Calcium imaging pluripotent stem cell, construction method thereof and application of calcium imaging pluripotent stem cell in differentiating pancreatic beta cells

Abstract

The invention discloses a calcium imaging pluripotent stem cell, a construction method thereof and application of the calcium imaging pluripotent stem cell in differentiating pancreatic beta cells. The calcium imaging pluripotent stem cell is obtained by inserting a calcium imaging gene GCaMP6f into an AAVS1 site of the pluripotent stem cell. The calcium imaging pluripotent stem cells differentiate the pancreas islet beta cells through in-vitro induction, calcium imaging fluorescence detection is carried out, and the functional maturation condition of the differentiated pancreas islet beta cells can be tracked and monitored. The calcium imaging pluripotent stem cells can be used for screening and optimizing an induced differentiation process in a process of differentiating the pluripotent stem cells into pancreas islet beta cells; the pancreatic beta cell fluorescence reporter line obtained by induced differentiation of the calcium imaging pluripotent stem cells can be used for monitoring the function level of insulin secretion of pancreatic beta cells, researching the mechanism or pathway of diabetes mellitus, or screening diabetes mellitus drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a calcium imaging pluripotent stem cell and a construction method thereof and an application thereof in differentiating pancreatic beta cells. Background Art

[0002] Diabetes has become one of the diseases that seriously threatens human health and quality of life worldwide. For patients with insulin-dependent diabetes, the main treatment method is to control blood sugar levels by injecting exogenous insulin. However, this treatment method is difficult to achieve precise regulation of blood sugar, which may lead to the occurrence of multiple complications and seriously affect the daily quality of life of diabetic patients.

[0003] HiPSCs are a type of multipotent cells with unlimited proliferation and self-renewal properties. Their research and application do not involve ethical and moral issues. Under specific culture conditions in vitro, hiPSCs can be induced to differentiate into pancreatic β cells. Implanting these differentiated pancreatic β cells into diabetic patients is expected to replace damaged pancreatic β cells and restore normal insulin secretion function. This treatment method is expected to cure diabetes fundamentally.

[0004] However, at the current stage in this research field, pancreatic β cells differentiated from human pluripotent stem cells are generally in a functionally immature state and cannot sensitively respond to changes in glucose concentration to secrete insulin, which is a major challenge facing their clinical application.

[0005] Conventional flow cytometer methods for detecting the positive proportion of β cells and immunofluorescence staining methods for detecting pancreatic β cell markers cannot dynamically display whether pancreatic β cells can secrete insulin in response to changes in glucose concentration.

[0006] A more accurate method is to monitor the changes in extracellular insulin content of pancreatic β cells in real time under the stimulation of continuous perfusion of different concentrations of glucose. However, this method is not only time-consuming but also very costly.

[0007] In the study of pancreatic β cells, real-time monitoring of pancreatic β cell activity is the key to studying their functional maturation. However, existing technologies lack simple visualization tools suitable for studying the functional maturation of pancreatic β cells during the differentiation of pluripotent stem cells, resulting in slow progress in the study of the functional maturation of differentiated pancreatic β cells.

[0008] It is necessary to develop a visualization tool that can monitor the activity of pancreatic β cells in real time, which will help study the functional maturation of pancreatic β cells during the differentiation of pluripotent stem cells and provide data basis for optimizing the process and improving the quality of in vitro differentiated pancreatic β cells. Visualization tools can also be used for mechanism research and drug screening of related diseases such as diabetes, simplify the steps of detecting cell insulin, and improve research and development efficiency. Summary of the invention

[0009] The purpose of the present invention is to provide a calcium imaging pluripotent stem cell and a construction method thereof, as well as the application of calcium imaging pluripotent stem cells in differentiating pancreatic β cells, which can track and monitor the functional maturation of pancreatic β cells through fluorescence imaging to achieve long-term, low-background, and high-sensitivity dynamic visualization monitoring of calcium signals.

[0010] Specifically, the technical solution adopted by the present invention is: A calcium imaging pluripotent stem cell is obtained by knocking the calcium imaging gene GCaMP6f into the AAVS1 site of the pluripotent stem cell.

[0011] Furthermore, the calcium imaging pluripotent stem cells are obtained by knocking the calcium imaging gene GCaMP6f into the AAVS1 site of pluripotent stem cells using CRISPR-Cas9 gene editing technology.

[0012] The nucleotide sequence of the calcium imaging gene GCaMP6f is shown in SEQ ID NO:1.

[0013] Furthermore, the present invention provides a calcium imaging pluripotent stem cell, wherein the calcium imaging pluripotent stem cell is obtained by the following method: The homologous template DNA of the calcium imaging gene GCaMP6f carrying a strong CAG promoter was designed and constructed, and the high-precision CRISPR-Cas9 gene editing technology was used to accurately introduce the efficient and specific sgRNA, eSpCas9 protein and homologous template DNA directed to the AAVS1 site into pluripotent stem cells. The calcium imaging gene GCaMP6f was knocked into the AAVS1 site of the pluripotent stem cells through a gene knock-in strategy to construct the calcium imaging pluripotent stem cells.

[0014] A plasmid containing sgRNA, a plasmid containing eSpCas9 gene, and a donor vector containing homologous template DNA can also be transferred into pluripotent stem cells to culture calcium imaging pluripotent stem cells in which the calcium imaging gene GCaMP6f is knocked into the AAVS1 site of pluripotent stem cells.

[0015] The homologous template DNA includes a left homology arm and a right homology arm that highly match the sequences at both ends of the AAVS1 site, and the calcium imaging gene GCaMP6f is located between the left homology arm and the right homology arm.

[0016] The preferred sequence of sgRNA is shown in SEQ ID NO: 2, and the sequence of the homologous template DNA is shown in SEQ ID NO: 3.

[0017] The method of knocking into pluripotent stem cells includes an electro-transfection method, that is, knocking sgRNA, eSpCas9 protein and homologous template DNA into pluripotent stem cells by electro-transfection.

[0018] The present invention also provides a method for constructing the calcium imaging pluripotent stem cells, which comprises knocking the calcium imaging gene GCaMP6f into the AAVS1 site of the pluripotent stem cells to obtain the calcium imaging pluripotent stem cells.

[0019] Preferably, the method is: designing and constructing a homologous template DNA of the calcium imaging gene GCaMP6f carrying a strong CAG promoter, using high-precision CRISPR-Cas9 gene editing technology, accurately introducing the efficient and specific sgRNA, eSpCas9 protein and homologous template DNA directed to the AAVS1 site into pluripotent stem cells, knocking the calcium imaging gene GCaMP6f into the AAVS1 site of the pluripotent stem cells, and constructing the calcium imaging pluripotent stem cells.

[0020] More specifically, the preferred method is: designing and constructing a homologous template DNA of the calcium imaging gene GCaMP6f carrying a strong CAG promoter, using CRISPR-Cas9 gene editing technology, and electro-transfecting the high-efficiency specific sgRNA, eSpCas9 protein and homologous template DNA directed to the AAVS1 site into pluripotent stem cells. Under the guidance of sgRNA, the eSpCas9 protein recognizes the AAVS1 site and cuts the DNA to produce double-stranded DNA breaks. During the DNA repair process, the homologous arms in the homologous template DNA are recognized, and the homologous arms are paired with the broken DNA ends to perform homologous recombination. The homologous template DNA fills the gap at the DNA break, thereby knocking the calcium imaging gene GCaMP6f into the AAVS1 site of pluripotent stem cells.

[0021] Generally, calcium imaging pluripotent stem cells with successful knock-in of the GCaMP6f gene are screened by fluorescent labeling.

[0022] Furthermore, the present invention also provides the use of calcium imaging pluripotent stem cells in differentiating pancreatic β cells with the ability to monitor visualized calcium signals.

[0023] Furthermore, the application method is that calcium imaging pluripotent stem cells are induced to differentiate into pancreatic beta cells in vitro, and the pancreatic beta cells have the ability to monitor visualized calcium signals. During the induction of differentiation, calcium imaging fluorescence detection is performed on the pancreatic beta cells to track and monitor the functional maturation of the differentiated pancreatic beta cells.

[0024] More specifically, the application method is as follows: calcium imaging pluripotent stem cells are induced to differentiate in vitro to obtain pancreatic beta cells with the ability to monitor visualized calcium signals. Under glucose stimulation, the fluorescence intensity will increase with the increase of intracellular calcium ion concentration. The fluorescence intensity is detected in real time to track and determine the functional maturation of differentiated pancreatic beta cells.

[0025] Calcium imaging pluripotent stem cells can be used to optimize the screening of induced differentiation processes during the differentiation of pluripotent stem cells into pancreatic β cells. In the prior art, pancreatic β cells induced and differentiated from human pluripotent stem cells in vitro are generally functionally immature and cannot sensitively respond to changes in glucose concentration to secrete insulin. This may be related to various factors such as the formulation of the induced differentiation medium and the culture process. The calcium imaging pluripotent stem cells provided by the present invention can be used to induce the differentiation of pancreatic β cells under different process conditions, and to track and monitor in real time the functional level of differentiated pancreatic β cells in secreting insulin in response to glucose stimulation, thereby screening the optimal process conditions for inducing differentiation.

[0026] The present invention also provides a method for optimizing the screening of induced differentiation technology during the differentiation of pluripotent stem cells into pancreatic β cells. The method comprises the following steps: inducing differentiation of calcium imaging pluripotent stem cells using different induced differentiation processes to obtain pancreatic β cells, wherein the pancreatic β cells have the ability to monitor visualized calcium signals, detect the fluorescence intensity of pancreatic β cells under glucose stimulation, and track and monitor in real time the functional level of differentiated pancreatic β cells in secreting insulin in response to glucose stimulation, thereby screening the optimal induced differentiation process.

[0027] The induced differentiation process includes the culture medium component formula, culture time, etc. at different differentiation stages.

[0028] The present invention also provides a pancreatic beta cell fluorescent reporter system with the ability of visualizing calcium signal monitoring obtained by inducing differentiation of calcium imaging pluripotent stem cells.

[0029] The pancreatic β cell fluorescence reporter system can be used to track the fluorescence intensity of pancreatic β cells in real time and monitor the functional level of pancreatic β cells in secreting insulin.

[0030] The pancreatic β cell fluorescence reporter system can be used for the study of the mechanism pathway of diabetes or the screening of diabetes drugs.

[0031] Specifically, the application method is to induce differentiation of calcium imaging pluripotent stem cells in vitro to obtain a pancreatic β cell fluorescent reporter system with the ability to monitor visualized calcium signals, stimulate the pancreatic β cells with different drugs or inducing agents, detect the fluorescence intensity in real time, track the changes in insulin secretion levels under different stimulation conditions, and study the effects of drugs or inducing agents on the insulin secretion function of pancreatic β cells.

[0032] The secretion of insulin by pancreatic β cells is affected by many factors, but there is currently no technology that can achieve the goal of finding a method that is easy to monitor and can intuitively reflect the function of pancreatic β cells. Specifically, the mechanism of insulin secretion by pancreatic β cells is: under the induction of glucose, pancreatic β cells produce ATP through glycolysis, and the increase in intracellular ATP levels causes the closure of ATP-sensitive potassium channels (KATP channels), which changes the potential of the cell membrane and depolarizes the cell membrane, that is, the potential difference between the inside and outside of the cell decreases. After the cell is depolarized, the voltage-gated calcium channel opens, allowing calcium ions to enter the cell. This influx of calcium ions increases the intracellular calcium concentration, and the increase in intracellular calcium concentration triggers the fusion of insulin vesicles with the cell membrane, resulting in insulin secretion outside the cell. Therefore, the present invention proposes to monitor the intracellular calcium ion concentration by using pancreatic β cells with the ability to monitor visualized calcium signals induced by calcium imaging pluripotent stem cells, thereby reflecting the activity level of pancreatic β cells.

[0033] GCaMP6f is a highly sensitive calcium indicator that can accurately capture changes in calcium ion concentration in β cell activity. The present invention knocks in the calcium imaging gene GCaMP6f in the cell safety site, expresses the GCaMP6f calcium signal protein, and monitors the intracellular calcium ion concentration. When the intracellular calcium ion concentration increases, the fluorescence intensity of the GCaMP6f calcium signal protein increases, and the activity level of pancreatic β cells can be visually observed through a fluorescence microscope to determine the functional maturity of pancreatic β cells.

[0034] Moreover, AAVS1 is a specific site on the human genome, which is called a "safe site" because gene knock-in at this safe site will not affect the normal function of the host cell. The GCaMP6f gene was knocked into the AAVS1 safe site of pluripotent stem cells using CRISPR-Cas9 technology to achieve stable expression of the GCaMP6f gene, and then combined with the in vitro differentiation strategy induced by chemical small molecules, the gene-edited pluripotent stem cells were differentiated into pancreatic β cells.

[0035] Existing technologies also use lentiviruses to insert calcium imaging genes, but since the sites where lentiviruses integrate into the genome are random, this method cannot ensure that the genes are accurately inserted into safe sites, which may potentially affect the normal functions of host cells.

[0036] The calcium imaging pluripotent stem cells of the present invention can be used in the study of differentiation of pancreatic β cells, and can perform visualized real-time monitoring with high sensitivity. The pancreatic β cell fluorescent reporter system with visualized calcium signal monitoring capability obtained by induced differentiation can be applied to the study of pancreatic β cell functional maturation and the mechanism study and drug screening of related diseases such as diabetes, etc., and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of GCaMP6f gene knock-in into the AAVS1 safety site.

[0038] Figure 2 This is a fluorescence microscope photograph of the pluripotent stem cell clone with the GCaMP6f gene knocked into it in Example 1.

[0039] Figure 3 These are the results of induced differentiation of pluripotent stem cells into directed endoderm in Example 2, wherein the left picture is a microscope bright field image, and the right picture is the FOXA2 / SOX17 double-positive detection result.

[0040] Figure 4 These are the results of inducing pluripotent stem cells to differentiate into pancreatic progenitor cells in Example 2, wherein the left figure is a microscope bright field image, and the right figure is the PDX1 / NKX6.1 double-positive detection result.

[0041] Figure 5 The diagram shows the result of inducing pluripotent stem cells to differentiate into pancreatic β cells in Example 2, wherein the left diagram is a microscope bright field image, and the right diagram is a C-peptide / NKX6.1 double-positive detection result.

[0042] Figure 6 These are fluorescence photographs of pancreatic β cells under different stimulation conditions in Example 3. The left picture is a fluorescence photograph under stimulation with 2.8 mM glucose-krebs buffer, and the right picture is a fluorescence photograph under stimulation with 2.8 mM glucose-krebs buffer + 30 mM KCl. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0044] Example 1 GCaMP6f gene knock-in pluripotent stem cells 1. Use mTeSR-Plus stem cell medium to culture pluripotent stem cells and replace fresh medium every day; 2. Observe the cloning status of pluripotent stem cells under a microscope. When the cell confluence reaches 70-80% under the microscope, replace the mTeSR-Plus medium containing 10 μM Y27632 ROCK inhibitor; 3. On the second day, remove the culture medium supernatant, add preheated Accutase digestion enzyme, and incubate for 5 minutes; 4. Use a pipette to add an equal volume of mTeSR-Plus medium containing 10 μM Y27632, and gently disperse the pluripotent stem cell clones into single cells; 5. Centrifuge at 300 g for 3 min and remove the supernatant; 6. The single cell pellet was resuspended in mTeSR-Plus medium containing 10% Clone R2, and the number of live cells was counted using trypan blue; 7. Take 500,000 cells and dispense them into each tube. Centrifuge at 300g for 3 minutes and aspirate the supernatant. 8. Prepare the commercial reagent Lonza P3 electroporation kit, eSpCas9 protein, and synthetic sgRNA (sequence as shown in SEQ ID NO: 2) and homologous template GCaMP6f HDR Template (HDRT, sequence as shown in SEQ ID NO: 3); 9. Add 240 pmol of sgRNA solution and 80 pmol of eSpCas9 protein to 9 μl of P3 electroporation solution (LONZA) and incubate at room temperature for 10-20 minutes; 10. Then add 500 ng of homologous template (HDRT), incubate at room temperature for two minutes, and then use P3 electroporation solution to make up the total volume to 20 μl. This solution is the RNP-HDRT mixture; 11. Add the above RNP-HDRT mixture to the tube and gently resuspend the pluripotent stem cells; 12. Add the resuspended single cell suspension into the electroporation cup, making sure there are no bubbles at the bottom; 13. After the Lonza-4D nuclear transfer instrument is turned on, start program CA-137 to start nuclear transfer; 14. After nuclear electroporation, add mTeSR-Plus medium containing 10% Clone R2 and gently resuspend; 15. Transfer all reaction solutions to a 24-well plate and add 1 µM HDR enhancer V2 to each well; 16. After 24 hours, replace with fresh mTeSR-Plus medium and observe cell survival status and fluorescence; 17. After 2-4 days of culture, subculture the pluripotent stem cells and mark the fluorescent clones with a marker pen; 18. After the labeled pluripotent stem cell clones grow up, single clones are selected under a stereomicroscope and transferred to a 12-well plate for amplification to obtain calcium imaging pluripotent stem cells. Fluorescence microscope photos of pluripotent stem cell clones are shown in Figure 1. Figure 2 The pluripotent stem cell clones showed green fluorescence, indicating that the GCaMP6f gene had been successfully knocked into the genome of human pluripotent stem cells and achieved efficient expression of calcium signal fluorescent protein, providing a reliable marker for subsequent pancreatic β function research.

[0045] Example 2 In vitro induction of differentiation of pancreatic β cells The pluripotent stem cells that have been successfully gene-edited are differentiated into pancreatic β cells using a chemical small molecule-induced differentiation strategy. In vitro induced differentiation of pancreatic β cells is prepared by the following steps: Stage 1: Definitive endoderm The first stage formula is based on MCDB131 (Life Technologies, catalog number 10372019) basal medium supplemented with 1% B-27 (Gibco, catalog number 12587010), 1% GlutaMAX (Gibco, catalog number 35050079), 4.5 mM glucose (Sigma, catalog number G7021), 1% penicillin / streptomycin (Pen / Strep), 100 ng / mL ActivinA, 0.25 mM ascorbic acid, 50 nMPI-103, 6 μM CHIR-99021 and 10 μM Y-27632 (only on the first day). The culture medium for the next 3 days was prepared as MCDB131 supplemented with 1% B-27, 1% GlutaMAX, 4.5 mM glucose, 1% Pen / Strep, 50 ng / mL Activin A, and 0.25 mM ascorbic acid, and fresh medium was replaced every day.

[0046] After 4 days of induction of the first stage of differentiation of successfully gene-edited pluripotent stem cells in vitro, the pluripotent stem cells transformed into oriented endoderm. Bright field imaging of the microscope shows ( Figure 3 The cells formed a uniform flat layer and were highly differentiated. Accutase was used to digest the flat layer cells into single cells, and flow cytometry was used to detect the directional endoderm markers FOXA2 / SOX17. The experimental results showed that the double positive ratio was 96.8% ( Figure 3 (right image).

[0047] Stage 2: primitive gut tube The second stage formula is to supplement MCDB131 basal medium with 1% B-27, 1% GlutaMAX, 4.5 mM glucose, 1% Pen / Strep, 50 ng / mL keratinocyte growth factor (KGF), 0.25 mM ascorbic acid, 100 nM WNT-C59 and 5 μM SB431542, and replace fresh medium every day for 2 days.

[0048] Stage 3: Posterior foregut In pancreatic development, the small molecule SANT-1 inhibits the Sonic Hedgehog (Shh) signaling pathway; Retinoic Acid is a bioactive vitamin A metabolite that regulates the structure and morphogenesis of posterior foregut cells; LDN193189 is a selective BMP signaling inhibitor that inhibits differentiation into the hepatocyte fate lineage; the combined use of key small molecules such as Retinoic Acid, LDN193189, and SANT-1 promotes high expression of the posterior foregut cell marker PDX1, driving cell fate to the posterior foregut cell specialization. The third stage formula is to supplement DMEM (Gibco, catalog number C11965500CP) basal medium with 1% B-27, 1% Pen / Strep, 2 μM Retinoic Acid, 0.1 μM LDN-193189, 0.25 μM SANT-1, and 100 nM WNT-C59, replace fresh medium every day, and culture for 4 days.

[0049] Stage 4: pancreatic progenitor In vitro induced differentiation cells were transformed from 2D planar culture to 3D suspension culture mode to achieve the 3D structure of pancreatic islets. PDX1 / NKX6.1 gene expression was activated by the combined action of epidermal growth factor (EGF) combined with nicotinamide and α-amyloid precursor protein regulator (TPB) to produce pancreatic progenitor cells. The fourth stage formula is to supplement DMEM basal medium with 1% B-27, 1% GlutaMAX, 1% Pen / Strep, 100 ng / mL epidermal growth factor (EGF), 0.2 μM α-amyloid precursor protein regulator (TPB), 10 mM nicotinamide, 0.25 μM SANT-1 and 0.25 mM ascorbic acid, and fresh medium was replaced every two days for 5 days.

[0050] In vitro induced differentiation of cells from 2D planar culture to 3D suspension culture mode, induced differentiation into pancreatic progenitor cells. Microscope bright field imaging shows ( Figure 4 The cell spheres were uniform and glossy. The pancreatic progenitor cell markers PDX1 / NKX6.1 were detected by flow cytometry. Figure 4 As shown in the right figure, the experimental results showed that the double-positive ratio was 80.6%, indicating that it had been efficiently induced to differentiate into pancreatic progenitor cells.

[0051] Stage 5: pancreatic endocrine progenitor ALK5 inhibitor II, as an ALK5 inhibitor, promotes the transformation of pancreatic endocrine cells; ISX-9 small molecules can induce upregulation of NEUROD1 gene expression; combined use of ALK5 inhibitor II, ISX-9 and WNT-C59 small molecules can synergistically promote the transformation of pancreatic endocrine progenitor cells (PDX1 + / NKX6.1 + / NEUROD1 + The fifth stage formula is MCDB131 basal medium supplemented with 1% B-27, 1% GlutaMAX, 1% Pen / Strep, 10 μM ALK5 inhibitor II, 0.3 μM LDN-193189, 1 μM triiodothyronine (T3), 10 μM ISX-9, 10 μg / mL heparin sodium, 0.1 μM γ-secretase inhibitor XX, 100 nM WNT-C59, 10 μM Y-27632 and 0.25 mM ascorbic acid, with fresh medium replaced every two days for 6 days.

[0052] Stage 6: Pancreatic β cells (hiPSC-β cells) Continuous treatment of cells with ALK5 inhibitor II can significantly enhance insulin gene expression; small molecule R428, an AXL tyrosine kinase inhibitor, effectively induces the expression of mature pancreatic β cell marker MAFA through combined action with ALK5 inhibitor II and T3; antioxidant NAC can effectively maintain intracellular MAFA protein levels; after treatment with small molecules such as ALK5inhibitor II, R428, T3 and NAC, pancreatic β cells were successfully induced to differentiate. The sixth stage formula is to supplement MCDB131 basal medium with 1% B-27, 1% Pen / Strep, 10 μM ALK5 inhibitor II, 1 μM T3, 10 μg / mL heparin, 0.25 mM ascorbic acid, 0.5 μM R428, 2 mM N-acetyl-L-cysteine ​​(NAC) and 10 μM zinc sulfate, and culture for 2 days.

[0053] Finally, pluripotent stem cells were successfully induced to differentiate into pancreatic β cells. Bright field microscopy showed ( Figure 5 The pancreatic β cell spheres formed were uniform and transparent. The pancreatic β cell marker C-peptide / NKX6.1 was detected by flow cytometry. Figure 5 As shown in the right figure, the experimental results show that the proportion of double-positive cells is 61.6%, indicating that they have been efficiently induced to differentiate into pancreatic β cells and can be used for subsequent pancreatic β cell functional maturation research.

[0054] Example 3 K + Ion stimulation of pancreatic β-cell function experiment KCl is widely used to simulate the depolarization during glucose-stimulated insulin secretion. Therefore, insulin secretion caused by KCl is generally considered to be equivalent to the first phase of glucose-induced insulin secretion. When pancreatic β cells are stimulated by high extracellular KCl concentrations, the cell membrane depolarizes, causing calcium ion channels to open, and extracellular calcium ions flow inward to stimulate insulin secretion into the extracellular space.

[0055] Static sugar stimulation solution formula (Krebs buffer): 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl2,1.2 mM MgCl2, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, 0.1% BSA.

[0056] K + The ion stimulation pancreatic β cell functional experiment is completed by the following steps: 1. Take about 50-100 differentiated pancreatic β-cell spheres, place them in a 6-well plate, and wash them twice with 0mM glucose-krebs buffer; 2. Add 1 mL of 0 mM glucose-krebs buffer to the well plate and place it in a 37°C, 5% CO2 incubator for 1 hour. 3. After 1 hour, remove the supernatant with a pipette; 4. Add 1 mL of 0 mM glucose-krebs buffer to the well plate and place it in a 37°C, 5% CO2 incubator for 30 min. 5. After 30 minutes, place the well plate on the stage of a fluorescence microscope, slowly add glucose along the well wall to a final concentration of 2.8 mM, and record the green fluorescence of the cell spheres using a fluorescence microscope; 6. Slowly add KCl along the well wall to a final concentration of 30 mM and record the green fluorescence of the cell spheres using a fluorescence microscope.

[0057] Under stimulation with 2.8 mM glucose in Krebs buffer, the fluorescence intensity of differentiated pancreatic β-cell spheres was dim ( Figure 6 When 30 mM KCl is added, the cell membrane depolarizes, causing calcium channels to open. Extracellular calcium ions flow in and combine with intracellular GCaMP6f calcium signal fluorescent protein, significantly enhancing the green fluorescence of pancreatic β cell spheres ( Figure 6 The right picture shows that pancreatic β cells differentiated from pluripotent stem cells with the GCaMP6f gene knocked in can respond to intracellular calcium ion concentration in real time, indicating that differentiated pancreatic β cells carrying the calcium signal fluorescent protein GCaMP6f gene can be used in the study of cell function maturation.

Claims

1. A calcium imaging pluripotent stem cell, characterized in that The calcium imaging pluripotent stem cells are obtained by the following method: designing and constructing a homologous template DNA of the calcium imaging gene GCaMP6f carrying a CAG strong promoter, using high-precision CRISPR-Cas9 gene editing technology, accurately introducing a highly efficient and specific sgRNA directed to the AAVS1 site, an eSpCas9 protein, and a homologous template DNA into the pluripotent stem cells, knocking the calcium imaging gene GCaMP6f into the AAVS1 site of the pluripotent stem cells, and constructing the calcium imaging pluripotent stem cells; The sequence of sgRNA is shown in SEQ ID NO:2, and the sequence of homologous template DNA is shown in SEQ ID NO:

3.

2. The calcium imaging pluripotent stem cell according to claim 1, characterized in that The nucleotide sequence of the calcium imaging gene GCaMP6f is shown in SEQ ID NO:

1.

3. A method for constructing calcium imaging pluripotent stem cells, characterized in that The method comprises: designing and constructing a homologous template DNA of a calcium imaging gene GCaMP6f carrying a strong CAG promoter, using a high-precision CRISPR-Cas9 gene editing technology, accurately introducing a highly efficient and specific sgRNA directed to the AAVS1 site, an eSpCas9 protein and a homologous template DNA into pluripotent stem cells, knocking the calcium imaging gene GCaMP6f into the AAVS1 site of the pluripotent stem cells, and constructing the calcium imaging pluripotent stem cells; The sequence of sgRNA is shown in SEQ ID NO:2, and the sequence of homologous template DNA is shown in SEQ ID NO:

3.

4. The method according to claim 3, characterized in that The method comprises the following steps: designing and constructing a homologous template DNA of a calcium imaging gene GCaMP6f carrying a strong CAG promoter, electro-transfecting a highly efficient and specific sgRNA directed to the AAVS1 site, an eSpCas9 protein and the homologous template DNA into pluripotent stem cells, wherein the eSpCas9 protein recognizes the AAVS1 site and cuts the DNA under the guidance of the sgRNA to generate double-stranded DNA breaks, and recognizes homologous arms in the homologous template DNA during the DNA repair process, wherein the homologous arms are paired with the broken DNA ends to perform homologous recombination, and the homologous template DNA fills the gap at the DNA break, thereby knocking the calcium imaging gene GCaMP6f into the AAVS1 site of the pluripotent stem cells.

5. Use of the calcium imaging pluripotent stem cell according to any one of claims 1 to 2 in differentiating pancreatic β cells with the ability to monitor visualized calcium signals.

6. The use according to claim 5, characterized in that The application method is that calcium imaging pluripotent stem cells are induced to differentiate into pancreatic beta cells in vitro. The pancreatic beta cells have the ability to monitor visualized calcium signals. During the induction of differentiation, calcium imaging fluorescence detection is performed on the pancreatic beta cells to track and monitor the functional maturation of the differentiated pancreatic beta cells.

7. A method for optimizing the screening of induced differentiation technology in the process of pluripotent stem cells differentiating into pancreatic β cells, characterized in that The method comprises: inducing differentiation of the calcium imaging pluripotent stem cells as described in any one of claims 1 to 2 by using different induction differentiation processes to obtain pancreatic β cells, wherein the pancreatic β cells have the ability to monitor visualized calcium signals, detect the fluorescence intensity of the pancreatic β cells under glucose stimulation, and track and monitor in real time the functional level of differentiated pancreatic β cells in secreting insulin in response to glucose stimulation, thereby screening the optimal induction differentiation process.

8. The pancreatic β cell fluorescent reporter system with the ability of visualizing calcium signal monitoring obtained by inducing differentiation of calcium imaging pluripotent stem cells as described in any one of claims 1 to 2.

9. Use of the pancreatic β cell fluorescence reporter system as claimed in claim 8 in monitoring the functional level of insulin secretion of pancreatic β cells, studying the mechanism pathway of diabetes or screening diabetes drugs.

10. The use according to claim 8, characterized in that The application method is to induce differentiation of calcium imaging pluripotent stem cells in vitro to obtain a pancreatic β cell fluorescent reporter system with the ability to monitor visualized calcium signals, stimulate the pancreatic β cells with different drugs or induction reagents, detect the fluorescence intensity in real time, track the changes in insulin secretion levels under different stimulation conditions, and study the effects of drugs or induction reagents on the insulin secretion function of pancreatic β cells.

Images