Umbilical cord mesenchymal stem cell adipogenic differentiation medium and culture method

Abstract

The present invention provides an umbilical cord mesenchymal stem cell adipogenic differentiation medium and a culture method, belonging to the technical field of stem cell induction differentiation. The medium comprises DMEM culture medium, fetal bovine serum, dexamethasone, insulin, indomethacin, IBMX, zardaverine, and glabridin. By adding zardaverine and glabridin to the umbilical cord mesenchymal stem cell adipogenic differentiation medium at a certain concentration, the synergistic effect of zardaverine, glabridin, and other components can better induce the umbilical cord mesenchymal stem cells to differentiate into adipocytes, increase the number of lipid droplets, increase the volume of lipid droplets, enhance the lipid storage capacity of adipocytes, shorten the adipogenic differentiation induction cycle, and solve the problems of low adipogenic differentiation rate, long induction time, few and small lipid droplets, and unstable adipogenic effect in the three-way differentiation identification of umbilical cord mesenchymal stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell induction differentiation, and specifically relates to a culture medium and a method for culturing umbilical cord mesenchymal stem cells into adipogenic differentiation. Background Art

[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells with high self-renewal capacity and multiple differentiation potentials. They are widely found in adipose tissue, bone marrow, umbilical cord, and placental tissue. They can proliferate in vitro and be induced to differentiate into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, pancreatic islet cells, vascular endothelial cells, and epidermal cells, among other cells. They are of great significance in the fields of tissue engineering and regenerative medicine. Human umbilical cord MSCs are widely used in experimental and clinical research in cell therapy and regenerative medicine due to their low immunogenicity, multipotential differentiation potential, and immunomodulatory and anti-inflammatory effects.

[0003] Mesenchymal stem cells can differentiate into mature lipid droplets when exposed to adipogenic induction medium, validating their biological properties of directed adipogenesis and providing a test basis for stem cell function evaluation. Adipose differentiation occurs in two main stages: the committed stage, in which mesenchymal stem cells become preadipocytes; and the terminal differentiation stage, in which preadipocytes differentiate into mature adipocytes.

[0004] Li Xiuying et al. (Li Xiuying, Bai Jinping, Li Xue, et al. Comparison of the in vitro proliferation and adipogenic abilities of three human mesenchymal stem cells [J]. Journal of Jilin University: Medical Edition, 2014(4):7. DOI:10.13481 / j.1671-587x.20140420.) showed that mesenchymal stem cells from different sources have significant differences in their adipogenic differentiation abilities. Specifically, umbilical cord-derived mesenchymal stem cells have the worst adipogenic differentiation abilities compared to adipose-derived and bone marrow-derived mesenchymal stem cells. Regarding the mechanism of adipogenic differentiation of mesenchymal stem cells, many important signaling molecules are involved in the regulatory process. During adipocyte differentiation, the most critical regulator of adipogenic differentiation is the nuclear peroxisome proliferator-activated receptor γ (PPARγ). This receptor is adipose tissue-specific, with expression increasing continuously from the initiation of differentiation to the formation of mature adipocytes. Its role in adipogenic differentiation has been elucidated and confirmed, making PPARγ a marker for adipogenic detection. Studies have shown that during adipogenic differentiation, PPARγ mRNA expression is highest in adipose-derived mesenchymal stem cells, followed by bone marrow-derived mesenchymal stem cells, and lowest in umbilical cord-derived mesenchymal stem cells, positively correlating with their adipogenic capacity. Further studies have revealed that approximately 20 miRNAs are involved in regulating adipogenic differentiation of mesenchymal stem cells. One of these, the miR-310b–miR-130b cluster, underlies PPARγ expression and exhibits a significant negative correlation with the adipogenic capacity of mesenchymal stem cells derived from three different tissues: adipose, bone marrow, and umbilical cord. Therefore, due to their inherent non-adipose developmental background, the basal mRNA expression level of the adipogenic core transcription factor PPARγ in umbilical cord-derived mesenchymal stem cells is significantly lower than that in adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells, and the threshold for initiating adipogenic differentiation is significantly increased, so the adipogenic differentiation ability is significantly reduced.

[0005] The current classic system for adipogenic induction is the "triple induction method," which combines insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX). Adipose-derived and bone marrow-derived mesenchymal stem cells can effectively differentiate into adipocytes under this classic induction system, accumulating significant lipid droplets in the cytoplasm. However, due to differences in gene expression profiles and differentiation potential, umbilical cord-derived mesenchymal stem cells exhibit significantly reduced adipogenic differentiation capacity under the same induction conditions. Furthermore, adipogenic differentiation is characterized by prolonged induction time, easy cell shedding and death during adipogenic differentiation, formation of few and small lipid droplets, low differentiation induction rate, and unstable adipogenic response. Summary of the Invention

[0006] To address the above-mentioned problems, the present invention provides an umbilical cord mesenchymal stem cell adipogenic differentiation medium and culture method. By adding zardaverine and glabridin to the umbilical cord mesenchymal stem cell adipogenic differentiation medium at certain concentrations, zardaverine, glabridin, and other ingredients synergistically improve the adipogenic differentiation rate of umbilical cord mesenchymal stem cells, increase the number and volume of lipid droplets, enhance the lipid storage capacity of adipocytes, shorten the adipogenic differentiation induction period, and facilitate operation and achieve stable induction results. This method solves the problems of low adipogenic differentiation rate of umbilical cord mesenchymal stem cells, small and few lipid droplets, long induction time, and unstable adipogenic effect.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] In one aspect, the present invention provides an umbilical cord mesenchymal stem cell adipogenic differentiation medium, comprising: DMEM medium, fetal bovine serum, dexamethasone, insulin, indomethacin, IBMX, Zardaverine and glabridin.

[0009] Furthermore, the umbilical cord mesenchymal stem cell adipogenic differentiation medium includes: DMEM medium, 10% (v / v) fetal bovine serum, 5-15 μg / ml insulin, 0.5-1.5 μM dexamethasone, 100-200 nM indomethacin, 0.2-0.6 mM IBMX, 8-12 μM Zardaverine and 5-20 μM glabridin.

[0010] Furthermore, the umbilical cord mesenchymal stem cell adipogenic differentiation medium includes: DMEM medium, 10% (v / v) fetal bovine serum, 8-12 μg / ml insulin, 0.8-1.2 μM dexamethasone, 125-175 nM indomethacin, 0.3-0.5 mM IBMX, 9-11 μM Zardaverine and 10-15 μM glabridin.

[0011] Furthermore, the umbilical cord mesenchymal stem cell adipogenic differentiation medium includes: DMEM medium, 10% (v / v) fetal bovine serum, 10 μg / ml insulin, 1.0 μM dexamethasone, 150 nM indomethacin, 0.4 mM IBMX, 10 μM Zardaverine and 10-15 μM glabridin.

[0012] On the other hand, the present invention provides the use of any of the above-mentioned umbilical cord mesenchymal stem cell adipogenic differentiation medium in the adipogenic differentiation of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, chorionic villus mesenchymal stem cells, dental pulp mesenchymal stem cells and uterine blood mesenchymal stem cells.

[0013] In another aspect, the present invention provides a method for culturing adipogenic differentiation of umbilical cord mesenchymal stem cells, comprising inoculating umbilical cord mesenchymal stem cells into any of the above-mentioned adipogenic differentiation medium for umbilical cord mesenchymal stem cells for induction culture.

[0014] Furthermore, the P3-P5 umbilical cord mesenchymal stem cells are inoculated into any of the above-mentioned umbilical cord mesenchymal stem cell adipogenic differentiation medium for induction culture.

[0015] Furthermore, the umbilical cord mesenchymal stem cells are inoculated into any of the above-mentioned umbilical cord mesenchymal stem cell adipogenic differentiation medium for induction culture, and the medium is replaced every 3-4 days. The induction differentiation cycle is 7-21 days.

[0016] Compared with the existing technology, the effective effects of the present invention include:

[0017] 1. The present invention provides an umbilical cord mesenchymal stem cell adipogenic differentiation medium. In addition to essential adipogenic differentiation induction components such as insulin, dexamethasone, and indomethacin, the innovative use of zardaverine and glabridin synergistically promotes the adipogenic differentiation of umbilical cord mesenchymal stem cells, shortens the induction time, and improves the adipogenic rate.

[0018] 2. The present invention provides a medium for adipogenic differentiation of umbilical cord mesenchymal stem cells. A significant number of lipid droplets can be observed under a microscope approximately three days after the first induction medium change. A large number of lipid droplets are formed after 14 days of induction, significantly shortening the detection period for the adipogenic differentiation function of umbilical cord mesenchymal stem cells.

[0019] 3. The lipid solvent decolorization method was used to measure the OD 510nm absorbance to quantitatively analyze the adipogenic differentiation results of umbilical cord mesenchymal stem cells. Within the same induction time, the OD 510nm absorbance of the present invention was at least 2 times higher than that of the control experimental group.

[0020] 4. The components of the umbilical cord mesenchymal stem cell adipogenic differentiation medium provided by the present invention are safe, non-cytotoxic, and have clear ingredients. Only one culture medium is used during the induction process, and the traditional A and B induction culture media do not need to be replaced in rotation, making the operation simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 This is a diagram of the cell morphology of human umbilical cord mesenchymal stem cells in Example 1;

[0023] Figure 2 This is a graph showing the results of flow cytometry detection of surface markers of human umbilical cord mesenchymal stem cells in Example 1;

[0024] Figure 3 The figures are graphs showing the results of human umbilical cord mesenchymal stem cells induced in the adipogenic differentiation induction medium of the present invention for 3, 7, and 14 days in Example 1, wherein A corresponds to the results of 3 days of induction; B corresponds to the results of 7 days of induction; and C corresponds to the results of 14 days of induction;

[0025] Figure 4 These are Oil Red O staining results of human umbilical cord mesenchymal stem cells induced for 2-3 weeks in a negative control and different adipogenic differentiation induction media in Example 1, wherein A corresponds to the negative control staining result; B corresponds to the staining result of the adipogenic differentiation induction medium provided by the present invention for 14 days; C corresponds to the staining result of the experimental control group induced with the adipogenic differentiation induction medium for 21 days; and D corresponds to the staining result of the commercial adipogenic differentiation induction medium for 21 days. DETAILED DESCRIPTION

[0026] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Unless otherwise specified, the reagents, methods and instruments used in the present invention are conventional reagents, methods and facilities in the art. In the embodiments, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. All reagents or instruments are not specified by manufacturer, and are conventional products that can be directly purchased commercially.

[0027] It should be noted that the technical solution provided by the present invention has significant advantages in inducing adipogenic differentiation of umbilical cord mesenchymal stem cells. Furthermore, the technical solution is also applicable to adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, chorionic villus-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and uterine blood-derived mesenchymal stem cells.

[0028] The present invention provides a culture medium and culture method for adipogenic differentiation of umbilical cord mesenchymal stem cells, belonging to the technical field of stem cell induction differentiation. The culture medium comprises DMEM culture medium, fetal bovine serum, dexamethasone, insulin, indomethacin, IBMX, zardaverine, and glabridin. The synergistic effect of zardaverine, glabridin, and other ingredients can better induce umbilical cord mesenchymal stem cells to differentiate into adipocytes, increase the number and volume of lipid droplets, enhance the lipid storage capacity of adipocytes, and shorten the adipogenic differentiation induction period.

[0029] IBMX plays a crucial role. Numerous studies have shown that removing IBMX from the adipogenic induction system significantly reduces adipogenesis or even prevents it. IBMX is a nonspecific inhibitor of phosphodiesterase, inhibiting the degradation of cAMP. cAMP is a crucial adipogenic factor that can increase PPARγ expression by activating protein kinase C (PKC), thereby promoting adipocyte differentiation.

[0030] Zardaverine is a dual phosphodiesterase inhibitor that simultaneously inhibits PDE3 / PDE4, synergistically increasing intracellular cAMP levels, activating cAMP response element binding protein, and regulating CCAAT enhancer binding protein-α (c / EBPα) expression, promoting adipocyte differentiation, similar to IBMX. Glabridin, on the other hand, increases the gene expression of PPAR-γ, c / EBPα, and lipoprotein lipase, thereby enhancing the lipid storage capacity of adipocytes. The synergistic effect of zardaverine and glabridin promotes adipocyte differentiation and lipid deposition.

[0031] In order to better illustrate the embodiments of the present invention, the present invention is further described in detail below through specific examples.

[0032] Example 1

[0033] Step (1): Passaging of umbilical cord mesenchymal stem cells:

[0034] The cells were seeded into T75cm2 cell culture flasks, an appropriate amount of mesenchymal stem cell complete culture medium was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0035] Umbilical cord mesenchymal stem cells Figure 1 As shown, the prepared umbilical cord mesenchymal stem cells were identified by flow cytometry, and the cell surface positive markers CD29, CD44, CD73, CD90, CD105 and negative markers CD14, CD19, CD34, CD45, CD90, HLA-DR were marked respectively. The flow cytometer detection results are shown in FIG. Figure 2As shown, the test results are consistent with the surface marker characteristics of umbilical cord mesenchymal stem cells, indicating that the separation and preparation of umbilical cord mesenchymal stem cells are successful.

[0036] Step (2): Adipogenic differentiation of umbilical cord mesenchymal stem cells:

[0037] Step (2.1): According to the experimental requirements, 4 groups of umbilical cord mesenchymal stem cell adipogenic differentiation culture media containing different components were set up, among which group A was a negative control group, group B was an experimental control group, group F was an experimental group using the culture medium provided by the present invention, and group M was a commercial induction culture medium control group.

[0038] Group A was the negative control group, which consisted of DMEM culture medium and 10% (v / v) fetal bovine serum.

[0039] Group B is the experimental control group, which includes B1-B5. The culture medium components are as follows:

[0040] Group B1: DMEM medium, 10% (v / v) fetal bovine serum, 5 μg / ml insulin, 0.5 μM dexamethasone, 100 nM indomethacin, and 0.2 mM IBMX.

[0041] Group B2: DMEM medium, 10% (v / v) fetal bovine serum, 15 μg / ml insulin, 1.5 μM dexamethasone, 200 nM indomethacin, and 0.6 mM IBMX.

[0042] Group B3: DMEM medium, 10% (v / v) fetal bovine serum, 8 μg / ml insulin, 0.8 μM dexamethasone, 125 nM indomethacin, and 0.3 mM IBMX.

[0043] Group B4: DMEM medium, 10% (v / v) fetal bovine serum, 12 μg / ml insulin, 1.2 μM dexamethasone, 175 nM indomethacin, and 0.5 mM IBMX.

[0044] Group B5: DMEM medium, 10% (v / v) fetal bovine serum, 10 μg / ml insulin, 1.0 μM dexamethasone, 150 nM indomethacin, and 0.4 mM IBMX.

[0045] Group F is the culture medium experimental group provided by the present invention, including F1-F6, and its culture medium components are respectively Zardaverine and glabridin added to the corresponding Group B experimental group at certain concentrations, as follows:

[0046] F1 is based on B1 with the addition of 8μM Zardaverine and 5μM glabridin.

[0047] F2 is based on B2 with the addition of 12 μM Zardaverine and 20 μM glabridin.

[0048] F3 is based on B3 with the addition of 9μM Zardaverine and 10μM glabridin.

[0049] F4 is based on B4 with the addition of 11 μM Zardaverine and 15 μM Glabridin.

[0050] F5 is based on B5 with the addition of 10 μM Zardaverine and 10 μM Glabridin.

[0051] F6 is based on B5 with the addition of 10 μM Zardaverine and 15 μM Glabridin.

[0052] Group M was a commercial induction medium control group purchased from STEMCELL (MesenCult™ Adipogenic Differentiation Kit (Human), 05412).

[0053] Step (2.2): When the cell confluence of the cells to be passaged reaches 80%-90%, the cells are harvested and seeded into a 6-well plate at a density of 5000 cells / cm2. An appropriate amount of mesenchymal stem cell complete medium is added to each well, and the cells are cultured in a 37°C, 5% CO2 incubator. When the cell confluence in the 6-well plate reaches 70%-90%, the complete medium is aspirated, and 2 ml of the experimental control group medium, the adipogenic differentiation induction medium of the present invention, the purchased commercial adipogenic differentiation induction medium, and the negative control medium are added to each well, and the cells are cultured in a 37°C, 5% CO2 incubator.

[0054] Step (2.3): Replace the experimental control group culture medium, the adipogenic differentiation induction culture medium of the present invention, the commercially available adipogenic differentiation induction culture medium, and the negative control culture medium with fresh culture medium every 3-4 days.

[0055] The results are as follows Figure 3 As shown, umbilical cord mesenchymal stem cells formed a large number of small lipid droplets under a microscope after 3 days of induction with the adipogenic differentiation induction medium of the present invention. As the induction time prolonged, the number of small lipid droplets formed increased and the lipid droplets became larger. In contrast, no obvious lipid droplets were observed in the experimental control group, the commercial experimental group, and the negative control group. After 14 days of induction, a small number of lipid droplets were observed in the experimental control group and the commercial experimental group, while the lipid droplets in the experimental group of the present invention were more obvious, more numerous, and larger in size.

[0056] Oil red O staining was performed on the umbilical cord mesenchymal stem cells induced by adipogenic differentiation of the culture medium experimental group provided by the present invention and the commercial experimental group. The staining results are as follows: Figure 4 As shown, the umbilical cord mesenchymal stem cells induced by the adipogenic differentiation induction medium provided by the present invention can produce more and larger lipid droplets after 14 days of induction than those induced by the commercial adipogenic differentiation induction medium for 21 days, indicating that the induction medium provided by the present invention can greatly shorten the adipogenic differentiation induction cycle, and has a higher adipogenesis rate and a more stable adipogenesis effect.

[0057] Step (2.4): Quantitatively assess the adipogenic differentiation properties of umbilical cord mesenchymal stem cells induced with different culture media. Decolorize the umbilical cord mesenchymal stem cells after staining with Oil Red O. Gently shake at room temperature for 20–30 minutes to fully dissolve the Oil Red O dye within the cells. Collect the extract and centrifuge to remove cellular debris. Transfer the extract to an ELISA plate, adding 200 μl to each well, with triplicate wells per group. Measure the OD value of each well at a wavelength of 510 nm using a microplate reader. The average value for each group was calculated and normalized. The grouped analysis results are shown in Table 1.

[0058] Table 1 Absorbance detection and analysis results of different groups of adipogenic differentiation cells after decolorization

[0059] Group A B1 B2 B3 B4 B5 M OD510nm (normalized to negative control) 1 2.4 2.3 2.6 2.8 3.1 3.4 Group F1 F2 F3 F4 F5 F6 - OD510nm (normalized to negative control) 5.4 5.3 6.6 6.7 7.7 7.8 -

[0060] The analysis results in Table 1 show that both the commercial experimental group and the experimental control group B produced relatively small numbers of lipid droplets after 14 days of induction. The OD510nm absorbance results after Oil Red O dye decolorization were consistent with those in random field images. The adipogenic induction effects of the experimental groups F1-F6 in the present invention were significantly superior to those of the experimental control group B and the commercial experimental group M. Furthermore, the differentiation effects of the experimental groups in the culture medium provided by the present invention were ranked from best to worst: F5 and F6, F3 and F4, and F1 and F2. Specifically, the induction groups supplemented with zardaverine and glabridin produced significantly more mature lipid droplets than those induced by the other experimental groups. The number of lipid droplets induced by F1 and F2 was 2.27 times that of the experimental control group B; the number of lipid droplets induced by F3 and F4 was 2.46 times that of the experimental control group B; and the number of lipid droplets induced by F5 and F6 was 2.5 times that of the experimental control group B. These results indicate a high number of lipid droplets, both large in size and high adipogenic differentiation efficiency.

[0061] In summary, the adipogenic differentiation medium with added Zardaverine and Glabridin can better induce human umbilical cord mesenchymal stem cells to differentiate into adipocytes. Compared with the classic adipogenic differentiation medium and commercial adipogenic differentiation medium, the adipogenic differentiation medium of the present invention has higher induction efficiency and better results.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A medium for adipogenic differentiation of umbilical cord mesenchymal stem cells, characterized in that: include: DMEM medium, 10% (v / v) fetal bovine serum, 5-15 μg / ml insulin, 0.5-1.5 μM dexamethasone, 100-200 nM indomethacin, 0.2-0.6 mM IBMX, 8-12 μM Zardaverine and 5-20 μM glabridin.

2. The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to claim 1, characterized in that include: DMEM medium, 10% (v / v) fetal bovine serum, 8-12 μg / ml insulin, 0.8-1.2 μM dexamethasone, 125-175 nM indomethacin, 0.3-0.5 mM IBMX, 9-11 μM Zardaverine and 10-15 μM glabridin.

3. The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to claim 2, characterized in that include: DMEM medium, 10% (v / v) fetal bovine serum, 10 μg / ml insulin, 1.0 μM dexamethasone, 150 nM indomethacin, 0.4 mM IBMX, 10 μM Zardaverine and 10-15 μM glabridin.

4. The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to any one of claims 1 to 3 is used in the adipogenic differentiation of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, amniotic-derived mesenchymal stem cells, chorionic-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and uterine blood-derived mesenchymal stem cells.

5. A method for culturing umbilical cord mesenchymal stem cells by inducing adipogenic differentiation, characterized by: The umbilical cord mesenchymal stem cells are inoculated into the umbilical cord mesenchymal stem cell adipogenic differentiation medium according to any one of claims 1 to 3 for induction culture.

6. The method for culturing adipogenic differentiation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that: The umbilical cord mesenchymal stem cells are P3-P5 generation mesenchymal stem cells.

7. The method for culturing adipogenic differentiation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that: The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to any one of claims 1 to 3 is replaced with a new one every 3 to 4 days, and the induction differentiation cycle is 7 to 21 days.

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