WO2023221438A1

WO2023221438A1 - Method for promoting differentiation of ipsc into islet cell by means of regulating ngn3 expression

Info

Publication number: WO2023221438A1
Application number: PCT/CN2022/133111
Authority: WO
Inventors: 顾雨春; 吴理达; 王贯乔; 安翠平
Original assignee: 呈诺再生医学科技(北京)有限公司
Priority date: 2022-05-20
Filing date: 2022-11-21
Publication date: 2023-11-23
Also published as: CN114703123B; CN114703123A

Abstract

Provided is a method for promoting differentiation of iPSC into an islet cell by means of regulating NGN3 expression. The method promotes the differentiation of iPSC into the islet cell by means of adding recombinant human activin A at a gradient concentration in the process of inducing cell differentiation and adding CK-6666 to up-regulate the NGN3 expression.

Description

A method to promote iPSC differentiation into islet cells by regulating NGN3 expression

Technical field

The invention belongs to the field of biomedicine, and specifically relates to a method for promoting the differentiation of iPSCs into islet cells by regulating NGN3 expression.

Background technique

Pancreatic islet β cells (i.e. islet B cells) are a type of islet cells and are endocrine cells, accounting for about 70% of the total number of islet cells. They are mainly located in the center of the islets and can secrete insulin and regulate blood sugar levels. Impaired pancreatic beta cell function and absolute or relative insufficient insulin secretion can lead to diabetes. The cause of type 1 diabetes is mainly due to a defect in the patient's own immune system that damages the insulin-producing pancreatic beta cells, resulting in the body's inability to produce enough insulin. .

As a method to treat diabetes, islet cell transplantation shows unique advantages over pancreas transplantation and islet transplantation in clinical applications guided by basic research, but its long-term effects are not ideal. Currently, islet cells obtained from brain-dead donors are mainly transplanted to patients. However, due to individual differences, the number of islet cells obtained from each donor is not stable. Generally, it takes 2-3 donors to transplant islet cells to one patient. , and due to problems such as rejection and insufficient blood supply and oxygen supply to the islets, the islet cells transplanted into the body will continue to be lost over time. In other words, islet cell transplantation faces the challenges of poor long-term results and a serious shortage of donor organs.

Therefore, developing methods to differentiate iPSC cells into pancreatic beta cells provides broad prospects for cell therapy in diabetes. Current differentiation protocols typically use various growth factors or small molecules to model pancreatic beta cell development and gradually induce iPSCs through various intermediate states (definitive endoderm, primitive gut, posterior foregut, pancreatic progenitors, and endocrine progenitors) Generates insulin-producing cells (pancreatic beta cells). However, there are currently problems in inducing pancreatic islet β cells, which mainly include: 1. The efficiency of generating β cells is low; 2. The generated cells are usually heterogeneous and contain many unnecessary cell types, including large amounts of insulin and glucagon. Dual hormone-positive cells.

Ngn3 is located on chromosome 10 and is a polypeptide sequence composed of 214 amino acids. It belongs to the basic helix-loop-helix (BHLH) structural class of transcription factors. It is an activator of endocrine stem cell gene transcription. During the development of the pancreas, a small number of pancreatic stem cells in the pancreas express Ngn3, differentiate into islet cells with endocrine effects, and aggregate to form islets. NGN3 is essential for the development and differentiation of pancreatic endocrine cells and is considered a marker protein of pancreatic progenitor cells.

Therefore, it is of great significance to promote the differentiation of iPSCs into islet cells by regulating NGN3 expression, thereby preparing a large number of islet cells and applying them to clinical treatment of diabetes.

Contents of the invention

The purpose of the present invention is to improve the differentiation efficiency of iPSCs into pancreatic progenitor cells by accurately regulating the expression of NGN3, reducing the proportion of dual hormone (insulin and glucagon dual hormone) positive cells, and improving the maturity of pancreatic islet cells.

The induction scheme of the present invention includes 1) Gradient addition of recombinant human Activin A (Activin A). The gradient concentration is 115ng/mL on the first day, 110ng/mL on the second day, and 100ng/mL on the third day, which can effectively increase the concentration of NGN3. expression, increasing the differentiation efficiency of iPSCs into pancreatic progenitor cells; 2) adding CK-666 inhibits cell adhesion, further upregulates NGN3 expression, and promotes iPSC differentiation into islet cells.

Medium combination

In a first aspect, the present invention provides a culture medium combination, which includes the following culture medium:

1) First culture medium A: composed of recombinant human Activin A (Activin A), CHIR-99021, defatted BSA (fetal bovine albumin) (BSA), glucose, glutamine (GlutaMax), sodium bicarbonate, and basic culture base composition;

Preferably, the working concentration of recombinant human activin A is 115ng/mL, the working concentration of CHIR-99021 is 3 μM, the working concentration of fetal bovine albumin is 0.5%, and the working concentration of glucose is 10mM , the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

2) First culture medium B: composed of recombinant human Activin A (Activin A), CHIR-99021, defatted BSA (fetal bovine albumin) (BSA), glucose, glutamine (GlutaMax), sodium bicarbonate, and basic culture base composition;

Preferably, the working concentration of recombinant human activin A is 110ng/mL, the working concentration of CHIR-99021 is 3 μM, the working concentration of fetal bovine albumin is 0.5%, and the working concentration of glucose is 10mM , the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

3) First culture medium C: composed of recombinant human Activin A (Activin A), CHIR-99021, defatted BSA (fetal bovine albumin) (BSA), glucose, glutamine (GlutaMax), sodium bicarbonate, and basic culture base composition;

Preferably, the working concentration of recombinant human activin A is 100ng/mL, the working concentration of CHIR-99021 is 3 μM, the working concentration of fetal bovine albumin is 0.5%, and the working concentration of glucose is 10mM , the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

4) Second medium: composed of ascorbic acid, fibroblast growth factor 7 (FGF-7), defatted BSA (fetal bovine albumin) (BSA), glucose, glutamine (GlutaMax), sodium bicarbonate, and basic medium MCDB131 composition;

Preferably, the working concentration of the ascorbic acid concentration is 0.25mM, the working concentration of the fibroblast growth factor 7 (FGF-7) is 50ng/mL, and the working concentration of the fetal bovine albumin is 0.5%. The working concentration of glucose is 10mM, the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

5) The third medium: composed of ascorbic acid, fibroblast growth factor 7 (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator TPPB, Composed of defatted BSA (fetal bovine albumin) (BSA), glucose, glutamine (GlutaMax), sodium bicarbonate, and basal medium MCDB131;

Preferably, the working concentration of the ascorbic acid concentration is 0.25mM, the working concentration of the fibroblast growth factor 7 (FGF-7) is 50ng/mL, the working concentration of the SANT-1 is 0.25μM, and the visual The working concentration of xanthate is 1 μM, the working concentration of LDN193189 is 100nM, the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%, the working concentration of the protein kinase C activator TPPB is 200nM, The working concentration of fetal bovine albumin is 2%, the working concentration of glucose is 10mM, the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 2.5g/L;

6) The fourth medium: composed of ascorbic acid, fibroblast growth factor 7 (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator TPPB, Composed of defatted BSA (fetal bovine albumin) (BSA), glucose, glutamine (GlutaMax), sodium bicarbonate, and basal medium MCDB131;

Preferably, the working concentration of the ascorbic acid concentration is 0.25mM, the working concentration of the fibroblast growth factor 7 (FGF-7) is 2ng/mL, the working concentration of the SANT-1 is 0.25μM, and the visual The working concentration of xanthate is 1 μM, the working concentration of LDN193189 is 100nM, the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%, the working concentration of the protein kinase C activator TPPB is 200nM, The working concentration of fetal bovine albumin is 2%, the working concentration of glucose is 10mM, the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 2.5g/L;

7) Fifth culture medium: SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, sodium bicarbonate, Composed of glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), and basal medium MCDB131;

Preferably, the working concentration of the SANT-1 concentration is 0.25 μM, the working concentration of the retinoic acid is 0.1 μM, the working concentration of the LDN193189 is 100 nM, and the insulin-transferrin-selenium-ethanolamine additive is The working concentration is 0.5%, the working concentration of triiodothyronine is 1 μM, the working concentration of the ALK5 inhibitor ALK5i II is 10 μM, the working concentration of zinc sulfate is 10 μM, and the working concentration of heparin is 10 μg/mL, the working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin is 2% ;

Preferably, the culture medium combination also includes the following culture media:

8) The sixth medium A: composed of cell adhesion inhibitor CK-666, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, γ-secretase Inhibitor GSi XX, heparin, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), and basal medium MCDB131;

Preferably, the working concentration of CK-666 in the culture medium is 100 μM, the working concentration of LDN193189 is 100 nM; the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%; the triiodothyronine The working concentration of orogenic acid is 1 μM; the working concentration of the ALK5 inhibitor ALK5i II is 10 μM; the working concentration of the zinc sulfate is 10 μM; the working concentration of the γ-secretase inhibitor GSi XX is 100 nM, The working concentration of heparin is 10 μg/mL, the working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin The working concentration is 2%;

9) The sixth medium B: composed of LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, γ-secretase inhibitor GSi XX, heparin, carbonic acid Composed of sodium hydrogen, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), and basal medium MCDB131;

Preferably, the working concentration of LDN193189 in the culture medium is 100 nM; the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%; the working concentration of triiodothyronine is 1 μM; The working concentration of the ALK5 inhibitor ALK5i II is 10 μM; the working concentration of the zinc sulfate is 10 μM; the working concentration of the γ-secretase inhibitor GSi XX is 100 nM, and the working concentration of the heparin concentration is 10 μg/mL, The working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin is 2%;

10) The sixth medium C: composed of LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, sodium bicarbonate, glutamine (GlutaMax) , glucose, defatted BSA (fetal bovine albumin) (BSA), and basal medium MCDB131;

Preferably, the working concentration of LDN193189 is 100 nM; the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%; the working concentration of triiodothyronine is 1 μM; and the ALK5 inhibitor The working concentration of ALK5i II is 10 μM; the working concentration of zinc sulfate is 10 μM; the working concentration of heparin is 10ug/mL, the working concentration of sodium bicarbonate is 1.5g/L, and the working concentration of glutamine The concentration is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin is 2%;

Preferably, the culture medium combination also includes a seventh culture medium, which is composed of insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, Heparin, acetyl-l-cysteine (N-Cys), water-soluble vitamin E and Axl inhibitor R428, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA) , composed of basal culture medium MCDB131.

Preferably, the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%, the working concentration of the triiodothyronine is 1 μM, and the working concentration of the ALK5 inhibitor ALK5i II is 10 μM, so The working concentration of zinc sulfate is 10 μM, the working concentration of heparin is 10 μg/mL, the working concentration of acetyl-l-cysteine (N-Cys) is 1 mM, and the working concentration of water-soluble vitamin E is 10 μM, the working concentration of the Axl inhibitor R428 is 2 μM, the working concentration of the sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, and the working concentration of the glucose is 20mM, so The working concentration of fetal bovine albumin is 2%.

Preferably, in any of the above culture media, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), and basal culture medium MCDB131 are common culture medium compositions. Common sense can be freely replaced and adjusted to other similar components.

Preferably, the basal medium MCDB131 of the present invention is a glutamine-free medium, and more specifically, the product used in the embodiment of the present invention is Gibco product No. 10372019. The MCDB131 culture medium does not contain protein or growth factors, and those skilled in the art can purchase commercial culture media or prepare it by themselves.

Reagent test kit

On the other hand, the present invention provides a kit, which contains reagents required for preparing any one of the aforementioned media or a combination of media composed of multiple media.

Preferably, the kit includes any one or more of the following reagents:

Recombinant human Activin A (Activin A), CHIR-99021, ascorbic acid, fibroblast growth factor 7 (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive , protein kinase C activator TPPB, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, CK-666, γ-secretase inhibitor GSi XX, acetyl-l-cysteine (N- Cys), vitamin E, R428, BSA, glucose, glutamine, sodium bicarbonate.

application

On the other hand, the present invention provides the application of the aforementioned culture medium combination and the aforementioned kit in inducing differentiation of stem cells into mature or immature pancreatic β cells.

On the other hand, the present invention provides cell adhesion inhibitor CK-666 and recombinant human activin A for inducing mature or immature pancreatic beta cells, increasing the expression of marker genes of pancreatic progenitor cells, and increasing the expression of marker genes of pancreatic beta cells. applications in.

Preferably, the marker gene of pancreatic progenitor cells includes NGN3.

Preferably, the marker genes of pancreatic β cells include GCG, INS, MAFA, and NKX6.1.

As described in the present invention, "stem cells" (SC) are a type of cells with self-renewing ability and multi-directional differentiation potential. Under certain conditions, they can differentiate into a variety of functional cells. According to the developmental stage of stem cells, they are divided into embryonic stem cells (ES cells) and adult stem cells (somatic stem cells). Stem cells are divided into three categories according to their developmental potential: totipotent stem cells (TSC), pluripotent stem cells (pluripotent stem cells) and unipotent stem cells (unipotent stem cells). According to the function of adult stem cells, they can be divided into neural stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, skin stem cells, adipose stem cells, etc. The sources of stem cells are embryos, bone marrow, peripheral blood, umbilical cord and umbilical cord blood.

Stem cells. In one embodiment, the iPSCs can be commercial cell lines or can be induced from donor cells. The sources of the donor cells include villus cells, skin (fibroblasts and keratinocytes). ), amniotic fluid, extraembryonic tissue (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, amniotic membrane-derived mesenchymal stem cells, amniotic membrane-derived peripheral blood cells, mammary epithelial cells, adipose stem cells, One or more of umbilical cord matrix and placenta.

Preferably, the stem cells are iPSC cells.

method

On the other hand, the present invention also provides a method for inducing mature or immature pancreatic beta cells, the method comprising at least one of the following steps:

1) Use the aforementioned first culture medium A to culture for 1 day,

2) Use the aforementioned first culture medium B to culture for 1 day,

3) Use the aforementioned first medium C to culture for 1 day,

4) Use the aforementioned second culture medium to culture for 2 days,

5) Use the aforementioned third culture medium to culture for 2 days,

6) Use the aforementioned fourth culture medium to culture for 3 days,

7) Use the aforementioned fifth culture medium to culture for 3 days,

8) Use the aforementioned sixth culture medium A to culture for 1 day,

9) Use the aforementioned sixth culture medium B to culture for 6 days,

10) Use the aforementioned sixth culture medium C to culture for 7 days,

Preferably, the method for inducing mature pancreatic β cells further includes culturing for 15 days using the aforementioned seventh culture medium.

Preferably, the culture time only gives an optimal solution, and those skilled in the art can adjust it according to the state of the cells.

Preferably, the mature pancreatic β cells are mature pancreatic β cells with high expression of NGN3.

More preferably, the mature pancreatic β cells are mature pancreatic β cells with high expression of GCG, INS, MAFA, and NKX6.1.

Preferably, the expression includes mRNA expression or protein expression; as Example 1 demonstrates, the expression of the marker mRNA is increased.

Preferably, the mature or immature pancreatic beta cells are derived from iPSC cells.

Preferably, the iPSC cells are obtained by the following methods:

1) After the iPSC polymerization degree reaches 70–80%, wash it twice with DPBS and then digest it with TrypLE Express;

2) Neutralize with DMEM/F12 at a ratio of 5:1 and then centrifuge;

3) Resuspend and seed in E8 complete medium + 10 μM Y-27632, and coat the cell plate with Matrigel in advance.

More specific iPSC cell processing methods are as applied in specific embodiments of the present invention.

Preferably, the method is a method of inducing pancreatic beta cells that highly express NGN3.

Preferably, the medium needs to be changed every day in the method.

The culture environment of mammalian cells is a well-known technology in the art. Standard mammalian cell culture conditions can be used in the above culture steps, such as 37°C, 21% oxygen, and 5% carbon dioxide.

cell

On the other hand, the present invention provides a cell that highly expresses NGN3 prepared by the aforementioned method.

Preferably, the cells are mature or immature pancreatic beta cells.

On the other hand, the present invention provides the use of the above-mentioned cells that highly express NGN3 in preparing drugs for treating diabetes.

Preferably, the diabetes is type 1 diabetes.

On the other hand, the present invention provides a method for treating diabetes, which method includes administering the above-mentioned NGN3-highly expressing cells to a patient.

Description of the drawings

Figure 1 is a summary of culture media used in the present invention.

Figure 2 is a flow chart for inducing pancreatic beta cells according to the present invention.

Figure 3 is a graph comparing the effects of using recombinant human activin A or not using recombinant human activin A on the expression of NGN3.

Figure 4 is a comparison of the effects of using CK-666 or not using CK-666 on NGN3 expression.

Figure 5 is a statistical result chart showing the effects of using recombinant human activin A and CK-666 simultaneously on pancreatic β-cell markers GCG, INS, MAFA, and NKX6.1. A: GCG, B: INS, C: MAFA, D: NKX6 .1.

Detailed ways

The present invention will be further described below in conjunction with the examples. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled person familiar with the art may make use of the technical content disclosed above. Changes to equivalent embodiments of equivalent variations. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention fall within the protection scope of the present invention.

Example 1. Comparison of different methods for inducing islet cell differentiation

The experimental materials involved in the present invention are shown in Table 1 below:

Table 1. Reagents used in the present invention

Method 1: Combination of recombinant human activin A (gradient) and CK-666

(The culture media statistics involved are shown in Figure 1, and the process is shown in Figure 2)

After the iPSCs polymerization degree reached 70–80%, they were washed twice with DPBS and then digested with TrypLE Express for 3–5 min at 37°C. Neutralize with DMEM/F12 at a ratio of 5:1 and centrifuge at 200g for 5 minutes. Resuspend and seed in E8 complete medium + 10 μM Y-27632, with a density of about 1.8-2.2×10 ⁵ cells/cm ² . The 12-well cell plate is coated with 0.13-0.2mg/mL Matrigel 24-48 hours in advance.

The first stage

iPSCs (prepared according to the method in the company's previous patent application 2019101107687, the full text of 2019101107687 is incorporated herein by reference.) was differentiated into definitive endoderm by basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, and defatted BSA (Fetal bovine albumin) (BSA), recombinant human Activin A (Activin A) and CHIR-99021 (referred to as the first culture medium or stage one culture medium in the present invention).

In this medium: the concentrations of recombinant human activin A are 115ng/mL (stage one medium A), 110ng/mL (stage one medium B), and 100ng/mL (stage one medium C), CHIR-99021 It is 3 μM, fetal bovine albumin is 0.5%, glucose is 10mM, glutamine is 2mM, and sodium bicarbonate is 1.5g/L.

The first stage of induction lasts for 3 days. On the first day of induction, use Stage 1 medium A; on the 2nd day of induction, use Stage 1 medium B; on the 3rd day of induction, use Stage 1 medium C. Change the fresh medium every day.

second stage

To induce the differentiation of definitive endoderm cells into primitive intestinal tubes, the basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), ascorbic acid and fibroblast growth factor 7 ( FGF-7) (referred to as the second medium or stage two medium in the present invention).

In this medium: the concentration of ascorbic acid is 0.25mM, fibroblast growth factor 7 (FGF-7) is 50ng/mL, fetal bovine albumin is 0.5%, glucose is 10mM, glutamine is 2mM, and sodium bicarbonate is 1.5g/L.

The duration of the second stage of induction is 2 days. On the 4th to 5th day of induction, fresh medium is replaced every day.

The third phase

To induce the differentiation of primitive intestinal tube cells into posterior foregut cells, the basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), ascorbic acid, and fibroblast growth factor 7 (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive and protein kinase C activator TPPB (referred to as the third medium or stage three medium in the present invention ).

In this medium: Ascorbic acid concentration is 0.25mM, Fibroblast Growth Factor 7 (FGF-7) is 50ng/mL, SANT-1 is 0.25μM, Retinoic acid is 1μM, LDN193189 is 100nM, Insulin-Transferrin - Selenium-ethanolamine additive is 0.5%, protein kinase C activator TPPB is 200nM, fetal bovine albumin is 2%, glucose is 10mM, glutamine is 2mM, and sodium bicarbonate is 2.5g/L.

In the third stage, the induction duration is 2 days. On the 6th to 7th day of induction, fresh medium is replaced every day.

Stage 4

After induction, the foregut cells differentiate into pancreatic progenitor cells, which is composed of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), ascorbic acid, and fibroblast growth factor 7. (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive and protein kinase C activator TPPB (referred to as the fourth medium or stage four medium in the present invention ).

In this medium: Ascorbic acid concentration is 0.25mM, Fibroblast Growth Factor 7 (FGF-7) is 2ng/mL, SANT-1 is 0.25μM, Retinoic acid is 1μM, LDN193189 is 100nM, Insulin-Transferrin - Selenium-ethanolamine additive is 0.5%, protein kinase C activator TPPB is 200nM, fetal bovine albumin is 2%, glucose is 10mM, glutamine is 2mM, and sodium bicarbonate is 2.5g/L.

The duration of the fourth stage of induction is 3 days. On the 8th to 10th day of induction, fresh medium is replaced every day.

fifth stage

Differentiation from pancreatic progenitor cells to endocrine progenitor cells, consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), SANT-1, retinoic acid, LDN193189 , insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate and heparin (referred to as the fifth culture medium or stage five culture medium in the present invention).

In this medium: SANT-1 concentration is 0.25 μM, retinoic acid is 0.1 μM, LDN193189 is 100 nM, insulin-transferrin-selenium-ethanolamine additive is 0.5%, triiodothyronine is 1 μM, ALK5 inhibition ALK5i II is 10 μM, zinc sulfate is 10 μM, heparin is 10ug/mL, sodium bicarbonate is 1.5g/L, glutamine is 2mM, glucose is 20mM, and fetal bovine albumin is 2%.

The duration of the fifth stage of induction is 3 days. On the 11th to 13th day of induction, fresh medium is replaced every day.

Stage six

To induce the differentiation of endocrine progenitor cells into immature beta cells, the basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), cell adhesion inhibitor CK-666, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, γ-secretase inhibitor GSi XX and heparin (referred to as the sixth culture in the present invention base or stage six medium).

In this medium: CK-666 is 100 μM, LDN193189 is 100 nM, insulin-transferrin-selenium-ethanolamine additive is 0.5%, triiodothyronine is 1 μM, ALK5 inhibitor ALK5i II is 10 μM, and zinc sulfate is 10 μM, γ-secretase inhibitor GSi XX is 100 nM, heparin is 10ug/mL, sodium bicarbonate is 1.5g/L, glutamine is 2mM, glucose is 20mM, and fetal bovine albumin is 2%.

The duration of the sixth phase of induction is 14 days.

On the 14th day of induction, the concentration of CK-666 in the culture medium was 100 μM, the concentration of LDN193189 was 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%; the concentration of triiodothyronine was 1 μM; The concentration of ALK5 inhibitor ALK5i II is 10 μM; the concentration of zinc sulfate is 10 μM; the concentration of γ-secretase inhibitor GSi XX is 100 nM, and the concentration of heparin is 10ug/mL. On the 15th day of induction, cells were plated into low-affinity 6-well culture plates using cell scrapers.

On days 15-20 of induction, the concentration of LDN193189 in the culture medium was 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%; the concentration of triiodothyronine was 1 μM; the concentration of ALK5 inhibitor ALK5i II The concentration is 10 μM; the concentration of zinc sulfate is 10 μM; the γ-secretase inhibitor GSi XX is 100 nM, and the concentration of heparin is 10ug/mL.

On days 21-27 of induction, the concentration of LDN193189 in the culture medium was 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%; the concentration of triiodothyronine was 1 μM; the concentration of ALK5 inhibitor ALK5i II The concentration is 10μM; the concentration of zinc sulfate is 10μM; the concentration of heparin is 10ug/mL, that is: use stage 6 medium A on the 14th day of induction, use stage 6 medium B on days 15-20 of induction, and use stage 6 medium B on days 21-20 of induction. Use stage six medium C for 27 days and replace with fresh medium every day.

Stage 7

To induce the differentiation of immature beta cells into mature beta cells, the basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine albumin) (BSA), insulin-transferrin-selenium- Ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, acetyl-l-cysteine (N-Cys), water-soluble vitamin E and Axl inhibitor R428 (in the present invention Called seventh medium or stage seven medium).

In this medium: insulin-transferrin-selenium-ethanolamine additive is 0.5%, triiodothyronine is 1μM, ALK5 inhibitor ALK5i II is 10μM, zinc sulfate is 10μM, heparin is 10ug/mL, acetyl- l-cysteine (N-Cys) is 1mM, water-soluble vitamin E is 10μM, Axl inhibitor R428 is 2μM, sodium bicarbonate is 1.5g/L, glutamine is 2mM, glucose 20mM, fetal bovine albumin is 2%.

The duration of the seventh stage of induction is 14 days. On the 28th to 35th day of induction, fresh medium is replaced every day.

Method 2: Recombinant human activin A (gradient) acts alone

Stages 1 to 4, 5, and 7 are the same as method 1, and CK-666 is not added in the sixth stage.

Method 3: Using fixed concentrations of recombinant human activin A and CK-666

The second to seventh stages are the same as method 1. In the first stage, recombinant human activin A1 is added at a fixed concentration of 100ng/mL.

Method 4: Control group

In the first stage, recombinant human activin A was added at a fixed concentration of 100ng/mL, and in the sixth stage, CK-666 was not added.

Compare cells prepared by the above different methods

1. Prepare cells according to method 2 and method 4 respectively, and detect the expression level of NGN3 on the 13th day of culture (that is, the completion of the first 5 stages of culture). The results are shown in Figure 3:

The expression of the pancreatic progenitor cell marker gene NGN3 in the recombinant human activin A gradient group increased by about 4.5 times. From this, we can conclude that the gradient addition of recombinant human activin A in the first stage of induction can significantly increase the expression level of NGN3 and promote iPSCs to Pancreatic progenitor cell differentiation;

2. Prepare cells according to methods three and four respectively, and detect the NGN3 expression level on the 13th day of culture (that is, the completion of the first five stages of culture). The results are shown in Figure 4:

The NGN3 mRNA level in the CK-666-treated group was 2.5 times higher than that in the non-CK-666-treated group. From this, we can conclude that on induction Day 14, using 100 μM CK-666 for 24 hours can significantly increase the expression level of NGN3 and promote iPSC transfer to the pancreas. progenitor cell differentiation;

3. Compare the cells prepared by Method 1 and Method 4, and detect the expression levels of GCG, INS, MAFA, and NKX6.1 mRNA. The results are shown in Figure 5:

After gradient action of recombinant human activin A combined with CK-666 induction until Day35, the expression levels of INS, GCG, MAFA, and NKX6.1 mRNA were all higher than those in the group without CK-666, increasing by 2.2 times, 1.6 times, 22 times, and 123 times respectively. times.

It is concluded that adding recombinant human activin A in the induction Day1-3 gradient and using 100 μM CK-666 for 24 hours on Day14 can promote islet cell maturation.

Claims

A culture medium combination, the culture medium combination includes the following culture media:

1) First culture medium A: composed of recombinant human activin A, CHIR-99021, fetal bovine albumin, glucose, glutamine, sodium bicarbonate, and basic culture medium;

Preferably, the working concentration of recombinant human activin A is 115ng/mL, the working concentration of CHIR-99021 is 3 μM, the working concentration of fetal bovine albumin is 0.5%, and the working concentration of glucose is 10mM , the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

2) First culture medium B: composed of recombinant human activin A, CHIR-99021, fetal bovine albumin, glucose, glutamine, sodium bicarbonate, and basic culture medium;

Preferably, the working concentration of recombinant human activin A is 110ng/mL, the working concentration of CHIR-99021 is 3 μM, the working concentration of fetal bovine albumin is 0.5%, and the working concentration of glucose is 10mM , the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

3) First culture medium C: composed of recombinant human activin A, CHIR-99021, fetal bovine albumin, glucose, glutamine, sodium bicarbonate, and basic culture medium;

Preferably, the working concentration of recombinant human activin A is 100ng/mL, the working concentration of CHIR-99021 is 3 μM, the working concentration of fetal bovine albumin is 0.5%, and the working concentration of glucose is 10mM , the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

4) Second culture medium: composed of ascorbic acid, FGF-7, fetal bovine albumin, glucose, glutamine, sodium bicarbonate, and basal culture medium;

Preferably, the working concentration of the ascorbic acid concentration is 0.25mM, the working concentration of the FGF-7 is 50ng/mL, the working concentration of the fetal bovine albumin is 0.5%, and the working concentration of the glucose is 10mM, so The working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 1.5g/L;

5) The third culture medium: composed of ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator TPPB, fetal bovine albumin, glucose, glutathione Composed of aminoamide, sodium bicarbonate, and basic culture medium;

Preferably, the working concentration of the ascorbic acid concentration is 0.25mM, the working concentration of the FGF-7 is 50ng/mL, the working concentration of the SANT-1 is 0.25μM, and the working concentration of the retinoic acid is 1μM, The working concentration of LDN193189 is 100nM, the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%, the working concentration of the protein kinase C activator TPPB is 200nM, and the working concentration of fetal bovine albumin The concentration is 2%, the working concentration of glucose is 10mM, the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 2.5g/L;

6) The fourth medium: composed of ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator TPPB, fetal bovine albumin, glucose, glutathione Composed of aminoamide, sodium bicarbonate, and basic culture medium;

Preferably, the working concentration of the ascorbic acid concentration is 0.25mM, the working concentration of the FGF-7 is 2ng/mL, the working concentration of the SANT-1 is 0.25μM, and the working concentration of the retinoic acid is 1μM, The working concentration of LDN193189 is 100nM, the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%, the working concentration of the protein kinase C activator TPPB is 200nM, and the working concentration of fetal bovine albumin The concentration is 2%, the working concentration of glucose is 10mM, the working concentration of glutamine is 2mM, and the working concentration of sodium bicarbonate is 2.5g/L;

7) Fifth culture medium: SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, sodium bicarbonate, Composed of glutamine, glucose, fetal bovine albumin, and basal culture medium;

Preferably, the working concentration of the SANT-1 concentration is 0.25 μM, the working concentration of the retinoic acid is 0.1 μM, the working concentration of the LDN193189 is 100 nM, and the insulin-transferrin-selenium-ethanolamine additive is The working concentration is 0.5%, the working concentration of triiodothyronine is 1 μM, the working concentration of the ALK5 inhibitor ALK5i II is 10 μM, the working concentration of zinc sulfate is 10 μM, and the working concentration of heparin is 10 μg/mL, the working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin is 2% ;

Preferably, the culture medium combination also includes the following culture media:

8) The sixth medium A: composed of cell adhesion inhibitor CK-666, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, γ-secretase Inhibitor GSi XX, heparin, sodium bicarbonate, glutamine, glucose, fetal bovine albumin, and basal culture medium;

Preferably, the working concentration of CK-666 in the culture medium is 100 μM, the working concentration of LDN193189 is 100 nM; the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%; the triiodothyronine The working concentration of orogenic acid is 1 μM; the working concentration of the ALK5 inhibitor ALK5i II is 10 μM; the working concentration of the zinc sulfate is 10 μM; the working concentration of the γ-secretase inhibitor GSi XX is 100 nM, The working concentration of heparin is 10 μg/mL, the working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin The working concentration is 2%;

9) The sixth medium B: composed of LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, γ-secretase inhibitor GSi XX, heparin, carbonic acid Composed of sodium hydrogen, glutamine, glucose, fetal bovine albumin, and basal culture medium;

Preferably, the working concentration of LDN193189 in the culture medium is 100 nM; the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%; the working concentration of triiodothyronine is 1 μM; The working concentration of the ALK5 inhibitor ALK5i II is 10 μM; the working concentration of the zinc sulfate is 10 μM; the working concentration of the γ-secretase inhibitor GSi XX is 100 nM, and the working concentration of the heparin concentration is 10 μg/mL, The working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin is 2%;

10) The sixth medium C: composed of LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, sodium bicarbonate, glutamine, glucose, Composed of fetal bovine albumin and basal culture medium;

Preferably, the working concentration of LDN193189 is 100 nM; the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%; the working concentration of triiodothyronine is 1 μM; and the ALK5 inhibitor The working concentration of ALK5i II is 10 μM; the working concentration of zinc sulfate is 10 μM; the working concentration of heparin is 10ug/mL, the working concentration of sodium bicarbonate is 1.5g/L, and the working concentration of glutamine The concentration is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin is 2%;

More preferably, the culture medium combination also includes a seventh culture medium, the seventh culture medium: consisting of insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, sulfuric acid Composed of zinc, heparin, acetyl-l-cysteine, water-soluble vitamin E and Axl inhibitor R428, sodium bicarbonate, glutamine, glucose, fetal bovine albumin, and basal culture medium;

Preferably, the working concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%, the working concentration of the triiodothyronine is 1 μM, and the working concentration of the ALK5 inhibitor ALK5i II is 10 μM, so The working concentration of zinc sulfate is 10 μM, the working concentration of heparin is 10 μg/mL, the working concentration of acetyl-l-cysteine is 1 mM, the working concentration of water-soluble vitamin E is 10 μM, and The working concentration of Axl inhibitor R428 is 2 μM, the working concentration of sodium bicarbonate is 1.5g/L, the working concentration of glutamine is 2mM, the working concentration of glucose is 20mM, and the working concentration of fetal bovine albumin The working concentration is 2%;

Preferably, the basal medium is MCDB131.
A kit containing reagents required for preparing the culture medium combination of claim 1.
The application of the culture medium combination according to claim 1 and the kit according to claim 2 in inducing differentiation of stem cells into mature or immature pancreatic beta cells.
The application of cell adhesion inhibitor CK-666 and recombinant human activin A in inducing mature or immature pancreatic beta cells, increasing the expression of marker genes of pancreatic progenitor cells, and increasing the expression of marker genes of pancreatic beta cells.
The application according to claim 4, wherein the marker gene of pancreatic progenitor cells includes NGN3;

Preferably, the marker genes of pancreatic β cells include GCG, INS, MAFA, and NKX6.1;

Preferably, the stem cells are iPSC cells.
A method for inducing mature or immature pancreatic beta cells, the method comprising at least one of the following steps:

1) Cultivate for 1 day using the first culture medium A described in claim 1,

2) Cultivate for 1 day using the first culture medium B described in claim 1,

3) Cultivate for 1 day using the first culture medium C described in claim 1,

4) Cultivate for 2 days using the second culture medium described in claim 1,

5) Cultivate for 2 days using the third culture medium described in claim 1,

6) Cultivate for 3 days using the fourth culture medium described in claim 1,

7) Cultivate for 3 days using the fifth culture medium described in claim 1,

8) Cultivate for 1 day using the sixth culture medium A described in claim 1,

9) Cultivate for 6 days using the sixth culture medium B described in claim 1,

10) Cultivate using the sixth culture medium C of claim 1 for 7 days.
The method of claim 6, wherein the method of inducing mature pancreatic beta cells further includes culturing for 15 days using the seventh culture medium of claim 1;

Preferably, the mature pancreatic beta cells are pancreatic beta cells with high NGN3 expression;

More preferably, the mature pancreatic beta cells are mature pancreatic beta cells with high expression of GCG, INS, MAFA, and NKX6.1;

Preferably, the expression includes mRNA expression level or protein expression level.
The method of claim 6, wherein the mature or immature pancreatic beta cells are derived from stem cells;

Preferably, the stem cells include embryonic stem cells and adult stem cells;

Preferably, the stem cells include totipotent stem cells, multipotent stem cells and unipotent stem cells;

Preferably, the stem cells include neural stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, skin stem cells, adipose stem cells, etc.;

Preferably, the source of the stem cells includes embryos, bone marrow, peripheral blood, umbilical cord and umbilical cord blood;

Preferably, the stem cells can be commercial cell lines, or can be induced from donor cells. The sources of the donor cells include villus cells, skin, amniotic fluid, extraembryonic tissues (placenta and umbilical cord), umbilical cord One or more of blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, amniotic membrane-derived mesenchymal stem cells, amniotic membrane-derived peripheral blood cells, mammary epithelial cells, adipose stem cells, umbilical cord matrix and placenta;

Preferably, the stem cells are iPSC cells;

More preferably, the iPSC cells are obtained by the following methods:

1) After the iPSC polymerization degree reaches 70-80%, wash it twice with DPBS, and then digest it with TrypLE Express;

2) Neutralize with DMEM/F12 at a ratio of 5:1 and then centrifuge;

3) Resuspend and seed in E8 complete medium + 10 μM Y-27632, and coat the cell plate with Matrigel in advance.
A cell highly expressing NGN3 prepared by any method of claims 6-8;

Preferably, the cells are mature or immature pancreatic beta cells.
The use of the cells described in claim 9 in the preparation of products for treating diabetes;

Preferably, the diabetes is type 1 diabetes.