CN112725264A - Method for inducing differentiation of human mesenchymal stem cells into lipid in vitro - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for inducing human mesenchymal stem cells to differentiate into lipid in vitro. The invention provides a method for inducing human mesenchymal stem cells to differentiate into lipid in vitro, which comprises the following steps: (1) culturing human mesenchymal stem cells by using a culture medium; (2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1); (3) adding a differentiation inducing agent to differentiate into lipid; (4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent; (5) repeating the steps (1) to (4) once or more times. Solves the phenomenon of cell reduction in the process of cell induced differentiation. In addition, the method developed by the application avoids the phenomenon of contracture caused by the reduction of the adherence capacity of the mesenchymal stem cells after differentiation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing human mesenchymal stem cells to differentiate into lipid in vitro.
Background
Mesenchymal stem cells are pluripotent stem cells that have all of the commonalities of stem cells, namely self-renewal and multipotential differentiation capacity. The application is most in clinical application, and the combined application with hematopoietic stem cells can improve the success rate of transplantation and accelerate hematopoietic reconstruction. The mesenchymal stem cells are clinically applied to the aspects of solving various blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, repair of partial resection injury of knee joint meniscus, autoimmune diseases and the like, and make a major breakthrough, thereby saving the lives of more patients.
In order to further improve the research of the differentiation performance of the mesenchymal stem cells, more and more applicants are engaged in the research of the mesenchymal stem cells. The invention patent of Chinese patent application No. 201910037685.X discloses a method for inducing differentiation by mesenchymal stem cell adipogenesis, and the method mainly discloses the composition and preparation of a mesenchymal stem cell adipogenesis induction differentiation culture medium.
However, the culture medium used in the above mesenchymal stem cell adipogenic induction differentiation process is still relatively complex, and for the aspect of cell culture in such biotechnology field, the change of micro conditions will affect the differentiation and growth of cells. A phenomenon that causes reduction of cells during the process of inducing differentiation. In order to solve the technical problems existing in the prior art, the development of a method for inducing the differentiation of human mesenchymal stem cells into lipids in vitro, which is simple to operate and easy to obtain culture medium, becomes an important research.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a method for inducing differentiation of human mesenchymal stem cells into lipids in vitro, comprising the steps of:
(1) culturing human mesenchymal stem cells by using a culture medium;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1);
(3) adding a differentiation inducing agent to differentiate into lipid;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) repeating the steps (1) to (4) once or more times.
As a preferable mode, the time for the culture in the step (1) is 1 day or more.
As a preferable technical scheme, the culture time in the step (1) is 1-2 days.
As a preferable technical scheme, the agent for inducing differentiation into lipid in the step (2) is replaced every 24-48 hours.
As a preferable technical scheme, the fat formation time in the step (3) is more than 7 days.
As a preferable technical scheme, the fat formation time in the step (3) is 7-9 days.
As a preferable technical scheme, the step (5) is repeated for 1-4 times from the step (1) to the step (4).
As a preferred technical scheme, the culture medium in the step (1) is selected from MM3 culture medium.
As a preferred technical scheme, the MM3 culture medium comprises a mesenchymal stem cell basic culture medium and/or a serum substitute.
As a preferable technical scheme, the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 15-25: 1.
has the advantages that: the smooth differentiation and adipogenesis of the mesenchymal stem cells in the process of differentiating the mesenchymal stem cells into the lipid are obtained through a great amount of creative experiments of the applicant, and the method developed by the application can be used for inducing and differentiating human mesenchymal stem cells of different sources such as umbilical cords, fat, placenta and amnion into the lipid, so that the phenomenon that the cells are withered and dead due to lack of nutrient substances in the differentiation process is avoided, and the phenomenon that the cells are reduced in the cell induction and differentiation process is solved. In addition, the method developed by the application avoids the phenomenon of contracture caused by the reduction of the adherence capacity of the mesenchymal stem cells after differentiation.
Drawings
Fig. 1 is a diagram illustrating a state in which mesenchymal stem cells are cultured using MM3 medium for 2 days according to example 1 of the present invention.
Detailed Description
The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
As used herein, a feature that does not define a singular or plural form is also intended to include a plural form of the feature unless the context clearly indicates otherwise. It will be further understood that the term "prepared from …," as used herein, is synonymous with "comprising," including, "comprising," "having," "including," and/or "containing," when used in this specification means that the recited composition, step, method, article, or device is present, but does not preclude the presence or addition of one or more other compositions, steps, methods, articles, or devices. Furthermore, the use of "preferred," "preferably," "more preferred," etc., when describing embodiments of the present application, is meant to refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In order to solve the above technical problems, the present invention provides a method for inducing differentiation of human mesenchymal stem cells into lipids in vitro, comprising the steps of:
(1) culturing human mesenchymal stem cells by using a culture medium;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1);
(3) adding a differentiation inducing agent to differentiate into lipid;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) repeating the steps (1) to (4) once or more times.
In some preferred embodiments, the time of the culture in step (1) is 1 day or more.
In some preferred embodiments, the culturing in step (1) is carried out for 1 to 2 days.
In some preferred embodiments, the differentiation-inducing agent in step (2) is replaced every 24 to 48 hours.
In the present application, the induced differentiation into lipid reagent is purchased from Israel Biological Industries (BioInd); the induced Differentiation lipid reagent comprises an Adipogenic Differentiation basic Medium, Adipogenic SF, XF Supplement Mix I, Adipogenic SF and XF Supplement Mix II.
An adaptive Differentiation basic Medium, model 05-330-1B; AdipogenicSF, XFSupplement Mix I, model 05-331-1-01; the model number of the AdipogenicSF and XF Supplement Mix II is 05-332-1-15.
The use method of the agent for inducing differentiation into lipid comprises the following steps:
1) thawing the Adipogen SF, XF Supplement Mix I and Adipogen Supplement SF, XF Supplement Mix II at room temperature for use
2) Adding 0.1mL of Adipogenic SF, XF Supplement Mix I, 1.5mL of LAdipogenicSF and XF Supplement Mix II into 100mL of Adipogenic Differentiation basic Medium, placing the mixture in an environment at 2-8 ℃, and storing the mixture for later use.
In some preferred embodiments, the time for lipolysis in step (3) is 7 days or more.
In some preferred embodiments, the time for the lipolysis in step (3) is 7 to 9 days.
In some preferred embodiments, the step (5) is repeated from 1 to 4 times in the steps (1) to (4).
In some preferred embodiments, the medium of step (1) is selected from the group consisting of MM3 medium.
In some preferred embodiments, the MM3 medium comprises mesenchymal stem cell basal medium and/or serum replacement.
In some preferred embodiments, the MM3 medium comprises a mesenchymal stem cell basal medium and a serum replacement.
Mesenchymal stem cell basal medium, model 05-200-1A, available from Biological Industries (BioInd) of Israel; serum replacement, model EPAGMP-500, was purchased from EliteCell biomedical group, Inc.
In some preferred embodiments, the mesenchymal stem cell basic medium and the serum substitute have a volume ratio of 15-25: 1.
in the experimental process, the applicant finds that in the experimental process of selecting a specific serum substitute, the control of the volume ratio of the serum substitute to the basic culture medium of the mesenchymal stem cells can ensure the differentiation of the human mesenchymal stem cells into lipid, and the applicant speculates possible reasons: when the volume ratio of the basic culture medium of the mesenchymal stem cells is 15-25 times of that of the serum substitute, the influence of the cells in the serum substitute can be better absorbed in the growth and differentiation process, the better energy supply of the culture medium is ensured, and the phenomena of crimping and adherence of the cells differentiated into lipid are avoided; however, if the mesenchymal stem cell basic culture medium is less than 15 times of the serum substitute, a large amount of nutrients are consumed in the cell growth and propagation process, and the differentiation speed of the mesenchymal stem cells into lipid is affected; on the other hand, when the mesenchymal stem cell basic culture medium is 25 times larger than the serum substitute, the concentration of the mesenchymal stem cell basic culture is higher, and the intracellular concentration is too high due to the fact that a large amount of nutrient substances in the culture medium are absorbed in the cell differentiation process, so that the success rate of cell adipogenesis is influenced, and the experimental effect is influenced.
Therefore, through a great deal of research of creative experiments by the applicant, in the system, the serum substitute and the mesenchymal stem cell basic culture medium must be ensured at a reasonable ratio to better ensure the differentiation of the cells into lipid.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
Example 1
A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro comprises the following steps:
(1) culturing human mesenchymal stem cells by using an MM3 culture medium for 2 days;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1), and replacing the reagent for inducing differentiation into lipid every 24 hours;
(3) adding a differentiation-inducing lipid reagent to differentiate into lipid, wherein the differentiation time into lipid is 7 days;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) if the experiment is ended when the fat drops appear, repeating the steps (1) to (4) if no fat drops appear;
the induced Differentiation lipid reagent comprises an Adipogenic Differentiation basic Medium, Adipogenic SF, XF Supplement Mix I, Adipogenic SF and XF Supplement Mix II.
An adaptive Differentiation basic Medium, model 05-330-1B; AdipogenicSF, XF Supplement Mix I, model 05-331-1-01; AdipogenicSF, XF Supplement Mix II, model 05-332-1-15, reagents for inducing differentiation into lipids were purchased from Biological Industries (BioInd) of Israel.
The use method of the agent for inducing differentiation into lipid comprises the following steps:
1) thawing the Adipogen SF, XF Supplement Mix I and Adipogen Supplement SF, XF Supplement Mix II at room temperature for use
2) Adding 0.1mL of Adipogenic SF, XF Supplement Mix I, 1.5mL of LAdipogenicSF and XF Supplement Mix II into 100mL of Adipogenic Differentiation basic Medium, placing the mixture in an environment at 2-8 ℃, and storing the mixture for later use.
The MM3 culture medium comprises a mesenchymal stem cell basic culture medium and a serum substitute; the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 20: 1.
mesenchymal stem cell basal medium, model 05-200-1A, available from Biological Industries (BioInd) of Israel; serum replacement, model EPAGMP-500, was purchased from EliteCell biomedical group, Inc.
Example 2
A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro comprises the following steps:
(1) culturing human mesenchymal stem cells by using an MM3 culture medium for 0.5 day;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1), and replacing the reagent for inducing differentiation into lipid every 24 hours;
(3) adding a differentiation-inducing lipid reagent to differentiate into lipid, wherein the differentiation time into lipid is 7 days;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) if the experiment is ended when the fat drops appear, repeating the steps (1) to (4) if no fat drops appear;
the induced Differentiation lipid reagent comprises an Adipogenic Differentiation basic Medium, Adipogenic SF, XF Supplement Mix I, Adipogenic SF and XF Supplement Mix II.
An adaptive Differentiation basic Medium, model 05-330-1B; AdipogenicSF, XF Supplement Mix I, model 05-331-1-01; AdipogenicSF, XF Supplement Mix II, model 05-332-1-15, reagents for inducing differentiation into lipids were purchased from Biological Industries (BioInd) of Israel.
The use method of the agent for inducing differentiation into lipid comprises the following steps:
1) thawing the Adipogen SF, XF Supplement Mix I and Adipogen Supplement SF, XF Supplement Mix II at room temperature for use
2) Adding 0.1mL of Adipogenic SF, XF Supplement Mix I, 1.5mL of LAdipogenicSF and XF Supplement Mix II into 100mL of Adipogenic Differentiation basic Medium, placing the mixture in an environment at 2-8 ℃, and storing the mixture for later use.
The MM3 culture medium comprises a mesenchymal stem cell basic culture medium and a serum substitute; the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 20: 1.
mesenchymal stem cell basal medium, model 05-200-1A, available from Biological Industries (BioInd) of Israel; serum replacement, model EPAGMP-500, was purchased from EliteCell biomedical group, Inc.
Example 3
A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro comprises the following steps:
(1) culturing human mesenchymal stem cells by using an MM3 culture medium for 5 days;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1), and replacing the reagent for inducing differentiation into lipid every 24 hours;
(3) adding a differentiation-inducing lipid reagent to differentiate into lipid, wherein the differentiation time into lipid is 7 days;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) if the experiment is ended when the fat drops appear, repeating the steps (1) to (4) if no fat drops appear;
the induced Differentiation lipid reagent comprises an Adipogenic Differentiation basic Medium, Adipogenic SF, XF Supplement Mix I, Adipogenic SF and XF Supplement Mix II.
An adaptive Differentiation basic Medium, model 05-330-1B; AdipogenicSF, XF Supplement Mix I, model 05-331-1-01; AdipogenicSF, XF Supplement Mix II, model 05-332-1-15, reagents for inducing differentiation into lipids were purchased from Biological Industries (BioInd) of Israel.
The use method of the agent for inducing differentiation into lipid comprises the following steps:
1) thawing the Adipogen SF, XF Supplement Mix I and Adipogen Supplement SF, XF Supplement Mix II at room temperature for use
2) Adding 0.1mL of Adipogenic SF, XF Supplement Mix I, 1.5mL of LAdipogenicSF and XF Supplement Mix II into 100mL of Adipogenic Differentiation basic Medium, placing the mixture in an environment at 2-8 ℃, and storing the mixture for later use.
The MM3 culture medium comprises a mesenchymal stem cell basic culture medium and a serum substitute; the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 20: 1.
mesenchymal stem cell basal medium, model 05-200-1A, available from Biological Industries (BioInd) of Israel; serum replacement, model EPAGMP-500, was purchased from EliteCell biomedical group, Inc.
Example 4
A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro comprises the following steps:
(1) culturing human mesenchymal stem cells by using an MM3 culture medium for 2 days;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1), and replacing the reagent for inducing differentiation into lipid every 48 hours;
(3) adding a differentiation-inducing lipid reagent to differentiate into lipid, wherein the differentiation time into lipid is 7 days;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) if the experiment is ended when the fat drops appear, repeating the steps (1) to (4) if no fat drops appear;
the induced Differentiation lipid reagent comprises an Adipogenic Differentiation basic Medium, Adipogenic SF, XF Supplement Mix I, Adipogenic SF and XF Supplement Mix II.
An adaptive Differentiation basic Medium, model 05-330-1B; AdipogenicSF, XF Supplement Mix I, model 05-331-1-01; AdipogenicSF, XF Supplement Mix II, model 05-332-1-15, reagents for inducing differentiation into lipids were purchased from Biological Industries (BioInd) of Israel.
The use method of the agent for inducing differentiation into lipid comprises the following steps:
1) thawing the Adipogen SF, XF Supplement Mix I and Adipogen Supplement SF, XF Supplement Mix II at room temperature for use
2) Adding 0.1mL of Adipogenic SF, XF Supplement Mix I, 1.5mL of LAdipogenicSF and XF Supplement Mix II into 100mL of Adipogenic Differentiation basic Medium, placing the mixture in an environment at 2-8 ℃, and storing the mixture for later use.
The MM3 culture medium comprises a mesenchymal stem cell basic culture medium and a serum substitute; the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 20: 1.
mesenchymal stem cell basal medium, model 05-200-1A, available from Biological Industries (BioInd) of Israel; serum replacement, model EPAGMP-500, was purchased from EliteCell biomedical group, Inc.
Example 5
A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro comprises the following steps:
(1) culturing human mesenchymal stem cells by using an MM3 culture medium for 1 day;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1), and replacing the reagent for inducing differentiation into lipid every 72 hours;
(3) adding a differentiation-inducing lipid reagent to differentiate into lipid, wherein the differentiation time into lipid is 7 days;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) if the experiment is ended when the fat drops appear, repeating the steps (1) to (4) if no fat drops appear;
the induced Differentiation lipid reagent comprises an Adipogenic Differentiation basic Medium, Adipogenic SF, XF Supplement Mix I, Adipogenic SF and XF Supplement Mix II.
An adaptive Differentiation basic Medium, model 05-330-1B; AdipogenicSF, XF Supplement Mix I, model 05-331-1-01; AdipogenicSF, XF Supplement Mix II, model 05-332-1-15, reagents for inducing differentiation into lipids were purchased from Biological Industries (BioInd) of Israel.
The use method of the agent for inducing differentiation into lipid comprises the following steps:
1) thawing the Adipogen SF, XF Supplement Mix I and Adipogen Supplement SF, XF Supplement Mix II at room temperature for use
2) Adding 0.1mL of Adipogenic SF, XF Supplement Mix I, 1.5mL of LAdipogenicSF and XF Supplement Mix II into 100mL of Adipogenic Differentiation basic Medium, placing the mixture in an environment at 2-8 ℃, and storing the mixture for later use.
The MM3 culture medium comprises a mesenchymal stem cell basic culture medium and a serum substitute; the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 20: 1.
mesenchymal stem cell basal medium, model 05-200-1A, available from Biological Industries (BioInd) of Israel; serum replacement, model EPAGMP-500, was purchased from EliteCell biomedical group, Inc.
Example 6
The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro is the same as in example 1, except that the differentiation time of step (3) into lipids is 9 days in example 1.
Example 7
The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro is the same as in example 1, except that the differentiation time of step (3) into lipids is 14 days in example 1.
Example 8
A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro, which is substantially the same as in example 1, except that the differentiation time of step (3) into lipids is 5 days.
Example 9
The specific implementation mode of the method for inducing the differentiation of the human mesenchymal stem cells into the lipid in vitro is the same as that in example 1, and the difference from the embodiment 1 is that the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 10: 1.
example 10
The specific implementation mode of the method for inducing the differentiation of the human mesenchymal stem cells into the lipid in vitro is the same as that in example 1, and the difference from the embodiment 1 is that the volume ratio of the mesenchymal stem cell basic culture medium to the serum substitute is 30: 1.
and (3) performance testing:
1. and (3) fat forming judgment: judging the fatting of the fatting experiments obtained by the methods of examples 1-10, and performing dyeing identification by using an oil red O dyeing agent; the cells appear bright red after dyeing to indicate that the adipogenic differentiation is mature; the cells appear light red after staining to indicate that the adipogenic differentiation is immature; no color change after staining indicates that the cells are undifferentiated; scoring according to the maturity of adipogenic differentiation, and marking 10 points when the color of the cells is bright red; the color of the cells is light red and is marked as 5 points; no color change is scored as 0 point; among them, it is necessary to say: scores 6-9 and 1-4 (border values included) were assigned according to the tendency of the color cells to stain from bright red to pale red and from pale red to colorless.
2. Cell growth in vitro: observing the in vitro culture growth condition of the cells by adopting an inverted microscope, and preferably recording the in vitro growth condition of the cells without crimp and adherence; the phenomenon that the cells grow and shrink and adhere to the wall is recorded as good in-vitro growth condition; recording the phenomena of severe crimpling and adherence of the cells during growth as difference; and the test results are tabulated below.
| Experiment of | Formation of fat | Growth of cells in vitro |
| Example 1 | 10 minutes | Superior food |
| Example 2 | 0 point (min) | \ |
| Example 3 | 6 minutes | Good wine |
| Example 4 | 10 minutes | Superior food |
| Example 5 | 8 is divided into | Good wine |
| Example 6 | 10 minutes | Good wine |
| Example 7 | 6 minutes | Good wine |
| Example 8 | 4 is divided into | Difference (D) |
| Example 9 | 3 points of | Good wine |
| Example 10 | 5 points of | Good wine |
The performance test results show that the in vitro induced human mesenchymal stem cells developed by the method have good lipid differentiation effect and good cell growth condition, and no phenomenon of crimping and adherence occurs.
Finally, it should be understood that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for inducing differentiation of human mesenchymal stem cells into lipids in vitro is characterized by comprising the following steps:
(1) culturing human mesenchymal stem cells by using a culture medium;
(2) adding a reagent for inducing differentiation into lipid into the culture medium obtained in the step (1);
(3) adding a differentiation inducing agent to differentiate into lipid;
(4) judging fatting after differentiation in the step (3), and performing dyeing identification by using an oil red O dyeing agent;
(5) repeating the steps (1) to (4) once or more times.
2. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 1, wherein the time for culturing in step (1) is 1 day or more.
3. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 2, wherein the time for culturing in step (1) is 1-2 days.
4. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 1, wherein the agent for inducing differentiation into lipids in step (2) is replaced every 24-48 hours.
5. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 1, wherein the time for differentiation into lipids in step (3) is 7 days or more.
6. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 5, wherein the time for differentiation into lipids in step (3) is 7-9 days.
7. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 1, wherein the step (5) is repeated from step (1) to step (4)1 to 4 times.
8. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro according to claim 1, wherein the medium of step (1) is selected from the group consisting of MM3 medium.
9. The method for inducing differentiation of human mesenchymal stem cells into lipid in vitro as claimed in claim 8, wherein the MM3 medium comprises mesenchymal stem cell basal medium and/or serum replacement.
10. The method for inducing differentiation of human mesenchymal stem cells into lipids in vitro as claimed in claim 9, wherein the volume ratio of the mesenchymal stem cell basic medium to the serum substitute is 15-25: 1.
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