A method for inducing muse cells to differentiate into pancreatic beta cell spheroids through alternating spheroidization and adhesion culture and application thereof

Abstract

The present application relates to the technical field of cell engineering, and particularly relates to a method for inducing Muse cells to differentiate into pancreatic beta cell spheroids through alternate spheroid formation and adhesion culture and application. Experimental results in the present application show that Muse cells cultured in static spheroids have obvious apoptosis due to lack of oxygen in the spheroids, and almost all cells are apoptotic after 23 days of culture; Muse cells directly induced by adhesion culture have strong proliferation ability, and the differentiation efficiency is 37.7%; and the Muse cells induced by alternate spheroid formation and adhesion culture not only can be stably passaged, but also have a differentiation efficiency of 62.4%; the alternate induction culture method effectively solves the problems of proliferation and differentiation efficiency, and realizes stable passage and induced differentiation of pancreatic beta cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell engineering, and in particular to a method for inducing Muse cells to differentiate into islet beta cell spheroids through alternating spheroidization and adhesion culture and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Diabetes is a metabolic disorder characterized by high blood sugar, and its main pathogenesis is the decrease and / or disorder of insulin secretion. Currently, diabetes has become the third largest threat to human health and life quality after tumors, cardiovascular and cerebrovascular diseases.

[0004] Patients with type I diabetes and type II diabetes often need to inject exogenous insulin to control blood glucose balance in the body. Although this method can effectively control disease progression, long-term injection of insulin cannot stably maintain the physiological balance of blood glucose in the body, and further leads 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 lack of donors, this method cannot become the main treatment for diabetes. The replacement of functional insulin-secreting cells differentiated from stem cells is the closest treatment to normal physiological conditions, which can effectively control blood glucose and prevent, reverse complications. At present, the induction of islet beta cells mainly comes from mesenchymal stem cells, embryonic stem cells and iPS cells, and the 2D adhesion or 3D spheroidization method is used for induction. However, the induction efficiency of mesenchymal stem cells is low, and the induction efficiency of embryonic stem cells and iPS cells is high, but the embryonic stem cells and iPS cells that are not completely differentiated or not completely separated and removed have the risk of tumorigenesis. At the same time, the 2D adhesion induction has a high proliferation rate, but the efficiency of differentiation into beta islets is low, and the 3D spheroidization induction has high efficiency, but the proliferation is slow. Therefore, there is an urgent need for a stem cell and its differentiation method that is rich in source, can efficiently induce islet beta cells, and has no risk of tumorigenesis. SUMMARY

[0006] In order to overcome the above problems, the present application provides a method for inducing Muse cells to differentiate into islet beta cell spheroids through alternating spheroidization and adhesion culture and application thereof.

[0007] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a method for inducing differentiation of Muse cells into pancreatic beta cell spheroids by alternating spheroidization and adhesion culture, comprising the following steps:

[0009] (1) preparing Muse cells;

[0010] (2) culturing the Muse cells obtained in step (1) with S1 medium to direct differentiation into definitive endoderm cells;

[0011] (3) culturing the definitive endoderm cells obtained in step (2) with S2 medium to induce differentiation into pancreatic precursor cells;

[0012] (4) culturing the pancreatic precursor cells obtained in step (3) with S3 medium to induce differentiation into pancreatic endocrine progenitor cells;

[0013] (5) culturing the pancreatic endocrine progenitor cells obtained in step (4) with S4 medium to induce differentiation into pancreatic endocrine cells;

[0014] (6) culturing the pancreatic endocrine cells obtained in step (5) with S5 medium to induce differentiation into pancreatic beta cell spheroids.

[0015] In steps (2) to (6), the cells are cultured by alternating spheroidization and adhesion culture.

[0016] In one or more embodiments, in step (1), the Muse cells are derived from mesenchymal tissue of bone marrow, fat, umbilical cord and dermis, preferably umbilical cord.

[0017] In one or more embodiments, in step (2), the S1 medium is a DMEM cell culture medium supplemented with recombinant human Activin A, GSK-3β selective inhibitor CHIR-99021 and endoderm inducer 1 (IDE1).

[0018] Preferably, the concentration of recombinant human Activin A is 95-105 ng / mL, preferably 100 ng / mL.

[0019] Preferably, the concentration of GSK-3β selective inhibitor CHIR-99021 is 2.8-3.2 μM, preferably 3 μM.

[0020] Preferably, the concentration of endoderm inducer 1 (IDE1) is 1.8-2.2 mM, preferably 2 mM.

[0021] In one or more embodiments, in step (2), the method for culturing the obtained Muse cells in S1 medium to direct differentiation into definitive endoderm cells specifically comprises:

[0022] The cells are cultured in suspension for 48 h at 37 °C, 5% CO2, and then directly adherent cultured for 24 h.

[0023] In one or more embodiments, in step (3), the S2 medium is a DMEM cell culture medium-based medium supplemented with recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7);

[0024] 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.

[0025] In one or more embodiments, in step (3), the method for culturing the obtained definitive endoderm cells in S2 medium to induce differentiation to obtain pancreatic precursor cells specifically comprises:

[0026] The cells are cultured in suspension for 48 h at 37 °C, 5% CO2, and then directly adherent cultured for 24 h.

[0027] In one or more embodiments, in step (4), the S3 medium is a DMEM cell culture medium-based medium 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);

[0028] 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;

[0029] Preferably, the concentration of retinoic acid is 0.08-0.12 μM, preferably 0.1 μM;

[0030] Preferably, the concentration of Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1 is 0.20-0.30 μM, preferably 0.25 μM;

[0031] Preferably, the concentration of the ROCK signaling pathway inhibitor Y-27632 is 8-12 μM, preferably 10 μM;

[0032] Preferably, the concentration of the selective BMP type I receptor inhibitor LDN193189 is 180-220 nM, preferably 200 nM;

[0033] Preferably, the concentration of the PKC activator PDBu is 450-550 nM, preferably 500 nM;

[0034] Preferably, the concentration of the recombinant human Activin A is 95-105 ng / mL, preferably 100 ng / mL.

[0035] In one or more embodiments, in step (4), the obtained pancreatic precursor cells are cultured in the S3 medium to induce differentiation to obtain pancreatic endocrine progenitor cells, and the method specifically comprises:

[0036] The cells are cultured in a suspension state to form spheroids at 37 °C, 5% CO2 for 48 h, and then directly adherent culture for 24 h.

[0037] In one or more embodiments, in step (5), the S4 medium is a DMEM cell culture medium-based medium, and the following are added: heparin sodium, 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;

[0038] Preferably, the concentration of heparin sodium is 8-12 μg / mL, preferably 10 μg / mL;

[0039] Preferably, the concentration of the ALK5 inhibitor A83-01 is 8-12 μM, preferably 10 μM;

[0040] Preferably, the concentration of the TGF-β receptor kinase inhibitor SB-431542 is 0.8-1.2 μM, preferably 1 μM;

[0041] Preferably, the concentration of the Betacellulin protein is 18-22 ng / mL, preferably 20 ng / mL;

[0042] Preferably, the concentration of triiodothyronine T3 is 0.8-1.2 μM, preferably 1 μM;

[0043] Preferably, the concentration of retinoic acid is 0.08-0.12 μM, preferably 0.1 μM;

[0044] Preferably, the concentration of Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1 is 0.20-0.30 μM, preferably 0.25 μM.

[0045] In one or more embodiments, in step (5), the method for culturing the obtained pancreatic endocrine progenitor cells using S4 medium to induce differentiation to obtain pancreatic endocrine cell spheres comprises:

[0046] Under the condition of 37 ℃ and 5% CO2, the cells are co-induced for 7 days, and each three days is a group. In each group, the cells are first cultured in a suspension state for 48 h, and then directly adherent cultured for 24 h. On the 7th day, the cells are cultured in a suspension state for 24 h.

[0047] In one or more embodiments, in step (6), the S5 medium is based on a mixed medium of DMEM and DMEM / F12, and ZnSO4, ALK5 inhibitor A83-01, triiodothyronine T3, and glucagon-like peptide-1 (GLP-1 / GCG protein) are added;

[0048] Preferably, in the mixed medium of DMEM and DMEM / F12, the volume ratio of DMEM to DMEM / F12 is 1:1;

[0049] Preferably, the concentration of ZnSO4 is 18-22 μg / mL, preferably 20 μg / mL;

[0050] Preferably, the concentration of ALK5 inhibitor A83-01 is 8-12 μM, preferably 10 μM;

[0051] Preferably, the concentration of triiodothyronine T3 is 0.8-1.2 μM, preferably 1 μM;

[0052] Preferably, the concentration of glucagon-like peptide-1 (GLP-1 / GCG protein) is 8-12 nM, preferably 10 nM.

[0053] In one or more embodiments, in step (6), the method for culturing the obtained pancreatic endocrine cell spheres using S5 medium to induce differentiation to obtain islet β cell spheres comprises:

[0054] Under the condition of 37 ℃ and 5% CO2, the cells are co-induced for 7 days, and each three days is a group. In each group, the cells are first cultured in a suspension state for 48 h, and then directly adherent cultured for 24 h. On the 7th day, the cells are cultured in a suspension state for 24 h.

[0055] In a second aspect of the present application, a functional mature pancreatic islet beta cell spheroid is provided, wherein the pancreatic islet beta cell spheroid is obtained by the method of the first aspect.

[0056] In one or more embodiments, the pancreatic islet beta cell spheroid is a functional, stable, mature pancreatic islet beta cell or cell population.

[0057] In a third aspect of the present application, a pharmaceutical composition for treating and / or preventing diabetes is provided, wherein the pharmaceutical composition comprises the functional mature pancreatic islet beta cell spheroid of the second aspect.

[0058] In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0059] In one or more embodiments, the diabetes comprises type I diabetes, type II diabetes, special type diabetes, and gestational diabetes.

[0060] More preferably, the diabetes is type I diabetes.

[0061] The present application has the following beneficial effects:

[0062] (1) Muse cells (multilineage differentiating stress enduring cells) are a kind of multipotent stem cells with differentiation stage between embryonic stem cells and tissue stem cells, which 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 three germ layers (endoderm, mesoderm and ectoderm). Studies have shown that no tumorigenicity was found in nude mice for up to 6 months. Muse cells mainly come from bone marrow, fat, umbilical cord and connective tissue of almost all organs, so Muse cells are widely sourced and have no tumorigenic risk, which are a better stem cell selection for inducing functional pancreatic islet beta cells.

[0063] (2) The experimental results in the present application show that Muse cells cultured in static spheroid (3D) have obvious apoptosis due to lack of oxygen inside the spheroid, and almost all cells are apoptotic after 23 days of culture. Muse cells directly induced by adherent culture (2D) have strong proliferation ability, and the differentiation efficiency is 37.7%. The Muse cells induced by alternating culture of spheroid (3D) and adherent culture (2D) not only realize stable subculture, but also have a differentiation efficiency of 62.4%. The alternating culture induction method effectively solves the problems of proliferation and differentiation efficiency, and realizes stable subculture and induction differentiation of pancreatic islet beta cells.

[0064] (3) The islet beta cell spheroids obtained by induction exhibit mature beta cell characteristics, and the islet beta cell spheroids obtained by inducing Muse cells by alternately culturing in a spheroid (3D) and adherent (2D) manner exhibit extremely high insulin secretion levels after being implanted subcutaneously into type I diabetic mice, and the insulin secretion levels of each group are positively correlated with the induction efficiency.

[0065] (4) The in vivo experiments of the islet beta cell spheroids obtained by induction prove that the islet beta cell spheroids obtained by inducing Muse cells by alternately culturing in a spheroid (3D) and adherent (2D) manner have a sustained hypoglycemic effect: after being transplanted into diabetic model mice, the blood glucose levels can be significantly reduced and long-term efficacy can be maintained. BRIEF DESCRIPTION OF DRAWINGS

[0066] The drawings accompanying the specification of this application form a part thereof, serve to further provide a further understanding of the application, and together with the description of the exemplary embodiments of the application, serve to explain the application, and do not constitute an improper limitation of the application.

[0067] Figure 1 is a flow cytometry graph of Muse cells in Example 1;

[0068] Figure 2 is a flow cytometry graph of islet beta cell spheroids obtained in Example 2;

[0069] Figure 3 is an immunofluorescence graph of islet beta cell spheroids obtained in Example 2;

[0070] Figure 4 is an immunofluorescence graph of islet beta cell spheroids obtained in Example 3;

[0071] Figure 5 is an immunofluorescence graph of islet beta cell spheroids obtained in Example 4;

[0072] Figure 6 is a flow cytometry graph of non-Muse cells after induction and differentiation in Comparative Example 1;

[0073] Figure 7 is a flow cytometry graph of Muse cells after adherent (2D) induction and differentiation by using a common adherent culture flask in Comparative Example 2;

[0074] Figure 8 is the apoptosis of Muse cells after spheroid (3D) induction and culture under suspension conditions in Comparative Example 3;

[0075] Figure 9 is the change in blood glucose in mice after STZ injection;

[0076] Figure 10 is the insulin secretion level in diabetic mice;

[0077] Figure 11 The change of blood glucose in vivo of the diabetic mice. DETAILED DESCRIPTION

[0078] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0079] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0080] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0081] The experimental reagents and experimental materials involved in the embodiments of the present application are shown in Table 1, and the names, manufacturers and item numbers of the experimental reagents involved in Table 1 are shown.

[0082] Table 1 Experimental reagents and experimental materials

[0083]

[0084] The DMEM cell culture medium is purchased from the vivacell company, and the item number is C3113-0500;

[0085] and the DMEM / F12 cell culture medium is purchased from the vivacell company, and the item number is C3132-0500.

[0086] In the present application, the cell culture conditions are all at 37℃, 5% CO2.

[0087] Example 1

[0088] Obtaining of Muse cells:

[0089] (1) Obtaining of umbilical cord mesenchymal stem cells: mesenchymal stem cells MSC are extracted from donated umbilical cords, and the third generation MSC is collected.

[0090] (2) Obtaining Muse cells and non-Muse cells: MSC cells were incubated with SSEA3 primary antibody for 35 min, centrifuged to remove the supernatant, and the unbound primary antibody was removed. Immunomagnetic beads (streptavidin-coated magnetic beads) were added and incubated for 20 min. The SSEA3 positive cells were sorted by magnetic sorting. The cells on the sorting column were Muse cells, and the cells flowing down were non-Muse cells. Flow cytometry showed that SSEA3 and CD105 were both positive. The flow cytometry graph of Muse cells is shown in FIG. 2. It can be determined from FIG. 2 that Muse cells are successfully obtained. Figure 1 Figure 1

[0091] Example 2

[0092] Inducing Muse cells to differentiate into pancreatic beta cell spheres

[0093] (1) Inducing differentiation of definitive endoderm cells:

[0094] Muse cells were seeded in low-adsorption culture bottles (containing S1 medium) and cultured in suspension for 48 h. Then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture bottles (containing S1 medium) for adherent culture for 24 h. S1 medium was a DMEM cell culture medium supplemented with 100 ng / mL recombinant human Activin-A, 3 μM GSK-3β selective inhibitor CHIR-99021, and 2 mM endoderm inducer 1 (IDE1).

[0095] (2) Inducing differentiation of pancreatic precursor cells:

[0096] The definitive endoderm cells obtained in step (1) were seeded in low-adsorption culture bottles (containing S2 medium) and cultured in suspension for 48 h. Then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture bottles (containing S2 medium) for adherent culture for 24 h. S2 medium was a DMEM cell culture medium supplemented with 50 ng / mL recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 (KGF-1 / FGF-7).

[0097] (3) Inducing differentiation of pancreatic endocrine progenitor cells:

[0098] ​​The pancreatic precursor cells obtained in step (2) were digested into single cells, which were then inoculated into low-adsorption culture bottles (containing S3 medium) for spheroid culture under suspension conditions for 48 h, and then, without trypsin digestion, the cell spheroids were directly transferred to ordinary adherent culture bottles (containing S3 medium) for adherent culture for 24 h; the S3 medium was a DMEM cell culture medium-based medium added 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, and 100 ng / mL recombinant human activin-A (Activin A);

[0099] (4) Induced differentiation of pancreatic endocrine cell spheroids:

[0100] The pancreatic endocrine progenitor cells obtained in step (3) were digested into single cells, which were then inoculated into low-adsorption culture bottles (containing S4 medium) for co-induction culture for 7 days, with each group being cultured for 48 h under suspension conditions, and then, without trypsin digestion, the cell spheroids were directly transferred to ordinary adherent culture bottles (containing S4 medium) for adherent culture for 24 h; the medium was replaced every three days; on the seventh day, the cells were still cultured under suspension conditions for 24 h; the S4 medium was a DMEM cell culture medium-based medium added with 10 μg / mL heparin sodium, 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;

[0101] (5) Induced differentiation of islet β cell spheroids:

[0102] The pancreatic endocrine cell spheres obtained in step (4) were digested into single cells and then inoculated into low-adhesion culture flasks (containing S5 culture medium) for a total of 7 days of induction culture, with each group consisting of three days. In each group, the spheres were first cultured in suspension for 48 hours, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture flasks (containing S5 culture medium) for adherent culture for 24 hours; on the 7th day, the spheres were still cultured in suspension for 24 hours; the culture medium was changed every three days; the S5 culture medium was based on a mixture of DMEM and DMEM / F12 (volume ratio of 1:1), with the addition of 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).

[0103] The flow cytometric images of the pancreatic β cell spheres obtained in this example are shown in FIG. Figure 2 As shown. Figure 2 It can be confirmed that pancreatic β cells were successfully obtained, and the single positive rate of pancreatic β cells was 62.4%.

[0104] The immunofluorescence images of pancreatic β cell spheres obtained in this example are as follows: Figure 3 As shown, from Figure 3 As can be seen from the results, the pancreatic β-cell spheroids obtained in this example express PDX1, INS (insulin), C-peptide, and ZO-1 (tight junction protein). PDX1 is a key regulator of pancreatic development and β-cell function and a key transcription factor of the pancreatic endoderm. Its expression begins early in pancreatic development (endodermal stage) and persists until mature β-cells (endodermal stage to mature β-islet cell stage). INS (insulin) and C-peptide are markers of mature β-cells. ZO-1 is a key protein of tight junctions that maintains epithelial / endothelial cell polarity. This suggests that the spheroids form a functional three-dimensional structure, with cell-cell interactions similar to those of natural pancreatic islets, which facilitates glucose-responsive secretion. This demonstrates that the spheroids obtained in this example possess β-cell-like properties and functional insulin secretion capacity.

[0105] Example 3

[0106] Inducing Muse cells to differentiate into pancreatic β-cell spheres:

[0107] (1) Induction of differentiation of definitive endoderm cells:

[0108] Muse cells were inoculated into low-adsorption culture flasks (containing S1 medium) for 48 h of spheroid culture under suspension conditions, and then, without trypsin digestion, the cell spheroids were directly transferred to ordinary adherent culture flasks (containing S1 medium) for 24 h of adherent culture; S1 medium was a DMEM cell culture medium-based medium added with 95 ng / mL of recombinant human Activin-A (Activin A) and 2.8 μM of GSK-3β (Glycogen synthase kinase-3β) selective inhibitor CHIR-99021 and 1.8 mM of endoderm inducer 1 (IDE1);

[0109] (2) Induced differentiation of pancreatic precursor cells:

[0110] The definitive endoderm cells obtained in step (1) were inoculated into low-adsorption culture flasks (containing S2 medium) for 48 h of spheroid culture under suspension conditions, and then, without trypsin digestion, the cell spheroids were directly transferred to ordinary adherent culture flasks (containing S2 medium) for 24 h of adherent culture; S2 medium was a DMEM cell culture medium-based medium added with 45 ng / mL of recombinant human KGF-1 / FGF-7 (Keratinocyte growth factor 1 / Fibroblast growth factor 7);

[0111] (3) Induced differentiation of pancreatic endocrine progenitor cells:

[0112] The pancreatic precursor cells obtained in step (2) were digested into single cells, and then inoculated into low-adsorption culture flasks (containing S3 medium) for 48 h of spheroid culture under suspension conditions, and then, without trypsin digestion, the cell spheroids were directly transferred to ordinary adherent culture flasks (containing S3 medium) for 24 h of adherent culture; S3 medium was a DMEM cell culture medium-based medium added with 45 ng / mL of recombinant human KGF-1 / FGF-7 (Keratinocyte growth factor 1 / Fibroblast growth factor 7), 0.08 μM of retinoic acid, 0.20 μM of Sonic Hedgehog (SHH) signaling pathway inhibitor SANT-1, 8 μM of ROCK signaling pathway inhibitor Y-27632, 180 nM of selective BMP type I receptor inhibitor LDN193189, 450 nM of PKC activator PDBu, and 95 ng / mL of recombinant human Activin-A (Activin A);

[0113] (4) Induced differentiation of pancreatic endocrine cell spheroids:

[0114] The pancreatic endocrine progenitor cells obtained in step (3) were digested into single cells and then inoculated into low-adhesion culture flasks (containing S4 medium) for a total of 7 days of induction culture, with each group consisting of three days. In each group, the cell spheres were first cultured in suspension for 48 hours, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture flasks (containing S4 medium) for adherent culture for 24 hours; the culture medium was changed every three days; on the 7th day, the cells were still cultured in suspension for 24 hours; the S4 medium was based on DMEM cell culture medium with the addition of 8 μg / mL sodium heparin, 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;

[0115] (5) Induction of differentiation of pancreatic β-cell spheres:

[0116] The pancreatic endocrine cell spheres obtained in step (4) were digested into single cells and then inoculated into low-adhesion culture flasks (containing S5 culture medium) for a total of 7 days of induction culture, with each group consisting of three days. In each group, the spheres were first cultured in suspension for 48 hours, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture flasks (containing S5 culture medium) for adherent culture for 24 hours; on the 7th day, the spheres were still cultured in suspension for 24 hours; the culture medium was changed every three days; the S5 culture medium was based on a mixture of DMEM and DMEM / F12 (volume ratio of 1:1), with the addition of 18 μg / mL ZnSO4, 8 μM ALK5 inhibitor A83-01, 0.8 μM triiodothyronine T3, and 8 nM glucagon-like peptide-1 (GLP-1 / GCG protein).

[0117] The immunofluorescence images of pancreatic β cell spheres obtained in this example are as follows: Figure 4 As shown, from Figure 4 It can be seen that the cell spheroids obtained in this example and the cell spheroids obtained in Example 2 both have pancreatic β cell-like characteristics and have functional insulin secretion capabilities.

[0118] Example 4

[0119] Inducing Muse cells to differentiate into pancreatic β-cell spheres:

[0120] (1) Induction of differentiation of definitive endoderm cells:

[0121] Muse cells were inoculated in low-attachment culture flasks (containing S1 medium) for 48 h in suspension, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture flasks (containing S1 medium) for adherent culture for 24 h; S1 medium was a DMEM cell culture medium-based medium added with 105 ng / mL recombinant human Activin-A (Activin A) and 3.2 μM GSK-3β (Glycogen synthase kinase-3β) selective inhibitor CHIR-99021 and 2.2 mM IDE1 (Endoderm inducer 1);

[0122] (2) Induced differentiation of pancreatic precursor cells:

[0123] The step (1) obtained definitive endoderm cells were inoculated in low-attachment culture flasks (containing S2 medium) for 48 h in suspension, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture flasks (containing S2 medium) for adherent culture for 24 h; S2 medium was a DMEM cell culture medium-based medium added with 55 ng / mL recombinant human KGF-1 / FGF-7 (Keratinocyte growth factor 1 / Fibroblast growth factor 7);

[0124] (3) Induced differentiation of pancreatic endocrine progenitor cells:

[0125] The step (2) obtained pancreatic precursor cells were digested into single cells, and then inoculated in low-attachment culture flasks (containing S3 medium) for 48 h in suspension, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture flasks (containing S3 medium) for adherent culture for 24 h; S3 medium was a DMEM cell culture medium-based medium added with 55 ng / mL recombinant human KGF-1 / FGF-7 (Keratinocyte growth factor 1 / Fibroblast growth factor 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);

[0126] (4) Induced differentiation of pancreatic endocrine cell spheres:

[0127] The pancreatic endocrine progenitor cells obtained in step (3) were digested into single cells, and then inoculated into low-adsorption culture bottles (containing S4 medium) for a total of 7 days of induction culture, with each three days as a group. In each group, the cells were first cultured into spheres under suspension conditions for 48 h, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture bottles (containing S4 medium) for adherent culture for 24 h. The medium was changed every three days. On the seventh day, the cells were still cultured into spheres under suspension conditions for 24 h. The S4 medium was a DMEM cell culture medium-based medium 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;

[0128] (5) Induced differentiation of islet β-cell spheres:

[0129] The pancreatic endocrine cell spheres obtained in step (4) were digested into single cells, and then inoculated into low-adsorption culture bottles (containing S5 medium) for a total of 7 days of induction culture, with each three days as a group. In each group, the cells were first cultured into spheres under suspension conditions for 48 h, and then, without trypsin digestion, the cell spheres were directly transferred to ordinary adherent culture bottles (containing S5 medium) for adherent culture for 24 h. On the seventh day, the cells were still cultured into spheres under suspension conditions for 24 h. The medium was changed every three days. The S5 medium was a DMEM and DMEM / F12 mixed medium (volume ratio 1:1) added with 22 μg / mL ZnSO4, 12 μM ALK5 inhibitor A83-01, 1.2 μM triiodothyronine T3, and 12 nM glucagon-like peptide-1 (GLP-1 / GCG protein).

[0130] The immunofluorescence image of the islet β-cell spheres obtained in this example is shown in Figure 5 As can be seen from Figure 5 , 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.

[0131] Comparative Example 1

[0132] Compared with Example 2, the Muse was replaced by non-Muse cells, and the other induction culture methods were exactly the same. The flow cytometry image of the cells after induction is shown in Figure 6 As can be seen from Figure 6It can be seen that the proportion of insulin-positive (INS+) cells in the non-Muse cell induction group was significantly lower than that in the Muse cell induction group. Therefore, Muse cells are significantly better than non-Muse cells in inducing differentiation into functional β-islet cells.

[0133] Comparative Example 2

[0134] Ordinary adherent culture flasks were used for direct adherence (2D) induced culture of Muse cells. Compared with Example 2, the induced differentiation of definitive endoderm cells, the induced differentiation of pancreatic precursor cells, the induced differentiation of pancreatic endocrine progenitor cells, the induced differentiation of pancreatic endocrine cells, and the induced differentiation of pancreatic β cells used exactly the same culture medium, with the following differences: when induced differentiation of definitive endoderm cells, the cells were cultured entirely in adherent culture flasks for 72 h; when induced differentiation of pancreatic precursor cells, the cells were cultured entirely in adherent culture flasks for 72 h; when induced differentiation of pancreatic endocrine progenitor cells, the cells were cultured entirely in adherent culture flasks for 72 h; when induced differentiation of pancreatic endocrine cells, the cells were cultured entirely in adherent culture flasks for 7 days; when induced differentiation of pancreatic β cells, the cells were cultured entirely in adherent culture flasks for 7 days.

[0135] The flow cytometric images of the cells induced by this comparative example are shown in FIG. Figure 7 As shown, from Figure 7 As can be seen from the results, after inducing Muse cell differentiation using direct adherent (2D) culture, the proportion of insulin-positive (INS+) cells in the cells was significantly lower than the result of inducing Muse cell differentiation using sphere / adherent alternating culture in Example 2. Compared with the traditional direct adherent (2D) culture induction differentiation system, the sphere / adherent alternating culture induction method in Example 2 showed significant advantages in inducing Muse cells to differentiate into functional β-islet cells.

[0136] Comparative Example 3

[0137] Muse cells were induced to form spheres (3D) under suspension conditions. Compared with Example 2, the induced differentiation of definitive endoderm cells, the induced differentiation of pancreatic precursor cells, the induced differentiation of pancreatic endocrine progenitor cells, the induced differentiation of pancreatic endocrine cells, and the induced differentiation of pancreatic β cells used exactly the same culture medium, with the following differences: when induced differentiation of definitive endoderm cells, they were completely induced to form spheres (3D) in low-adsorption culture flasks for 72 h; when induced differentiation of pancreatic precursor cells, they were completely induced to form spheres (3D) in low-adsorption culture flasks for 72 h; when induced differentiation of pancreatic endocrine progenitor cells, they were completely induced to form spheres (3D) in low-adsorption culture flasks for 72 h, and when induced differentiation of pancreatic endocrine cells, they were completely induced to form spheres (3D) in low-adsorption culture flasks for 7 days; when induced differentiation of pancreatic β cells, they were completely induced to form spheres (3D) in low-adsorption culture flasks for 7 days.

[0138] Due to the slow cell proliferation rate under the pure sphere (3D) induction culture condition, a large number of apoptosis occurs in the cell sphere with the prolongation of the culture time, which does not support flow detection. After the induction is completed, the cell number is 0.17±0.015×10 6 , and the results are shown in Figure 8 .

[0139] Experimental Example 1

[0140] Induction of type I diabetic mice: Before the experiment, the C57BL / 6 mice need to be adaptively fed for 3-7 days, and they are allowed to freely eat and drink water. Before injection of STZ, the blood glucose is measured once. A single high-dose STZ induction method is used to establish a rapid diabetes model. The mice are fasted for 4-6 hours to enhance the targeting of STZ to β cells, and then a freshly prepared 0.1 M citric acid buffer solution is used to dissolve the STZ solution, which is injected intraperitoneally at a dose of 100 mg / kg. After 6 hours, the mice are allowed to eat. On the 3rd, 5th, 7th and 9th day after injection, the random blood glucose level is detected. When the blood glucose level is ≥16.7 mM for 3 consecutive days, it is considered that the modeling is successful. The blood glucose test results are shown in Figure 9 .

[0141] The cells induced by Example 2 and Comparative Examples 1-3 are used to treat the above-mentioned type I diabetic mice. Specifically, the induced cells are transplanted into mice for 10 days, and then an ELISA kit specific for human insulin (which does not cross-react with mouse endogenous insulin) is used to detect the level of human insulin in the mice. As shown in Figure 10 , the serum human insulin level of the mice transplanted with the functional β islet cells obtained by inducing the Muse cells of Example 2 under the sphere / adherent cell alternating culture is significantly higher than that of the other control groups, indicating that the induction method can effectively promote the secretion of human insulin in diabetic mice.

[0142] At the same time, after the induced cells are transplanted into mice, the random blood glucose level is detected on the 3rd, 5th, 7th and 9th day. The results are shown in Figure 11 , and it can be seen from Figure 11 that the blood glucose of the mice transplanted with the functional β islet cells obtained by inducing the Muse cells of Example 2 under the sphere / adherent cell alternating culture continuously decreases, and the effect of reducing blood glucose is significantly better than that of the other groups, and the blood glucose approaches the normal level (about 5.5-6.5 mM) on the 9th day.

[0143] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for inducing differentiation of Muse cells into pancreatic beta cell spheroids by alternating spheroidization / adherence culture, characterized by, The method comprises the following steps: (1) preparing Muse cells; (2) culturing the Muse cells obtained in step (1) in S1 medium to differentiate the Muse cells into definitive endoderm cells; (3) culturing the definitive endoderm cells obtained in step (2) in S2 medium to induce differentiation of the definitive endoderm cells into pancreatic precursor cells; (4) culturing the pancreatic precursor cells obtained in step (3) in S3 medium to induce differentiation of the pancreatic precursor cells into pancreatic endocrine progenitor cells; (5) culturing the pancreatic endocrine progenitor cells obtained in step (4) in S4 medium to induce differentiation of the pancreatic endocrine progenitor cells into pancreatic endocrine cell spheroids; (6) culturing the pancreatic endocrine cell spheroids obtained in step (5) in S5 medium to induce differentiation of the pancreatic endocrine cell spheroids into islet beta cell spheroids; In steps (2)-(6), the cells are alternately cultured in a spheroid culture mode and an adherent culture mode; The S1 medium is a DMEM cell culture medium to which recombinant human activin-A, glycogen synthase kinase-3 beta selective inhibitor CHIR-99021 and endoderm inducer 1 are added; The S2 medium is a DMEM cell culture medium to which recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 is added; The S3 medium is a DMEM cell culture medium to which 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 are added; The S4 medium is a DMEM cell culture medium to which heparin sodium, ALK5 inhibitor A83-01, TGF-beta receptor kinase inhibitor SB-431542, Betacellulin protein, triiodothyronine T3, retinoic acid and Sonic Hedgehog signaling pathway inhibitor SANT-1 are added; The S5 medium is a DMEM / DMEM / F12 mixed medium to which ZnSO4, ALK5 inhibitor A83-01, triiodothyronine T3 and glucagon-like peptide-1 are added; In step (2), the Muse cells obtained in step (1) are cultured in the S1 medium to differentiate the Muse cells into definitive endoderm cells, and the method comprises the following steps: culturing the Muse cells in a suspension state to form spheroids for 48 h at 37 DEG C under a 5% CO2 condition, and then directly culturing the spheroids adherently for 24 h; In step (3), the definitive endoderm cells obtained in step (2) are cultured in the S2 medium to induce differentiation of the definitive endoderm cells into pancreatic precursor cells, and the method comprises the following steps: culturing the definitive endoderm cells in a suspension state to form spheroids for 48 h at 37 DEG C under a 5% CO2 condition, and then directly culturing the spheroids adherently for 24 h; In step (4), the pancreatic precursor cells obtained in step (3) are cultured in the S3 medium to induce differentiation of the pancreatic precursor cells into pancreatic endocrine progenitor cells, and the method comprises the following steps: culturing the pancreatic precursor cells in a suspension state to form spheroids for 48 h at 37 DEG C under a 5% CO2 condition, and then directly culturing the spheroids adherently for 24 h; In step (5), the method for culturing the obtained pancreatic endocrine progenitor cells using S4 medium to induce differentiation to obtain pancreatic endocrine cell spheres comprises: co-inducing culture at 37℃, 5% CO2 for 7 days, every three days as a group, and in each group, first culturing in a suspension state for 48 h, and then directly adhering to culture for 24 h; and culturing in a suspension state for 24 h on the 7th day; In step (6), the method for culturing the obtained pancreatic endocrine cell spheres using S5 medium to induce differentiation to obtain islet β cell spheres comprises: co-inducing culture at 37℃, 5% CO2 for 7 days, every three days as a group, and in each group, first culturing in a suspension state for 48 h, and then directly adhering to culture for 24 h; and culturing in a suspension state for 24 h on the 7th day; In step (1), the Muse cells are derived from umbilical cords; In step (2), in the S1 medium, the concentration of recombinant human activin-A is 95-105 ng / mL; the concentration of glycogen synthase kinase-3β selective inhibitor CHIR-99021 is 2.8-3.2 μM; and the concentration of endoderm inducer 1 is 1.8-2.2 mM.

2. The method of claim 1, wherein, In step (3), in the S2 medium, the concentration of recombinant human keratinocyte growth factor 1 / fibroblast growth factor 7 is 45-55 ng / mL.

3. The method of claim 1, wherein, In step (4), in the S3 medium, 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; and the concentration of PKC activator PDBu is 450-550 nM. The concentration of recombinant human activin-A is 95-105 ng / mL.

4. The method of claim 1, wherein, In step (5), in the S4 medium, 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; and the concentration of Sonic Hedgehog signaling pathway inhibitor SANT-1 is 0.20-0.30 μM.

5. The method of claim 1, wherein, In step (6), the volume ratio of DMEM and DMEM / F12 in the mixed medium in the S5 medium is 1:1; the concentration of ZnSO4 is 18-22 μg / mL; the concentration of ALK5 inhibitor A83-01 is 8-12 μM; and the concentration of triiodothyronine T3 is 0.8-1.2 μM. The concentration of glucagon-like peptide-1 is 8-12 nM.

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