A method for in vitro induced differentiation of induced pluripotent stem cells to form intestinal smooth muscle cells

Abstract

The application relates to a method for forming intestinal smooth muscle cells by in-vitro induced differentiation based on induced pluripotent stem cells, which comprises the following steps: (1) induced culture: inoculating induced pluripotent stem cells into a well plate coated with basement membrane for incubation, and then performing induced culture after the incubation to obtain mature gastric-like mesoderm; (2) combined culture of cells: using a Transwell non-contact co-culture device, inoculating the gastric-like mesoderm obtained in the step (1) into a lower chamber of the Transwell device, inoculating primary human intestinal smooth muscle cells into an upper chamber of the Transwell device, and performing combined culture to obtain the intestinal smooth muscle cells. In the combined culture system, the adult primary human intestinal smooth muscle cells in the upper chamber and the gastric-like mesoderm cells in the lower chamber are completely physically isolated by a polyester porous membrane barrier, the possibility of spatial mixing or physical fusion of the two types of cells is completely cut off, and the purity of the cells obtained in the lower chamber is ensured.

Description

Technical Field

[0001] This application belongs to the field of microbial technology and relates to a method for in vitro induced differentiation of intestinal smooth muscle cells based on induced pluripotent stem cells. Background Technology

[0002] The intestines, as vital digestive and absorptive organs, rely on the synergistic effects of the epithelial barrier, immune homeostasis, and smooth muscle-driven dynamic processes for their complex physiological functions. Clinically, insufficient bowel resection or bypass surgery can lead to severe short bowel syndrome (SBS). This syndrome is characterized by malabsorption of nutrients, intractable diarrhea, and severe malnutrition, significantly impacting patients' quality of life and prognosis. Current traditional treatments for SBS include small bowel transplantation, enteral rehabilitation, drug therapy, and long-term parenteral nutrition. However, these methods are not only expensive but also face core challenges such as severe donor shortages, high risk of immune rejection, and the fact that most only slow disease progression rather than achieving a fundamental cure.

[0003] Intestinal tissue engineering is an emerging regenerative medicine strategy that reconstructs key structures and functions of the intestine in vitro using biomaterial scaffolds and human cells. However, current research largely focuses on constructing a single-layer intestinal mucosal epithelial barrier, with insufficient attention paid to the in vitro reconstruction of the intestinal muscular layer and its neuromuscular integration unit. The circular, longitudinal, and enteric nervous systems in the natural intestine work synergistically to regulate intestinal peristalsis and propulsion; abnormalities in the development or function of this system can lead to various serious intestinal diseases. Therefore, establishing an intestinal muscular layer model with three-dimensional structure and neural microenvironment regulation is of great significance for elucidating disease mechanisms, drug screening, and the clinical application of engineered intestinal tissues.

[0004] The key to constructing an intestinal muscular layer model lies in obtaining a sufficient quantity of high-quality, uniformly viable intestinal smooth muscle seed cells. Currently, these cells are mainly obtained directly from clinically surgically removed intestinal tissue. Although tissue-derived human intestinal smooth muscle cells (hISMCs) can be used to construct in vitro intestinal muscular layer models, they suffer from low proliferative capacity, significant donor variability, and phenotypic drift (conversion from contractile to synthetic phenotypes) during long-term culture, leading to loss of contractile function. These limitations hinder the application of hISMCs in large-scale, standardized intestinal muscular layer construction. Therefore, there is an urgent need to develop alternative sources of human smooth muscle cells that can follow in vivo developmental trajectories, be scalable, and phenotypically stable. Human induced pluripotent stem cells (iPSCs), with their unlimited self-renewal potential and multi-lineage differentiation potential, provide an ideal cell source for overcoming this bottleneck. iPSCs not only theoretically allow for unlimited expansion in vitro, thus avoiding the limitations of primary cell proliferation, but also maintain a highly consistent genetic background and stable differentiation potential, laying a solid cellular foundation for constructing standardized, high-throughput in vitro tissue engineering models. Furthermore, using iPSCs to prepare seed cells can effectively circumvent the ethical controversies and sample scarcity issues involved in obtaining clinical primary tissues. However, there are currently few reports of successfully inducing iPSCs to differentiate directly into hISMCs in vitro. Recent attempts have involved using a combination of small molecule factors to induce iPSCs to the gastric-like mesoderm (GM) stage, followed by transplantation back into mice, where the complex in vivo microenvironment is required for further induction into hISMCs. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this application is to provide a method for in vitro induced differentiation of induced pluripotent stem cells into intestinal smooth muscle cells.

[0006] To achieve this objective, the present application adopts the following technical solution: In a first aspect, this application provides a method for in vitro induced differentiation of intestinal smooth muscle cells based on induced pluripotent stem cells, the method comprising: (1) Induction culture: Induced pluripotent stem cells were seeded into well plates coated with base colloid and incubated. After incubation, they were induced to obtain mature gastric mesoderm. (2) Cell co-culture: Using the Transwell non-contact co-culture device, the gastric mesodermal cells obtained in step (1) were seeded into the lower chamber of the Transwell device, and the primary human intestinal smooth muscle cells were seeded into the upper chamber of the Transwell device for co-culture to obtain the intestinal smooth muscle cells.

[0007] This application creatively designs a method for inducing intestinal smooth muscle cells. First, induced pluripotent rod cells are induced to differentiate into gastric-like mesoderm. Then, the gastric-like mesoderm and intestinal smooth muscle cells are co-cultured in a Transwell apparatus. In this co-culture system, adult primary human intestinal smooth muscle cells (hISMCs) in the upper chamber and gastric-like mesoderm cells (GM) in the lower chamber are completely physically isolated by a porous polyester membrane barrier, completely eliminating the possibility of spatial mixing or physical fusion between the two cell types, ensuring the purity of the cells obtained from the lower chamber. However, healthy adult cells in the upper chamber continuously and relentlessly release endogenous soluble paracrine factors (such as the TGF-β family, PDGF, and Wnt pathway signaling molecules) with naturally occurring physiological proportions and complex compositions into the culture medium. These factors freely diffuse and exchange fluid across the membrane between the upper and lower chambers through the polyester membrane pores, thereby creating a highly efficient physiological-level microenvironment driving stimulation in the GM cell layer of the lower chamber. This application creatively introduces the Transwell non-contact co-culture system, which utilizes the natural physiological soluble paracrine signals continuously released by healthy adult hISMCs. Under the premise of completely severing the physical mixing and cell fusion of the two types of cells, it successfully drives GM to evolve into a functionally mature smooth muscle phenotype under pure cell culture conditions in vitro, thus opening up a complete route for the preparation of intestinal smooth muscle cells that is completely safe and autonomously closed in vitro.

[0008] In some embodiments, the culture medium used for incubation in step (1) is iPSC medium.

[0009] In some embodiments, the seeding density of the induced pluripotent stem cells in step (1) is 2 × 10⁻⁶. 4 ~3.5×10 4 cell / cm 2 (For example, it could be 2×10) 4 cell / cm 2 2.5×10 4 cell / cm 2 3×10 4 cell / cm 2 3.5×10 4 cell / cm 2 wait).

[0010] In some embodiments, the incubation temperature in step (1) is 35-40°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.), and the incubation time is 12-24 h (e.g., 12 h, 14 h, 16 h, 20 h, 24 h, etc.).

[0011] And / or, the induction culture temperature in step (1) is 35-40℃ (e.g., it can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.).

[0012] In some embodiments, the seeding density of primary human intestinal smooth muscle cells in step (2) is 1 × 10⁻⁶. 3 ~1×10 5 cell / cm 2 (For example, it could be 1×10) 3 cell / cm 2 1×10 4 cell / cm 2 1×10 5 cell / cm 2 wait).

[0013] In some embodiments, the temperature of the co-culture in step (2) is 35-40℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.), and the time is 6-8 days (e.g., 6 days, 7 days, 8 days, etc.).

[0014] In some embodiments, in the co-culture described in step (2), the culture medium used in the upper chamber and the lower chamber is independently a smooth muscle complete culture medium containing 8-12% (e.g., 8%, 9%, 10%, 11%, 12%, etc.) fetal bovine serum.

[0015] In some embodiments, the induction culture time in step (1) is 7-10 days (e.g., 7 days, 8 days, 9 days, 10 days, etc.).

[0016] In some embodiments, the induction culture in step (1) is divided into 5 stages, wherein the first stage lasts for 20-28 hours (e.g., 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, etc.), the second stage lasts for 20-28 hours (e.g., 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, etc.), the third stage lasts for 70-80 hours (e.g., 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, etc.), the fourth stage lasts for 45-50 hours (e.g., 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, etc.), and the fifth stage lasts for 20-28 hours (e.g., 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, etc.).

[0017] In some embodiments, the induction medium used in the five stages of the induction culture is specifically: In the first induction stage, the induction medium used included iPSC medium, and activin A, bone morphogenetic protein 4, CHIR99021, fibroblast growth factor 2 and PIK90 added to the iPSC medium. The second induction stage used an induction medium that included a basal medium and A8301, bone morphogenetic protein 4, Wnt-C59 and retinoic acid added to the basal medium. The third induction stage used an induction medium that included a basal medium and added A8301, bone morphogenetic protein 4, Wnt-C59, retinoic acid and fibroblast growth factor 2 to the basal medium. The fourth induction stage uses an induction medium that includes a basal medium and retinoic acid and phorbol ester added to the basal medium. The fifth induction stage uses an induction medium that includes a basal medium and retinoic acid, phorbol ester, and head protein added to the basal medium.

[0018] In some embodiments, the basal culture medium consists of Advanced DMEM / F12 and 1×B27, 1×N2, 10-20 mM HEPES (e.g., 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, etc.), 1-3 mM L-glutamine (e.g., 1 mM, 2 mM, 3 mM, etc.), 80-120 U / mL penicillin (e.g., 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, etc.), and 80-120 μg / mL streptomycin (e.g., 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, etc.).

[0019] In some embodiments, the induction medium used in the first induction stage consists of iPSC medium and the following components added thereto: 20-40 ng / mL activin A (e.g., 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, etc.), 30-50 ng / mL bone morphogenetic protein 4 (e.g., 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc.), 4-8 μM CHIR99021 (e.g., 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, etc.), 10-30 ng / mL fibroblast growth factor 2 (e.g., 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, etc.), and 80-120 nM PIK90 (e.g., 80 nM, 85 nM, 90 nM, etc.). nM, 95 nM, 100 nM, 110 nM, 120 nM, etc.).

[0020] In some embodiments, the induction medium used in the second induction stage consists of a basal medium and the following components added thereto: 0.5–2 μM A8301 (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, etc.), 20–40 ng / mL bone morphogenetic protein 4 (e.g., 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, etc.), 0.5–2 μM Wnt-C59 (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, etc.), and 1–3 μM retinoic acid (e.g., 1 μM, 2 μM, 3 μM, etc.).

[0021] In some embodiments, the induction medium used in the third induction stage consists of a basal medium and the following components added thereto: 0.5–2 μM A8301 (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, etc.), 20–40 ng / mL bone morphogenetic protein 4 (e.g., 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, etc.), 0.5–2 μM Wnt-C59 (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, etc.), 1–3 μM retinoic acid (e.g., 1 μM, 2 μM, 3 μM, etc.), and 10–30 ng / mL fibroblast growth factor 2 (e.g., 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, etc.).

[0022] In some embodiments, the induction medium used in the fourth induction stage consists of a basal medium and the following components added thereto: 1–3 μM retinoic acid (e.g., 1 μM, 2 μM, 3 μM, etc.) and 3–5 μM phorbol ester (e.g., 3 μM, 4 μM, 5 μM, etc.).

[0023] In some embodiments, the induction medium used in the fifth induction stage consists of a basal medium and the following components added thereto: 1–3 μM retinoic acid (e.g., 1 μM, 2 μM, 3 μM, etc.), 3–5 μM phorbol ester (e.g., 3 μM, 4 μM, 5 μM, etc.), and 180–220 ng / mL head protein (e.g., 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, etc.).

[0024] All other specific point values ​​not listed above within the numerical ranges mentioned above are optional and are all within the scope of protection of this application. For the sake of brevity, they will not be elaborated here.

[0025] Compared with the prior art, this application has the following beneficial effects: This application creatively designs a method for inducing intestinal smooth muscle cells. First, induced pluripotent rod cells are induced to differentiate into gastric-like mesoderm. Then, the gastric-like mesoderm and intestinal smooth muscle cells are co-cultured in a Transwell apparatus. In this co-culture system, adult primary human intestinal smooth muscle cells (hISMCs) in the upper chamber and gastric-like mesoderm cells (GM) in the lower chamber are completely physically isolated by a porous polyester membrane barrier, completely eliminating the possibility of spatial mixing or physical fusion between the two cell types, ensuring the purity of the cells obtained from the lower chamber. However, healthy adult cells in the upper chamber continuously and relentlessly release endogenous soluble paracrine factors (such as the TGF-β family, PDGF, and Wnt pathway signaling molecules) with naturally occurring physiological proportions and complex compositions into the culture medium. These factors freely diffuse and exchange fluid across the membrane between the upper and lower chambers through the polyester membrane pores, thereby creating a highly efficient physiological-level microenvironment driving stimulation in the GM cell layer of the lower chamber. This application creatively introduces the Transwell non-contact co-culture system, which utilizes the natural physiological soluble paracrine signals continuously released by healthy adult hISMCs. Under the premise of completely severing the physical mixing and cell fusion of the two types of cells, it successfully drives GM to evolve into a functionally mature smooth muscle phenotype under pure cell culture conditions in vitro, thus opening up a complete route for the preparation of intestinal smooth muscle cells that is completely safe and autonomously closed in vitro. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the in vitro induction of induced pluripotent stem cells to form intestinal smooth muscle cells according to this application. Figure 2 This image shows the results of cell morphology, ISL1 protein immunofluorescence, and RT-qPCR quantitative detection of key genes at different initial seeding densities induced to the third stage of differentiation in Test Example 1 of this application (Examples 1-3). The upper part of the image is a bright field image, the left side is an immunofluorescence image, and the right side of the image shows the relative mRNA expression levels of VIM, HOXA5, CYP26A1, FOXF1, GLI1, and PICH1 genes detected by RT-qPCR in the iPSC control group and differentiated cells at the three seeding densities. Figure 3 The images show the morphology of cells induced to the fifth stage of differentiation at different initial seeding densities in Examples 1-3 of this application, as well as the immunofluorescence results of CTNNB1 (β-catenin) protein and the RT-qPCR quantitative detection results of key genes. The upper part of the image is a bright field image, the left side is an immunofluorescence image, and the right side of the image shows the relative mRNA expression levels of VIM, FOXF1, NKX3-2, and MSC genes detected by RT-qPCR in the iPSC control group and the three seeding densities. Figure 4In Test Example 2 of this application, the expression levels of genes such as VIM, BARX1, NKX3-2, FOXF1 and MSC were detected by RT-qPCR in different PMA and Noggin concentration groups of Preparation Example 1 and Preparation Examples 4-6 to comprehensively evaluate the lineage specificity and population purity of each group. Figure 5 In Test Example 3 of this application, the immunofluorescence of characteristic proteins of cell lineages, Western blot of FOXF1 protein, and RT-qPCR quantitative detection results of characteristic genes at different differentiation time points of the induction system of Example 1 were used to verify the efficiency of the induction system in directing differentiation into GM cell lineages. The immunofluorescence imaging areas at the top are the cell fluorescence images of the second, third, and fifth induction stages, the Western blot protein blot image of the third induction stage is in the upper right corner, and the RT-qPCR bar chart is at the bottom. Figure 6 Bright-field images of cells induced by planar co-culture for 3 days, as shown in Comparative Example 1 of this application; Figure 7 The results of RT-qPCR quantitative detection and cell fluorescence detection were performed on the induction results of Example 1 in Test Example 5 of this application. The upper left of the figure is a schematic diagram of the Transwell co-culture device, the upper right of the figure is the RT-qPCR quantitative detection result, and the lower part of the figure is the fluorescence image of gastric mesoderm and intestinal smooth muscle cells. Detailed Implementation

[0027] To further illustrate the technical means and effects adopted in this application, the following describes the technical solution of this application in conjunction with preferred embodiments, but this application is not limited to the scope of the embodiments.

[0028] The iPSC culture medium used in the embodiments and comparative examples of this application was NcTarget. TM hPSC Medium (Catalog No.: RP01020, Anhui Shouning Biotechnology Co., Ltd., Hefei, China); the complete smooth muscle culture medium was prepared by adding 10% fetal bovine serum (FBS, Catalog No.: 10099-141C, Australian source, Gibco, USA) to DMEM / F-12 basal medium (Catalog No.: 11330-032, Gibco, USA).

[0029] Induced pluripotent stem cells were obtained from Qijia Technology Co., Ltd.; primary human intestinal smooth muscle cells were obtained from human intestinal tissue.

[0030] The tissue samples involved in this patent were provided by the Fourth Affiliated Hospital of Soochow University and have been approved by the hospital's ethics committee. All participants have signed written informed consent forms (ethics review number: 250039).

[0031] The complete flowchart of the in vitro induced differentiation of induced pluripotent stem cells into intestinal smooth muscle cells in this application is as follows: Figure 1 As shown.

[0032] Preparation Example 1 This preparation example provides a method for in vitro induced differentiation of induced pluripotent stem cells to form gastric-like mesoderm, the specific steps of which are as follows: (1) iPSC seeding: iPSC cells were seeded at a density of 2.5 × 10⁶ cells / year. 4 cell / cm 2 The samples were inoculated at a density of 100% into 24-well plates pre-coated with Matrigel substrate, using iPSC medium, and incubated at 37°C for 24 h. (2) Induction culture: After culturing iPSCs for 24 h, the original culture medium was discarded, and the cells were gently rinsed 3 times with preheated DMEM / F12 culture medium. Then, the culture medium was replaced to start the induction culture, as follows: Induction Phase 1: 30 ng / mL Activin A, 40 ng / mL BMP4, 6 μM CHIR99021, 20 ng / mL FGF2 and 100 nM PIK90 were added to iPSC medium as induction medium. After induction at 37℃ for 24 h, the medium was rinsed 3 times with preheated DMEM / F12 medium. Second induction stage: The induction medium was changed to basal medium supplemented with 1 μM A8301, 30 ng / mL BMP4, 1 μM C59, and 2 μM RA. After induction at 37°C for 24 h, the medium was rinsed three times with preheated DMEM / F12 medium. (Basal medium: prepared by adding 1× B27, 1× N2, 15 mM HEPES, 2 mM L-glutamine, and 1× penicillin-streptomycin to Advanced DMEM / F12; this medium was used for all differentiation cultures induction stages two through five.) The third stage of induction: change the induction medium, that is, add 1 μM A8301, 30 ng / mL BMP4, 1 μM C59, 20 ng / mL FGF2 and 2 μM RA to the basal medium as the induction medium, and induce at 37℃ for 72 h. Then wash 3 times with preheated DMEM / F12 medium. Fourth stage of induction: Change the induction medium, that is, add 2 μM RA and 4 μM PMA to the basal medium as the induction medium, induce at 37℃ for 48 h, and then rinse 3 times with preheated DMEM / F12 medium. Fifth stage of induction: Change the induction medium, that is, add 2 μM RA, 4 μM PMA and 200 ng / mL Noggin to the basal medium, and induce at 37℃ for 24 h to obtain mature gastric mesoderm.

[0033] Preparation Example 2 This preparation example provides a method for in vitro induced differentiation of induced pluripotent stem cells to form gastric mesoderm, which differs from Preparation Example 1 only in that the seeding density of iPSCs in step (1) is changed to 3.75 × 10⁻⁶. 4 cell / cm 2 All other conditions remain unchanged.

[0034] Preparation Example 3 This preparation example provides a method for in vitro induced differentiation of induced pluripotent stem cells to form gastric mesoderm. The only difference from preparation example 1 is that the seeding density of iPSCs in step (1) is changed to 5 × 10⁻⁶ cells. 4 cell / cm 2 All other conditions remain unchanged.

[0035] Preparation Example 4 This preparation example provides a method for in vitro induced differentiation of induced pluripotent stem cells to form gastric mesoderm. The only difference from preparation example 1 is that the concentration of PMA in the fourth and fifth induction stages in step (2) is changed to 2 μM, and the concentration of Noggin in the fifth stage is changed to 100 ng / mL, while other conditions remain unchanged.

[0036] Preparation Example 5 This preparation example provides a method for in vitro induction of intestinal smooth muscle cells based on induced pluripotent stem cells. The only difference from preparation example 1 is that the concentration of PMA in the fourth and fifth induction stages in step (2) is changed to 2 μM, while other conditions remain unchanged.

[0037] Preparation Example 6 This preparation example provides a method for in vitro induction of intestinal smooth muscle cells based on induced pluripotent stem cells. The only difference from preparation example 1 is that the concentration of Noggin in the fifth induction stage in step (2) is changed to 100 ng / mL, while all other conditions remain unchanged.

[0038] Test Example 1 This test case evaluates different inoculation densities in preparation examples 1-3; and evaluates cell morphology, gene expression levels, and protein expression levels based on the third and fifth induction stages.

[0039] When different initial inoculation densities were used in Preparation Examples 1-3 (Preparation Example 1: 2.5 × 10⁻⁶),4 cells / cm 2 Preparation Example 2: 3.75 × 10 4 cell / cm 2 Preparation Example 3: 5 × 10 4 cell / cm 2 At the end of the third induction phase, the cells from each preparation were subjected to immunofluorescence staining, and the results are as follows: Figure 2 As shown in the figure, the iPSC group was treated as follows: after passaging, it was allowed to proliferate normally until the fusion rate reached approximately 80%, at which point it was ready for further treatment. Figure 2 ISL1 was not expressed in the iPSC group, serving as a negative control.

[0040] Bright-field cell observation results showed that the number of induced cells increased with increasing iPSC seeding density. Immunofluorescence staining results showed that at this stage, the positive signal of ISL1 (a marker of cardiac mesodermal cells) was highest in the high-density group (3.75 × 10⁻⁶ cells / year). 4 and 5×10 4 cells / cm 2 DAPI-labeled nuclei showed significant expression in [the cell type], and the nuclei of DAPI-labeled cells exhibited good co-localization with ISL1 (red) fluorescence. In contrast, 2.5 × 10 [cells / cells / etc.] showed [significant expression]. 4 cells / cm 2 The low-density group showed lower ISL1 expression levels, a pattern consistent with the molecular characteristics of visceral mesoderm, where ISL1 is highly expressed in cardiac mesoderm but lowly expressed in pFG-SpM. Therefore, the low ISL1 expression in the low-density group indicates successful cell specialization into pFG-SpM, effectively preventing shift towards the cardiac mesoderm cell lineage.

[0041] RT-qPCR results showed that, compared with the iPSC control group, the relative expression levels of GM-related genes (VIM, HOXA5, CYP26A1, FOXF1) were significantly upregulated in all groups, while GLI1 and PITH1 genes were not expressed or expressed at extremely low levels. This result indicates that iPSCs have been successfully induced to differentiate into pFG-SpM, without deviating from the differentiation direction of cardiac mesoderm and anterior foregut splanchnic mesoderm (aFG-SpM). Specifically, HOXA5, FOXF1 (a key transcription factor in lateral mesoderm / gastric mesenchyme), and CYP26A1 (a retinoic acid metabolic enzyme) were significantly elevated in all groups, consistent with the molecular characteristics of pFG-SpM development.

[0042] When cells from Preparation Examples 1-3 were cultured at different initial seeding densities until the end of the fifth induction phase, immunofluorescence staining was performed on the cells from each preparation example. The results are as follows: Figure 3As shown.

[0043] The results showed that CTNNB1 (β-catenin) was significantly expressed in all three groups, and was mainly localized in the cell membrane region. This result indicates that the Wnt signaling pathway is activated during GM specialization. Specifically, 2.5 × 10⁻⁶ CTNNB1 was expressed in all three groups. 4 cells / cm 2 The CTNNB1 membrane localization was most clearly observed in group 1 (Preparation Example 1), indicating that the signaling pathway was activated under this density condition. RT-qPCR results showed that, compared to iPSCs, the relative expression levels of GM-related genes (VIM, FOXF1) were significantly upregulated in all groups of cells, but the gene NKX3-2, which should be highly expressed, was not expressed, indicating that the induction conditions need further optimization. At 2.5 × 10⁻⁶ 4 cells / cm 2 In this group, MSC expression was significantly downregulated, indicating that there was no shift towards a mesenchymal stem cell-like direction.

[0044] In 5×10 4 cells / cm 2 In this group, the expression levels of GM-related genes in MSCs were consistent with those in the iPSC group, indicating that high-density conditions may induce cells to shift towards a mesenchymal stem cell or fibroblast-like phenotype. This contradicts the requirements for inducing GM and may increase the risk of fibrosis or lead to differentiation heterogeneity in subsequent organoid construction.

[0045] Based on comprehensive morphological, protein level, and gene expression levels, 2.5 × 10⁻⁶ 4 cells / cm 2 Under the specified planting density, cells exhibited uniform morphology, adequate expression of key mesodermal markers (such as VIM, CYP26A1, and FOXF1), and minimal shift towards MSC-like cells, effectively balancing induction efficiency and cell fate specificity. Therefore, the 2.5 × 10⁻⁶ cells prepared in Example 1... 4 cells / cm 2 The planting density was determined to be the optimal inoculation density.

[0046] Test Example 2 This test case screens and evaluates different induction methods used in the fourth and fifth induction stages of Preparation Example 1 and Preparation Examples 4-6.

[0047] After induction, RT-qPCR was used to detect the expression levels of genes such as VIM, BARX1, NKX3-2, FOXF1, and MSC to comprehensively assess the lineage specificity and population purity of each group. The test results are as follows: Figure 4As shown in the figure, the iPSC group served as the control group in qPCR, i.e., labeled 1. The Normal group was the PMA and Noggin concentration reduction group of Preparation Example 4, the PMA group was the Noggin concentration reduction group of Preparation Example 6, the Noggin group was the PMA concentration reduction group of Preparation Example 5, and the PMA-NOGGIN group was Preparation Example 1 of this application.

[0048] RT-qPCR results showed that, compared to the iPSC group, VIM and FOXF1 maintained high expression levels across all induction groups, indicating that the optimized cells retained their basic visceral mesoderm identity. Among late-stage GM markers, NKX3-2 showed the most sensitive response to various optimization conditions, exhibiting the most significant expression differences. Compared to the iPSC group, the expression of NKX3-2 was most significantly upregulated in Preparation Example 1 (PMA-NOGGIN group), significantly higher than in the Normal group and both individually doubled groups. This result demonstrates that the combined enhancement of the two factors can effectively drive the induction system from the established visceral mesoderm state (induction stage 3) towards a more mature GM phenotype closer to the target.

[0049] In summary, simultaneously increasing the concentrations of PMA and NOGGIN in the culture medium during the fourth and fifth induction stages is the optimal cytokine optimization scheme, which can be applied to the subsequent establishment and large-scale expansion of the GM differentiation system.

[0050] Test Example 3 This test case systematically validated the lineage differentiation efficacy of the induction system prepared in Example 1. Validation was conducted from three aspects: the formation of PS, pFG-SpM, and GM characteristics; validation at the key protein level; and exclusion of lineage shifts in the terminal stage. The feasibility of this induction system for constructing a GM cell population was assessed, and the test results are as follows: Figure 5 As shown.

[0051] Immunofluorescence staining results showed that TBXT exhibited a significant positive staining signal after the second stage of differentiation induction, indicating that the original stripes were successfully induced.

[0052] RT-qPCR results showed that, compared with the iPSC control group, cells induced in the third stage underwent significant transcriptional remodeling: (1) Enhanced cytoskeleton and migration-related programs: VIM expression was significantly upregulated, indicating that cells began to transition from a pluripotent state to a mesodermal state. This is a common manifestation of cytoskeleton remodeling and enhanced migration-related programs after mesodermal induction; (2) Establishment of foregut visceral mesodermal identity: HOXA5 and CYP26A1 are typical retinoic acid response genes. During the establishment of pFG-SpM, they increased with the RA-mediated anterior-posterior axis patterning, which is important molecular evidence that cells further shift from visceral mesodermal to foregut-associated visceral mesodermal. The significant increase in HOXA5 and CYP26A1 expression levels indicates that the induction system successfully established the basic identity of the foregut visceral mesoderm; (3) Visceral mesoderm lineage orientation: In multiple developmental studies, it has been confirmed that it is closely related to the visceral mesoderm or lateral plate mesoderm lineage and its mesenchymal characteristics, indicating that the cell population has been further oriented to the visceral mesoderm-related branches based on the mesoderm; (4) Inhibition of the Hedgehog signaling pathway: The decreased expression levels of GLI1 and PICH1 indicate that the transcription downstream of the Hedgehog signaling pathway was inhibited, and the cell development direction did not shift towards aFG-SpM. Therefore, the downregulation of GLI1 and PICH1 corroborates that the system is in a dynamic process of advancing from the mesoderm to pFG-SpM.

[0053] Immunofluorescence staining results showed that VIM exhibited a significant positive staining signal and a reticular fiber structure after the third stage of induced differentiation, indicating that the cells had acquired mesenchymal characteristics. Low expression of ISL1 indicated that the cells successfully differentiated into the pFG-SpM fate, effectively avoiding the shift towards the cardiac mesodermal cell lineage.

[0054] To avoid potential biases from relying solely on gene-level analysis, this study further employed Western blotting (WB) to detect the protein expression level of FOXF1. The results showed that the FOXF1 protein band was weak or invisible in the iPSC control group, while a clear, specific band (with β-actin as the internal control) appeared in the samples from the third induction stage. This result confirms that FOXF1 is not only upregulated at the mRNA level but also effectively expressed as a protein, indicating that the relevant transcriptional program was successfully executed within the cell. In establishing the differentiation system, validation at the protein level has more direct biological significance. Firstly, it demonstrates that the induction signal is sufficient to drive the translation of key transcription factors and ensure stable protein expression. Secondly, it signifies that the downstream gene regulatory network has the foundation for activation, thereby improving the reproducibility and translational application value of the induction system.

[0055] RT-qPCR results at the fifth stage of induction showed that VIM, BARX1, NKX3-2, and FOXF1 continued to be upregulated, while MSC expression levels decreased significantly. During the differentiation of pluripotent stem cells into mesenchymal-associated lineages, improper signal control can easily lead to the proliferation of cells into broad-spectrum MSC-like cells. The significant decrease in MSCs in this system indicates that the induction process did not simply produce "generalized MSCs," but rather successfully formed a GM cell population with a specific developmental identity. CTNNB1, as a core molecule in cell connectivity and the Wnt signaling pathway, often reflects the structural integrity of the cell population, intercellular adhesion, and the activity of related signaling pathways through its distribution characteristics on the cell membrane. At the fifth stage of induction, CTNNB1 showed significant fluorescence staining, exhibiting clear cellular localization, and the fluorescence signal was continuous, indicating that the cell population obtained at the final stage of induction possessed tissue-like structures and intercellular connectivity characteristics.

[0056] In summary, through optimization of the induction system, a cell population exhibiting GM characteristics was successfully obtained: early TBXT positivity confirmed the origin of mesodermal development; changes in the mRNA level of genes such as FOXF1 and FOXF1 protein expression in the third stage confirmed the establishment of pFG-SpM; and the immunofluorescence results of CTNNB1 and the decrease in MSC gene expression in the fifth stage provided evidence of "structural stability" and "exclusion of non-target deviations." These results are consistent with the developmental trajectories of PS, pFG-SpM, and GM reported in the literature, providing a mature GM system for the subsequent construction of smooth muscle cells.

[0057] Example 1 This embodiment provides a method for in vitro induced differentiation of intestinal smooth muscle cells based on induced pluripotent stem cells, the specific steps of which are as follows: (1) Using the Transwell non-contact co-culture device, gastric mesoderm was induced to differentiate in the lower chamber of the Transwell device according to the induction method of Preparation Example 1; (2) Healthy, stable-expansion-phase adult primary human intestinal smooth muscle cells isolated from human clinical intestinal tissue were subjected to a 1×10⁻⁶ PCR reaction. 4 Cells were seeded at a density per well on the inner surface of the upper chamber polyester porous membrane of a Transwell. The upper and lower chambers were simultaneously cultured in a smooth muscle complete medium containing 10% fetal bovine serum (FBS). Fresh medium was replaced every 24 hours. After culturing at 37°C for 7 days, the intestinal smooth muscle cells were obtained.

[0058] Comparative Example 1 This comparative example provides a method for in vitro induced differentiation of intestinal smooth muscle cells based on induced pluripotent stem cells, the specific steps of which are as follows: After counting the gastric mesodermal cells induced from differentiation in Example 1, they were seeded with primary human intestinal smooth muscle cells at a 1:1 ratio in 24-well plates and co-cultured in complete smooth muscle medium containing 10% fetal bovine serum (FBS). The medium was replaced with fresh medium every 24 hours and cultured at 37°C for 7 days, while keeping other conditions constant.

[0059] Test Example 4 This test case evaluates the induction method used in Comparative Example 1.

[0060] Planar co-culture was performed using the method described in Comparative Example 1. A bright-field photograph taken on the third day of culture is shown below. Figure 6 As shown in the figure. The results showed that on the third day of planar co-culture, spindle-shaped intestinal smooth muscle cells filled the entire plate, and gastric mesodermal cells were competed for and unable to successfully differentiate into intestinal smooth muscle cells. The above results indicate that planar co-culture cannot achieve the technical effect achieved by Transwell co-culture as described in Example 1 of this application.

[0061] Test Example 5 This test case verifies the induction results of Example 1.

[0062] The results of induced differentiated gastric mesoderm and intestinal smooth muscle cells were detected by RT-qPCR and cell fluorescence imaging. The test results are as follows: Figure 7 As shown, after 7 days of continuous co-culture with exogenous paracrine stimulation, the GM cells in the lower chamber completely detached from their ordinary primary mesenchymal or myofibroblast state, undergoing a profound transformation in cell fate. Morphologically, the cells were completely remodeled and stretched from flattened, disordered roundness into a typical long spindle-shaped, highly parallel smooth muscle-like fibrous microfilament framework. In terms of molecular and protein spatial phenotype, not only were the early markers α-SMA and SM22 strongly expressed positively, but the high-fidelity dense assembly and spatial parallel arrangement of MYH11 (smooth muscle myosin heavy chain) and CNN1 (Calponin), the gold standard indicators for determining the maturity of functional contractile smooth muscle, were also achieved, giving them true mechanical contractile potential and completing the induction from pluripotent stem cells into fully human, scalable, and functionally mature intestinal smooth muscle seed cells.

[0063] The applicant declares that this application illustrates the technical solution of this application through the above embodiments, but this application is not limited to the above embodiments, that is, it does not mean that this application must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of this application's products, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

[0064] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0065] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. A method for in vitro induction of intestinal smooth muscle cells based on induced pluripotent stem cells, characterized in that, The method includes: (1) Induction culture: Induced pluripotent stem cells were seeded into well plates coated with base colloid and incubated. After incubation, they were induced to obtain mature gastric mesoderm. (2) Cell co-culture: Using the Transwell non-contact co-culture device, the gastric mesodermal cells obtained in step (1) were seeded into the lower chamber of the Transwell device, and the primary human intestinal smooth muscle cells were seeded into the upper chamber of the Transwell device for co-culture to obtain the intestinal smooth muscle cells.

2. The method according to claim 1, characterized in that, The culture medium used for incubation in step (1) is iPSC medium.

3. The method according to claim 1 or 2, characterized in that, The seeding density of the induced pluripotent stem cells in step (1) is 2 × 10⁻⁶. 4 ~3.5×10 4 cell / cm 2 .

4. The method according to any one of claims 1-3, characterized in that, The incubation temperature in step (1) is 35-40℃, and the incubation time is 12-24 h; And / or, the induction culture temperature in step (1) is 35-40℃.

5. The method according to any one of claims 1-4, characterized in that, The seeding density of primary human intestinal smooth muscle cells in step (2) is 1×10⁻⁶. 3 ~1×10 5 cell / cm 2 .

6. The method according to any one of claims 1-5, characterized in that, The temperature for the co-culture in step (2) is 35-40℃, and the time is 6-8 days; And / or, in the co-culture described in step (2), the culture medium used in the upper and lower chambers is independently a smooth muscle complete culture medium containing 8-12% fetal bovine serum.

7. The method according to any one of claims 1-6, characterized in that, The induction culture time in step (1) is 7-10 days; And / or, the induction culture described in step (1) is divided into 5 stages, wherein the first stage lasts for 20-28 h, the second stage lasts for 20-28 h, the third stage lasts for 70-80 h, the fourth stage lasts for 45-50 h, and the fifth stage lasts for 20-28 h.

8. The method according to claim 7, characterized in that, The induction medium used in the five stages of the induction culture is specifically as follows: In the first induction stage, the induction medium used included iPSC medium, and activin A, bone morphogenetic protein 4, CHIR99021, fibroblast growth factor 2 and PIK90 added to the iPSC medium. The second induction stage used an induction medium that included a basal medium and A8301, bone morphogenetic protein 4, Wnt-C59 and retinoic acid added to the basal medium. The third induction stage used an induction medium that included a basal medium and added A8301, bone morphogenetic protein 4, Wnt-C59, retinoic acid and fibroblast growth factor 2 to the basal medium. The fourth induction stage uses an induction medium that includes a basal medium and retinoic acid and phorbol ester added to the basal medium. The fifth induction stage uses an induction medium that includes a basal medium and retinoic acid, phorbol ester, and head protein added to the basal medium.

9. The method according to claim 8, characterized in that, The basal culture medium consists of Advanced DMEM / F12 and 1× B27, 1× N2, 10-20 mM HEPES, 1-3 mM L-glutamine, 80-120 U / mL penicillin, and 80-120 μg / mL streptomycin added thereto.

10. The method according to claim 8, characterized in that, The induction medium used in the first induction stage consists of iPSC medium and the following components added thereto: 20-40 ng / mL activin A, 30-50 ng / mL bone morphogenetic protein 4, 4-8 μM CHIR99021, 10-30 ng / mL fibroblast growth factor 2 and 80-120 nM PIK90; And / or, the induction medium used in the second induction stage consists of a basal medium and the following components added thereto: 0.5–2 μM A8301, 20–40 ng / mL bone morphogenetic protein 4, 0.5–2 μM Wnt-C59 and 1–3 μM retinoic acid; And / or, the induction medium used in the third induction stage consists of a basal medium and the following components added thereto: 0.5–2 μM A8301, 20–40 ng / mL bone morphogenetic protein 4, 0.5–2 μM Wnt-C59, 1–3 μM retinoic acid and 10–30 ng / mL fibroblast growth factor 2; And / or, the induction medium used in the fourth induction stage consists of a basal medium and the following components added thereto: 1–3 μM retinoic acid and 3–5 μM phorbol ester; And / or, the induction medium used in the fifth induction stage consists of a basal medium and the following components added thereto: 1–3 μM retinoic acid, 3–5 μM phorbol ester and 180–220 ng / mL head protein.

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