Method for differentiating mesenchymal stem cells into fat cells and application

Abstract

The invention relates to a method for differentiating mesenchymal stem cells into adipose cells, which comprises the following steps: (1) culturing early passage mesenchymal stem cells to a confluence degree close to 100%, and ensuring enough basic cell quantity; (2) replacing the basic culture medium with a fat induction culture medium, and performing induced differentiation for 14-21 days; (3) observing a saturated oil red O dyeing result; and (4) the method can be applied to differentiation of mesenchymal stem cells in animal cell culture meat into adipocytes to form lipids, and is suitable for differentiation of mesenchymal stem cells into adipocytes in stem cell treatment and health care.

Description

Technical field:

[0003] It involves cell culture and cell differentiation technology, especially fat differentiation induction, which is used to differentiate animal mesenchymal stem cells in animal cell cultured meat into fat cells to form lipids, and is also suitable for the differentiation of mesenchymal stem cells into fat cells in stem cell therapy and health care. Background technology:

[0004] Mesenchymal stem cells (MSCs) are a type of multipotent stem cells that can be isolated from a variety of tissues and induced to differentiate into osteoblasts, chondrocytes, adipocytes, neurons, muscle cells and other functional cells under specific conditions in vitro. Fat cells can be used in many fields such as plastic surgery, medical beauty, tissue repair and artificial meat, and are a very valuable cell material. The fat cells obtained by liposuction surgery are limited, and they cause serious damage to the donor and even bring the risk of infection. In addition, fat cell transplantation is limited to autologous transplantation. Mesenchymal stem cells have multi-differentiation characteristics and can be used as an effective source of fat cells. However, there are many problems that need to be solved in effectively differentiating mesenchymal stem cells into adipocytes: complex induction factors have uncertainty in induction, and differentiation efficiency cannot be guaranteed. Mesenchymal stem cells are a type of adult stem cells derived from bone marrow stroma with high self-renewal ability and multidirectional differentiation potential. Because of its advantages such as easy acquisition and convenient and rapid in vitro expansion, it has become an ideal cell for animal cell artificial meat and stem cell beauty treatment. The differentiation of bone marrow mesenchymal stem cells into myogenic and adipogenic factors has a direct impact on the composition of muscle and fat in animals, and is therefore closely related to meat quality and human health. Summary of the invention:

[0005] The present invention provides a method for differentiating mesenchymal stem cells into fat cells. The method can effectively induce mesenchymal stem cells to differentiate into fat cells and can be used for inducing fat differentiation of various mesenchymal stem cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] (1) Mesenchymal stem cells passaged to P3 were used for adipogenic differentiation induction. Before induction, the cells were observed under a microscope to ensure that they were in a good growth state. After the cells grew to 100% confluence, they were washed twice with PBS to remove floating dead cells;

[0008] (2) replacing the basal medium with adipogenic differentiation induction medium and inducing differentiation for 14 to 21 days to ensure sufficient time for adipogenesis;

[0009] (3) After differentiation was completed, saturated Oil Red O staining was used to verify the differentiation results.

[0010] The beneficial effect of the present invention is that the fat differentiation inducing factor used can greatly promote the uptake and utilization of glucose by mesenchymal stem cells, and stimulate the expression of fat synthesis genes to promote fat synthesis. The present method can effectively induce fat differentiation of various mesenchymal stem cells.

[0011] The components of the adipogenic differentiation induction medium include high-glucose DMEM medium, hydrocortisone, insulin, IBMX, and sodium ascorbate.

[0012] High-glucose DMEM culture medium can provide sufficient glucose to provide sufficient energy and raw materials for fat synthesis for the adipogenic differentiation of mesenchymal stem cells.

[0013] Hydrocortisone is a type of adrenal glucocorticoid drug that acts as a growth factor in the adipose differentiation of mesenchymal stem cells. Compared with the commonly used dexamethasone, it has a milder effect, fewer side effects, and is more cell-friendly.

[0014] Insulin is the only hormone in the body that lowers blood sugar. It can promote the uptake of glucose by cells and stimulate the synthesis of intracellular fat.

[0015] IBMX acts like hydrocortisone and can stimulate the division and proliferation of mesenchymal stem cells.

[0016] As an antioxidant, sodium ascorbate can reduce the oxidative stress on cells during adipose differentiation and increase cell survival during differentiation.

[0017] The contents of the components in the adipogenic differentiation medium are as follows:

[0018] Hydrocortisone: 1nM-1μM

[0019] Insulin: 10 ng / mL-10 μg / mL

[0020] IBMX: 0.5μM-0.5mM

[0021] Sodium ascorbate: 1ng / mL-20ng / mL

[0022] Preferred:

[0023] Hydrocortisone: 50nM

[0024] Insulin: 50 ng / mL

[0025] IBMX: 1 μM

[0026] Sodium ascorbate: 10 ng / mL Description of the drawings:

[0027] Figure 1.Mesenchymal stem cell isolation and morphological observation results of adipocyte differentiation.

[0028] Figure 2 .Staining results of mesenchymal stem cells differentiated into adipocytes after induction. Specific implementation method:

[0029] Embodiment 1:

[0030] (1) Mesenchymal stem cells passaged to P3 were used for adipogenic differentiation induction. Before induction, the cells were observed under a microscope to ensure that they were in a good growth state. After the cells grew to 100% confluence, they were washed twice with PBS to remove floating dead cells;

[0031] (2) replacing the basal medium with adipogenic differentiation induction medium and inducing differentiation for 14 to 21 days to ensure sufficient time for adipogenesis;

[0032] (3) After differentiation is complete, saturated Oil Red O staining is performed to verify the differentiation results ( Figure 1 A).

[0033] Adipogenic differentiation medium used:

[0034] Hydrocortisone: 10nM

[0035] Insulin: 10 ng / mL

[0036] IBMX: 0.5 μM

[0037] Sodium ascorbate: 1 ng / mL

[0038] Embodiment 2:

[0039] (1) Mesenchymal stem cells passaged to P3 were used for adipogenic differentiation induction. Before induction, the cells were observed under a microscope to ensure that they were in a good growth state. After the cells grew to 100% confluence, they were washed twice with PBS to remove floating dead cells;

[0040] (2) replacing the basal medium with adipogenic differentiation induction medium and inducing differentiation for 14 to 21 days to ensure sufficient time for adipogenesis;

[0041] (3) After differentiation is complete, saturated Oil Red O staining is performed to verify the differentiation results ( Figure 1 B).

[0042] Adipogenic differentiation medium used:

[0043] Hydrocortisone: 50nM

[0044] Insulin: 50 ng / mL

[0045] IBMX: 1 μM

[0046] Sodium ascorbate: 10 ng / mL

[0047] Embodiment three:

[0048] (1) Mesenchymal stem cells passaged to P3 were used for adipogenic differentiation induction. Before induction, the cells were observed under a microscope to ensure that they were in a good growth state. After the cells grew to 100% confluence, they were washed twice with PBS to remove floating dead cells;

[0049] (2) replacing the basal medium with adipogenic differentiation induction medium and inducing differentiation for 14 to 21 days to ensure sufficient time for adipogenesis;

[0050] (3) After differentiation is complete, saturated Oil Red O staining is performed to verify the differentiation results ( Figure 1 C).

[0051] Adipogenic differentiation medium used:

[0052] Hydrocortisone: 100nM

[0053] Insulin: 150 ng / mL

[0054] IBMX: 100 μM

[0055] Sodium ascorbate: 20 ng / mL

[0056] It can be seen from the result diagram that under the induction of adipogenic differentiation inducer, mesenchymal stem cells maintain good cell viability and can differentiate into adipocytes, but the adipogenic differentiation is not obvious in Example 3. It is speculated that this is because a large amount of reactive oxygen species is generated during the adipogenic differentiation process, and a certain amount of reactive oxygen species is required to maintain adipogenic differentiation. In Example 3, a high concentration of antioxidants was added, resulting in the inability to differentiate. Therefore, Example 2 is preferably used as the adipogenic differentiation medium.

[0057] Embodiment 4:

[0058] Since the precursor cells of intramuscular fat are more inclined to adipose-derived stem cells, cell culture experiments use myostatin to treat animal muscle satellite cells and adipose-derived stem cells differentiated from mesenchymal stem cells, and then co-culture them to form meat tissue with a mixture of muscle and adipose tissue. This will be closer to natural meat products, and it is also a new strategy for cell-cultured meat.

[0059] Bone marrow was extracted from 1-3 month old pigs, and the mononuclear cell layer was extracted by density gradient centrifugation for adherent culture. Primary (P0) pig BMSCs were obtained and then subcultured, and the multidirectional differentiation ability was identified. The third generation (P3), fourth generation (P4), and seventh generation (P7) pig BMSCs were taken respectively, and adipocyte differentiation induction culture medium was added to the experimental group, and conventional culture medium was added to the control group.

[0060] The changes in cell morphology were observed under an inverted microscope. After 3 weeks of induction, the adipocyte differentiation rate was calculated using Oil Red O staining, and the changes in adipocyte differentiation rate between different generations were compared. Results The porcine bone marrow mononuclear cells extracted in the experiment had the ability to differentiate into adipocytes, indicating that they were pluripotent stem cells. On the 3rd to 7th day after adipogenic induction and differentiation culture, lipid droplets could be observed in the cytoplasm of a few cells in the experimental group. Subsequently, the number and size of the lipid droplets in the cytoplasm and the number of cells containing lipid droplets gradually increased, reaching a peak at about 2-4 weeks. Figure 2 As shown in the figure, the lipid droplets were orange-red by Oil Red O staining; the adipocyte differentiation rate was: P3 (44.2±10.0)%, P4 (48.8±4.6)%, P7 (56.5±4.2)%, the difference was statistically significant (P<0.05). No lipid droplet formation was observed in the control group, and Oil Red O staining was negative.

[0061] The results showed that the adipocyte differentiation medium derived from pig BMSCs can induce pig BMSCs to differentiate into adipocytes in vitro. The differentiation rate of adipocytes increases with the number of passages, and adipose tissue can be formed, becoming a source of fat in cultured meat.

Claims

1. A method for differentiating mesenchymal stem cells into adipocytes and its application, It is characterized in that The following steps are involved: (1) Cultivate early passage mesenchymal stem cells to nearly 100% confluence to ensure sufficient basal cell numbers; (2) replacing the basal medium with adipogenic induction medium and inducing differentiation for 7-21 days; (3) Observation results of saturated oil red O staining. This method can effectively differentiate mesenchymal stem cells into adipocytes and is applicable to the adipogenic differentiation of various mesenchymal stem cells.

2. A method for differentiating mesenchymal stem cells into adipocytes according to claim 1, It is characterized in that The components of the fat differentiation induction medium include high-glucose DMEM medium, hydrocortisone, insulin, IBMX, and sodium ascorbate.

3. A method for differentiating mesenchymal stem cells into adipocytes according to claim 2, It is characterized in that The contents of the components of the adipose differentiation induction medium are as follows: Hydrocortisone: 1nM-1μM Insulin: 10 ng / mL-10 μg / mL IBMX: 0.5μM-0.5mM Sodium ascorbate: 1ng / mL-20ng / mL.

4. A method for differentiating mesenchymal stem cells into adipocytes according to claim 3, It is characterized in that The contents of the components of the adipose differentiation induction medium are as follows: Hydrocortisone: 50nM Insulin: 50 ng / mL IBMX: 1 μM Sodium ascorbate: 10 ng / mL.

5. A method for differentiating mesenchymal stem cells into adipocytes according to claim 1, It is characterized in that In step (2), the amount of adipogenic differentiation medium added is 1 mL per well of each 24-well plate.

6. A method for differentiating mesenchymal stem cells into adipocytes according to claim 1, It is characterized in that In step (2), the adipogenic differentiation medium is replaced every 1-3 days.

7. This method can be applied to the differentiation of animal mesenchymal stem cells in animal cell cultured meat into fat cells to form lipids, and is also applicable to the differentiation of mesenchymal stem cells into fat cells in stem cell therapy and health care.

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