Method for inducing Muse cells to differentiate into pancreatic beta cell spheres through balling / adherent alternate culture and application

Abstract

The invention relates to the technical field of cell engineering, in particular to a method for inducing Muse cells to differentiate into pancreatic beta cell spheres through balling / adherent alternate culture and application. Experimental results in the invention show that the Muse cells subjected to static balling culture are obvious in apoptosis due to hypoxia in spheres, and almost all the cells are apoptotic after being cultured for 23 days; the Muse cells subjected to direct adherent induction culture are high in proliferation capacity, and the differentiation efficiency is 37.7%; a balling and adherent alternate culture induction mode is adopted, so that stable passage can be realized, and the differentiation efficiency is 62.4%; the alternative culture induction method effectively solves the problems of proliferation and differentiation efficiency, and realizes stable passage and induced differentiation of the pancreatic beta cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and particularly relates to a method and application for inducing Muse cells to differentiate into islet β cell spheres by alternate culture of spheroid formation / adhesion. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Diabetes is a metabolic disorder disease characterized by elevated blood glucose levels clinically, and its main pathogenesis is the reduction of insulin secretion and / or utilization disorder. Currently, diabetes has become the third major disease threatening human health and affecting people's quality of life after tumors and cardiovascular and cerebrovascular diseases.

[0004] Late-stage patients with type I diabetes and type II diabetes often need to inject exogenous insulin to control the blood glucose balance in the body. Although this method can effectively control the development of the disease, long-term insulin injection cannot stably maintain the physiological blood glucose balance in the body, thereby leading to the occurrence of some high-risk complications. Therefore, a treatment strategy that can reduce or even eliminate long-term complications is needed for the treatment of diabetes.

[0005] The method of islet transplantation can fundamentally treat diabetes, but due to the extreme shortage of donors, this method cannot become the main treatment means for diabetes. The replacement of functional insulin-secreting cells differentiated from stem cells is the treatment closest to normal physiological conditions, which can not only effectively control blood glucose but also prevent and reverse complications. Currently, the induction of islet β cells mainly comes from mesenchymal stem cells, embryonic stem cells, and iPS cells, and 2D adhesion or 3D spheroid formation methods are used for induction. However, the induction efficiency of mesenchymal stem cells is relatively low, and the induction efficiency of embryonic stem cells and iPS cells is relatively high, but there are tumorigenic risks in embryonic stem cells and iPS cells that are not fully differentiated or not fully separated and removed. At the same time, the 2D adhesion induction has a high proliferation rate but a low efficiency of differentiating into β islets, while the 3D spheroid formation induction has a high efficiency but a slow proliferation rate. Therefore, there is an urgent need for a stem cell and its differentiation method that is rich in sources, can efficiently induce the acquisition of islet β cells, and has no tumorigenic risk. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides a method and application for inducing Muse cells to differentiate into islet β cell spheres by alternate culture of spheroid formation / adhesion.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions: In the first aspect of the present invention, a method for inducing the differentiation of Muse cells into islet β cell spheres by alternating spheroid formation / adhesion culture is provided, comprising the following steps: (1) Prepare and obtain Muse cells; (2) Culture the obtained Muse cells using S1 medium to direct their differentiation into definitive endoderm cells; (3) Culture the obtained definitive endoderm cells using S2 medium to induce their differentiation to obtain pancreatic progenitor cells; (4) Culture the obtained pancreatic progenitor cells using S3 medium to induce their differentiation to obtain pancreatic endocrine progenitor cells; (5) Culture the obtained pancreatic endocrine progenitor cells using S4 medium to induce their differentiation to obtain pancreatic endocrine cell spheres; (6) Culture the obtained pancreatic endocrine cell spheres using S5 medium to induce their differentiation to obtain islet β cell spheres; During the cell culture process of steps (2) to (6), the method of alternating spheroid formation / adhesion culture is adopted.

[0008] In one or more embodiments, in step (1), the Muse cells are derived from mesenchymal tissues of bone marrow, adipose tissue, umbilical cord, and dermis, preferably umbilical cord.

[0009] In one or more embodiments, in step (2), the S1 medium is based on DMEM cell culture medium, and recombinant human activin - A (Activin A), a selective inhibitor of glycogen synthase kinase - 3β (GSK - 3β) CHIR - 99021, and endoderm inducer 1 (IDE1) are added; Preferably, the concentration of recombinant human activin - A (Activin A) is 95 - 105 ng / mL, preferably 100 ng / mL; Preferably, the concentration of the selective inhibitor of glycogen synthase kinase - 3β (GSK - 3β) CHIR - 99021 is 2.8 - 3.2 μM, preferably 3 μM; Preferably, the concentration of endoderm inducer 1 (IDE1) is 1.8 - 2.2 mM, preferably 2 mM.

[0010] In one or more embodiments, in step (2), the method for culturing the obtained Muse cells using S1 medium to direct their differentiation into definitive endoderm cells specifically includes: Under the conditions of 37 °C and 5% CO2, perform spheroid formation culture in suspension for 48 h, and then directly perform adherent culture for 24 h.

[0011] In one or more embodiments, in step (3), the S2 medium is based on DMEM cell medium and supplemented with recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7). Preferably, the concentration of recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7) is 45 - 55 ng / mL, preferably 50 ng / mL.

[0012] In one or more embodiments, in step (3), the method for inducing the differentiation of the definitive endoderm cells cultured with the S2 medium to obtain pancreatic progenitor cells specifically includes: Under the conditions of 37 °C and 5% CO2, spheroid culture is carried out under suspension conditions for 48 h, and then direct adherent culture is carried out for 24 h.

[0013] In one or more embodiments, in step (4), the S3 medium is based on DMEM cell medium and supplemented with recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7), retinoic acid, Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1, ROCK signaling pathway inhibitor Y-27632, selective BMP type I receptor inhibitor LDN193189, PKC activator PDBu, and recombinant human activin-A (Activin A); Preferably, the concentration of recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7) is 45 - 55 ng / mL, preferably 50 ng / mL; Preferably, the concentration of retinoic acid is 0.08 - 0.12 μM, preferably 0.1 μM; Preferably, the concentration of Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1 is 0.20 - 0.30 μM, preferably 0.25 μM; Preferably, the concentration of ROCK signaling pathway inhibitor Y-27632 is 8 - 12 μM, preferably 10 μM; Preferably, the concentration of selective BMP type I receptor inhibitor LDN193189 is 180 - 220 nM, preferably 200 nM; Preferably, the concentration of PKC activator PDBu is 450 - 550 nM, preferably 500 nM; Preferably, the concentration of recombinant human activin-A (Activin A) is 95 - 105 ng / mL, preferably 100 ng / mL.

[0014] In one or more embodiments, in step (4), the method for culturing the obtained pancreatic progenitor cells in S3 medium and inducing their differentiation to obtain pancreatic endocrine progenitor cells specifically includes: At 37 °C and 5% CO2, culture in suspension to form spheres for 48 h, and then directly culture adherently for 24 h.

[0015] In one or more embodiments, in step (5), the S4 medium is based on DMEM cell culture medium and supplemented with sodium heparin, ALK5 inhibitor A83-01, TGF-β receptor kinase inhibitor SB-431542, Betacellulin protein, triiodothyronine T3, retinoic acid, and Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1; Preferably, the concentration of sodium heparin is 8-12 μg / mL, preferably 10 μg / mL; Preferably, the concentration of ALK5 inhibitor A83-01 is 8-12 μM, preferably 10 μM; Preferably, the concentration of TGF-β receptor kinase inhibitor SB-431542 is 0.8-1.2 μM, preferably 1 μM; Preferably, the concentration of Betacellulin protein is 18-22 ng / mL, preferably 20 ng / mL; Preferably, the concentration of triiodothyronine T3 is 0.8-1.2 μM, preferably 1 μM; Preferably, the concentration of retinoic acid is 0.08-0.12 μM, preferably 0.1 μM; Preferably, the concentration of Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1 is 0.20-0.30 μM, preferably 0.25 μM.

[0016] In one or more embodiments, in step (5), the method for culturing the obtained pancreatic endocrine progenitor cells in S4 medium and inducing their differentiation to obtain pancreatic endocrine cell spheres includes: At 37 °C and 5% CO2, co-induce culture for 7 days. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h, and then directly culture adherently for 24 h; on the 7th day, culture in suspension to form spheres for 24 h.

[0017] In one or more embodiments, in step (6), the S5 medium is based on the medium mixed with DMEM and DMEM / F12, and supplemented with ZnSO4, ALK5 inhibitor A83-01, triiodothyronine T3, and glucagon-like peptide-1 (GLP-1 / GCG protein); Preferably, in the culture medium after mixing DMEM and DMEM / F12, the volume ratio of DMEM to DMEM / F12 is 1:1; Preferably, the concentration of ZnSO4 is 18 - 22 μg / mL, preferably 20 μg / mL; Preferably, the concentration of the ALK5 inhibitor A83 - 01 is 8 - 12 μM, preferably 10 μM; Preferably, the concentration of triiodothyronine T3 is 0.8 - 1.2 μM, preferably 1 μM; Preferably, the concentration of glucagon - like peptide - 1 (GLP - 1 / GCG protein) is 8 - 12 nM, preferably 10 nM.

[0018] In one or more embodiments, in step (6), the method for inducing the pancreatic endocrine cell spheres obtained by culturing with S5 medium to differentiate into islet β - cell spheres includes: Under the conditions of 37 °C and 5% CO2, co - induction culture is carried out for 7 days. Every three days is a group. In each group, first spheroid culture is carried out under suspension conditions for 48 h, then direct adherent culture is carried out for 24 h, and on the 7th day, spheroid culture is carried out under suspension conditions for 24 h.

[0019] In the second aspect of the present invention, a functionally mature islet β - cell sphere is provided, and the islet β - cell sphere is induced and differentiated by the method described in the first aspect.

[0020] In one or more embodiments, the islet β - cell sphere is a functional, stable, and mature islet β - cell or cell population.

[0021] In the third aspect of the present invention, a pharmaceutical composition for treating and / or preventing diabetes is provided, characterized in that the pharmaceutical composition contains the functionally mature islet β - cell sphere described in the second aspect.

[0022] In one or more embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable carrier and / or excipient.

[0023] In one or more embodiments, the diabetes includes type I diabetes, type II diabetes, special - type diabetes, and gestational diabetes; More preferably, the diabetes is type I diabetes.

[0024] The beneficial effects of the present invention are as follows: (1) Muse cells (multilineage differentiating stress enduring cells) are pluripotent stem cells at a differentiation stage between embryonic stem cells and tissue stem cells. These cells specifically express SSEA-3 and also express pluripotent genes such as Oct3 / 4, Nanog, and Sox2, and have the ability to differentiate into cells of the three germ layers (endoderm, mesoderm, and ectoderm). Research has shown that no tumorigenicity was found in in-vivo experiments on nude mice lasting up to 6 months. Moreover, Muse cells are mainly derived from bone marrow, adipose tissue, umbilical cord, and connective tissue of almost all organs. Therefore, Muse cells have a wide source and no tumorigenic risk, making them a better stem cell choice for inducing the formation of functional pancreatic islet β cells.

[0025] (2) The experimental results in the present invention show that Muse cells cultured in static spheroids (3D) undergo significant apoptosis due to hypoxia inside the spheroids. After 23 days of culture, almost all the cells have apoptosed. Muse cells induced by direct adherent (2D) culture have a strong proliferation ability, and the differentiation efficiency is 37.7%. While using the method of alternating culture induction of spheroids (3D) and adherent (2D) not only enables stable passage, but also has a differentiation efficiency of 62.4%. The alternating culture induction method effectively solves the problems of proliferation and differentiation efficiency, achieving the stable passage and induced differentiation of pancreatic islet β cells.

[0026] (3) The pancreatic islet β cell spheres induced in the present invention exhibit mature β cell characteristics. After the pancreatic islet β cell spheres obtained by alternating culture induction of spheroids (3D) and adherent (2D) of Muse cells are implanted subcutaneously in type I diabetic mice, they exhibit extremely high insulin secretion levels, and the insulin secretion levels of each group are positively correlated with the induction efficiency.

[0027] (4) The in-vivo experiments of the pancreatic islet β cell spheres induced in the present invention confirm that the pancreatic islet β cell spheres obtained by alternating culture induction of spheroids (3D) and adherent (2D) of Muse cells have a continuous hypoglycemic effect: after being transplanted into diabetic model mice, they can significantly reduce blood glucose levels and maintain long-term efficacy. Brief Description of the Drawings

[0028] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0029] Figure 1 It is the flow cytometry diagram of Muse cells in Example 1; Figure 2 It is the flow cytometry diagram of the pancreatic islet β cell spheres obtained in Example 2; Figure 3Immunofluorescence image of pancreatic islet β cell spheres obtained in Example 2; Figure 4 Immunofluorescence image of pancreatic islet β cell spheres obtained in Example 3; Figure 5 Immunofluorescence image of pancreatic islet β cell spheres obtained in Example 4; Figure 6 Flow cytometry plot of non-Muse cells after induced differentiation in Comparative Example 1; Figure 7 Flow cytometry plot of Muse cells after adherent (2D) induction culture in a common adherent culture flask in Comparative Example 2; Figure 8 Apoptosis of Muse cells cultured under suspension conditions for sphere formation (3D) induction in Comparative Example 3; Figure 9 Changes in blood glucose levels in mice after STZ injection; Figure 10 Insulin secretion levels in diabetic mice; Figure 11 Changes in blood glucose levels in diabetic mice. Detailed implementation manners

[0030] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners of the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific examples.

[0033] The experimental reagents and experimental materials involved in the embodiments of the present invention are shown in Table 1, which includes the names, manufacturers, and catalog numbers of the experimental reagents.

[0034] Table 1 Experimental reagents and experimental materials

[0035] The DMEM cell culture medium was purchased from vivacell, with the product number C3113-0500; and the DMEM / F12 cell culture medium was purchased from vivacell, with the product number C3132-0500.

[0036] In this invention, the cell culture conditions were all at 37 °C and 5% CO2.

[0037] Example 1 Obtaining of Muse cells: (1) Obtaining of umbilical cord mesenchymal stem cells: Mesenchymal stem cells MSC were extracted from donated umbilical cords, and the third-generation MSC were collected.

[0038] (2) Obtaining of Muse cells and non-Muse cells: The MSC cells were incubated with the SSEA3 primary antibody for 35 min, centrifuged to remove the supernatant, the unbound primary antibody was removed, immunomagnetic beads (streptavidin nanomagnetic beads) were added and incubated for 20 min, and the SSEA3-positive cells were sorted magnetically. The cells on the sorting column were Muse cells, and the cells flowing down were non-Muse cells. Flow cytometry detected that both SSEA3 and CD105 were positive. The flow cytometry plot of Muse cells is as Figure 1 shown. It can be determined from Figure 1 that Muse cells were successfully obtained.

[0039] Example 2 Inducing Muse cells to differentiate into pancreatic islet β cell spheres: (1) Inductive differentiation of definitive endoderm cells: The Muse cells were inoculated into a low-attachment culture flask (containing S1 medium), cultured in suspension to form spheres for 48 h, and then, without trypsin digestion, the cell spheres were directly transferred to a common adherent culture flask (containing S1 medium) and cultured adherently for 24 h; The S1 medium was based on the DMEM cell culture medium and added with 100 ng / mL recombinant human activin-A (ActivinA) and 3 μM glycogen synthase kinase-3β (GSK-3β) selective inhibitor CHIR-99021 and 2 mM endoderm inducer 1 (IDE1); (2) Inductive differentiation of pancreatic progenitor cells: The definitive endoderm cells obtained in step (1) were seeded in a low-attachment culture flask (containing S2 medium) and cultured in suspension to form spheres for 48 h. Subsequently, without trypsin digestion, the cell spheres were directly transferred to a normal adherent culture flask (containing S2 medium) and cultured adherently for 24 h; The S2 medium was based on DMEM cell culture medium supplemented with 50 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7). (3) Inductive differentiation of pancreatic endocrine progenitor cells: The pancreatic progenitor cells obtained in step (2) were digested into single cells and then seeded in a low-attachment culture flask (containing S3 medium) and cultured in suspension to form spheres for 48 h. Subsequently, without trypsin digestion, the cell spheres were directly transferred to a normal adherent culture flask (containing S3 medium) and cultured adherently for 24 h; The S3 medium was based on DMEM cell culture medium supplemented with 50 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7), 0.1 μM retinoic acid, 0.25 μM Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1, 10 μM ROCK signaling pathway inhibitor Y-27632, 200 nM selective BMP type I receptor inhibitor LDN193189, 500 nM PKC activator PDBu, 100 ng / mL recombinant human activin-A (Activin A). (4) Inductive differentiation of pancreatic endocrine cell spheres: The pancreatic endocrine progenitor cells obtained in step (3) were digested into single cells and then seeded in a low-attachment culture flask (containing S4 medium) and co-induced for 7 days. Every three days was a group. In each group, the cells were first cultured in suspension to form spheres for 48 h, and then, without trypsin digestion, the cell spheres were directly transferred to a normal adherent culture flask (containing S4 medium) and cultured adherently for 24 h; The medium was changed every three days; On the 7th day, the cells were still cultured in suspension to form spheres for 24 h; The S4 medium was based on DMEM cell culture medium supplemented with 10 μg / mL sodium heparin, 10 μM ALK5 inhibitor A83-01, 1 μM TGF-β receptor kinase inhibitor SB-431542, 20 ng / mL Betacellulin protein, 1 μM triiodothyronine T3, 0.1 μM retinoic acid, and 0.25 μM Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1. (5) Inductive differentiation of islet β cell spheres: The pancreatic endocrine cell spheres obtained in step (4) were digested into single cells and then inoculated into a low-attachment culture flask (containing S5 medium). They were co-induced for 7 days, with each three days as a group. In each group, they were first cultured as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, the cell spheres were directly transferred to a common adherent culture flask (containing S5 medium) for adherent culture for 24 h. On the 7th day, they were still cultured as spheres under suspension conditions for 24 h. The medium was changed every three days. The S5 medium was based on a mixed medium of DMEM and DMEM / F12 (volume ratio 1:1), and 20 μg / mL ZnSO4, 10 μM ALK5 inhibitor A83-01, 1 μM triiodothyronine T3, and 10 nM glucagon-like peptide-1 (GLP-1 / GCG protein) were added.

[0040] The flow cytometry diagram of the islet β cell spheres obtained in this example is as Figure 2 shown. It can be determined from Figure 2 that islet β cells were successfully obtained, and the single positive rate of islet β cells was 62.4%.

[0041] The immunofluorescence diagram of the islet β cell spheres obtained in this example is as Figure 3 shown. It can be seen from Figure 3 that the islet β cell spheres obtained in this example expressed PDX1, INS (insulin), C-peptide (C-Peptide), and ZO-1 (tight junction protein). PDX1 is a key regulatory factor for pancreatic development and β cell function, a key transcription factor in the pancreatic endoderm, which begins to be expressed in the early stage of pancreatic development (endoderm stage) and continues until mature β cells (endoderm period - mature β islet cell period); INS (insulin) and C-peptide (C-Peptide) are markers of mature β cells; ZO-1 is a key protein of tight junctions, maintaining the polarity of epithelial / endothelial cells, indicating that the cell spheres form a functional three-dimensional structure, similar to the cell-cell interaction of natural islets, which helps glucose-responsive secretion. This further proves that the cell spheres obtained in this example have islet β cell-like characteristics and possess functional insulin secretion ability.

[0042] Example 3 Inducing Muse cells to differentiate into islet β cell spheres: (1) Inducing and differentiating definitive endoderm cells: Inoculate Muse cells into a low-attachment culture flask (containing S1 medium), and culture them as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a normal adherent culture flask (containing S1 medium) and culture them adherently for 24 h; The S1 medium is based on DMEM cell medium and added with 95 ng / mL recombinant human activin-A (Activin A), 2.8 μM selective inhibitor of glycogen synthase kinase-3β (GSK-3β) CHIR-99021, and 1.8 mM endoderm inducer 1 (IDE1). (2) Inductive differentiation of pancreatic progenitor cells: Inoculate the definitive endoderm cells obtained in step (1) into a low-attachment culture flask (containing S2 medium), and culture them as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a normal adherent culture flask (containing S2 medium) and culture them adherently for 24 h; The S2 medium is based on DMEM cell medium and added with 45 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7). (3) Inductive differentiation of pancreatic endocrine progenitor cells: Digest the pancreatic progenitor cells obtained in step (2) into single cells, then inoculate them into a low-attachment culture flask (containing S3 medium), and culture them as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a normal adherent culture flask (containing S3 medium) and culture them adherently for 24 h; The S3 medium is based on DMEM cell medium and added with 45 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7), 0.08 μM retinoic acid, 0.20 μM Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1, 8 μM ROCK signaling pathway inhibitor Y-27632, 180 nM selective BMP type I receptor inhibitor LDN193189, 450 nM PKC activator PDBu, 95 ng / mL recombinant human activin-A (Activin A). (4) Inductive differentiation of pancreatic endocrine cell spheres: Digest the pancreatic endocrine progenitor cells obtained in step (3) into single cells, and then inoculate them into a low-attachment culture flask (containing S4 medium), and co-induce and culture for 7 days. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a common adherent culture flask (containing S4 medium) for adherent culture for 24 h; change the medium every three days; on the 7th day, still culture in suspension to form spheres for 24 h; The S4 medium is based on DMEM cell medium and added with 8 μg / mL heparin sodium, 8 μM ALK5 inhibitor A83-01, 0.8 μM TGF-β receptor kinase inhibitor SB-431542, 18 ng / mL Betacellulin protein, 0.8 μM triiodothyronine T3, 0.08 μM retinoic acid, and 0.20 μM Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1; (5)Induction and differentiation of pancreatic islet β cell spheres: Digest the pancreatic endocrine cell spheres obtained in step (4) into single cells, and then inoculate them into a low-attachment culture flask (containing S5 medium), and co-induce and culture for 7 days. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a common adherent culture flask (containing S5 medium) for adherent culture for 24 h; on the 7th day, still culture in suspension to form spheres for 24 h; change the medium every three days; The S5 medium is based on the mixed medium of DMEM and DMEM / F12 (volume ratio 1:1), and added with 18 μg / mL ZnSO4, 8 μM ALK5 inhibitor A83-01, 0.8 μM triiodothyronine T3, 8 nM glucagon-like peptide-1 (GLP-1 / GCG protein).

[0043] The immunofluorescence image of the pancreatic islet β cell spheres obtained in this example is as Figure 4 shown. It can be seen from Figure 4 that the cell spheres obtained in this example and the cell spheres obtained in Example 2 both have pancreatic islet β cell-like characteristics and have functional insulin secretion ability.

[0044] Example 4 Induce Muse cells to differentiate into pancreatic islet β cell spheres: (1)Induction and differentiation of definitive endoderm cells: Inoculate Muse cells into a low-attachment culture flask (containing S1 medium), and culture them as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a normal adherent culture flask (containing S1 medium) and culture them adherently for 24 h; S1 medium is based on DMEM cell culture medium and supplemented with 105 ng / mL recombinant human activin-A (Activin A), 3.2 μM selective inhibitor of glycogen synthase kinase-3β (GSK-3β) CHIR-99021, and 2.2 mM endoderm inducer 1 (IDE1). (2) Inductive differentiation of pancreatic progenitor cells: Inoculate the definitive endoderm cells obtained in step (1) into a low-attachment culture flask (containing S2 medium), and culture them as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a normal adherent culture flask (containing S2 medium) and culture them adherently for 24 h; S2 medium is based on DMEM cell culture medium and supplemented with 55 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7). (3) Inductive differentiation of pancreatic endocrine progenitor cells: Digest the pancreatic progenitor cells obtained in step (2) into single cells, then inoculate them into a low-attachment culture flask (containing S3 medium), and culture them as spheres under suspension conditions for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a normal adherent culture flask (containing S3 medium) and culture them adherently for 24 h; S3 medium is based on DMEM cell culture medium and supplemented with 55 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7), 0.12 μM retinoic acid, 0.30 μM Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1, 12 μM ROCK signaling pathway inhibitor Y-27632, 220 nM selective BMP type I receptor inhibitor LDN193189, 550 nM PKC activator PDBu, 105 ng / mL recombinant human activin-A (Activin A). (4) Inductive differentiation of pancreatic endocrine cell spheres: Digest the pancreatic endocrine progenitor cells obtained in step (3) into single cells, and then inoculate them into a low-attachment culture flask (containing S4 medium), and co-induce and culture for 7 days. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a common adherent culture flask (containing S4 medium) for adherent culture for 24 h; change the medium every three days; on the 7th day, still culture in suspension to form spheres for 24 h; The S4 medium is based on DMEM cell medium and added with 12 μg / mL heparin sodium, 12 μM ALK5 inhibitor A83-01, 1.2 μM TGF-β receptor kinase inhibitor SB-431542, 22 ng / mL Betacellulin protein, 1.2 μM triiodothyronine T3, 0.12 μM retinoic acid and 0.30 μM Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1; (5)Induction and differentiation of islet β cell spheres: Digest the pancreatic endocrine cell spheres obtained in step (4) into single cells, and then inoculate them into a low-attachment culture flask (containing S5 medium), and co-induce and culture for 7 days. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h. Subsequently, without trypsin digestion, directly transfer the cell spheres to a common adherent culture flask (containing S5 medium) for adherent culture for 24 h; on the 7th day, still culture in suspension to form spheres for 24 h; change the medium every three days; The S5 medium is based on the medium after mixing DMEM and DMEM / F12 (volume ratio 1:1), and added with 22 μg / mL ZnSO4, 12 μM ALK5 inhibitor A83-01, 1.2 μM triiodothyronine T3, 12 nM glucagon-like peptide-1 (GLP-1 / GCG protein).

[0045] The immunofluorescence image of the islet β cell spheres obtained in this example is as Figure 5 shown. It can be seen from Figure 5 that the cell spheres obtained in this example and the cell spheres obtained in Example 2 both have islet β cell-like characteristics and have functional insulin secretion ability.

[0046] Comparative Example 1 Compared with Example 2, replace Muse with non-Muse cells, and the other induction culture methods are exactly the same. The flow cytometry image of the induced cells is as Figure 6 shown. It can be seen from Figure 6 that the proportion of insulin-positive (INS+) cells in the non-Muse cell induction group is significantly lower than that in the Muse cell induction group. Therefore, Muse cells are significantly superior to non-Muse cells in inducing differentiation into functional β islet cells.

[0047] Comparative Example 2 Muse cells were directly adherently (2D) induced and cultured using a common adherent culture flask. Compared with Example 2, the same culture medium was used for the induced differentiation of definitive endoderm cells, pancreatic progenitor cells, pancreatic endocrine progenitor cells, pancreatic endocrine cells, and pancreatic islet β cells. The difference lies in that: during the induced differentiation of definitive endoderm cells, they were adherently cultured in the adherent culture flask for 72 h; during the induced differentiation of pancreatic progenitor cells, they were adherently cultured in the adherent culture flask for 72 h; during the induced differentiation of pancreatic endocrine progenitor cells, they were adherently cultured in the adherent culture flask for 72 h, during the induced differentiation of pancreatic endocrine cells, they were adherently cultured in the adherent culture flask for 7 days; during the induced differentiation of pancreatic islet β cells, they were adherently cultured in the adherent culture flask for 7 days.

[0048] The flow cytometry plot of the cells induced in this comparative example is as Figure 7 shown. It can be seen from Figure 7 that after inducing the differentiation of Muse cells by direct adherent (2D) culture, the proportion of insulin-positive (INS+) cells in the cells was significantly lower than the result of inducing the differentiation of Muse cells by the alternate sphere formation / adherent culture in Example 2. Compared with the traditional direct adherent (2D) culture-induced differentiation system, the method of alternate sphere formation / adherent culture in Example 2 showed significant advantages in inducing the differentiation of Muse cells into functional β islet cells.

[0049] Comparative Example 3 Muse cells were induced and cultured in spheres (3D) under suspension conditions. Compared with Example 2, the same culture medium was used for the induced differentiation of definitive endoderm cells, pancreatic progenitor cells, pancreatic endocrine progenitor cells, pancreatic endocrine cells, and pancreatic islet β cells. The difference lies in that: during the induced differentiation of definitive endoderm cells, they were induced and cultured in spheres (3D) in a low-attachment culture flask for 72 h; during the induced differentiation of pancreatic progenitor cells, they were induced and cultured in spheres (3D) in a low-attachment culture flask for 72 h; during the induced differentiation of pancreatic endocrine progenitor cells, they were induced and cultured in spheres (3D) in a low-attachment culture flask for 72 h, during the induced differentiation of pancreatic endocrine cells, they were induced and cultured in spheres (3D) in a low-attachment culture flask for 7 days; during the induced differentiation of pancreatic islet β cells, they were induced and cultured in spheres (3D) in a low-attachment culture flask for 7 days.

[0050] Due to the slow cell proliferation rate under the pure sphere formation (3D) induced culture conditions, with the extension of the culture time, a large number of apoptotic phenomena occurred inside the cell spheres, which did not support flow cytometry detection. After the induction ended, the number of cells was: 0.17 ± 0.015×10 6 , and the result is asFigure 8 as shown

[0051] Experimental Example 1 Inducing type I diabetic mice: Before the experiment, C57BL / 6 mice need to be adaptively fed for 3 - 7 days, allowing them to freely eat and drink water, and their blood glucose levels are measured once before injecting STZ. A rapid diabetes model is established using the single high-dose STZ induction method. First, the mice are fasted for 4 - 6 hours to enhance the targeting of STZ to β cells. Then, an STZ solution dissolved in 0.1 M citrate buffer is freshly prepared and intraperitoneally injected at a dose of 100 mg / kg. Feeding is resumed 6 hours after injection. The random blood glucose levels are detected on the 3rd, 5th, 7th, and 9th days after injection. When the blood glucose levels are ≥16.7 mM for 3 consecutive days, it indicates successful model establishment. The blood glucose test results are as Figure 9 as shown

[0052] The above-mentioned type I diabetic mice are treated with the cells induced in Example 2 and Comparative Examples 1 - 3 respectively. Specifically, 10 days after transplanting the induced cells into the mice, an ELISA kit specifically recognizing human insulin (without cross-reaction with murine endogenous insulin) is used to detect the human insulin level in the mice. As Figure 10 shown, in the mice transplanted with the functional β islet cells induced by the alternating culture of spheroid formation / adhesion in Example 2, the serum human insulin level is significantly higher than that of other control groups, indicating that this induction method can effectively promote the secretion of human insulin in diabetic mice.

[0053] Meanwhile, after transplanting the induced cells into the mice, the random blood glucose levels are detected on the 3rd, 5th, 7th, and 9th days respectively. The results are as Figure 11 shown. As can be seen from Figure 11 it, in the mice transplanted with the functional β islet cells induced by the alternating culture of spheroid formation / adhesion in Example 2, their blood glucose continuously decreases, and the hypoglycemic effect is significantly better than that of other groups, and the blood glucose level approaches the normal level (about 5.5 - 6.5 mM) on the 9th day.

[0054] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for inducing Muse cells to differentiate into islet β cell spheres by alternating suspension culture and adherent culture, characterized in that, It includes the following steps: (1) Prepare and obtain Muse cells; (2) Culture the obtained Muse cells using S1 medium to direct their differentiation into definitive endoderm cells; (3) Culture the obtained definitive endoderm cells using S2 medium to induce their differentiation to obtain pancreatic progenitor cells; (4) Culture the obtained pancreatic progenitor cells using S3 medium to induce their differentiation to obtain pancreatic endocrine progenitor cells; (5) Culture the obtained pancreatic endocrine progenitor cells using S4 medium to induce their differentiation to obtain pancreatic endocrine cell spheres; (6) Culture the obtained pancreatic endocrine cell spheres using S5 medium to induce their differentiation to obtain islet β cell spheres; During the cell culture process from step (2) to step (6), a method of alternating sphere formation / adhesion culture is adopted.

2. The method according to claim 1, characterized in that In step (1), the Muse cells are derived from mesenchymal tissues of bone marrow, adipose tissue, umbilical cord, and dermis.

3. The method according to claim 1, wherein In step (2), the S1 medium is based on DMEM cell culture medium and added with recombinant human activin-A, a selective inhibitor of glycogen synthase kinase-3β, CHIR-99021, and endoderm inducer 1; wherein, the concentration of recombinant human activin-A is 95 - 105 ng / mL; the concentration of the selective inhibitor of glycogen synthase kinase-3β, CHIR-99021, is 2.8 - 3.2 μM; the concentration of endoderm inducer 1 is 1.8 - 2.2 mM; In step (2), the method of using S1 medium to culture the obtained Muse cells to direct their differentiation into definitive endoderm cells specifically includes: Under the conditions of 37 °C and 5% CO2, perform sphere formation culture in suspension for 48 h, and then directly perform adherent culture for 24 h.

4. The method according to claim 1, wherein In step (3), the S2 medium is based on DMEM cell culture medium and added with recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7; the concentration of recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 is 45 - 55 ng / mL; In step (3), the method of using S2 medium to culture the obtained definitive endoderm cells to induce their differentiation to obtain pancreatic progenitor cells specifically includes: Under the conditions of 37 °C and 5% CO2, perform sphere formation culture in suspension for 48 h, and then directly perform adherent culture for 24 h.

5. The method according to claim 1, characterized in that In step (4), the S3 medium is based on DMEM cell culture medium and supplemented with recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7, retinoic acid, Sonic Hedgehog signaling pathway inhibitor SANT-1, ROCK signaling pathway inhibitor Y-27632, selective BMP type I receptor inhibitor LDN193189, PKC activator PDBu, and recombinant human activin-A; the concentration of recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 is 45-55 ng / mL; the concentration of retinoic acid is 0.08-0.12 μM; the concentration of Sonic Hedgehog signaling pathway inhibitor SANT-1 is 0.20-0.30 μM; the concentration of ROCK signaling pathway inhibitor Y-27632 is 8-12 μM; the concentration of selective BMP type I receptor inhibitor LDN193189 is 180-220 nM; the concentration of PKC activator PDBu is 450-550 nM; the concentration of recombinant human activin-A is 95-105 ng / mL; In step (4), the method for culturing the obtained pancreatic progenitor cells with the S3 medium to induce their differentiation into pancreatic endocrine progenitor cells specifically includes: At 37 °C and 5% CO2, culture in suspension to form spheres for 48 h, and then directly culture adherently for 24 h.

6. The method according to claim 1, characterized in that In step (5), the S4 medium is based on DMEM cell culture medium and supplemented with sodium heparin, ALK5 inhibitor A83-01, TGF-β receptor kinase inhibitor SB-431542, Betacellulin protein, triiodothyronine T3, retinoic acid, and Sonic Hedgehog signaling pathway inhibitor SANT-1; the concentration of sodium heparin is 8-12 μg / mL; the concentration of ALK5 inhibitor A83-01 is 8-12 μM; the concentration of TGF-β receptor kinase inhibitor SB-431542 is 0.8-1.2 μM; the concentration of Betacellulin protein is 18-22 ng / mL; the concentration of triiodothyronine T3 is 0.8-1.2 μM; the concentration of retinoic acid is 0.08-0.12 μM; the concentration of Sonic Hedgehog signaling pathway inhibitor SANT-1 is 0.20-0.30 μM; In step (5), the method for culturing the obtained pancreatic endocrine progenitor cells with the S4 medium to induce their differentiation into pancreatic endocrine cell spheres includes: At 37 °C and 5% CO2, co-induce culture for 7 days. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h, and then directly culture adherently for 24 h; on the 7th day, culture in suspension to form spheres for 24 h.

7. The method according to claim 1, wherein In step (6), the S5 medium is based on the medium after mixing DMEM and DMEM / F12, and ZnSO4, ALK5 inhibitor A83-01, triiodothyronine T3, and glucagon-like peptide-1 are added; in the medium after mixing DMEM and DMEM / F12, the volume ratio of DMEM to DMEM / F12 is 1:1; the concentration of ZnSO4 is 18-22 μg / mL; the concentration of ALK5 inhibitor A83-01 is 8-12 μM; the concentration of triiodothyronine T3 is 0.8-1.2 μM; the concentration of glucagon-like peptide-1 is 8-12 nM; In step (6), the method for inducing the pancreatic endocrine cell spheres obtained by culturing with the S5 medium to differentiate into islet β cell spheres includes: Co-inducing and culturing for 7 days at 37 °C and 5% CO2. Every three days is a group. In each group, first culture in suspension to form spheres for 48 h, and then directly culture adherently for 24 h; on the 7th day, culture in suspension to form spheres for 24 h.

8. A functionally mature islet β-cell sphere, characterized in that, The islet β cell spheres are induced and differentiated by the method according to any one of claims 1 to 7.

9. A pharmaceutical composition for treating and / or preventing diabetes, characterized in that, The pharmaceutical composition contains the functionally mature islet β cell spheres according to claim 8.

10. The pharmaceutical composition according to claim 9, characterized in that, The diabetes includes type 1 diabetes, type 2 diabetes, special type diabetes, and gestational diabetes.

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