Method for iPSC directed induction of secreting functional cells and use thereof

Abstract

The application belongs to the technical field of cell engineering and cell differentiation regulation, and discloses a method for directionally inducing secretion function cells from iPSCs and application thereof. The method takes the initial state of iPSCs and the potential of secretion function as cores, and constructs a pretreatment-induction differentiation integrated system. By screening and passing, iPSC clones with specific adhesion characteristics and enzyme sensitivity are enriched, and an iPSC population with enhanced secretion function potential is obtained. In the differentiation initiation stage, a culture system containing a Rock inhibitor is used in combination with laminin modified culture surface to maintain the intercellular connection state and realize the synchronous response of the induction signal. Then, the directionally induced differentiation is carried out by adding induction factors in stages, and the obtained cells are screened and purified to prepare high-purity secretion function cells. The prepared cells can be widely applied to drug screening, cell therapy and disease model construction, and provide technical support for the large-scale preparation of clinical-grade secretion function cells.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering and cell differentiation regulation technology, specifically relating to a method and application of iPSC-directed induction of secretory cells. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) have shown great promise in regenerative medicine, cell therapy, and disease model construction due to their ability to be obtained through somatic cell reprogramming and their multipotent differentiation potential similar to embryonic stem cells. Obtaining cells with specific secretory functions, such as pancreatic β cells, hepatocytes, or neurosecreting cells, through in vitro induced differentiation is considered a crucial technological approach for addressing endocrine, metabolic, and nervous system diseases. Therefore, how to efficiently and stably induce iPSCs into mature, functional cells with secretory functions has remained a research hotspot in this field.

[0003] In existing technologies, methods for inducing iPSCs into secretory cells mainly focus on optimizing the culture system during the differentiation stage. For example, this involves regulating the types, concentrations, and duration of growth factors and signaling pathway molecules to simulate embryonic development and achieve targeted differentiation. While these methods can obtain target cell types to some extent, they generally suffer from low differentiation efficiency, high cell population heterogeneity, unstable secretory function, and poor batch-to-batch reproducibility. Especially with the increasing demand for large-scale preparation and clinical translation, these shortcomings severely restrict the further application of iPSC differentiation technology. Further research has revealed that even using the same differentiation protocol, iPSCs from different sources or passage states still exhibit significant differences in differentiation results and functional performance. However, existing technologies generally attribute this difference to imprecise parameter control during differentiation, paying less attention to the initial state characteristics of iPSCs before entering the differentiation stage, such as clonal composition, cell viability, and population homogeneity. Furthermore, in the differentiation initiation stage, conventional practices often involve removing survival-promoting factors to trigger differentiation, which can easily lead to disruption of intercellular connections and asynchronous signal responses, further exacerbating functional heterogeneity during differentiation. Summary of the Invention

[0004] To address the shortcomings mentioned in the background art, the present invention aims to provide a method and application for the targeted induction of secretory cells by iPSCs. This method enriches an iPSC population with enhanced secretory potential before differentiation through selective passage, and employs a culture system containing Rock inhibitors combined with laminin modification of the culture surface during the differentiation initiation stage to achieve synchronous differentiation of the cell population. Combined with staged induction and screening purification steps, this method significantly improves the yield, purity, and functional stability of secretory cells, making it suitable for the preparation and application of various secretory cell types.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for targeted induction of secretory cells by iPSCs includes the following steps:

[0007] (1) Selective passage treatment was used for induced pluripotent stem cells. By controlling the digestion conditions, iPSC clones with specific adherent properties and enzyme sensitivity were selectively retained to obtain an iPSC population with enhanced secretory potential.

[0008] (2) The iPSCs obtained in step (1) were seeded onto a culture surface modified with laminin and cultured in a differentiation initiation medium containing Rock inhibitor for 24–48 h.

[0009] (3) In the system obtained in step (2), inducing factors for inducing the differentiation of secretory cells are added in stages according to a predetermined order, and directed induction culture is carried out to obtain secretory cells;

[0010] (4) The induced cells were screened and purified to obtain high-purity secretory cells.

[0011] More preferably, the secretory cells are selected from one or more of pancreatic β cells, hepatocytes, and neurosecretory cells.

[0012] More preferably, the screening passage treatment in step (1) includes: digesting iPSCs with 0.25% trypsin at 37°C for 1.5–2.0 min, collecting cell clones in suspension and semi-adherent state, performing monoclonal culture by limiting dilution, screening clones with an Oct4 to Sox2 gene expression ratio of not less than 1.2, enriching an iPSC population with enhanced secretory function potential, wherein the proportion of high-potential clones in the population is not less than 85%.

[0013] More preferably, the Rock inhibitor in step (2) is Y-27632, and its concentration in the differentiation initiation medium is 10–15 μmol / L; the surface modification concentration of the laminin is 5–8 μg / mL, and the modification conditions are incubation at 37°C for 1–2 h.

[0014] More preferably, the differentiation initiation medium in step (2) is based on DMEM / F12, with the addition of 2% B27 additive, 1% penicillin-streptomycin mixture, 1 mmol / L glutamine and 5 mmol / L glucose.

[0015] More preferably, when the secretory cells are pancreatic β cells, the directed induction differentiation in step (3) includes the following stages: on induction days 1–3, 100 ng / mL Activin A and 25 ng / mL Wnt3a are added to the culture system; on induction days 4–6, 50 ng / mL FGF2 and 2 μmol / L retinoic acid are added to the culture system; on induction days 7–10, 10 μmol / L DAPT and 10 nmol / L Exendin-4 are added to the culture system; the total induction time is 14–16 days; when the secretory cells are hepatocytes, the directed induction differentiation in step (3) includes the following stages: on induction days 1–3, 80 ng / mL Activin A and 20 ng / mL BMP4 are added; on induction days 4–7, 20 ng / mL HGF is added; on induction days 8–14, 10 ng / mL OSM and 1 μmol / L dexamethasone are added.

[0016] More preferably, the screening and purification in step (4) is performed by flow cytometry; when the secretory cells are pancreatic β cells, CD200 and CD319 are used as positive markers; when the secretory cells are hepatocytes, CD26 and CD13 are used as positive markers; and a cell population with a positive rate of not less than 80% is selected.

[0017] More preferably, the iPSC in step (1) is an iPSC obtained by reprogramming human peripheral blood mononuclear cells, and its passage number is controlled at 5-10 generations.

[0018] The use of secretory cells prepared by a method for iPSC-directed induction of secretory cells in drug screening, cell therapy or disease model construction.

[0019] The beneficial effects of this invention are:

[0020] This invention enriches iPSC clones with specific adherent properties and enzyme sensitivity through selective passaging, effectively reducing the intrinsic heterogeneity of the cell population. This ensures that cells entering the differentiation stage maintain a high degree of consistency in developmental potential and responsiveness, laying a stable foundation for subsequent targeted induction. Compared with conventional indiscriminate passaging, this source quality control strategy significantly improves the uniformity of cell response to induction signals during differentiation and reduces the generation of non-target cell types. Simultaneously, by retaining Rock inhibitors and modifying the culture surface with laminin during the differentiation initiation stage, the integrity of intercellular connections and population structure is maintained, effectively preventing premature cell dispersion or stress-induced death. This creates a synchronized signal transduction microenvironment at the population level, allowing inducing factors to act uniformly and stably on each cell. This synchronized initiation mechanism not only improves differentiation efficiency but also significantly enhances the maturity and functional consistency of secretory cells. Based on this, through staged targeted induction simulating in vivo development and subsequent screening and purification, this invention can obtain a target cell population with high purity and stable secretory capacity, significantly improving the problems of large batch-to-batch variations and significant functional fluctuations commonly found in existing technologies. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: Based on the selection and passage enrichment of iPSCs with enhanced secretory potential, and combined with the cell connectivity state regulation and staged directed induction process during the differentiation initiation stage, target cells with stable secretory function were prepared.

[0023] (1) Induced pluripotent stem cells (iPSCs) obtained by reprogramming human peripheral blood mononuclear cells were selected and passaged to the 5th generation for use. The iPSCs were seeded in a conventional feeder-free culture system, and selective passage was performed when the cell confluence reached about 70%. 0.25% (w / v) trypsin solution was added to the culture system, and the cells were digested at 37°C for 1.5 min. The cells were gently pipetted to detach some of them, and cell clones in suspension and semi-adherent state were collected. The obtained cells were cultured as monoclonal cells using the limiting dilution method, and the gene expression levels of Oct4 and Sox2 were detected by real-time quantitative PCR. Clones with an expression ratio of ≥1.2 were screened for expansion culture to obtain an iPSC population with enhanced secretory potential, of which high-potential clones accounted for 85% of the population.

[0024] (2) The preprocessed iPSCs are processed at 2×10 4 Cells were seeded at a density of 10 cells / cm² into laminin-modified culture plates. The laminin solution concentration was 5 μg / mL, and the modification conditions were incubation at 37°C for 1 h. Differentiation initiation medium was based on DMEM / F12, with 2% B27 additive and 1% penicillin-streptomycin mixture added sequentially by volume fraction. Glutamine was supplemented to a final concentration of 1 mmol / L, glucose to a final concentration of 5 mmol / L, and the rock inhibitor Y-27632 to a final concentration of 10 μmol / L. Cells were cultured under these conditions for 24 h to maintain intercellular connections.

[0025] (3) Then, directed differentiation of pancreatic β cells was performed. On days 1–3 of induction, Activin A was added to the culture system to a final concentration of 100 ng / mL and Wnt3a was added to a final concentration of 25 ng / mL; on days 4–6 of induction, FGF2 was added to a final concentration of 50 ng / mL and retinoic acid was added to a final concentration of 2 μmol / L; on days 7–10 of induction, DAPT was added to a final concentration of 10 μmol / L and Exendin-4 was added to a final concentration of 10 nmol / L; the total induction time was 14 days.

[0026] (4) After induction, the obtained cells were screened and purified. Flow cytometry was used to screen cell populations with a double positive rate of ≥80% using CD200 and CD319 as positive markers to obtain high-purity secretory cells.

[0027] Example 2: Based on Example 1, this example further strengthens the cell pretreatment conditions and differentiation initiation regulation intensity. By extending the treatment time and increasing the level of related factors, target cells with stable secretory function and high purity are obtained.

[0028] (1) Human peripheral blood mononuclear cells (iPSCs) obtained through reprogramming were selected and passaged to the 10th generation as starting cells. The iPSCs were seeded in a feeder-free culture system and cultured. When the cell confluence reached approximately 75%, selective passage was performed. 0.25% (w / v) trypsin solution was added to the culture system and digested at 37°C for 2.0 min. The cells were then gently pipetted to fully disperse them, and cell clones in suspension and semi-adherent states were collected. The resulting cells were cultured as monoclonal cells using limiting dilution. The gene expression levels of Oct4 and Sox2 were detected by real-time quantitative PCR. Clones with an expression ratio ≥1.2 were screened for amplification culture to obtain an iPSC population with enhanced secretory potential, of which high-potential clones accounted for 85%.

[0029] (2) The preprocessed iPSCs are processed at 3×10 4 Cells were seeded at a density of 10 cells / cm² into laminin-modified culture plates. The laminin solution concentration was 8 μg / mL, and the cells were incubated at 37°C for 2 h to complete the surface modification. Differentiation initiation medium was based on DMEM / F12, with 2% B27 additive and 1% penicillin-streptomycin mixture added sequentially by volume fraction. Glutamine was supplemented to a final concentration of 1 mmol / L, glucose to a final concentration of 5 mmol / L, and the rock inhibitor Y-27632 to a final concentration of 15 μmol / L. Cells were cultured under these conditions for 48 h to stabilize the cell population structure and maintain intercellular connections.

[0030] (3) After the differentiation initiation was completed, pancreatic β-cells were induced to differentiate in a directed manner. On days 1–3 of induction, Activin A was added to the culture system to a final concentration of 100 ng / mL, and Wnt3a was added to a final concentration of 25 ng / mL. On days 4–6 of induction, FGF2 was added to the culture system to a final concentration of 50 ng / mL, and retinoic acid was added to a final concentration of 2 μmol / L. On days 7–10 of induction, DAPT was added to the culture system to a final concentration of 10 μmol / L, and Exendin-4 was added to a final concentration of 10 nmol / L. The entire induction process lasted for 16 days.

[0031] (4) After induction, the obtained cells were screened and purified. Flow cytometry was used to detect the expression of cell surface markers. CD200 and CD319 were used as positive markers. Cell populations with double positive rate ≥80% were screened to obtain high-purity secretory cells with stable secretory function.

[0032] Example 3: In this example, under the condition that the key process parameters are moderate, iPSCs are pretreated, differentiation is initiated and directed, and combined with screening and purification steps to obtain target cells with stable secretory function and high purity.

[0033] (1) Human peripheral blood mononuclear cells (iPSCs) obtained through reprogramming were selected and passaged to the 7th generation as starting cells. The iPSCs were seeded in a feeder-free culture system and cultured. When the cell confluence reached about 70%–75%, selective passage was performed. 0.25% (w / v) trypsin solution was added to the culture system and digested at 37°C for 1.75 min. Cells were partially detached by gentle pipetting, and cell clones in suspension and semi-adherent state were collected. The cells were cultured as monoclonal cells using the limiting dilution method, and the gene expression levels of Oct4 and Sox2 were detected by real-time quantitative PCR. Clones with an expression ratio ≥ 1.35 were screened for amplification culture to obtain an iPSC population with enhanced secretory potential, of which high-potential clones accounted for 85%.

[0034] (2) The preprocessed iPSCs were subjected to a 2.5×10⁻⁶ ppm. 4 Cells were seeded at a density of 10 cells / cm² into laminin-modified culture plates. The laminin solution concentration was 6.5 μg / mL, and the cells were incubated at 37°C for 1.5 h to complete the surface modification. The differentiation initiation medium was DMEM / F12 basal medium, with 2% B27 additive and 1% penicillin-streptomycin mixture added by volume, and glutamine and glucose were supplemented to a final concentration of 1 mmol / L and 5 mmol / L, respectively. Rock inhibitor Y-27632 was added to a final concentration of 12.5 μmol / L. Cells were cultured under these conditions for 36 h to maintain intercellular connections and synchronize differentiation initiation.

[0035] (3) After the differentiation initiation was completed, pancreatic β cells were directed to differentiate. On days 1–3 of induction, Activin A was added to the culture system to a final concentration of 100 ng / mL, and Wnt3a was added to a final concentration of 25 ng / mL; on days 4–6 of induction, FGF2 was added to the culture system to a final concentration of 50 ng / mL, and retinoic acid was added to a final concentration of 2 μmol / L; on days 7–10 of induction, DAPT was added to the culture system to a final concentration of 10 μmol / L, and Exendin-4 was added to a final concentration of 10 nmol / L; the entire induction process lasted 15 days.

[0036] (4) After induction, the obtained cells were screened and purified. The expression of cell surface markers was detected by flow cytometry. CD200 and CD319 were used as positive markers. Cell populations with double positive rate ≥80% were screened to obtain high-purity secretory cells with stable secretory function.

[0037] Example 4: In this example, iPSCs were pretreated and their differentiation initiation was synchronously regulated. Targeted hepatocyte induction and screening purification steps were then used to prepare hepatocytes with stable secretory function and high purity.

[0038] (1) Human peripheral blood mononuclear cells (iPSCs) obtained through reprogramming were selected and passaged to the 7th generation as starting cells. The iPSCs were seeded in a feeder-free culture system and cultured. When the cell confluence reached about 70%–75%, selective passage was performed. 0.25% (w / v) trypsin solution was added to the culture system and digested at 37°C for 1.75 min. Cells were partially detached by gentle pipetting, and cell clones in suspension and semi-adherent state were collected. The obtained cells were cultured as monoclonal cells using the limiting dilution method, and the gene expression levels of Oct4 and Sox2 were detected by real-time quantitative PCR. Clones with an expression ratio of ≥1.35 were screened for amplification culture to obtain an iPSC population with enhanced secretory potential, of which high-potential clones accounted for 85% of the population.

[0039] (2) The preprocessed iPSCs were subjected to a 2.5×10⁻⁶ ppm. 4 Cells were seeded at a density of 100 cells / cm² into laminin-modified culture plates. The laminin solution concentration was 6.5 μg / mL, and the culture was incubated at 37°C for 1.5 h to complete the surface modification. The differentiation initiation medium was DMEM / F12 basal medium, with 2% B27 additive and 1% penicillin-streptomycin mixture added by volume, and glutamine and glucose were supplemented to a final concentration of 1 mmol / L and 5 mmol / L, respectively. Rock inhibitor Y-27632 was added to a final concentration of 12.5 μmol / L. The cells were cultured under the above conditions for 36 h to maintain intercellular connections and synchronize differentiation initiation.

[0040] (3) After the differentiation initiation is completed, hepatocytes are directionally induced to differentiate. On days 1–3 of induction, Activin A is added to the culture system to a final concentration of 80 ng / mL and BMP4 is added to a final concentration of 20 ng / mL to induce cells to differentiate toward the endoderm. On days 4–7 of induction, HGF is added to the culture system to a final concentration of 20 ng / mL to promote the formation of hepatic progenitor cells. On days 8–14 of induction, OSM is added to the culture system to a final concentration of 10 ng / mL and dexamethasone is added to a final concentration of 1 μmol / L to promote hepatocyte maturation. The entire induction process lasts for 14 days.

[0041] (4) After induction, the obtained cells were screened and purified. Flow cytometry was used for detection. When the secretory cells were hepatocytes, CD26 and CD13 were used as positive markers to screen cell populations with a positive rate of ≥80% to obtain high-purity hepatocytes with stable secretory function.

[0042] Comparative Example 1: A method for preparing iPSC-directed induced secretory cells without selective passage.

[0043] (1) iPSCs obtained by reprogramming human peripheral blood mononuclear cells were selected and passaged to the 7th generation as starting cells. The iPSCs were not subjected to selective passage treatment, but were expanded and cultured using conventional indiscriminate passage method. That is, when the cell confluence reached about 70%-75%, they were directly digested with 0.25% (w / v) trypsin at 37°C for 1.75 min to cause the cells to detach as a whole. After centrifugation, they were collected and re-inoculated for culture. No selection was made for suspension or semi-adherent clones, nor was single-clonal culture or Oct4 to Sox2 expression ratio screening performed.

[0044] (2) The above-mentioned unfiltered iPSCs were processed at a concentration of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / cm² into laminin-modified culture plates. The laminin solution concentration was 6.5 μg / mL, and the cells were incubated at 37°C for 1.5 h to complete the surface modification. The differentiation initiation medium was DMEM / F12 basal medium, with 2% B27 additive and 1% penicillin-streptomycin mixture added by volume, and glutamine and glucose were supplemented to a final concentration of 1 mmol / L and 5 mmol / L, respectively. Rock inhibitor Y-27632 was added to a final concentration of 12.5 μmol / L. Cells were cultured under these conditions for 36 h to initiate the differentiation process.

[0045] (3) After the differentiation initiation was completed, pancreatic β cells were induced to differentiate in a directed manner. On days 1–3 of induction, Activin A was added to the culture system to a final concentration of 100 ng / mL, and Wnt3a was added to a final concentration of 25 ng / mL; on days 4–6 of induction, FGF2 was added to the culture system to a final concentration of 50 ng / mL, and retinoic acid was added to a final concentration of 2 μmol / L; on days 7–10 of induction, DAPT was added to the culture system to a final concentration of 10 μmol / L, and Exendin-4 was added to a final concentration of 10 nmol / L; the entire induction process lasted for 15 days.

[0046] (4) After induction, the obtained cells were screened and purified. The expression of cell surface markers was detected by flow cytometry. CD200 and CD319 were used as positive markers. Cell populations with double positive rate ≥80% were screened to obtain comparative secretory cells.

[0047] Comparative Example 2: A method for preparing iPSCs with directed secretory function without the addition of Rock inhibitors during the differentiation initiation phase.

[0048] (1) The iPSC pretreatment and screening method is the same as step (1) in Example 3, to obtain an iPSC population with enhanced secretory function potential.

[0049] (2) The preprocessed iPSCs were subjected to a 2.5×10⁻⁶ ppm. 4 Cells were seeded at a density of 10 cells / cm² into laminin-modified culture plates. The laminin solution was at a concentration of 6.5 μg / mL, and the cells were incubated at 37°C for 1.5 h to complete the surface modification. The differentiation initiation medium was DMEM / F12 as the basal medium, with 2% B27 additive and 1% penicillin-streptomycin mixture added by volume, and glutamine and glucose were supplemented to a final concentration of 1 mmol / L and 5 mmol / L, respectively, without the addition of Rock inhibitors. Cells were cultured under these conditions for 36 h to initiate the differentiation process.

[0050] (3) After the differentiation initiation was completed, pancreatic β cells were induced to differentiate in a directed manner. On days 1–3 of induction, Activin A was added to the culture system to a final concentration of 100 ng / mL and Wnt3a was added to a final concentration of 25 ng / mL; on days 4–6 of induction, FGF2 was added to the culture system to a final concentration of 50 ng / mL and retinoic acid was added to a final concentration of 2 μmol / L; on days 7–10 of induction, DAPT was added to the culture system to a final concentration of 10 μmol / L and Exendin-4 was added to a final concentration of 10 nmol / L; the entire induction process lasted for 15 days.

[0051] (4) After induction, the obtained cells were screened and purified. The expression of cell surface markers was detected by flow cytometry. CD200 and CD319 were used as positive markers. Cell populations with double positive rate ≥80% were screened to obtain comparative secretory cells.

[0052] Performance testing

[0053] 1. Differentiation efficiency and target cell ratio test

[0054] Cells induced in Examples 1–4 and Comparative Examples 1–2 were collected, and single-cell suspensions were prepared by trypsin digestion. After washing with PBS, the suspensions were prepared at a concentration of 1×10⁻⁶ cells / mL. 6 Samples were taken from each cell / tube. Fluorescently labeled antibodies were added, and the cells were incubated at 4°C in the dark for 30 min. Pancreatic β-cells were sampled using CD200 and CD319 antibodies, and hepatocytes using CD26 and CD13 antibodies. After incubation, the cells were washed and resuspended, and flow cytometry was used for detection. The proportion of double-positive cells in the total cell count was calculated, and the results are shown in Table 1 below.

[0055] Table 1. Proportion of target secretory cells

[0056] sample Target cell type Double positive markers Target cell percentage (%) Example 1 pancreatic β cells CD200⁺ / CD319⁺ 80–85 Example 2 pancreatic β cells CD200⁺ / CD319⁺ 88–93 Example 3 pancreatic β cells CD200⁺ / CD319⁺ 90–95 Example 4 hepatocytes CD26⁺ / CD13⁺ 85–90 Comparative Example 1 pancreatic β cells CD200⁺ / CD319⁺ 55–65 Comparative Example 2 pancreatic β cells CD200⁺ / CD319⁺ 60–70

[0057] As shown in Table 1, the proportion of target secretory cells in Examples 1–4 prepared using the method of this invention was significantly higher than that in Comparative Examples 1–2, indicating that selective passage and synchronous regulation of differentiation initiation can effectively improve the efficiency of directed differentiation. Comparative Example 1 showed a significantly reduced differentiation efficiency due to the high heterogeneity of the initial iPSC population; Comparative Example 2 also suffered from unstable cell connections and insufficient differentiation synchronicity during the differentiation initiation stage, thus affecting the proportion of target cells. Although Example 2 further enhanced the pretreatment and initiation conditions compared to Example 3, the higher seeding density and stronger control intensity increased cell stress and competition, affecting the response of some cells to induction signals and resulting in a slightly lower proportion of target cells.

[0058] 2. Secretory function test

[0059] Collect secretory cells induced and purified in Examples 1–4 and Comparative Examples 1–2, and administer at a ratio of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates and cultured for 24 h. After culture, the culture supernatant was collected, and the secretory product content was detected by ELISA, strictly following the kit instructions. Results are expressed as ng / 10 6 The results are expressed as cells / 24 h. Specifically, insulin and C-peptide secretion were measured in pancreatic β-cells, albumin secretion and urea synthesis in hepatocytes, and in the stimulus-responsive secretion assay, pancreatic β-cells were cultured for 1 h under low glucose (2.8 mmol / L) and high glucose (16.7 mmol / L) conditions, respectively, and the high glucose / low glucose insulin secretion ratio was calculated. Changes in albumin and urea production in hepatocytes after the addition of metabolic substrates were measured. The results are shown in Table 2 below.

[0060] Table 2. Basic secretory capacity and stimulus-responsive secretion test results of secretory cells.

[0061] sample <![CDATA[Basal secretion (ng / 10 6 cells / 24 h)]]> Stimulus-responsive secretion Example 1 Insulin 200, C-peptide 45 High sugar / low sugar ratio 2.2 Example 2 Insulin 220, C-peptide 50 High sugar / low sugar ratio 2.4 Example 3 Insulin 250, C-peptide 58 High sugar / low sugar ratio 2.8 Example 4 Albumin 17; Urea 14 Albumin secretion increased by 35%, and urea synthesis increased by 30%. Comparative Example 1 Insulin 110, C-peptide 22 High sugar / low sugar ratio 1.5 Comparative Example 2 Insulin 130, C-peptide 26 High sugar / low sugar ratio 1.7

[0062] As shown in Table 2, compared with Comparative Examples 1 and 2, the secretory cells prepared in Examples 1–4 of this invention showed significantly improved basal secretion levels and stimulus-responsive secretion, indicating a marked enhancement in their secretory capacity and functional maturity. Comparative Example 1, due to the lack of selective passage, had insufficient initial cell potential, resulting in a lower secretion level; Comparative Example 2 lacked synchronous regulation during the differentiation initiation stage, limiting its stimulus-responsiveness. Example 3 obtained the highest secretion level and response ratio under suitable process conditions, demonstrating that the synergistic effect of source quality control and synchronous differentiation regulation helps to achieve optimal secretory function.

[0063] 3. Group functional consistency and heterogeneity testing

[0064] Collect secretory cells induced and purified in Examples 1–4 and Comparative Examples 1–2, and administer at a ratio of 1×10⁻⁶. 6 Individual cells / samples were collected. Single-cell secretion levels were detected using flow cytometry (sorting purity ≥98%) combined with ELISA. Insulin secretion from pancreatic β-cells or albumin secretion from hepatocytes were used as evaluation indicators. The standard deviation (SD) and coefficient of variation (CV) of endocrine levels within the same population were calculated. By comparing the SD and CV values ​​of the examples with those of the comparative examples, the concentration and variability of endocrine function distribution within the cell population were assessed. The results are shown in Table 3.

[0065] Table 3 Group functional consistency and heterogeneity

[0066] sample Target cell type SD CV (%) Example 1 pancreatic β cells 28 14 Example 2 pancreatic β cells 24 11 Example 3 pancreatic β cells 18 8 Example 4 hepatocytes 20 9 Comparative Example 1 pancreatic β cells 55 32 Comparative Example 2 pancreatic β cells 42 24

[0067] As shown in Table 3, the secretory cells prepared in Examples 1–4 of this invention are significantly superior to the comparative examples in terms of population functional consistency. The standard deviation and coefficient of variation of secretion levels in the cell populations of the examples are significantly reduced, indicating that secretory function is more concentrated and less volatile within the population. Example 3 shows the lowest SD and CV, indicating that under suitable process conditions, the cells respond most synchronously to the induction signal. Comparative Example 1 exhibits significantly increased cell population heterogeneity due to the lack of selective passage; Comparative Example 2 still shows significant population functional volatility due to insufficient synchronicity during the differentiation initiation stage.

[0068] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0069] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for targeted induction of secretory cells by iPSCs, characterized in that, Includes the following steps: (1) Selective passage treatment was used for induced pluripotent stem cells. By controlling the digestion conditions, iPSC clones with specific adherent properties and enzyme sensitivity were selectively retained to obtain an iPSC population with enhanced secretory potential. (2) The iPSCs obtained in step (1) were seeded onto a culture surface modified with laminin and cultured in a differentiation initiation medium containing Rock inhibitor for 24–48 h. (3) In the system obtained in step (2), inducing factors for inducing the differentiation of secretory cells are added in stages according to a predetermined order, and directed induction culture is carried out to obtain secretory cells; (4) The induced cells were screened and purified to obtain high-purity secretory cells.

2. The method for targeted induction of secretory cells using iPSCs according to claim 1, characterized in that, The secretory cells are selected from one or more of pancreatic β cells, hepatocytes, and neurosecretory cells.

3. The method for targeted induction of secretory cells using iPSCs according to claim 1, characterized in that, The screening passage process described in step (1) includes: digesting iPSCs with 0.25% trypsin at 37°C for 1.5–2.0 min, collecting cell clones in suspension and semi-adherent states, performing monoclonal culture by limiting dilution, screening clones with an Oct4 to Sox2 gene expression ratio of not less than 1.2, enriching an iPSC population with enhanced secretory potential, wherein the proportion of high-potential clones in the population is not less than 85%.

4. The method for targeted induction of secretory cells using iPSCs according to claim 1, characterized in that, The Rock inhibitor mentioned in step (2) is Y-27632, and its concentration in the differentiation initiation medium is 10–15 μmol / L; the surface modification concentration of the laminin is 5–8 μg / mL, and the modification conditions are incubation at 37℃ for 1–2 h.

5. The method for targeted induction of secretory cells using iPSCs according to claim 1, characterized in that, The differentiation initiation medium described in step (2) is based on DMEM / F12, with the addition of 2% B27 additive, 1% penicillin-streptomycin mixture, 1 mmol / L glutamine and 5 mmol / L glucose.

6. The method for targeted induction of secretory cells using iPSCs according to claim 2, characterized in that, When the secretory cells are pancreatic β cells, the directed differentiation in step (3) includes the following stages: on days 1-3 of induction, 100 ng / mL Activin A and 25 ng / mL Wnt3a are added to the culture system; on days 4-6 of induction, 50 ng / mL FGF2 and 2 μmol / L retinoic acid are added to the culture system; on days 7-10 of induction, 10 μmol / L DAPT and 10 nmol / L Exendin-4 are added to the culture system; the total induction time is 14-16 days. When the secretory cells are hepatocytes, the directed differentiation in step (3) includes the following stages: on days 1-3 of induction, 80 ng / mL Activin A and 20 ng / mL BMP4 are added; on days 4-7, 20 ng / mL HGF is added; on days 8-14, 10 ng / mL OSM and 1 μmol / L dexamethasone are added.

7. The method for targeted induction of secretory cells using iPSCs according to claim 1, characterized in that, The screening and purification described in step (4) uses flow cytometry; when the secretory cells are pancreatic β cells, CD200 and CD319 are used as positive markers; when the secretory cells are hepatocytes, CD26 and CD13 are used as positive markers; and a cell population with a positive rate of not less than 80% is selected.

8. The method for targeted induction of secretory cells by iPSCs according to claim 1, characterized in that, The iPSCs mentioned in step (1) are iPSCs obtained by reprogramming human peripheral blood mononuclear cells, and their passage number is controlled at 5-10 generations.

9. The use of a secretory functional cell prepared by any one of the methods of claims 1-8 in drug screening, cell therapy or disease model construction.