 2 was cultured for 21 days to finally obtain neuron-like cells.

[0098] Experimental test:

[0099] 1. Characteristic analysis of human amniotic epithelial stem cells:

[0100] The following cell surface antigens were detected by flow cytometry: CD34, CD45, CD31, CD144, CD73 and CD90. The third generation human amniotic epithelial stem cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were rinsed once with D-PBA to adjust the cell density to 1×10 6 / mL, take 200μL of cell suspension according to the combination scheme, add 10μL of fluorescently labeled CD34, CD45, CD31, CD144, CD73, CD90 and HLA-DR antibodies, shake and mix, incubate at room temperature and avoid light for 25min, add 2mL of PBS containing 0.1% NaN3 to each tube, shake and mix, centrifuge at 1000rpm for 5min, discard the supernatant, and oscillate the suspended cells. Add 200μL of PBS containing 1% paraformaldehyde to each tube, shake and mix, place at 4℃ and avoid light for 24h, and then use flow cytometer (Becton Dickinson) for detection and analysis. The number of cells collected for each sample is ≥2×10 4 Phenotypic analysis was performed using FlowJo analysis software (Becton Dickinson).

[0101] Undifferentiated human amniotic epithelial stem cells were cultured in IMDM medium containing 10% fetal bovine serum, supplemented with 4 mM glutamine, 1 mM sodium pyruvate, 50 U / mL penicillin, 50 μg / mL streptomycin, and 1% non-essential amino acids at 37°C and 5% CO2, with the medium replaced every 3 days.

[0102] The fine structure of human amniotic epithelial stem cells Figure 1 , Figure 1 Phase contrast microscope image. Figure 1 It can be seen that human amniotic epithelial stem cells are pebble-shaped, which is a typical epithelial cell state. Figure 2 The results of flow cytometric analysis of the characteristics of human amniotic epithelial stem cells were obtained by Figure 2 It can be seen that human amniotic epithelial stem cells are negative for CD34 and CD45 (hematopoietic lineage markers), CD31 and CD144 (endothelial markers), and human amniotic epithelial stem cells express mesenchymal markers, such as CD73 and CD90. In addition, human amniotic epithelial stem cells are also positive for pluripotent markers, such as NANOG, OCT-4 ( Figure 3 As shown, it proves that the human amniotic epithelial stem cells obtained in Example 1 have multidirectional differentiation potential. The expression level of SOX-2 in human amniotic epithelial stem cells is also high, indicating that they have neuronal plasticity ( Figure 3 As shown). Thus, human amniotic epithelial stem cells with multidirectional differentiation potential were isolated and obtained in Example 1, which supported the subsequent induction and differentiation.

[0103] Hepatocyte-like cell differentiation assay:

[0104] 1) Real-time fluorescence quantitative PCR detection:

[0105] TRI RNA isolation reagent was used to treat human amniotic epithelial stem cells (passage 3) that were not induced to differentiate and hepatocyte-like cells that were induced to differentiate (obtained in Example 8). The concentration and purity of the isolated RNA were detected by spectrophotometry at 260 nm and 280 nm. 5 μg of isolated total RNA was reverse transcribed for 1 hour using the EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix kit to obtain cDNA. The primer sequences involved in real-time fluorescence quantitative PCR are as follows:

[0106] Table 1 Primer sequences involved in real-time fluorescence quantitative PCR

[0107]

[0108] The real-time fluorescence quantitative PCR was performed using the PerfectStart Green qPCR SuperMix kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The total reaction system of the real-time fluorescence quantitative PCR was 25 μL, including 0.4 μL of 10 μM forward primer, 0.4 μL of 10 μM reverse primer, and 3 μL of cDNA. The reaction procedure was 94°C pre-denaturation for 30 s, 1 cycle, 94°C denaturation for 5 s, 60°C annealing for 30 s, 45 cycles, and 2-ΔΔCt The results were statistically analyzed by the PCR method and normalized to housekeeping genes (CYCLOPHILIN and GAPDH).

[0109] Western blot:

[0110] Human amniotic epithelial hepatocytes (passage 3) without induced differentiation and induced differentiated hepatocyte-like cells (obtained in Example 8) were fully washed with PBS solution precooled at 4°C, and an appropriate amount of RIPA mixed lysis solution was added, and ultrasonic lysis was performed on ice, followed by centrifugation at 6000g centrifugal force for 15 minutes at 4°C, and the supernatant was collected. The supernatant was mixed with Laemmli sample buffer containing 2% sodium dodecyl sulfate and 30mM β-mercaptoethanol, boiled for 5 minutes, separated by SDS-PAGE on a 12% gel, and then electrophoretically transferred to a nitrocellulose membrane, equilibrated in 1×PBS, 5% skim milk powder was added at room temperature, and incubated on a shaking table for 1 hour. The incubation of the primary antibody was performed by washing the membrane 3 times with 1×TBST, each time for 10 minutes, and incubating on a shaking table at 4°C for 12 hours after adding the primary antibody. The incubation of the secondary antibody was similar, and the secondary antibody was selected according to the primary antibody, and incubated on a shaking table at room temperature for 1 hour. The membrane was washed three times with 1×TBST for 10 min each time, and the ultrasensitive ECL luminescent solution A and solution B were mixed in equal volumes, and the nitrocellulose membrane was soaked for 1 min, and the membrane was developed using a gel imager. The primary antibodies involved anti-ALB mouse antibody, anti-CYP3A4 rabbit antibody, anti-CYP7A1 rabbit antibody, and anti-p53 sheep antibody, all of which were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0111] 3) Urea content detection:

[0112] According to QuantiChrom TM Urea Assay Kit (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) instructions, place the samples to be tested (cell supernatants without differentiation induction and after differentiation induction) and reagents at room temperature to rewarm, dilute the 50 mg / dL standard to 5 mg / dL with culture medium, and mix equal volumes of solution A and solution B to form a working solution (use within 20 min);

[0113] 1. In a 96-well plate, add 50 µL of culture medium (blank), 50 µL (5 mg / dL) standard, and 50 µL of the sample to be tested to each well, with 2 replicate wells for each sample;

[0114] 2. Add 200 µL of working solution to each well, tap gently to mix, and incubate at room temperature for 50 min;

[0115] 3. Use an enzyme-labeled instrument to detect the OD value at 430 nm and calculate the urea concentration according to the formula in the instruction manual.

[0116] 5) Result evaluation:

[0117] Figure 3-Figure 5 In the horizontal axis, A represents cells that have not been induced to differentiate, B represents cells that have been induced to differentiate, B-1 represents the first day of induced differentiation, and similarly, A-1 represents the first day of uninduced differentiation in the same period, and so on, A-7, A-15, A-20 and B-7, B-15, B-20.

[0118] Depend on Figure 3 It can be seen that with the extension of culture time, the expression of pluripotent markers of cells cultured in induced differentiation medium will gradually decrease, especially at 15 days and 20 days of culture, the relative expression of pluripotent markers is close to that at 1 day of culture, or even lower, proving that after culture in culture medium I, human amniotic epithelial stem cells are directionally induced into specific cells.

[0119] The cells obtained in Example 8 were observed under a microscope. After 21 days of induction and differentiation, the cells were polygonal and had a large number of granules in the cytoplasm, which was similar to the liver morphology. Real-time quantitative PCR was used to analyze whether these cells expressed liver molecular markers and to determine the expression of specific liver mRNA. The results are shown in Figure 2. Figure 4 and Figure 5 As shown by Figure 4 It can be seen that compared with human amniotic epithelial stem cells that were not induced to differentiate, the relative expression levels of ALB, CYP7A1, and AFP in the cells cultured in Example 8 gradually increased with the extension of the culture time. Figure 5 It can also be seen that the relative expression of CYP3A4 has the same regular characteristics, that is, liver markers are all significantly expressed.

[0120] p21 and p53 are two key proteins involved in the cell cycle. p21 is the main downstream target of p53. p21 is the main control factor of the G1 phase, and p53 regulates the transition between G1 / S and G2 / M. Figure 5 It can be seen that during the culture process, the relative expression of p21 in the cells obtained in Example 8 gradually increased and then dropped to a lower level, while the relative expression of p53 continued to decrease, and the relative expression of p21 and p53 in human amniotic epithelial stem cells that were not induced to differentiate had a similar pattern. Therefore, it is proved that the culture medium I used in Example 8 promotes the cell cycle process and is conducive to the proliferation of cells during differentiation.

[0121] Western blot showed the expression of each protein on the 1st and 20th day of culture ( Figure 6 ), and further converted into a column chart ( Figure 7ALB (albumin) is a protein synthesized mainly in the liver, CYP7A1 (cholesterol 7α-enhancing enzyme) is the rate-limiting enzyme for bile acid synthesis, and CYP3A4 (cytochrome P450 3A4 enzyme) belongs to the CYP3 gene family and is one of the most abundant enzymes in the liver. Figure 6 In the experiment, the expression of the above three proteins increased significantly with the extension of culture time, and the relative band intensity was the highest at 20 days of culture, which was obviously different from the human amniotic epithelial hepatocytes that were not induced to differentiate. It can be seen that after inducing differentiation using culture medium I, human amniotic epithelial stem cells showed obvious characteristics of hepatocyte-like cells, which was specifically described in the Western blot results as a significant increase in the content of three proteins closely related to the liver. When the protein p53 content was determined by Western blotting, the expression of p53 gradually decreased during the liver differentiation process starting from the first day of culture.

[0122] The urea production of the cells obtained in Example 8 during the culture process is as follows: Figure 8 As shown by Figure 8 It can be seen that as the culture time increases, the urea concentration increases significantly, which confirms that the cells obtained in Example 8 gradually exert the function of hepatocytes.

[0123] In addition, the cells obtained in Example 8 were also subjected to immunofluorescence experiments to further confirm the cell characteristics and functional status. The final results were similar to those above, and both confirmed that the cells obtained in Example 8 exhibited hepatocyte characteristics.

[0124] As mentioned above, multiple detection methods were used to verify that the cells obtained in Example 8 were indeed hepatocyte-like cells, that culture medium I could promote the differentiation of human amniotic epithelial stem cells into hepatocyte-like cells, and that the obtained hepatocyte-like cells expressed hepatocyte-specific proteins and had the physiological functions of hepatocytes.

[0125] Similarly, the cells obtained in Example 9 were also subjected to real-time fluorescence quantitative PCR, western blot and other tests to verify the gene expression, protein expression, functional characteristics, immunological characteristics, etc. of the obtained cells. Multiple results determined that the cells obtained in Example 9 were indeed neuron-like cells and had the physiological functions of neuron-like cells. Fig. 9 The cell morphology of the cells obtained in Example 9 is shown. The neuron-like cell morphology is obvious, which is different from human amniotic epithelial stem cells ( Figure 1 ).

[0126] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A culture medium for multidirectional induction differentiation of amniotic epithelial stem cells, characterized in that: Culture medium relates to culture medium I; Culture medium I is a culture medium for inducing differentiation of amniotic epithelial stem cells into hepatocyte-like cells, specifically DMDM / F12 culture medium supplemented with 80-100 μL / mL of stem cell induction stimulator I, 10-15 ng / mL of plant-derived isorhamnetin, and 1-2 ng / mL of hepatocyte growth factor; The preparation method of the stem cell induction stimulator I is as follows: L1. miRNA-141-3p was obtained by chemical synthesis and stored in the form of lyophilized powder for future use. When used, sterilized RNase-free H2O was used to prepare a miRNA-141-3p solution with a mass volume concentration of 70-80 mg / L; L2. Chitosan was dissolved in acetic acid solution to obtain a chitosan solution with a mass volume concentration of 4-5 mg / mL, myristic acid was dissolved in 75% ethanol to obtain a myristic acid solution with a mass volume concentration of 2-3 mg / mL, EDC.HCl was added to the myristic acid solution, stirred to obtain solution 1, solution 1 was dropped into the chitosan solution, maintained at room temperature to obtain solution 2, solution 2 was dialyzed with deionized water, and then vacuum freeze-dried to obtain substance 1; L3. The substance 1 obtained in step L2 is dissolved in sterilized RNase-free H2O to obtain a solution 3 with a mass volume concentration of 25-30 mg / L, and the miRNA-141-3p solution obtained in step L1 is mixed with solution 3 at a volume ratio of 1:1-1.4, and subjected to ultrasonic treatment. After the treatment, the solution is heated and maintained, and then immediately subjected to an ice water bath to obtain a stem cell induction stimulator I, which is stored at 4°C for future use; The plant-derived isorhamnetin is extracted from natural plants. The purity of the isorhamnetin is ≥95%, and the natural plants are seabuckthorn or elaeagnus.

2. The culture medium for multidirectional induction differentiation of amniotic epithelial stem cells according to claim 1, characterized in that: The sequence of miRNA-141-3p in step L1 was 5′-UAACACUGUCUGGUAAAGAUGG-3′.

3. The culture medium for multidirectional induction differentiation of amniotic epithelial stem cells according to claim 2, characterized in that: In step L2, the chitosan is a product with a deacetylation degree of ≥95% and a MW in the range of 5000-6000Da, the amount of EDC.HCl added is 5-8% of the mass of the myristic acid solution, and the volume ratio of solution 1:chitosan solution is 1:1-1.

2.

4. A method for preparing a culture medium for multidirectional induction differentiation of amniotic epithelial stem cells according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: Add stem cell induction stimulator I, plant-derived isorhamnetin, and hepatocyte growth factor to DMDM / F12 culture medium in sequence, and stir until dispersed evenly to obtain culture medium I.

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