Methods for the production of sound-sensitive stem cell-derived beta functional cells and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to a preparation method of sound-sensitive stem cell-derived beta functional cells and application thereof. The preparation method provided by the present application introduces a prokaryote-derived engineered mechanosensitive ion channel MscL-G22S into human pluripotent stem cells, and combines with a directional differentiation technology to obtain pancreatic islet beta-like cells with ultrasonic response function. The cell product not only has a natural glucose-dependent insulin secretion capacity, but also has an additional function of being activated by low-intensity focused ultrasound in a non-invasive manner through the introduction of an exogenous ion channel. This double regulation mechanism significantly improves the functional maturity and response sensitivity of the transplanted cells in vivo, and can achieve a better blood glucose control effect under the same number of cells, thereby providing a technical possibility for reducing the amount of transplanted cells, reducing the volume of transplanted cells and expanding the transplanted site, and being suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing β-functional cells derived from acoustically sensitive stem cells and their applications. Background Technology

[0002] Type 1 diabetes is a chronic metabolic disease caused by the destruction of pancreatic beta cells by an autoimmune system, requiring lifelong exogenous insulin therapy. In recent years, beta cell transplantation derived from stem cells has been considered a promising curative strategy. Inducing human pluripotent stem cells to differentiate into pancreatic beta cells in vitro can address the shortage of donor cells. However, existing stem cell-derived beta cells still have functional limitations, including: slow response to glucose stimulation and insufficient insulin secretion; lack of physiological regulatory mechanisms, hindering precise and timely blood glucose control; and the current technology primarily relies on chemical induction, lacking external physical regulation methods, limiting the ways to enhance function and thus restricting clinical therapeutic effects.

[0003] Currently used methods for directed differentiation of stem cells into pancreatic β cells typically employ a multi-stage growth factor induction strategy. For example, they first induce differentiation into endoderm cells, then into pancreatic progenitor cells, and finally into pancreatic β cells. While these methods can produce cells expressing insulin and C-peptide, their secretory function remains significantly lower than that of normal human pancreatic β cells, particularly in dynamic glycemic regulation. Furthermore, there is currently no systematic method in the art to enhance the function of stem cell-derived β cells through physical stimulation. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing β-functional cells derived from acoustically sensitive stem cells and its application. The present invention provides a method for preparing β-functional cells derived from acoustically sensitive stem cells that can respond to ultrasound stimulation and enhance insulin secretion by combining acoustic genetics technology with stem cell directed differentiation technology.

[0005] This invention provides a method for preparing β-functional cells derived from acoustically sensitive stem cells, comprising the following steps:

[0006] Step 1: Introduce the vector expressing the high-conductivity mechanosensitive ion channel MscL-G22S into pluripotent stem cells to construct and obtain acoustically sensitive pluripotent stem cells;

[0007] Step 2: Take the acoustically sensitive pluripotent stem cells obtained in Step 1 and induce their differentiation through endoderm to obtain endoderm cells;

[0008] Step 3: Take the endoderm cells obtained in Step 2 and induce them to differentiate into pancreatic progenitor cells to obtain pancreatic progenitor cells;

[0009] Step 4: Take the pancreatic progenitor cells obtained in Step 3 and perform endocrine-directed induction and early maturation culture to obtain endocrine precursor cells;

[0010] Step 5: Take the endocrine precursor cells described in Step 4, perform three-dimensional aggregation culture, and then culture in terminal maturation medium to obtain β-functional cells derived from acoustic-sensitive stem cells.

[0011] In some embodiments, in step 1, the vector of the high-conductivity mechanosensitive ion channel MscL-G22S comprises a viral vector having at least one of a promoter, a fluorescent protein tag encoding gene, and an resistance gene.

[0012] In some embodiments, the promoter is selected from any one of the CAG promoter, EF1α promoter, CMV promoter, PGK promoter, UBC promoter, and Tet-On / Off inducible promoter;

[0013] The fluorescent protein tag encoding gene is selected from any one of EGFP, mCherry, tdTomato, EYFP, and ZsGreen1;

[0014] The resistance gene is selected from any one of the following: puromycin resistance gene, hygromycin resistance gene, bleomycin resistance gene, and blast fungicide resistance gene.

[0015] In some specific embodiments, the promoter is the CAG promoter, the fluorescent protein tag encoding gene is EGFP, and the resistance gene is the puromycin resistance gene.

[0016] In some embodiments, in step 2, the initial seeding density of the acoustically sensitive pluripotent stem cells is 60% to 70%.

[0017] The time for inducing differentiation of the endoderm is 3-6 days;

[0018] The culture medium for inducing endoderm differentiation includes 50% IMDM, 50% Ham's F-12, 1×ITS-A, 400~500 μM Monothioglycerol, 3~10 mg / mL Albumin Fraction V, and 25~60 ng / mL Activin A.

[0019] In some embodiments, in step 2, the endoderm differentiation is induced for 4 to 5 days;

[0020] The culture medium for inducing endoderm differentiation included 50% IMDM, 50% Ham's F-12, 1×ITS-A, 425~475 μM Monothioglycerol, 4~6 mg / mL Albumin Fraction V, and 40~55 ng / mL Activin A.

[0021] In some specific embodiments, in step 2, the endoderm differentiation induction time is 5 days;

[0022] The culture medium for inducing endoderm differentiation included 50% IMDM, 50% Ham's F-12, 1×ITS-A, 450 μMmonothioglycerol, 5 mg / mL Albumin Fraction V, and 50 ng / mL Activin A.

[0023] In some embodiments, in step 3, the pancreatic progenitor cells are induced to differentiate for 3 to 6 days;

[0024] The culture medium for inducing differentiation of pancreatic progenitor cells includes 50% IMDM, 50% Ham's F-12, 1×ITS-A, 400~500 μM Monothioglycerol, 3~10 mg / mL Albumin Fraction V and 0.25~1.5 μM Metinoic Acid.

[0025] In some embodiments, in step 3, the pancreatic progenitor cells are induced to differentiate for 4 to 5 days;

[0026] The culture medium for inducing differentiation of pancreatic progenitor cells includes 50% IMDM, 50% Ham's F-12, 1×ITS-A, 425~475 μM Monothioglycerol, 4~6 mg / mL Albumin Fraction V and 0.5~1.25 μM Metinoic Acid.

[0027] In some embodiments, in step 3, the pancreatic progenitor cells are induced to differentiate for 4 days;

[0028] The culture medium for inducing differentiation of pancreatic progenitor cells included 50% IMDM, 50% Ham's F-12, 1×ITS-A, 450 μM Monothioglycerol, 5 mg / mL Albumin Fraction V, and 1 μM Retinoic Acid.

[0029] In some embodiments, in step 4, the endocrine-directed induction and early maturation culture includes: taking the pancreatic progenitor cells and culturing them in pancreatic islet cell maturation culture medium 1 for 2-5 days, and then culturing them in pancreatic islet cell maturation culture medium 2 for 1-3 days;

[0030] The pancreatic islet cell maturation culture medium 1 comprises DMEM / F-12, 1×ITS-A, 5~15 ng / mL bFGF and 1~3 mg / mL Albumin Fraction V;

[0031] The pancreatic islet cell maturation culture medium 2 includes DMEM / F-12, 1×ITS-A, 5~15 mM Nicotinamide and 1~3 mg / mL Albumin Fraction V.

[0032] In some embodiments, in step 4, the endocrine-directed induction and early maturation culture includes: taking the pancreatic progenitor cells and culturing them in pancreatic islet cell maturation culture medium 1 for 3-4 days, and then culturing them in pancreatic islet cell maturation culture medium 2 for 1-2 days;

[0033] The islet cell maturation culture medium 1 comprises DMEM / F-12, 1×ITS-A, 7.5~12.5 ng / mL bFGF and 1.5~2.5 mg / mL Albumin Fraction V;

[0034] The pancreatic islet cell maturation culture medium 2 comprises DMEM / F-12, 1×ITS-A, 7.5~12.5 mM Nicotinamide, and 1.5~2.5 mg / mL Albumin Fraction V.

[0035] In some specific embodiments, step 4, the endocrine-directed induction and early maturation culture includes: taking the pancreatic progenitor cells, culturing them in islet cell maturation culture medium 1 for 3 days, and then culturing them in islet cell maturation culture medium 2 for 2 days;

[0036] The pancreatic islet cell maturation culture medium 1 comprises DMEM / F-12, 1×ITS-A, 10 ng / mL bFGF and 2 mg / mL Albumin Fraction V;

[0037] The pancreatic islet cell maturation medium 2 comprises DMEM / F-12, 1×ITS-A, 10 mM Nicotinamide and 2 mg / mL Albumin Fraction V.

[0038] In some embodiments, in step 5, the three-dimensional aggregation culture includes taking the endocrine precursor cells, digesting them, and then seeding the resulting cell clumps in a low-adhesion culture container and culturing them in pancreatic islet cell maturation medium 2 for 6-10 days.

[0039] The pancreatic islet cell maturation culture medium 2 includes DMEM / F-12, 1×ITS-A, 5~15 mM Nicotinamide and 1~3 mg / mL Albumin Fraction V.

[0040] In some embodiments, in step 5, the terminal maturation medium is pancreatic islet cell maturation medium 2, and the culture time in the terminal maturation medium is 6 to 10 days;

[0041] The pancreatic islet cell maturation medium 2 comprises DMEM / F-12, 1×ITS-A, 10 mM Nicotinamide and 2 mg / mL Albumin Fraction V.

[0042] In some embodiments, step 5, the three-dimensional aggregation culture includes taking the endocrine precursor cells, digesting them, and then seeding the resulting cell clumps in a low-adhesion culture container and culturing them in pancreatic islet cell maturation medium 2 for 7-9 days;

[0043] The terminal maturation medium is pancreatic islet cell maturation medium 2, and the culture time in the terminal maturation medium is 7-9 days;

[0044] The pancreatic islet cell maturation culture medium 2 comprises DMEM / F-12, 1×ITS-A, 7.5~12.5 mM Nicotinamide, and 1.5~2.5 mg / mL Albumin Fraction V.

[0045] In some specific embodiments, in step 5, the three-dimensional aggregation culture includes taking the endocrine precursor cells, digesting them, and then seeding the resulting cell clumps in a low-adhesion culture container and culturing them in pancreatic islet cell maturation medium 2 for 8 days.

[0046] The terminal maturation medium is pancreatic islet cell maturation medium 2, and the culture time in the terminal maturation medium is 7 days;

[0047] The pancreatic islet cell maturation medium 2 comprises DMEM / F-12, 1×ITS-A, 10 mM Nicotinamide and 2 mg / mL Albumin Fraction V.

[0048] In some embodiments, the pluripotent stem cells include human pluripotent stem cells.

[0049] This invention provides the application of acoustically sensitive stem cell-derived β-functional cells prepared by the aforementioned method in the preparation of drugs for the prevention and treatment of diabetes.

[0050] This invention provides a drug for the prevention and treatment of diabetes, comprising acoustically sensitive stem cell-derived β-functional cells prepared by the method described above, and pharmaceutically acceptable excipients.

[0051] The present invention provides a method for preventing and treating diabetes, comprising: based on the drug.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] 1. The preparation method provided by this invention introduces the engineered mechanosensitive ion channel MScL-G22S from prokaryotes into human pluripotent stem cells and combines it with directed differentiation technology to prepare pancreatic β-like cells with ultrasound-responsive function. This cell product not only possesses the natural glucose-dependent insulin secretion capacity, but also acquires additional functions that can be non-invasively activated by low-intensity focused ultrasound through the introduction of exogenous ion channels. This dual regulatory mechanism significantly enhances the functional maturity and responsiveness of transplanted cells in vivo, achieving better glycemic control with the same number of cells. It provides technical possibilities for reducing the amount of transplanted cells, decreasing transplant volume, and expanding transplant sites, making it suitable for widespread application.

[0054] 2. The preparation method provided by this invention includes a complete "gene editing-directed differentiation-functional regulation" technology chain, achieving standardization of the entire process from vector construction to end product. Compared with existing cell regulation technologies such as optogenetics and chemogenetics, the ultrasound regulation mode adopted in this invention has significant advantages in clinical application: ultrasound can penetrate deep tissues, eliminating the need for implanting optical fibers or long-term administration of exogenous chemical inducers, fundamentally avoiding the infection risks associated with invasive procedures and the safety issues caused by long-term drug use. Furthermore, compared with traditional stem cell-derived β-cell therapy, the cell products of this invention exhibit superior insulin secretion kinetics in both in vivo and in vitro functional verification, providing an effective technical path to solve the industry problem of insufficient in vivo functional maturity of differentiated stem cells. Attached Figure Description

[0055] Figure 1 This image shows the morphological features of the initial hiPSC after resuscitation in Example 1 (4X).

[0056] Figure 2 The image shows the fluorescence morphology of hiPSCs after lentivirus transfection in Example 1 (40X). The left image is ipsc-GFP-PURO (control group), and the right image is ipsc-MSCL-GFP-PURO (experimental group).

[0057] Figure 3 The image shows the morphology of hiPSCs after puromycin screening in Example 1 (4X), where from left to right are Control, ipsc-GFP-PURO and ipsc-MSCL-GFP-PURO;

[0058] Figure 4 The fluorescence morphology (40X) of the purified monoclonal antibody in Example 1 is shown. The left image is ipsc-GFP-PURO (control group), and the right image is ipsc-MSCL-GFP-PURO (experimental group).

[0059] Figure 5 The fluorescence morphology (40X) of the monoclonal amplification in Example 1 is shown. The left image is ipsc-GFP-PURO (control group), and the right image is ipsc-MSCL-GFP-PURO (experimental group).

[0060] Figure 6 The diagram shows a comparison of cell growth status after 1 to 10 days of culture at different initial densities in Example 2. The left column represents the low-density group, and the right column represents the suitable density group. Detailed Implementation

[0061] This invention provides a method for preparing β-functional cells derived from acoustically sensitive stem cells and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0062] The present invention, “Preparation method of β-functional cells derived from acoustically sensitive stem cells,” is an interdisciplinary systematic solution integrating synthetic biology, developmental biology, ultrasound engineering, and transplantation medicine.

[0063] 1. Summary of core innovations in the technical solution

[0064] A creative combination of target and tool: For the first time, the mechanically sensitive ion channel MscL-G22S, derived from prokaryotes and engineered, was stably introduced into human pluripotent stem cells and successfully applied to the preparation of therapeutic cell products.

[0065] The closed-loop design of the process route: from gene vector design, viral packaging, stem cell editing, directed differentiation, to functional quality control and end product preparation, a standardized and scalable complete process flow has been formed.

[0066] The non-invasive and precise nature of the regulation mode: By using low-intensity focused ultrasound as an external regulatory switch, deep tissue, non-invasive, and spatiotemporally precise remote control of transplanted cell function is achieved. This is a core feature that distinguishes it from any chemical or gene-based internal regulation method.

[0067] A revolutionary treatment philosophy: Moving from static cell replacement and supplementation to intelligent cell replacement, supplementation, and dynamic regulation. This allows doctors or patients to activate cells to secrete more insulin "on demand" via external devices based on real-time blood glucose levels, achieving personalized and dynamic blood glucose management.

[0068] 2. Significant advantages compared to existing technologies

[0069] Compared to traditional stem cell differentiation into β cells, this method addresses the issues of low functional maturity and slow response in vivo. With ultrasound enhancement, it can reduce the number of cells required for a cure, thus reducing limitations on transplant volume and location.

[0070] Compared to other cell function regulation technologies (such as optogenetics and chemogenetics), ultrasound has excellent tissue penetration capabilities (centimeter level), does not require the implantation of optical fibers or long-term administration of exogenous drugs, and has significant advantages in non-invasiveness and ease of clinical application.

[0071] Compared to medication or insulin pump therapy alone, it provides a physiological, glucose-dependent basis for insulin secretion. Combined with on-demand ultrasound enhancement, it is expected to achieve more stable blood glucose control with less risk of hypoglycemia.

[0072] 3. Safety and Forward-looking Considerations

[0073] Security:

[0074] Gene safety: Using self-inactivating lentiviruses, the risk of insertional mutations is low. MscL-G22S is an ion channel that does not interfere with core cellular metabolic pathways.

[0075] Ultrasound safety: The low-intensity focused ultrasound parameters used are within the diagnostic ultrasound energy range, and numerous studies have confirmed their safety for biological tissues.

[0076] Cell safety: Strict cell quality control (sterility, mycoplasma, karyotype) ensures the safety of the final product.

[0077] Forward-looking applications:

[0078] This technology platform can be extended to other cell therapies that require modulated secretory functions, such as dopaminergic neurons (regulating neurotransmitter release) for Parkinson's disease and coagulation factor secreting cells for hemophilia.

[0079] By combining wearable ultrasound devices with continuous glucose monitoring systems, a truly "artificial intelligence biological pancreas" could be built in the future, achieving closed-loop, fully automated glucose regulation.

[0080] In summary, this invention not only provides a detailed and feasible technical solution, but also demonstrates its scientific validity, effectiveness, and innovation through multi-level empirical data (from molecules and cells to whole animals), laying a solid foundation for the development of next-generation intelligent cell therapy.

[0081] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0082] Example 1: Establishment of a stable human pluripotent stem cell line expressing the mechanosensitive ion channel MScL-G22S

[0083] This embodiment forms the basis of the entire technology, with the goal of obtaining "sound-sensitive seed cells" that are genotype-stable, phenotype-uniform, and free from contamination.

[0084] 1. Technical Principles and Carrier Design

[0085] Selection of acoustic-sensitive mediator: MscL-G22S was selected as the acoustic-sensitive effector. MscL (high-conductivity mechanosensitive channel) is derived from *E. coli*, and its G22S point mutant (glycine at position 22 replaced with serine) has been shown in multiple studies to significantly enhance its sensitivity to mechanical forces (including ultrasonic radiation). Furthermore, it is safe to express in mammalian cells and is not easily desensitized by continuous stimulation. The nucleotide sequence of MscL-G22S is shown at https: / / www.addgene.org / 107455 / sequences / .

[0086] Gene editing strategy: Lentiviral transduction is used to achieve stable integration and long-term expression of genes. This method is highly efficient, suitable for difficult-to-transfect human pluripotent stem cells, and can achieve continuous expression of exogenous genes during differentiation.

[0087] Vector design: Two lentiviral expression vectors were constructed: the experimental vector (pLV-MscL-G22S-GFP-PURO): containing a CAG promoter-driven fusion gene of MscL-G22S and Enhanced Green Fluorescent Protein (EGFP), and an independent puromycin resistance gene (PURO). The control vector (pLV-GFP-PURO): containing only the GFP and PURO genes, used to exclude nonspecific effects.

[0088] Viral Packaging: The above-described vector, along with packaging plasmid psPAX2 and envelope plasmid pMD2.G, was co-transfected into HEK293T cells (cultured in DMEM + 10% FBS). Supernatant containing viral particles was collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm filter, and concentrated using ultracentrifugation (e.g., 50,000 × g, 2 hours, 4°C). The virus was then resuspended in an appropriate amount of PBS. The viral titer was determined using an infection titer assay (measured on HT1080 cells), with a target titer ≥ 1 × 10^8 TU / mL.

[0089] 2. Human pluripotent stem cell culture and preparation

[0090] Cell lines: Commercially available or laboratory-developed human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) can be used. This protocol uses hiPSCs as an example.

[0091] Training system:

[0092] Culture medium: Use a well-defined, heterologous hiPSC-specific culture medium, such as mTeSR™1 or StemFlex™.

[0093] Matrix: Coat the culture surface with Matrigel® (hESC-qualified, Corning). Procedure: Melt Matrigel on ice, dilute with pre-chilled DMEM / F-12 medium at a ratio of 1:100, and evenly cover the surface of the culture dish (1 mL per well in a 6-well plate). Incubate at room temperature for at least 1 hour. Aspirate excess liquid before use; drying is not required.

[0094] Standard passage: Perform when cell confluence reaches 80-90%. Discard the old culture medium and gently wash once with D-PBS free of Ca²⁺ / Mg²⁺. Add an appropriate amount of ReLeSR™ or a similar mild dissociation reagent (e.g., 0.5 mM EDTA / PBS) and incubate at room temperature for 5-7 minutes until the cell edges curl up. Discard the dissociation solution, add fresh culture medium, and gently pipette the cells into small clumps of approximately 50-200 μm using a pipette tip or cell scraper. Transfer the cell clump suspension to centrifuge tubes, allow them to stand briefly to allow larger clumps to settle, and seed suitable clumps from the supernatant into new Matrigel-coated plates at a ratio of 1:6 to 1:20. Within 24 hours after passage, add 10 μM Y-27632 (ROCK inhibitor) to the culture medium to reduce apoptosis; after 24 hours, replace with regular culture medium.

[0095] 3 Lentiviral transduction and establishment of stable cell lines

[0096] Preliminary experiments - determining optimal infection conditions:

[0097] The hiPSCs were digested into small clumps and inoculated into 24-well plates at a low density (e.g., covering 30-40% of the culture floor area).

[0098] After 24 hours, cell adhesion was restored (e.g. Figure 1 As shown, the initial hiPSC status is good, and the clones are typical. Prepare a series of virus dilutions (e.g., corresponding to MOI=1, 5, 10, 20) and mix them with fresh medium containing 5 μg / mL Polybrene. Discard the old medium from the well plate and add the virus-medium mixture. Incubate at 37°C in a 5% CO2 incubator for 12–16 hours. Replace with fresh complete medium (without Polybrene).

[0099] Seventy-two hours after transduction, EGFP expression was observed under a fluorescence microscope. The lowest effective MOI (Minimum Active Cell Indicator) with a high proportion of fluorescently positive cells (target >70%) and well-maintained cell morphology (no obvious differentiation or death) was selected for large-scale experiments. Multiple ipsc-MSCL-GFP-PURO (experimental group) and ipsc-GFP-PURO (control group) monoclonal cell lines were successfully obtained. Figure 2 As shown, both the experimental and control groups exhibited extensive and intense green fluorescence, demonstrating efficient transduction.

[0100] Formal transduction and drug screening:

[0101] Prepare a large number of hiPSCs using the method described above and inoculate them into multiple 6-well plates.

[0102] Dilute the virus with a polybrene-containing medium according to the optimal MOI determined in the preliminary experiment, and infect the cells.

[0103] Change the fluid 12-16 hours after infection.

[0104] Forty-eight hours post-infection, puromycin was added for selection. The initial concentration could be 0.5 μg / mL. Cell status was observed and the medium changed every other day.

[0105] Over 3-5 days, gradually increase the puromycin concentration to 1.0-1.5 μg / mL (this concentration needs to be determined through preliminary experiments and is sufficient to kill untransduced wild-type hiPSCs within 3 days). Continue screening for 7-10 days until all uninfected control group cells die, and discrete, strongly fluorescent resistant clones are visible in the experimental wells. Results are as follows. Figure 3 As shown, all cells in the control group died; the cells successfully transduced in the experimental group and the control group formed surviving clones.

[0106] Monoclonal isolation, amplification, and library construction:

[0107] The selected mixed cell pools were digested into single cells or very small clumps (which may be briefly treated with Accutase) and resuspended in a medium containing Y-27632.

[0108] Perform limiting dilution: Serially dilute the cell suspension and seed it into 96-well plates (Matrigel-coated) to theoretically obtain 0.5–1 cells per well. Culture and observe regularly.

[0109] Approximately 10-14 days later, wells exhibiting single, bright fluorescence and typical, uniform morphology are marked under a fluorescence microscope, such as... Figure 4 As shown.

[0110] Labeled single clones were transferred to 24-well plates for independent amplification using either a cloning pick loop or direct digestion.

[0111] Each amplified monoclonal cell was numbered, cryopreserved, and a portion of the cells was used for identification.

[0112] 4. Comprehensive evaluation of gene editing effects (key quality control point)

[0113] Morphology and fluorescence expression: Daily observation confirmed that monoclonal cells maintained the typical morphology of hiPSCs, and that the EGFP fluorescence signal was strong and stable, with no spontaneous differentiation regions. Figure 5 As shown.

[0114] Genome integration validation:

[0115] Genomic DNA extraction: Genomic DNA was extracted from monoclonal cells using a kit (such as the DNeasy Blood & Tissue Kit, Qiagen).

[0116] PCR identification: Specific primers were designed. One pair of outer primers amplified a partial sequence of MScL-G22S (e.g., ~500 bp), and one pair of inner primers (or primers spanning the integration site) confirmed specific integration. Wild-type hiPSC DNA was used as a negative control, and the experimental vector plasmid was used as a positive control.

[0117] To verify the transduction and expression of the MscL-G22S gene in iPSC cells, total RNA was extracted and reverse transcribed into cDNA. GAPDH was used as an internal reference gene, and the transcription level of the target gene was detected by SYBR Green qPCR. The experiment was performed in triplicate, and the relative expression level was calculated using the 2^-ΔCt method. The results are as follows:

[0118] Table 1. qPCR validation of the transcriptional expression of the MscL-G22S gene in iPSC cells

[0119]

[0120] Target gene amplification: In iPSC cells transfected with MscL-G22S (ipsc-MSC1-GFP group), a clear exponential amplification curve of the MscL-G22S gene was detected, with an average Ct value of 26.24 ± 0.17 (n=3), a ΔCt value of 7.91, and a relative expression level (2^-ΔCt) of 0.0041, indicating that the target gene was successfully transcribed.

[0121] The negative control was valid: no MscL-G22S gene amplification was detected in the negative control group (ipsc-GFP) that was transfected with GFP only, ruling out the possibility of non-specific amplification or cross-contamination.

[0122] Stable internal reference gene: The Ct value of the internal reference gene GAPDH amplification in all samples was stable between 18.32 and 19.21, and the coefficient of variation (CV) within the group was <2%, indicating that the RNA quality, reverse transcription efficiency and sample loading consistency were good.

[0123] Transfection efficiency reference: Simultaneous detection of GFP gene expression showed that the Ct value of GFP in the ipsc-MSC1-GFP group was 23.81, which was lower than the Ct value of MscL-G22S (26.24), consistent with expectations.

[0124] The above qPCR data confirm that the MscL-G22S gene has been successfully transferred into iPSC cells and expressed at the transcriptional level, laying the foundation for subsequent functional studies.

[0125] Aseptic and mycoplasma detection: Culture supernatant was collected and tested using commercial mycoplasma detection kits based on PCR or culture methods. All results were negative.

[0126] In this embodiment, a hiPSC cell line and its control line that stably expresses MScL-G22S and has undergone comprehensive quality identification were successfully established, fully meeting the requirements for subsequent differentiation experiments.

[0127] Example 2: Directing the differentiation of acoustically sensitive hiPSCs into functional pancreatic β-like cells

[0128] This embodiment aims to efficiently and reproducibly induce the "seed cells" obtained in Example 1 into clusters of pancreatic β-like cells with insulin secretion function and retained acoustic sensitivity through a differentiation protocol with clearly defined chemical composition and distinct stages. Specific experimental methods include:

[0129] 1. Precise Preparation of Differentiation Media All media were prepared using ultrapure water and cell culture-grade reagents, and sterilized by filtration through a 0.22 μm filter membrane. Key media formulations are shown in Table 2 below.

[0130] Table 2. Culture medium composition and description

[0131]

[0132] 2. Staged differentiation induction process (using a 6-well plate as an example, the entire process is carried out in a 37°C, 5% CO2, high humidity incubator)

[0133] Day -1: Plate formation: Digest qualified acoustically sensitive hiPSC monoclonal cells (or control cells) into suitable small clumps and seed them at a high density (approximately covering 60-70% of the bottom area) into Matrigel-coated 6-well plates. Add 2 mL of mTeSR1 medium containing 10 μM Y-27632 to each well. Mark this as Day 0 of differentiation.

[0134] Day 0-1: Adaptation and Consolidation: After 24 hours of culture, replace with fresh mTeSR1 cells that do not contain Y-27632. Continue culturing until the cells reach 90-95% confluence (usually at the end of day 1).

[0135] Phase 1: Induction of the finalized endoderm (days 1-5)

[0136] Day 1: Discard mTeSR1 and add 2 mL of modified CDM to each well. This step replaces the pluripotency maintenance factor and initiates the differentiation program.

[0137] Day 2: Discard the modified CDM and add 2 mL of endoderm induction medium to each well. Change this medium daily for 4 consecutive days (days 2, 3, 4, and 5).

[0138] Expected morphology: Cells gradually change from a compact clonal structure to a uniform epithelial-like monolayer with blurred boundaries.

[0139] Phase 2: Pancreatic progenitor cell induction (days 6-9)

[0140] Day 6: Discard the endoderm induction medium and add 2 mL of pancreatic progenitor cell induction medium to each well. Change this medium daily for 4 consecutive days (days 6, 7, 8, and 9).

[0141] Expected morphology: Local thickening occurs in the cell layer, forming a "rose knot" or tubular structure, which is the typical morphology of pancreatic progenitor cells.

[0142] Phase 3: Endocrine Orientation and Early Maturation (Days 10-14)

[0143] Day 10: Discard the pancreatic progenitor cell induction medium and add 2 mL of pancreatic islet cell maturation medium 1 to each well. Change this medium daily for 3 consecutive days (days 10, 11, and 12).

[0144] Day 13: Discard islet cell maturation medium 1 and add 2 mL of islet cell maturation medium 2 to each well. Change this medium daily for two consecutive days (days 13 and 14).

[0145] Expected morphology: The epithelial lamellae gradually disintegrate, and cells begin to detach from the bottom of the culture dish, forming irregular aggregates.

[0146] Phase 4: Three-dimensional aggregation and terminal maturation (days 15-22)

[0147] Day 15: Aggregation culture is carried out.

[0148] Discard the pancreatic islet cell maturation culture medium 2.

[0149] Add 1 mL of dispersase (Dispase, Gibco, 17105041) solution (0.5-1 mg / mL in DMEM / F-12) to each well and incubate at 37°C for 10-15 minutes, or until the cell sheet edges curl up.

[0150] Gently aspirate the dispersing enzyme and wash once with DMEM / F-12.

[0151] Add 2 mL of pancreatic islet cell maturation medium 2. Using a 1 mL pipette tip (with the tip blunted) or a cell scraper, very gently scrape the cells from the bottom of the well plate and pipette them into clumps of varying sizes. Avoid pipetting into single cells.

[0152] Transfer the suspension containing cell clumps to a 15 mL centrifuge tube and let it stand for 1-2 minutes to allow large tissue fragments to settle.

[0153] Transfer the supernatant (containing appropriately sized clumps) to a 6-well plate or petri dish with ultra-low attachment.

[0154] Supplement pancreatic islet cell maturation medium by 2 to 3-4 mL per well / plate.

[0155] Days 16-22: Suspension culture. Place the cells in an incubator; daily medium changes are not required. Every other day, allow the cell clumps to settle naturally (or centrifuge at 100×g for 1 minute), carefully aspirate half of the old culture medium, and add an equal volume of fresh, pre-warmed islet cell maturation medium 2. This process promotes further compaction, vascularization (simulating), and functional maturation of the cell clumps.

[0156] Expected morphology: It will eventually form a rounded, dense three-dimensional islet-like cell cluster with a size of about 100-300 μm, which has strong refractive properties under a microscope.

[0157] 3. Quality control and biomarker identification:

[0158] Morphological monitoring: Regularly observe the morphological evolution of cells from clones to epithelial layers to tubular structures to three-dimensional cell clusters.

[0159] Key biomarker immunofluorescence detection:

[0160] Stage: Pancreatic progenitor cell stage (D9), PDX-1 detection.

[0161] Endpoint: Mature cell cluster (D21), detection of C-peptide (β cell-specific marker) and GFP (indicating MscL-G22S expression).

[0162] Expected experimental results:

[0163] This study aims to verify cell differentiation efficiency and gene expression through morphological observation and immunofluorescence staining, and expects to obtain the following results:

[0164] Morphological evolution during differentiation (4X, 10X): Cell morphology photographs are expected to be taken on days 0, 5, 9, 14, and 22 to show the typical morphological evolution from stem cells to mature cells.

[0165] Immunofluorescence colocalization images (high-resolution confocal):

[0166] The expected expression of PDX1 (pancreatic-duodenal homeobox protein 1, red fluorescence) in the cell nucleus and colocalization with DAPI (blue) confirmed that the cells had been directed to differentiate into pancreatic progenitor cells.

[0167] It is expected that in mature cell cluster slices, C-peptide (insulin secretion marker, red) and GFP (green reporter gene) will show high colocalization (appearing yellow after merging), and DAPI (blue) will stain the nucleus.

[0168] Expected results: At the differentiation endpoint, C-peptide-positive cell clusters should be observed, with most of these cells simultaneously expressing GFP, demonstrating successful expression of the acoustic-sensitive channel protein in the target β-like cells.

[0169] This embodiment also explored the effect of differentiation initiation density on cell survival and differentiation process, specifically including:

[0170] I. Technical Issues and Objectives

[0171] In the process of developing a technology for the directed differentiation of acoustically sensitive human pluripotent stem cells into pancreatic β-like cells, this invention discovered that the cell seeding density at the start of differentiation is one of the key factors determining the success of differentiation. If the starting density is too low, the number of cells will continuously decrease in the early stages of differentiation, making it impossible to maintain a normal growth state, ultimately leading to differentiation failure. This experiment aims to clarify the optimal starting density range for achieving stable differentiation.

[0172] II. Experimental Design

[0173] The grouping is designed as shown in Table 3 below.

[0174] Table 3

[0175]

[0176] Both groups used the same batch of cells and culture medium, operated in parallel, and recorded 10× bright-field imaging daily.

[0177] III. Experimental Results (10× Bright Field)

[0178] Low-density group (30–40% initial confluence)

[0179] Days 0-4: Cell proliferation is slow, and a complete cell layer is never formed;

[0180] Day 5 and thereafter: The number of cells decreased significantly compared to the previous day, and the adherent cells became increasingly sparse;

[0181] Days 9-10: Very few adherent cells remain, making further differentiation impossible, and the differentiation process terminates.

[0182] Suitable density group (60–70% initial confluence)

[0183] Days 0-4: Cells proliferate rapidly, and by day 4 a dense and uniform cell layer has been formed;

[0184] Day 5 and thereafter: Cell count increases steadily, and cell layer remains intact;

[0185] Days 9-10: The cell layer thickens, showing a typical morphology indicating that it is about to enter the next differentiation stage, and the differentiation process is progressing smoothly.

[0186] The results are as follows Figure 6 As shown, the differences between the two groups are clearly discernible to the naked eye under 10× bright field conditions, where:

[0187] Left column (low-density group): Representative images from day 5 and day 9. It can be seen that the cells were already significantly sparse on day 5, and only a very small number of adherent cells remained on day 9.

[0188] Right column (appropriate density group): Representative images of day 5 and day 9. It can be seen that the cell layer is dense and intact on day 5, and the cell layer is thickened and has a typical morphology on day 9.

[0189] Conclusion: The low-density group showed a clear and irreversible decrease in cell number as early as day 5 of differentiation, ultimately leading to differentiation failure; the appropriate density group grew well throughout the differentiation process, with smooth progress. These differences were clearly discernible under a 10× bright-field microscope, requiring no staining or complex analysis.

[0190] Example 3: Functional Verification of Acoustic Sensitivity and the Enhancing Effect of Ultrasound on Insulin Secretion

[0191] This embodiment rigorously verifies the "sound-sensitive" functional characteristics of the prepared cells and the precise enhancement effect of ultrasound stimulation on insulin secretion function in vitro and in vivo through multi-level experiments from molecules to whole animals.

[0192] 1. In vitro functional verification experiment

[0193] Experimental design: Differentiated and mature cell clusters were divided into three groups: MScL group (experimental group), GFP control group, and wild-type group.

[0194] Ultrasound stimulation parameters: Low-intensity focused ultrasound was used at a frequency of 650 kHz and an intensity of 2 W / cm². Single pulse (for calcium imaging, lasting 10-30 seconds) or repetitive pulse (for secretion experiments, such as a 1 kHz pulse repetition frequency, 10% duty cycle, and a total stimulation duration of 1 hour).

[0195] Detection method:

[0196] Experiment 1: Calcium Imaging Detection of Ultrasound-Triggered Ca²⁺ Influx

[0197] Principle: Ultrasound activates the MScL-G22S channel, causing an influx of cations (including Ca²⁺). The increase in intracellular Ca²⁺ concentration is a key signal for the initiation of the insulin secretion chain.

[0198] step:

[0199] The cell clusters were transferred to a confocal microscope culture dish and loaded with a calcium ion fluorescent indicator (e.g., Fluo-4AM, 5 μM, incubated at 37°C for 30 minutes).

[0200] After washing with phenol red-free buffer, place it on the microscope stage and connect it to the ultrasonic stimulation system.

[0201] Record a baseline fluorescence signal (approximately 30 seconds).

[0202] A single pulse of ultrasound stimulation (e.g., 650 kHz, 2 W / cm², lasting 20 seconds) is applied while fluorescence images are acquired at high speed (e.g., 1-5 frames per second).

[0203] Continue recording the post-stimulation signal for 2-3 minutes.

[0204] Data Analysis: Define the region of interest (ROI) and analyze the average fluorescence intensity (F) of each cell cluster. Calculate the relative fluorescence change ΔF / F0 = (F - F0) / F0, where F0 is the baseline average fluorescence intensity. Plot the curve of ΔF / F0 over time.

[0205] Expected results:

[0206] In the MscL group, ΔF / F0 increased rapidly and significantly within seconds of the start of ultrasound stimulation, reaching a peak (e.g., peak increase of 80-150%), and then slowly decreased, exhibiting typical calcium transients.

[0207] GFP control group and WT group: The ΔF / F0 curves were basically flat with only minor fluctuations (<10%), indicating no specific calcium response.

[0208] Experiment 2: Glucose-stimulated insulin secretion experiment and ultrasound enhancement effect

[0209] Principle: To assess the functional output of cells under physiological stimulation (glucose) and physical stimulation (ultrasound).

[0210] Steps (Static Incubation Method):

[0211] Collect uniformly sized cell clusters and wash with Krebs-Ringer Bicarbonate HEPES buffer.

[0212] Low glucose incubation: Place cell clusters in a buffer containing 2.8 mM glucose and incubate at 37°C for 1 hour. Collect the supernatant (Basal sample).

[0213] High glucose incubation: Cells were transferred to a buffer solution containing 20 mM glucose and incubated for 1 hour. This group was then divided into two halves:

[0214] High sugar group: routine incubation.

[0215] High sugar + US group: Repetitive pulsed ultrasound stimulation was applied during incubation (parameters as above).

[0216] Collect the cleared data under various conditions.

[0217] Use a highly sensitive human insulin or C-peptide ELISA kit and strictly follow the instructions to detect the concentration of insulin / C-peptide in each sample.

[0218] After the experiment, the total DNA content of the cell clusters was measured using a DNA quantification kit for data normalization (e.g., secretion volume is expressed as ng / mg DNA / hour).

[0219] Data Analysis and Expected Results:

[0220] Calculate the Stimulation Index (SI): SI = High glucose secretion / Low glucose secretion. A well-functioning β-cell SI should be >2.

[0221] Calculate the ultrasound enhancement factor: EF = (high glucose + US secretion) / (high glucose secretion).

[0222] This study aims to verify the enhancing effect of MScL-G22S combined with ultrasound stimulation on insulin release through a glucose-stimulated insulin secretion assay. Based on the mechanosensitive properties of the MScL channel, the expected results are shown in Table 4.

[0223] Table 4. Expected Insulin Secretion Outcomes

[0224]

[0225] 2. In vivo functional verification

[0226] 2.1 Establishment of Diabetic Animal Models and Optimization and Screening of Dosage Parameters

[0227] I. Technical Issues and Objectives

[0228] Before conducting in vivo functional validation of β cells derived from acoustically sensitive stem cells, it is necessary to establish a stable, reliable, and applicable type 1 diabetes model suitable for immunodeficient mice.

[0229] Existing literature reports that STZ induction protocols mostly involve a single high-dose intraperitoneal injection, but different strains exhibit significant differences in STZ tolerance. Immunodeficient mice routinely used for human cell transplantation (such as NSG and NOD-scid) are far more sensitive to STZ toxicity than ordinary strains. Directly applying the dosage (180-200 mg / kg) of C57BL / 6 or BALB / c from the literature can easily lead to animal death or near-death experiences, failing to meet the survival requirements for subsequent cell transplantation.

[0230] To address the aforementioned technical issues, prior to formal in vivo experiments, this invention systematically conducted multi-dose, single-STZ modeling parameter screening experiments on NOD and NSG strains to determine the optimal modeling scheme that balances modeling rate, stability, and survival rate, thus laying a reproducible foundation for the in vivo efficacy evaluation of the cell preparations of this invention.

[0231] II. Experimental Design

[0232] The strains and dosage groups of the experimental animals are shown in Table 5 below.

[0233] Table 5

[0234]

[0235] Modeling methods

[0236] STZ should be prepared and used immediately, dissolved in sodium citrate buffer (pH 4.5);

[0237] A single intraperitoneal injection, with the injection volume calculated based on body weight;

[0238] Continuous monitoring of body weight and random / fasting blood glucose (D1–D6) after injection.

[0239] Successful model criteria: two consecutive random blood glucose levels >16.7 mmol / L (300 mg / dL);

[0240] Toxicity assessment indicators: weight loss rate, near-death / fatal events.

[0241] III. The experimental results are shown in Table 6 below. The NSG strain has extremely low tolerance to STZ and is not suitable for long-term transplantation evaluation in this invention.

[0242] Table 6

[0243]

[0244] in conclusion:

[0245] NSG mice showed >20% weight loss and near-death events at a single dose of 150 mg / kg; definite deaths occurred in dose groups of 170 mg / kg and above, which could not meet the requirements for animal survival time in subsequent cell transplantation; the NSG strain is not suitable for the long-term transplantation evaluation system of this invention under the experimental conditions.

[0246] The dose response results of the NOD strain (reference control) are shown in Table 7 below.

[0247] Table 7

[0248]

[0249] The results showed that the NOD strain had better tolerance to STZ than NSG; a single injection of 140 mg / kg could stably establish the model without any deaths, and the weight loss was controllable.

[0250] IV. The Preferred Modeling Scheme Finally Determined in This Invention

[0251] Based on the above system screening results, the subsequent in vivo functional verification experiments of this invention adopt the optimization scheme shown in Table 8 below:

[0252] Table 8

[0253]

[0254] Special Note: This invention, through the aforementioned preliminary experiments, clearly demonstrates for the first time that the NSG strain poses a significant toxicity risk within the dose range of 150–190 mg / kg under a single STZ injection regimen, failing to meet the requirements for subsequent cell transplantation evaluation. This finding provides important technical guidance for subsequent researchers and directly guides the selection of NOD-scid / NRG as the formal experimental strain in this invention.

[0255] The above content systematically discloses for the first time the dose tolerance boundary of the NSG strain to a single STZ injection, filling the data gap in the field of diabetes modeling of this strain; it clarifies the basis for strain selection in subsequent in vivo experiments of this invention, making the technical solution empirically supported and not arbitrarily selected; and it provides a defined basis at the animal model level for further verification of the "therapeutic effective dose" and "glycemic control" of the acoustically sensitive stem cell-derived β cells prepared by this invention.

[0256] 2.2 Based on the optimized parameters of the diabetic animal model determined in 2.1, this section systematically evaluates the survival, functional maintenance, and therapeutic effect of ultrasound-regulated insulin secretion of the cells prepared in this invention under physiological conditions by transplanting β cells derived from acoustically sensitive stem cells into diabetic immunodeficient mice and combining them with non-invasive ultrasound stimulation.

[0257] Establishment of experimental animal and diabetes models

[0258] Animal strain: NOD-scid or NRG mice (6–8 weeks old, male), which have both immunodeficiency and good STZ tolerance, making them suitable for long-term transplantation of human cells.

[0259] Modeling method: Single intraperitoneal injection of streptozotocin (STZ) at a dose of 120–140 mg / kg (adjusted according to batch pre-experiment, with the principle of weight loss <15% and stable hyperglycemia).

[0260] Successful modeling criteria: Two consecutive random blood glucose levels >16.7 mmol / L (300 mg / dL) on days 7–10 after injection are considered as successful diabetes modeling.

[0261] Inclusion criteria: Mice that have undergone a weight loss of <15%, are not in a near-death state, and have stable blood glucose levels are eligible to proceed to the subsequent cell transplantation experiment.

[0262] Cell transplantation

[0263] Cell preparation: Collect cell clusters on day 22 of differentiation and wash with buffer. Under a stereomicroscope, extract approximately 5-10 × 10^6 cells using a grafting needle or capillary tube. 5 One cell equivalent (approximately 100-200 cell clusters).

[0264] Surgery: After anesthetizing the mice, an incision was made in the left rib area to expose the left kidney. A cell suspension was slowly injected under the renal capsule using a microinjector, forming a small vesicle. The incision was then sutured.

[0265] Experimental grouping and ultrasound intervention:

[0266] Grouping (n=8 animals / group):

[0267] MscL+US group: MscL group cells were transplanted and ultrasound intervention was performed postoperatively.

[0268] MscL group: Transplanted with MscL group cells, without ultrasound intervention (sham stimulation or shielding).

[0269] GFP+US group: GFP control group cells were transplanted and subjected to ultrasound intervention.

[0270] Disease control group: received only sham surgery (injection of buffer solution), and did not receive cell transplantation.

[0271] Intervention plan: Begin in the 4th week post-transplantation, after the graft has become vascularized and stabilized.

[0272] Ultrasound equipment: In vivo ultrasound system equipped with a transcutaneous focused transducer.

[0273] Methods: Mice were lightly anesthetized or immobilized, and the hair in the left kidney area was shaved and an ultrasound coupling agent was applied. The transducer was aimed at the transplanted kidney area, with parameters the same as in vitro (650 kHz, 2 W / cm²). Intervention was performed once daily for 10 minutes each time, for 14 consecutive days.

[0274] Monitoring and evaluation:

[0275] Weight and blood sugar: Weight was measured twice a week, and non-fasting blood glucose was measured three times a week by tail vein blood sampling.

[0276] Fasting blood glucose and serum insulin were measured before intervention (day 0), during intervention (day 7), and after intervention (day 14).

[0277] Intraperitoneal glucose tolerance test: conducted after the intervention. Mice were fasted for 6 hours, and fasting blood glucose (0 minutes) was measured before intraperitoneal injection of glucose (2 g / kg). Blood glucose was measured at 15, 30, 60, 90, and 120 minutes after injection, and serum insulin was measured at 0 and 30 minutes.

[0278] Endpoint histological analysis:

[0279] After the final intervention, the mice were euthanized and the transplanted kidney was removed.

[0280] General observation: Take photos to record the location and size of the graft.

[0281] Tissue processing: Kidneys were fixed with 4% paraformaldehyde, embedded in paraffin, and serially sectioned.

[0282] Staining analysis:

[0283] H&E staining: to observe the overall structure of the graft, cell survival, and infiltration of host immune cells.

[0284] Immunofluorescence multiple staining:

[0285] Functional biomarkers: human C-peptide, insulin.

[0286] Reporter gene: GFP (or anti-MscL antibody).

[0287] Vascularization markers, such as CD31, are used to assess graft angiogenesis.

[0288] Apoptosis detection: TUNEL staining.

[0289] Expected histological results: Obvious, vascularized grafts will be visible under the renal capsule in both the MScL+US and MScL groups. Numerous clusters of human C-peptide-positive cells will be visible under high magnification, with most of these cells expressing GFP, indicating graft survival and continued expression of functional proteins. TUNEL-positive cells should be fewer.

[0290] Expected in vivo experimental results:

[0291] Non-fasting blood glucose changes (expected): The MScL+US group was expected to see a rapid decrease in blood glucose after ultrasound intervention and maintain it at near-normal levels; the MScL group saw a slight decrease in blood glucose, but the effect was less pronounced; the GFP+US group and the disease control group had persistently high blood glucose levels.

[0292] Intraperitoneal glucose tolerance test (expected): The MScL+US group was expected to show significant improvement in glucose tolerance, with blood glucose returning to near baseline after 120 minutes; the MScL group showed the second-best improvement; the control group showed no significant improvement.

[0293] The key metabolic parameters expected for different groups are shown in Table 9 below.

[0294] Table 9 Comparison of expected key metabolic parameters (Day 14 of intervention)

[0295]

[0296] Note: * indicates expected significant improvement compared to the disease control group; * indicates expected further significant improvement compared to the MScL group. This table represents expected results**, and the final results are subject to actual experimental data.

[0297] Calcium imaging and GSIS experiments provided direct mechanistic evidence demonstrating that ultrasound can specifically and efficiently activate the MScL-G22S channel and synergistically enhance glucose-induced insulin secretion. Diabetic mouse model experiments provided crucial evidence regarding therapeutic efficacy, demonstrating that the acoustically sensitive β-like cells prepared in this invention can survive and function in vivo after transplantation, and that their therapeutic efficacy can be remotely, precisely, and significantly enhanced via non-invasive ultrasound.

[0298] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing β-functional cells derived from acoustically sensitive stem cells, characterized in that, Includes the following steps: Step 1: Introduce the vector expressing the high-conductivity mechanosensitive ion channel MscL-G22S into pluripotent stem cells to construct and obtain acoustically sensitive pluripotent stem cells; Step 2: Take the acoustically sensitive pluripotent stem cells obtained in Step 1 and induce their differentiation through endoderm to obtain endoderm cells; Step 3: Take the endoderm cells obtained in Step 2 and induce them to differentiate into pancreatic progenitor cells to obtain pancreatic progenitor cells; Step 4: Take the pancreatic progenitor cells obtained in Step 3 and perform endocrine-directed induction and early maturation culture to obtain endocrine precursor cells; Step 5: Take the endocrine precursor cells described in Step 4, perform three-dimensional aggregation culture, and then culture in terminal maturation medium to obtain β-functional cells derived from acoustic-sensitive stem cells.

2. The preparation method according to claim 1, characterized in that, In step 1, the vector of the high-conductivity mechanosensitive ion channel MscL-G22S includes a viral vector having at least one of a promoter, a fluorescent protein tag encoding gene, and an resistance gene.

3. The preparation method according to claim 1, characterized in that, In step 2, the initial seeding density of the acoustically sensitive pluripotent stem cells is 60%~70%. The time for inducing differentiation of the endoderm is 3-6 days; The culture medium for inducing endoderm differentiation includes 50% IMDM, 50% Ham's F-12, 1×ITS-A, 400~500 μMmonothioglycerol, 3~10 mg / mL Albumin Fraction V, and 25~60 ng / mL Activin A.

4. The preparation method according to claim 1, characterized in that, In step 3, the pancreatic progenitor cells are induced to differentiate over a period of 3 to 6 days. The culture medium for inducing differentiation of pancreatic progenitor cells includes 50% IMDM, 50% Ham's F-12, 1×ITS-A, 400~500 μM Monothioglycerol, 3~10 mg / mL Albumin Fraction V and 0.25~1.5 μM Retinoic Acid.

5. The preparation method according to claim 1, characterized in that, In step 4, the endocrine-directed induction and early maturation culture includes: taking the pancreatic progenitor cells and culturing them in pancreatic islet cell maturation culture medium 1 for 2-5 days, and then culturing them in pancreatic islet cell maturation culture medium 2 for 1-3 days; The pancreatic islet cell maturation culture medium 1 comprises DMEM / F-12, 1×ITS-A, 5~15 ng / mL bFGF and 1~3 mg / mL Albumin Fraction V; The pancreatic islet cell maturation culture medium 2 includes DMEM / F-12, 1×ITS-A, 5~15 mM Nicotinamide and 1~3 mg / mL Albumin Fraction V.

6. The preparation method according to claim 1, characterized in that, In step 5, the three-dimensional aggregation culture includes taking the endocrine precursor cells, digesting them, and then seeding the resulting cell clumps into a low-adhesion culture container and culturing them in pancreatic islet cell maturation medium 2 for 6-10 days. The pancreatic islet cell maturation culture medium 2 includes DMEM / F-12, 1×ITS-A, 5~15 mM Nicotinamide and 1~3 mg / mL Albumin Fraction V.

7. The preparation method according to claim 1, characterized in that, In step 5, the terminal maturation medium is pancreatic islet cell maturation medium 2, and the culture time in the terminal maturation medium is 6-10 days. The pancreatic islet cell maturation culture medium 2 includes DMEM / F-12, 1×ITS-A, 5~15 mM Nicotinamide and 1~3 mg / mL Albumin Fraction V.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The pluripotent stem cells include human pluripotent stem cells.

9. The use of acoustically sensitive stem cell-derived β-functional cells prepared by the method according to any one of claims 1 to 8 in the preparation of a drug for the prevention and treatment of diabetes.

10. A drug for the prevention and treatment of diabetes, characterized in that, It includes acoustically sensitive stem cell-derived β-functional cells prepared by the preparation method according to any one of claims 1 to 8, and pharmaceutically acceptable excipients.

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