Original digestive tract partition universal type induction method

Abstract

The invention provides a general induction method for original digestive tract partition. The method comprises the following steps: high-quality human pluripotent stem cell verification, pluripotent stem cell sensitivity test, cell inoculation and start convergence degree verification, endoderm-mesoderm co-induction, original digestive tract induction and original digestive tract partition. Compared with a traditional method, on the premise of not depending on serum, original digestive tract partition derivatives (in a 3D sphere form) of different organs such as lungs, stomachs, livers, pancreas, small intestines and large intestines can be efficiently generated, and subsequent batch preparation of final products is facilitated. The method is helpful for promoting large-scale industrial transformation and application of respiratory digestive tract organs.

Description

Technical Field

[0001] The present invention belongs to the field of cell differentiation, and particularly relates to a general induction method for partitioning the primitive gut tube. Background Art

[0002] The primitive gut tube is the initial development site of most visceral organs in the human body (especially respiratory and digestive tract organs). In the early stage of embryonic development, under the precise regulation of the "anterior-posterior" axis and the "ventral-dorsal" axis, the primitive gut tube rapidly forms different sub-regions with unique molecular characteristic spectra; subsequently, it differentiates and generates a series of organs such as the thyroid gland, pharynx, lung, liver, gallbladder, pancreas, and intestine. In the past decade, with the rapid development of human pluripotent stem cells (PSC) and organoid technology, induction methods for differentiating PSC into organ-specific sub-regions of the primitive gut tube have been successively established. As the developmental progenitors of organs, the induction efficiency of these specific sub-region derivatives directly determines the yield of downstream organoids.

[0003] Currently, the induction efficiency of derivatives of different organoid primitive gut tube sub-regions is generally low and faces stability challenges. For example, in the construction of small intestine and large intestine organoids, the method established by the JR Spence team in 2011 is still mainly used; in the S1 endoderm induction stage and the S2 primitive gut tube intestinal sub-region induction stage (middle and posterior segments), fetal bovine serum (FBS) is used as nutritional support (DOI: 10.1038 / nprot.2011.410; 10.1016 / bs.mcb.2020.03.007). Due to the unclear composition and xenogeneic origin of FBS, the induction efficiency is low (DOI: 10.1016 / j.stemcr.2017.11.004). Similarly, there are the same problems in a group of organoids such as the esophagus, stomach, and lung (DOI: 10.1016 / j.stem.2018.08.008; 10.1038 / s41596-018-0080-z; 10.7554 / eLife.05098). For the construction of liver organoids, the method proposed by T Takebe et al. in 2019 is mainly used; although it successfully replaces FBS with serum substitutes N2 and B27 in the S2 primitive gut tube liver sub-region induction stage (rear part of the front segment), the use of serum cannot be avoided in the S1 endoderm stage (DOI: 10.1016 / j.cmet.2019.05.007). In addition, due to the huge differences in induction methods for different sub-regions, it is still impossible to form a generally compatible general technology base, making the industrialization process extremely difficult. Summary of the Invention

[0004] In view of this, the present invention aims to propose a general induction method for partitioning the primitive gut tube. Compared with traditional methods, without relying on serum, the present invention widely supports the efficient induction (in the form of 3D spheres) of partitioning the primitive gut tube of different types of organoids such as the lung, stomach, liver, pancreas, small intestine, and large intestine, facilitating the subsequent batch preparation of end-product organoids. The present invention will contribute to promoting the large-scale industrial transformation and application of respiratory and digestive tract organoids.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows: A general induction method for partitioning the primitive gut tube, the method comprising the following steps: S1. Preparation stage: Perform flow cytometry on pluripotent stem cells, take the PSCs that pass the pluripotency test, digest and inoculate them on the basement matrices LN521 and LN111, add mTeSR1 medium and culture until the required confluence is reached, then change the stem cell medium to a medium containing ActA, and continue to culture until the confluence decreases by more than 50% (decreases by more than 50% of the original confluence), which is regarded as passing the sensitivity test; take the PSCs that pass the sensitivity test and inoculate them on a differentiation plate containing the basement matrices LN521 and LN111, continue to use mTeSR1 medium to culture until the required confluence is reached, and then initiate differentiation; S2. Differentiation stage: Co-induce the PSCs into endoderm and mesoderm using a medium containing the serum replacement KSR, and then change the medium to perform primitive gut tube induction and primitive gut tube partitioning culture respectively, so as to form 3D derivatives of the lung region or stomach region or liver region or pancreas region or small intestine region or large intestine region.

[0006] Furthermore, in S1, the standard for pluripotency test is SSEA4 + NANOG + ≥90%.

[0007] Furthermore, in S1, the volume ratio of the basement matrices LN521 and LN111 is 1:3, and the final concentration of laminin is 5 - 15 μg / ml.

[0008] Preferably, the final concentration of laminin is 5 μg / ml.

[0009] Furthermore, in S1, the concentration of ActA is 100 - 300 ng / ml. Non-limiting examples can be 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, and preferably 100 ng / ml.

[0010] Furthermore, in S2, culture for 1 - 2 days in medium 1, medium 2, and medium 3 in sequence respectively.

[0011] Furthermore, the volume ratio of KSR added to Medium 2 is 0.4 - 0.8%, and the volume ratio of KSR added to Medium 3 is 4 - 8%.

[0012] The volume ratio of KSR added to Medium 2 can be exemplified non - restrictively as 0.4%, 0.5%, 0.6%, 0.7%, 0.8%; The volume ratio of KSR added to Medium 3 can be exemplified non - restrictively as 4%, 5%, 6%, 7%, 8%; Preferably, the volume ratio of KSR added to Medium 2 is 0.4%, and the volume ratio of KSR added to Medium 3 is 4%.

[0013] Furthermore, Medium 1 includes additives BMP4 and ActA, and both Medium 2 and Medium 3 include additives ActA, FGF2 and KSR.

[0014] Specifically, Medium 1 includes 10 - 50 ng / ml BMP4 and 100 - 200 ng / ml ActA; the concentration of BMP4 can be exemplified non - restrictively as 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, preferably 50 ng / ml; the concentration of ActA can be exemplified non - restrictively as 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml, preferably 100 ng / ml; Medium 2 and Medium 3 include 100 - 200 ng / ml ActA and 10 - 30 ng / ml FGF2; the concentration of ActA can be exemplified non - restrictively as 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml, preferably 100 ng / ml; the concentration of FGF2 can be exemplified non - restrictively as 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, preferably 10 ng / ml.

[0015] Furthermore, in S2, the medium for inducing the primitive digestive tract includes additives FGF7 and vitamin C, and Ad - DMEM / F12 added with KSR at a volume ratio of 1.5 - 4.5%; the differentiation time at this stage is 2 - 3 days.

[0016] Specifically, the concentration of FGF7 is 20 - 50 ng / ml, and the concentration of vitamin C is 0.5 - 2 mM.

[0017] Non-limiting examples of the concentration of FGF7 can be 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 50 ng / ml, preferably 50 ng / ml; non-limiting examples of the concentration of vitamin C can be 0.5 mM, 1 mM, 1.5 mM, 2 mM, preferably 0.5 mM.

[0018] Non-limiting examples of the volume ratio of KSR can be 1.5%, 2%, 2.5%, 3%, 3.5%, 4%; preferably, the volume ratio of KSR is 1.5%.

[0019] Furthermore, in S2, the culture medium for the primary digestive tract regional culture comprises Ad-DMEM / F12 supplemented with 2-8% volume ratio of KSR.

[0020] Non-limiting examples of the volume ratio of KSR can be 2%, 3%, 4%, 5%, 6%, 7%, 8%; preferably, the volume ratio of KSR can be 2%.

[0021] Furthermore, in S1, when the confluence reaches 70-90%, the culture medium containing ActA is replaced; when the confluence reaches 70-90%, differentiation is initiated.

[0022] Compared with the prior art, the primary digestive tract regional general induction method described in the present invention has the following advantages: The primary digestive tract regional general induction method described in the present invention is free of animal-derived components and serum throughout the process, improving stability and reducing the risk of contamination, and having the potential for clinical translation; it has strong generality and can support the generation of at least 6 different regional derivatives of the primary digestive tract (such as lung, stomach, liver, pancreas, small intestine, large intestine, etc.); the induction efficiency is high, and compared with the original method, the derivative generation efficiency is increased by 20% - 420% varyingly. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 It is a schematic diagram of the primary digestive tract regional general induction method; Figure 2Comparison of induction efficiencies of derivatives of each region of the primitive gut tube; The left two columns are respectively: representative bright-field images of 3D-derived spheres of each organ region of each classical induction method and this method. Among them, a single well of a 24-well plate is used, and the image acquisition point is the induction time point required by each method; The middle column is the immunofluorescence test results of derivatives of each region produced by this method. Among them, NKX2.1 is a marker for lung epithelial progenitor cells, HNF1β is a marker for gastric epithelial progenitor cells, PROX1 and HHEX are markers for hepatic primordium (hepatic progenitor cells), NKX6.1 and PDX1 are markers for pancreatic epithelial progenitor cells, GATA4 is a marker for small intestinal epithelial progenitor cells, SATB2 is a marker for colonic epithelium, SOX2 is a marker for the anterior segment of the primitive gut tube, CDX2 is a marker for the middle-posterior segment of the primitive gut tube, scale bar = 50 μm; The rightmost column is the relative comparison of induction efficiencies of each region of the primitive gut tube. Among them, each classical regional induction method is used as a control, and its induction efficiency value is set to 1 (i.e., 100%). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Biological replicates N = 3 times; And the number of replicate wells n = 3 is counted; Figure 3 Effect of failure to pass the sensitivity test on the regional derivation efficiency of the primitive gut tube. A is a cell image of PSC-a strain that failed to pass the sensitivity test (PSC-a, after being stimulated with ActA for 24 h, the confluence decreased by no more than 50%) and PSC-b strain that passed the test (the confluence decreased by more than 50% after 24 h); B is the relative induction efficiency of the lung region produced by PSC-a and PSC-b; C is the relative induction efficiency of the small intestinal region produced by PSC-a and PSC-b, scale bar = 50 μm, ***, P < 0.001, Biological replicates N = 3 times, and the number of replicate wells n = 3 is counted; Figure 4 Effect of the underlying matrix at the differentiation stage on the induction efficiency of the primitive gut tube regions; The above bright-field pictures are shown as representative pictures; In the actual test, biological replicates N = 2 times, replicate wells n = 3, scale bar = 100 μm; Figure 5 Effect of serum replacement on the induction efficiency of the primitive gut tube regions; The above bright-field pictures are shown as representative pictures; In the actual test, biological replicates N = 2 times, replicate wells n = 3, scale bar = 100 μm; Figure 6 Effect of the induction stage of the primitive gut tube on the regional induction efficiency, KO 分化阶段2 For omitting the induction stage of the primitive gut tube, KO FGF7 And KO Vc For not adding FGF7 and Vc; The above bright-field pictures are shown as representative pictures; In the actual test, biological replicates N = 2 times, replicate wells n = 3, scale bar = 100 μm. Detailed implementation methods

[0024] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention.

[0026] The reagents used in the examples and comparative examples are shown in Table 1-2.

[0027] Example 1 General induction of the primitive gut tube partition The process of the general induction method of the primitive gut tube partition is as Figure 1 shown.

[0028] S1. Preparation stage: S11. Preparation stage 1: PSC pluripotency identification (differentiation day -5) Take pluripotent stem cells for flow cytometry assay. When SSEA4 + NANOG + ≥90%, it is regarded as passing the test.

[0029] S12. Preparation stage 2: PSC sensitivity test (differentiation day -3) The sensitivity test method is as follows: Take the PSC that has passed the pluripotency assay, digest and inoculate it on the bottom matrix LN521 and LN111 (volume ratio 1:3, final concentration 5 μg / ml), add mTeSR1 medium and culture until the confluence reaches 70-90%. Then replace the stem cell medium with RPMI-1640 containing 100 ng / ml ActA. Observe after 24 hours. If the confluence decreases by more than 50%, it is regarded as passing the test.

[0030] This step is extremely crucial for balling. Otherwise, it is almost impossible to form balls.

[0031] S13. Preparation stage 3: Differentiation inoculation and start confluence test (differentiation day -1-0) Take the PSC that has passed the sensitivity test and inoculate it on the differentiation plate (the culture matrix is the same as that in S12, that is, LN521 and LN111 with a volume ratio of 1:3), and continue to use mTeSR1 medium to culture until the confluence reaches 70%-90%, and then start differentiation.

[0032] The use and ratio of the bottom matrix LN521 and LN111 are extremely crucial for efficient balling.

[0033] The underlying matrix for cell culture plays a crucial role in cell culture. It is laid before inoculation on the differentiation plate until the end of differentiation. During this period, the cultures rely on this matrix for differentiation.

[0034] S2. Differentiation stage: S21. Differentiation stage 1: Co-induction of endoderm and mesoderm (differentiation days 1 - 3) Culture for 1 day in Medium 1, which includes additives and a basal medium. The additives include 50 ng / ml BMP4 and 100 ng / ml ActA, and the basal medium is RPMI - 1640.

[0035] Then culture for 1 day in Medium 2, which includes additives and a basal medium. The additives include 100 ng / ml ActA and 10 ng / ml FGF2, and the basal medium is RPMI - 1640 supplemented with 0.4% (v / v) KSR.

[0036] Then culture for 1 day in Medium 3, which includes additives and a basal medium. The additives include 100 ng / ml ActA and 10 ng / ml FGF2, and the basal medium is RPMI - 1640 supplemented with 4% (v / v) KSR.

[0037] Change the medium daily.

[0038] The serum replacement KSR and its concentration used in this stage are extremely crucial for efficient spheroid formation.

[0039] S22. Differentiation stage 2: Induction of the primitive gut tube (differentiation days 4 - 5) The medium used in this stage includes additives and a basal medium. The additives include 50 ng / ml FGF7 and 0.5 mM Vc (vitamin C), and the basal medium is Ad - DMEM / F12 supplemented with 1.5% (v / v) KSR. Change the medium daily during this period.

[0040] S23. Differentiation stage 3: Partitioning of the primitive gut tube The basal medium used in this stage is: Ad - DMEM / F12 supplemented with 2% (v / v) KSR.

[0041] The partitioning culture of the primitive gut tube adopts the factor combination method in the existing method.

[0042] The inducing factors for each partition are: 1. Anterior part of the anterior primitive gut tube (lung region): Differentiation days 6 - 8: 10 μM SB431542, 1 μM SAG, 200 ng / ml NOG, 500 ng / ml FGF4, 3 μM CHIR99021.

[0043] 2. Posterior part of the anterior primitive gut tube (stomach region): Differentiation day 6 - 7: 200 ng / ml NOG, 500 ng / ml FGF4, 3 μM CHIR99021; Differentiation day 8: 2 μM RA, 200 ng / ml NOG, 500 ng / ml FGF4, 3 μM CHIR99021.

[0044] 3. Posterior part of the anterior primitive gut tube (liver region): Differentiation day 6 - 7: 500 ng / ml FGF4, 3 μM CHIR99021.

[0045] 4. Posterior part of the anterior primitive gut tube (pancreas region): Differentiation day 6 - 9: 500 ng / ml FGF4, 3 μM CHIR99021, 50 ng / ml FGF7, 0.25 μM SANT - 1, 0.25 μM TPPB, 0.1 μM LDN193189.

[0046] 5. Middle - posterior part of the primitive gut tube (small intestine region): Differentiation day 6 - 9: 500 ng / ml FGF4, 500 ng / ml WNT3a.

[0047] 6. Posterior part of the primitive gut tube (colon region): Differentiation day 6 - 11: 500 ng / ml FGF4, 500 ng / ml WNT3a; Differentiation day 12 - 14: 100 ng / ml BMP2.

[0048] Comparative Example 1 Efficiency comparison of this method with various methods The classic regional induction methods selected for comparison are: Lung region: BR Dye et al., 2015 (doi: 10.7554 / eLife.05098.001); Stomach region: TR Broda et al., 2019 (doi: 10.1038 / s41596 - 018 - 0080 - z); Liver region: R Ouchi et al., 2019 (doi: 10.1016 / j.cmet.2019.05.007); Pancreas region: No method for generating 3D spheres of the primitive digestive tract partition has been reported; Small intestine region: KW McCracken et al., 2011 (doi: 10.1038 / nprot.2011.410); Colon region: JO Múnera et al., 2017 (doi: 10.1016 / j.stem.2017.05.020).

[0049] Using this method to induce derivatives of each partition of the primitive digestive tract and comparing the induction efficiency with each classical method in parallel, the results show that ( Figure 2 ): 1) In the comparison of the derivation efficiency in the lung region (NKX2.1 + SOX2 + 3D spheres in the anterior part of the anterior primitive digestive tract), the efficiency is increased by 69.3%; 2) In the comparison of the derivation efficiency in the stomach region (HNF1β + SOX2 + 3D spheres in the anterior part of the anterior primitive digestive tract), the efficiency is increased by 114.1%; 3) In the comparison of the derivation efficiency in the liver region (PROX1 + HHEX + 3D spheres in the posterior part of the anterior primitive digestive tract), the efficiency is increased by 965.0%; 4) In the pancreas region, the derivation efficiency is 174 NKX6.1 + PDX1 + 3D spheres / well of 24-well plate (for the pancreatic partition, there is no reported protocol for generating 3D spheres of this organ partition); 5) In the comparison of the derivation efficiency in the small intestine region (CDX2 + GATA4 + 3D spheres in the middle-posterior part of the primitive digestive tract), the efficiency is increased by 429.5%; 6) In the comparison of the derivation efficiency in the colon region (SATB2 + CDX2 + 3D spheres in the posterior part of the primitive digestive tract), the efficiency is increased by 50.8%.

[0050] In summary, compared with the mainstream methods, this method has an average increase in the derivation efficiency of the primitive digestive tract partitions of about 325.74%.

[0051] Comparative Example 2 Sensitivity screening of PSCs is a prerequisite for the effectiveness of this method Taking the induction of the lung region and small intestine region of the primitive digestive tract as an example, a comparison is made on whether the sensitivity test is passed.

[0052] PSC-a strain that failed the sensitivity test: After being stimulated with ActA for 24 h, the confluence decreased by no more than 50%.

[0053] PSC-b strain that passed the test: After being stimulated with ActA for 24 h, the confluence decreased by more than 50%.

[0054] The results are as Figure 3 shown. If PSC-a that failed the sensitivity test is used (such as Figure 3 A in, after being treated with 100 ng / ml ActA for 24 h, the remaining confluence > 50%), and the subsequent operation is carried out according to this method, the induction efficiency of the 3D spheres of the two cannot be maintained and both show a very significant decrease ( Figure 3 B in and Figure 3 C in).

[0055] Comparative Example 3 The underlying matrix used in the differentiation stage has a significant impact on the induction efficiency of 3D spheres in each partition Taking the induction of the pancreatic region of the primitive digestive tract as an example, in the differentiation stage, the combination of the underlying culture media of LN521 and LN111 is replaced with other common matrices, such as Fibronectin, Matrigel, Collagen I, Vitronectin, Gelatin, etc.; or directly use 100% LN521 and LN111 instead of using them in combination.

[0056] Except for the different underlying matrices, the same technical parameters are used in each group to induce the pancreatic partition of the primitive digestive tract. In the comparison groups of other underlying matrices, the selected concentrations all refer to their common concentrations in the PSC induction differentiation experiment. Specifically, the concentration of Fibronectin is 5 μg / ml; the concentration of Matrigel is 0.02% volume ratio; the concentration of Collagen I is 12.5 μg / ml; the concentration of Vitronectin is 5 μg / ml; the concentration of Gelatin is 0.2% volume ratio; the concentration of LN521 is 10 μg / ml; the concentration of LN111 is 30 μg / ml.

[0057] The results are as Figure 4 shown. It can be seen that after replacing the combination of LN521 and LN111 in the differentiation stage, the production efficiency of 3D spheres will be significantly reduced.

[0058] Comparative Example 4 The serum substitute in the differentiation stage has a significant impact on the induction efficiency of 3D spheres in each partition Taking the induction of the gastric region of the primitive digestive tract as an example, other common serum substitutes such as B27, N2, and ITS were used in differentiation stage 1.

[0059] Except for the different serum substitutes, the same technical parameters were used to induce the primitive digestive tract gastric region in each group. In the comparison groups of other serum substitutes, the selected concentrations were all referenced from the commonly used concentrations in the PSC induction and differentiation experiments; specifically, the concentration of B27 was 2% by volume; the concentration of N2 was 1% by volume; in the B27-N2 combination, the used concentration was 2% by volume B27 + 1% by volume N2; the used concentration of ITS was 1% by volume.

[0060] The results are as Figure 5 shown. If KSR is replaced with other serum substitutes, the differentiation efficiency decreases extremely significantly, indicating the importance of KSR in differentiation.

[0061] Comparative Example 5 Influence of adding primitive digestive tract induction on the generality and efficiency of the method compared with other methods Taking the induction of the gastric region, pancreatic region, and hepatic region of the primitive digestive tract as examples, the primitive digestive tract induction stage was respectively omitted, FGF7 or Vc (vitamin C) was removed, and compared with this method.

[0062] The results are as Figure 6 shown. If this induction stage is omitted, the pancreatic region cannot be formed, and the production efficiency of the 3D spheres in the gastric and hepatic regions decreases significantly; while removing the induction factors in this differentiation stage, such as FGF7 or Vc, the induction efficiency of these three regions all shows a significant decrease. It can be seen that this step is very crucial for generality.

[0063] Table 1 Reagent List

[0064] Table 2. Antibody List

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A general induction method for partitioning the primitive digestive tract, characterized in that: The method includes the following steps: S1. Preparation stage: Perform flow cytometry on pluripotent stem cells, take the PSCs that pass the pluripotency test, digest and inoculate them on the underlying substrates LN521 and LN111, add mTeSR1 medium and culture until the required confluence is reached, then change the stem cell medium to a medium containing ActA, and continue to culture until the confluence decreases by more than 50%, which is regarded as passing the sensitivity test; take the PSCs that pass the sensitivity test, inoculate them on a differentiation plate containing the underlying substrates LN521 and LN111, continue to use mTeSR1 medium to culture until the required confluence is reached, and then initiate differentiation; S2. Differentiation stage: Use a medium with a gradually increasing content gradient of the serum substitute KSR to co-induce the endo-mesoderm of PSCs, and the endo-mesoderm co-induction is carried out in medium 1, medium 2, and medium 3 in sequence. KSR is not added in medium 1, and the content of KSR added in medium 2 is lower than that added in medium 3; then change the medium and perform primitive gut tube induction and primitive gut tube regional culture respectively, so as to form 3D derivatives of the lung region, stomach region, liver region, pancreatic region, small intestine region, or large intestine region.

2. The general induction method for partitioning the primitive digestive tract according to claim 1, wherein: In S1, the standard for pluripotency assay is SSEA4 + NANOG + ≥90%.

3. The general induction method for partitioning the primitive digestive tract according to claim 1, characterized in that: In S1, the volume ratio of the underlying substrates LN521 and LN111 is 1:3, and the final concentration of laminin is 5 - 15 μg / ml.

4. The general induction method for partitioning the primitive digestive tract according to claim 1, characterized in that: In S1, the concentration of ActA is 100 - 300 ng / ml.

5. The general induction method for partitioning the primitive digestive tract according to claim 1, characterized in that: In S2, culture in medium 1, medium 2, and medium 3 for 1 - 2 days in sequence.

6. The general induction method for partitioning the primitive digestive tract according to claim 5, characterized in that: In S2, the volume ratio of KSR added in medium 2 is 0.4 - 0.8%, and the volume ratio of KSR added in medium 3 is 4 - 8%.

7. The general induction method for partitioning the primitive digestive tract according to claim 6, characterized in that: Medium 1 includes additives BMP4 and ActA, and both medium 2 and medium 3 include additives ActA, FGF2, and KSR.

8. The general induction method for partitioning the primitive digestive tract according to claim 1, characterized in that: In S2, the medium for primitive gut tube induction includes additives FGF7 and vitamin C, and Ad-DMEM / F12 added with KSR at a volume ratio of 1.5 - 4.5%; the differentiation time at this stage is 2 - 3 days.

9. The general induction method for partitioning the primitive digestive tract according to claim 8, wherein: In S2, the medium for primitive gut tube regional culture includes Ad-DMEM / F12 added with KSR at a volume ratio of 2 - 8%.

10. The general induction method for partitioning the primitive digestive tract according to claim 1, characterized in that: In S1, when the confluence reaches 70 - 90%, change to the medium containing ActA; when the confluence reaches 70 - 90%, initiate differentiation.

Images