CN107858331B - Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells - Google Patents
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Abstract
The invention discloses a method for inducing human pluripotent stem cells to differentiate into spinal cord motor nerve precursor cells, which comprises the following steps: 1) culturing human pluripotent stem cells: digesting human pluripotent stem cells into single cells, inoculating the single cells into a coated culture dish for adherent culture, wherein the culture solution is pluripotent stem cell culture solution and 5% CO at 37 DEG C2Culturing in a saturated humidity incubator for 20-28 hr; 2) differentiation induction of spinal motor precursor cells: culturing the human pluripotent stem cells obtained in the step 1) in a basic complete culture medium, adding different space-time specific signal paths at different times to regulate and control small molecules and/or growth factors to induce differentiation, changing the medium once every two days, and changing the medium at 37 ℃ by 5% CO2Culturing in saturated humidity incubator for 8-20 days. The method is efficient, rapid, stable, safe and simple and convenient to operate, and more than 90% of spinal cord motor nerve precursor cells can be obtained on the eighth day of differentiation.
Description
Technical Field
The invention relates to the technical field of stem cell differentiation culture. And more particularly, to a method of inducing differentiation of human pluripotent stem cells into spinal motor precursor cells.
Background
The human pluripotent stem cells have unlimited proliferation capacity and the potential of differentiating into various somatic cells of human bodies, and are important seed resource cells for researching a development regulation mechanism, a disease pathogenesis and regenerative medicine. However, the problems of long differentiation time and low differentiation efficiency of the prior method for differentiating the human pluripotent stem cells into specific functional cells generally exist, and the application of the prior method in the biomedical field is seriously influenced.
In recent years, the number of patients with spinal cord injury due to traffic accidents, falls, contusions, and the like tends to increase year by year. Because nerves inside the spinal cord are damaged, patients with spinal cord injury are generally paralyzed in lower limbs, and life is difficult to take care of themselves. Under normal physiological conditions, injured nerve cells in the human body cannot repair themselves, so that the disease is difficult to be completely cured by traditional medical treatment means. The cell replacement therapy provides a new treatment way for the patients with spinal cord injury. Spinal cord motor precursor cells have the potential to differentiate into spinal cord motor neurons and glial cells, and are one of the ideal cell types for disease treatment of spinal cord injury.
The conventional method for differentiating pluripotent stem cells into spinal motor precursor cells has the following problems: (1) the operation process is complicated, and relates to the steps of performing suspension culture and differentiation on human pluripotent stem cells into embryoid bodies, performing adherent culture on the embryoid bodies to obtain rosette-like neural tube structures, screening the rosette-like structures, performing suspension culture on the rosette-like structures to form neurospheres, and performing adherent culture and differentiation on the neurospheres to obtain spinal motor nerve precursor cells; (2) the time is long, the efficiency is low, the time span for obtaining the spinal cord motor nerve precursor cells by inducing and differentiating the human pluripotent stem cells reaches 28 days, and the differentiation efficiency is only about 60 percent; (3) the culture solution used in the differentiation process is added with animal-derived components.
Therefore, it is required to provide a method for efficiently and rapidly inducing differentiation of human pluripotent stem cells into spinal motor precursor cells, so that the spinal motor cells for clinical application have (1) cell availability convenience; (2) the cell purity is high; (3) the cell culture solution has the characteristics of simple components, no animal-derived components and the like, and ensures the safety, effectiveness and traceability of clinically applied cells.
Disclosure of Invention
The invention aims to provide a method for inducing human pluripotent stem cells to differentiate into spinal cord motor nerve precursor cells efficiently, quickly, stably and safely, wherein the differentiation efficiency of the method can reach more than 90%.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for inducing human pluripotent stem cells to differentiate into spinal cord motor nerve precursor cells, which comprises the following steps:
1) culturing human pluripotent stem cells: digesting human pluripotent stem cells into single cells, inoculating the single cells into a coated culture dish for adherent culture, wherein the culture solution is pluripotent stem cell culture solution and 5% CO at 37 DEG C2Culturing in a saturated humidity incubator for 20-28 hr;
2) differentiation induction of spinal motor precursor cells: culturing the human pluripotent stem cells obtained in the step 1) in a basic complete culture medium, adding different space-time specific signal paths at different times to regulate and control small molecules and/or growth factors to induce differentiation, changing the medium once every two days, and changing the medium at 37 ℃ by 5% CO2Culturing in saturated humidity incubator for 8-20 days.
Further, the space-time specific signal pathway regulation small molecule and/or growth factor comprises a signal regulation small molecule and/or growth factor related to neuroectodermal specialization, a signal regulation small molecule and/or growth factor related to spinal cord motor precursor cell dorsoventral axis specialization and a signal regulation small molecule and/or growth factor related to spinal cord motor precursor cell anteroposterior axis specialization.
Wherein the small signaling regulatory molecule and/or growth factor associated with neuroectodermal specification is an inhibitor of the TGF-beta signaling pathway; the TGF beta signal channel inhibitor and final use concentration are SB431542, 1-100 μ M; LDN193189, 0.001-10 μ M; a77-01, 1-50 μ M; a83-01, 1-50 μ M; chebulinic acid, 0.1-50 μ M; disitertide, 0.5-80 mu M and EW-7197, 1-50 mu M, preferably SB431542, 1-100 mu M; LDN193189, 0.001-10 μ M; one or more of A77-01, 1-50 μ M and A83-01, 1-50 μ M, most preferably SB431542, 1-100 μ M and LDN193189, 0.001-10 μ M; the action time is differentiation D0-2, D2-4, D0-4, D0-6 or D2-6, preferably D0-6, most preferably D0-4.
The signal regulation small molecule and/or the growth factor related to the dorsal-ventral axis specialization of the spinal cord motor nerve precursor cell is a Wnt signal pathway activator; the Wnt signal pathway activator and the final use concentration are WNT3a, 1-100 ng/mL; 6-BIO, 1-20 μ M; CHIR-98014, 0.1-20 μ M; TDZD-8, 10-100 μ M; AZD1080, 0.1-50 μ M; CHIR99021, 0.1-20 μ M, preferably 6-BIO, 1-20 μ M and CHIR99021, 0.1-20 μ M, most preferably CHIR99021, 0.1-20 μ M; the action time is differentiation D0-2, D2-4, D0-4, D0-6 or D2-6, preferably D0-6, most preferably D2-6.
The signal regulation and control small molecules and/or growth factors related to the anterior-posterior axis specialization of the spinal cord motor nerve precursor cells are SHH signal pathway activators and retinoic acid signal pathway activators; the SHH signal pathway activator and final use concentration are one or a combination of more of SHH, 1-500ng/mL, SAG, 0.1-20 μ M and purmorphamine, 0.1-20 μ M, preferably SAG, 0.1-20 μ M and SHH, 1-500ng/mL combination or SAG, 0.1-20 μ M and purmorphamine, 0.1-20 μ M combination; most preferably SAG, 0.1-20. mu.M and purmorphamine, 0.1-20. mu.M, for a time period such that the differentiation is D0-10, D2-20, D0-8, D2-8, D2-16, D0-16, preferably D0-16, most preferably D2-16; the retinoic acid signal pathway activator and the final use concentration are 10-10000nM of retinoic acid; preferably, retinoic acid, 10-1000 nM; most preferably, retinoic acid, 10-100 nM; the action time is differentiation D0-10, D2-20, D0-8, D2-8, D2-16, D0-16, preferably D0-8, most preferably D2-16.
In the present invention, the differentiation D0-6 represents the 0 th day to the 6 th day of differentiation, and D2-4 represents the 2 nd day to the 4 th day of differentiation, and so on, and the description thereof is omitted.
Further, the pluripotent stem cell culture solution is a mTeSR and E8 culture solution. Preferably, it is E8 culture solution.
Further, when the pluripotent stem cells are digested into single cells, a ROCK signal pathway inhibitor may be added to the culture solution in order to prevent the death of the pluripotent stem cells; the ROCK signal pathway inhibitor was Y27632 at a final use concentration of 10 μ M.
Further, the coated culture dish is a Poly ornithine (Poly-L-ornithine, PLO) -Laminin (Laminin) coated culture dish; the method comprises the following specific steps: diluting PLO with PBS to 15 μ g/mL, adding into the culture dish to submerge the bottom of the culture dish, and incubating at 37 ℃ for 2h or 4 ℃ overnight without allowing the bottom of the culture dish to dry off during the incubation; the PLO is discarded, and the mixture is rinsed twice with PBS and once with DMEM/F12; diluting Laminin to 5. mu.g/mL with DMEM/F12, adding to the PLO coated petri dish until the bottom of the dish is submerged, and incubating at 37 ℃ for 2h or 4 ℃ overnight.
Further, the basic complete culture medium in the step 2) consists of a basic culture medium and a nutrient additive;
wherein the basic culture medium is one or two of DMEM/F12medium and Neurobasal medium; preferably, the volume ratio of the DMEM/F12medium to the Neurobasal medium is 1: 1;
the nutritional additive comprises the following components in percentage by weight: 0.1-20mg/L of human insulin, 10-200mg/L of vitamin C, 10-100mg/L of glutathione, 0.05-5mg/L of linolenic acid, 0.2-20mg/L, N of carnitine, 5-500 mu M of acetylcysteine, 0.01-10mg/L of ethanolamine and 0.05-5mg/L of linoleic acid;
preferably, the composition and final use concentration of the nutritional additive are respectively as follows: 0.5-15mg/L of human insulin, 20-100mg/L of vitamin C, 20-90mg/L of glutathione, 0.1-4mg/L of linolenic acid, 0.5-15mg/L, N of carnitine-10-400 mu M of acetylcysteine, 0.05-8mg/L of ethanolamine and 0.1-4mg/L of linoleic acid;
most preferably, the nutritional supplement has a composition and end-use concentration, respectively, of: 1-10mg/L of human insulin, 30-80mg/L of vitamin C, 30-80mg/L of glutathione, 1-3mg/L of linolenic acid, 1-10mg/L, N of carnitine, 50-100 mu M of acetylcysteine, 0.1-5mg/L of ethanolamine and 1-3mg/L of linoleic acid.
The invention has the following beneficial effects:
firstly, high efficiency and high speed. Regulation of small molecules and/or growth factor regulation signals by adding different spatio-temporal specific signal pathways at different timesChannel, simulating the specialization process of the developing spinal cord motor nerve precursor cells, and obtaining Olig2 of which the concentration is more than 90 percent on the eighth day of differentiation+Spinal motor precursor cells.
Secondly, the stability and safety are realized. The basic complete culture medium used in the method has definite chemical components, no animal-derived components and no extract or hydrolysate, can prevent the system instability caused by batch quality difference of extracted proteins such as serum albumin and the like, and can prevent the possibility of being polluted by various human-derived and animal-derived pathogenic microorganisms.
Thirdly, the operation is simple and convenient. The cells are cultured in an adherent way in the whole differentiation process, the operation is simple and convenient, the repeatability is good, and the requirement on the technical level of an operator is low.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Figure 1 shows a light mirror image of human pluripotent stem cells, the left image being of well grown colony sizes just suitable for differentiating human pluripotent stem cells, and the right image being of human pluripotent stem cells that have not been grown too densely for differentiation without timely passaging.
Fig. 2 shows morphology of spinal cord motor precursor cells on day eight of differentiation (experimental group, control group one, control group two).
FIG. 3 shows that the expression ratio of Olig2 positive cells was measured by flow cytometry at day eight of differentiation (experimental group, control group one, control group two).
FIG. 4 shows immunofluorescence staining pattern of spinal cord motor precursor cell marker gene Olig2 on day eight of differentiation (experimental group).
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Basal media DMEM/F12medium and Neurobasal medium, E8 broth, mTeSR broth, B27 used in the examples of use of the invention were purchased from Life Technologies; the space-time specific signal path regulating small molecules and growth factors are purchased from Sigma, Stemgent, Enzo and Tocris, and other materials are common materials in the field.
EXAMPLE 1 differentiation of human pluripotent Stem cells into spinal motor precursor cells
1. Preparation of the culture Medium
1.1 coating of Petri dishes
1.1.1 coating of PLO-Lamin Petri dishes
Poly-ornithine (Poly-L-ornithline, PLO) was diluted to 15 μ g/mL with PBS, added to the petri dish to submerge the bottom of the dish in the amount shown in table 1, incubated at 37 ℃ for 2h or overnight at 4 ℃, without allowing the bottom of the dish to dry off during incubation; the PLO is discarded, and the mixture is rinsed twice with PBS and once with DMEM/F12; laminin (Laminin) was diluted to 5. mu.g/mL with DMEM/F12, added to the PLO-coated dishes to the bottom of the dishes and incubated at 37 ℃ for 2h or at 4 ℃ overnight, as shown in Table 1.
1.1.2 coating of Vitronect Petri dishes
Vitronectin (Life technologies) was diluted to 5. mu.g/mL with DMEM/F12, added to the dishes to be coated in the amounts shown in Table 1, incubated at 37 ℃ for 1-2h or overnight at 4 ℃ taking care not to dry the dishes during the incubation period, and the media was discarded from the inoculated cells at the time of use.
TABLE 1 amount of PLO, Lamin or Vitronecin required to coat the dishes
| Culture dish specification | Growth area (cm)2) | Amount added (mL) |
| 6 orifice plate | 10cm2/well | 1mL/well |
| 12-hole plate | 4cm2/well | 0.4mL/well |
| 24-hole plate | 2cm2/well | 0.2mL/well |
| 35mm culture dish | 10cm2 | 1mL |
| 60mm culture dish | 20cm2 | 2mL |
| 100mm culture dish | 60cm2 | 6mL |
1.2 basal complete Medium consisting of
Basal medium (volume ratio): 50% DMEM/F12medium and 50% Neurobasal medium.
The nutrient additive comprises the following components: the nutritional additive comprises the following components in percentage by weight: 10mg/L of human insulin, 50mg/L of vitamin C, 50mg/L of glutathione, 1mg/L of linolenic acid, 20mg/L, N mu M of carnitine-acetylcysteine, 5mg/L of ethanolamine and 1mg/L of linoleic acid.
1.3 pluripotent Stem cell culture solution
E8 culture solution
1.4 acquisition of human pluripotent Stem cells
1.4.1 Resuscitation of human pluripotent Stem cells
1) Preparing DMEM/F12 with the volume 10 times that of the frozen stock solution in a 15mL centrifuge tube in advance;
2) taking out the cells from the liquid nitrogen, and rapidly shaking and melting the cells in a water bath kettle at 37 ℃;
3) the cells from the frozen tube were transferred to DMEM/F12 prepared in advance, centrifuged at 0.4 Xg for 3min, the supernatant was discarded, the cells were resuspended in E8 medium preheated at 37 ℃ and seeded into a Petri dish coated with Vitronectin.
Note that: to increase cell viability, 10 μ M Y27632 can be added to the culture broth.
1.4.2 passages of human pluripotent Stem cells
When the colony of the human pluripotent stem cells is large enough or when the confluency of the human pluripotent stem cells reaches 90%, subculture is required, the general passage ratio is 1:6-1:10, and the passage ratio of different cell lines is different.
Note that: all liquids used for passaging required preheating at 37 ℃.
1) Discarding the old culture medium, and washing with PBS without calcium and magnesium;
2) adding appropriate amount of 0.5mM EDTA, and digesting at 37 deg.C for 3 min;
3) EDTA was carefully discarded and gently washed once with DMEM/F12;
4) the cells were resuspended in E8 medium and plated onto previously Vitronectin-coated petri dishes.
1.4.3 cryopreservation of human pluripotent Stem cells
The formula of the frozen stock solution is as follows: e8+ 10% DMSO; note that: the frozen stock solution needs to be pre-cooled at 4 ℃.
1) Discarding the old culture medium, and washing with PBS without calcium and magnesium;
2) adding appropriate amount of 0.5mM EDTA, and digesting at 37 deg.C for 3 min;
3) EDTA was carefully discarded and gently washed once with DMEM/F12;
4) resuspend the cells in DMEM/F12, centrifuge at 0.4 Xg for 3min, discard the supernatant;
5) resuspending the cells with the cryopreservation solution, and subpackaging the cells into cryopreservation tubes, wherein the cells of a 100mm culture dish can be cryopreserved for 8-10 tubes generally;
6) placing the freezing tube into a programmed cooling box, placing the box in a refrigerator at minus 80 ℃ for overnight, and transferring the box to a liquid nitrogen tank the next day.
2. Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells
1) Culture of human pluripotent stem cells
Human pluripotent stem cells (FIG. 1) with good growth status were digested into single cells with Accutase according to 105cells/mL were inoculated into a PLO-Lamin-coated petri dish at a density of 10. mu. M Y27632 in a culture solution of pluripotent stem cells with 5% CO at 37 ℃2Culturing in a saturated humidity incubator for 24 hours; the differentiation procedure, differentiation D0, was initiated.
2) Differentiation induction of spinal motor precursor cells
Culturing the human pluripotent stem cells obtained in step 1) in a basic complete culture medium, adding different space-time specific signal pathways to regulate small molecules and/or growth factors at different times, and 5% CO at 37 ℃2The cells were differentiated by changing the medium every two days in a saturated humidity incubator according to the amount of the medium shown in Table 2.
Different space-time specific signal paths are added at different times in the differentiation process to regulate and control small molecules and/or growth factors, and the action time and the final use concentration of the space-time specific signal paths to regulate and control the small molecules and/or the growth factors are respectively as follows:
the TGF beta signal pathway inhibitor is SB 43154240 mu M, and the action time is differentiation D0-4;
the Wnt signal pathway activator is 6-BIO 5 mu M, and the action time is differentiation D0-4;
the SHH signal pathway activator is SHH 100ng/mL, and the action time is differentiation D2-16;
the retinoic acid signaling pathway activator is retinoic acid 100nM, and the action time is differentiation D2-16.
TABLE 2 amount of culture fluid required during differentiation
| Culture dish specification | Growth area (cm)2) | Amount added (mL) |
| 6 orifice plate | 10cm2/well | 2mL/well |
| 12-hole plate | 4cm2/well | 1mL/well |
| 24-hole plate | 2cm2/well | 0.5mL/well |
| 35mm culture dish | 10cm2 | 2mL |
| 60mm culture dish | 20cm2 | 4mL |
| 100mm culture dish | 60cm2 | 12mL |
When the D6 cells are differentiated, the cell density is too high, and subculture is needed, and the specific method is as follows:
to prevent cell death, 10 μ M Y27632 was added to the basal complete medium.
Discarding the original basic complete culture medium, washing with PBS, adding appropriate amount of accutase, digesting at 37 deg.C for 3min to make the cells in loose adherent state, discarding accutase carefully, re-suspending the cells in basic complete culture medium, counting the cells, and counting the number of cells according to 105cells/mL were plated in pre-coated dishes.
EXAMPLE 2 differentiation of human pluripotent Stem cells into spinal motor precursor cells
1. Preparation of the culture Medium
1.1 coating of Petri dishes As in example 1
1.2 basal complete Medium consisting of
Basal medium (volume ratio): 50% DMEM/F12medium and 50% Neurobasal medium;
the nutrient additive comprises the following components: the nutritional additive comprises the following components in percentage by weight: 0.1mg/L of human insulin, 10mg/L of vitamin C, 10mg/L of glutathione, 0.05mg/L of linolenic acid, 0.2mg/L, N-acetylcysteine 5 mu M of carnitine, 0.01mg/L of ethanolamine and 0.05mg/L of linoleic acid.
1.3 pluripotent Stem cell culture solution
mTeSR culture solution
2. Induction of differentiation of human pluripotent stem cells into spinal motor precursor cells
1) Culture of human pluripotent stem cells
Digesting the human pluripotent stem cells with good growth condition into single cells by using Accutase according to the proportion of 105cells/mL are inoculated into a culture dish coated with PLO-Lamin in advance, and the culture solution is pluripotent stem cellsCulture broth + 10. mu. M Y27632, 5% CO at 37 ℃2Culturing in a saturated humidity incubator for 24 hours; the differentiation procedure, differentiation D0, was initiated.
2) Differentiation induction of spinal motor precursor cells
Culturing the human pluripotent stem cells obtained in step 1) in a basic complete culture medium, adding different space-time specific signal pathways to regulate small molecules and/or growth factors at different times, and 5% CO at 37 ℃2The cells were differentiated by changing the medium every two days in a saturated humidity incubator according to the amount of the medium shown in Table 2.
The action time and the final use concentration of the space-time specificity signal path for regulating and controlling the small molecules and/or the growth factors in the differentiation process are respectively as follows:
the TGF beta signal channel inhibitor is LDN 1931890.001 mu M, and the action time is differentiation D2-6;
the Wnt signal pathway activator is CHIR 990210.1 μ M, and the action time is differentiation D2-4;
the SHH signal pathway activator is SAG100nM, and the action time is differentiation D2-16;
the retinoic acid signaling pathway activator is retinoic acid 10nM, and the action time is differentiation D2-16.
EXAMPLE 3 differentiation of human pluripotent Stem cells into spinal motor precursor cells
1. Preparation of the culture Medium
1.1 coating of Petri dishes As in example 1
1.2 basal complete Medium consisting of
Basal medium (volume ratio): 50% DMEM/F12medium and 50% Neurobasal medium
The nutrient additive comprises the following components: the nutritional additive comprises the following components in percentage by weight: human insulin 20mg/L, vitamin C200 mg/L, glutathione 100mg/L, linolenic acid 5mg/L, carnitine 20mg/L, N-acetylcysteine 500 mu M, ethanolamine 10mg/L and linoleic acid 5 mg/L.
1.3 pluripotent Stem cell culture solution
E8 culture solution
2. Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells
1) Culture of human pluripotent stem cells
Digesting the human pluripotent stem cells with good growth condition into single cells by using Accutase according to the proportion of 105cells/mL are inoculated into a culture dish coated with PLO-Lamin in advance, the culture solution is pluripotent stem cell culture solution +10 mu M Y27632, and the temperature is 37 ℃ and the CO content is 5 percent2Culturing in a saturated humidity incubator for 24 hours; the differentiation procedure, differentiation D0, was initiated.
2) Differentiation induction of spinal motor precursor cells:
culturing the human pluripotent stem cells obtained in step 1) in a basic complete culture medium, adding different space-time specific signal pathways to regulate small molecules and/or growth factors at different times, and 5% CO at 37 ℃2The cells were differentiated by changing the medium every two days in a saturated humidity incubator according to the amount of the medium shown in Table 2.
Different space-time specific signal paths are added at different times in the differentiation process to regulate and control small molecules and/or growth factors, and the action time and the final use concentration of the space-time specific signal paths to regulate and control the small molecules and/or the growth factors are respectively as follows:
the TGF beta signal pathway inhibitor is LDN 193189100 nM and SB 43154210. mu.M, and the action time is differentiation D2-4 (day 2 to day 4);
wnt signal pathway activators are CHIR 990211 μ M and 6-BIO 2 μ M, and the action time is differentiation D2-6;
the SHH signal channel activator is purmorphamine 2 mu M, and the action time is differentiation D0-16;
the retinoic acid signaling pathway activator is retinoic acid 500nM, and the action time is differentiation D0-16.
Example 4 comparative experiment for efficiently and rapidly inducing differentiation of human pluripotent stem cells into spinal motor precursor cells
The method used in the experimental group was the method in example 3;
control group one the method used was the method of example 3 except that no Wnt signaling pathway activator was added;
the method used in control group two was the method of example 3, except that the formulation of the basal complete medium was changed to the following formulation:
basal medium (volume ratio): 50% DMEM/F12medium and 50% Neurobasal medium;
the nutrient additive comprises the following components: B27.
the differentiation results are shown in FIGS. 2 to 4, and FIG. 2 shows the morphology of the differentiation of human pluripotent stem cells into spinal motor precursor cells on the eighth day of differentiation, from which it can be seen that the growth states of the cells of the experimental group, the first control group and the second control group are equivalent on the eighth day of differentiation. Fig. 3 shows the expression ratio of the human spinal cord motor precursor cell marker gene Olig2 of the eighth day of differentiation of human pluripotent stem cells into spinal cord motor precursor cells, so that it can be seen that the differentiation efficiency of the experimental group is significantly higher than that of the control group i and the control group ii, and it can be seen that the use of the Wnt signaling pathway activator and the additive described in the invention are necessary conditions for efficient differentiation during the differentiation process. FIG. 4 shows the immunofluorescence pictures of spinal cord motor precursor cell marker gene Olig2 of experimental group human pluripotent stem cells differentiating into spinal cord motor precursor cells at the eighth day of differentiation.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (7)
1. A method for inducing differentiation of human pluripotent stem cells into spinal cord motor precursor cells, comprising the steps of:
1) culturing human pluripotent stem cells: digesting human pluripotent stem cells into single cells, inoculating the single cells into a coated culture dish for adherent culture, wherein the culture solution is a pluripotent stem cell culture solution，37℃5％CO2Culturing in a saturated humidity incubator for 20-28 hr;
2) differentiation induction of spinal motor precursor cells: culturing the human pluripotent stem cells obtained in the step 1) in a basic complete culture medium, adding different space-time specific signal paths at different times to regulate and control small molecules or growth factors to induce differentiation, changing the medium once every two days, and changing the medium at 37 ℃ by 5% CO2Culturing in a saturated humidity incubator for 8-20 days;
wherein the basic complete culture medium consists of a basic culture medium and a nutrient additive;
the nutritional additive comprises the following components in percentage by weight: human insulin 20mg/L, vitamin C200 mg/L, glutathione 100mg/L, linolenic acid 5mg/L, carnitine 20mg/L, N-acetylcysteine 500 mu M, ethanolamine 10mg/L, linoleic acid 5 mg/L;
the adding of different time-space specific signal channels at different times to regulate the small molecules or growth factors is specifically as follows: the TGF beta signal pathway inhibitor is LDN 1931890.1 mu M and SB 43154210 mu M, and the action time is differentiation D2-D4; wnt signal pathway activators are CHIR 990211 μ M and 6-BIO 2 μ M, and the action time is differentiation D2-D6; the SHH signal pathway activator is purmorphamine 2 mu M, and the action time is differentiation D0-D16; the retinoic acid signaling pathway activator is retinoic acid 500nM, and the action time is differentiation D0-D16.
2. The method of claim 1, wherein the basal medium is one or both of DMEM/F12medium, Neurobasal medium.
3. The method of claim 2, wherein the basal medium is a mixed culture of DMEM/F12medium and Neurobasal medium at a volume ratio of 1: 1.
4. The method according to claim 1, wherein the pluripotent stem cell culture fluid is mTeSR and E8 culture fluid.
5. The method according to claim 1, wherein the pluripotent stem cell culture solution is E8 culture solution.
6. The method of claim 4, wherein a ROCK signaling pathway inhibitor is further added to the culture broth of said pluripotent stem cells.
7. The method of claim 6, wherein the ROCK signaling pathway inhibitor is Y27632 at a final use concentration of 10 μ Μ.
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| CN115125210B (en) * | 2022-08-31 | 2022-12-02 | 华科星河(北京)生物科技有限公司 | Culture medium and method for lumbosacral segment spinal cord neural stem cells induced from iPSC |
| CN115125209B (en) * | 2022-08-31 | 2022-12-06 | 华科星河(北京)生物科技有限公司 | Method for inducing cervical spinal cord neural stem cells from induced pluripotent stem cells |
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