A method for obtaining intestinal mesenchymal stem cells from pluripotent stem cells through vagal neural crest cell induction

Abstract

The application relates to a method for obtaining intestinal mesenchymal stem cells from pluripotent stem cells through vagal neural crest cell induction; through limited differentiation paths, specific vagal neural crest cells are obtained, high purity and spatial identity of the starting cells are ensured, and vagal neural crest-derived mesenchymal stem cells with clear developmental origin and single lineage are obtained. Further, intestinal mesenchymal stem cells derived from vagal neural crest cells are obtained. The differentiation path is clear, the developmental origin is clear, and the existing mesenchymal stem cells with different developmental origins are supplemented; not only have the basic biological characteristics of mesenchymal stem cells, but also can be massively expanded; have strong multi-directional differentiation and immunomodulatory capacity, can effectively repair intestinal damage, and become an important cell source for the treatment of intestinal damage-related diseases. This tissue homologous or functionally matched co-culture mode provides an ideal stromal cell source for constructing a high-simulation and high-quality intestinal organoid model.

Description

Technical Field

[0001] This application relates to the field of stem cell induction technology, and in particular to a method for inducing intestinal mesenchymal stem cells from pluripotent stem cells via vagus nerve crest cell induction. Background Technology

[0002] Intestinal mesenchymal cells (IMCs) are widely distributed in the lamina propria and submucosa of the intestine, playing a crucial role in maintaining intestinal tissue structure, regulating the microenvironment of epithelial stem cells, and participating in tissue repair. Recent studies have found that IMCs can participate in the proliferation, differentiation, and tissue remodeling of intestinal epithelial cells by secreting various growth factors, cytokines, and extracellular matrix molecules. For example, IMCs expressing Foxl1 are considered an important component of the microenvironment of intestinal epithelial stem cells, regulating their proliferation and tissue homeostasis by secreting signaling molecules such as Wnt and R-spondin (Cellular and Molecular Gastroenterology and Hepatology, 2016, 2(2):175-188). Furthermore, recent studies have shown that IMCs play an important role in maintaining intestinal epithelial regeneration capacity, regulating inflammatory responses, and participating in intestinal damage repair (Nature Reviews Gastroenterology & Hepatology, 2019, 16:133-147). However, most existing intestinal mesenchymal cells rely on primary tissue isolation for acquisition, which not only limits cell sources but also makes it difficult to control cell purity and batch stability, thus failing to meet the needs of large-scale preparation and standardized application.

[0003] Currently, mesenchymal stem cells (MSCs) used for research and treatment are mainly derived from tissues such as bone marrow, adipose tissue, umbilical cord, and placenta. However, these traditionally derived MSCs typically belong to a systemic population of mesenchymal cells, exhibiting weak tissue specificity and limited adaptability to the intestinal microenvironment. Therefore, developing a stable, scalable, and intestinal tissue-specific source of MSCs is of great significance for advancing research in intestinal regenerative medicine and cell therapy.

[0004] Pluripotent stem cells (PSCs) possess unlimited self-renewal capacity and multi-lineage differentiation potential, and can differentiate into various somatic cell types under appropriate induction conditions. During embryonic development, neural crest cells are a transient cell population with high migration capacity and multi-lineage differentiation potential. Vagal neural crest cells (vNCCs), as an important subset of neural crest cells, migrate along the anterior-posterior axis of the intestine into the intestinal wall during embryonic development and ultimately form the enteric nervous system. Studies have shown that vNCCs differentiate into enteric neurons and glial cells during the formation of the enteric nervous system and are the main cellular source for the development of the enteric nervous system (Neurogastroenterology and Motility, 2004, 16(Suppl 1):1-15). In addition, neural crest cells are considered to have strong developmental plasticity and can differentiate into various cell types under certain conditions, including neurons, glial cells, and some cell populations with mesenchymal-like characteristics. This suggests that neural crest cells may have the potential to generate mesenchymal-like cells under specific induction conditions.

[0005] The above information suggests that in vitro differentiation of human pluripotent stem cells (hiPSCs) may yield intestinal mesenchymal stem cells derived from the vagus nerve crest. However, there are currently no reports of successfully inducing intestinal mesenchymal stem cells using hiPSCs through the neural crest stage. Furthermore, the existence of mesenchymal cells derived from the neural crest in the intestines of mammals (including mice) has long lacked clear in vivo evidence.

[0006] The prior art (PMID: 36109564) reported a method for inducing vagal nerve crest using pluripotent stem cells. However, the developmental lineage of the cranial nerve crest derived from MSCs is strictly limited to the head and face tissues, and they lack the ability to differentiate into the intestinal mesenteric tissue. Therefore, they cannot be used to treat digestive tract-related diseases such as enteritis, intestinal fibrosis, and gastrointestinal mucosal damage.

[0007] Therefore, it is necessary to develop a new technical method that simulates the formation and differentiation of neural crest cells during embryonic development, starting from pluripotent stem cells, and then obtaining a cell population with intestinal mesenchymal cell characteristics through vagal crest lineage-directed induction. This would establish a stable, high-purity, and scalable intestinal mesenchymal stem cell preparation technology system, providing a new cell source for intestinal damage repair and cell therapy for related diseases. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for obtaining intestinal mesenchymal stem cells from pluripotent stem cells through vagus nerve crest cell induction.

[0009] A first object of the present invention is to provide a culture medium composition.

[0010] A second object of the present invention is to provide the use of the culture medium composition in inducing pluripotent stem cells to differentiate into vagal crest cells or inducing pluripotent stem cells to differentiate into mesenchymal stem cells.

[0011] A third objective of this invention is to provide a method for inducing pluripotent stem cells to differentiate into vagal crest cells.

[0012] A fourth object of the present invention is to provide a method for culturing intestinal mesenchymal stem cells.

[0013] A fifth objective of this invention is to provide the application of intestinal mesenchymal stem cells prepared by the method.

[0014] This invention establishes a standardized induction differentiation process, in which pluripotent stem cells (PSCs) are specifically induced into vagal crest cells (hiPSC-vNCCs) through three different stages of specific induction culture medium. After purification, the vNCCs are continuously cultured in mesenchymal stem cell culture medium or commercially available MSC culture medium (StemFit), and their phenotype is detected to obtain vagal neural crest-derived intestinal mesenchymal stromal cells (IMSCs).

[0015] The “vNCCs” used in this invention are vagal crest cells.

[0016] The “hiPSC-VNCs” used in this invention refer to vagal crest cells specifically induced by human induced pluripotent stem cells.

[0017] The “IMSCs” used in this invention refer to intestinal mesenchymal stem cells derived from the vagus crest and specifically induced by pluripotent stem cells.

[0018] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims a culture medium composition comprising Stage I culture medium, Stage II culture medium, and Stage III culture medium; Stage I culture medium is a basic culture medium containing N-2 additive (100×), B-27 neural culture additive, NEAA cell culture additive, glutamine, β-mercaptoethanol, FGF signaling pathway agonist and ROCK signaling pathway inhibitor. Stage II culture medium is a basic culture medium containing N-2 additive (100×), Wnt pathway agonist, TGF-β pathway inhibitor, and Hedgehog pathway agonist; Stage III culture medium is Stage II culture medium containing retinoic acid.

[0019] Preferably, the FGF signaling pathway agonist is recombinant human basic fibroblast growth factor bFGF; the ROCK signaling pathway inhibitor is one or more of Y27632 or GSK429286A; the TGF-β pathway inhibitor is one or more of SB431542, A83-01, RepSox, SB505124 or LY364947; and the Hedgehog pathway agonist is one or more of SAG, Purmorphamine, Sonic Hedgehog protein or Hh-Ag1.5.

[0020] More preferably, the ROCK signaling pathway inhibitor is Y27632; the TGF-β pathway inhibitor is SB431542; and the Hedgehog pathway agonist is SAG.

[0021] More preferably, it contains Stage I culture medium, Stage II culture medium, and Stage III culture medium; Stage I culture medium is a basic culture medium containing N-2 additive (100×), B-27 neural culture additive, NEAA cell culture additive, glutamine, β-mercaptoethanol, bFGF and Y27632; Stage II culture medium is a basic culture medium containing N-2 additive (100×), CHIR99021, SB431542, and SAG; Stage III culture medium is Stage II culture medium containing retinoic acid.

[0022] More preferably, it contains Stage I culture medium, Stage II culture medium, and Stage III culture medium; The Stage I culture medium is a basal culture medium containing 1–5% (V / V) N-2 additive (100×), 1–5% (V / V) B-27 neural culture additive, 1–5% (V / V) NEAA cell culture additive, 1–3 mM glutamine, 50–150 μM β-mercaptoethanol, 1–100 ng / ml bFGF and 5–10 μM Y27632. Stage II culture medium is a basic culture medium containing 1-5% (V / V) N-2 additive (100×), 1-10 μM CHIR99021, 1-10 μM SB431542, and 1-10 μM SAG; Stage III culture medium is Stage II culture medium containing 1–10 μM retinoic acid.

[0023] More preferably, the Stage I culture medium is a basal culture medium containing 1% (V / V) N-2 additive (100×), 2% (V / V) B-27 neural culture additive, 1% (V / V) NEAA cell culture additive, 2 mM glutamine, 110 μM β-mercaptoethanol, 10 ng / ml bFGF and 10 μM Y27632.

[0024] More preferably, the Stage II culture medium is a basal culture medium containing 1% (V / V) N-2 additive (100×), 2μM CHIR99021, 2μM SB431542, and 2μM SAG.

[0025] More preferably, the Stage III culture medium is a Stage II culture medium containing 1 μM retinoic acid.

[0026] More preferably, the basic culture medium is DMEM-F12 culture medium.

[0027] Preferably, the culture medium composition further contains mesenchymal stem cell culture medium.

[0028] Preferably, the mesenchymal stem cell culture medium is: serum-free complete culture medium for mesenchymal stem cells or commercially available MSC culture medium, wherein the serum-free complete culture medium for mesenchymal stem cells is a basal culture medium containing fetal bovine serum and FGF signaling pathway agonists.

[0029] More preferably, the basal culture medium is DMEM (L) culture medium or α-DMEM culture medium.

[0030] More preferably, the basal culture medium is DMEM (L) culture medium.

[0031] More preferably, the amount of fetal bovine serum used is 1–10% (V / V).

[0032] More preferably, the amount of fetal bovine serum used is 5% (V / V).

[0033] More preferably, the bFGF signaling pathway agonist is recombinant human basic fibroblast growth factor bFGF.

[0034] More preferably, the amount of bFGF used is 1 to 100 ng / mL.

[0035] More preferably, the amount of bFGF used is 10 ng / mL.

[0036] More preferably, the commercially available MSC culture medium is: STEMCELL Technologies' commercially available MSC culture medium (ACF) or Ajinomoto's commercially available MSC culture medium (STEMFIT).

[0037] The present invention also claims the use of the culture medium composition described in the claims in inducing pluripotent stem cells to differentiate into vagal crest cells or inducing pluripotent stem cells to differentiate into mesenchymal stem cells.

[0038] This invention also claims a method for inducing pluripotent stem cells to differentiate into vagal nerve crest cells, comprising the following steps: S1. Pluripotent stem cells, which are resuspended in Stage I culture medium of the culture medium composition and digested into single cells or cell clusters, are cultured in an adherent environment. S2. After adhering culture for 1 to 4 days, replace with Stage II culture medium from the culture medium composition and culture for 1 to 10 days; S3. Replace with Stage III culture medium from the culture medium composition and culture for 2-10 days to obtain vagal crest cells.

[0039] Preferably, the method for preparing pluripotent stem cells by digesting them into single cells or cell clusters using the digestive solution is as follows: cultured pluripotent stem cells are digested with a cell digestive solution. More preferably, the cell digestive solution is Accutase cell digestive solution.

[0040] Preferably, the adherent culture is carried out in a culture vessel inoculated with Matrigel or fibronectin.

[0041] Preferably, the inoculation density for the adherent culture is 1×10⁻⁶. 4 ~1×10 5 / cm 2 .

[0042] Most preferably, the inoculation density of the adherent culture is 1×10⁻⁶. 5 / cm2 .

[0043] More preferably, it includes the following steps: S1. Pluripotent stem cells, which are resuspended in Stage I culture medium of the culture medium composition and digested into single cells or cell clusters, are cultured in an adherent environment. S2. After adhering culture for 1-2 days, replace with Stage II culture medium from the culture medium composition and culture for 2-4 days; S3. Replace with Stage III culture medium from the culture medium composition and culture for 3-6 days to obtain vagal crest cells.

[0044] More preferably, S1. Pluripotent stem cells are digested into single cells or cell clusters by resuspending the digestive solution in Stage I culture medium of the culture medium composition, and then cultured in an adherent culture medium. S2. After adhering culture for 24 hours, replace with Stage II culture medium from the culture medium composition and culture for 4 days; S3. Replace with Stage III culture medium from the culture medium composition and culture for 6 days to obtain vagal crest cells.

[0045] More preferably, the step further includes sorting the product of step S3 using a flow cytometer to sort CD49d and CD217 double-positive cells, thereby obtaining purified vagal crest cells.

[0046] The present invention also claims a method for culturing intestinal mesenchymal stem cells, wherein vagal crest cells are obtained by adhering to and passaged in a mesenchymal stem cell culture medium.

[0047] Preferably, the vagal crest cells are obtained according to the method described above.

[0048] Preferably, according to 4×10 4 ~1×10 5 / cm 2 Continuous culture at the desired density for 3-4 weeks.

[0049] Preferably, the mesenchymal stem cell culture medium is: serum-free complete culture medium for mesenchymal stem cells or commercially available MSC culture medium, wherein the serum-free complete culture medium for mesenchymal stem cells is a basal culture medium containing fetal bovine serum and FGF signaling pathway agonists.

[0050] More preferably, the basal culture medium is DMEM (L) culture medium or α-DMEM culture medium.

[0051] More preferably, the basal culture medium is DMEM (L) culture medium.

[0052] More preferably, the amount of fetal bovine serum used is 1–10% (V / V).

[0053] More preferably, the amount of fetal bovine serum used is 5% (V / V).

[0054] More preferably, the bFGF signaling pathway agonist is recombinant human basic fibroblast growth factor bFGF.

[0055] More preferably, the amount of bFGF used is 1 to 100 ng / mL.

[0056] More preferably, the amount of bFGF used is 10 ng / mL.

[0057] More preferably, the commercially available MSC culture medium is STEMCELL Technologies' commercially available MSC culture medium (ACF) or Ajinomoto's commercially available MSC culture medium (STEMFIT).

[0058] This invention also claims protection for the application of the intestinal mesenchymal stem cells prepared by the method, wherein the application is one or more of the following: Prepared immunomodulatory drugs; To prepare drugs for treating intestinal damage; To prepare drugs for the treatment of inflammatory bowel disease; It acts as a trophoblast to promote organoid development.

[0059] Preferably, the immunomodulatory drug inhibits inflammatory factors IFN-γ and / or TNFα.

[0060] Compared with the prior art, the present invention has the following beneficial effects: This invention defines the differentiation pathway from pluripotent stem cells to vagal crest cells by varying the concentration, combination, and duration of action of different signaling pathways. The proposed induction method, through a standardized differentiation process, obtains specific vagal crest cells (vNCCs), ensuring high purity and precise spatial identification of the starting cells, resulting in vagal crest-derived mesenchymal stem cells with a clear developmental origin and a single lineage. Simultaneously, this invention refines the developmental origin of MSCs, exploring and successfully obtaining intestinal mesenchymal stem cells (IMSCs) derived from vagal crest cells (vNCCs). These IMSCs, obtained through cell differentiation pathways, not only have clear differentiation pathways but also a defined developmental origin, supplementing existing mesenchymal stem cells from different developmental origins. The obtained intestinal mesenchymal stem cells possess the basic biological characteristics of mesenchymal stem cells, exhibiting not only the ability to proliferate in large quantities and strong multi-lineage differentiation and immunomodulatory capabilities but also the ability to effectively repair intestinal damage, becoming an important cell source for the treatment of intestinal injury-related diseases. Furthermore, this co-culture mode based on tissue homology or functional adaptation provides an ideal source of stromal cells for constructing highly realistic and high-quality intestinal organoid models. Attached Figure Description

[0061] Figure 1 A. Morphological image of cultured human induced pluripotent stem cells (hiPSCs) induced into vagal crest cells (vNCCs); B. Schematic diagram of the induction of human induced pluripotent stem cells (hiPSCs) into vagal crest cells (vNCCs); C. Cell morphology of hiPSCs after digestion and adherence to Stage I culture medium for 1 day, after stage II culture medium for 4 days, and after stage III culture medium for 6 days; D. Immunofluorescence staining images of vagal crest cell origin markers (HOXB4) and neural crest markers (SOX10 and P75) after stage III culture medium for 6 days during the induction of hiPSCs into vNCCs.

[0062] Figure 2 Purification of vagal crest cells derived from human induced pluripotent stem cells (hiPSC-vNCCs); A is a schematic diagram of flow cytometry sorting of human induced pluripotent stem cells after culturing in Stage I, Stage II, and Stage III media using vagal crest cell markers (CD271 and CD49d); B is a morphological diagram of sorted vagal crest cells (vNCCs); C is an immunofluorescence staining image of vagal origin markers (HOXB4) and neural crest markers (SOX10 and P75) of sorted vNCCs.

[0063] Figure 3A shows the induction of vagal crest cells into intestinal mesenchymal stem cells; A is a cell morphology diagram of vagal crest cells induced by human induced pluripotent stem cells (hiPSC-vNCCs) after continuous passage and induction in complete mesenchymal stem cell culture medium (CMM) for 3 weeks; B is a flow cytometry diagram of cell surface markers after passage and induction of IMSCs in CMM for 3 weeks; C is an image of Alizarin Red S staining, Oil Red O staining, and Alcian Blue staining of cells cultured in CMM after osteogenic, adipogenic, and chondrogenic differentiation.

[0064] Figure 4 The immunomodulatory capacity of intestinal mesenchymal stem cells (IMSCs) is shown in Figure A. The immunomodulatory capacity of IMSCs derived from the vagus crest obtained in Example 5 is compared with that of BMSCs isolated from long bone marrow of control individuals. Flow cytometry results were used to verify the inhibitory effect of IMSCs on the proliferation of peripheral blood mononuclear cells (PBMCs). Figure B shows the inhibition of T cell secretion of pro-inflammatory factors IFN-γ and TNF-α by IMSCs derived from the vagus crest obtained in Example 5 and BMSCs isolated from long bone marrow of control individuals, along with the corresponding statistical analysis.

[0065] Figure 5 This document presents the following diagrams for the treatment of mouse inflammatory bowel disease with intestinal mesenchymal stem cells (IMSCs): A shows the flowcharts for the use of vagal crest-derived mesenchymal stem cells (vNCC-MSCs, or IMSCs) obtained through induced differentiation in Example 5, BMSCs isolated from long bone marrow in Control Example 1, and human skin fibroblasts (HDFs) in a mouse ulcerative colitis (IBD) injury model and cell therapy, including TNBS pre-sensitization, rectal administration for model establishment, cell infusion therapy, and sample collection; B shows the dynamic changes in body weight of mice in each group during the model establishment and treatment period of 7 days; C shows the dynamic scoring curves of the Disease Activity Index (DAI) for each group of mice; D shows the statistical results of colon morphology and length after sacrifice in each group of mice; E shows the detection of myeloperoxidase (MPO) levels in different treatment groups after acute intestinal injury; and F shows the pathological and injury scoring results of colon tissue in each group of mice.

[0066] Figure 6 Intestinal mesenchymal stem cells (IMSCs) were used to treat inflammatory bowel disease in mice; A shows the levels of pro-inflammatory factors (IL-1β, IL-12α, TNF-α, and IL-17) and chemokines (MIP-2 and Rantes) in the colon tissue of mice in each group; B shows the levels of CD4+ in the colon tissue of mice in each group detected by immunofluorescence staining. + T cells, CD8 + The results of T cell infiltration and the statistical analysis of positive cell counts are shown in the figure.

[0067] Figure 7 The image shows the role of intestinal mesenchymal stem cells (IMSCs) in supporting the growth of colonic organoids. A is a schematic diagram of 3D colonic organoids induced in vitro after isolating mouse colonic crypts and indirectly co-culturing them with mesenchymal stem cells from different sources via Transwell. B shows the colonic organoid formation efficiency after 1 day of co-culture in the control group (CON), vagal crest-derived mesenchymal stem cells (Co-IMSC), and bone marrow-derived mesenchymal stem cells (Co-BMSC). C shows the diameter of colonic organoids in each group after 3 days of co-culture. D shows the white light morphology of colonic organoids after 6 days of co-culture. E shows the budding rate statistics for each group.

[0068] Figure 8 A shows the growth support role of intestinal mesenchymal stem cells (IMSCs) for colonic organoids; A represents the regulatory effect of mesenchymal stem cells from different sources on the proliferation and epithelial differentiation function of colonic organoids, with immunofluorescence staining images of colonic organoid proliferation marker (Ki67) and goblet cell-specific differentiation marker (MUC2) in the control group (Con), VNC-MSC co-culture group (Co-IMSC), and BMSC co-culture group (Co-BMSC); B shows the relative mRNA expression levels of key genes for intestinal epithelial differentiation in colonic organoids of each group, including goblet cell marker (MUC2), downstream secretory differentiation regulatory gene (HES1) of enteroendocrine cell markers (ChgA and Notch), absorptive epithelial differentiation regulatory gene (Atoh1), and intestinal stem cell marker (ASCL2). Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0070] 1. The primers used for real-time PCR in the following examples are shown in Table 1.

[0071] Table 1:

[0072] 2. The antibodies used for immunofluorescence staining in the following examples are shown in Table 2.

[0073] Table 2:

[0074] 3. The antibodies used for immunofluorescence staining in the following examples are shown in Table 3.

[0075] Table 3:

[0076] 4. Methods of Immunofluorescence Staining for Cells Cell clones were fixed with 4% paraformaldehyde (PFA), washed three times with PBS, blocked with 2% BSA for 30 minutes, and washed twice with PBS. Primary antibody was added, and the cells were incubated overnight at 4°C, followed by three 5-minute washes with PBS. Secondary antibody was added, and the cells were incubated at room temperature for 30–45 minutes. After four washes with PBS, 0.5 μg / ml DAPI was added for staining for 10 minutes. After removing DAPI, the cells were washed three times with PBS. The cells were then mounted with mounting medium and photographed using confocal microscopy.

[0077] 5. Quantitative Real-Time PCR Method Cells were collected by digestion with Accutase, washed three times with PBS, and then lysed with 1 ml of RNAzol. Total RNA was extracted and reverse transcribed into cDNA using a quantitative reverse transcription kit (Qiagen) in a 10 μl reaction system. Quantitative real-time PCR was performed using the DyNAmoColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific) on a LightCycler 480 detection system. The thermal cycling conditions were: 95℃ denaturation for 10 s, 60℃ annealing for 20 s, and 72℃ extension for 30 s, for a total of 45 cycles. GAPDH was used as an internal control, and gene expression levels were calculated using the 2-ΔΔCT method.

[0078] 6. Flow cytometry detection method Cells were digested with Accutase, centrifuged at 1100 rpm for 4 min, and the supernatant was discarded. Cells were resuspended in 50 μL of PBS, and a homologous antibody (BD Pharmingen) was used as a negative control. In separate tubes, flow cytometry antibodies were added and stained for 20 min. Cells were washed twice with PBS for 5 min each time, centrifuged at 1100 rpm for 4 min, and the supernatant was discarded. Cells were resuspended in 200 μL of PBS and the proportion of positive cells was detected by flow cytometry.

[0079] Example 1: A culture medium composition for inducing pluripotent stem cells into specific vagal crest cells. Stage I culture medium was DMEM-F12 containing 1% (V / V) N-2 additive (100×), 2% (V / V) B-27 neural culture additive, 1% (V / V) NEAA cell culture additive, 2 mM glutamine, 110 μM β-mercaptoethanol, 10 ng / ml bFGF, 10 μM MY27632 and 1% (V / V) penicillin-streptomycin.

[0080] Stage II culture medium was DMEM-F12 medium containing 1% (V / V) N-2 additive (100×), 2μM CHIR99021, 2μMSB431542, 2μM SAG and 1% (V / V) penicillin-streptomycin.

[0081] Stage III culture medium was DMEM-F12 medium containing 1% (V / V) N-2 additive (100×), 2 μM CHIR99021, 2 μM MSB431542, 2 μM SAG, 1 μM retinoic acid (RA), and 1% (V / V) penicillin-streptomycin.

[0082] SAG, or moothened agonist, is a highly potent, cell-permeable Smo receptor agonist.

[0083] Example 2: A complete culture medium for mesenchymal stem cells A complete mesenchymal stem cell culture medium (CMM): a low-glucose DMEM culture medium containing 5% (V / V) fetal bovine serum and 10 ng / mL bFGF.

[0084] Example 3: Induction of human induced pluripotent stem cells (hiPSCs) into vagal neural crest cells (vNCCs) I. Expand pluripotent stem cells while maintaining their undifferentiated state. 1. Experimental Methods The pluripotent stem cells used in the following examples are induced pluripotent stem cells (hiPSCs) or embryonic stem cells (H1-ES), wherein the induced pluripotent stem cells (hiPSCs) are induced by human skin fibroblasts (HEF).

[0085] For pluripotent stem cells induced by human skin fibroblasts, see the existing technology: Fan Y, Huang S, Li S, Wu B, Zhao Q, Huang L, Zheng Z, Xie X, Liu J, Huang W, Sun J, Zhu X, Zhu J, Xiang AP, Li W. The adipose-neural axis is involved in epicardial adipose tissue-related cardiac arrhythmias. Cell Rep Med. 2024 May 21;5(5):101559.doi: 10.1016 / j.xcrm.2024.101559. Epub 2024 May 13. PMID: 38744275; PMCID:PMC11148799.

[0086] Pluripotent stem cells need to be expanded on a large scale without a feeder layer and kept in an undifferentiated state. Cells can be selected using STEM CELL's mTeSR series products and E8 culture medium. The culture dishes containing adherent pluripotent stem cells need to be pre-coated with Matrigel or laminin to ensure high-quality, well-preserved pluripotent stem cells, which is crucial for the next step of induction. At this stage, the pluripotent stem cells grow in a flattened, clonal pattern with tightly packed cells.

[0087] The specific amplification operation steps are as follows: (1) Coating culture dish: After melting the Matrigel stock solution on ice, dilute it with cold DMEM-F12 at a volume ratio of 1:100 and add it to the well plate. Coat overnight at 37°C for later use.

[0088] (2) Resuscitation of pluripotent stem cells: Human induced pluripotent stem cells (hiPSCs) were rapidly thawed in a 37℃ water bath and taken out at -80℃. They were then transferred to a 15ml centrifuge tube containing 5ml mTeSR culture medium and centrifuged at 300g for 5min to collect the cells.

[0089] (3) Culture of pluripotent stem cells: Remove the coating Matrigel, resuspend the cells in 2 ml mTeSR, seed them evenly, and place them in an incubator at 37°C, 5% CO2 and 95% humidity for static culture. Change the culture medium daily, observe the cell status, and culture until the clones grow to a density of 80-90% before passage.

[0090] (4) Cell passage: Wash cells twice with PBS, add 0.5 mM EDTA, incubate at 37°C for 1 to 10 min, discard EDTA, gently pipette cells with PBS, collect cell clumps into 15 ml centrifuge tubes, centrifuge at 300 g for 5 min to collect cells. Resuspend cells in 1 ml mTeSR, and seed cells evenly into Matrigel-coated wells at a volume of 100 μl per well. Incubate in a 37°C, 5% CO2 and 95% humidity incubator for static adhesion culture.

[0091] (5) Photographs: Take clonal morphology photos of hiPSCs using a Leica microscope.

[0092] (6) Repeat the above culture and amplification steps to obtain a sufficient number of cells for the next step of differentiation induction.

[0093] 2. Experimental Results Figure 1 A in the image is a white light image of the morphology of human induced pluripotent stem cells (hiPSCs) obtained from expansion culture. The cells show a tight clonal growth. The cell culture plate was coated with laminin LN, which has a similar effect to Matrigel, maintaining the pluripotency of pluripotent stem cells and preserving their clonal morphology.

[0094] II. Induction of human induced pluripotent stem cells (hiPSCs) into vagal crest cells Figure 1 B in this embodiment is a schematic diagram of the induction of human induced pluripotent stem cells (hiPSCs) into vagal crest cells.

[0095] 1. Experimental Methods (1) Digesting hiPSC clones: Cover the hiPSC clones obtained from the previous amplification and culture with 1 ml of Accutase (STEMCELL Technologies) cell digestion solution and digest them in a 37°C incubator for 3 min. Then dilute with 5 ml of PBS, collect hiPSC single cells into a 15 ml centrifuge tube, centrifuge at 1100 rpm for 4 min and collect the cell pellet.

[0096] (2) Preparation of cell suspension: Discard the supernatant and resuspend the cell pellet in 1 ml of Stage I culture medium from Example 1.

[0097] Stage I culture medium consisted of DMEM-F12 medium supplemented with 1% (V / V) N-2 additive (100×), 2% (V / V) B-27 neural culture additive, 1% (V / V) NEAA cell culture additive, 2 mM glutamine, 110 μM β-mercaptoethanol, 10 ng / ml bFGF, 1% (V / V) penicillin-streptomycin, and 10 μM Y27632 (Calbiochem, San Diego, CA).

[0098] (3) Adherent culture for 24 h: The cells resuspended in Stage I culture medium in the previous step were cultured at 1×10⁻⁶ cells / mL. 5 cm 2 Cells were seeded at a density in Matrigel or fibronectin LN-coated culture dishes and cultured for 24 hours. Cell morphology was photographed using a Leica microscope.

[0099] (4) Stage II: The culture medium of adherent cells for 24 hours was replaced with the Stage II culture medium in Example 1 and cultured for 4 days, with the Stage II culture medium being replaced every other day. After 4 days, cell morphology photographs were taken using a Leica microscope.

[0100] The Stage II culture medium consisted of DMEM-F12 medium supplemented with 1% (V / V) N-2 additive (100×), 2μM CHIR99021, 2μM SB431542, and 2μM SAG and 1% (V / V) penicillin-streptomycin.

[0101] (5) Stage III: After culturing in Stage II medium for 4 days, the culture medium was changed to Stage III medium from Example 1 and cultured for 6 days, with the Stage III medium being changed daily. After 6 days of Stage III culture, cell morphology photographs were taken using a Leica microscope, and the cells were fixed with 4% paraformaldehyde (PFA) and then immunofluorescence staining was performed to identify markers of vagal crest cells (HOXB4, SOX10, and P75).

[0102] The Stage III culture medium contained DMEM-F12 culture medium and added 1% (V / V) N-2 additive (100×), 2μM CHIR99021, 2μM SB431542, 2μM SAG and 1μM retinoic acid (RA) and 1% (V / V) penicillin-streptomycin mixture.

[0103] 2. Experimental Results Cell morphology after hiPSC digestion and adherence to Stage I culture medium for 1 day, cell morphology after stage II culture medium for 2 days, and cell morphology after stage III culture medium for 3 days are shown in the figures. Figure 1 Immunofluorescence staining images of C. hiPSCs after 6 days of culture in Stage III medium during the induction of vNCCs are shown in the image. Figure 1 D in the middle.

[0104] The results showed that after staged induction, pluripotent stem cells gradually lost their typical clonal morphology and transformed into adherent, long spindle-shaped cells. This indicates that human induced pluripotent stem cells can be efficiently and specifically induced to produce vagal crest cells after three-stage induction with culture medium.

[0105] The above evidence indicates that the pluripotent stem cells in this invention are specifically and efficiently induced to produce human induced pluripotent stem cell-derived vagus nerve crest cells (hiPSC-vNCCs) through the Stage I, Stage II, and Stage III culture media of Example 1. However, the current induction product is a mixed cell population (a mixture of cells containing hiPSC-vNCCs), and further enrichment of target cells through flow cytometry and other methods is needed to improve cell purity.

[0106] Example 4: Purification of vagal crest cells (hiPSC-vNCCs) derived from human induced pluripotent stem cells 1. Experimental Methods (1) Purification of vagal crest cells derived from human induced pluripotent stem cells The mixed cells containing hiPSC-vNCCs induced in Example 3 were digested into a single-cell suspension. The cells were washed twice with 1×PBS, and Accutase was added to cover the cell surface. The cells were digested at 37°C for 5–10 min and observed under a microscope. When the cells were digested into single cells, an appropriate amount of PBS was added, and the cell suspension was collected into a 15 mL centrifuge tube and centrifuged at 1100 rpm for 5 min. The supernatant was discarded, and the cells were stained at room temperature in the dark for 20 min according to the flow cytometry chromosome volume and antibody volume as specified by the reagent manufacturer. The cells were then washed once with PBS and centrifuged at 1100 rpm for 5 min. After discarding the supernatant, 2 mL of PBS was added to resuspend the cells. The cells were then filtered through a 40 μm sieve into a new flow cytometry sample tube and sorted by flow cytometer for double-positive cells of the specific surface markers CD271 and CD49d.

[0107] Collect the complete mesenchymal stem cell culture medium (CMM) of Example 2 containing 1% amphotericin B (2.5 μg / mL) and 1‰ moxifloxacin (3 μg / mL).

[0108] (2) Photographing and staining identification: The morphological images of the vagal crest cells (vNCCs) obtained in step (1) above were photographed using a Leica microscope. After the cells were fixed with 4% paraformaldehyde (PFA), the expression of SOX10 and HOXB4 markers was identified by immunofluorescence staining.

[0109] 2. Experimental Results Figure 2 In Example 3, A represents the results of flow cytometry (FACS) analysis and sorting of the hiPSC-vNCC hybrid cells obtained by induction using the vNCC-specific surface markers CD271 and CD49d. Figure 2 B in the diagram represents the cell morphology of vNCCs. Figure 2 In the image, C represents the immunofluorescence staining pattern of vNCCs markers (SOX10 and HOXB4).

[0110] The results showed that the mixed cells containing hiPSC-vNCCs induced in Example 3 were successfully processed into highly pure human induced pluripotent stem cell-derived vagal crest cells (hiPSC-vNCCs) using a CD271 and CD49d dual-marker stream sorting technique. The obtained cells not only morphologically conformed to the characteristics of neural crest cells, but also simultaneously expressed the broad-spectrum neural crest marker (SOX10) and the location-specific marker (HOXB4) at the molecular level, demonstrating their accurate spatial identity as vagal crest cells. This invention provides a stable and scalable hiPSC-vNCCs acquisition protocol, offering sufficient seed cells for subsequent downstream differentiation (such as intestinal neurons and mesenchymal stem cells).

[0111] Example 5: Method for inducing differentiation of purified vagal crest-derived intestinal mesenchymal stromal cells (vNCC-MSCs, IMSCs) I. Experimental Methods (1) Induction of intestinal mesenchymal stem cells by IMSCs: The purified human induced pluripotent stem cell-derived vagal crest cells (hiPSC-vNCCs) obtained in Example 4 were processed at a concentration of 1×10⁻⁶. 4 cm 2The culture medium was seeded into Matrigel or fibronectin-coated culture dishes at a density of 1000 mg / mL. The culture was then incubated in a 37°C, 5% CO2 and 95% humidity incubator with DMEM (L) medium, 5% fetal bovine serum and 10 ng / mL bFGF. The culture medium was changed every 2-3 days. After 21 days, IMSCs were obtained.

[0112] The CMM culture medium contained DMEM (L) culture medium and added 5% fetal bovine serum and 10 ng / mL bFGF. (2) Cell passage: After the cells reached 80-90% confluence, they were digested and passaged using Accutase cell digestion solution. The cells were then transferred into 15ml centrifuge tubes and centrifuged at 300g for 5min to collect the cells. Subsequently, cells cultured in mesenchymal stem cell culture medium (Complete Mesenchymal Stem Cell Culture Medium (CMM) of Example 2, commercially available MSC culture medium (ACF; STEM CELL Technologies), or commercially available MSC culture medium (StemFit; Ajinomoto)) were seeded into new cell culture dishes at a density of 1 passage to 3 and continued to be cultured. (3) Flow cytometry detection: Repeat step (2) above. The cells induced and cultured in the mesenchymal stem cell culture medium used in the previous step (complete mesenchymal stem cell culture medium (CMM) of Example 2 or commercially available MSC culture medium (ACF; STEM CELL Technologies) or commercially available MSC culture medium (StemFit, Ajinomoto)) are digested into single cells with Accutase cell digestion solution. A portion of the cells are then flow cytometry detected for CD34, CD45, CD11b, CD73, CD90, CD105, etc. The detection is repeated during the induction and passage process until CD105, CD73, and CD90 reach more than 99%, and CD34, CD45, and CD11b are less than 1%, thus obtaining intestinal mesenchymal stem cells derived from the vagal crest.

[0113] III. Experimental Results Figure 3 In Example 4, A is a cell morphology diagram of vagal crest cells (hiPSC-vNCCs) induced by human induced pluripotent stem cells after continuous passage in complete mesenchymal stem cell culture medium (CMM) for 3 weeks. Figure 3 B in the figure represents the flow cytometry analysis of cell surface markers in hiPSC-vNCCs after 3 weeks of passage and induction in complete mesenchymal stem cell culture medium (CMM).

[0114] The results showed that vNCCs in complete mesenchymal stem cell culture medium (CMM) underwent a process where the cell morphology changed to spindle shape, adhered to the wall, and grew in a vortex pattern. Flow cytometry analysis revealed that CD44, CD105, CD73, and CD90 were above 99%, while CD34, CD45, and CD11b were below 1%, indicating that the induced cells met the MSC criteria. Intestinal mesenchymal stem cells derived from the vagal crest were successfully obtained for subsequent experiments.

[0115] Example 6: Detection of the multi-lineage differentiation capacity of induced intestinal mesenchymal stem cells I. Experimental Methods (1) Osteogenic differentiation: Intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 were collected by digestion with Accutase and seeded in six-well plates until the density reached 80-90%. The culture medium was then replaced with osteogenic induction differentiation culture medium (DMEM (L) + 10% (V / V) FBS + 10 mM β-glycerophosphate + 50 μg / ml vitamin C and 100 nM dexamethasone). The medium was changed every 3 days. After 21 days of induction culture, Alizarin Red S staining was performed.

[0116] (2) Adipogenic differentiation: Intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 were collected by digestion with Accutase and seeded in six-well plates until they were fully confluent. At this time, the culture medium was replaced with adipogenic differentiation induction culture medium (DMEM(H) + 10%(V / V)FBS + 100 nM Indo + 0.5 mM IBMX and 1 mM dexamethasone). The medium was changed every 3 days. Oil Red O staining was performed after 21 days of induction culture.

[0117] (3) Chondrogenic differentiation: Intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 were collected by Accutase digestion and then chondrogenic differentiation was carried out at a concentration of 2-3 × 10⁻⁶. 5 Cells were aliquoted from 1 ml centrifuge tubes into 15 ml centrifuge tubes, centrifuged at 300 × g for 5 min, and the supernatant was discarded. 1 ml of chondrocyte differentiation induction medium (DMEM(H) + 10 ng / ml recombinant human transforming growth factor (TGF-β3), 100 nM dexamethasone, 50 μg / ml vitamin C, 1 mM sodium pyruvate, 40 μg / ml proline, and ITS supplement + premix) was added, and the mixture was incubated. The medium was changed every 3 days. After 21 days of induction culture, aspen blue staining was performed.

[0118] III. Experimental Results Figure 3In the figure, C represents the Alizarin Red S staining, Oil Red O staining, and Asin Blue staining images of IMSCs cultured in complete mesenchymal stem cell culture medium (CMM) after osteogenic, adipogenic, and chondrogenic differentiation.

[0119] The results showed that the intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 showed a deep red color when stained with Alizarin Red S, indicating the formation of calcium nodules, which indicates that they can be induced to differentiate into osteoblasts; the intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 showed an orange-red or bright red color when stained with Oil Red O, indicating the formation of lipid droplets, which confirms that IMSCs can differentiate into adipocytes; the intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 showed a purple-blue color when stained with Asin Blue, indicating abundant cartilage matrix, which confirms that IMSCs can differentiate into chondrocytes.

[0120] In summary, the preparation method of the present invention can not only efficiently obtain intestinal mesenchymal stem cells, but the prepared intestinal mesenchymal stem cells (IMSCs) also meet the criteria for mesenchymal stem cells in terms of morphology, expression of surface markers and multi-lineage differentiation potential. The method establishes a stable and reproducible induction system, which can realize large-scale preparation and is suitable for standardized production.

[0121] Comparative Example 1: Preparation and Culture Methods of Bone Marrow Mesenchymal Stem Cells (BMSCs) This comparative example provides a method for isolating, preparing, and culturing bone marrow mesenchymal stem cells (BMSCs) used in subsequent examples. Specifically, mesenchymal stem cells were isolated from human long bone bone marrow using density gradient centrifugation; and expanded culture was performed using mesenchymal stem cell culture medium, wherein the mesenchymal stem cell culture medium was the complete mesenchymal stem cell culture medium (CMM) from Example 2.

[0122] The specific steps are as follows: (1) Dilute 5 mL of human bone marrow fluid with an equal volume of PBS, mix well, and centrifuge at 1500 rpm for 10 minutes at room temperature. (2) After discarding the fat layer, slowly add the remaining bone marrow fluid along the centrifuge tube wall onto an equal volume of Ficoll separation solution (1.077 g / mL), and centrifuge at 2500 rpm for 30 minutes at room temperature; (3) Take the mononuclear cells with a white membrane layer at the interface from the separation solution, suspend them in 40 ml of PBS, and centrifuge at 1500 rpm for 10 minutes at room temperature; (4) Discard the supernatant and wash twice with 50ml PBS.

[0123] (5) Resuspend the cells in complete mesenchymal stem cell culture medium (CMM) for expansion culture at a concentration of 2×10⁻⁶. 5 At a cell density of / mL, cells were seeded in a substrate with a base area of ​​25cm². 2The culture flasks were placed in a 37°C, 5% CO2 incubator for incubation.

[0124] (6) The medium was changed for the first time after 3 days, and the suspended cells were discarded. The medium was then changed every 2 to 3 days thereafter. Cell morphology and growth changes were observed under a microscope daily. After 14 days, there were basically no suspended cells in the culture flask, and all cells grew in a fibrous adherent manner and were in good condition.

[0125] (7) When the cells reach 80% to 90% confluence, they are digested with Accutase and passaged at a ratio of 1:3. The resulting bone marrow mesenchymal stem cells (BMSCs) are used for subsequent experiments.

[0126] Example 7: Immunomodulatory capacity of intestinal mesenchymal stem cells (IMSCs) I. Experimental Methods In this embodiment, the intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 and cultured in the complete mesenchymal stem cell culture medium (CMM) of Example 2 were used, along with the bone marrow mesenchymal stem cells (BMSCs) isolated in Control Example 1 and obtained in the complete mesenchymal stem cell culture medium (CMM) of Example 2.

[0127] 1. Detect the inhibitory effect of intestinal mesenchymal stem cells (IMSCs) on PBMC cell proliferation. (1) CFSE (5, 6-carboxyfluorescein diacetate, succinimidyl ester) labeling PBMC cells were resuspended in 10 ml of PBS, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cells were then resuspended in PBS containing 0.1% BSA, pre-warmed to 37°C, to a concentration of 10. 6 Add 5 μM of pre-prepared working concentration CFSE dye stock solution to each ml of the culture medium. Vortex and mix thoroughly. Place the mixture in a 37°C water bath for 10 minutes. Immediately after the water bath, add 5 volumes of pre-cooled RPMI-1640 culture medium (complete medium) containing 10% (V / V) FBS. Place the mixture on ice for 5 minutes to stop staining. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and resuspend the cells in 10 ml of RPMI-1640 complete medium. Wash once more to remove excess CFSE dye. Incubate overnight in RPMI-1640 complete medium at 37°C, 5% CO2, and 95% humidity. (2) Co-culture and proliferation detection: On the second day, the labeled PBMC cells were co-cultured with the intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 and the BMSCs obtained in Control Example 1 at a ratio of 5:1. The control group was not co-cultured. PHA was added to each group to a final concentration of 3 μg / ml to stimulate proliferation, and IL-2 at 100 μg / ml to support PBMC cell survival. On the sixth day, the PBMC cells were washed twice with physiological saline and then transferred to flow cytometry for detection.

[0128] 2. Detect the immunomodulatory capacity of intestinal mesenchymal stem cells (IMSCs) on T lymphocytes. (1) Intestinal mesenchymal stem cells (IMSCs) prepared in complete mesenchymal stem cell culture medium (CMM) in Example 5 and BMSCs in Control Example 1 were routinely digested. The cells were resuspended in complete mesenchymal stem cell culture medium (CMM) to prepare 4×10⁶ cells / mL. 4 / cm 2 The suspension was spread into 24-well plates and left overnight; (2) Flow cytometry sorting CD3 + T cells were resuspended in RPMI-1640 complete medium; (3) T cells were seeded into 24-well plates pre-coated with IMSCs at a ratio of 5:1 and placed in an incubator at 37°C, 5% CO2 and 95% humidity for 3 days. After 3 days, stimulants were added 6 hours in advance for pretreatment, and T cells were collected into 15ml centrifuge tubes and centrifuged at 500g for 10min to collect the cells; (4) After resuspending the cell pellet with 100 μl PBS, add 100 μl 4% (V / V) paraformaldehyde fixative (PFA) and fix at room temperature for 15 min; (5) Add 0.2% (V / V) saponin for penetration, and add appropriate amounts of IFN-γ and TNFα antibodies for staining at room temperature for 20 min; (6) Wash the antibody twice with PBS, resuspend the cell pellet in 300 μl PBS, and use a flow cytometer to detect the positive ratio of IFN-γ and TNFα.

[0129] II. Experimental Results Figure 4 This is a comparison of the immunomodulatory capacity of intestinal mesenchymal stem cells (IMSCs) obtained in Example 5 and BMSCs obtained in Control Example 1.

[0130] The results showed that the IMSCs obtained in Example 5 in complete mesenchymal stem cell culture medium (CMM) had a comparable inhibitory effect on PBMC proliferation compared to the BMSCs obtained in Control Example 1. Compared to the PBMC-only culture group (control group), the CFSE dilution ratio (i.e., proliferation ratio) was significantly reduced after co-culture. Statistical analysis indicated that the proliferation ratio in the PBMC group was approximately 43%, while it was significantly reduced to approximately 26% in the IMSC co-culture group. These results demonstrate that IMSCs possess a strong ability to inhibit the activation and proliferation of immune cells.

[0131] Compared with T cells cultured alone, co-culture with IMSCs significantly reduced the proportion of positive expression of IFNγ and TNFα in T cells, showing a statistically significant difference and exhibiting a better inhibitory effect on the secretion of inflammatory factors by T cells. This indicates that the induced intestinal mesenchymal stem cells have significant efficacy in regulating the immune microenvironment and suppressing inflammatory responses.

[0132] The above evidence suggests that intestinal mesenchymal stem cells (IMSCs) have excellent immunomodulatory capabilities, effectively inhibiting the secretion of inflammatory factors and reducing immune stress under inflammatory conditions. Example 8: Intestinal mesenchymal stem cells (IMSCs) for the treatment of inflammatory bowel disease in mice I. Experimental Methods In this embodiment, intestinal mesenchymal stem cells (IMSCs) cultured in the complete mesenchymal stem cell culture medium (CMM) of Example 2 were used in Example 5, and bone marrow mesenchymal stem cells (BMSCs) isolated in Control Example 1 and obtained in the complete mesenchymal stem cell culture medium (CMM) of Example 2 were used. A schematic diagram of the mouse ulcerative colitis (IBD) injury model and cell therapy process is shown below. Figure 5 A in the middle.

[0133] 1. Experimental Methods (1) Pre-sensitization: On the first day of the experiment, a 1cm long and 1cm wide area was shaved off the back of an 8-week-old male BALB / c mouse. Then, 150μl of TNBS sensitization solution was evenly applied to this area, and the mouse was put back into the cage for rearing.

[0134] (2) On day 7 of the experiment, the mice were randomly divided into 5 groups of 5 mice each. The mice were fasted for 24 hours but allowed water. On day 8 of the experiment, the mice in each group were weighed and their initial weight was recorded. Then, rectal enema was started. The experimental procedure is as follows: Figure 5 A in the middle.

[0135] The model control group received no treatment. Model group: 100 µl of 2.5% TNBS solution was instilled via rectal cannula; IMSCs group: 100 µl of 2.5% TNBS solution was instilled into the mice via rectal cannula. 12 h later, the IMSCs obtained in Example 5 were injected into the tail vein of mice. 1×10 6 cells / 200μl cell suspension; BMSCs group: 100 µl of 2.5% TNBS solution was instilled into the mice via rectal cannula. 12 h later, mice were injected via tail vein with BMSCs obtained from control example 1, 1×10 6 cells / 200μl cell suspension; HDFs group: 100 µl of 2.5% TNBS solution was instilled via rectal cannula. 12 h later, human skin fibroblasts (HDFs) were injected via tail vein into mice. 6 cells / 200μl cell suspension.

[0136] Control group: 100µl of 2.5% TNBS solution was instilled through anal cannula, and 200µl of PBS was injected into the tail vein of mice 12 hours later.

[0137] (3) After successful modeling, the weight changes, diarrhea status, and survival status of the mice were observed and recorded for 7 consecutive days (day 14 of the experiment). 24 hours after cell therapy, the colon from the cecum to the anus was removed, and the colon length of each group was measured. The colon was scored based on the degree of inflammation, etc. The fecal morphology and presence of bleeding were used as indicators for evaluating the disease progression. The specific evaluation criteria were as follows: normal fecal morphology 0 points; loose fecal morphology 1 point; soft fecal morphology 2 points; diarrhea 3 points; diarrhea with anal bleeding 4 points.

[0138] (4) Subsequently, a portion of the tissue was taken and the myeloperoxidase level was measured using the MPO colorimetric assay kit (Elabscience); (5) Partial fixation for sectioning. After sectioning the colon, H&E staining was used to observe the infiltration of inflammatory cells in the colon at the histological level and to score it. The specific evaluation criteria were as follows: no obvious inflammation 0 points, low level of inflammatory cell infiltration 1 point, moderate level of inflammatory cell infiltration 2 points, high level of inflammatory cell infiltration, moderate degree of fibrosis, high vascular density, thickening of the colonic wall, moderate degree of loss of goblet cells and loss of central pit 3 points, and full-thickness inflammatory cell infiltration, large amount of loss of goblet cells, inflammatory fibrosis and diffuse loss of central pit 4 points.

[0139] (6) Simultaneously, CD4 in the colon was observed using immunofluorescence staining. + and CD8 +The proportion of T cells was used to reflect the infiltration of T cell subsets in the colon. Images were acquired using a Zeiss LSM710 confocal microscope. The percentage of TUNEL-positive cells was counted in 10 random regions. Total RNA was extracted from some tissues using RNAzol, and then reverse transcribed into cDNA using a reverse transcription kit according to standard procedures. Subsequently, the expression of chemokines and inflammatory factors was detected by quantitative real-time PCR.

[0140] 2. Experimental Results See results Figure 5 After modeling, body weight and clinical manifestations were observed for 7 consecutive days. Figure 5 B in the text refers to the disease activity index (Colitis score) reflected in the gross colon score. Figure 5 C, the gross morphology and length statistics of the colon are shown in [reference needed]. Figure 5 The levels of myeloperoxidase (MPO) in different treatment groups after acute intestinal injury are shown in the figure. Figure 5 E; staining and histological scoring results were used to further determine the structural integrity of the intestinal mucosa (see E; staining and histological scoring results). Figure 5 F in the middle.

[0141] The results showed that the IMSCs prepared in Example 5 had a good ability to repair intestinal inflammatory damage in mice. Mice exhibited significant weight loss and colonic shortening after IBD modeling, while the weight recovery curve of the IMSC-treated group was basically the same as that of the BMSC group, significantly better than the untreated group and the HDF control group. Furthermore, IMSCs effectively inhibited pathological shortening of the colon, with a colonic length significantly higher than that of the IBD and HDF groups. The Colitis score showed that IMSCs significantly reduced the severity of colitis. Biochemical tests further confirmed that IMSCs significantly downregulated MPO (myeloperoxidase) activity in colonic tissue, suggesting that they could effectively block the recruitment of neutrophils to the inflammatory site. H&E staining showed widespread crypt loss and inflammatory cell infiltration in the intestinal mucosa of the IBD group, while IMSC treatment significantly maintained the integrity of the intestinal structure and reduced the histological damage score.

[0142] qPCR results showed that ( Figure 6 In the A section, TNBS modeling triggered an explosive expression of multiple pro-inflammatory factors in colonic tissue, including chemokines (MIP-2, Rantes) and typical inflammatory factors (IL-17, IL-1β, IL-12-α, TNF-α). IMSC treatment significantly inhibited the transcriptional levels of these factors and showed superior immunosuppressive efficacy compared to HDF on most indicators. Immunofluorescence staining analysis showed ( Figure 6 (B) IMSC treatment significantly reduced CD4 levels in the intestinal mucosa. +T cell infiltration density, while CD8 + The number of T cells was not significantly affected, but CD4+ was effectively suppressed. + An excessive immune response characterized by T cell infiltration protects the intestinal mucosal barrier and alleviates the progression of acute colitis.

[0143] The above results indicate that intestinal mesenchymal stem cells (IMSCs) have good damage repair capabilities in intestinal injury repair, and they may be a new and high-quality cell source for the clinical treatment of intestinal injury and related diseases.

[0144] Example 9: Growth support of intestinal mesenchymal stem cells (IMSCs) for colonic organoids I. Extraction of mouse colonic crypts and in vitro culture of organoids 1. Experimental Methods (1) Mice were euthanized by cervical dislocation. Under sterile conditions, the abdominal cavity was opened, a 5-6 cm colon segment was cut off, and the intestinal cavity was rinsed clean with pre-cooled DPBS buffer. (2) Cut the intestinal segment longitudinally to fully unfold the colonic tissue, with the mucosal surface facing upwards, and gently scrape away the surface mucus and part of the villous tissue with a glass slide; (3) Cut the processed colon tissue into tissue fragments of about 3-4 mm in size, transfer them into 15 ml centrifuge tubes containing 6-8 ml of 5 mM EDTA digestion solution, and place them in a shaker at 4°C for slow shaking digestion for 25-30 min to separate the crypt structure from the basement membrane. (4) After digestion, discard the supernatant. Then add DPBS containing double antibiotics and wash gently once to remove residual EDTA. Repeatedly pipette with DPBS to fully separate the crypts from the attached epithelial tissue. Then filter a sufficient amount of pipetteed crypts and fluid crypts through a 70 μm cell sieve and transfer to a 15 ml centrifuge tube; (5) Centrifuge the filtrate at 300×g for 5 min, discard the supernatant, and retain the precipitate; (6) Add an appropriate amount of matrix gel to resuspend the crypts according to the amount of sediment. Preferably, add about 30 μl of Corning Matrigel matrix gel to every 200-250 crypts and mix well; (7) IMSCs prepared in Example 5 and BMSCs prepared in Control Example 1 were planted in 24-well plates in advance to achieve a healing rate of 80% and used as a trophoblast layer for subsequent co-culture with organoids. They were designated as Co-IMSC group and Co-BMSC group, respectively. The control group had no trophoblast layer, i.e., the Con group. (8) Add the mixture of crypt and matrix gel to the center of the 24-well Transwell chamber, about 30 μl of the mixture to each well, and incubate in a 37°C incubator for 10-15 min to allow the matrix gel to solidify completely. (9) After the matrix gel solidifies, place the chamber on a 24-well plate with feeder cells, add 500 μl of colon organoid culture medium (any brand available commercially) to each well, and incubate at 37°C, 5% CO2 and 95% humidity in an incubator, changing the culture medium every 2 to 3 days. (10) On the second day of culture, the crypt formation rate of the three groups of organoids was counted, the diameter of the organoids was counted on the fourth day, and the budding rate of the organoids was counted on the sixth day.

[0145] II. Detection of organoid function after co-culture (1) On day 7 of organoid culture, remove the original culture medium. Slowly add pre-cooled DPBS along the well wall and repeatedly blow the matrix gel with a pipette tip to fully disperse and separate the matrix gel from the organoids. Then transfer the collected organoids and washing solution mixture to a new 15 mL centrifuge tube. (2) Settle at low temperature: Settle at -20℃ for 6 min or at 4℃ for 30 min to allow the matrix gel to separate, centrifuge at 300×g for 5 min, discard the supernatant and collect the organoid precipitate; (3) Photographing and staining identification: The organoid morphology obtained in step (2) above was photographed using a Leica microscope. After fixing the cells with 4% paraformaldehyde (PFA), the expression of the proliferation marker Ki67 and secretory cell MUC2 was identified by immunofluorescence staining.

[0146] (4) After extracting total RNA from the organoids obtained in step (2) using RNAzol, reverse transcription was performed on the obtained RNA using a reverse transcription kit according to the standard procedure. After reverse transcription into cDNA, the expression of secretory lineage marker genes (MUC2, ChgA and Atoh1), absorption lineage marker gene (HES1), and stem cell characteristic marker gene (ASCL2) was detected by quantitative real-time PCR.

[0147] III. Experimental Results Figure 7 In the diagram, A represents a schematic of an indirect co-culture system of mesenchymal stem cells from different sources and colon organoids extracted from mouse crypts.

[0148] The results showed that, compared with the control group (Con) cultured alone, co-culture of IMSCs and BMSCs significantly improved the formation efficiency of colonic organoids. Figure 7(B in the text). Regarding morphological and volumetric development, the diameter of organoids in the co-culture groups significantly increased, with the Co-IMSC group showing the most significant promoting effect, its organoid diameter being much larger than that of the Co-BMSC group (…). Figure 7 (C in the text). Bright-field microscopy further revealed the complex differences in organoid morphology. Figure 7 (D) In ​​the control group (Con), organoids mostly exhibited small, simple spherical or sac-like structures; while the organoids in the co-culture groups (especially the Co-IMSC group) were large and formed complex multi-layered crypt-like budding structures. Statistical results showed that the organoid budding rate of the IMSC group exceeded 50%, significantly higher than that of the Co-BMSC group and the control group (D). Figure 7 (E in the text).

[0149] Figure 8 In this context, A represents the detection of cell proliferation and secretory capacity in colonic organoids using immunofluorescence staining. Figure 8 In this context, B represents the influence of gene expression level analysis on internal cell dynamics and lineage differentiation.

[0150] Immunofluorescence staining of the cell proliferation marker Ki67 showed that, compared with the control group, the number of Ki67-positive cells (green) in the co-cultured organoids was significantly increased, and highly concentrated strong positive expression was observed in the Co-IMSC group, indicating that IMSCs strongly promoted the proliferation activity of organoid epithelial cells. Meanwhile, staining results for the goblet cell marker MUC2 showed that MUC2-positive cells (red) were extremely abundant in the Co-IMSC group, while expression was minimal in the control group, suggesting that IMSCs significantly induced differentiation into goblet cells.

[0151] qPCR gene expression analysis further confirmed this phenotype. Compared with the control group and the Co-BMSC group, IMSC co-culture significantly upregulated the relative mRNA expression levels of goblet cell marker MUC2 and endocrine cell marker ChgA. In addition, the expression of stem cell marker ASCL2 was relatively low in the IMSC group, while the expression of the absorptive cell line-related gene HES1 showed no significant difference among the groups.

[0152] The results showed that the IMSCs prepared in Example 5 could effectively support the in vitro expansion of colonic organoids, and the enterogenic IMSCs had a superior effect in promoting organoid morphogenesis and complex budding. Combined immunofluorescence and gene expression data indicated that IMSCs not only maintained the highly proliferative state of colonic organoids, but also specifically and strongly drove the differentiation and maturation of intestinal epithelial cells into secretory lineages (such as goblet cells and endocrine cells). The decrease in stem cell markers suggested that IMSCs primarily drove the differentiation of stem cells into mature functional cells. This demonstrates the high tissue-specific induction ability of IMSCs, precisely regulating the organoid microenvironment and solving the problems of insufficient organoid functional maturity and imbalanced cell component ratios in traditional culture methods. Furthermore, IMSCs have irreplaceable technical advantages in intestinal mucus barrier repair and mucosal immunity.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A culture medium composition, characterized in that, It contains Stage I culture medium, Stage II culture medium, and Stage III culture medium; Stage I culture medium is a basic culture medium containing N-2 additive, B-27 neural culture additive, NEAA cell culture additive, glutamine, β-mercaptoethanol, FGF signaling pathway agonist and ROCK signaling pathway inhibitor; Stage II culture medium is a basic culture medium containing N-2 additives, Wnt pathway agonists, TGF-β pathway inhibitors, and Hedgehog pathway agonists; Stage III culture medium is Stage II culture medium containing retinoic acid.

2. The culture medium composition according to claim 1, characterized in that, The FGF signaling pathway agonist is recombinant human basic fibroblast growth factor bFGF; the ROCK signaling pathway inhibitor is one or more of Y27632 or GSK429286A; the TGF-β pathway inhibitor is one or more of SB431542, A83-01, RepSox, SB505124 or LY364947; and the Hedgehog pathway agonist is one or more of SAG, Purmorphamine, Sonic Hedgehog protein or Hh-Ag1.

5.

3. The culture medium composition according to claim 2, characterized in that, The ROCK signaling pathway inhibitor is Y27632; the TGF-β pathway inhibitor is SB431542; and the Hedgehog pathway agonist is SAG.

4. The culture medium composition according to claim 3, characterized in that, It contains Stage I culture medium, Stage II culture medium, and Stage III culture medium; Stage I culture medium is a basic culture medium containing N-2 additive, B-27 neural culture additive, NEAA cell culture additive, glutamine, β-mercaptoethanol, bFGF and Y27632; Stage II culture medium is a basic culture medium containing N-2 additive, CHIR99021, SB431542, and SAG; Stage III culture medium is Stage II culture medium containing retinoic acid.

5. The culture medium composition according to claim 4, characterized in that, It contains Stage I culture medium, Stage II culture medium, and Stage III culture medium; The Stage I culture medium is a basal culture medium containing 1–5% (V / V) N-2 additive (100×), 1–5% (V / V) B-27 neural culture additive, 1–5% (V / V) NEAA cell culture additive, 1–3 mM glutamine, 50–150 μM β-mercaptoethanol, 1–100 ng / ml bFGF and 5–10 μM Y27632. Stage II culture medium is a basic culture medium containing 1-5% (V / V) N-2 additive (100×), 1-10 μM CHIR99021, 1-10 μM MSB431542, and 1-10 μM SAG; Stage III culture medium is Stage II culture medium containing 1–10 μM retinoic acid.

6. The use of the culture medium composition of claim 5 in inducing pluripotent stem cells to differentiate into vagal crest cells or inducing pluripotent stem cells to differentiate into mesenchymal stem cells.

7. A method for inducing pluripotent stem cells to differentiate into vagal nerve crest cells, characterized in that, Includes the following steps: S1. Pluripotent stem cells, which are resuspended in the Stage I culture medium of the culture medium composition according to claim 1, are digested into single cells or cell clusters and then cultured in an adherent culture vessel; S2. After adhering culture for 1 to 4 days, replace with Stage II culture medium from the culture medium composition and culture for 1 to 10 days; S3. Replace with Stage III culture medium from the culture medium composition and culture for 2-10 days to obtain vagal crest cells.

8. A method for culturing intestinal mesenchymal stem cells, characterized in that, Vagus crest cells were obtained by adhering to and passaged in mesenchymal stem cell culture medium.

9. The method according to claim 8, characterized in that, The vagal crest cells are obtained according to the method described in claim 7.

10. The application of intestinal mesenchymal stem cells prepared by the method of claim 8 or 9, characterized in that, The application is one or more of the following: Prepared immunomodulatory drugs; To prepare drugs for treating intestinal damage; To prepare drugs for the treatment of inflammatory bowel disease; It acts as a trophoblast to promote organoid development.

Images