CN115261308A

CN115261308A - Method for inducing direct transdifferentiation of sheep fibroblasts into myotubes

Info

Publication number: CN115261308A
Application number: CN202211007047.1A
Authority: CN
Inventors: 郑勇; 赖良学; 叶升
Original assignee: Chaoji Liangshi Shenzhen Biotechnology Co ltd
Current assignee: Shantou Debao Investment Co ltd
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2022-11-01
Anticipated expiration: 2042-08-22
Also published as: CN115261308B

Abstract

The invention provides a method for inducing the direct transdifferentiation of sheep fibroblasts into myotubes, which comprises the following steps: (1) Inoculating sheep fibroblasts, and performing primary culture by using a basic culture medium; (2) Removing the basal culture medium, and performing secondary culture by using an induction culture medium, wherein the induction culture medium contains a small molecule compound, and the small molecule compound comprises any one or a combination of at least two of CHIR99021, forsklin or Repsox; (3) Removing the induction culture medium, and culturing with differentiation culture medium for three times. The method successfully realizes the direct transdifferentiation of the sheep fibroblasts into the myotubes without involving the transfer of exogenous genes, without the need of firstly inducing the fibroblasts to reprogram into pluripotent stem cells and then differentiating to obtain the myotubes, and has the advantages of convenient operation, easy standardization and good safety of the obtained myotubes.

Description

Method for inducing direct transdifferentiation of sheep fibroblasts into myotubes

Technical Field

The invention belongs to the technical field of biology, and relates to a method for inducing direct transdifferentiation of sheep fibroblasts into myotubes.

Background

The skeletal muscle satellite cell is a dry cell in skeletal muscle, plays a key role in the growth, development and regeneration of animal muscle, and is one of seed cells of the existing cell culture meat (by utilizing the technologies of cell culture engineering, tissue engineering and the like, animal muscle tissue is cultured in vitro to be used as an edible material).

There are many reports related to the isolation of skeletal muscle satellite cells from chicken, pig, sheep, cattle, etc., but there are problems such as: 1. the capability of proliferation and differentiation to form myotubes is easily lost when the skeletal muscle satellite cells obtained by separation are cultured in vitro, 2, because the content of the skeletal muscle satellite cells in the muscle is low, the methods can only obtain few cells by separation each time, and the extraction efficiency is low. Therefore, the strategy of obtaining a large amount of myotubes by inducing differentiation of skeletal muscle satellite cells in animal muscle by isolation and obtaining a large amount of cell culture meat still faces a great challenge.

Another solution is to induce pluripotent stem cells to differentiate into muscle progenitor cells, which are further differentiated to give myotubes. However, the method has the disadvantages of long induction time, complicated operation and difficult source of pluripotent stem cells. Therefore, it is more advantageous to transdifferentiate fibroblasts directly into myotubes without going through the pluripotent stem cell stage. There are studies based on mouse experiments to obtain myotubes by overexpressing the Myod1 gene in fibroblasts, but this operation requires the use of transgenic technology, with safety risks.

Therefore, how to provide a method for directly transdifferentiating fibroblasts into myotubes without transferring exogenous genes or undergoing a pluripotent stem cell stage, which is simple and convenient to operate, easy to standardize and high in safety, becomes an urgent problem in the field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for inducing the direct transdifferentiation of sheep fibroblasts into myotubes.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, the method comprising the steps of:

(1) Inoculating sheep fibroblasts, and performing primary culture by using a basic culture medium;

(2) Removing the basal culture medium, and performing secondary culture by using an induction culture medium, wherein the induction culture medium contains a small molecule compound, and the small molecule compound comprises any one or a combination of at least two of CHIR99021, forsklin or Repsox;

(3) Removing the induction culture medium, and performing three times of culture with differentiation culture medium.

Preferably, the small molecule compound includes CHIR99021, forsklin and Repsox.

Preferably, the induction medium further contains bFGF (basic fibroblast growth factor) and/or vitamin C.

Preferably, the components in the induction medium comprise DMEM 70-90% w/w, serum replacement 5-15% w/w, FBS 5-15% w/w, bFGF 5-15ng/mL, vitamin C40-60 μ g/mL, CHIR99021 1-5 μ M, forsklin 5-50 μ M and Repsox 5-50 μ M in concentration.

The w/w in the invention refers to a mass ratio.

Specific values of 70 to 90% are, for example, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, etc.

Specific values of 5 to 15% are, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

Specific values of 5 to 15ng/mL are, for example, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 14ng/mL, 15ng/mL, and the like.

Specific values of the above-mentioned 40 to 60. Mu.g/mL include, for example, 40. Mu.g/mL, 42. Mu.g/mL, 44. Mu.g/mL, 46. Mu.g/mL, 48. Mu.g/mL, 50. Mu.g/mL, 52. Mu.g/mL, 54. Mu.g/mL, 56. Mu.g/mL, 58. Mu.g/mL, 60. Mu.g/mL, and the like.

Specific values among the above 1 to 5. Mu.M include, for example, 1. Mu.M, 1.5. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M, and the like.

Specific values among the above 5 to 50. Mu.M include, for example, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M, 30. Mu.M, 35. Mu.M, 40. Mu.M, 45. Mu.M, and 50. Mu.M.

Preferably, the basal medium contains DMEM, double antibody and FBS.

The double antibody of the invention refers to a mixture of penicillin and streptomycin.

Preferably, the basic culture medium comprises, by mass, 84-94% of DMEM, 0.5% -1.5% of double antibodies and 5-15% of FBS.

Specific values of 84 to 94% are, for example, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and the like.

Specific values of the above-mentioned 0.5% to 1.5% are, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, etc.

Preferably, the differentiation medium is DMEM/F12 medium containing horse serum.

Preferably, the horse serum is 1.5-2.5% by mass in the differentiation medium, such as 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5% and the like.

Preferably, the temperature of the primary, secondary and tertiary cultures is 35-40 deg.C, such as 35 deg.C, 35.5 deg.C, 36 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C, 38 deg.C, 38.5 deg.C, 39 deg.C, 39.5 deg.C, 40 deg.C, etc., independently.

Preferably, the time for one culture is 0.5 to 1.5 days.

Preferably, the time of the secondary culture is 4 to 7 days.

Preferably, the time for the three cultivations is 2-4 days.

Specific values of the above-mentioned 0.5 to 1.5 days include, for example, 0.5 day, 1 day, 1.5 days, etc.

Specific values of the above 4 to 7 days include, for example, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, etc.

Specific values of the above 2 to 4 days include, for example, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, etc.

Preferably, the seeding is on gelatin coated cell culture plates.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

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

the method successfully realizes the direct transdifferentiation of the sheep fibroblasts into the myotubes without involving the transfer of exogenous genes, without the need of firstly inducing the fibroblasts to reprogram into pluripotent stem cells and then differentiating to obtain the myotubes, and has the advantages of convenient operation, easy standardization and good safety of the obtained myotubes.

According to the invention, CHIR99021, forsklin and Repsox are creatively added into the induction culture medium at the same time, and three small molecular compounds are mutually matched, so that the direct transdifferentiation of the sheep fibroblasts into myotubes is realized, the improvement of the transdifferentiation efficiency is remarkably promoted, and the CHIR99021, the Forsklin and the Repsox have a synergistic effect in the aspect of inducing the direct transdifferentiation of the sheep fibroblasts into myotubes.

The invention unexpectedly discovers that the bFGF and the vitamin C play a role in assisting the micromolecular compound of the invention to induce the direct transdifferentiation of the sheep fibroblasts into myotubes.

Drawings

FIG. 1 is a view showing the microscopic observation results of sheep fibroblasts, and the scale in the upper right lower part of the figure indicates 100. Mu.m.

FIG. 2 is a graph showing microscopic observations of myotubes obtained by transdifferentiation of sheep fibroblasts induced by the method of example 1, the arrows indicating the myotubes, and the upper right lower part of the graph showing 100 μm.

Detailed Description

The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.

In the following examples, unless otherwise specified, reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise indicated, all experimental methods and technical means used are those conventional in the art.

The following examples and comparative examples have the following raw material information:

CHIR99021: a GSK-3 inhibitor available from Selleck, cat # S1263.

Forsklin: adenylate cyclase activator, available from Selleck under the trade name S2449.

Repsox: a TGF β R-1/ALK5 inhibitor, available from Selleck, inc., under item number S7223.

Gelatin was purchased from Stemcell, cat # 7903.

Horse serum was purchased from Gibco, cat # 26050088.

bFGF (basic fibroblast growth factor) was purchased from PeproTech, inc., cat # 100-18B.

The following examples and comparative examples relate to the preparation of sheep fibroblasts (SFF) as follows:

taking down the sheep fetus together with uterus, putting into PBS, and transporting to the laboratory at low temperature. Carefully stripping the fetus and fetal membranes by using sterilized scissors, placing the fetus in PBS, taking the skin tissue of the fetus of the goat by using the scissors, washing the skin tissue of the fetus of the goat for three times by using the PBS, shearing the skin tissue of the fetus until no obvious tissue blocks exist, adding 10-15 mL of freshly prepared digestive juice, putting the digestive juice into an incubator for digesting for 2-4h, observing the digestion condition in the period, transferring the digestive juice into a 15mL centrifuge tube after digestion is finished, centrifuging at 1000rpm for 8min, collecting cells, removing supernatant, washing the cells twice by using DMEM +10 FBS (fetal bovine serum), centrifuging at 200g for 5min, collecting the cells, and suspending and paving the cell sediment by using DMEM +10 FBS.

The sheep fibroblasts were observed under a microscope and photographed, as shown in fig. 1.

Example 1

The embodiment provides a method for inducing the direct transdifferentiation of sheep fibroblasts into myotubes, which comprises the following specific steps:

prepared sheep fibroblasts (SFF) were used to inoculate 50000 cells per well in 6-well plates previously coated with 0.1% gelatin, mediumBasal medium (high glucose DMEM (glucose content 4.5 g/L), 1% diabase (penicillin-streptomycin), 10% FBS) was placed in an incubator with 5% carbon dioxide, 95% humidity, 37 ℃. After culturing for 24 hours, removing the basal culture medium, washing with PBS for three times, and replacing with an induction culture medium, wherein the formula of the culture medium is as follows: knockOut ^TM DMEM、10％FBS、10％KnockOut ^TM Serum Replacement, 10ng/mL bFGF (basic fibroblast growth factor), 50 μ g/mL vitamin C and three small molecule compounds (3 μ M CHIR99021, 20 μ M Forsklin, 20 μ M MRepsox), with the induction medium changed every other day; after induction culture for six days, removing the induction culture medium, washing with PBS for three times, and replacing with a differentiation culture medium, wherein the formula of the differentiation culture medium is as follows: DMEM/F12,2% horse serum, cultured for 3 days to obtain transdifferentiated myotubes.

The specific method for coating the 0.1% gelatin comprises the following steps: 0.1% gelatin is added to the culture plate in advance, and after being placed in an incubator with 5% carbon dioxide, humidity of 95% and temperature of 37 ℃ for 30 minutes, 0.1% gelatin is discarded, and the plate is washed three times with PBS, and then the plate can be used for inoculating cells.

The transdifferentiation condition was observed under a microscope after the experiment, and the result is shown in fig. 2, and it can be seen from comparison with fig. 1 that the cells become elongated and fused together, and present the shape of myotubes, which fully confirms that the method of this example successfully induces the sheep fibroblasts to transdifferentiate into myotubes directly.

Example 2

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which is different from example 1 only in that the formula of the induction medium is different, and "3 μ M CHIR99021, 20 μ M Forsklin, and 20 μ M Repsox" are replaced with "1 μ M CHIR99021, 5 μ M Forsklin, and 5 μ M Repsox", and other conditions are not changed.

This example successfully induced direct transdifferentiation of sheep fibroblasts into myotubes.

Example 3

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which is different from example 1 only in that the formulation of the induction medium is different, and "3 μ M CHIR99021, 20 μ M Forsklin, 20 μ M Repsox" is replaced with "5 μ M CHIR99021, 50 μ M Forsklin, 50 μ M Repsox", and other conditions are not changed.

This example successfully induced direct transdifferentiation of ovine fibroblasts into myotubes.

Example 4

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which is different from example 1 only in that the formulation of the induction medium is different, and "3 μ M CHIR99021, 20 μ M Forsklin, and 20 μ M Repsox" are replaced with "21.5 μ M Forsklin and 21.5 μ M Repsox", and other conditions are not changed.

Example 5

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which is different from example 1 only in that the formulation of the induction medium is different, and "3 μ M CHIR99021, 20 μ M Forsklin, 20 μ M Repsox" is replaced by "3 μ M CHIR99021, 20 μ M Forsklin", and the amount of the deficiency is equal to the mass of KnockOut ^TM DMEM supplemented, other conditions were unchanged.

Example 6

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which differs from example 1 only in that the formulation of the induction medium is different, and "3. Mu.M CHIR99021, 20. Mu.M Forsklin, 20. Mu.M Repsox" is replaced with "3. Mu.M CHIR99021, 20. Mu.M Repsox", and the amount of deficiency is equal mass of KnockOut ^TM DMEM supplemented with no other conditions.

Example 7

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which is different from example 1 only in that the formulation of the induction medium is different, and "3 μ M CHIR99021," 20 μ M Forsklin, "20 μ M Repsox" is replaced by "3 μ M CHIR99021," and the amount of the deficiency is equal to the mass of KnockOut ^TM DMEM supplemented with no other conditions.

Example 8

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which differs from example 1 only in that the formulation of the induction medium is different, that is, "3. Mu.M CHIR99021, 20. Mu.M Forsklin, 20. Mu.M Repsox" is replaced by "20. Mu.M Forsklin", and that the amount of deficiency is equal to the mass of KnockOut ^TM DMEM supplemented with no other conditions.

Example 9

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which differs from example 1 only in that the formulation of the induction medium is different, that "3. Mu.M CHIR99021, 20. Mu.M Forsklin, 20. Mu.M Repsox" is replaced with "20. Mu.M Repsox", and that the amount of deficiency is equal to the mass of KnockOut ^TM DMEM supplemented with no other conditions.

Example 10

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which differs from example 1 only in that the induction medium formulation lacks bFGF and the amount of bFGF used is equal to the mass of KnockOut ^TM DMEM supplemented, other conditions were unchanged.

Example 11

This example provides a method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, which differs from example 1 only in the absence of vitamin C in the formulation of the induction medium, the amount of vitamin C in the formulation being equal to the amount of KnockOut ^TM DMEM supplemented with no other conditions.

Comparative example 1

The comparative example provides a method for inducing the direct transdifferentiation of sheep fibroblasts into myotubes, which is different from the method in example 1 only in that the formula of an induction medium is different, CHIR99021, forsklin and Repsox are not contained, and the amount of the deficiency is equal to the mass of KnockOut ^TM DMEM supplemented with no other conditions.

This comparative example did not allow for the induction of direct transdifferentiation of sheep fibroblasts into myotubes.

Test example

Identification of myotubes using immunofluorescence

Further immunofluorescence identification was performed on myotubes obtained in the above examples in which the microscopic observation result was "successful transdifferentiation", and the expression of a labeled antigen of myotubes, i.e., myosin heavy chain (MyHC), was detected.

Washed 3 times with PBS, fixed for 15min with paraformaldehyde (4%), the fixed cells were washed 3 times 5min with PBS, permeabilized for 10min with TritionX-100 at room temperature (25 ℃), washed 3 times 5min with PBS. 5% sheep serum was added and blocked at room temperature for 1h, washed 3 times with PBS, 5min each time. The primary antibody was diluted with 1% sheep serum at a ratio of 1. The next day, the cells were allowed to re-incubate at room temperature and then washed 3 times with PBS for 5min each time. The secondary antibody was also diluted with 1% BSA (bovine serum albumin) according to the dilution ratio of the antibody, and then washed 3 times with PBS for 5min each. Observed by a fluorescence microscope and recorded by photographing.

Calculating transdifferentiation efficiency by the following formula: transdifferentiation efficiency = number of nuclei contained in MyHC-positive myotubes/total number of nuclei.

The test results are shown in Table 1.

TABLE 1

The results show that: from the comparison results of the embodiment 1 and the embodiments 4 to 9, it can be seen that the invention simultaneously adds CHIR99021, forsklin and Repsox in the induction culture medium, and the three small molecular compounds are mutually matched, so that the direct transdifferentiation of the sheep fibroblasts into myotubes is induced, and the improvement of the transdifferentiation efficiency is remarkably promoted. From the comparison results of example 1 and examples 10-11, it can be seen that the addition of bFGF and vitamin C to the induction medium further promotes the improvement of transdifferentiation efficiency, indicating that bFGF and vitamin C play a role in assisting the small molecule compound of the present invention in inducing the direct transdifferentiation of sheep fibroblasts into myotubes.

In addition, as can be seen from the comparison results of example 1 and examples 2-3, the amounts of the three small molecule compounds have a certain effect on the transdifferentiation efficiency, and when the amounts are too large or too small, the transdifferentiation efficiency is reduced to some extent, and when the amounts are 2-5 μ M CHIR99021, 10-50 μ M Forsklin, and 10-50 μ M Repsox, the transdifferentiation efficiency can be more than 10%.

The applicant states that the present invention is illustrated by the above examples to describe the method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes, but the present invention is not limited to the above examples, which does not mean that the present invention is necessarily dependent on the above examples to be implemented. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.

It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

Claims

1. A method for inducing the direct transdifferentiation of sheep fibroblasts into myotubes, comprising the steps of:

(3) Removing the induction culture medium, and culturing with differentiation culture medium for three times.

2. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to claim 1, wherein the small molecule compounds comprise CHIR99021, forsklin and Repsox.

3. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to claim 1 or 2, wherein the induction medium further comprises bFGF and/or vitamin C.

4. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to any one of claims 1-3, wherein the components in the induction medium comprise DMEM 70-90% w/w, serum replacement 5-15% w/w, FBS 5-15% w/w, bFGF 5-15ng/mL, vitamin C40-60 μ g/mL, CHIR99021 1-5 μ M, forsklin 5-50 μ M and Repsox 5-50 μ M in concentration.

5. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to any one of claims 1 to 4, wherein the basal medium comprises DMEM, diabody and FBS.

6. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to claim 5, wherein the basic medium comprises 84-94% of DMEM, 0.5% -1.5% of double antibody and 5-15% of FBS by mass percentage.

7. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to any one of claims 1 to 6, wherein the differentiation medium is DMEM/F12 medium containing horse serum.

8. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to claim 7, wherein the horse serum is present in the differentiation medium in an amount of 1.5-2.5% by weight.

9. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to any one of claims 1 to 8, wherein the temperature of the primary culture, the secondary culture and the tertiary culture is 35 to 40 ℃ independently.

10. The method for inducing the direct transdifferentiation of ovine fibroblasts into myotubes according to any one of claims 1 to 9, wherein the time of the primary culture is 0.5 to 1.5 days;

preferably, the time of the secondary culture is 4 to 7 days;

preferably, the time of the three times of culture is 2-4 days;

preferably, the seeding is on gelatin coated cell culture plates.