CN113637630B

CN113637630B - Islet-like cell mass, and preparation method and application thereof

Info

Publication number: CN113637630B
Application number: CN202010394320.5A
Authority: CN
Inventors: 程新; 肖伊妮
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2024-03-29
Anticipated expiration: 2040-05-11
Also published as: CN113637630A; WO2021228104A1

Abstract

The invention provides an artificially cultured islet-like cell mass, which is characterized in that the cell mass is obtained by differentiation of stem cells and comprises the following components in the absence of enrichment: (a) islet beta cells account for 20% -90%; and (b) islet alpha cells account for 5% -70%; and a culture system for obtaining the islet-like cell mass by culturing the human pluripotent stem cells. The invention also provides a model for constructing an islet microenvironment by using the islet-like cell mass, an artificial pancreas device prepared by using the islet-like cell mass and a pharmaceutical composition comprising the islet-like cell mass. The artificially cultured islet-like cell mass provided by the invention can effectively release insulin under the stimulation of secretagogues such as glucose and the like, and has good effects in static and dynamic GSIS experiments; is used for in vivo model of type I diabetes, and can maintain blood sugar of type I diabetes mice in normal interval for a long period.

Description

Islet-like cell mass, and preparation method and application thereof

Technical Field

The invention belongs to the field of stem cell differentiation, and in particular relates to a preparation method and application of islet-like cell mass.

Background

Diabetes is a disease affecting the health of about 4.6 million people worldwide, caused by abnormal regulation of blood glucose due to insufficient insulin secretion or insulin resistance. All type one diabetics and a fraction of advanced type two diabetics require insulin replacement therapy to lower hyperglycemia to sustain life, such as subcutaneous insulin. The method needs to frequently monitor the change of blood sugar to adjust the dosage of insulin, but can not realize precise regulation and control of blood sugar like pancreas islet, so complications such as retinopathy, nephropathy, neurological diseases, vascular diseases and the like caused by hyperglycemia and coma and even death caused by hypoglycemia can not be avoided. On the other hand, insulin abuse can produce adverse side effects such as hypoglycaemic response, insulin edema, etc.

Islet transplantation can solve the above problems, but faces the problems of scarcity of donors and the need to take anti-rejection drugs for a long period of time. Human pluripotent stem cells have great potential in the field of cell therapy due to their ability to proliferate indefinitely and differentiate in all directions, and thus are a good source of cells for preparing islet beta cells. For achieving cell therapy of diabetes, it is of great importance to prepare islet-like cell masses with a cell composition ratio similar to that of human islets. However, all current studies fail to produce both alpha and beta cells in the same culture system at a similar ratio to the true islet cell population.

There is an urgent need in the art to create a method of constructing islet-like cell mass that is high in yield and that can induce complete secretory function.

Disclosure of Invention

The invention aims to provide a method for constructing islet-like cell mass, which has high yield and can induce complete secretion function.

In a first aspect, the present invention provides an islet-like cell mass obtained by in vitro differentiation, said islet-like cell mass comprising the following components:

(a) Islet beta cells;

(b) Islet alpha cells; and;

(c) Optionally other endocrine cells (including but not limited to endocrine cells expressing other endocrine hormones such as SST or PPY);

wherein the islet beta cells account for 20% -90% of the total number of cells in the cell mass;

the islet alpha cells account for 5% -70% of the total number of cells in the cell mass.

In a preferred embodiment, the islet-like cell mass is an unenriched islet-like cell mass.

In a preferred embodiment, the islet-like cell mass comprises islet-like cell masses comprising corresponding proportions of islet beta cells and islet alpha cells, e.g., similar proportions to a real islet cell mass, obtained by sorting to obtain islet beta cells and islet alpha cells, respectively, followed by repolymerization.

In a specific embodiment, the islet beta cells in the islet-like cell mass comprise 30% -80%; and islet alpha cells account for 6% -40%; more preferably, islet beta cells in the islet-like cell mass account for 40% -70%; and islet alpha cells account for 10% -35%.

In a preferred embodiment, the islet-like cell mass expresses the endocrine cell marker NEUROD1 in an amount equivalent to that of islets.

In a preferred embodiment, the pancreatic islet beta cells express specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8 as detected by immunofluorescent staining; preferably, specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8, MAFA are expressed; more preferably, specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8, MAFA, IAPP are expressed;

preferably, the proportion of β cells expressing MAFA based on at least expression of the above specific marker gene is 70-85% of all β cells, more preferably at least 80% of all β cells; the proportion of IAPP-expressing beta cells is 30-60% of the total beta cells, more preferably at least 50% of the total beta cells; the alpha cell expresses a marker gene GCG, ARX, PCSK2;

In another preferred embodiment, the β cells expressing both INS and IAPP account for 30-45%, preferably 32-40%, more preferably 35-38% of all β cells as detected by single cell sequencing; beta cells expressing INS, IAPP and MAFA simultaneously account for 15-30%, preferably 18-25%, more preferably 20-24% of all beta cells.

In a preferred embodiment, the islet-like cell mass is capable of secreting insulin upon stimulation with glucose; preferably, the islet-like cell mass releases at least 1.5 times, preferably at least 2 times, more preferably at least 3 times, even more preferably at least 5 times, more preferably at least 10 times more human insulin under high sugar stimulation than under low sugar stimulation.

In a preferred embodiment, the islet-like cell mass releases at least 2 times, preferably at least 3 times, more preferably at least 4 times, most preferably at least 5 times more human glucagon under low sugar stimulation than under high sugar stimulation; the low sugar stimulus is 1.5mM-3.5mM glucose, preferably 2mM glucose; the high sugar stimulus is 15mM-35mM glucose, preferably 20mM glucose.

In a preferred embodiment, the islet-like cell mass exhibits an insulin secretion pattern similar to that of islets in an insulin secretion assay under glucose stimulation.

In a preferred embodiment, the insulin secretion assay under glucose stimulation includes static GSIS and dynamic GSIS.

In a preferred embodiment, the islet-like insulin secretion pattern comprises the following reactions:

(I) Insulin secretion under high sugar stimulation is higher than insulin secretion under low sugar stimulation in each stimulation cycle of static GSIS experiment, and insulin is released to the greatest extent under potassium chloride stimulation.

(II) throughout the stimulation of the dynamic GSIS assay, insulin secretion patterns are:

(i) Insulin exhibits a lower background secretion pattern under low sugar stimulation;

(ii) Insulin secretion increases rapidly and rapidly after stimulation from low to high sugar and reaches the peak of insulin secretion within 5 min;

(iii) The insulin secretion drops drastically to near the background secretion amount within 2min after reaching the peak of insulin secretion, after which the insulin secretion slowly rises and continues;

(iv) After the high sugar phase enters the stimulation of high sugar and 10nM Exendin-4, insulin secretion begins to increase significantly again and decreases within 2-3 min;

(v) Insulin secretion was maintained at a lower background level after reentry into the low-sugar stimulation phase from high sugar and 10nM Exendin-4; and

(vi) Insulin secretion increases sharply upon entering the KCl stimulation phase and decreases rapidly around 5min after reaching the peak.

In a preferred embodiment, the islet-like cell mass exhibits an insulin secretion pattern similar to that of islets in both static and dynamic GSIS experiments.

In a preferred embodiment, the islet-like cell mass is obtained by in vitro induced differentiation of stem cells.

In a preferred embodiment, the stem cells are selected from the group consisting of: human embryonic stem cells, human induced pluripotent stem cells (hipscs), or combinations thereof.

In a preferred embodiment, the human induced pluripotent stem cells comprise human induced pluripotent stem cells, a genetically engineered cell line thereof, or a combination thereof.

In a preferred embodiment, the human induced pluripotent stem cells are reprogrammed from various somatic cells such as human peripheral blood mononuclear cells or human mesenchymal stem cells or human fibroblasts.

In a preferred embodiment, the human embryonic stem cells are selected from the group consisting of: mel-1 embryonic stem cells or a genetically edited cell line thereof, HES3 embryonic stem cells or a genetically edited cell line thereof, HUES8 embryonic stem cells or a genetically edited cell line thereof, H1 embryonic stem cells or a genetically edited cell line thereof, H9 embryonic stem cells or a genetically edited cell line thereof, or a combination thereof.

In a preferred embodiment, the human embryonic stem cells are INS ^GFP/w NKX6.1 ^{mcherry/mcherry} Mel-1 embryonic stem cell line.

In a preferred embodiment, the human embryonic stem cells are INS ^GFP/w HES3 embryonic stem cell line.

In a second aspect, the present invention provides a method of preparing an islet-like cell mass, the method comprising:

(1) Performing differentiation treatment on the stem cells in the form of adherent cells; and

(2) Performing differentiation treatment in the form of cell spheres on the adherent cells obtained in the step (1);

wherein BMP signaling pathway inhibitor, nicotinamide, and Epidermal Growth Factor (EGF) are utilized at the differentiation stage of the adherent cell form; preferably, the BMP signaling pathway inhibitor is Noggin, LDN-193189 or Dorsomophin or a combination thereof

In a preferred embodiment, the culture system uses different culture conditions and media at different stages of culture, depending on the state of the cells in the system.

In a preferred embodiment, no PKC signaling pathway activator is utilized during the differentiation stage of the adherent cell form; preferably, the PKC signal pathway activator is TPB (- (2 s,5 s) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadioylamino) benzolactam).

In a preferred embodiment, nicotinamide, epidermal Growth Factor (EGF) or a combination thereof is utilized at the differentiation stage of the cell pellet form.

In a specific embodiment, at the end of the differentiation stage of the adherent cell form, an adherent cell digestion treatment is performed in order to disperse the adherent cells into cell clusters; preferably, the adherent cell digestion treatment is performed using a combination of dispase and pancreatin.

In a preferred embodiment, the cultivation stage comprises seven stages S1, S2, S3, S4, S5, S6 and S7; each culture stage comprises:

the S1 stage refers to the 0 th to 2 nd days of culturing initial cells in the culture system;

the S2 stage refers to the 3 rd to 5 th days of culturing initial cells in the culture system;

the S3 stage refers to the 6 th to 7 th days of culturing initial cells in the culture system;

the S4 stage refers to 8 th to 9 th days of culturing initial cells in the culture system;

the S5 stage refers to 10 th to 12 th days of culturing initial cells in the culture system;

the S6 stage refers to the 13 th to 19 th days of culturing initial cells in the culture system; and

The S7 phase is a phase starting on day 20 when the initial cells are cultured in the culture system.

In a preferred embodiment, adherent cell digestion is performed at the end of the S4 stage.

In a preferred embodiment, the digestion treatment refers to dispersing the adherent cells into cell clusters.

In a preferred embodiment, the cell mass is a uniform size, high-activity cell mass with spheronization capability.

In a preferred embodiment, the cell pellet is a cell pellet expressing PDX1 and NKX 6.1.

In a preferred embodiment, the cell mass contains 4 to 500 cells; preferably, 4 to 100 cells are contained; more preferably, 10 to 50 cells are contained; optimally, 20 cells are contained.

In a preferred embodiment, the digestion treatment at the end of the S4 stage uses a combined digestion solution.

In a preferred embodiment, the combined digestive juice is a combination digestive juice of dispase and pancreatin.

In a preferred embodiment, the digestion process comprises the steps of:

1) Removing the supernatant;

2) Rinsing;

3) Adding a dispersing enzyme;

4) Standing for 5-30 min at 37 ℃.

5) Discarding the dispersed enzyme, and adding pancreatin at normal temperature to digest for 30s at room temperature; and

6) Stop pancreatin reaction and centrifuge.

In a preferred embodiment, the dispase is prepared as-is with DPBS that is free of calcium and magnesium.

In a preferred embodiment, the digestion time is shorter when the ratio of S4 cells pdx1+nkx6.1+ is higher; the lower the pdx1+nkx6.1+ ratio in S4 cells, the longer the digestion time.

In a preferred embodiment, the Dispase is present in an amount of 1mg/ml to 5mg/ml Dispase, preferably 2mg/ml Dispase.

In a preferred embodiment, the pancreatin is 0.25% trypsin pancreatin.

In a preferred embodiment, the digestion treatment is 2mg/ml Dispase-dispersing enzyme for 8min followed by 0.25% Trypsin pancreatin for 30s.

In a preferred embodiment, the medium used in the S5 stage contains MCDB131 basic, RA, SANT-1, LDN193189, ALK5iII, and T3.

In a preferred embodiment, the medium used in the S5 stage is supplemented with an EGF component.

In a preferred embodiment, nicotinamide fraction is added to the medium used in step S5.

In a preferred embodiment, the pancreatic precursor cells of stage S5 express PDX1, NKX6.1, NGN3, PAX4, ARX and INS.

In another preferred embodiment, 60-98%, preferably 70-98% of the pancreatic precursor cells of the S5 stage express PDX1 and NKX6.1.

In a preferred embodiment, the medium used in stage S6 contains MCDB131 basic, LDN193189, ALK5iII, and T3.

In a preferred embodiment, the medium used in stage S6 further comprises any one of the following components: GSI-XX, DAPT, compound-E, and GSI-X.

In a preferred embodiment, nicotinamide fraction is added to the medium used in stage S6.

In a preferred embodiment, the S6 stage is divided into S6.1 and S6.2.

In a preferred embodiment, the S6 stage is 7 days, wherein the S6.1 time is 0 to 3 days.

In a preferred embodiment, the medium used in S6.1 comprises the following components: MCDB131 basic, LDN193189, ALK5iII, T3, GSI-XX, and EGF.

In a preferred embodiment, EGF component is added to the medium used in stage S6.1.

In a preferred embodiment, EGF component is added to the medium used in the S5 and S6.1 stages.

In a preferred embodiment, the medium used in stage S7 contains MCDB131 basic, ALK5iII, T3, N-acetyl-cysteine, trolox, and R428.

In a preferred embodiment, nicotinamide fraction is added to the medium used in step S7.

In a preferred embodiment, nicotinamide components are added to the medium used in the S5, S6 and S7 stages.

In a preferred embodiment, the culture environment for stages S1-S6 is 5% oxygen and 5% carbon dioxide, and the culture environment for stage S7 is 21% oxygen and 5% carbon dioxide.

In a preferred embodiment, the culture environment for stages S1-S5 is 5% oxygen and 5% carbon dioxide, and the culture environment for stages S6-S7 is 21% oxygen and 5% carbon dioxide.

In a preferred embodiment, the culture environment for stages S1-S4 is 5% oxygen and 5% carbon dioxide, and the culture environment for stages S5-S7 is 21% oxygen and 5% carbon dioxide.

In a preferred embodiment, the three-dimensional culture vessel is a low-adsorption petri dish.

In a preferred embodiment, the three-dimensional culture vessel is a large-scale roller bottle.

In a third aspect, the present invention provides a culture system in which primary cells are subjected to differentiation culture to obtain islet-like cell masses as described in the first aspect.

In a preferred embodiment, the starting cell is selected from the group consisting of: human embryonic stem cells, human induced pluripotent stem cells (hipscs), or combinations thereof.

In a preferred embodiment, the primary cells are differentiated according to the method of the second aspect.

In a fourth aspect, the invention provides an islet microenvironment model comprising an islet-like cell mass according to the first aspect.

In a preferred embodiment, the model does not contain microvascular structures.

In a preferred embodiment, cells such as endothelial cells, fibroblasts or mesenchymal stem cells are added to the model to form islet-like tissue.

In a fifth aspect, the present invention provides a use of the islet microenvironment model according to the fourth aspect, wherein the use is for simulating the effects and links between islet cells in normal physiological functions and pathological states of islets.

In a sixth aspect, the invention provides a pharmaceutical composition comprising the islet-like cell mass of the first aspect and a pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutical composition of the invention further comprises other functional cells; the functional cells include, but are not limited to: vascular endothelial cells, mesenchymal cells, immune cells.

In a seventh aspect, the invention provides the use of the islet-like cell mass of the first aspect for the preparation of an artificial pancreatic device or a medicament for the treatment of diabetes.

In a preferred embodiment, the artificial pancreatic device is capable of secreting insulin upon glucose stimulation.

In a preferred embodiment, the artificial pancreatic device is capable of secreting insulin upon stimulation by secretagogues L-arginine, tolbutamide, IBMX, exendin-4 and the like.

In a preferred embodiment, the artificial pancreatic device secretes insulin under glucose stimulation in static GSIS and dynamic GSIS experiments.

In a preferred embodiment, the artificial pancreatic device secretes insulin under glucose stimulation in a patient in need thereof.

In a preferred embodiment, the diabetes is type I diabetes.

In an eighth aspect, the invention provides an artificial pancreas device comprising a container comprising the islet-like cell mass of the first aspect and/or the islet microenvironmental model of the fourth aspect.

In a preferred embodiment, the container provides oxygen and energy supply for islet-like cell mass.

In a preferred embodiment, the container is a capsule.

In a ninth aspect, the present invention provides a method of obtaining insulin, comprising the steps of: (a) Stimulating the islet-like cell mass of the first aspect with glucose.

In a preferred embodiment, the method further comprises the step of (b) isolating and concentrating to obtain insulin that can be used directly.

In a preferred embodiment, the insulin is present in an amount of 50-99% wt, preferably 75-99% wt; more preferably 85-99% wt.

In a preferred embodiment, the insulin further comprises an amount of glucagon.

In a preferred embodiment, the glucagon is present in an amount of 0.001 to 10% wt, preferably 0.01 to 10% wt; more preferably 0.1-10% wt.

In a tenth aspect, the present invention provides a method of treating diabetes comprising administering to a subject in need thereof a therapeutically effective amount of the islet-like cell mass of the first aspect or the pharmaceutical composition of the sixth aspect.

In a preferred embodiment, the diabetes is insulin dependent diabetes mellitus.

In a preferred embodiment, the diabetes is type I diabetes.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a schematic diagram of a method for producing islet cells.

FIG. 2 shows gene expression under treatment with TPB or EGF+Nico.

FIG. 3 shows a comparison of gene expression of S7 islet-like cells and human islets.

Fig. 4 shows flow cytometric analysis of islet-like cells. Here, the graph (a) is INS vs GCG (n=5), and the graph (B) is INS vs NKX6.1 (n=5). INS-GFP is the fluorescent protein with GFP after the INS gene; the final fate of ins+gcg+ cells is α cells (INS-gcg+ cells).

FIG. 5 shows (A) CPEP, PDX1, NKX6.1, GCG expression (scale bar 50 μm); (B) And (C) expression of functional transcription factors, MAFA, etc. (scale: upper panel 50 μm, lower panel 10 μm).

FIG. 6 shows the vesicle structure of beta cells under electron microscopy (scale bar: islet-like cell mass 200nm; human islets: 500 nm).

Figure 7 shows the static GSIS response of islet-like cells.

Figure 8 shows the dynamic GSIS response of islet-like cells.

FIG. 9 compares differentiated islet cells obtained by other culture methods of the prior art with islet-like cells obtained by the present invention.

Fig. 10 shows that the human glucagon release of the inventive human islet-like cell pellet upon 2mM glucose stimulation is significantly higher than upon 20mM glucose stimulation (p=0.0236).

Figure 11 shows the decreased insulin secretion fold of the islet-like cell mass of the invention following inhibition of the glucagon receptor by small molecule drugs.

FIG. 12 shows UMAP diagrams after single cell sequencing of islet-like cell clusters of the present invention.

Detailed Description

The present inventors have conducted extensive and intensive studies and have unexpectedly found that islet-like cell populations can be obtained from stem cells by a specific differentiation method using a specific differentiation medium. The islet-like cell population contains both islet alpha cells and islet beta cells with physiological functions, and the proportion of islet alpha cells and islet beta cells is similar to that of the real islet cell population. On this basis, the inventors completed the present invention.

Description of the terms

Unless defined otherwise, 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 invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Embryonic stem cells

The term "embryonic stem cells" (embryonic stem cell, ESCs, ES or ESC for short) as used herein is a class of cells isolated from the Inner Cell Mass (ICM) of a blastula (blastocyst) prior to implantation of an early embryo, which has the properties of cultured immortalization, self-renewal and differentiation into three germ layers in vitro. ES cells can be induced to differentiate into almost all cell types of the body, both in an in vitro and in vivo environment. Wherein the INS ^GFP/w Mel-1 embryonic stem cells "supplied by E.Stanley and A.Elefany ¹ The method comprises the steps of carrying out a first treatment on the surface of the The INS ^GFP/w ；NKX6.1 ^{mCherry/mCherry} Mel-1 embryonic stem cells "from INS ^GFP/w The Mel-1 embryonic stem cells were obtained by CRISPR/CAS9 mediated homologous recombination after insertion of the mCherry fluorescence after the NKX6.1 gene.

The term "Pluripotent Stem Cells (PSC)" as used herein refers to a class of cells that possess both self-renewing and multipotent differentiation potential, including but not limited to: (1) Embryonic-derived pluripotent stem cells, (2) somatic cell reprogramming resulting pluripotent stem cells.

The term "induced pluripotent stem cells (ipscs)" as used herein refers to a class of stem cells that the somatic cells develop back into a state of multipotent differentiation potential via a reprogramming process. The multi-energy expression mode is as follows: has the ability to differentiate into three germ layers and multiple cell types.

The term "differentiation" as used herein refers to the process by which stem cells are transformed into a cell type of interest in a specific context.

The term "differentiation efficiency" as used herein refers to the frequency of transformation of pluripotent stem cells into a target cell type in a particular context. Such as: a differentiation efficiency of 90% means that at least 90% of the differentiated cell population is the target cell type.

Three-dimensional cell culture

The term "three-dimensional cell culture" (three-dimensional cell culture, TDCC) as used herein refers to co-culturing a carrier having a three-dimensional structure of different materials with various different kinds of cells in vitro, so that the cells can migrate and grow in the three-dimensional spatial structure of the carrier, constituting a three-dimensional cell-carrier complex. The conventional cell culture gradually loses the original properties due to the proliferation of cells in an in-vitro changed environment, often does not coincide with in-vivo conditions, and animal experiments are completely carried out in vivo, but the conventional cell culture is complicated due to the restriction of various factors in vivo and the mutual influence of in-vivo and external environments, so that a single process is difficult to study, and an intermediate process is difficult to study. The three-dimensional cell culture technology is a technology between monolayer cell culture and animal experiments, not only can simulate the in-vivo environment to the greatest extent, but also can show the advantages of intuitiveness and condition controllability of cell culture.

The conversion of cells from two dimensions to three dimensions can be divided into the following ways:

1) The cells are dispersed into single cells and then are gathered into cell clusters; 2) The cells are dispersed into a plurality of cell clusters with cells together, and the reagglomeration is not needed; 3) The cells are dispersed into a plurality of clusters of cells together, which are reaggregated. The inventors found in actual practice that: mode 1) the cell is digested into single cell, which requires long-time treatment of pancreatin and has a certain damage to the cell; modes 2) and 3) are milder for cells but require exploration of digestive enzyme selection and digestion methods.

Islet-like cells and methods of making same

The pancreas is both an endocrine and an exocrine gland, which is located behind the stomach. The pancreas contains groups of specific cells called langerhans islets (islets). Islet cells include B cells (beta cells), accounting for about 60% -80% of islet cells, secreting insulin that can lower blood glucose, and the lack of islet B cells can lead to the development of diabetes; a cell (alpha cell) accounting for about 24% -40% of islet cells, and secreting glucagon, which has the action opposite to insulin and can raise blood sugar; d cells (delta cells), accounting for about 6% -15% of the total number of islet cells, secrete somatostatin; islet PP cells, about 1% of islet cells, secrete pancreatic polypeptides. These cells constitute the endocrine part of the pancreas (ductless), and their function is distinct from the exocrine gland part (ducted), which, during digestion, endocrine digestive enzymes into the small intestine. The pancreas functions as an endocrine gland to secrete hormones directly into the blood stream, with the most important hormones being insulin and glucagon. Therefore, it is of great clinical importance to be able to obtain alpha and beta cells in a proportion similar to that of the actual islet cell population.

The terms "islet-like cells" and "islet-like cell mass" as used herein refer to an islet-like cell mass obtained by optimized differentiation culture of embryonic stem cells using the methods of the present invention that is functionally equivalent to a real islet cell mass in a similar proportion.

In a specific embodiment, the islet-like cell mass obtained by in vitro differentiation of the present invention comprises: (a) islet beta cells; and (b) islet alpha cells; and (c) optionally other endocrine cells;

In a preferred embodiment, the islet beta cells in the islet-like cell mass of the invention comprise 30% to 80%; and islet alpha cells account for 6% -40%; more preferably, islet beta cells in the islet-like cell mass account for 40% -70%; and islet alpha cells account for 10% -35%.

The inventors noted that almost all of the islet-like cell masses of the present invention were endocrine cells. For example, in addition to islet beta cells and islet alpha cells, the islet-like cell mass of the invention also comprises small amounts, e.g., 1% to 10%, of other endocrine cells (e.g., delta cells), including, but not limited to, other endocrine cells expressing SST or PPY.

In contrast, other islet-like cell clusters obtained by prior art methods contain a substantial proportion of undifferentiated non-endocrine cells.

"refolding", "refolding" or "enriching" as used herein have the same meaning; that is, these terms refer to the digestion of islet-like cell mass into single cells, sorting out, e.g., by flow cytometry, of cells bearing beta cell markers (e.g., INS and NKX 6.1), and then polymerizing the sorted single cells into a cell mass. Thus, in a specific embodiment, the islet-like cell mass of the present invention comprises islet-like cell masses comprising a corresponding proportion of islet beta cells and islet alpha cells, e.g., similar to a real islet cell population, after sorting to obtain islet beta cells and islet alpha cells, respectively, and then repolymerizing. In a preferred embodiment, the islet-like cell mass obtained by the repolymerization is capable of having the same physiological function as the actual islet cell mass.

The islet-like cell mass of the invention not only has a similar proportion to the actual islet cells, but also expresses the specific markers of the islet cells, thereby being capable of having the functions equivalent to the actual islet cells.

For example, the islet-like cell mass of the invention expresses the endocrine cell marker NEUROD1 in an amount equivalent to that of islets. For another example, the islet beta cells of the present invention express specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8; preferably, specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8, MAFA are expressed; preferably, specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8, MAFA, IAPP are expressed; the alpha cells express the marker gene GCG, ARX, PCSK2.

In a specific embodiment, the proportion of β cells expressing MAFA, as measured by immunofluorescent staining, based on at least expression of the specific marker gene described above, comprises 70-85% of all beta cells, more preferably at least 80% of all β cells; the proportion of IAPP-expressing beta cells is 30-60% of the total beta cells, more preferably at least 50% of the total beta cells; the alpha cell expresses a marker gene GCG, ARX, PCSK2;

in another preferred embodiment, the β cells expressing both INS and IAPP account for 30-45%, preferably 32-40%, more preferably 35-38% of all β cells as detected by single cell sequencing; beta cells expressing both INS, IAPP and MAFA account for 15-30%, preferably 18-25%, more preferably 20-24% of all beta cells.

The islet-like cell mass of the invention is capable of secreting insulin upon glucose stimulation; preferably, the islet-like cell mass releases at least 1.5 times, preferably at least 2 times, more preferably at least 3 times, even more preferably at least 5 times, more preferably at least 10 times more human insulin under high sugar stimulation than under low sugar stimulation.

The glucagon release of the islet-like cell mass of the invention upon low sugar stimulation is at least 2 times, preferably at least 3 times, more preferably at least 4 times, most preferably at least 5 times the release of human glucagon upon high sugar stimulation.

Insulin secretion experiments under glucose stimulation of the islet-like cell mass of the present invention, including static GSIS and dynamic GSIS, also exhibit insulin secretion patterns similar to islets.

The islet-like cell mass of the present invention is produced by subjecting stem cells to differentiation treatment, which divides the differentiation treatment of stem cells into differentiation treatment in the form of adherent cells; and differentiation treatment in the form of a cell sphere.

In the differentiation processing stage of the adherent cell form, the inventors used BMP signaling pathway inhibitors (including but not limited to Noggin), nicotinamide, epidermal Growth Factor (EGF) or combinations thereof to obtain pancreatic precursor cells expressing PDX1, NKX6.1, NGN3, PAX4, ARX;

during the differentiation treatment stage in the form of a cell pellet, the present inventors switched the cell culture from two dimensions to three dimensions. The conversion of cells from two dimensions to three dimensions can be divided into the following ways: 1) The cells are dispersed into single cells and then are gathered into cell clusters; 2) The cells are dispersed into a plurality of cell clusters with cells together, and the reagglomeration is not needed; 3) The cells are dispersed into a plurality of clusters of cells together, which are reaggregated. Modes 1) to 3) each have advantages and disadvantages. The inventors have creatively determined the best way to switch cell culture from two-dimensional to three-dimensional by combining pancreatin with dispase.

As used herein, "pancreatic precursor cells" refers to precursor states of islet alpha and beta cells in islet-like cell mass differentiation culture. The "pancreatic precursor cells" of the present invention are capable of expressing pancreatic precursor marker genes well. In a preferred embodiment, the pancreatic precursor cells of the invention express pancreatic precursor marker genes PDX1, NKX6.1, NGN3, PAX4, ARX and INS. In another preferred embodiment, 60-98%, preferably 70-98%, of the pancreatic precursor cells of the invention express the pancreatic precursor marker genes PDX1 and NKX6.1.

Based on the islet-like cell mass of the invention, the invention also comprises a culture system in which the primary cells are subjected to the differentiation method of the invention to obtain the islet-like cell mass of the invention, as will also be appreciated by the person skilled in the art. Based on the teachings of the present invention, it will be appreciated by those skilled in the art that the starting cells refer to cells that can be differentiated to give islet-like cell mass. In specific embodiments, the starting cell is selected from the group consisting of: human embryonic stem cells, human induced pluripotent stem cells (hipscs), or combinations thereof. Based on the teachings of the present invention in combination with the prior art, one skilled in the art will appreciate that the culture system of the present invention may be used for the study of gene function. In particular embodiments, cells may be genetically edited at various stages of the culture process of the culture system of the present invention, such as for example, initial cells (e.g., human pluripotent stem cells) may be genetically edited (e.g., knocked out or knockdown of genes), cells during the culture process may be genetically edited, or cells in the islet-like cell mass obtained by differential culture may be genetically edited to investigate the function of the gene. The method of gene editing may be a method of gene editing known to those skilled in the art, such as CRISPR/CAS9 gene editing, and the like.

On the basis of the islet-like cell mass, the invention also provides an islet microenvironment model containing the islet-like cell mass. The model may not include microvascular structures therein; alternatively, cells such as endothelial cells, fibroblasts, or mesenchymal stem cells may be added to the model to form islet-like tissue. The islet microenvironment model provided by the invention can simulate the normal physiological functions of islets and the actions and the relations among islet cells in pathological states.

The islet-like cell mass of the invention can be used for the preparation of an artificial pancreatic device that can secrete insulin under glucose stimulation or for the treatment of diabetes, such as type I diabetes.

Thus, on the basis of the islet-like cell mass of the invention, the invention also provides a pharmaceutical composition comprising said islet-like cell mass and a pharmaceutically acceptable excipient; and methods of treating diabetes, such as type I diabetes, using the islet-like cell mass or pharmaceutical composition of the invention. Based on the teachings of the present invention and the teachings already in the art, one skilled in the art will also appreciate that other functional cells may also be included in the pharmaceutical compositions of the present invention, thereby enabling different functions to be performed simultaneously. The functional cells include, but are not limited to: vascular endothelial cells, mesenchymal cells, immune cells.

Based on the teachings of the present invention, it will also be appreciated by those skilled in the art that the islet-like cell mass of the present invention produces insulin upon stimulation with glucose. Thus, the invention also provides a method of using glucose to stimulate the islet-like cell mass of the invention to obtain insulin.

Basal medium

The term "basal medium" as used herein includes, but is not limited to, "basal medium SFD", "basal medium MCDB131 basic".

Wherein, the formulation of the basal medium SFD and serum free medium is 75%IMDM,25%Ham's F12,0.5 XN 2 additive, 0.5 XB 27 additive (without vitamin A), 0.1% BSA (A1470) and 0.5 XStreptomyces lividans.

The formula of the basal medium MCDB131 basic is MCDB131,2% BSA,1:200 ITS-X,1 XGlutaMax, 14.5mM glucose, 10mM nicotinamide, 10 mu M zinc sulfate, 10 mu g/ml heparin and 0.5 XQing streptomycin.

Experimental materials

1. Cell lines

1.1 Mel-1 embryonic stem cells or genetically edited cell lines thereof, including INSFP/w Mel-1 reporter embryonic stem cell lines supplied by E.Stanley and A.Elefant laboratories and Nkx6.1mCherry obtained by inserting T2A and mCherry fluorophores after the NKX6.1 gene by INSFP/w Mel-1 embryonic stem cells via CRISPR/CAS 9-mediated homologous recombination.

1.2 HES3 embryonic stem cell line or INSFGFP/w HES3 embryonic stem cell line or other HES3 genetically engineered cell line

1.3 HUES8 embryonic stem cell line or genetically edited cell line thereof

1.4 H1 embryonic stem cell line or genetically edited cell line thereof

1.5 H9 embryonic stem cell line or a genetically edited cell line.

1.6 INS ^GFP/w Mel-1 embryonic stem cells are provided by e.stanley and a.elefanty; INS (inertial navigation System) ^GFP/w ；NKX6.1 ^{mCherry/mCherry} Mel-1 embryonic stem cells were derived from INS ^GFP/w The Mel-1 embryonic stem cells were obtained by CRISPR/CAS9 mediated homologous recombination after insertion of the mCherry fluorescence after the NKX6.1 gene.

2. Experimental reagent

3. Basic culture medium formula

1) Basal medium SFD (serum free medium) contained 75%IMDM,25%Ham's F12,0.5 ×n2 additive, 0.5×b27 additive (without vitamin a), 0.1% bsa (a 1470), 0.5×penicillin.

2) The basal medium MCDB131 basic contains MCDB131,2% BSA,1:200ITS-X,1 XGlutaMax, 14.5mM glucose, 10mM nicotinamide, 10. Mu.M zinc sulfate, 10. Mu.g/ml heparin, 0.5 XStreptomyces lividans.

4. Antibodies to

Name of the name	Company (Corp)	Goods number	Species of genus	Dilution ratio
					PDX1	R&D	AF2419	Goat	1:50
NKX6.1	DSHB	F55A12	Mouse IgG1	1:36
					C-peptide	CST	4593S	Rabbit	1:33
Insulin	DAKO	A0564	Guinea pigs	1:200
					Glucagon(K79bB10)	Sigma	G2654	Mouse IgG1	1:1000
Somatostatin	Santa Cruz	sc-74556	Mouse IgG1	1:100
					MAFA	Abcam	ab26405	Rabbit	1:100

qRT-PCR primers

Experimental method

1. Cell culture

Maintenance and passaging of pluripotent stem cells are consistent with literature reports (Kennedy et al, 2007)

The general flow of differentiation of pluripotent stem cells into islet-like cells is shown in FIG. 1, and the steps are as follows:

1) Pluripotent stem cells are passaged onto matrigel (3-fold diluted by IMDM) and grown to 80% density within 1-2 days to begin differentiation;

2) Differentiation is divided into 7 stages (S1-S7), wherein S1-S4 are differentiated in the form of adherent cells, and each time the cell is switched from the previous stage to the next stage, the cell needs to be rinsed 2 times by using the basic culture medium of the next stage, and then the cell is changed into a freshly prepared culture medium; S5-S7 are differentiated in a cell sphere shape, liquid exchange adopts 70-90 g for 1-3 min for centrifugation or natural sedimentation, and fresh culture medium is added after supernatant is absorbed. S5, changing liquid every day, S6, changing liquid every day or every two days, and S7, changing liquid every two days. The media of S5 and S6.1 were supplemented with EGF (Rezania et al 2014), and S5-S7 were supplemented with Nicotinamide (Nicotinamide). The medium composition at each stage was as follows:

3) And S4, after finishing, special treatment is needed, so that the adherent cells are uniformly dispersed into cell aggregates of about 20 cells under the condition of ensuring good activity, and the cells smoothly enter subsequent three-dimensional culture, wherein the three-dimensional culture is favorable for the formation of endocrine cells. The specific treatment method comprises the following steps: s4, sucking the supernatant from the cells, washing the cells once with PBS, adding 2mg/ml of disperse enzyme (dispese, which is prepared by using DPBS without calcium and magnesium), and standing the cells at 37 ℃ for 5 to 30 minutes. Regarding digestion time, the higher the S4 cell pdx1+nkx6.1+ efficiency, the shorter the digestion time; when more non-target cells are mixed in the S4 cells, the digestion time is longer. The adherent cells marked S4, judged to be completely digested by Dispase, were either separated from the dish or mostly separated with only a small portion of the center remaining, assuming jellyfish-like. After the completion of the enzyme dispersion digestion, the enzyme was aspirated, and the mixture was rinsed with DPBS twice the volume of the enzyme to be digested, and the same volume of normal temperature or cold 0.25% Trypsin was added for a second digestion for a period of less than 30 seconds, and then the mixture was blown 1 to 3 times with a 1ml pipette to form a pellet of about 20 cells, and after that, the enzyme and cell mixture was rapidly transferred to DPBS containing 10% serum to terminate the digestion. Centrifuging for 3min at normal temperature of 90-150 g. The supernatant was blotted and gently flicked to disperse the pellet at the bottom of the tube, then resuspended in S5 medium, transferred to a low adsorption 6 well plate (Costar, # 3471) and placed on a horizontal shaker at 90-100 rpm for subsequent three-dimensional culture.

4) S1-S6 cells are placed in an incubator containing 5% of oxygen and 5% of carbon dioxide, S7 cells are placed in an incubator containing 21% of oxygen and 5% of carbon dioxide, and the purpose is to induce high expression of GCG, IAPP, SLC A8 isogenes in the final maturation process of islet cells.

2. Quantitative reverse transcription PCR (qRT-PCR)

1 to 5 multiplied by 10 ⁵ Extracting cells of the stem cells with a root trace total RNA extraction kit, carrying out reverse transcription on 1 mu g of RNA with a Promega reverse transcription kit to obtain cDNA, diluting the cDNA by 1:9, carrying out gradient dilution on the genomic DNA of the human pluripotent stem cells to obtain a standard, and carrying out qRT-PCR with Roche SYBR GREEN.

3. Immunofluorescence

The induced cell pellet was washed twice with PBS, fixed with 4% PFA at room temperature for 20min, washed twice with PBS, dried, embedded with OCT, and stored at-80 ℃. The embedded block was cut into 5 μm slices with an ice cutter, attached to a glass slide, and the tissue was enclosed with a histochemical pen, then blocked with blocking solution (PBS containing 10% FBS, 0.1% Triton-100) at room temperature for 1 hour, then incubated with the primary antibody diluted with blocking solution overnight at 4 degrees, washed three times with PBS containing 0.1% Triton-100 the next day, incubated with the secondary antibody diluted with blocking solution at room temperature for 1 hour, then washed three times with PBS containing 0.1% Triton-100, and photographed with a Leica SP8 confocal fluorescence microscope after dropping DAPI.

4. Transmission electron microscope

And (3) after the islet-like cells of the S7 are rinsed twice by DPBS, transferring the islet-like cells to 2.5% glutaraldehyde for preservation, fixing, embedding, slicing and staining an Shanghai biochemical and cell institute electron microscope experiment platform, and then placing the islet-like cells in an FEI Tecnai G2 spirat electron microscope for shooting.

5. Flow cytometry analysis

Cells were digested with 0.25% Trypsin-EDTA into single cells, washed twice with PBS, fixed with 1.6% PFA at 37 degrees for 30min, then washed three times with FACS buffer, and stored at 4 degrees in FACS buffer. The cells were permeabilized by washing twice with 1X Saponin buffer, incubated for 30min with 1X Saponin buffer diluted primary antibody, washed twice with 1X Saponin buffer, incubated for 30min with 1X Saponin buffer diluted secondary antibody, washed twice with 1X Saponin buffer, resuspended in 300. Mu.l FACS buffer, analyzed on BD LSRII flow analyser, and further analyzed by Flowjo software.

6. Static GSIS

Islet cell pellets and islets were washed three times with KREB buffer (containing 129mM NaCl,5mM NaHCO3,4.8mM KCl,2.5mM CaCl2,1.2mM MgSO4,1.2mM KH2PO4, 10mM HEPES,0.1%BSA), transferred to KREB buffer containing 2mM glucose for 1 hour, then washed three times with KREB buffer, transferred to KREB buffer containing 2mM glucose, collected after 30min, washed three times with KREB buffer, transferred to KREB buffer containing 20mM glucose, collected after 30min, and so cycled three times. All stimulation steps were completed in a 37 degree, 5% carbon dioxide incubator. The supernatants collected each time were stored at-20℃and the content of C-peptide was determined using the super-sensitive C-peptide Elisa kit.

7. Dynamic GSIS (Perifusion)

Experiments were performed using Biorep perfusion apparatus, firstly islet cell pellets and islets were resuspended in Bio-Rad Bio-Gel P4 beads, loaded in a chamber, then the chamber, flow guide tube, peristaltic pump, multi-directional valve were assembled together, KREB buffer and each stimulator configured in the KREB buffer were added, and the procedure was set: the flow rate was 100. Mu.l/min, first starved in 2mM glucose for 1 hour, followed by 2mM low sugar 15min,20mM high sugar 30min, 20mM high sugar 15min with 10nM Exendin-4, 2mM low sugar 15min,2mM low sugar 10min with 30mM KCl. The effluent liquid was received into a 96-well plate at one well per minute. Cells were recovered after perfusion, shaken in 75% ethanol solution containing 1.5% hydrochloric acid, and stored at-20℃for insulin content and DNA quantification. Insulin was detected using the hypersensitive C peptide Elisa kit or the insulin Elisa kit.

The invention has the beneficial effects that:

1. the invention establishes an optimized culture medium formula and a combined action method of BMP signal channel inhibitor Noggin, nicotinamide and epidermal growth factor (epidermal growth factor, EGF) to successfully prepare pancreatic precursor cells with high expression of PDX1 and NKX 6.1.

2. The invention also establishes a method for combined digestion of the digestive enzymes of the dispease-pancreatin, and islet-like cell clusters with three-dimensional structures similar to the composition of human islet cells can be obtained simultaneously in the same differentiation system without enrichment.

3. The islet-like cell mass obtained by differentiation of the invention requires shorter maturation time, and can have mature insulin function about the 33 th day of differentiation initiation.

4. The islet-like cell mass obtained by differentiation of the invention has strong insulin release capability under the stimulation of glucose and the like, and has good effect in static and dynamic GSIS experiments.

5. The islet-like cell mass obtained by the invention can be used for an in-vivo model of type I diabetes, so that the blood sugar of a type I diabetes mouse can be maintained in a normal interval for a long time.

The invention is further described below in conjunction with the specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1: optimization of differentiation methods

1.1S3 and S4 recipe optimization

200nM and 100nM PKC signal pathway activator TPB was added at S3 and S4, respectively, as described in the experimental methods section. To prepare pancreatic precursor cells with high differentiation potential, the optimized group used EGF and nicotinamide instead of TPB.

Results: as shown in FIG. 2, it was unexpectedly found that the addition of TPB did not induce very good expression of pancreatic precursor marker genes PDX1, NKX6-1, pancreatic endocrine precursor marker genes NGN3, ARX, PAX4, and conversely, non-target intestinal related gene CDX 2. The optimized group uses EGF and nicotinamide to replace TPB, so that PDX1, NKX6.1, NGN3, PAX4, ARX and INS can be well induced. On cell yield, the optimized group was 0.5X10 at initial differentiation ⁶ A plurality of pluripotent stem cells capable of obtaining 2.13X10S 4 ⁶ Left and right cells, 1.23×10 cells were obtained in S5 ⁶ Left and right cells, 2.73X10 at S6 ⁶ The left and right cells were obtained at S7 with a size of 0.80X 10 ⁶ Left and right cells (i.e., islet-like cells). Overall, the yield of the optimized group of islet-like cells was 1.6 times that of the starting cells. In the schemes reported in the prior art and literature, the data provided by Kieffer laboratories resulted in 1.2S 6 or S7 cells per pluripotent stem cell (Rezania, A.et al, reverse of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells Nature Biotechnology 32,1121-1133, doi:10.1038/nbt.3033 (2014)); the result of the enrichment method in the Melton laboratories is a yield of 1/15 to 1/10 of the starting cells (Veres, A.et al. Charting cellular identity during human in vitro beta-cell differentiation. Nature 569,368- +, doi:10.1038/s41586-019-1168-5 (2019)). Therefore, the scheme of the invention greatly improves the level of the differentiation of the pluripotent stem cells stimulated by the culture medium.

1.2 cell two-to-three dimensional transition culture formulation optimization

1.2.1 according to the prior art, the production of endocrine cells requires that they fall off the pancreatic epithelium and migrate to aggregate into endocrine cell clusters. To select a suitable method for digesting S4 phase cells, the inventors compared the various combinations and modes of three common modes of Trypsin pancreatin, trypLE pancreatin substitutes, dispase and the optimization schemes of the invention in the prior art, and other treatment modes were identical or substantially identical, and then evaluated the effects of each scheme by detecting the gene expression of S5 phase cells and the number of S5 phase cells. The specific operation is shown in the materials and methods, the formula and the corresponding results are shown in the following table:

remarks: the gene expression level is "gene expression level relative to housekeeping gene TBP"; s5 cell yield represents the number of S5 cells obtained from the initial 1 pluripotent stem cells.

Results: from the above data, it can be seen that the digestion mode of treating with Dispase for 8min and then with Trypsin for 30S greatly improves the cell yield at the transformation stage from S4 stage to S5 stage.

1.2.2 in order to find a culture method for promoting the formation of endocrine cell clusters by S5-phase cells, the inventors compared several culture medium formulations, and other treatment modes were consistent or approximately consistent, and then evaluated the effects of each regimen by detecting the gene expression status and the cytoball morphology of S5-phase cells. The formulations and corresponding results are shown in the following table:

Remarks: the gene expression level was "Gene expression level relative to housekeeping Gene TBP"

Results: the most suitable time point for switching cells from two-dimensional to three-dimensional culture is at the end of S4. The most suitable method of digesting S4 cells is to use 2mg/ml Dispase in combination with 0.25% pancreatin. Nicotinamide is preferably added to the culture medium of S5-S7, and EGF is further added in the first two days of S5 and S6, which is most favorable for the cells in the S5 phase to form endocrine cell clusters. EGF may then act to promote cell proliferation.

Example 2: analysis of cell composition of islet-like cell mass

The resulting islet-like cell mass was differentiated using the optimized formulation described in example 1 to further analyze its cell composition.

2.1 Gene expression in islet-like cells

As shown in fig. 3, the qRT-PCR analysis results showed that, compared with human adult islets (complete islets of all endocrine cells including islet β cells, α cells, δ cells, etc.), the islet-like cell mass of S7 stage expressed endocrine cell markers, resured 1, expressing β cell specific marker genes PDX1, NKX6-1, INS, PCSK1, ABCC8, MAFB, and the functional genes MAFA, SLC30A8 were approximately equivalent in level to human adult islets. Furthermore, the inventors have unexpectedly found that islet-like cell mass at stage S7 also expresses the α -cell marker gene GCG, ARX, PCSK2, which is comparable to human adult islets.

2.2 cell composition of islet-like cell clusters

Analyzing the cell composition of islet-like cell mass by flow cytometry, wherein INS-GFP is the INS gene and then carries GFP fluorescent protein; INS (inertial navigation System) ⁺ GCG ⁺ The final fate of the cells is alpha cells (INS ^- GCG ⁺ Cells).

As shown in FIG. 4 (A), 36.0% -67.7% islet beta cells (INS) can be obtained simultaneously without enrichment ⁺ GCG ^- ) And 6.33% -34.9% islet alpha cells (INS) ^- GCG ⁺ ). The data prove that the composition of the islet-like cell mass obtained by induction by using the optimized culture scheme provided by the invention is similar to that of human islets (28% -75% of beta cells, 38% of alpha cells and 7% of delta cells). The core transcription factor NKX6.1 is expressed in the islet beta cells with secretion function, and as shown in fig. 4 (B), 36.4% -64.2% of islet beta cells can be obtained by the invention.

2.3 immunofluorescence of islet-like cell clusters

As shown in FIG. 5, at the protein expression level, the beta cells in the S7 islet-like cell mass obtained by the optimization scheme express INS and transcription factors PDX1 and NKX6-1, and most of the beta cells express functional transcription factors MAFA. Furthermore, most endocrine cells in the S7 islet-like cell mass express only a single hormone (INS) ⁺ GCG ^- Or INS ^- GCG ⁺ )。

2.4 vesicle Structure under electronic microscope

The results are shown in FIG. 6, where the left panel shows the vesicles of the beta cells in the islet-like cell mass of S7 and the right panel shows the vesicles of the beta cells in human islets. The electron microscope observes that the S7 islet-like cell mass obtained by the optimization scheme has a structure similar to that of beta cells in human islets and also has a typical compact core. The S7 islet-like cell mass was suggested to have a secretory function resembling that of beta cells in human islets.

Example 3: analysis of cell composition of islet-like cell clusters by single cell sequencing

In this example, the inventors performed single cell sequencing of the islet-like cell mass of the invention. UMAP diagrams after single cell sequencing are shown in FIG. 12, which reveals all cell groupings 1-5. 1beta-cell is beta cell in islet-like cell mass, and the other three figures are the gene displays in UMAP. Wherein INS is a marker gene of all beta cells, MAFA and IAPP are marker genes of mature beta cells, and the beta cells expressed by INS and IAPP account for 36.7% of all beta cells; the β cells expressed by INS, IAPP and MAFA all account for 21.4% of all β cells.

Example 4: insulin secretion test under glucose stimulation

1) Static GSIS

Results As shown in FIG. 7, in static GSIS experiments, S7 islet-like cells exhibited low sugar (2 mM glucose) in the first stage (1 st), high sugar (20 mM glucose) and low sugar (2 mM glucose) in the second stage (2 nd), high sugar (20 mM glucose), and potassium chloride (30 mM KCl) in the final stage stimulated the reaction. In each stage, the insulin secretion under high sugar stimulation is higher than that under low sugar stimulation, and the insulin is released to the greatest extent under potassium chloride stimulation. The S7 islet-like cell mass has secretion function similar to beta cells in human islets under the stimulation of secretagogues such as glucose.

2) Dynamic GSIS (perifusion)

As a result, as shown in FIG. 8, in the experiment of dynamic GSIS, insulin had a background secretion amount under the stimulation of 2mM glucose, and reached the peak of insulin secretion within 1 to 5 minutes after entering 20mM glucose, which was one-phase (phase 1) insulin secretion. The insulin secretion then drops drastically within 2min until 30min falls to a minimum, after which 3min insulin secretion again rises slightly, which is biphasic (phase 2) insulin secretion. Insulin secretion again began to increase significantly upon entry into 10nM Exendin-4 high sugar, and decreased significantly upon entry into 2mM glucose. Insulin secretion is further increased during the final KCl stimulation phase.

Throughout the stimulation, insulin secretion patterns for a total of 4 periods (high glucose phase1, high glucose phase2, ex-4, kcl) were similar to islets. The S7 islet-like cell mass has secretion and secretion functions similar to those of beta cells in human islets under the stimulation of secretagogues such as glucose.

Example 5: comparison of multiple islet cell differentiation methods

In order to compare the effect of the main islet cell differentiation method in the prior art with that of the islet cell differentiation method provided by the invention, control experiments are carried out according to the methods, and other treatments are ensured to be consistent or approximately consistent. The gene expression is shown in FIG. 9, and the treatment mode and the comparison result are shown in the following table:

1.Rezania,A.et al.Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.Nature Biotechnology 32,1121-1133,doi:10.1038/nbt.3033(2014).

2.Pagliuca,F.W.,Millman,J.R.,Gurtler,M.,Segel,M.,Van Dervort,A.,Ryu,J.H.,Peterson,Q.P.,Greiner,D.,and Melton,D.A.(2014).Generation of functional human pancreatic beta cells in vitro.Cell 159,428-439.

3.Velazco-Cruz,L.,Song,J.,Maxwell,K.G.,Goedegebuure,M.M.,Augsornworawat,P.,Hogrebe,N.J.,and Millman,J.R.(2019).Acquisition of Dynamic Function in Human Stem Cell-Derived beta Cells.Stem Cell Rep 12,351-365.

4.Nair,G.G.,Liu,J.S.,Russ,H.A.,Tran,S.,Saxton,M.S.,Chen,R.,Juang,C.,Li,M.L.,Nguyen,V.Q.,Giacometti,S.,et al.(2019).Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived beta cells.Nat Cell Biol 21,263-274.

5.Veres,A.,Faust,A.L.,Bushnell,H.L.,Engquist,E.N.,Kenty,J.H.,Harb,G.,Poh,Y.C.,Sintov,E.,Gurtler,M.,Pagliuca,F.W.,et al.(2019).Charting cellular identity during human in vitro beta-cell differentiation.Nature 569,368-373.

6.Rezania,A.,Riedel,M.J.,Wideman,R.D.,Karanu,F.,Ao,Z.,Warnock,G.L.,and Kieffer,T.J.(2011).Production of functional glucagon-secreting alpha-cells from human embryonic stem cells.Diabetes 60,239-247.

Example 6: human glucagon release from islet-like cell mass under glucose stimulation

The inventors further validated the human glucagon release profile of the islet-like cell pellet of the invention under low and high concentration glucose stimulation. The specific steps are as follows:

human islet-like cell clusters (50) obtained in example 1 were stimulated in 500 μl of a low concentration glucose (2 mM) KREB solution for 30 minutes, cell supernatants were collected, and Elisa detected for human glucagon concentration (2 mM on abscissa, unit pmol/L); the human islet-like cell mass was rinsed and stimulated with 500. Mu.l of a high concentration glucose (20 mM) KREB solution for 30 minutes, and the cell supernatant was collected, and the concentration of human glucagon (indicated by 20mM on the abscissa, unit pmol/L) was measured by Elisa; finally, the cells were collected in a low sugar KREB solution containing 30mM potassium chloride (KCl) for 30min, and the concentration of human glucagon (expressed in pmol/L on the abscissa) was measured by Elisa.

Results (as shown in fig. 10): the human glucagon release from the human islet-like cell mass stimulated with 2mM glucose was significantly higher than that stimulated with 20mM glucose (p=0.0236). Indicating that alpha cells in the human islet-like cell mass of the invention are capable of secreting glucagon under low sugar stimulation.

Example 7: verification of alpha cell function in islet-like cell clusters

Glucagon (GCG) was secreted by islet alpha cells in human islet-like cell mass to demonstrate that Glucagon would affect insulin secretion by islet beta cells, the inventors treated the islet-like cell mass obtained in example 1 with a 1 μm concentration of the small molecule drug L168,049, (4- [3- (5-Bromo-2-propxyphenyl) -5- (4-chlorophenyl) -1H-pyrrol-2-yl ] pyridine). The small molecule is capable of inhibiting the GCGR receptor of GCG, while beta cells express GCGR. The fold of insulin secretion (Stimulation index) is the ratio of insulin secretion at high-glucose stimulation to low-glucose stimulation, the higher the fold indicating better beta cell function.

The results are shown in FIG. 11, which shows that the insulin secretion fold of islet-like cells is decreased after the small molecule drug inhibits the receptor of GCG. The presence of alpha cells in the islet-like cell mass of the invention is therefore essential for the function of the islet-like cell mass.

Discussion:

in vitro induced differentiation of human pluripotent stem cell-derived islet beta cells has progressed mainly in the last 20 years. However, the initial approach using embryoid bodies is inefficient in obtaining insulin-secreting cells during spontaneous differentiation of human embryonic stem cells (hESCs) and lacks functional verification. After that, human pluripotent stem cells are taken as a starting point in vitro by utilizing the thought of simulating in-vivo development process, and differentiated into islet beta cells through several steps of definitive endoderm, rear foregut, pancreatic precursor cells, endocrine precursor cells, immature beta cells and mature beta cells, the method describes the cell composition and gene expression of each step of differentiation, but the most obvious disadvantage is that the whole process is two-dimensional culture, and the final beta cell efficiency is only 12%.

All current studies of islet cell differentiation focus on specifically inducing beta cells or alpha cells, and although some studies have resulted in beta cells with gene expression similar to human islets, both studies utilized the enrichment of beta cell markers, resulting in a lower proportion of alpha cells in the final cell fraction than in authentic islets. While, studies by Rodriguez-Diaz, r.et al (Paracrine Interactions within the Pancreatic Islet Determine the Glycemic Set point. Cell meta 27,549- +, doi:10.1016/j. Cmet.2018.01.015 (2018)) indicate that interactions of various endocrine cells in the islets together affect insulin secretion and regulation of blood glucose, the ratio of alpha cells in the islet cell mass transplanted into a type one diabetic host affects the blood glucose set point after treatment of diabetes, and the blood glucose set point is lower as the alpha cell ratio is higher. Therefore, in order to achieve cell therapy of diabetes, it is of great importance to prepare islet-like cell masses having a cell composition ratio similar to that of human islets. However, all current studies fail to produce both alpha and beta cells in the same culture system at a similar ratio to the actual islet cell population.

The invention discloses a method for combining two digestive enzymes to digest by optimizing culture medium components, which can simultaneously obtain islet-like cells with similar compositions to human islet cells in the same differentiation system, has high yield for 30-33 days, and has good functions compared with other methods by reacting under static and dynamic GSIS. The preliminary results at present show that the islet-like cell mass obtained by the invention can save the blood sugar of mice with type I diabetes, and the experiments are planned to be carried out in large animals so as to lay a foundation for future preclinical researches.

In the development process, the fate determination of beta cells and alpha cells of islet endocrine cell populations is an important scientific problem, and it is currently found that transcription factors PAX4 and ARX jointly control the occurrence of beta cells and alpha cells through mutual antagonism, but the upstream regulation mechanism of the transcription regulation network is still unclear, and the differentiation system can be used as a good in vitro model to help the research of the fate determination mechanism of beta cells and alpha cells.

Currently, studies on islet-like cell differentiation mainly focus on beta cells therein, and only Kieffer methods can realize expression of functional gene MAFA in many studies, and all the obtained beta cells can not respond to glucose stimulation in the period experiment of dynamic GSIS without enrichment and refocusing (rese) and other operations. There is only one study of alpha cell differentiation and the process is long. Therefore, it is of great significance to construct islet-like cell masses that are similar in proportion to real islet cells and functionally equivalent.

Although the existing diabetes treatment is basically monopoly by commercial insulin, and insulin secreted by artificial pancreatic cells has short half-life, and glucagon is rapidly degraded in human body after concentration, so that the glucagon is not used for treating diseases. The invention provides a treatment idea, islet-like cell clusters with secretion function can be obtained through in vitro culture, insulin and a small amount of glucagon can be secreted simultaneously by the islet-like cell clusters, and the islet-like cell clusters are transplanted into a patient, so that the insufficiency of the islet cells can be effectively improved, and adverse reactions caused by improper use of insulin can be avoided.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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Claims

1. A method for preparing an islet-like cell mass, wherein the method induces differentiation of stem cells into an islet-like cell mass by 7 stages (S1-S7), comprising the steps of:

(1) The stem cells are subjected to differentiation treatment in the form of adherent cells in the S1-S4 stage; and

(2) S5-S7, performing differentiation treatment in a cell sphere form on the adherent cells obtained in the step (1);

wherein no PKC signaling pathway activator is added during the S3-S4 differentiation stage of the adherent cell form;

Adding Noggin, nicotinamide and Epidermal Growth Factor (EGF) at the S4 differentiation stage of the adherent cell form;

at the S5 differentiation stage of the pellet form, nicotinamide and Epidermal Growth Factor (EGF) are added;

adding nicotinamide during the S6 differentiation stage of the pellet form;

adding nicotinamide during the S7 differentiation stage of the pellet form;

at the end of the differentiation stage of the adherent cell form, performing adherent cell digestion treatment with a combination of a dispersing enzyme and a pancreatin to disperse the adherent cells into cell clumps;

the digestion treatment process comprises the following steps: the cells were treated with 2mg/mL of the dispersion enzyme for 8min and then with 0.25% pancreatin for 30s.

2. The method of claim 1, wherein the stem cells are selected from the group consisting of: human embryonic stem cells, human induced pluripotent stem cells (hipscs), or combinations thereof.

3. The method of claim 1, wherein the PKC signaling pathway activator is TPB (- (2 s,5 s) - (E, E) -8- (5- (4- (trifluoromethyl) phenyl) -2, 4-pentadienoamino) benzolactam).

4. The method of claim 1, wherein nicotinamide, epidermal Growth Factor (EGF), or a combination thereof is utilized during the differentiation stage of the cell pellet form.

5. An islet-like cell mass obtainable by in vitro differentiation according to the method of any one of claims 1 to 4, characterized in that said islet-like cell mass comprises the following components:

(a) Islet beta cells;

(b) Islet alpha cells; and;

(c) Optionally other endocrine cells, including but not limited to endocrine cells expressing other endocrine hormones such as SST or PPY;

wherein the islet beta cells account for 36.0% -67.7% of the total number of cells in the cell mass;

the islet alpha cells account for 6.33% -34.9% of the total number of cells in the cell mass;

expressing specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8, MAFA and IAPP in the islet beta cells;

based on the expression of at least the specific marker gene, the proportion of beta cells expressing MAFA accounts for 70-85% of all beta cells; the proportion of IAPP-expressing beta cells is 30-60% of the total beta cells.

6. The islet-like cell mass of claim 5, wherein the islet beta cells express specific marker genes PDX1, NKX6.1, INS, ABCC8, MAFB, SLC30A8, MAFA, IAPP, and wherein the proportion of beta cells expressing MAFA comprises 80% of all beta cells and the proportion of beta cells expressing IAPP comprises 50% of all beta cells based on expression of at least the specific marker genes.

7. The islet-like cell mass of claim 5, wherein islet beta cells in the islet-like cell mass comprise 30% -80%; and 6% -40% of islet alpha cells.

8. The islet-like cell mass of claim 7, wherein islet beta cells in the islet-like cell mass comprise 40% -70%; and islet alpha cells account for 10% -35%.

9. The islet-like cell mass of claim 5, wherein the islet-like cell mass expresses the equivalent amount of the endocrine cell marker plurod 1 to islets.

10. The islet-like cell mass of claim 5, wherein the alpha cells express marker gene GCG, ARX, PCSK.

11. The islet-like cell mass of claim 5, wherein the β cells that simultaneously express INS and IAPP comprise from 30% to 45% of all β cells as detected by single cell sequencing; beta cells expressing INS, IAPP and MAFA simultaneously account for 15-30% of all beta cells.

12. The islet-like cell mass of claim 5, wherein the islet-like cell mass is capable of secreting insulin upon stimulation with glucose, and wherein the islet-like cell mass releases at least 1.5 times more human insulin upon stimulation with high glucose than upon stimulation with low glucose.

13. The islet-like cell mass of claim 12, wherein the islet-like cell mass releases at least 2 times more human insulin under high-sugar stimulation than under low-sugar stimulation.

14. The islet-like cell mass of claim 12, wherein the islet-like cell mass releases at least 3 times more human insulin under high-sugar stimulation than under low-sugar stimulation.

15. The islet-like cell mass of claim 12, wherein the islet-like cell mass releases at least 5 times more human insulin under high-sugar stimulation than under low-sugar stimulation.

16. The islet-like cell mass of claim 12, wherein the islet-like cell mass releases at least 10 times more human insulin under high-sugar stimulation than under low-sugar stimulation.

17. The islet-like cell mass of claim 5, wherein the islet-like cell mass releases at least 2 times greater human glucagon under low sugar stimulation than under high sugar stimulation; the low sugar stimulation refers to 1.5mM-3.5 mM glucose; the high sugar stimulation refers to 15 mM-35 mM glucose.

18. The islet-like cell mass of claim 17, wherein the islet-like cell mass releases at least 3 times greater human glucagon upon low sugar stimulation than upon high sugar stimulation.

19. The islet-like cell mass of claim 17, wherein the islet-like cell mass releases at least 4 times greater human glucagon under low sugar stimulation than under high sugar stimulation.

20. The islet-like cell mass of claim 17, wherein the islet-like cell mass releases at least 5 times greater human glucagon under low sugar stimulation than under high sugar stimulation.

21. The islet-like cell mass of claim 17, wherein the low-sugar stimulus is 2 mM glucose.

22. The islet-like cell mass of claim 17, wherein the low-sugar stimulus is 20 mM glucose.

23. The islet-like cell mass of claim 5, wherein the islet-like cell mass is derived via in vitro induced differentiation of stem cells.

24. The islet-like cell mass of claim 23, wherein the stem cell is selected from the group consisting of: human embryonic stem cells, human induced pluripotent stem cells (hipscs), or combinations thereof.

25. A culture system in which primary cells are subjected to differentiation culture to obtain the islet-like cell mass of any one of claims 5 to 24.

26. The culture system of claim 25, wherein the starter cells are selected from the group consisting of: human embryonic stem cells, human induced pluripotent stem cells (hipscs), or combinations thereof.

27. The culture system of claim 25 or 26, wherein the primary cells are differentiated according to the method of claim 1.

28. An islet microenvironment model comprising an islet-like cell mass according to any one of claims 5-24.

29. Use of the islet microenvironment model of claim 28 for modeling the effects and relationships between islet cells in normal physiological function and pathological states of the islets.

30. A pharmaceutical composition comprising the islet-like cell mass of any one of claims 5-24 and a pharmaceutically acceptable excipient.

31. Use of the islet-like cell mass of any one of claims 5-24 for the preparation of an artificial pancreatic device or a medicament for the treatment of diabetes.

32. The use according to claim 31, wherein the diabetes is type I diabetes.

33. An artificial pancreas device comprising a container containing an islet-like cell mass according to any of claims 5 to 24 and/or an islet microenvironment model according to claim 28.

34. A method of obtaining an insulin-containing product, comprising the steps of: (a) Stimulating the islet-like cell mass of any one of claims 5-24 with glucose.

35. The method of claim 34, further comprising the step of (b) isolating and concentrating to obtain an insulin-containing product that can be used directly.

36. The method of claim 34 or 35, wherein the insulin-containing product produced by the method has an insulin content of 50 to 99% wt.

37. The method of claim 34 or 35, wherein the insulin-containing product produced by the method has an insulin content of 75 to 99% wt.

38. A method according to claim 34 or 35, wherein the insulin-containing product produced by the method has an insulin content of 85-99% wt.

39. The method of claim 34 or 35, wherein the method further comprises the step of producing the insulin-containing product in the presence of an amount of glucagon.

40. The method of claim 39, wherein the glucagon is present in an amount of 0.001 to 10% by weight.

41. The method of claim 39, wherein the glucagon is present in an amount of 0.01 to 10% wt.

42. The method of claim 39, wherein the glucagon is present in an amount of 0.1 to 10% wt.