Universal induction method for primitive digestive tract partitioning

Abstract

This invention provides a universal method for inducing primitive digestive tract compartments. The method comprises the following steps: high-quality human pluripotent stem cell assay, pluripotent stem cell sensitivity testing, cell seeding and initial confluence testing, endomesoderm co-induction, primitive digestive tract induction, and primitive digestive tract compartmentalization. Compared to traditional methods, this method, without relying on serum, can efficiently generate primitive digestive tract compartmentalized derivatives (in the form of 3D spheroids) of various organoids, such as the lung, stomach, liver, pancreas, small intestine, and large intestine, facilitating subsequent mass production of the final product. This invention will help promote the large-scale industrial application and application of aerodigestive tract organoids.

Description

Technical Field

[0001] The invention belongs to the field of cell differentiation, and in particular relates to a universal induction method for primitive digestive tract partitioning. Background Art

[0002] The primitive digestive tract is the initial developmental site for most human internal organs, particularly the aerodigestive tract. During early embryonic development, the primitive digestive tract rapidly forms distinct subregions with unique molecular profiles, meticulously regulated by the anterior-posterior and ventral-dorsal axes. These subregions subsequently specialize and give rise to a series of organs, including the thyroid gland, pharynx, lungs, liver, gallbladder, pancreas, and intestine. Over the past decade, with the rapid development of human pluripotent stem cell (PSC) and organoid technology, methods have been established to induce organ-specific subregions from PSCs. The efficiency of these subregional derivatives, which serve as the developmental progenitors of organs, directly determines the yield of downstream organoids.

[0003] Currently, the induction efficiency of primitive gut compartment derivatives in various organoids is generally low and faces stability challenges. For example, the construction of small and large intestinal organoids still primarily relies on the method established by J.R. Spence's team in 2011. Fetal bovine serum (FBS) is used as a nutritional support during the S1 endoderm induction stage and the S2 primitive gut intestinal compartment induction stage (mid and posterior segments) (DOI: 10.1038 / nprot.2011.410; 10.1016 / bs.mcb.2020.03.007). Due to the unknown composition and xenogeneic origin of FBS, induction efficiency is low (DOI: 10.1016 / j.stemcr.2017.11.004). Similarly, organoids of the esophagus, stomach, and lung also face similar challenges (DOI: 10.1016 / j.stem.2018.08.008; 10.1038 / s41596-018-0080-z; 10.7554 / eLife.05098). The construction of liver organoids is primarily based on the method proposed by T. Takebe et al. in 2019. Although they successfully replaced FBS with serum substitutes N2 and B27 during the induction of the S2 primitive digestive tract liver compartment (the anterior and posterior parts), the use of serum during the S1 endoderm stage is still necessary (DOI: 10.1016 / j.cmet.2019.05.007). Furthermore, due to the significant differences in induction methods for different compartments, a universally compatible technical foundation has yet to be established, making the path to industrialization fraught with difficulties. Summary of the Invention

[0004] In light of this, the present invention aims to develop a universal method for inducing primitive digestive tract compartments. Compared to traditional methods, this method, without relying on serum, supports the efficient induction (in the form of 3D spheroids) of primitive digestive tract compartments from diverse organoids, such as the lung, stomach, liver, pancreas, small intestine, and large intestine, facilitating the subsequent mass production of final organoids. This invention will help promote the large-scale industrial application and transformation of aerodigestive tract organoids.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A universal method for inducing primitive digestive tract partitioning, comprising the following steps:

[0007] S1. Preparation stage:

[0008] Pluripotent stem cells were tested by flow cytometry. PSCs that passed the pluripotency test were digested and plated on LN521 and LN111 substrates. After culture in mTeSR1 medium until the desired confluence was reached, the stem cell culture medium was replaced with a medium containing ActA and cultured until the confluence decreased by more than 50% (a decrease of more than 50% of the original confluence). These cells were considered to have passed the sensitivity test. PSCs that passed the sensitivity test were plated on differentiation plates containing LN521 and LN111 substrates and cultured in mTeSR1 medium until the desired confluence was reached, at which point differentiation was initiated.

[0009] S2, differentiation stage:

[0010] PSCs are co-induced into endomesoderm using a culture medium containing the serum substitute KSR, and then the culture medium is changed for primitive digestive tract induction and primitive digestive tract partitioning culture, thereby forming 3D derivatives of the lung region, stomach region, liver region, pancreas region, small intestine region, or large intestine region.

[0011] Furthermore, in S1, the pluripotency test standard is SSEA4 + NANOG + ≥90%.

[0012] Furthermore, in S1, the volume ratio of the bottom matrix LN521 and LN111 was 1:3, and the final concentration of laminin was 5-15 μg / ml.

[0013] Preferably, the final concentration of laminin is 5 μg / ml.

[0014] Furthermore, in S1, the concentration of ActA is 100-300 ng / ml, non-limiting examples include 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, and 300 ng / ml, preferably 100 ng / ml.

[0015] Furthermore, in S2, the cells were cultured in culture medium 1, culture medium 2, and culture medium 3, respectively, for 1-2 days.

[0016] Furthermore, the volume ratio of KSR added to culture medium 2 is 0.4-0.8%, and the volume ratio of KSR added to culture medium 3 is 4-8%.

[0017] The volume ratio of KSR added to culture medium 2 can be, for example, non-limitingly 0.4%, 0.5%, 0.6%, 0.7%, or 0.8%;

[0018] The volume ratio of KSR added to culture medium 3 can be, for example, 4%, 5%, 6%, 7%, or 8% by volume, but is not limited thereto.

[0019] Preferably, the volume ratio of KSR added to culture medium 2 is 0.4%, and the volume ratio of KSR added to culture medium 3 is 4%.

[0020] Furthermore, culture medium 1 includes the additives BMP4 and ActA, and culture medium 2 and culture medium 3 both include the additives ActA, FGF2 and KSR.

[0021] Specifically, culture medium 1 includes 10-50 ng / ml BMP4 and 100-200 ng / ml ActA; non-limiting examples of the concentration of BMP4 include 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, preferably 50 ng / ml; non-limiting examples of the concentration of ActA include 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml, preferably 100 ng / ml;

[0022] Culture medium 2 and culture medium 3 include 100-200 ng / ml ActA, 10-30 ng / ml FGF2; non-limiting examples of the concentration of ActA can be 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml, preferably 100 ng / ml; non-limiting examples of the concentration of FGF2 can be 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, preferably 10 ng / ml.

[0023] Furthermore, in S2, the culture medium for primitive digestive tract induction includes additives FGF7 and vitamin C, as well as Ad-DMEM / F12 supplemented with 1.5-4.5% volume ratio of KSR; the differentiation time at this stage is 2-3 days.

[0024] Specifically, the concentration of FGF7 is 20-50 ng / ml, and the concentration of vitamin C is 0.5-2 mM.

[0025] Non-limiting examples of the concentration of FGF7 include 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 include 0.5 mM, 1 mM, 1.5 mM, 2 mM, preferably 0.5 mM.

[0026] Non-limiting examples of the volume ratio of KSR may be 1.5%, 2%, 2.5%, 3%, 3.5%, and 4%; preferably, the volume ratio of KSR is 1.5%.

[0027] Furthermore, in S2, the culture medium for the original digestive tract partition culture includes Ad-DMEM / F12 supplemented with 2-8% volume ratio of KSR.

[0028] Non-limiting examples of the volume ratio of KSR may be 2%, 3%, 4%, 5%, 6%, 7%, and 8%; preferably, the volume ratio of KSR may be 2%.

[0029] 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.

[0030] Compared with the existing technology, the universal induction method of original digestive tract partitioning described in the present invention has the following advantages:

[0031] The universal induction method for original digestive tract partitioning described in the present invention contains no animal-derived ingredients and serum throughout the process, which improves stability, reduces contamination risks, and has clinical translation potential; it has strong versatility and supports the production of at least six derivatives of different original digestive tract partitions (lung, stomach, liver, pancreas, small intestine, large intestine, etc.); it has high induction efficiency, and compared with the original method, the efficiency of derivative production is increased by 20% to 420%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 This is a schematic diagram of the universal induction method for the original digestive tract partitioning;

[0034] Figure 2The figure shows the comparison of the induction efficiency of the derivatives of each partition of the original digestive tract; the two columns on the left are respectively: representative bright field images of the 3D-derived spheroids of each organ partition by each classic induction method and this method, among which a single well of a 24-well plate was used, and the sampling point was the induction time point required by each method; the middle column is the immunofluorescence assay results of the derivatives of each partition produced by this method, among which NKX2.1 is a marker of lung epithelial progenitor cells, HNF1β is a marker of gastric epithelial progenitor cells, PROX1 and HHEX are markers of liver primordium (liver progenitor cells), NKX6.1 and PDX1 are markers of pancreatic epithelial progenitor cells, and NKX2.1 is a marker of lung epithelial progenitor cells, HNF1β is a marker of gastric epithelial progenitor cells, PROX1 and HHEX are markers of liver primordium (liver progenitor cells), and NKX6.1 and PDX1 are markers of pancreatic epithelial progenitor cells. Markers of epithelial progenitor cells, GATA4 for small intestinal epithelial progenitor cells, SATB2 for colonic epithelial cells, SOX2 for the anterior segment of the primitive digestive tract, and CDX2 for the middle-posterior segment of the primitive digestive tract. Scale bar = 50 μm. The rightmost column shows the relative comparison of the induction efficiency of each compartment of the primitive digestive tract. Each classic compartment induction method was used as a control, and its induction efficiency was set to 1 (i.e., 100%). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Biological replicates N = 3; and replicate wells n = 3.

[0035] Figure 3 Figure 3: Effect of failure in sensitivity testing on the derivation efficiency of original digestive tract compartments. A shows cells from the PSC-a strain that failed the sensitivity test (PSC-a, confluence did not decrease by more than 50% after 24 hours of ActA stimulation) and the PSC-b strain that passed the test (confluence decreased by more than 50% after 24 hours). B shows the relative induction efficiency of lung compartments generated by PSC-a and PSC-b, and C shows the relative induction efficiency of small intestine compartments generated by PSC-a and PSC-b. Scale bar = 50 μm. ***, P < 0.001. Biological replicates N = 3, with n = 3 replicates.

[0036] Figure 4 The effect of the underlying matrix on the efficiency of primitive digestive tract partitioning during the differentiation phase. The bright field images above are representative images. In actual testing, biological replicates (N) = 2, replicates (n = 3), and scale bars = 100 μm.

[0037] Figure 5 The effect of serum replacement on the efficiency of induction of primary digestive tract partitioning is shown above. The bright field images above are representative images. In actual testing, biological replicates N = 2, replicate wells n = 3, scale bar = 100 μm.

[0038] Figure 6 The effect of the original digestive tract induction stage on the efficiency of partition induction, KO 分化阶段2 To discard the original digestive tract induction stage, KO FGF7 and KO VcWithout the addition of FGF7 and Vc; the above bright field images are shown as representative images; in actual testing, biological replicates N = 2 times, replicates n = 3, scale bar = 100 μm. DETAILED DESCRIPTION

[0039] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] The reagents used in the examples and comparative examples are shown in Table 1-2.

[0042] Example 1 Universal Induction of Primitive Digestive Tube Partitions

[0043] The process of the universal induction method for the original digestive tract partitioning is as follows Figure 1 shown.

[0044] S1. Preparation stage:

[0045] S11. Preparation stage 1: PSC pluripotency determination (differentiation day -5)

[0046] Pluripotent stem cells were taken for flow cytometry assay. + NANOG + When the value is ≥90%, the test is considered passed.

[0047] S12. Preparation Stage 2: PSC Sensitivity Test (Differentiation Day -3)

[0048] The sensitivity test method is as follows:

[0049] PSCs that pass the pluripotency test are digested and plated onto LN521 and LN111 substrates (volume ratio 1:3, final concentration 5 μg / ml). Culture in mTeSR1 medium until confluence reaches 70-90%. Then, the stem cell medium is replaced with RPMI-1640 containing 100 ng / ml ActA. After 24 hours of observation, cells are considered to have passed the test if confluence decreases by more than 50%.

[0050] This step is extremely critical for pilling, otherwise it will be almost impossible to pill.

[0051] S13. Preparation Stage 3: Differentiation Seeding and Initiation of Confluence Testing (Differentiation Day 1-0)

[0052] PSCs that have passed the sensitivity test were seeded onto differentiation plates (the culture medium is the same as for S12, i.e., LN521 and LN111 at a volume ratio of 1:3). Culture in mTeSR1 medium until the confluence reaches 70%-90% and then initiate differentiation.

[0053] The use and proportion of the base substrates LN521 and LN111 are critical for efficient pilling.

[0054] The cell culture substrate plays a crucial role in cell culture. It is laid before inoculation on the differentiation plate and remains in place until the differentiation is complete. During this period, the culture relies on this substrate for differentiation.

[0055] S2, differentiation stage:

[0056] S21, Differentiation Stage 1: Endomesoderm Co-induction (Differentiation Days 1-3)

[0057] The cells were cultured for 1 day in Medium 1, which included supplements and a basal medium. The supplements included 50 ng / ml BMP4 and 100 ng / ml ActA, and the basal medium was RPMI-1640.

[0058] The cells were then cultured for 1 day in medium 2, which included additives and a basal medium. The additives included 100 ng / ml ActA and 10 ng / ml FGF2, and the basal medium was RPMI-1640 supplemented with 0.4% by volume KSR.

[0059] The cells were then cultured for 1 day in medium 3, which included additives and a basal medium. The additives included 100 ng / ml ActA and 10 ng / ml FGF2, and the basal medium was RPMI-1640 supplemented with 4% by volume KSR.

[0060] The culture medium was changed daily.

[0061] The serum substitute KSR and its concentration used in this stage are extremely critical for efficient pilling.

[0062] S22, Differentiation Stage 2: Primitive Digestive Tube Induction (Differentiation Days 4-5)

[0063] The culture medium used during this phase included supplements (50 ng / ml FGF7, 0.5 mM Vitamin C) and a basal medium (Ad-DMEM / F12 supplemented with 1.5% KSR by volume). The culture medium was changed daily.

[0064] S23, Differentiation stage 3: Primitive digestive tract division

[0065] The basal culture medium used at this stage was Ad-DMEM / F12 supplemented with 2% by volume of KSR.

[0066] The original digestive tract partition culture adopts the factor combination method in the existing method.

[0067] The induction factors for each partition are:

[0068] 1. Anterior part of the primitive digestive tract (lung area):

[0069] Differentiation Days 6-8:

[0070] 10μM SB431542, 1μM SAG, 200ng / ml NOG, 500ng / ml FGF4, 3μM CHIR99021.

[0071] 2. The posterior part of the anterior segment of the primitive digestive tract (stomach area):

[0072] Differentiation Days 6-7:

[0073] 200ng / ml NOG, 500ng / ml FGF4, 3μM CHIR99021;

[0074] Differentiation Day 8:

[0075] 2μM RA, 200ng / ml NOG, 500ng / ml FGF4, 3μM CHIR99021.

[0076] 3. The posterior part of the anterior segment of the primitive digestive tract (liver area):

[0077] Differentiation Days 6-7:

[0078] 500ng / ml FGF4, 3μM CHIR99021.

[0079] 4. The posterior part of the anterior segment of the primitive digestive tract (pancreatic area):

[0080] Differentiation Days 6-9:

[0081] 500ng / ml FGF4, 3μM CHIR99021, 50ng / ml FGF7, 0.25μM SANT-1, 0.25μM TPPB, 0.1μM LDN193189.

[0082] 5. Middle and posterior part of the primitive digestive tract (small intestine):

[0083] Differentiation Days 6-9:

[0084] 500ng / ml FGF4, 500ng / ml WNT3a.

[0085] 6. Posterior part of the primitive digestive tract (colon area):

[0086] Differentiation Days 6-11:

[0087] 500ng / ml FGF4, 500ng / ml WNT3a;

[0088] Differentiation Days 12-14:

[0089] 100ng / ml BMP2.

[0090] Comparative Example 1 Comparison of the efficiency of this method with other methods

[0091] The classical partition-inducing methods selected for comparison are:

[0092] Lung: BR Dye et al., 2015 (doi:10.7554 / eLife.05098.001);

[0093] Gastric region: TR Broda et al., 2019 (doi:10.1038 / s41596-018-0080-z);

[0094] Liver region: R Ouchi et al., 2019 (doi:10.1016 / j.cmet.2019.05.007);

[0095] Pancreas: No method has been reported to generate 3D spheroids of the original digestive tract partitions;

[0096] Small intestine: KW McCracken et al., 2011 (doi: 10.1038 / nprot.2011.410);

[0097] Colon: JO Múnera et al., 2017 (doi: 10.1016 / j.stem.2017.05.020).

[0098] This method was used to induce derivatives of each partition of the original digestive tract, and the induction efficiency was compared with that of various classical methods. The results showed that ( Figure 2 ):

[0099] 1) In the comparison of the derivation efficiency in the lung region (the anterior part of the primitive digestive tract, NKX2.1 + SOX2 + 3D sphere), efficiency increased by 69.3%;

[0100] 2) In the comparison of the derivation efficiency in the gastric region (the anterior part of the primitive digestive tract), HNF1β + SOX2 +3D sphere), efficiency increased by 114.1%;

[0101] 3) Comparison of the derivation efficiency of the liver region (the anterior and posterior parts of the primitive digestive tract, PROX1 +

[0102] HHEX + 3D sphere), efficiency increased by 965.0%;

[0103] 4) In the pancreas, the derivation efficiency was 174 NKX6.1 + PDX1 + 3D spheroids / single well of a 24-well plate (for pancreatic partitioning, there are currently no reported protocols for deriving 3D spheroids of this organ's partitioning);

[0104] 5) In the comparison of derivation efficiency in the small intestine (middle-posterior segment of the primitive digestive tract) + GATA4 + 3D sphere), efficiency increased by 429.5%;

[0105] 6) Comparison of derivation efficiency in the colon region (posterior segment of the original digestive tract) + CDX2 + 3D sphere), efficiency increased by 50.8%.

[0106] In summary, compared with mainstream methods, this method improves the derivation efficiency of original digestive tract partitions by an average of about 325.74%.

[0107] Comparative Example 2 PSC passing sensitivity screening is a prerequisite for the effectiveness of this method

[0108] Taking the induction of the original digestive tract lung area and small intestine area as an example, a comparison is made to see whether the sensitivity test is passed.

[0109] PSC-a strains that failed the sensitivity test: After 24 hours of ActA stimulation, the confluence did not decrease by more than 50%.

[0110] The PSC-b strain that passed the test: After 24 hours of ActA stimulation, the confluence decreased by more than 50%.

[0111] The results are as follows Figure 3 As shown, if PSC-a that fails the sensitivity test is used (such as Figure 3 In Figure A, after 24 hours of treatment with 100 ng / ml ActA, the remaining confluence was >50%). When the method was subsequently used, the induction efficiency of the 3D spheroids could not be maintained and both showed a very significant decrease ( Figure 3 Middle B and Figure 3 Middle C).

[0112] Comparative Example 3 The bottom matrix used in the differentiation stage has a significant impact on the induction efficiency of 3D spheroids in each partition

[0113] Taking the induction of the primitive digestive tract pancreatic region as an example, during the differentiation stage, the underlying culture matrix combination of LN521 and LN111 is replaced with other common matrices, such as Fibronectin, Matrigel, Collegen I, Vitronectin, Gelatin, etc.; or 100% LN521 and LN111 are directly used instead of using them in combination.

[0114] Aside from the differences in the underlying matrix, all groups used the same technical parameters for induction of pancreatic differentiation into the primitive digestive tract. For the control groups using other underlying matrices, the concentrations used were based on those commonly used in PSC differentiation experiments. Specifically, Fibronectin was 5 μg / ml; Matrigel was 0.02% v / v; Collegen I was 12.5 μg / ml; Vitronectin was 5 μg / ml; Gelatin was 0.2% v / v; LN521 was 10 μg / ml; and LN111 was 30 μg / ml.

[0115] The results are as follows Figure 4 As shown, it can be seen that replacing the combination of LN521 and LN111 during the differentiation stage will significantly reduce the efficiency of 3D sphere generation.

[0116] Comparative Example 4: Serum replacement in differentiation stage 1 significantly affects the induction efficiency of 3D spheroids in each partition

[0117] Taking the induction of the gastric region of the primitive digestive tract as an example, other common serum replacements such as B27, N2, and ITS are used in differentiation stage 1.

[0118] Aside from the serum replacements used, all groups used the same technical parameters for induction of the primary digestive organ, the stomach. For the other serum replacement control groups, the concentrations used were based on those commonly used in PSC differentiation induction experiments: specifically, B27 at 2% volume; N2 at 1% volume; the B27-N2 combination used a 2% volume B27 + 1% volume N2 concentration; and ITS at 1% volume.

[0119] The results are as follows Figure 5 As shown in the figure, if KSR is replaced with other serum substitutes, the differentiation efficiency decreases significantly, which shows the importance of KSR in differentiation.

[0120] Comparative Example 5: Effect of adding original digestive tract induction on the versatility and efficiency of the method compared with other methods

[0121] Taking the induction of the gastric, pancreatic and liver regions of the primitive digestive tract as an example, the primitive digestive tract induction stage, FGF7 or Vc (vitamin C) were discarded respectively, and compared with this method.

[0122] The results are as follows Figure 6 As shown, if this induction stage is omitted, the pancreatic compartment fails to form, and the efficiency of generating 3D spheroids in the stomach and liver compartments decreases significantly. Furthermore, if induction factors such as FGF7 or Vc are removed during this differentiation stage, the induction efficiency of all three compartments decreases significantly. This step is crucial for versatility.

[0123] Table 1 Reagent list

[0124]

[0125] Table 2. Antibody List

[0126]

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A universal method for inducing primitive digestive tract partitioning, characterized by: The method comprises the following steps: S1. Preparation stage: S11. Preparation stage 1: PSC pluripotency determination on differentiation day 5 Pluripotent stem cells were taken for flow cytometry assay. + NANOG + When ≥90%, it is considered to have passed the test; S12. Preparation stage 2: PSC sensitivity test on differentiation day 3 The sensitivity test method is as follows: PSCs that have passed the pluripotency test were digested and seeded onto LN521 and LN111 substrates at a 1:3 volume ratio, at a final concentration of 5 µg / ml. mTeSR1 medium was added and cultured until confluence reached 70-90%. The stem cell culture medium was then replaced with RPMI-1640 supplemented with 100 ng / ml ActA. After 24 hours, cells were observed and considered to have passed the test if confluence decreased by more than 50%. S13. Preparation stage 3: On differentiation day 1-0, differentiation seeding and initiation of confluence test PSCs that have passed the sensitivity test are seeded onto differentiation plates. The culture medium is the same as for S12, i.e., LN521 and LN111 at a volume ratio of 1:

3. Continue culturing in mTeSR1 medium until the confluence reaches 70%-90%, and then initiate differentiation. S2, differentiation stage: S21, Differentiation stage 1: Endomesoderm co-induction on differentiation days 1-3 Culture for 1 day in Medium 1, which includes supplements and basal medium. The supplements include 50 ng / ml BMP4 and 100 ng / ml ActA, and the basal medium is RPMI-1640. The cells were then cultured for 1 day in medium 2, which included supplements and a basal medium. The supplements included 100 ng / ml ActA and 10 ng / ml FGF2, and the basal medium was RPMI-1640 supplemented with 0.4% by volume KSR. Then, the cells were cultured for 1 day in medium 3, which included supplements and a basal medium. The supplements included 100 ng / ml ActA and 10 ng / ml FGF2, and the basal medium was RPMI-1640 supplemented with 4% by volume KSR. The culture medium was changed daily; S22, differentiation stage 2: on differentiation day 4-5, primitive digestive tract induction The culture medium used in this stage includes additives and basal medium. The additives include 50 ng / ml FGF7 and 0.5mM Vc. The basal medium is Ad-DMEM / F12 supplemented with 1.5% volume ratio KSR. The culture medium is changed daily during this period. S23, Differentiation stage 3: Primitive digestive tract division The basal medium used in this stage was: Ad-DMEM / F12 supplemented with 2% volume ratio of KSR; The induction factors for each partition are:

1. Anterior part of the primitive digestive tract, lung area: Differentiation Days 6-8: 10µM SB431542, 1µM SAG, 200ng / ml NOG, 500ng / ml FGF4, 3µM CHIR99021; 2. The posterior part of the primitive digestive tract, the stomach area: Differentiation Days 6-7: 200ng / ml NOG, 500ng / ml FGF4, 3µM CHIR99021; Differentiation Day 8: 2µM RA, 200ng / ml NOG, 500ng / ml FGF4, 3µM CHIR99021; 3. The posterior part of the anterior segment of the primitive digestive tract, the liver area: Differentiation Days 6-7: 500ng / ml FGF4, 3µM CHIR99021; 4. The posterior part of the primitive digestive tract, pancreatic area: Differentiation Days 6-9: 500ng / ml FGF4, 3µM CHIR99021, 50ng / ml FGF7, 0.25µM SANT-1, 0.25µM TPPB, 0.1µMLDN193189; 5. Middle and posterior segments of the primitive digestive tract, small intestine: Differentiation Days 6-9: 500ng / ml FGF4, 500ng / ml WNT3a; 6. The posterior part of the primitive digestive tract, the colon area: Differentiation Days 6-11: 500ng / ml FGF4, 500ng / ml WNT3a; Differentiation Days 12-14: 100ng / ml BMP2.

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