CN112342189B

CN112342189B - Method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells

Info

Publication number: CN112342189B
Application number: CN202011254170.4A
Authority: CN
Inventors: 费文勇; 曹世超; 刘明生; 庞而凯
Original assignee: Northern Jiangsu Peoples Hospital
Current assignee: Northern Jiangsu Peoples Hospital
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2021-08-03
Anticipated expiration: 2040-11-11
Also published as: CN112342189A

Abstract

The invention discloses a method for inducing myogenic differentiation of adipose-derived stem cells (ADSCs), which comprises the step of placing the adipose-derived stem cells (ADSCs) in a medium containing an inducer H₂The culture environment of (2) is induced at a temperature of 35-40 ℃ for 3-9 days. The invention discovers thatAfter 9 days of induction, the cells expressed troponin, myosin, MyoD and MHC, while the sarcomeric alpha-actin expression and myoblast differentiation rate were significantly higher than those of the control group. The invention adopts hydrogen for the first time to effectively promote the myogenic differentiation of ADSCs, compared with the application of 5-azacytidine (5-Aza), hydrogen exposure is a simpler, nontoxic and cheap differentiation method, and simultaneously, the hydrogen can also increase the effect of 5-Aza on the differentiation of ADSC myoblasts. Therefore, hydrogen is a recommended inducer for realizing differentiation of MSC into muscle fibers, and has a wide application prospect.

Description

Method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells

Technical Field

The invention belongs to the technical field of myogenic differentiation of stem cells, and particularly relates to a method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells.

Background

Loss of skeletal muscle function caused by trauma, nerve injury, tumor resection or degenerative muscle disease (e.g., fatty infiltration after rotator cuff tear) is a serious clinical problem that apparently affects people's daily life and work, but few solutions. Stem cell transplantation is a promising technology developed in the 1990's, providing promise for the treatment of skeletal muscle diseases.

In recent years, research on human MSCs has received much attention, most of which are bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs). As shown by in vitro, ex vivo and in vivo evidence, these pluripotent cells can differentiate into mature adipocytes, chondrocytes, osteoblasts, cardiomyocytes, hepatocytes, neuronal-like and endothelial cells, and other lineages. However, bone marrow extraction is painful and the number of bone marrow cells is extremely small, thus requiring in vitro expansion to obtain sufficient cells. Currently, the lengthy amplification process limits the clinical use of BMSCs. ADSCs obtained from adipose tissue by minimally invasive liposuction have numerous advantages and are readily available from renewable cell sources. ADSCs have great advantages over BMSCs because they are easy to harvest, have multi-differentiation potential and are easy to expand, and have genomic stability and low immunogenicity. ADSCs can also be used to produce many pluripotent stem cells with good viability and are seed cells for stem cell bioengineering studies. Because adipose tissue and bone marrow develop from mesoderm and contain easily separable matrix components, ADSCs and BMSCs have many similar biological properties and can retain the properties of stem cells after multiple passages and the degree of senescence is relatively low, which makes them ideal seed cells for tissue engineering.

In 2001, Zuk obtained ADSCs from human adipose tissue for the first time by fat digestion and isolation and stem cell extraction, and research on adipose stem cells continued to be developed. The ADSCs have multidirectional differentiation potential and self-renewal function; in addition, these cells can be induced to differentiate into myoblasts, adipoblasts, osteoblasts and chondroblasts under certain conditions. In this study, adipose tissue was obtained from the groin of new zealand white rabbits by surgery and ADSCs were obtained by enzymatic digestion.

Currently, 5-azacytidine (5-Aza) is the most commonly used chemical agent for inducing differentiation of MSCs. However, this method has some degree of cytotoxicity, and to some extent, cell proliferation is affected, resulting in morphological changes upon cell differentiation. Most importantly, the mechanism of myoblast differentiation remains unclear. Hydrogen is a colorless, odorless, biologically active, reducing, small molecule gas that reacts with reactive oxygen species in the body. Meanwhile, the antioxidant, anti-inflammatory and anti-apoptotic effects of hydrogen have been demonstrated in various animal disease models and human studies. Thanks to the invention of hydrogen-containing incubators, it is now possible to assess the effect of hydrogen on cell proliferation and differentiation in vitro. However, the prior art does not relate to the field of hydrogen used for stem cells, and the effect of hydrogen on differentiation of myoblasts induced by ADSCs is not reported.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to research whether hydrogen has a beneficial effect on myogenic differentiation of ADSC by comparing two methods of ADSC induction and differentiation into muscle cells under different culture conditions.

In a first aspect, the present invention provides a method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs), the method comprising placing cells to be induced in a culture environment containing an inducing agent to induce myogenic differentiation, the inducing agent comprising hydrogen.

Adopt above-mentioned technical scheme's effect: under the culture condition containing hydrogen, the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSC) can be successfully induced, the hydrogen is colorless, tasteless, nontoxic and bioactive reductive small molecule gas, and can react with active oxygen in vivo, and the method can avoid the cell morphological change generated by adopting an inducer 5-azacytidine (5-Aza).

The technical scheme can be further changed into the following technical scheme:

in certain embodiments, the culture environment is hydrogen H₂The concentration of (A) is 20 to 30%, preferably 25%.

By adopting the hydrogen with the concentration, the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSC) can be efficiently induced.

In certain embodiments, the temperature of induction is 35-38 ℃.

In certain embodiments, the temperature of the induction is 37 ℃.

In certain embodiments, the hydrogen gas H₂The induction time is at least 1 day, preferably at least 3 days, more preferably at least 5 days, more preferably at least 9 days.

By adopting the culture conditions, the optimum myogenic differentiation temperature and time can be provided for the cells to be induced, the differentiation rate of the cells to be induced is effectively improved, and the abnormal differentiation generated by the differentiated cells is effectively avoided.

In certain embodiments, the inducing agent further comprises 5-azacytidine (5-Aza).

In certain embodiments, the inducer 5-azacytidine (5-Aza) is at a concentration of 10 μmol/L.

In certain embodiments, the inducer 5-azacytidine (5-Aza) is treated for 24 hours.

5-azacytidine (5-Aza) serving as an inducer is added in an induced differentiation environment at the same time, a synergistic effect is generated between the 5-azacytidine and hydrogen, and the hydrogen can effectively promote myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by the 5-azacytidine.

In certain embodiments, the culture environment further comprises oxygen O₂Carbon dioxide CO₂And nitrogen gas N₂。

In certain embodiments, the culture environment is oxygen O₂The concentration of (A) is 20 to 25%, preferably 21%.

In certain embodiments, the culture environment is carbon dioxide CO₂In a concentration of 3-8% CO₂Preferably 5%.

In certain embodiments, the culture environment is nitrogen N₂The concentration of (A) is 45-50%, preferably 49%.

In certain embodiments, the humidity in the culture environment is maintained above 90%.

By adopting the technical scheme, the culture environment contains oxygen O with certain concentration₂Carbon dioxide CO₂And nitrogen gas N₂Can provide proper gas environment for cell differentiation, and can regulate hydrogen H by the gas₂The concentration is used.

In certain embodiments, the cells to be induced may be subcultured cells, and the cells are cultured using an ADSCs cell culture medium commonly used in the art, such as high glucose DMEM, low glucose DMEM, and the like, preferably high glucose DMEM containing 15% FBS.

In a second aspect, the present invention provides the use of hydrogen as an inducer for inducing myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs).

The antioxidant, anti-inflammatory and anti-apoptotic effects of hydrogen have been demonstrated in a variety of animal disease models and in human studies. Thanks to the invention of hydrogen-containing incubators, it is now possible to assess the effect of hydrogen on cell proliferation and differentiation in vitro. The invention realizes that the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSC) is induced by adopting hydrogen as a single inducer, and provides the application of the hydrogen as the myogenic differentiation inducer.

Compared with the prior art, the invention has the beneficial effects that:

1) by adopting the method, 9 days after the myogenic differentiation of the ADSCs, the RT-PCR result of MyoD expression shows that the expression level of the upstream gene for regulating and controlling the myoblast differentiation in the induction group is higher than that of the control group, and the expression of the sarcomere alpha-actin and troponin, the myosin, MyoD and MHC genes proves the result. There was no significant difference in MyoD expression between the hydrogen group and the 5-Aza group, and these results indicate that the mechanism by which hydrogen and 5-Aza induce myoblast differentiation is similar, and hydrogen exposure is a simpler, non-toxic, and inexpensive method of differentiation compared to 5-Aza administration.

2) The invention proves that two inducers of hydrogen H are obtained through experiments₂And 5-azacytidine (5-Aza) may increase MyoD expression. After 9 days of induction, actin expression rates of

groups

1, 2, 3 and 4 were 3.2% + -0.3%, 48.5% + -5%, 41.1% + -3.4% and 62.8% + -3.5%, respectively. The proportion of myogenic differentiated cells in

groups

2 and 3 was significantly higher than that in group 1 (average p)<0.01), group 2 is lower than group 4 (P)<0.05). The proportion of myogenic differentiated cells in group 2 was increased but not significantly different compared to group 3 (P)>0.05). These results indicate that hydrogen can not only promote myogenic differentiation of ADSCs alone, but also improve myogenic differentiation of 5-Aza-induced cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 is a cell morphology chart of group 1-4 third generation ADSCs before induction, 1 day after induction, and 9 days after induction.

Figure 2 flow cytometry analysis results and expression of cell surface CD markers for passage 3 ADSCs. The x-axis is fluorescence intensity and the y-axis is cell number.

FIG. 3 alizarin red, oil red O and toluidine blue staining and differentiation of ADSCs after induction.

FIG. 4 MTT assay results. Effect of different culture conditions on the viability of ADSCs at passage 3 after 1, 3, 5, 7 and 9 days of exposure. The x-axis is time (days) and the y-axis is the average absorbance (A) value.

FIG. 5 immunofluorescence analysis of ADSC.

FIG. 6 myogenic differentiation rates are expressed as percent actin expression as measured by immunohistochemistry.

FIG. 7 results of troponin, myosin, MyoD and Myosin Heavy Chain (MHC) expression.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

The ADSCs of the invention obtained from rabbits were distributed to two different differentiation inducing incubators: containing 5% CO₂Standard incubator (control group (group 1) and 5-azacytidine (group 2)) and 25% H₂Hydrogen incubator (H)₂(group 3)) and 5-Aza + H₂(group 4)). The morphology of ADSCs was observed using bright field and phase contrast microscopy and confocal laser scanning microscopy. Expression of CD31, CD44, CD45 and CD90 was analyzed by flow cytometry. Viability was assessed by MTT assay, immunocytochemistry and semi-quantitative analysis of sarcomeric alpha-actin expression, and troponin, myosin, MyoD and Myosin Heavy Chain (MHC) expression were measured by RT-PCR. Flow cytometry results showed that ADSCs exhibited typical fusiform morphology and expressed CD90, CD44, but not CD31 and CD 45.

After 9 days of hydrogen and 5-Aza induction, the cells expressed troponin, myosin, MyoD and MHC, but there was no significant difference between

groups

3 and 2. Sarcomere alpha-actin expression and myoblast differentiation rate were significantly higher than those of the control group but lower than those of the 4 th group. The hydrogen can not only increase the effect of 5-Aza on the differentiation of the ADSC myoblasts, but also induce the differentiation of the myoblasts when being singly applied, thereby having wide application prospect.

Example 1

An eight-week-old male New Zealand white rabbit (animal experiment center of Jiangsu university) weighing 2.0kg was used to raise and feed the rabbits in a single-cage feeding condition. The temperature (25 ℃) and humidity (40-60%) and illumination (12h, light and dark cycle) of the house are controlled. Animals were observed for one week prior to surgery to confirm that they were healthy and disease free.

To obtain ADSCs, animals were euthanized by passive inhalation of isoflurane in an anesthesia chamber until overdose occurred. Death of rabbits was assessed by monitoring cardiac-respiratory arrest. Hairs were cut from the abdominal region, an incision was made to expose the peritoneum, and groin fat was removed. The adipose tissue is then placed in a cell laminar flow chamber. Adipose tissues were washed 3 times with Phosphate Buffered Saline (PBS) to remove erythrocytes. The collected adipose tissue was cut into small pieces and transferred to a 20mL centrifuge tube and a mixture of equal volumes of 0.25% trypsin (Gibco, usa) and 0.1% collagenase type I (Sigma, usa) was added. The tissue was incubated on a shaker at 37 ℃ for about 15 minutes with constant agitation. The liquid was then separated into three layers: the upper layer contains yellow oily adipocytes, the middle layer contains adipose tissue, and the bottom layer contains monocytes. The bottom layer was extracted and transferred to centrifuge tubes containing 15% fetal bovine serum (FBS, Gibco, usa) and high glucose DMEM (Sigma, usa). The remaining matrix fraction was treated with 3mL of red blood cell lysis buffer (Sigma, usa) for 10 minutes at room temperature, filtered through a 100mm nylon mesh, and centrifuged at 1200 × g for 10 minutes; then, the supernatant was removed. The cell pellet was then suspended in high glucose DMEM medium containing 15% FBS, 100U/mL penicillin and 100mg/mL streptomycin (Gibco Company, St. Louis, MO, USA). Cells were incubated at 37 ℃ and 5.0% CO₂The whole culture medium was changed every three days. When the cells reached 80% confluence, they were digested with a mixture of 0.25% trypsin and 0.04% EDTA (chinese shanghai reagent) and passaged for later use.

Example 2 evaluation of isolated cells

There are no specific antigens in MSCs. Therefore, there is a need to simultaneously analyze multiple surface antigens and cell morphology to determine characteristics of mesenchymal cells. ADSC surface markers were examined by flow cytometry. Using 0.25% pancreatic eggsPassage 3 adherent cells were treated with white enzyme (Gibco, usa) and washed twice with PBS. Cells were incubated with rabbit anti-CD 31, anti-CD 44, anti-CD 45, and anti-CD 90 antibodies (Invitrogen, usa and Gibco, usa) overnight at 4 ℃. Unbound antibody was removed by washing three times with PBS. After washing, the cells were incubated with Cy 3-labeled anti-goat/anti-rabbit secondary antibody for 45 minutes at room temperature in the dark, and then resuspended in PBS for FACS analysis. Each sample was analyzed by flow cytometry (BD FACSVerse, USA) at least 1X 10⁶And (4) cells.

In this study, cells isolated from adipose tissue have the following characteristics: (1) growth in spindle-shaped adhesion and in colony formation (2) higher expression (> 95%) of CD44, CD90 and CD105, while lower expression (< 5%) of CD31 and CD45 and (3) have multilineage differentiation potential. These characteristics conform to the standards specified for the MSC by ISCT. In combination with the method of isolation, immunophenotypic characterization and cell morphology, the isolated cells are indeed ADSCs. We examined the expression of CD31, CD44, CD45 and CD90 in 3 rd generation ADSCs, CD44 in 95.2% of cells, CD90 in 97.4% of cells, CD31 in 1.6% of cells and CD45 in 1.1% of cells (as shown in fig. 1).

The multi-lineage differentiation potential of ADSCs is characterized by osteogenic, adipogenic, and chondrogenic differentiation. Briefly, ADSCs are applied at 5X 10⁵Density of/mL resuspended and seeded in 6-well plates. When the cultured cells reached 80% confluence, the cells were cultured using osteogenic, adipogenic and chondrogenic differentiation kit. After washing with PBS and fixation with 4% paraformaldehyde for 20 minutes, the stained areas were observed under a fluorescence microscope (Leica, Wetzlar, germany) using alizarin red, oil red O and toluidine blue when differentiation reached the desired number of days, according to the manufacturer's instructions. Multilineage differentiation was confirmed by in vitro induction of osteogenic, chondrogenic and adipogenic lineages (as shown in figure 2). All results indicate that these cells meet MSC evaluation criteria as described by the international society for cell therapy.

Example 3

The third generation cells were digested with a mixture of trypsin and EDTA to give 10⁴cells/mL of single cell suspension, then seeded into 6-well plates. Cells were divided into 4 groups. Group 1 ofA control group; group 2 was treated with inducer 5-Aza; group 3 Induction Agents H₂Processing; group 4 Induction agent 5-Aza + H₂And (6) processing. Cells in

groups

2 and 4 were induced with 10. mu. mol/L5-Aza (Sigma, USA) for 24h and washed with D-Hanks balanced salt solution; then, the medium was replaced with high glucose DMEM containing 15% FBS.

Group

1 and 2 were incubated at 37 ℃ and 5% CO in a conventional incubator₂Incubated together, while

group

3 and 4 were incubated at 37 ℃ in a hydrogen-containing incubator with 25% H₂Incubation; oxygen concentration is 21%, humidity is maintained at 90% or more, and the culture environment contains 5% CO₂And N at a concentration of 49%₂. The medium was replaced with fresh DMEM and FBS every 3 days until the test was started after 9 days.

As shown in fig. 3, the third generation ADSCs before induction were generally spindle shaped with uniform hair distribution and good cell growth. On the first day after induction, neither the proliferating cells in the control group nor the hydrogen group showed significant changes in cell morphology. However, in

groups

2 and 4, partial cell death occurred, the cell shape was slightly larger than before induction, and the cell growth was not as good as before induction. On the 9 th day after induction, the cell density of each group reaches more than 80%, and the cells are distributed in a fusiform spiral shape. The three treatment groups had larger and fewer cells than group 1.

Example 4 cell viability

Cell viability was quantified using the 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-tetrabromoammonium bromide (MTT) method. MTT is a yellow tetrazolium dye that responds to metabolic activity, and a reductase in living cells reduces MTT from a pale yellow compound to a deep blue formazan maz crystal. The cells were cultured at 1X 10⁴Density per well 100 μ L DMEM with FBS seeded in 96-well plates. The groups were tested on

days

1, 3, 5, 7 and 9, at which time 100 μ LMTT (Beyotime Biotechnology, china) was added to each well. The plates were incubated at 37 ℃ for 4 hours. The supernatant was then removed and 200 μ L of dimethyl sulfoxide (DMSO, Merck, germany) was added to each well to dissolve the blue material. The absorbance (OD) at 570nm was read using a microplate reader (Biotek, USA).

The MTT results showed a significant reduction in growth rate in

groups

2 and 4 compared to

groups

1 and 3. Notably, the growth rates of

groups

2 and 4 have not reached stationary phase and both groups show a sustained tendency to proliferate. However, the trend is relatively smooth. As shown by the MTT proliferation curves, the growth rate of group 3 was less than that of group 1 on the first 5 days of culture under different culture conditions. However, after day 5, the growth rate of group 3 increased and exceeded group 1. Group 2 grew faster from day two than group 3 (as shown in fig. 4).

Example 5 intracellular smooth muscle sarcomere alpha-actin expression and percent expression

After inducing cell differentiation, the plates were washed with PBS solution and the cells were fixed with 4% paraformaldehyde in PBS for 15 minutes. Then, the cells were washed twice with PBS for 10 minutes each, and permeabilized with 0.25% Triton X-100(Roche, germany), and then washed 3 times with PBS only. Coverslips containing treated cells were blocked with PBS containing 1% Bovine Serum Albumin (BSA) and 5% goat serum. Then, the cells were incubated with primary antibody in a humidified chamber at 4 ℃ for 2 hours. Then, the cells were incubated overnight at 4 ℃ under a rabbit anti-sarcomeric protein- α -actin antibody (1: 200) (Proteintech, USA), a rabbit anti-connexin 43 antibody, a monoclonal GJA1 antibody (1: 400 dilution) (CX-43, AbCam), and goat anti-rabbit and goat anti-rat Alexa Fluor 555-conjugated secondary antibodies. Hoechst 33258(Beyotime Biotechnology, china) was added to each slide and the slides were incubated in the dark for 30 minutes at room temperature. After washing, the slides were sealed and micrographs were taken using a fluorescence microscope (Leica, germany).

The confocal laser scanning microscope can be used for quantifying fluorescence of the marked tissue specimen and displaying fluorescence change in the Z-axis direction. Images were viewed and captured using 420 to 460nm (blue), 510 to 560nm (green) and 560 to 660nm (red) filters, respectively. Photographs were analyzed using ImageJ Software (Rawak Software, germany) and data were statistically analyzed using prism demo Software (GraphPad Software, usa).

All ADSC groups induced differentiation expressed sarcomere alpha-actin. However, the control cells were hardly differentiated. Sarcomeric protein-alpha-actin, which is normally expressed in skeletal muscle cells, is expressed in the exoskeleton of the cells in all three differentiation protocols. There was no significant difference in actin expression between

groups

3 and 2, but the expression level was highest in group 4. Cell density can be approximated by observing the density of blue-stained nuclei. The cell density of group 3 was similar to that of group 1, higher than that of the two groups induced by 5-aza. However, the nuclei of group 1 were larger in volume than the other groups (as shown in fig. 5).

Myogenic differentiation rate was expressed as percent actin expression by immunohistochemistry. After 9 days of induction, the expression rates of the

groups

2 and 3 was significantly higher than in group 1 (mean P <0.01), and group 2 was lower than group 4 (P < 0.05). The proportion of myogenic differentiated cells in group 2 was increased compared to group 3, but the difference was not significant (P >0.05) (as shown in FIG. 6). These results indicate that hydrogen can not only promote myogenic differentiation of ADSCs alone, but also improve myogenic differentiation of 5-Aza-induced cells.

Example 6 troponin, myosin, MyoD and Myosin Heavy Chain (MHC) expression (RT-PCR)

Total RNA from untreated and induced differentiated ADSCS was isolated using RNeasy Mini kit (Invitrogen, USA), including digestion with DNase I. The reverse transcription reaction system was prepared using the reverse transcription kit according to the instructions. Primers for reverse transcription PCR were designed and synthesized using Primer Premier 5.0 software, with housekeeping gene GAPDH as an internal reference. The reaction system was put into a real-time detector (Applied Biosystems, USA) for detection using SYBR Green PCR kit (Thermo Scientific, USA) according to the instructions. Each set had two duplicate wells and the following temperature program was used: 40 cycles of 94 ℃ for 10 minutes and 94 ℃ for 20 seconds, 55 ℃ for 20 seconds and 72 ℃ for 20 seconds.

Quantitative RT-PCR results showed that the mRNA expression levels of troponin, myosin and MyoD were significantly higher in

groups

2 and 3 than in group 1 (average p < 0.01). Furthermore, MHC mRNA expression was increased in

groups

2 and 3 compared to group 1 (P < 0.05). Furthermore, the expression levels of all tested mRNAs in group 4 were significantly higher than those in group 2 (mean P < 0.05). The expression level of these mRNAs in group 2 was higher than that in group 3, but there was no significant difference between the two groups (both P >0.05) (as shown in FIG. 7).

The RT-PCR result of MyoD expression 9 days after the myogenic differentiation of the ADSCs shows that the expression level of the upstream gene for regulating and controlling the myoblast differentiation in the induction group is higher than that of the control group. There was no significant difference in MyoD expression between the hydrogen group and the 5-aza group, but synergy of the two inducers could increase MyoD expression. RT-PCR analysis showed similar results for the secondary regulatory genes troponin and myosin and the terminal differentiation gene MHC. These results indicate that the mechanism by which hydrogen and 5-Aza induce myoblast differentiation is similar.

Therefore, hydrogen and 5-Aza can induce myogenic differentiation of adipose tissue-derived MSCs. This result is demonstrated by the expression of sarcomeric alpha-actin and troponin, myosin, MyoD and MHC genes. Like 5-aza, hydrogen effectively promotes myogenic differentiation. Furthermore, hydrogen exposure is a simpler, non-toxic and inexpensive method of differentiation compared to 5-aza administration. Therefore, hydrogen is the recommended inducer to achieve differentiation of MSCs into muscle fibers.

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.

Claims

1. A method for inducing myogenic differentiation of adipose-derived mesenchymal stem cells, which is characterized in that the method comprises the step of placing cells to be induced in a culture environment containing an inducer, wherein the inducer comprises hydrogen with the concentration of 25%.

2. The method of claim 1, wherein the temperature of induction is 35-38 ℃.

3. The method of claim 2, wherein the temperature of induction is 37 ℃.

4. The method of claim 1, wherein the hydrogen induction time is at least 1 day.

5. The method of claim 4, wherein the hydrogen induction time is at least 3 days.

6. The method of claim 4, wherein the hydrogen induction time is at least 5 days.

7. The method of claim 1, wherein the inducing agent further comprises 5-azacytidine.

8. The method according to claim 7, wherein the concentration of the inducer 5-azacytidine is 10 μmol/L.

9. The method of claim 7, wherein the inducer 5-azacytidine is treated for 24 hours.

10. The method of claim 1, wherein the culture environment further comprises oxygen, carbon dioxide, and nitrogen.

11. The application of hydrogen as an inducer in inducing myogenic differentiation of adipose-derived mesenchymal stem cells is characterized in that the concentration of the hydrogen is 25%.