CN113388582A - Method, culture medium and system for promoting iPSC to differentiate into peripheral neural stem cells - Google Patents
Method, culture medium and system for promoting iPSC to differentiate into peripheral neural stem cells Download PDFInfo
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Abstract
The invention discloses a method, a culture medium and a system for promoting induced pluripotent stem cells to differentiate into peripheral nerve stem cells, wherein a cell culture vessel treated by vitronectin is used in the culture process of the induced pluripotent stem cells, a cell culture vessel treated by recombinant laminin is used in the process of inducing the pluripotent stem cells to differentiate into the peripheral nerve stem cells, and meanwhile, animal-origin-free peripheral nerve induction culture medium-01 is used for culture. Compared with the prior art, the method has the advantages that the process is integrally optimized, the chemical components of the culture medium are clear, the method has obvious advantages in the quality control of the culture medium and the quality control of the final product, the process is integrally free from animal sources, the pollution risk of animal-derived pathogenic factors is avoided, the method is suitable for cell culture vessels with large surface areas, the yield of peripheral nerve stem cells of each batch is increased, and the industrial production is facilitated.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a method, a culture medium and a system for promoting induced pluripotent stem cells to differentiate into peripheral neural stem cells.
Background
Neural stem cells (NPs) of the Peripheral Nervous System have great application potential in the fields of disease treatment, drug development, scientific research and the like, and a large amount of high-purity NPs are required for disease treatment and drug development. The conventional method for obtaining NP is mainly a Stem Cell differentiation method, in which Embryonic Stem Cells (ESC) or Induced Pluripotent Stem cells (iPSC) are differentiated into NP by an in vitro differentiation means. However, most of the existing in vitro differentiation methods are under-mature in process, a culture medium with incompletely defined chemical components is used, the concentration of key components of the culture medium is not deeply researched, the quality difference of the obtained NP batches is large, the industrial production is not facilitated, and the application of the NP is limited. The reagents and materials used in the differentiation process contain a large amount of animal-derived components such as bovine serum albumin, murine cell secretions (Matrigel) and the like, which bring risks such as animal pathogen contamination and are not beneficial to various applications including clinical treatment. In view of the above, there is still a great distance between the production of NPs by the existing stem cell differentiation method and the industrial scale production and practical clinical application. On the other hand, there is some controversy that acquisition of ESCs themselves requires the consumption of embryonic tissue. In addition, the prior art is mostly used for scientific research, and a method for searching for mass production of NP is not attempted, so that the requirements of industrial production and therapeutic application cannot be met.
In conclusion, the NP has limited acquisition routes, an immature preparation process, and the use of reagents and culture media containing animal-derived components with incompletely defined chemical components, and the obtained NP has large quality difference among the batches, high preparation cost and low yield, thus limiting the application of NP, and urgently requiring a differentiation method and culture media which have simplified preparation process, short preparation time, small pollution risk, high yield and purity and are beneficial to industrial production.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method, a culture medium and a system for inducing pluripotent stem cells to differentiate into peripheral nerve stem cells, wherein a cell culture vessel treated by vitronectin is used in the culture process of the induced pluripotent stem cells, a cell culture vessel treated by recombinant laminin is used in the process of inducing the pluripotent stem cells to differentiate into the peripheral nerve stem cells, and meanwhile, animal origin-free peripheral nerve induction culture medium-01 is used for culture.
The invention provides a method for promoting induced pluripotent stem cells to differentiate into peripheral neural stem cells, which comprises the following steps:
s1: culturing induced pluripotent stem cells;
the method comprises primary culture and subculture, wherein cell culture vessels used in the primary culture and the subculture are both treated by vitronectin;
s2: inducing differentiation of pluripotent stem cells into Peripheral Neural stem cells (pluripotent Neural System Neural promoter, NP);
before differentiation is started, inoculating induced pluripotent stem cells into a cell culture vessel treated by recombinant laminin, culturing by using an animal-origin-free peripheral nerve induction culture medium-01, replacing the culture medium, culturing for 8-10 days, treating the peripheral nerve stem cells obtained after differentiation of the induced pluripotent stem cells by using a pancreatin substitute, and collecting the peripheral nerve stem cells.
Further, the seeding density of the induced pluripotent stem cells in the primary culture and/or the subculture in the step S1 is 1.0-2.5 × 104Per cm2Preferably 1.5X 104Per cm2. At 1.5X 104Per cm2The relative yield of induced pluripotent stem cells is highest at the seeding density of (a).
Further, the seeding density of the induced pluripotent stem cells in the step S2 is 1.0-4.0 × 104Per cm2Preferably 2.0X 104Per cm2. At 2.0X 104Per cm2The relative yield of peripheral neural stem cells was highest at the seeding density of (a).
Further, the animal-origin-free peripheral nerve induction medium-01 in the step S2 includes the following components: DMEM/F12 medium, non-essential amino acids, insulin, holotransferrin, putrescine, human serum albumin, superoxide dismutase, glutathione, progesterone, retinol, vitamin A, dl-alpha-tocopheryl acetate, vitamin E, linoleic acid, alpha-linolenic acid, lipoic acid, LDN193189, SB431542, CHIR99021, RO4902097, SU5402, all-trans vitamin a acid, Y27632. Wherein, LDN193189, SB431542, CHIR99021, RO4902097, SU5402 and Y27632 are codes of potential candidate drugs.
Further, the non-essential amino acid content in the non-animal-derived peripheral nerve induction culture medium-01 is 0.1-5%, and the content is in volume ratio; the concentration of the insulin is 0.1-10 mug/mL; the concentration of the total transferrin is 2-100 mug/mL; the concentration of the putrescine is 5-200 mug/mL; the concentration of the human serum albumin is 250-2500 mug/mL; the concentration of the superoxide dismutase is 1-10 mug/mL; the concentration of the glutathione is 0.1-10 mug/mL; the concentration of the progesterone is 1-10 ng/mL; the concentration of the retinal is 0.01-2 mug/mL; the concentration of the vitamin A is 0.01-2 mug/mL; the concentration of the dl-alpha-tocopheryl acetate is 0.1-10 mu g/mL; the concentration of the vitamin E is 0.1-10 mug/mL; the concentration of the linoleic acid is 0.1-10 mug/mL; the concentration of the alpha-linolenic acid is 0.1-10 mug/mL; the concentration of the lipoic acid is 1-10 ng/mL; the concentration of the LDN193189 is 0.1-1 mu g/mL; the concentration of the SB431542 is 1-10 mug/mL; the concentration of CHIR99021 is 2.5-12.5 mug/mL, the concentration of RO4902097 is 0.5-5 mug/mL, the concentration of SU5402 is 0.5-5 mug/mL, the concentration of all-trans vitamin A acid is 1-20 ng/mL, and the concentration of Y27632 is 1-10 mug/mL.
The invention also provides an animal origin-free peripheral nerve induction culture medium-01 for promoting the differentiation of induced pluripotent stem cells into peripheral nerve stem cells, which comprises the following components: DMEM/F12 medium, non-essential amino acids, insulin, holotransferrin, putrescine, human serum albumin, superoxide dismutase, glutathione, progesterone, retinol, vitamin A, dl-alpha-tocopheryl acetate, vitamin E, linoleic acid, alpha-linolenic acid, lipoic acid, LDN193189, SB431542, CHIR99021, RO4902097, SU5402, all-trans vitamin a acid, Y27632. The animal origin-free peripheral nerve induction culture medium-01 is completely animal origin-free. Wherein, LDN193189, SB431542, CHIR99021, RO4902097, SU5402 and Y27632 are codes of potential candidate drugs.
Further, the content of the non-essential amino acid is 0.1-5%, and the content is in a volume ratio; the concentration of the insulin is 0.1-10 mug/mL; the concentration of the total transferrin is 2-100 mug/mL; the concentration of the putrescine is 5-200 mug/mL; the concentration of the human serum albumin is 250-2500 mug/mL; the concentration of the superoxide dismutase is 1-10 mug/mL; the concentration of the glutathione is 0.1-10 mug/mL; the concentration of the progesterone is 1-10 ng/mL; the concentration of the retinal is 0.01-2 mug/mL; the concentration of the vitamin A is 0.01-2 mug/mL; the concentration of the dl-alpha-tocopheryl acetate is 0.1-10 mu g/mL; the concentration of the vitamin E is 0.1-10 mug/mL; the concentration of the linoleic acid is 0.1-10 mug/mL; the concentration of the alpha-linolenic acid is 0.1-10 mug/mL; the concentration of the lipoic acid is 1-10 ng/mL; the concentration of the LDN193189 is 0.1-1 mu g/mL; the concentration of the SB431542 is 1-10 mug/mL; the concentration of CHIR99021 is 2.5-12.5 mug/mL, the concentration of RO4902097 is 0.5-5 mug/mL, the concentration of SU5402 is 0.5-5 mug/mL, the concentration of all-trans vitamin A acid is 1-20 ng/mL, and the concentration of Y27632 is 1-10 mug/mL.
The invention also provides a system for promoting the differentiation of induced pluripotent stem cells into peripheral neural stem cells, which comprises the following components:
(1) a vitronectin-treated cell culture vessel;
(2) recombinant laminin treated cell culture vessel;
(3) the animal-derived peripheral nerve-free induction culture medium-01.
Wherein, the vitronectin and recombinant laminin treated cell culture vessel avoids animal-derived materials on the culture vessel level.
Further, the surface area of the cell culture vessel treated by the recombinant laminin is 9.5-75 cm2。75cm2The large vessel can realize the preparation of the peripheral nerve stem cells with large volume, and is beneficial to industrial production.
In summary, compared with the prior art, the invention achieves the following technical effects:
1. the method improves the reagent combination, the formula of the culture medium can be definite in chemical components, the concentration range of the key reagent is successfully searched, all raw materials can be controlled when the culture medium of each batch is prepared, the quality fluctuation of the final product caused by the unclear components of the culture medium or the uncertain concentration of the reagent is reduced, the method has obvious advantages in the quality control of the culture medium and the quality control of the final product, and the improvement enables the preparation process of the peripheral nerve stem cells to be integrally optimized, thereby being beneficial to industrial production.
2. The method of the invention uses non-animal-derived materials to partially or completely replace bovine-derived and murine-derived materials, develops an animal-origin-free culture system, and reduces the risks of animal pathogen pollution and the like. Among them, the prior art often uses B27 as a serum substitute, but B27 still contains animal origin, and the concentrations of the raw materials are not disclosed, which is not favorable for quality control. The above improvements are useful for a wide variety of applications including clinical treatments.
3. The method of the invention searches conditions such as cell inoculation density, size of culture vessel, usage amount of culture medium, etc., is beneficial to mass production of peripheral nerve stem cells, and meets the requirements of industrial production and treatment application.
4. The peripheral nerve stem cells obtained by the method have high purity, and the peripheral nerve stem cells with the purity of more than 90 percent can be obtained.
5. The method of the present invention can shorten the time for differentiating into peripheral nerve stem cells and obtain high purity peripheral nerve stem cells in 8 days.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the NP purity after differentiation of iPSCs of different seeding densities by each medium formulation.※: comparative inoculation Density 4.0X 104，P≤0.05。
FIG. 2 shows the NP yield after different inoculum densities of iPSCs were differentiated for each media formulation.§: the inoculation density is 1.0X 10 compared with that of the culture medium4P is less than or equal to 0.05, which has statistical significance;§to: the inoculation density is 1.0X 10 compared with that of the culture medium4And 2.0X 104P is less than or equal to 0.05, which has statistical significance;＄: pMiN is compared with Control, the inoculation density is the same, P is less than or equal to 0.05, and the statistical significance is achieved.
FIG. 3 shows the NP purity after differentiation of iPSCs seeded on dishes of varying surface area with pNiM 01-AF.
FIG. 4 shows the NP yield from differentiation of iPSC seeded on dishes with different surface areas by pNiM 01-AF.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
The method specifically comprises the following steps:
(1) culture of iPSC
The iPSC is added at a ratio of 1.0-2.5 × 104Per cm2Inoculating the cells into a cell culture vessel treated by Vitronectin (Vc, a material without animal origin and replacing matrix glue Matrigel and the like), performing primary culture by using an Essential 8 culture medium (E8), replacing the culture medium every 24 hours, treating the iPSC by using a pancreatin substitute (TrypLE) or EDTA (the concentration is 0.5mM) when the fusion degree of the iPSC reaches 60-80% after 3-4 days, collecting the iPSC, performing subculture, and performing subculture when the subculture is subcultured by using 1.0-2.5 multiplied by 104Per cm2Was seeded on vitronectin-treated cell culture dishes.
(2) NP differentiation of iPSC
The iPSC is added at 1.0-4.0 × 10 for 24 hours before the initiation of differentiation4Per cm2The method is characterized in that the cells are inoculated in a cell culture vessel treated by recombinant Laminin (materials such as Lamin, Lm, animal origin-free materials and matrix glue Matrigel and the like instead of mouse origin), an animal origin-free peripheral nerve induction culture medium-01 (pNiM-01AF) is used for culturing, the culture medium is replaced every 36-48 hours, NP obtained after iPSC differentiation is treated by TrypLE after 8-10 days of culture, and the collected NP can be used for analysis, next step test or frozen storage.
(3) NP differentiation of iPSC-larger Scale NP preparation exploration
Mixing iPSC at 2.0 × 104Per cm2The cells were inoculated in cell culture vessels of different surface areas treated with recombinant laminin (Lm), cultured with pNiM-01AF, and tested for their ability to produce NP at different surface areas (sizes).
Example 1 cultivation of iPSC
The iPSC is added at a ratio of 1.0-2.5 × 104Per cm2Density of (example seed density: 1.5X 10)4Per cm2) Seeded on vitronectin (Vc) -treated cell culture dishes (example dishes: 6-well plate), cultured using E8 (example amount: 2mL per well of a 6-well plate), replacing the culture medium every 24 hours, and treating the iPSC with EDTA (0.5mM) after 3-4 days when the fusion degree of the iPSC reaches 60-80% (EDTA treatment time range: 3-8 minutes, example treatment time: 5 minutes), collecting iPSC, then carrying out passage, wherein the passage is carried out at the same time of 1.0-2.5 multiplied by 104Per cm2Density of (example seed density: 1.5X 10)4Per cm2) The density of (b) was seeded on the Vc-treated cell culture dish.
Example 2 NP differentiation of iPSC
The iPSC is added at 1.0-4.0 × 10 for 24 hours before the initiation of differentiation4Per cm2(ii) was inoculated in a cell culture vessel treated with recombinant laminin (Lm) (optimum inoculation density in example: 2.0X 10)4Per cm2See table 3 below for details), animal origin-free peripheral nerve inductionThe medium-01 (pNiM-01AF) was cultured (amount used in example: 2mL per well in 6-well plate).
The final formulation of pNiM-01AF was determined by comparative experiments, and "animal origin-free peripheral nerve induction medium-01 (pNiM-01 AF)" was designed first, and then compared with the animal origin component-containing medium (Control) in the prior art, specifically as in tables 1-2 below, the components from serial No. 2 to the last row were added to DMEM/F12 minimal medium with serial No. 1.
TABLE 1 existing peripheral nerve induction medium (Control) containing animal-derived components
*: b27 is of animal origin.
TABLE 2 non-animal derived peripheral nerve Induction Medium-01 (pNiM-01AF)
| Serial number | Composition (I) | Range of |
| 1 | DMEM/F12 | / |
| 2 | Non-essential amino acids (100x) | 0.1 to 5x, i.e., 0.1 to 5% (by volume) |
| 3 | Insulin | 0.1～10μg/mL |
| 4 | All- |
2～100μg/mL |
| 5 | Putrescine | 5～200μg/mL |
| 6 | Human serum albumin | 250～2500μg/mL |
| 7 | |
1～10μg/mL |
| 8 | Glutathione | 0.1～10μg/mL |
| 9 | |
1～10ng/ |
| 10 | Retinal preparation | 0.01～2μg/mL |
| 11 | Vitamin A | 0.01～2μg/mL |
| 12 | dl-alpha-tocopheryl acetate | 0.1～10μg/mL |
| 13 | Vitamin E | 0.1～10μg/mL |
| 14 | Linoleic acid | 0.1～10μg/mL |
| 15 | Alpha-linolenic acid | 0.1～10μg/mL |
| 16 | |
1～10ng/mL |
| 17 | LDN193189 | 0.1～1μg/mL |
| 18 | |
1～10μg/mL |
| 19 | CHIR99021 | 2.5～12.5μg/ |
| 20 | RO4902097 | 0.5～5μg/mL |
| 21 | SU5402 | 0.5～5μg/mL |
| 22 | All-trans |
1～20ng/mL |
| 23 | Y27632** | 1～10μg/mL |
**: y27632 is added during the first 36-48 hours of culture.
LDN193189, SB431542, CHIR99021, RO4902097, SU5402 and Y27632 are the code numbers of potential candidate drugs.
In order to verify the effectiveness of inducing iPSC to differentiate into NP by 2 formulas, iPSC is inoculated according to the following table 3, the differentiation culture is carried out for 8-10 days by using the peripheral nerve induction culture medium shown in the tables 1-2, the culture medium is replaced every 36-48 hours during the period, and the purity of NP is counted after the completion, so that the optimal iPSC inoculation density is optimized.
TABLE 3 iPSC inoculation Density and NP differentiation results for each media formulation
Note: NP purity was calculated by counting the percentage positivity of the peripheral nervous system neural stem cell marker p 75.
※: the inoculation density is 4.0X 10 compared with that of the culture medium4P is less than or equal to 0.05, which has statistical significance.
§: the inoculation density is 1.0X 10 compared with that of the culture medium4P is less than or equal to 0.05, which has statistical significance.
§§: the inoculation density is 1.0X 10 compared with that of the culture medium4And 2.0X 104P is less than or equal to 0.05, which has statistical significance.
＄: pMiN-01AF is compared with Control, the inoculation density is the same, P is less than or equal to 0.05, and the statistical significance is achieved.
As a result of the data shown in Table 3, the animal origin-free peripheral nerve induction medium-01 was found to be 1.0X 104Per cm2、2.0×104Per cm2And 4.0X 104Per cm2At a seeding density of (2), NPs with a purity of 80% or more can be harvested, wherein the purity is 1.0X 104Per cm2And 2.0X 104Per cm2The purity of the seed is more than or equal to 90 percent (figure 1). "animal origin-free peripheral nerve induction culture medium-01" at 1.0X 104Per cm2And 2.0X 104Per cm2The number of NPs harvested at the inoculation density of (a) was higher than that of the existing peripheral nerve induction medium containing animal-derived components, and the fluctuation of yield among batches was reduced (fig. 2). In the above conclusion, the "animal origin-free peripheral nerve induction medium-01" was named pNiM-01 AF.
By using the formulation of 'pNiM-01 AF', NP with the purity of more than or equal to 90 percent can be differentiated from the iPSC in the shortest 8 days. The "pNiM-01 AF" formulation is a prerequisite for successful preparation of high purity NP. Wherein the pNiM-01AF completely does not contain animal derived components, has definite chemical components and can be used for preparing clinical treatment NP. In the prior art, a culture medium with incompletely-defined chemical components is often used, the process is immature as a whole, the quality difference of NP batches is large, the industrial production is not facilitated, and the application of NP is limited.
Example 3 NP differentiation of iPSC-larger Scale NP preparation exploration
Mixing iPSC at 2.0 × 104Per cm2The cells were seeded in recombinant laminin (Lm) -treated cell culture vessels of varying surface areas and cultured with pNiM-01AF, as detailed in Table 4 below.
TABLE 4 differentiation results of culture vessel NP at different surface areas
The data in Table 4 lead to conclusions of 9.5, 25 and 75cm2Three surface areas, pNiM-01AF differentiation of iPSC, resulted in the harvesting of NPs with purity greater than or equal to 90% (FIG. 3), while the surface area size did not significantly affect the yield of peripheral neural stem cells (FIG. 4). The inoculation density of the iPSC and the feasible surface area of the culture vessel are obtained by the method, and the method is favorable for industrial production.
In summary, in the method of the present invention, the iPSC culture vessel is treated with vitronectin, the culture vessel is treated with recombinant Laminin (Laminin) during NP differentiation, and the culture system is treated with vitronectin and recombinant Laminin instead of Matrigel, and animal-derived materials are firstly avoided on the culture vessel level. The NP was harvested by treatment with pancreatin replacement, and the medium "pNiM-01 AF" did not contain animal origin. Therefore, the invention can realize the complete process without animal source.
Using "pNiM-01 AF" to inoculate with density of 1.0X 104Per cm2、2.0×104Per cm2And 4.0X 104Per cm2At a seeding density of (2), NPs with a purity of 80% or more can be harvested, wherein the purity is 1.0X 104Per cm2And 2.0X 104Per cm2At an inoculation density of > 90% purity, based on at 2.0X 104Per cm2NP production efficiency is higher when iPSC is inoculated, 2.0 multiplied by 10 is selected4Per cm2The seeding density of (1).
Compared with the existing culture medium formula, the use of the pNiM-01AF can obviously increase the yield of NP, reduce the fluctuation of the yield among batches and be beneficial to industrial production.
The density of the inoculated seeds was 2.0X 10 using "pNiM-01 AF4Per cm2Seeded in 6-well plates (9.5cm surface area per well)2)、25cm2Petri dish and 75cm2NP with purity of more than or equal to 90 percent can be harvested by iPSC differentiation of the culture dish, and the result shows that the purity of the pNiM-01AF is smaller (9.5 cm)2) To a larger size (75 cm)2) Of surface areaThe cell culture vessel can promote the iPSC to be differentiated into NP with the purity of more than or equal to 90 percent.
pNiM-01AF completely avoids animal origin, and the chemical composition of the culture medium formula is definite, when the culture medium of each batch is prepared, all raw materials can be controlled, and the quality control of the culture medium has obvious advantages, and is suitable for the preparation of NP for clinical treatment or research.
By combining the above embodiments, the present invention has obvious advantages and advances over the prior art:
(1) optimization of iPSC differentiation to NP process
By using the culture medium formula of 'pNiM-01 AF' and the corresponding culture method, the invention not only can prepare high-purity NP, but also can obviously improve the absolute yield and reduce the difference of the yield among batches, thereby being beneficial to industrial production.
(2) The chemical components of the culture medium formula 'pNiM-01 AF' are definite
Some existing NP preparation methods use serum or additives with undefined chemical components, but the culture medium formula 'pNiM-01 AF' of the invention has defined chemical components, all raw materials can be controlled when the culture medium of each batch is prepared, and the quality control of the culture medium has obvious advantages.
(3) The whole process is animal origin-free
Some existing NP preparation methods use bovine serum, murine cell secretion (Matrigel) and the like, and introduce animal-derived pathogenic factor pollution risks.
(4) NP production in larger culture vessels
The application range of the scheme of the invention is verified to be smaller (9.5 cm)2) To a larger size (75 cm)2) The cell culture vessel with the surface area increases the NP yield of each batch, is beneficial to industrial production, and is an important progress of the invention, because the prior art does not precedent that the peripheral nerve stem cells are prepared in large volume and can achieve high purity and yield.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A method of promoting differentiation of an induced pluripotent stem cell into a peripheral neural stem cell, comprising the steps of:
s1: culturing induced pluripotent stem cells;
the method comprises primary culture and subculture, wherein cell culture vessels used in the primary culture and the subculture are both treated by vitronectin;
s2: inducing the differentiation of pluripotent stem cells into peripheral neural stem cells;
before differentiation is started, inoculating induced pluripotent stem cells into a cell culture vessel treated by recombinant laminin, culturing by using an animal-origin-free peripheral nerve induction culture medium-01, replacing the culture medium, culturing for 8-10 days, treating the peripheral nerve stem cells obtained after differentiation of the induced pluripotent stem cells by using a pancreatin substitute, and collecting the peripheral nerve stem cells.
2. The method according to claim 1, wherein the seeding density of the induced pluripotent stem cells in the primary culture and/or the subculture in the step S1 is 1.0-2.5 x 104Per cm2Preferably 1.5X 104Per cm2。
3. The method according to claim 1, wherein the induced pluripotent stem cells are seeded at a density of 1.0 to 4.0 x 10 in the step S24Per cm2Preferably 2.0X 104Per cm2。
4. The method according to claim 1, wherein the animal-derived peripheral nerve-free induction medium-01 of step S2 comprises the following components: DMEM/F12 medium, non-essential amino acids, insulin, holotransferrin, putrescine, human serum albumin, superoxide dismutase, glutathione, progesterone, retinol, vitamin A, dl-alpha-tocopheryl acetate, vitamin E, linoleic acid, alpha-linolenic acid, lipoic acid, LDN193189, SB431542, CHIR99021, RO4902097, SU5402, all-trans vitamin a acid, Y27632.
5. The method according to claim 4, wherein the non-animal derived peripheral nerve induction medium-01 contains the non-essential amino acid in an amount of 0.1 to 5% by volume; the concentration of the insulin is 0.1-10 mug/mL; the concentration of the total transferrin is 2-100 mug/mL; the concentration of the putrescine is 5-200 mug/mL; the concentration of the human serum albumin is 250-2500 mug/mL; the concentration of the superoxide dismutase is 1-10 mug/mL; the concentration of the glutathione is 0.1-10 mug/mL; the concentration of the progesterone is 1-10 ng/mL; the concentration of the retinal is 0.01-2 mug/mL; the concentration of the vitamin A is 0.01-2 mug/mL; the concentration of the dl-alpha-tocopheryl acetate is 0.1-10 mu g/mL; the concentration of the vitamin E is 0.1-10 mug/mL; the concentration of the linoleic acid is 0.1-10 mug/mL; the concentration of the alpha-linolenic acid is 0.1-10 mug/mL; the concentration of the lipoic acid is 1-10 ng/mL; the concentration of the LDN193189 is 0.1-1 mu g/mL; the concentration of the SB431542 is 1-10 mug/mL; the concentration of CHIR99021 is 2.5-12.5 mug/mL, the concentration of RO4902097 is 0.5-5 mug/mL, the concentration of SU5402 is 0.5-5 mug/mL, the concentration of all-trans vitamin A acid is 1-20 ng/mL, and the concentration of Y27632 is 1-10 mug/mL.
6. An animal-free peripheral nerve induction medium-01 for inducing differentiation of induced pluripotent stem cells into peripheral nerve stem cells, comprising the following components: DMEM/F12 medium, non-essential amino acids, insulin, holotransferrin, putrescine, human serum albumin, superoxide dismutase, glutathione, progesterone, retinol, vitamin A, dl-alpha-tocopheryl acetate, vitamin E, linoleic acid, alpha-linolenic acid, lipoic acid, LDN193189, SB431542, CHIR99021, RO4902097, SU5402, all-trans vitamin a acid, Y27632.
7. The culture medium according to claim 6, wherein the content of the non-essential amino acid is 0.1 to 5% by volume; the concentration of the insulin is 0.1-10 mug/mL; the concentration of the total transferrin is 2-100 mug/mL; the concentration of the putrescine is 5-200 mug/mL; the concentration of the human serum albumin is 250-2500 mug/mL; the concentration of the superoxide dismutase is 1-10 mug/mL; the concentration of the glutathione is 0.1-10 mug/mL; the concentration of the progesterone is 1-10 ng/mL; the concentration of the retinal is 0.01-2 mug/mL; the concentration of the vitamin A is 0.01-2 mug/mL; the concentration of the dl-alpha-tocopheryl acetate is 0.1-10 mu g/mL; the concentration of the vitamin E is 0.1-10 mug/mL; the concentration of the linoleic acid is 0.1-10 mug/mL; the concentration of the alpha-linolenic acid is 0.1-10 mug/mL; the concentration of the lipoic acid is 1-10 ng/mL; the concentration of the LDN193189 is 0.1-1 mu g/mL; the concentration of the SB431542 is 1-10 mug/mL; the concentration of CHIR99021 is 2.5-12.5 mug/mL, the concentration of RO4902097 is 0.5-5 mug/mL, the concentration of SU5402 is 0.5-5 mug/mL, the concentration of all-trans vitamin A acid is 1-20 ng/mL, and the concentration of Y27632 is 1-10 mug/mL.
8. A system for promoting differentiation of induced pluripotent stem cells into peripheral neural stem cells, comprising the following components:
(1) a vitronectin-treated cell culture vessel;
(2) recombinant laminin treated cell culture vessel;
(3) the animal origin-free peripheral nerve induction medium-01 according to any one of claims 6 to 7.
9. The system of claim 8, wherein the recombinant laminin-treated cell culture vessel has a surface area of 9.5-75 cm2。
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